About this Author
Derek Lowe, an Arkansan by birth, got his BA from Hendrix College and his PhD in organic chemistry from Duke before spending time in Germany on a Humboldt Fellowship on his post-doc. He's worked for several major pharmaceutical companies since 1989 on drug discovery projects against schizophrenia, Alzheimer's, diabetes, osteoporosis and other diseases.
To contact Derek email him directly: derekb.lowe@gmail.com
Twitter: Dereklowe
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Category Archives
November 5, 2009
Posted by Derek
Resveratrol's a mighty interesting compound. It seems to extend lifespan in yeast and various lower organisms, and has a wide range of effects in mice. Famously, GlaxoSmithKline has expensively bought out Sirtris, a company whose entire research program started with resveratrol and similar compound that modulate the SIRT1 pathway.
But does it really do that? The picture just got even more complicated. A group at Amgen has published a paper saying that when you look closely, resveratrol doesn't directly affect SIRT1 at all. Interestingly, this conclusion has been reached before (by a group at the University of Washington), and both teams conclude that the problem is the fluorescent peptide substrate commonly used in sirtuin assays. With the fluorescent group attached, everything looks fine - but when you go to the extra trouble of reading things out without the fluorescent tag, you find that resveratrol doesn't seem to make SIRT1 do anything to what are supposed to be its natural substrates.
"The claim of resvertraol being a SIRT1 activator is likely to be an experimental artifact of the SIRT1 assay that employs the Fluor de Lys-SIRT1 peptide as a substrate. However, the beneficial metabolic effects of resveratrol have been clearly demonstrated in diabetic animal models. Our data do not support the notion that these metabolic effects are mediated by direct SIRT1 activation. Rather, they could be mediated by other mechanisms. . ."
They suggest activation of AMPK (an important regulatory kinase that's tied in with SIRT1) as one such mechanism, but admit that they have no idea how resveratrol might activate it. Does that process still require SIRT1 at all? Who knows? One thing I think I do know is that this has something to do with this Amgen paper from 2008 on new high-throughput assays for sirtuin enzymes.
One wonders what assay formats Sirtris has been using to evaluate their new compounds, and one also wonders what they make of all this now at GSK. Does one not? We can be sure, though, that there are plenty of important things that we don't know yet about sirtuins and the compounds that affect them. It's going to be quite a ride as we find them out, too.
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+ TrackBacks (0) | Category: Aging and Lifespan | Biological News | Drug Assays
October 28, 2009
Posted by Derek
Now here's a completely weird idea: a group in Korea has encapsulated individual living yeast cells in silica. They start out by coating the cells with some charged polymers that are known to serve as a good substrate for silication, and then expose the yeast to silicic acid solution. They end up with hard-shell yeast, sort of halfway to being a bizarre sort of diatom.
The encapsulated cells behave rather differently, as no doubt would we all under such conditions. After thirty days in the cold with no nutrients, the silica-coated yeast is at least three times more viable than wild-type cells (as determined by fluorescent staining). On the other hand, when exposed to a warm nutrient broth, the silica-coated yeast does not divide, as opposed to wild-type yeast, which of course takes off like a rocket under such conditions. They're still alive, but just sitting around - which makes you wonder what signals, exactly, are interrupting mitosis.
The authors tried the same trick on E. coli bacteria, but found that the initial polymer coating step killed them off. That's disappointing, but not surprising, given that disruption of the bacterial membrane with charged species is the mode of action of several broad-spectrum antibiotics.
"Hmmm. . .so what?" might be one reaction to this work. But stop and think about it for a minute. This provides a new means to an biological/inorganic interface, a way to stich cell biology and chemical nanotechnology together. If you can layer yeast cells with silica and they survive (and are, in fact, fairly robust), you can imagine gaining more control over the process and extending it to other substances. A layer that could at least partially conduct electricity would be very interesting, as would layers with various-sized pores built into them. The surfaces could be further functionalized with all sorts of other molecules as well for more elaborate experiments. No, this could keep a lot of people busy for a long time, and I suspect it will.
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+ TrackBacks (0) | Category: Biological News
October 16, 2009
Posted by Derek
There have been several reports over the years of people engineering receptor proteins to make them do defined tasks. They've generally been using the bacterial periplasmic binding proteins (PBPs) as a starting point, attaching some sort of fluorescent group onto one end, so that when a desired ligand binds, the protein folds in on itself in a way to set off a fluorescent resonance energy transfer (FRET). That's a commonly used technique to see if two proteins are in close proximity to each other; it's robust enough to be used in many high-throughput screening assays.
So the readout isn't the problem. But something else certainly is. In a new PNAS paper, a group at the Max Planck Institute in Tübingen has gone back and taken a look at these receptors, which are reported to bind a number of interesting ligands such as serotonin, lactate, and even TNT and a model for nerve gas agents. You can see the forensic applications for those latter two if the technique worked well, and the press releases were rather breathless, as they tend to be. But not only did these workers claim a very interesting sensor system, but they also went out of their way to emphasize that they arrived at these results computationally:
Computational design offers enormous generality for engineering protein structure and function. Here we present a structure-based computational method that can drastically redesign protein ligand-binding specificities. This method was used to construct soluble receptors that bind trinitrotoluene, l-lactate or serotonin with high selectivity and affinity. These engineered receptors can function as biosensors for their new ligands; we also incorporated them into synthetic bacterial signal transduction pathways, regulating gene expression in response to extracellular trinitrotoluene or l-lactate. The use of various ligands and proteins shows that a high degree of control over biomolecular recognition has been established computationally.
The Max Planck group would like to disagree with that. Their PNAS paper is entitled "Computational Design of Ligand Binding is Not a Solved Problem". They were able to get crystals of the serotonin-binding protein, but could not get any X-ray structures that showed any serotonin binding in the putative ligand pocket. They then turned to a well-known suite of techniques to characterize ligand binding. One of these is thermal stability: when a protein is binding a high-affinity ligand, it tends to show a higher melting point, since its structure is often more settled-down than the open form. None of the reported receptors showed any such behavior, and all of them were substantially less thermally stable than the wild-type proteins. Strike one.
They then tried ITC, a calorimetry measurement to look for heat of binding. A favorable binding event releases heat - it's a lower-energy state - but none of the engineered receptors showed any changes at all when their supposed ligands were introduced. Strike two. And finally, they turned to NMR experiments, which are widely used to determine protein structure and characterize binding of small molecules. WIld-type proteins of this sort showed exactly what they should have: big conformational changes when their ligands were present. But the engineered proteins showed almost no changes at all. Strike three, and as far as I'm concerned, these pieces of evidence absolutely close the case. These so-called receptors aren't binding anything.
So why do they show FRET signals? The authors suggest that this is some sort of artifact, not related to real receptor binding and note dryly that "Our analysis shows the importance of experimental and structural validation to improve computational design methodologies".
I should also note a very interesting sidelight: the same original research group also published a paper in Science on turning these computationally engineered PBPs into a functional enzyme. Unfortunately, this was retracted last year, when it turned out that the work could not be reproduced. Some wild-type enzyme was still present as an impurity, and when the engineered protein was rigorously purified, the activity went away. (Update: more on this retraction here, and there is indeed more to it). It appears that some other results from this work may be going away now, too. . .
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+ TrackBacks (0) | Category: Biological News
October 7, 2009
Posted by Derek
This was another Biology-for-Chemistry year for the Nobel Committee. Venkatraman Ramakrishnan (Cambridge), Thomas Steitz (Yale) and Ada Yonath (Weizmann Inst.) have won for X-ray crystallographic studies of the ribosome.
Ribosomes are indeed significant, to put it lightly. For those outside the field, these are the complex machines that ratchet along a strand of messenger RNA, reading off its three-letter codons, matching these with the appropriate transfer RNA that's bringing in an amino acid, then attaching that amino acid to the growing protein chain that emerges from the other side. This is where the cell biology rubber hits the road, where the process moves from nucleic acids (DNA going to RNA) and into the world of proteins, the fundamental working units of a day-to-day living cell.
The ribosome has a lot of work to do, and it does it spectacularly quickly and well. It's been obvious for decades that there was a lot of finely balanced stuff going on there. Some of the three-letter codons (and some of the tRNAs) look very much like some of the others, so the accuracy of the whole process is very impressive. If more proofs were needed, it turned out that several antibiotics worked by disrupting the process in bacteria, which showed that a relatively small molecule could throw a wrench into this much larger machinery.
Ribosomes are made out of smaller subunits. A huge amount of work in the earlier days of molecular biology showed that the smaller subunit (known as 30S for how it spun down in a centrifuge tube) seemed to be involved in reading the mRNA, and the larger subunit (50S) was where the protein synthesis was taking place. Most of this work was done on bacterial ribosomes, which are relatively easy to get ahold of. They work in the same fashion as those in higher organisms, but have enough key differences to make them of interest by themselves (see below).
During the 1980s and early 1990s, Yonath and her collaborators turned out the first X-ray structures of any of the ribosomal subunits. Fuzzy and primitive by today's standards, those first data sets got better year by year, thanks in part to techniques that her group worked out first. (The use of CCD detectors for X-ray crystallography, a technology that was behind part of Tuesday's Nobel in Physics, was another big help, as was the development of much brighter and more focused X-ray sources). Later in the 1990s, Steitz and Ramakrishnan both led teams that produced much higher-resolution structures of various ribosomal subunits, and solved what's known as the "phase problem" for these. That's a key to really reconstructing the structure of a complex molecule from X-ray data, and it is very much nontrivial as you start heading into territory like this. (If you want more on the phase problem, here's a thorough and comprehensive teaching site on X-ray crystallography from Cambridge itself).
By the early 2000s, all three groups were turning out ever-sharper X-ray structures of different ribosomal subunits from various organisms. The illustration above, courtesy of the Nobel folks, shows the 50S subunit at 9-angstrom (1998), 5-angstrom (1999) and 2.4-angstrom (2000) resolution, and shows you how quickly this field was advancing. Ramakrishnan's group teased out many of the fine details of codon recognition, and showed how some antibiotics known to cause the ribosome to start bungling the process were able to to work. It turned out that the opening and closing behavior of the 30S piece was a key for this whole process, with error-inducing antibiotics causing it to go out of synch. And here's a place where the differences between bacterial ribosomes and eukaryotic ones really show up. The same antibiotics can't quite bind to mammalian ribosomes, fortunately. Having the protein synthesis machinery jerkily crank out garbled products is just what you'd wish for the bacteria that are infecting you, but isn't something that you'd want happening in your own cells.
At the same time, Steitz's group was turning out better and better structures of the 50S subunit, and helping to explain how it worked. One surprise was that there was a highly ordered set of water molecules and hydrogen bonds involved - in fact, protein synthesis seems to be driven (energetically) almost entirely by changes in entropy, rather than enthalpy. Both his group and Ramakrishnan's have been actively turning out structures of the ribosome subunits in complex with various proteins that are known to be key parts of the process, and those mechanisms of action are still being unraveled as we speak.
The Nobel citation makes reference to the implications of all this for drug design. I'm of two minds on that. It's certainly true that many important antibiotics work at the ribosomal level, and understanding how they do that has been a major advance. But we're not quite to the point where we can design new drugs to slide right in there and do what we want. I personally don't think we're really at that stage with most drug targets of any type, and trying to do it against structures with a lot of nucleic acid character is particularly hard. The computational methods for those are at an earlier stage than the ones we have for proteins.
One other note: every time a Nobel is awarded, the thoughts go to the people who worked in the same area, but missed out on the citation. The three-recipients-max stipulation makes this a perpetual problem. This is outside my area of specialization, but if I had to list some people that just missed out here, I'd have to cite Harry Noller of UC-Santa Cruz and Marina Rodnina of Göttingen. Update: add Peter Moore of Yale as well. All of them work in this exact same area, and have made many real contributions to it - and I'm sure that there are others who could go on this list as well.
One last note: five Chemistry awards out of the last seven, by my count, have gone to fundamental discoveries in cell or protein biology. That's probably a reasonable reflection of the real world, but it does rather cut down on the number of chemists who can expect to have their accomplishments recognized. The arguing about this issue is not be expected to cease any time soon.
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+ TrackBacks (0) | Category: Analytical Chemistry | Biological News | Current Events | Infectious Diseases
October 5, 2009
Posted by Derek
As many had expected, a Nobel Prize has been awarded to Elizabeth Blackburn (of UCSF), Carol Greider (of Johns Hopkins), and Jack Szostak (of Harvard Medical School/Howard Hughes Inst.) for their work on telomerase. Blackburn had been studying telomeres since her postdoc days in the late 1970s, and she and Szostak worked together in the field in the early 1980s, collarborating from two different angles. Greider (then a graduate student in Blackburn's lab) discovered the telomerase enzyme in 1984. She's continued to work in the area, as well she might, since it's been an extremely interesting and important one.
Telomeres, as many readers will know, are repeating DNA stretches found on the end of chromosomes. It was realized in the 1970s that something of this kind needed to be there, since otherwise replication of the chromosomes would inevitably clip off a bit from the end each time (the enzymes involved can't go all the way to the ends of the strands). Telomeres are the disposable buffer regions, which distinguish the natural end of a chromosome from a plain double-stranded DNA break.
What became apparent, though was that the telomerase complex often didn't quite compensate for telomere shortening. This provides a mechanism for limiting the number of cell divisions - when the telomeres get below a certain length, further replication is shut down. Telomerase activity is higher in stem cells and a few other specialized lines. This means that the whole area must be a key part of both cellular aging and the biology of cancer. In a later post, I'll talk about telomerase as a drug target, a tricky endeavour that straddles both of those topics.
It's no wonder that this work has attracted the amount of attention it has, and it's no wonder either that it's the subject of a well deserved Nobel. Congratulations to the recipients!
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+ TrackBacks (0) | Category: Aging and Lifespan | Biological News | Cancer | Current Events
September 11, 2009
Posted by Derek
Readers may remember a study from earlier this year that suggested that taking antioxidants canceled out some of the benefits of exercise. It seems that the reactive oxygen species themselves, which everyone's been assuming have to be fought, are actually being used to signal the body's metabolic changes.
Now there's another disturbing paper on a possible unintended effect of antioxidant therapy. Joan Brugge and her group at Harvard published last month on what happens to cells when they're detached from their normal environment. What's supposed to happen, everyone thought, is apoptosis, programmed cell death. Apoptosis, in fact, is supposed to be triggered most of the time when a cell detects that something has gone seriously wrong with its normal processes, and being detached from its normal signaling environment (and its normal blood supply) definitely qualifies. But cancer cells manage to dodge that difficulty, and since it's known that they also get around other apoptosis signals, it made sense that this was happening here, too.
But there have been some recent reports that cast doubt on apoptosis being the only route for detached cell death. This latest study confirms that, but goes on to a surprise. When this team blocked apoptotic processes, detached cells died anyway. A closer look suggested that the reason was, basically, starvation. The cells were deprived of nutrients after being dislocated, ran out of glucose, and that was that. This process could be stopped, though, if a known oncogene involved in glucose uptake (ERBB2) was activated, which suggests that one way a cancer cells survive their travels is by keeping their fuel supply going.
So far, so good - this all fits in well with what we already know about tumor cells. But this study found that there was another way to keep detached cells from dying: give them antioxidants. (They used either N-acetylcysteine or a water-soluble Vitamin E derivative). It appears that oxidative stress is one thing that's helping to kill off wandering cells. On top of this effect, reactive oxygen species also seem to be inhibiting another possible energy source, fatty acid oxidation. Take away the reactive oxygen species, and the cells are suddenly under less pressure and have access to a new food source. (Here's a commentary in Nature that goes over all this in more detail, and here's one from The Scientist).
They went on to use some good fluorescence microscopy techniques to show that these differences in reactive oxygen species are found in tumor cell cultures. There are notable metabolic differences between the outer cells of a cultured tumor growth and its inner cells (the ones that can't get so much glucose), but that difference can be smoothed out by. . .antioxidants. The normal process is for the central cells in such growths to eventually die off (luminal clearance), but antioxidant treatment kept this from happening. Even more alarmingly, they showed that tumor cells expressing various oncogenes colonized an in vitro cell growth matrix much more effectively in the presence of antioxidants as well.
This looks like a very strong paper to me; there's a lot of work in it and a lot of information. Taken together, these results suggest a number of immediate questions. Is there something that shuts down normal glucose uptake when a cell is detached, and is this another general cell-suicide mechanism? How exactly does oxidative stress keep these cells from using their fatty acid oxidation pathway? (And how does that relate to normally positioned cells, in which fatty acid oxidation is actually supposed to kick in when glucose supplies go down?)
The biggest questions, though, are the most immediate: first, does it make any sense at all to give antioxidants to cancer patients? Right now, I'd very much have to wonder. And second, could taking antioxidants actually have a long-term cancer-promoting effect under normal conditions? I'd very much like to know that one, and so would a lot of other people.
After this and that exercise study, I'm honestly starting to think that oxidative stress has been getting an undeserved bad press over the years. Have we had things totally turned around?
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+ TrackBacks (0) | Category: Biological News | Cancer
September 8, 2009
Posted by Derek
Imagine a drug molecule, and imagine it's a really good one. That is, it's made it out of the gut just fine, out into the bloodstream, and it's even slipped in through the membrane of the targeted cells. Now what?
Well, "cells are gels", as Arthur Kornberg used to say, and he was right. There's not a lot of bulk water sloshing around in there. It's all stuck to and sliding around with enzymes, structural proteins, carbohydrates, and the like, and that's what any drug molecule has to be able to do as well. And there's no particular reason for most of them to go anywhere particular inside the cell, once they're inside. They just diffuse around until they hit their targets, to which they stick (which is something they'd better do).
What if things didn't work this way? What if you could micro-inject your drug right into a particular cell compartment, or have it target a particular cell structure, instead of having to mist it all over the place? We now have a good answer to that question, but how much good it's going to do us drug discoverers is another thing entirely.
I'm referring to this paper from JACS, from a group at the University of Tokyo. They're targeting the important signaling enzyme PI3K. That's downstream of a lot of things, and in this case they used the PDGFR receptor in the cells, and a phosphorylated peptide that's a known ligand. To make the peptide go where they wanted, though, they further engineered both the ligand and the cells. The cells got modified by expression of dihydrofolate reductase (DHFR) in their plasma membranes, and the peptide ligand was conjugated to trimethoprim (TMP). TMP has a very strong association with DHFR, so this system was being used as an artificial targeting method. (It's as if the cell had been built up with hook-bearing Velcro on the inside of its plasma membrane, and the PI3K ligand was attached to a strip of the fuzzy side). Then to see what was going on, they also attached a fluorescent ligand to the peptide ligand as well.
Of course, this ligand-TMP-fluorescent fusion beast wasn't the best candidate for getting into a cell on its own, so the team microinjected it. And the results were dramatic. Normally, stimulating the PDGFR receptor in these cells led to downstream signaling in less than one minute. In cells that didn't have the DHFR engineered into their membranes, the fluorescent ligand could be seen diffusing through the whole cytosol, and giving a very weak PDGFR response. But in the cells with the targeting system built in, the ligand immediately seemed to stick to the inside of the plasma membrane, as planned, and a very robust, quick response was seen.
The paper details a number of control experiments that I'm not going into here, and I invite the curious to read the whole thing. I'm convinced, though, that the authors are seeing what they hoped to see. In other words, ligands which aren't worth much when they have to diffuse around on their own can be real tigers when they're dragged directly to their site of action. It makes sense that this would be true, but it's nice to see it demonstrated for real. I'll quote the last paragraph of the paper, though, because that's where I have some misgivings:
In summary, we have demonstrated that it is feasible to rapidly and efficiently activate an endogenous signaling pathway by placing a synthetic ligand at a specific location within a cell. The strategy should be applicable to other endogenous proteins and pathways through the choice of appropriate ligand molecules. More significantly, this proof-of-principle study highlights the importance of controlling the subcellular locales of molecules in the design of new synthetic modulators of intracellular biological events. There might be a number of compounds (not only activators but also inhibitors) that have been dismissed but may acquire potent biological activities when they are endowed with subcellular-targeting functions. Our next challenge is to develop cell-permeable carriers capable of delivering cargo ligands to specifically defined regions or organelles inside cells.
Where they lost me was in pointing out how important this is in designing new compounds. The problem is, these are very artificial, highly engineered cells. Everything's been set up to make them do just what you want them to do. If you don't cause them to express boatloads of DHFR in their membrane, nothing works. So what lessons does this have for a drug discovery guy like me? I'm not targeting cells that have been striped with convenient Velco patches.
And even if I find something endogenous that I can use, I can't make molecules that have to be delivered through the cell membrane by microinjection. You can see from the last sentence, though, that the authors realize that part as well. But that "next challenge" they speak of is more than enough to keep them occupied for the rest of their working lives. These kinds of experiments are important - they teach us a lot about cell biology, and there's sure a lot more of that to be learned. But the cells won't give up their secrets without a fight.
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August 20, 2009
Posted by Derek
It's hard to think of a more important class of drug targets than the G-protein coupled receptors (GPCRS). And back about fifteen years ago, I thought I had a reasonable understanding of how they worked. I was quite wrong, even given the standards of knowledge at the time, but since then the GPCR world has become gradually crazier and crazier.
The classic way of thinking about these receptors is that they live up on the cell surface, with part of the protein on the outside and part on the inside. The inside face is associated with various G-proteins, and the outside face has a binding site for some sort of signaling molecule. If the right molecule shows up and slots in the correct way into this binding cavity, the transmembrane helices of the protein rearrange, sliding around to change the shape and binding properties down there at the G-protein interface. This sets off some intracellular messaging - often by affecting levels of the messenger molecule cyclic-AMP. Thus is a signal from outside the cell relayed through the membrane to the inside.
Pretty nearly makes sense, doesn't it? Well, take a look at this new report from PLoS Biology. The authors rigged up living cells with a built-in fluorescent sensor system to monitor cAMP, and then studied the behavior of the thyroid-stimulating-hormone (TSH) receptor. That's a perfectly reasonable protein-ligand GPCR, but it turns out that it does things that are not (to us) perfectly reasonable.
This paper shows that when a TSH molecule binds, that the receptor gets taken back down through the membrane into the cell. That's certainly a known process (internalization), and was thought to be a regulatory process, a standard method for taking a specific GPCR out of the signaling business. Some receptors seem to do this right after they're used, and of those, some of them later resurface and some are broken up. (Other types hang around for many cycles until they're somehow worn out). But the ones that internalize quickly still set off their intracellular message before they get pulled back down. That's their purpose in life.
TSH does that. But the weird part is that the authors saw the receptor internalize along with its G-protein partners, and then continue signaling from inside the cell. Not only that, this extra signaling behavior set off somewhat different responses as compared to the first "normal" burst, and seems to be a necessary part of the usual TSH signaling pathway. It's a very odd thought, if you're used to thinking about GPCRs - it's like finding out that your cell phone works when it's turned off.
Now this sort of behavior has been demonstrated for a different class of signaling proteins (the tyrosine kinase receptors). And even GPCRs have been found, over the last few years, to be capable of setting off a different signaling regime (the MAP kinase pathway) after they've been internalized. (That's one of the weird findings of recent years that I mentioned in the introductory paragraph, and we still don't know what to do with that one as far as drug discovery goes). But everyone agreed that at least the good ol' cyclic AMP pathway worked the way we thought it did, through signaling at the cell surface, and thank goodness there was something you could still count on in this world.
Hah. Now we're going to have to see how many other GPCRs show this kind of behavior, and under what circumstances, and why. It may well turn out to be different for different cells or for different signaling ligands, or only occur under certain conditions. And we'll have to see how this relates to the other strange things that are being unraveled about GPCR behavior - they way that they can dimerize, with themselves or even other receptors, out on the cell surface, and the way that some of them seem to work in an opposite-sign signaling regime (always on, until something turns them off). Do these things still signal from beneath the waves, too?
Oh, this will keep the receptor folks busy, as if they weren't already. And, as usual when something like this shows up, it should serve as a reminder to anyone who thinks that we understand even the well-worked-out parts of cell biology. Hah!
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August 18, 2009
Posted by Derek
I see that there's a serious effort underway to standardize biochemical diagrams. About time! As a chemist, I don't mind admitting that I've been confused by many of these things over the years. As the current task force points out, one reason for that is that there are too many processes that all get drawn the same way: with a curved arrow. Enzymatic cleavage? Allosteric regulation? Product inhibition? Nucleic acid splicing? Enzyme activation? A curvy arrow should do nicely. And if the same scheme includes several of those phenomena at once, then we'll just use more arrows, making sure, of course, that they're all exactly the same size and style.
The new proposal seems to be based on the ideas behind electrical circuit diagrams and flow-chart conventions, and will attempt to convey information through several means (box shapes, arrow styles, etc.) I hope it, or something like it, actually catches on, although it'll take me a while to get used to translating it. Actually, what will take a while is getting used to the idea that biological diagrams are supposed to be imparting information at all. I've been trained in the other direction for too long.
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+ TrackBacks (0) | Category: Biological News
August 11, 2009
Posted by Derek
I was looking over a paper in PNAS, where a group at Stanford describes finding several small molecules that inhibit Hedgehog signaling. That's a very interesting (and ferociously complex) area, and the more tools that are available to study it, the better.
But let me throw something out to those who have read (or will read) the paper. (Here's the PDF, which is open access). The researchers seem to have done a screen against about 125,000 compounds, and come up with four single-digit micromolar hits. Characterizing these against a list of downstream assays showed that each of these acts in a somewhat different manner on the Hedgehog pathway.
And that's fine - the original screen would have picked up a variety of mechanisms, and there certainly are a variety out there to be picked up. I can believe that a list of compounds would differentiate on closer inspection. What I keep looking for, though, is (first) a mention that these compounds were run through some sort of general screening panel for other enzyme and/or receptor activities. They did look for three different kinase activities that had been shown to interfere (and didn't see them), but I'd feel much better about using some new structures as probes if I'd run them through a big panel of secondary assays first.
Second, I've been looking for some indication that there might have been some structure-activity relationships observed. I assume that each of these compounds might well have been part of a series - so how did the related structures fare? Having a one-off compound doesn't negate the data, naturally, although it certainly does make it harder to build anything from the hit you've found. But SAR is another factor that I'd immediately look for after a screen, and it seems strange to me that I can't find any mention of it.
Have I missed these things, or are they just not there? If they aren't, is that a big deal, or not? Thoughts?
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+ TrackBacks (0) | Category: Biological News | Drug Assays
July 7, 2009
Posted by Derek
While we're on the topic of hydrogen bonds and computations, there's a paper coming out in JACS that attempts to answer an old question. Why, exactly, does every living thing on earth use so much ribose? It's the absolute, unchanging carbohydrate backbone to all the RNA on Earth, and like the other things in this category (why L amino acids instead of D?), it's attracted a lot of speculation. If you subscribe to the RNA-first hypothesis of the origins of life, then the question becomes even more pressing.
A few years ago, it was found that ribose, all by itself, diffuses through membranes faster than the other pentose sugars. This results holds up for several kinds of lipid bilayers, suggesting that it's not some property of the membrane itself that's at work. So what about the ability of the sugar molecules to escape from water and into the lipid layers?
Well, they don't differ much in logP, that's for sure, as the original authors point out. This latest paper finds, though, by using molecular dynamic simulations that there is something odd about ribose. In nonpolar environments, its hydroxy groups form a chain of hydrogen-bond-like interactions, particularly notable when it's in the beta-pyranose form. These aren't a factor in aqueous solution, and the other pentoses don't seem to pick up as much stabilization under hydrophobic conditions, either.
So ribose is happier inside the lipid layer than the other sugars, and thus pays less of a price for leaving the aqueous environment, and (both in simulation and experimentally) diffuses across membranes ten times as quickly as its closely related carboyhydate kin. (Try saying that five times fast!) This, as both the original Salk paper and this latest one note, leads to an interesting speculation on why ribose was preferred in the origins of life: it got there firstest with the mostest. (That's a popular misquote of Nathan Bedford Forrest's doctrine of warfare, and if he's ever come up before in a discussion of ribose solvation, I'd like to hear about it).
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+ TrackBacks (0) | Category: Biological News | In Silico | Life As We (Don't) Know It
June 22, 2009
Posted by Derek
We organic chemists have it easy compared to the cell culture people. After all, our reactions aren't alive. If we cool them down, they slow down, and if we heat them up, they'll often pick up where they left off. They don't grow, they don't get infected, and they don't have to be fed.
Cells, though, are a major pain. You can't turn your back on 'em. Part of the problem is that there are, as yet, no cells that have evolved to grow in a dish or a culture bottle. Everything we do to them is artificial, and a lot of it what we ask cultured cells to do is clearly not playing to their strengths. Ask Genzyme: they use the workhorse CHO (Chinese Hamster Ovary) cells to produce their biologics, but they've been having variable yield problems over the past few months. Now it turns out that their production facilities are infected with Vesivirus 2117 - I'd never heard of that one, but it interferes with CHO growth, and that's bringing Genzyme's workflow to a halt. (No one's ever reported human infection with that one, just to make that clear).
I assume that the next step is a complete, painstaking cleanup and decontamination. That's going to affect supplies of Cerezyme (imiglucarase) and Frabazyme (agalsidase) late in the summer and into the fall, although it's not clear yet how long the outage will be. Any cell culture lab that's had to toss things due to mycoplasms or other nasties will sympathize, and shudder at the thought of cleaning things up on this scale.
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May 13, 2009
Posted by Derek
Now, this is an example of an idea being followed through to its logical conclusion. Here’s where we start: the good effects of exercise are well known, and seem to be beyond argument. Among these are marked improvements in insulin resistance (the hallmark of type II diabetes) and glucose uptake. In fact, exercise, combined with losing adipose weight, is absolutely the best therapy for mild cases of adult-onset diabetes, and can truly reverse the condition, an effect no other treatment can match.
So, what actually causes these exercise effects? There has to be a signal (or set of signals) down at the molecular level that tells your cells what’s happening, and initiates changes in their metabolism. One good candidate is the formation of reactive oxygen species (ROS) in the mitochondria. Exercise most certainly increases a person’s use of oxygen, and increases the work load on the mitochondria (since that’s where all the biochemical energy is coming from, anyway). Increased mitochondrial formation of ROS has been well documented, and they have a lot of physiological effects.
Of course, ROS are also implicated in many theories of aging and cellular damage, which is why cells have several systems to try to soak these things up. That’s exactly why people take antioxidants, vitamin C and vitamin E especially. So. . .what if you take those while you’re exercising?
A new paper in PNAS askes that exact question. About forty healthy young male volunteers took part in the study, which involved four weeks of identical exercise programs. Half of the volunteers were already in athletic training, and half weren’t. Both groups were then split again, and half of each cohort took 1000 mg/day of vitamin C and 400 IU/day vitamin E, while the other half took no antioxidants at all. So, we have the effects of exercise, plus and minus previous training, and plus and minus antioxidants.
And as it turns out, antioxidant supplements appear to cancel out many of the beneficial effects of exercise. Soaking up those transient bursts of reactive oxygen species keeps them from signaling. Looked at the other way, oxidative stress could be a key to preventing type II diabetes. Glucose uptake and insulin sensitivity aren't affected by exercise if you're taking supplementary amounts of vitamins C and E, and this effect is seen all the way down to molecular markers such as the PPAR coactivator proteins PGC1 alpha and beta. In fact, this paper seems to constitute strong evidence that ROS are the key mediators for the effects of exercise, and that this process is mediated through PGC1 and PPAR-gamma. (Note that PPAR-gamma is the target of the glitazone class of drugs for type II diabetes, although signaling in this area is notoriously complex).
Interestingly, exercise also increases the body's endogenous antioxidant systems - superoxide dismutase and so on. These are some of the gene targets of PPAR-gamma, suggesting that these are downstream effects. Taking antioxidant supplements kept these from going up, too. All these effects were slightly more pronounced in the group that hadn't been exercising before, but were still very strong across the board.
This confirms the suspicions raised by a paper from a group in Valencia last year, which showed that vitamin C supplementation seemed to decrease the development of endurance capacity during an exercise program. I think that there's enough evidence to go ahead and say it: exercise and antioxidants work against each other. The whole take-antioxidants-for-better-health idea, which has been taking some hits in recent years, has just taken another big one.
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+ TrackBacks (0) | Category: Aging and Lifespan | Biological News | Cardiovascular Disease | Diabetes and Obesity
May 1, 2009
Posted by Derek
One of Merck’s less wonderful recent experiences was the rejection of Cordaptive, which was an attempt to make a niacin combination for the cardiovascular market. Niacin would actually be a pretty good drug to improve lipid profiles if people could stand to take the doses needed. But many people experience a burning, itchy skin flush that’s enough to make them give up on the stuff. And that’s too bad, because it’s the best HDL-raising therapy on the market. It also lowers LDL, VLDL, free fatty acids, and tryglycerides, which is a pretty impressive spectrum. So it’s no wonder that Merck (and others) have tried to find some way to make it more tolerable.
A new paper suggests that everyone has perhaps been looking in the wrong place for that prize. A group at Duke has found that the lipid effects and the cutaneous flushing are mechanistically distinct, way back at the beginning of the process. There might be a new way to separate the two.
Niacin’s target seems to be the G-protein coupled receptor GPR109A – and, unfortunately, that seems to be involved in the flushing response, since both that and the lipid effects disappear if you knock out the receptor in a mouse model. The current model is that activation of the receptor produces the prostaglandin PGD2 (among other things), and that’s what does the skin flush, when it hits its own receptor later on. Merck’s approach to the side effect was the block the PGD2 receptor by adding an antagonist drug for it along with the niacin. But taking out the skin flush at that point means doing it at nearly the last possible step.
The Duke team has looked closely at the signaling of the GPR109A receptor and found that beta-arrestins are involved (they’ve specialized in this area over the last few years). The arrestins are proteins that modify receptor signaling through a variety of mechanisms, not all of which are well understood. Wew’ve known about signaling through the G-proteins for many years (witness the name of the whole class of receptors), but beta-arrestin-driven signaling is a sort of alternate universe. (GPCRs have been developing quite a few alternate universes – the field was never easy to understand, but it’s becoming absolutely baroque).
As it turns out, mice that are deficient in either beta-arrestin 1 or beta-arrestin 2 show the same lipid effects in response to niacin dosing as normal mice. But the mice lacking much of their beta-arrestin 1 protein show a really significant loss of the flushing response, suggesting that it’s mediated through that signaling pathway (as opposed to the “normal” G-protein one). And a known GPR109A ligand that doesn’t seem to cause so much skin flushing (MK-0354) fit the theory perfectly: it caused G-protein signaling, but didn’t bring in beta-arrestin 1.
So the evidence looks pretty good here. This all suggests that screening for compounds that hit the receptor but don’t activate the beta-arrestin pathway would take you right to the pharmacology you want. And I suspect that several labs are going to now put that idea to the test, since beta-arrestin assays are also being looked at in general. . .
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April 29, 2009
Posted by Derek
What a mess! Science has a retraction of a 2005 paper, which is always a nasty enough business, but in this case, the authors can’t agree on whether it should be retracted or not. And no one seems to be able to agree on whether the original results were real, and (even if they weren’t) whether the technique the paper describes works anyway. Well.
The original paper (free full text), from two Korean research groups, described a drug target discovery technique with the acronym MAGIC (MAGnetism-based Interaction Capture). It’s a fairly straightforward idea in principle: coat a magnetic nanoparticle with a molecule whose target(s) you’re trying to identify. Now take cell lines whose proteins have had various fluorescent tags put on them, and get the nanoparticles into them. If you then apply a strong magnetic field to the cells, the magnetic particles will be pulled around, and they’ll drag along whichever proteins have associated with your bait molecule. Watch the process under a microscope, and see which fluorescent spots move in which cells.
Papers were published (in both Science and Nature Chemical Biology), patent applications were filed (well, not in that order!), startup money was raised for a company to be called CGK. . .and then troubles began. Word was that the technique wasn’t reproducible. One of the authors (Yong-Weon Yi) asked that his name be removed from the publications, which was rather problematic of him, considering that he was also an inventor on the patent application. Early last year, investigations by the Korean Advanced Institute of Science and Technology came to the disturbing conclusion that the papers “do not contain any scientific truth”, and the journals flagged them.
The Nature Chemical Biology paper was retracted last July, but the Science paper has been a real rugby scrum, as the journal details here. The editorial staff seems to have been unable to reach one of the authors (Neoncheol Jung), and they still don’t know where he is. That’s disconcerting, since he’s still listed as the founding CEO of CGK. A complex legal struggle has erupted between the company and the KAIST about who has commercial rights to the technology, which surely isn’t being helped along by the fact that everyone is disagreeing about whether it works at all, or ever has. Science says that they’ve received parts of the KAIST report, which states that the authors couldn’t produce any notebooks or original data to support any of the experiments in the paper. This is Most Ungood, of course, and on top of that, two of the authors also appear to have stated that the key experiments (where they moved the fluorescent proteins around) were not carried out as the paper says. Meanwhile, everyone involved is now suing everyone else back in Korea for fraud, for defamation, and who knows. The target date for all this to be resolved is somewhere around the crack of doom.
Emerging from the fiery crater, CGK came up with another (very closely related) technique, which they published late last year in JACS. (If nothing else, everyone involved is certainly getting their work into an impressive list of journals. If only the papers wouldn’t keep sliding right back out. . .) That one has stood up so far, but it’s only April. I presume that the editorial staff at JACS asked for all kinds of data in support, but (as this whole affair shows) you can’t necessarily assume that everyone’s doing the job they’re supposed to do.
The new paper, most interestingly, does not reference the previous work at all, which I suppose makes sense on one level. But if you just came across it de novo, you wouldn't realize that people (at the same company!) had already been (supposedly) working on magnetic particle assays in living cells. Looking over this one and comparing it to the original Science paper, one of the biggest differences seems to be how the magnetic particles are made to expose themselves to the cytoplasm. The earlier work mentioned coating the particles with a fusogenic protein (TAT-HA2) that was claimed to help with this process; that step is nowhere to be found in the JACS work. Otherwise, the process looks pretty much identical to me.
Let’s come up for air, then, and ask how well useful these ideas could be, stipulating (deep breath) that they work. Clearly, there’s some utility here. But I have to wonder how useful this protocol will be for general target fishing expeditions. Fluorescent labeling of proteins is indeed one of the wonders of the world (and was the subject of a recent a well-deserved Nobel prize). But not all proteins can be labeled without disturbing their function – and if you don’t know what the protein’s up to in the first place, you’re never sure if you’ve done something to perturb it when you add the glowing parts. There are also a lot of proteins, of course, to put it mildly, and if you don’t have any idea of where to start looking for targets, you still have a major amount of work to do. The cleanest use I can think of for these experiments is verifying (or ruling out) hypotheses for individual proteins.
But that's if it works. And at this point, who knows? I'll be very interested to follow this story, and to see if anyone else picks up this technique and gets it to work. Who's brave enough?
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+ TrackBacks (0) | Category: Biological News | Drug Assays | The Dark Side | The Scientific Literature
April 17, 2009
Posted by Derek
So I see that the headlines are that it’s proving difficult to relate gene sequences to specific diseases. (Here's the NEJM, free full-text). I can tell you that the reaction around the drug industry to this news is a weary roll of the eyes and a muttered “Ya don’t say. . .”
That’s because we put our money down early on the whole gene-to-disease paradigm, and in a big way. As I’ve written here before, there was a real frenzy in the industry back in the late 1990s as the genomics efforts started really revving up. Everyone had the fear that all the drug targets that ever were, or ever could be, were about to be discovered, annotated, patented – and licensed to the competition, who were out there fearless on the cutting edge, ready to leap into the future, while we (on the other hand) lounged around like dinosaurs looking sleepily at that big asteroidy thing up there in the sky.
No, that’s really how it felt. Every day brought another press release about another big genomics deal. The train (all the trains!) were loudly leaving the station. A lot of very expensive deals were cut, sometimes in great haste, but (as far as I can tell) they yielded next to nothing – at least in terms of drug candidates, or even real drug targets themselves.
So yeah, we’ve already had a very expensive lesson in how hard it is to associate specific gene sequences with specific diseases. The cases where you can draw a dark, clear line between the two increasingly look like exceptions. There are a lot of these (you can read about them
in these texts
), but they tend to affect small groups of people at a time. The biggest diseases (diabetes, cardiovascular in general, Alzheimer’s, most cancers) seem to be associated with a vast number of genetic factors, most of them fairly fuzzy, and hardly any of them strong enough on their own to make a big difference one way or another. Combine that with the nongenetic (or epigenetic) factors like nutrition, lifestyle, immune response, and so on, and you have a real brew.
On that point, I like E. O. Wilson’s metaphor for nature versus nurture. He likened a person’s genetic inheritance to a photographic negative. Depending on how it’s developed and printed, the resulting picture can turn out a lot of different ways – but there’s never going to be more than was in there to start with. (These days, I suppose that we’re going to have to hunt for another simile – Photoshop is perhaps a bit too powerful to let loose inside that one).
But I've been talking mostly about variations in proteins as set by their corresponding DNA sequences. The real headscratcher has been this:
One observation that has taken many observers by surprise is that most loci that have been discovered through genomewide association analysis do not map to amino acid changes in proteins. Indeed, many of the loci do not even map to recognizable protein open reading frames but rather may act in the RNA world by altering either transcriptional or translational efficiency. They are thus predicted to affect gene expression. Effects on expression may be quite varied and include temporal and spatial effects on gene expression that may be broadly characterized as those that alter transcript levels in a constitutive manner, those that modulate transcript expression in response to stimuli, and those that affect splicing.
That's really going to be a major effort to understand, because we clearly don't understand it very well now. RNA effects have been coming on for the last ten or fifteen years as a major factor in living systems that we really weren't aware of, and it would be foolish to think that the last fireworks have gone off.
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March 26, 2009
Posted by Derek
So, people like me spend their time trying to make small molecules that will bind to some target protein. So what happens, anyway, when a small molecule binds to a target protein? Right, right, it interacts with some site on the thing, hydrogen bonds, hydrophobic interactions, all that – but what really happens?
That’s surprisingly hard to work out. The tools we have to look at such things are powerful, but they have limitations. X-ray crystal structures are great, but can lead you astray if you’re not careful. The biggest problem with them, though (in my opinion) is that you see this beautiful frozen picture of your drug candidate in the protein, and you start to think of the binding as. . .well, as this beautiful frozen picture. Which is the last thing it really is.
Proteins are dynamic, to a degree that many medicinal chemists have trouble keeping in mind. Looking at binding events in solution is more realistic than looking at them in the crystal, but it’s harder to do. There are various NMR methods (here's a recent review), some of which require specially labeled protein to work well, but they have to be interpreted in the context of NMR’s time scale limitations. “Normal” NMR experiments give you time-averaged spectra – if you want to see things happening quickly, or if you want to catch snapshots of the intermediate states along the way, you have a lot more work to do.
Here’s a recent paper that’s done some of that work. They’re looking at a well-known enzyme, dihydrofolate reductase (DHFR). It’s the target of methotrexate, a classic chemotherapy drug, and of the antibiotic trimethoprim. (As a side note, that points out the connections that sometimes exist between oncology and anti-infectives. DHFR produces tetrahydrofolate, which is necessary for a host of key biosynthetic pathways. Inhibiting it is espccially hard on cells that are spending a lot of their metabolic energy on dividing – such as tumor cells and invasive bacteria).
What they found was that both inhibitors do something similar, and it affects the whole conformational ensemble of the protein:
". . .residues lining the drugs retain their μs-ms switching, whereas distal loops stop switching altogether. Thus, as a whole, the inhibited protein is dynamically dysfunctional. Drug-bound DHFR appears to be on the brink of a global transition, but its restricted loops prevent the transition from occurring, leaving a “half-switching” enzyme. Changes in pico- to nanosecond (ps-ns) backbone amide and side-chain methyl dynamics indicate drug binding is “felt” throughout the protein.
There are implications, though, for apparently similar compounds having rather different effects out in the other loops:
. . .motion across a wide range of timescales can be regulated by the specific nature of ligands bound. Occupation of the active site by small ligands of different shapes and physical characteristics places differential stresses on the enzyme, resulting in differential thermal fluctuations that propagate through the structure. In this view, enzymes, through evolution, develop sensitivities to ligand properties from which mechanisms for organizing and building such fluctuations into useful work can arise. . .Because the affected loop structures are primarily not in contact with drug, it is reasonable to envision inhibitory small-molecule drugs that act by allosterically modulating dynamic motions."
There are plenty of references in the paper to other investigations of this kind, so if this is your sort of thing, you'll find plenty of material there. One thing to take home, though, is to remember that not only are proteins mobile beasts (with and without ligand bound to them), but that this mobility is quite different in each state. And keep in mind that the ligand-bound state can be quite odd compared to anything else the protein experiences otherwise. . .
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March 24, 2009
Posted by Derek
I’ve written here before about the "click" triazole chemistry that Barry Sharpless’s group has pioneered out at Scripps. This reaction has been finding a lot of uses over the last few years (try this category for a few, and look for the word "click"). One of the facets I find most interesting is the way that they’ve been able to use this Huisgen acetylene/azide cycloaddition reaction to form inhibitors of several enzymes in situ, just by combining suitable coupling partners in the presence of the protein. Normally you have to heat that reaction up quite a bit to get it to go, but when the two reactants are forced into proximity inside the protein, the rate speeds up enough to detect a product.
Note that I said “inside the protein”. My mental picture of these things has involved binding-site cavities where the compounds are pretty well tied down. But a new paper from Jim Heath’s group at Cal Tech, collaborating with Sharpless and his team, demonstrates something new. They’re now getting this reaction to work out on protein surfaces, and in the process making what are basically artificial antibody-type binding agents.
To start with, they prepared a large library of hexapeptides out of the unnatural D-amino acids, in a one-bead-one-compound format. (Heath’s group has been working in this area for a while, and has experience dealing with these - see this PDF presentation for an overview of their research). Each peptide had an acetylene-containing amino acid at one end, for later use. They exposed these to a protein target: carbonic anhydrase II, the friend of every chemist who’s trying to make proteins do unusual things. The oligopeptide that showed the best binding to the protein’s surface was then incubated with the target CA II protein and another library of diverse hexapeptides. These had azide-containing amino acids at both ends, and the hope was that some of these would come close enough, in the presence of the protein, to react with the anchor acetylene peptide.
Startlingly, this actually worked. A few of the azide oligopeptides did do the click triazole-forming reaction. And the ones that worked all had related sequences, strongly suggesting that this was no fluke. What impresses me here is that (1) these things were lying on top of the protein, picking up what interactions they could, not buried inside a more restrictive binding site, and (2) the click reaction worked even though the binding constants of the two partners must not have been all the impressive. The original acetylene hexapeptide, in fact, bound at only 500 micromolar, and the other azide-containing hexapeptides that reacted with them were surely in the same ballpark.
The combined beast, though, (hexapeptide-triazole-hexapeptide) was a 3 micromolar compound. And then they took the thing through another round of the same process, decorating the end with a reactive acetylene and exposing it to the same azide oligopeptide library in the presence of the carbonic anhydrase target. The process worked again, generating a new three-oligopeptide structure which now showed 50 nanomolar binding. This increase in affinity over the whole process is impressive, but it’s just what you’d expect as you start combining pieces that have some affinity on their own. Importantly, when they made a library on beads by coupling the whole list of azide-containing hexapeptides with the biligand (through the now-standard copper-catalyzed reaction), the target CA II protein picked out the same sequences that were generated by the in situ experiment.
So what you have, in the end, is a short protein-like thing (actually three small peptides held together by triazole linkers) that has been specifically raised to bind a protein target – thus the comparison to antibodies above. What we don't know yet, of course, is just how this beast is binding to the carbonic anhydrase protein. It would appear to be stretched across some non-functional surface, though, because the triligand didn't seem to interfere with the enzyme's activity once it was bound. I'd be very interested in seeing if an X-ray structure could be generated for the triligand complex or any of the others. Heath's group is now apparently trying to generate such agents for other proteins and to develop assays based on them. I look forward to seeing how general the technique is.
This result makes a person wonder if the whole in situ triazole reaction could be used to generate inhibitors of protein-protein interactions. Doing that with small molecules is quite a bit different than doing it with hexapeptide chains, of course, but there may well be some hope. And there's another paper I need to talk about that bears on the topic; I'll bring that one up shortly. . .
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March 4, 2009
Posted by Derek
Well, here’s another crack at open-source science. Stephen Friend, the previous head of Rosetta (before and after being bought by Merck), is heading out on his own to form a venture in Seattle called Sage. The idea is to bring together genomic studies from all sorts of laboratories into a common format and database, with the expectation that interesting results will emerge that couldn’t be found from just one lab’s data.
I’ll be interested to see if this does yield something worthwhile – in fact, I’ll be interested to see if it gets off the ground at all. As I’ve discussed before, the analogy with open-source software doesn’t hold up so well with most scientific research these days, since the entry barriers (facilities, equipment, and money) are significantly higher than they are in coding. Look at genomics – the cost of sequencing has been dropping, for sure, but it’s still very expensive to get into the game. That lowered cost is measured per base sequenced – today’s technology means that you sequence more bases, which means that the absolute cost hasn’t come down as much as you might think. I’m sure you can get ten-year-old equipment cheap, but it won’t let you do the kind of experiments you might want to do, at least not in the time you’ll be expected to do them in.
But even past that issue, once you get down to the many labs that can do high-level genomics (or to the even larger number that can do less extensive sequencing), the problems will be many. Sage is also going to look at gene expression levels, something that's easier to do (although we're still not in weekend-garage territory yet). Some people would say that it's a bit too easy to do: there are a lot of different techniques in this field, not all of which always yield comparable data, to put it mildly. There have been several attempts to standardize things, along with calls for more control experiments, but getting all these numbers together into a useful form will still not be trivial.
Then you've got the really hard issues: intellectual property, for one. If you do discover something by comparing all these tissues from different disease states, who gets to profit from it? Someone will want to, that's for sure, and if Sage itself isn't getting a cut, how will they keep their operation going? Once past that question (which is a whopper), and past all the operational questions, there's an even bigger one: is this approach going to tell us anything we can use at all?
At first thought, you'd figure that it has to. Gene sequences and gene expression are indeed linked to disease states, and if we're ever going to have a complete understanding of human biology, we're going to have to know how. But. . .we're an awful long way from that. Look at the money that's been poured into biomarker development by the drug industry. A reasonable amount of that has gone into gene expression studies, trying to find clear signs and correlations with disease, and it's been rough sledding.
So you can look at this two ways: you can say fine, that means that the correlations may well be there, but they're going to be hard to find, so we're going to have to pool as much data as possible to do it. Thus Sage, and good luck to them. Or the systems may be so complex that useful correlations may not even be apparent at all, at least at our current level of understanding. I'm not sure which camp I fall into, but we'll have to keep making the effort in order to find out who's right.
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November 11, 2008
Posted by Derek
I wrote a while back about the problem of compounds sticking to labware. That sort of thing happens more often than you’d think, and it can really hose up your assay data in ways that will send you running around in circles. Now there’s a report in Science of something that’s arguably even worse. (Here's a good report on it from Bloomberg, one of the few to appear in the popular press).
The authors were getting odd results in an assay with monoamine oxidase B enzyme, and tracked it down to two compounds leaching out of the disposable plasticware (pipette tips, assay plates, Eppendorf vials, and so on). Oleamide is used as a “slip agent” to keep the plastic units from sticking to each other, but it’s also a MAO-B inhibitor. Another problem was an ammonium salt called DiHEMDA, which is put in as a general biocide – and it appears to be another MAO-B inhibitor.
Neither of them are incredibly potent, but if you’re doing careful kinetic experiments or the like, it’s certainly enough to throw things off. The authors found that just rinsing water through various plastic vessels was enough to turn the solution into an enzyme inhibitor. Adding organic solvents (10% DMSO, methanol) made the problem much worse; presumably these extract more contaminants.
And it’s not just this one enzyme. They also saw effects on a radioligand binding assay to the GABA-A receptor, and they point out that the biocides used are known to show substantial protein and DNA binding. These things could be throwing assay data around all over the place – and as we work in smaller and smaller volumes, with more complex protocols, the chances of running into trouble increase.
What to do about all this? Well, at a minimum, people should be sure to run blank controls for all their assays. That’s good practice, but sometimes it gets skipped over. This effect has probably been noted many times before as some sort of background noise in such controls, and many times you should be able to just subtract it out. But there are still many experiments where you can’t get away from the problem so easily, and it’s going to make your error bars wider no matter what you do about it. There are glass inserts for 96-well plates, and there are different plastics from different manufacturers. But working your way through all that is no fun at all.
As an aside, this sort of thing might still make it into the newspapers, since there have been a lot of concerns about bisphenol A and other plastic contaminants. In this case, I think the problem is far greater for lab assays than it is for human exposures. I’m not so worried about things like oleamide, since these are found in the body anyway, and can easily be metabolized. The biocides might be a different case, but I assume that we’re loaded with all kinds of substances, almost all of them endogenous, that are better inhibitors of enzymes like MAO-B. And at any rate, we’re exposed to all kinds of wild stuff at low levels, just from the natural components of our diet. Our livers are there to deal with just that sort of thing, but that said, it’s always worth checking to make sure that they’re up to the job.
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November 7, 2008
Posted by Derek
Systems biology – depending on your orientation, this may be a term that you haven’t heard yet, or one from the cutting edge of research, or something that’s already making you roll your eyes at its unfulfilled promise. There’s a good spread of possible reactions.
Broadly, I’d say that the field is concerned with trying to model the interactions of whole biological systems, in an attempt to come up with come explanatory power. It’s the sort of thing that you could only imagine trying to do with modern biological and computational techniques, but whether these are up to the job is still an open question. This gets back to a common theme that I stress around here, that biochemical networks are hideously, inhumanly complex. There’s really no everyday analogy that works to describe what they’re like, and if you think you really understand them, then you’re in the same position as all those financial people who thought they understood their exposure to mortgage-backed security risks.
You’ll have this enzyme, you see, that phosphorylates another enzyme, which increases its activity. But that product of that second enzyme inhibits another enzyme that acts to activate the first one, and each of them also interacts with fourteen (or forty-three) others, some of which are only expressed under certain conditions that we don’t quite understand, or are localized in the cell in patterns that aren’t yet clear, and then someone discovers a completely new enzyme in the middle of the pathway that makes hash out of what we thought we knew about
So my first test for listening to systems biology people is whether they approach things with the proper humility. There’s a good article in Nature on the state of the field, which does point out that some of the early big-deal-big-noise articles in the field alienated many potential supporters through just this effect. But work continues, and a lot of drug companies are putting money into it, under the inarguable “we need all the help we can get” heading.
One of the biggest investors has been Merck, a big part of that being their purchase a few years ago of Rosetta Inpharmatics. That group published an interesting paper earlier this year (also in Nature) on some of the genetic underpinnings of metabolic disease. A phrase from the article's abstract emphasizes the difficulties of doing this work: "Our analysis provides direct experimental support that complex traits such as obesity are emergent properties of molecular networks that are modulated by complex genetic loci and environmental factors." Yes, indeed.
But here’s a worrisome thing that didn’t make the article: Merck recently closed the Seattle base of the Rosetta team, in its latest round of restructuring and layoffs. One assumes that many of them are being transitioned to the Merck mothership, and that the company is still putting money into this approach, but there is room to wonder. Update: here's an article on this very subject). There is this quote from the recent overview:
Stephen Friend, Merck's vice-president for oncology, thinks that any hesitancy will be overcome when the modelling becomes so predictive that the toxicity and efficacy of a potential drug can be forecast very accurately even before an experimental animal is brought out if its cage. "The next three to five years will provide a couple such landmark predictions and wake everyone up," he says.
Well, we’ll see if he’s right about that timeframe, and I hope he is. I fear that the problem is one of those that appears large, and as you get closer to it, does nothing but get even larger. My opinion, for what it’s worth, is that it’s very likely too early to be able to come up with any big insights from the systems approach. But I can’t estimate the chances that I’m wrong about that, and the potential payoffs are large. For now, I think the best odds are in the smaller studies, narrowing down on single targets or signaling networks. That cuts down on the possibility that you’re going to find something revolutionary, but it increases the chance that anything you find is actually real. Talk of “virtual cells” and “virtual genomes” is, to my mind, way premature, and anyone who sells the technology in those terms should, I think, be regarded with caution.
But that said, any improvement is a big one. Our failure rates due to tox and efficacy problems are so horrendous that just taking some of these things down 10% (in real terms) would be a startling breakthrough. And we’re definitely not going to get this approach to work if we don’t plow money and effort into it; it’s not going to discover itself. So press on, systems people, and good luck. You’re going to need it; we all do.
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October 31, 2008
Posted by Derek
Let’s talk sugar, and how you know if you’ve eaten enough of it. Just in time for Halloween! This is a field I’ve done drug discovery for in the past, and it’s a tricky business. But some of the signals are being worked out.
Blood glucose, as the usual circulating energy source in the body, is a good measure of whether you’ve eaten recently. If you skip a meal (or two), your body will start mobilizing fatty acids from your stored supplies, and circulate them for food. But there’s one organ that runs almost entirely on sugar, no matter what the conditions: the brain. Even if you’re fasting, your liver will make sugar from scratch for your brain to use.
And as you’d expect, brain glucose levels are one mechanism the body uses to decide whether to keep eating or not. A cascade of enzyme signals has been worked out over the years, and the current consensus seems to be that high glucose in the brain inactivates AMP kinase (AMPK). (That’s a key enzyme for monitoring the energy balance in the brain – it senses differences in concentration between ATP, the energy currency inside every cell, and its product and precursor, AMP). Losing that AMPK enzyme activity then removes the brakes on the activity of another enzyme, acetyl CoA-carboxylase (ACC). (That one’s a key regulator of fatty acid synthesis – all this stuff is hooked together wonderfully). ACC produces malonyl-CoA, and that seems to be a signal to the hypothalamus of the brain that you’re full (several signaling proteins are released at that point to spread the news).
You can observe this sort of thing in lab rats – if you infuse extra glucose into their brains, they stop eating, even under conditions when they otherwise would keep going. A few years ago, an odd result was found when this experiment was tried with fructose: instead of lowering food intake, infusing fructose into the central nervous system made the animals actually eat more. That’s not what you’d expect, since in the end, fructose ends up metabolized to the same thing as glucose does (pyruvate), and used to make ATP. So why the difference in feeding signals?
A paper in PNAS (open access PDF) from a team at Johns Hopkins and Ibaraki University in Japan now has a possible explanation. Glucose metabolism is very tightly regulated, as you’d expect for the main fuel source of virtually every living cell. But fructose is a different matter. It bypasses the rate-limiting step of the glucose pathway, and is metabolized much more quickly than glucose is. It appears that this fast (and comparatively unregulated) process actually uses up ATP in the hypothalamus – you’re basically revving up the enzyme machinery early in the pathway (ketohexokinase in particular) so much that you’re burning off the local ATP supply to run it.
Glucose, on the other hand, causes ATP levels in the brain to rise – which turns down AMPK, which turns up ACC, which allows malonyl-CoA to rise, and turns off appetite. But when ATP levels fall, AMPK is getting the message that energy supplies are low: eat, eat! Both the glucose and fructose effects on brain ATP can be seen at the ten-minute mark and are quite pronounced at twenty minutes. The paper went on to look at the activities of AMPK and ACC, the resulting levels of malonyl CoA, and everything was reversed for fructose (as opposed to glucose) right down the line. Even expression of the signaling peptides at the end of the process looks different.
The implications for human metabolism are clear: many have suspected that fructose could in fact be doing us some harm. (This New York Times piece from 2006 is a good look at the field: it's important to remember that this is very much an open question). But metabolic signaling could be altered by using fructose as an energy source over glucose. The large amount of high-fructose corn syrup produced and used in the US and other industrialized countries makes this an issue with very large political, economic, and public health implications.
This paper is compelling story – so, what are its weak points? Well, for one thing, you’d want to make sure that those fructose-metabolizing enzymes are indeed present in the key cells in the hypothalamus. And an even more important point is that fructose has to get into the brain. These studies were dropping it in directly through the skull, but that’s not how most people drink sodas. For this whole appetite-signaling hypothesis to work in the real world, fructose taken in orally would have to find its way to the hypothalamus. There’s some evidence that this is the case, but that fructose would have to find its way past the liver first.
On the other hand, it could be that this ATP-lowering effect could also be taking place in liver cells, and causing some sort of metabolic disruption there. AMPK and ACC are tremendously important enzymes, with a wide range of effects on metabolism, so there's a lot of room for things to happen. I should note, though, that activation of AMPK out in the peripheral tissues is thought to be beneficial for diabetics and others - this may be one route by which Glucophage (metformin) works. (Now some people are saying that there may be more than one ACC isoform out there, bypassing the AMPK signaling entirely, so this clearly is a tangled question).
I’m sure that a great deal of effort is now going into working out these things, so stay tuned. It's going to take a while to make sure, but if things continue along this path, there could be reasons for a large change in the industrialized human diet. There are a lot of downstream issues - how much fructose people actually consume, for one, and the problem of portion size and total caloric intake, no matter what form it's in, for another. So I'm not prepared to offer odds on a big change, but the implications are large enough to warrant a thorough check.
Update: so far, no one has been able to demonstrate endocrine or satiety differences in humans consuming high-fructose corn syrup vs. the equivalent amount of sucrose. See here, here, and here.
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October 9, 2008
Posted by Derek
I’ve spoken before about the acetylene-azide “click” reaction popularized by Barry Sharpless and his co-workers out at Scripps. This has been taken up by the chemical biology field in a big way, and all sorts of ingenious applications are starting to emerge. The tight, specific ligation reaction that forms the triazole lets you modify biomolecules with minimal disruption (by hanging an azide or acetylene from them, both rather small groups), and tag them later on in a very controlled way.
Adrian Salic and co-worker Cindy Yao have just reported an impressive example. They’ve been looking at ethynyluracil (EU), the acetylene-modified form of the ubiquitous nucleotide found in RNA. If you feed this to living organisms, they take it up just as if it were uracil, and incorporate it into their RNA. (It’s uracil-like enough to not be taken up into DNA, as they’ve shown by control experiments). Exposing cells or tissue samples later on to a fluorescent-tagged azide (and the copper catalyst needed for quick triazole formation) lets you light up all the RNA in sight. You can choose the timing, the tissue, and your other parameters as you wish.
For example, Salic and Yao have exposed cultured cells to EU for varying lengths of time, and watched the time course of transcription. Even ten minutes of EU exposure is enough to see the nuclei start to light up, and a half hour clearly shows plenty of incoporation into RNA, with the cytoplasm starting to show as well. (The signal increases strongly over the first three hours or so, and then more slowly).
Isolating the RNA and looking at it with LC/MS lets you calibrate your fluorescence assays, and also check to see just how much EU is getting taken up. Overall, after a 24-hour exposure to the acetylene uracil, it looks like about one out of every 35 uracils in the total RNA content has been replaced with the label. There’s a bit less in the RNA species produced by the RNAPol1 enzyme as compared to the others, interestingly.
There are some other tricks you can run with this system. If you expose the cells for 3 hours, then wash the EU out of the medium and let them continue growing under normal conditions, you can watch the labeled RNA disappear as it turns over. As it turns out, most of it drops out of the nucleus during the first hour, while the cytoplasmic RNA seems to have a longer lifetime. If you expose the cells to EU for 24 hours, though, the nuclear fluorescence is still visible – barely – after 24 hours of washout, but the cytoplasmic RNA fluorescence never really goes away at all. There seems to be some stable RNA species out there – what exactly that is, we don’t know yet.
Finally, the authors tried this out on whole animals. Injecting a mouse with EU and harvesting organs five hours later gave some very interesting results. It worked wonderfully - whole tissue slices could be examined, as well as individual cells. Every organ they checked showed nuclear staining, at the very least. Some of the really transcriptionally active populations (hepatocytes, kidney tubules, and the crypt cells in the small intestine) were lit up very brightly indeed. Oddly, the most intense staining was in the spleen. What appear to be lymphocytes glowed powerfully, but other areas next to them were almost completely dark. The reason for this is unknown, and that’s very good news indeed.
That’s because when you come up with a new technique, you want it to tell you things that you didn’t know before. If it just does a better or more convenient job of telling you what you could have found out, that’s still OK, but it’s definitely second best. (And, naturally, if it just tells you what you already knew with the same amount of work, you’ve wasted your time). Clearly, this click-RNA method is telling us a lot of things that we don’t understand yet, and the variety of experiments that can be done with it has barely been sampled.
Closely related to this work is what’s going on in Carolyn Bertozzi’s lab in Berkeley. She’s gone a step further, getting rid of the copper catalyst for the triazole-forming reaction by ingeniously making strained, reactive acetylenes. They’ll spontaneously react if they see a nearby azide, but they’re still inert enough to be compatible with biomolecules. In a recent Science paper, her group reports feeded azide-substituted galactosamine to developing zebrafish. That amino sugar is well known to be used in the synthesis of glycoproteins, and the zebrafish embryos seemed to have no problem accepting the azide variant as a building block.
And they were able to run these same sorts of experiments – exposing the embryos to different concentrations of azido sugar, for different times, with different washout periods before labeling all gave a wealth of information about the development of mucin-type glycans. Using differently labled fluorescent acetylene reagents, they could stain different populations of glycan, and watch time courses and developmental trafficking – that’s the source of the spectacular images shown.
Losing the copper step is convenient, and also opens up possibilities for doing these reactions inside living cells (which is definitely something that Bertozzi’s lab is working on). The number of experiments you can imagine is staggering – here, I’ll do one off the top of my head to give you the idea. Azide-containing amino acids can be incorporated at specific places in bacterial proteins – here’s one where they replaced a phenylalanine in urate oxidase with para-azidophenylalanine. Can that be done in larger, more tractable cells? If so, why not try that on some proteins of interest – there are thousands of possibilities – then micro-inject one of the Bertozzi acetylene fluorescence reagents? Watching that diffuse through the cell, lighting things up as it found azide to react with would surely be of interest – wouldn’t it?
I’m writing about this the day after the green fluorescent protein Nobel for a reason, of course. This is a similar approach, but taken down to the size of individual molecules – you can’t label uracil with GFP and expect it to be taken up into RNA, that’s for sure. Advances in labeling and detection are one of the main things driving biology these days, and this will just accelerate things. (It’s also killing off a lot of traditional radioactive isotope labeling work, too, not that anyone’s going to miss it). For the foreseeable future, we’re going to be bombarded with more information than we know what to do with. It’ll be great – enjoy it!
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October 8, 2008
Posted by Derek
So it was green fluorescent protein after all! We can argue about whether this was a pure chemistry prize or another quasi-biology one, but either way, the award is a strong one. So, what is the stuff and what’s it do?
Osamu Shimomura discovered the actual protein back in 1962, isolating it from the jellyfish Aequoria victoria. These were known to be luminescent creatures, but when the light-emitting protein was found (named aequorin), it turned out to give off blue light. That was strange, since the jellyfish were known for their green color. Shimomura then isolated another protein from the same jellyfish cells, which turned out to absorb the blue light from aequorin very efficiently and then fluoresce in the green: green fluorescent protein. The two proteins are a coupled system, an excellent example of a phenomenon known as FRET (fluorescence resonance energy transfer), which has been engineered into many other useful applications over the years.
Fluorescence is much more common in inorganic salts and small organic molecules, and at first it was a puzzle how a protein could emit light in the same way. As it turns out, there’s a three-amino-acid sequence right in the middle of its structure (serine-tyrosine-glycine) that condenses with itself when the protein is folded properly and makes a new fluorescent species. (The last step of the process is reaction with ambient oxygen). The protein has a very pronounced barrel shape to it, and lines up these key amino acids in just the orientation needed for the reaction to go at a reasonable rate (on a time scale of tens of minutes at room temperature). This is well worked out now, but it was definitely not obvious at the time.
In the late 1980s, for example, the gene for GFP was cloned by Doug Prasher, but he and his co-workers believed that they could well express a non-fluorescent protein that would need activation by some other system. He had the idea that this could be used as a tag for other proteins, but was never able to get to the point of demonstrating it, and will join the list of people who were on the trail of a Nobel discovery but never quite got there. Update: Here's what Prasher is doing now - this is a hard-luck story if I've ever heard one Prasher furnished some of the clone to Martin Chalfie at Columbia, who got it to express in E. coli and found that the bacteria indeed glowed bright green. (Other groups were trying the same thing, but the expression was a bit tricky at the time). The next step was to express it in the roundworm C. elegans (naturally enough, since Chalfie had worked with Sydney Brenner). Splicing it in behind a specific promoter caused the GFP to express in definite patterns in the worms, just as expected. This all suggested that the protein was fluorescing on its own, and could do the same in all sorts of organisms under all sorts of conditions.
And so it’s proved. GFP is wonderful stuff for marking proteins in living systems. Its sequence can be fused on to many other proteins without disturbing their function, it folds up just fine with no help to its active form, and it’s bright and very photoefficient. Where Roger Tsien enters the picture is in extending this idea to a whole family of proteins. Tsien worked out the last details of the fluorescent structure, showing that oxygen is needed for the last step. He and his group then set out to make mutant forms of the protein, changing the color of its fluorescence and other properties. He’s done the same thing with a red fluorescent protein from coral, and this work (which continues in labs all over the world) has led to a wide variety of in vivo fluorescent tags, which can be made to perform a huge number of useful tricks. They can sense calcium levels or the presence of various metabolites, fluoresce only when they come into contact with another specifically labeled protein, used in various time-resolved techniques to monitor the speed of protein trafficking, and who knows what else. A lot of what we’ve learned in the last fifteen years about the behavior of real proteins in living cells has come out of this work – the prize is well deserved.
I want to close with a bit of an interview with Martin Chalfie, which is an excellent insight into how things like this get discovered (or don't!)
Considering how significant GFP has been, why do you think no one else came up with it, while you were waiting for Doug Prasher to clone it?
"That’s a very important point. In hindsight, you wonder why 50 billion people weren’t working on this. But I think the field of bioluminescence or, in general, the research done on organisms and biological problems that have no immediate medical implications, was not viewed as being important science. People were working on this, but it was slow and tedious work, and getting enough protein from jellyfish required rather long hours at the lab. They had to devise ways of isolating the cells that were bioluminescent and then grinding them up and doing the extraction on them. It’s not like ordering a bunch of mice and getting livers out and doing an experiment. It was all rather arduous. It’s quite remarkable that it was done at all. It was mostly biochemists doing it, and they were not getting a lot of support. In fact, as I remember it, Doug Prasher had some funding initially from the American Cancer Society, and when that dried up he could not get grants to pursue the work. I never applied for a grant to do the original GFP research. Granting agencies would have wanted to see preliminary data and the work was outside my main research program. GFP is really an example of something very useful coming from a far-outside-the-mainstream source. And because this was coming from a non-model-organism system, these jellyfish found off the west coast of the U.S., people were not jumping at the chance to go out and isolate RNAs and make cDNAs from them. So we’re not talking about a field that was highly populated. It was not something that was widely talked about. At the time, there was a lot of excitement about molecular biology, but this was biochemistry. The discovery really was somewhat orthogonal to the mainstream of biological research."
Here's an entire site dedicated to the GFP story, full of illustrations and details. That interview with Chalfie is here, with some background on his part in the discovery. Science background from the Nobel Foundation is here (PDF), for those who want even more).
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September 9, 2008
Posted by Derek
As I’ve noted here, and many others have elsewhere, we have very little idea how many important central nervous system drugs actually work. Antidepressants, antipsychotics, antiseizure medications for epilepsy – the real workings of these drugs are quite obscure. The standard explanation for this state of things is that the human brain is extremely complicated and difficult to study, and that’s absolutely right.
But there’s an interesting paper on antipsychotics that’s just come out from a group at Duke, suggesting that there’s an important common mechanism that has been missed up until now. One thing that everyone can agree on is that dopamine receptors are important in this area. Which ones, and how they should be affected (agonist, antagonist, inverse partial what-have-you) – now that’s a subject for argument, but I don’t think you’ll find anyone who says that the dopaminergic system isn’t a big factor. Helping to keep the argument going is the fact that the existing drugs have a rather wide spectrum of activity against the main dopamine receptors.
But for some years now, the D2 subtype has been considered first among equals in this area. Binding affinity to D2 correlates as well as anything does to clinical efficacy, but when you look closer, the various drugs have different profiles as inverse agonists and antagonists of the receptor. What this latest study shows, though, is that a completely different signaling pathway – other than the classic GPCR signaling one – might well be involved. A protein called beta-arrestin has long been known to be important in receptor trafficking – movement of the receptor protein to and from the cell surface. A few years ago, it was shown that beta-arrestin isn’t just some sort of cellular tugboat in these systems, but can participate in another signaling pathway entirely.
Dopamine receptors were already complicated when I worked on them, but they’ve gotten a lot hairier since then. The beta-arrestin work makes things even trickier: who would have thought that these GPCRs, with all of their well-established and subtle signaling modes, also participated in a totally different signaling network at the same time? It’s like finding out that all your hammers can also drive screws, using some gizmo hidden in their handles that you didn’t even know was there.
When this latest team looked at the various clinical antipsychotics, what they found was that no matter what their profile in the traditional D2 signaling assays, they all are very good at disrupting the D2/beta-arrestin pathway. Since some of the downstream targets in that pathway (a protein called Akt and a kinase, GSK-3) have already been associated with schizophrenia, this may well be a big factor behind antipsychotic efficacy, and one that no one in the drug discovery business has paid much attention to. As soon as someone gets this formatted for a high-throughput assay, though, that will change – and it could lead to entirely new compound classes in this area.
Of course, there’s still a lot that we don’t know. What, for example, does beta-arrestin signaling actually do in schizophrenia? Akt and GSK-3 are powerful signaling players, involved in all sorts of pathways. Untangling their roles, or the roles of other yet-unknown beta-arrestin driven processes, will keep the biologists busy for a good long while. And the existing antipsychotics hit quite a few other receptors as well – what’s the role of the beta-arrestin system in those interactions? The brain will keep us busy for a good long while, and so will the signaling receptors.
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August 26, 2008
Posted by Derek
As all organic chemists who follow the literature know, over the last few years there’s been a strong swell of papers using Barry Sharpless’s “click chemistry” triazole-forming reactions. These reaction let you form five-membered triazole rings from two not-very-reactive partners, an azide and an acetylene, and people have been putting them to all kinds of uses, from the trivial to the very interesting indeed.
In the former category are papers that boil down to “We made triazoles from some acetylenes and azides that no one else has gotten around to using yet, and here they are, for some reason”. There are fewer of those publications than there were a couple of years ago, but they’re still out there. For its part, the latter (interesting) category is really all over the place, from in vivo biological applications to nanotechnology and materials science.
One recent paper in Organic Letters which was called to my attention starts off looking as if it’s going to be another bit of flotsam from the first group, but by the end it’s a very different thing indeed. The authors (from the Isobe group at Tohoku University in Japan, with collaborators from Tokyo) have made an analog of thymine, the T in the genetic code, where the 2-deoxyribose part has both an azide and an acetylene built onto it.
So far, so good, and at one point you probably could have gotten a paper out of things right there – let ‘em rip to make a few poly-triazole things and send off the manuscript. But this is a more complete piece of work. For one thing, they’ve made sure that their acetylenes can have removable silyl groups on them. That lets you turn their click reactivity on and off, since the copper-catalyzed reaction needs a free alkyne out there. So starting from a resin-supported sugar, they did one triazole click reaction after another in a controlled fashion – it took some messing around with the conditions, but they worked it out pretty smoothly.
And since the acetylene was at the 5 position of the sugar, and the azide was at the 3, they built a sort of poly-T oligonucleotide – but one that’s linked together by triazoles where instead of the phosphate groups found in DNA. People have, of course, made all sorts of DNA analogs, with all sorts of replacements for the phosphates, but they vary in how well they mimic the real thing. Startlingly, when they took a 10-mer of their “TL-DNA” (triazole-linked) and exposed it to a complementary 10-residue strand of good ol' poly-A DNA, the two zipped right up. In fact, the resulting helix seems to be significantly stronger than native DNA, as measured by a large increase in melting point. (That's their molecular model of the complex below left).
Well, after reading this paper, my first thought was that it might eventually make me eat some of my other words. Because just last week I was saying things about the prospects for nucleic acid therapies (RNAi, antisense) - mean, horrible, nasty things, according to a few of the comments that piled up, about how these might be rather hard to implement. But when I saw the end of this paper, the first thing that popped into my head was "stable high-affinity antisense DNA backbone. Holy cow". I assume that this also crossed the minds of the authors, and of some of the paper's other readers. Given the potential of the field, I would also assume that eventually we'll see that idea put to a test. It's a long way from being something that works, but it sure looks like a good thing to take a look at, doesn't it?
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July 16, 2008
Posted by Derek
At various points in my drug discovery career, I’ve worked on G-protein-coupled receptor (GPCR) targets. Most everyone in the drug industry has at some point – a significant fraction of the known drugs work through them, even though we have a heck of a time knowing what their structures are like.
For those outside the field, GPCRs are a ubiquitous mode of signaling between the interior of a cell and what’s going on outside it, which accounts for the hundreds of different types of the things. They’re all large proteins that sit in the cell membrane, looped around so that some of their surfaces are on the outside and some poke through to the inside. The outside folds have a defined binding site for some particular ligand - a small molecule or protein – and the inside surfaces interact with a variety of other signaling proteins, first among them being the G-proteins of the name. When a receptor’s ligand binds from the outside, that sets off some sort of big shape change. The protein’s coils slide and shift around in response, which changes its exposed surfaces and binding patterns on the inside face. Suddenly different proteins are bound and released there, which sets off the various chemical signaling cascades inside the cell.
The reason we like GPCRs is that many of them have binding sites for small molecules, like the neurotransmitters. Dopamine, serotonin, acetylcholine – these are molecules that medicinal chemists can really get their hands around. The receptors that bind whole other proteins as external ligands are definitely a tougher bunch to work with, but we’ve still found many small molecules that will interact with some of them.
Naturally, there are at least two modes of signaling a GPCR can engage in: on and off. A ligand that comes in and sets off the intracellular signaling is called an agonist, and one that binds but doesn’t set off those signals is called an antagonist. Antagonist molecules will also gum up the works and block agonists from doing their things. We have an easier time making those, naturally, since there are dozens of ways to mess up a process compared to the ways there are of running it correctly!
Now, when I was first working in the GPCR field almost twenty years ago, it was reasonably straightforward. You had your agonists and you had your antagonists – well, OK, there were those irritating partial agonists, true. Those things set off the desired cellular signal, but never at the levels that a full agonist would, for some reason. And there were a lot of odd behaviors that no one quite knew how to explain, but we tried to not let those bother us.
These days, it’s become clear that GPCRs are not so simple. There appear to be some, for example, whose default setting is “on”, with no agonist needed. People are still arguing about how many receptors do this in the wild, but there seems little doubt that it does go on. These constituitively active receptors can be turned off, though, by the binding of some ligands, which are known as inverse agonists, and there are others, good old antagonists, that can block the action of the inverse agonists. Figuring out which receptors do this sort of thing - and which drugs - is a full time job for a lot of people.
It’s also been appreciated in recent years that GPCRs don’t just float around by themselves on the cell surface. Many of them interact with other nearby receptors, binding side-by-side with them, and their activities can vary depending on the environment they’re in. The search is on for compounds that will recognize receptor dimers over the good ol’ monomeric forms, and the search is also on for figuring out what those will do once we have them. To add to the fun, these various dimers can be with other receptors of their own kind (homodimers) or with totally different ones, some from different families entirely (heterodimers). This area of research is definitely heating up.
And recently, I came across a paper which looked at how a standard GPCR can respond differently to an agonist depending on where it's located in the membrane. We're starting to understand how heterogeneous the lipids in that membrane are, and that receptors can move from one domain to another depending on what's binding to them (either on their outside or inside faces). The techniques to study this kind of thing are not trivial, to put it mildly, and we're only just getting started on figuring out what's going on out there in the real world in real time. Doubtless many bizarre surprises await.
So, once again, the "nothing is simple" rule prevails. This kind of thing is why I can't completely succumb to the gloom that sometimes spreads over the industry. There's just so much that we don't know, and so much to work on, and so many people that need what we're trying to discover, that I can't believe that the whole enterprise is in as much trouble as (sometimes) it seems. . .
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May 22, 2008
Posted by Derek
Benjamin Cravatt at Scripps has another interesting paper out this week – by my standards, he hasn’t published very many dull ones. I spoke about some earlier work of his here, where his group tried to profile enzymes in living cells and found that the results they got were much different than the ones seen in their model systems.
This latest paper is in the same vein, but addresses some more general questions. One of his group members (Eranthi Weerapana, who certainly seems to have put in some lab time) started by synthesizing five simple test compounds. Each of them had a reactive group on them, and each molecule had an acetylene on the far end. The idea was to see what sorts of proteins combined with the reactive head group. After labeling, a click-type triazole reaction stuck a fluorescent tag on via the acetylene group, allowing the labeled proteins to be detected.
All this is similar to the previous paper I blogged about, but in this case they were interested in profiling these varying head groups: a benzenesulfonate, an alpha-chloroamide, a terminal enone, and two epoxides – one terminal on a linear chain, and the other a spiro off a cyclohexane. All these have the potential to react with various nucleophilic groups on a protein – cysteines, lysines, histidines, and so on. Which reactive groups would react with which sorts of protein residues, and on which parts of the proteins, was unknown.
There have been only a few general studies of this sort. The most closely related work is from Daniel Liebler at Vanderbilt, who's looking at this issue from a toxicology perspective ( try here , here, and here). And an earlier look at different reactive groups from the Sames lab at Columbia is here, but that was much less extensive.
Cravatt's study reacted these probes first with a soluble protein mix from mouse liver – containing who knows how many different proteins – and followed that up with similar experiments with protein brews from heart and kidney, along with the insoluble membrane fraction from the liver. A brutally efficient proteolysis/mass spectroscopy technique, described by Cravatt in 2005, was used to simultaneously identify the labeled proteins and the sites at which they reacted. This is clearly the sort of experiment that would have been unthinkable not that many years ago, and it still gives me a turn to see only Cravatt, Weerapana, and a third co-author (Gabriel Simon) on this one instead of some lab-coated army.
Hundreds of proteins were found to react, as you might expect from such simple coupling partners. But this wasn’t just a blunderbuss scatter; some very interesting patterns showed up. For one thing, the two epoxides hardly reacted with anything, which is quite interesting considering that functional group’s reputation. I don’t think I’ve ever met a toxicologist who wouldn’t reject an epoxide-containing drug candidate outright, but these groups are clearly not as red-hot as they’re billed. The epoxide compounds were so unreactive, in fact, that they didn’t even make the cut after the initial mouse liver experiment. (Since Cravatt’s group has already shown that more elaborate and tighter-binding spiro-epoxides can react with an active-site lysine, I’m willing to bet that they were surprised by this result, too).
The next trend to emerge was that the chloroamide and the enone, while they labeled all sorts of proteins, almost invariably did so on their cysteine (SH) residues. Again, I think if you took a survey of organic chemists or enzymologists, you’d have found cysteines at the top of the expected list, but plenty of other things would have been predicted to react as well. The selectivity is quite striking. What’s even more interesting, and as yet unexplained, is that over half the cysteine residues that were hit only reacted with one of the two reagents, not the other. (Leibler has seen similar effects in his work).
Meanwhile, the sulfonate went for several different sorts of amino acid residues – it liked glutamates especially, but also aspartate, cysteine, tyrosine, and some histidines. One of the things I found striking about these results is how few lysines got in on the act with any of the electrophiles. Cravatt's finely tuned epoxide/lysine interaction that I linked to above turns out, apparently, to be a rather rare bird. I’ve always had lysine in my mind as a potentially reactive group, but I can see that I’m going to have adjust my thinking.
Another trend that I found thought-provoking was that the labeled residues were disproportionately taken from the list of important ones, amino acids that are involved in the various active sites or in regulatory domains. The former may be intrinsically more reactive, in an environment that has been selected to increase their nucleophilicity. And as for the latter, I’d think that’s because they’re well exposed on the surfaces of the proteins, for one thing, although they may also be juiced up in reactivity compared to their run-of-the-mill counterparts.
Finally, there’s another result that reminded me of the model-system problems in Cravatt’s last paper. When they took these probes and reacted them with mixtures of amino acid derivatives in solution, the results were very different than what they saw in real protein samples. The chloroamide looked roughly the same, attacking mostly cysteines. But the sulfonate, for some reason, looked just like it, completely losing its real-world preference for carboxylate side chains. Meanwhile, the enone went after cysteine, lysine, and histidine in the model system, but largely ignored the last two in the real world. The reasons for these differences are, to say the least, unclear – but what’s clear, from this paper and the previous ones, is that there is (once again!) no substitute for the real world in chemical biology. (In fact, in that last paper, even cell lysates weren’t real enough. This one has a bit of whole-cell data, which looks similar to the lysate stuff this time, but I’d be interested to know if more experiments were done on living systems, and how close they were to the other data sets).
So there are a lot of lessons here - at least, if you really get into this chemical biology stuff, and I obviously do. But even if you don't, remember that last one: run the real system if you're doing anything complicated. And if you're in drug discovery, brother, you're doing something complicated.
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May 19, 2008
Posted by Derek
OK, drugs generally bind to some sort of cavity in a protein. So what’s in that cavity when the drug isn’t there? Well, sometimes it’s the substance that the drug is trying to mimic or block, the body’s own ligand doing what it’s supposed to be doing. But what about when that isn’t occupying the space – what is?
A moment’s thought, and most chemists and biologists will say “water”. That’s mostly true, although it can give a false impression. When you get X-ray crystal structures of enzymes, there’s always water hanging around the protein. But at this scale, any thoughts of bulk water as we know it are extremely misleading. Those are individual water molecules down there, a very different thing.
There seem to be several different sorts of them, for one thing. Some of those waters are essential to the structure of the protein itself – they form hydrogen bonds between key residues of its backbone, and you mess with them at your peril. Others are adventitious, showing up in your X-ray structure in the same way that pedestrians show up in a snapshot of a building’s lobby. (That’s a good metaphor, if I do say so myself, but to work that first set of water molecules into it, you’d have to imagine people stuck against the walls with their arms spread, helping to hold up the building).
And in between those two categories are waters that can interact with both the protein and your drug candidate. They can form bridges between them, or they can be kicked out so that your drug interacts directly. Which is better? Unfortunately, it’s hard to generalize. There are potent compounds that sit in a web of water molecules, and there are others that cozy right up to the protein at every turn.
But there's one oddity that just came out in the literature. This one's weird enough to deserve its own paper: the protein beta-lactoglobulin appears to have a large binding site that's completely empty of water molecules. It's a site for large lipids to bind, so it makes sense that it would be a greasy environment that wouldn't be friendly to a lot of water, but completely empty? That's a first, as far as I know. When you think about it, that's quite weird: inside that protein is a small zone that's a harder vacuum than anything even seen in the lab: there's nothing there at all. It's a small bit of interstellar space, sitting inside a protein from cow blood. Nature abhors a vacuum, but apparently not this one.
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May 16, 2008
Posted by Derek
A good rule to follow: hold onto your wallet when two exciting, complicated fields of research are combined. Nature reported earlier this spring on a good example of this, the announcement by a small biotech called Primegen that they'd used carbon nanotubes to reprogram stem cells. (Here's a good article from VentureBeat on the same announcement, and there's an excellent piece on the announcement and the company in Forbes).
Stem cells and nanostructures are two undeniably hot areas of research. And also undeniable is that fact that they're both in their very early days - the amount of important information we don't know about both of these topics must be really impressive, which is why so many people are beavering away at them. So what are the odds of getting them to work together? Not as good as the odds that someone thought the combination would make a good press release, I'm afraid.
The PrimeGen web site, though a bit better than that VentureBeat article describes it, still has some odd notes to it. I particularly like this phrase: "PrimeGen’s broad intellectual property portfolio is founded on groundbreaking platform technologies invented by our team of dedicated and visionary scientists." Yep, we talk that way all the time in this business. You also have to raise an eyebrow at this part: "Disease and injury applications of PrimeCell™ include Alzheimer’s Disease, Cardiac Disease, Diabetes, Lupus, Multiple Sclerosis, Leukemia, Muscular Dystrophy, Parkinson’s Disease, Rheumatoid Arthritis, Spinal Cord Injury, Autoimmune Disease, Stroke, Skin Regeneration and Wound Healing." It'll mow your yard, too, if you're willing to participate in the next funding round.
The next sentence is the key one: "The extent to which stem cells can be used to treat injury and illness has yet to be fully evaluated. . ." You can say that again! In fact, I wouldn't mind seeing that in 36-point bold across the top of every stem cell company web page and press release. But what are the chances of that? As good as the chance that nanotechnology will suddenly going provide us a way to make the stem cells do what we want, I'm afraid. . .
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March 28, 2008
Posted by Derek
It’s been a while since I talked about RNA interference here. It’s still one of those tremendously promising therapeutic ideas, and it’s still having a tremendously hard time proving itself. Small RNA molecules can do all sorts of interesting and surprising things inside cells, but the trick is getting them there. Living systems are not inclined to let a lot of little nucleic acid sequences run around unmolested through the bloodstream.
The RNA folks can at least build on the experience (long, difficult, expensive) of the antisense DNA people, who have been trying to dose their compounds for years now and have tried out all sorts of ingenious schemes. But even if all these micro-RNAs could be dosed, would we still know what they’re going to do?
A report in the latest Nature suggests that the answer is “not at all”. This large multi-university group was looking at macular degeneration, a natural target for this sort of technology. It’s a serious disease, and it occurs in a privileged compartment of the body, the inside of the eye. You can inject your new therapy directly in there, for example (I know, it gives me the shivers, too, but it sure beats going blind). That bypasses the gut, the liver, and the bloodstream, and that humoral fluid of the eye is comparatively free of hostile enzymes. (It’s no coincidence that the antisense and aptamer people have gone after this and other eye diseases as well).
Angiogenesis is a common molecular target for macular regeneration, since uncontrolled formation of new capillaries is a proximate cause of blindness in such conditions. (That target has the added benefit of giving your therapy a possible entry into the oncology world, should you figure out how to get it to work well here). VEGF is the prototype angiogenesis target, so you’d figure that RNA interference targeting VEGF production or signaling would work as well as anything could, as a first guess.
And so it does, as this team found out. But here comes the surprise: when the researchers checked their control group, using a similar RNA that should have been ineffective, they found that it was working just fine, too – just as well as the VEGF-targeted ones, actually. Baffled, they went on to try a host of other RNAs. Reading the paper, you can just see the disbelief mounting as they tried various sequences against other angiogenic targets (success!), nonangiogenic proteins (success!?), proangiogenic ones that should make the disease worse (success??), genes for proteins that aren’t even expressed in the eye (success!), sequences against RNAs from plants and microbes that don’t even exist in humans at all (oh God, success again), totally random RNAs (success, damnit), and RNAs that shouldn’t be able to silence anything because they’ve got completely the wrong sort of sequence (oh the hell with it, success). Some of these even worked when injected i.p., into the gut cavity, instead of into the eye at all, suggesting that this was a general mechanism that had nothing to do with the retina.
As it turns out, these things are acting through hitting a cell surface receptor, TLR3. And all you need, apparently, is a stretch of RNA that’s at least 21 units long. Doesn’t seem to matter much what the sequence is – thus all that darn success with whatever they tried. Downstream of TLR3 come induction of gamma-interferon and IL-12, and those are what are doing the job of shutting down angiogenesis. (Off-target effects involving these have been noted before with siRNA, but now I think we’re finally figuring out why).
What does this all mean? Good news and bad news. The companies that are already dosing RNAi therapies for macular degeneration have just discovered that there's an awful lot that they don't know about what they're doing, for one thing. On the flip side, there are a lot of human cell types with TLR3 receptors on them, and a lot of angiogenic disorders that could potentially be treated, at least partially, by targeting them in this manner. That’s some good news. The bad news is that most of these receptors are present in more demanding environments than the inside of the eye, so the whole problem of turning siRNAs into drugs still looms large.
And the other bad news is that if you do figure out a way to dose these things, you may well set off TLR3 effects whether you want them or not. Immune system effects on the vasculature are not the answer to everything, but that may be one of the answers you always get. And this sort of thing makes you wonder what other surprising things systemic RNA therapies might set off. We will, in due course, no doubt find out. More here from John Timmer at Nobel Intent, who correctly tags this as a perfect example of why you want to run a lot of good control experiments. . .
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February 14, 2008
Posted by Derek
I’ve been reading an interesting paper from JACS with the catchy title of “Optimization of Activity-Based Probes for Proteomic Profiling of Histone Deacetylase Complexes”. This is work from Benjamin Cravatt's lab at Scripps, and it says something about me, I suppose, that I found that title of such interest that I immediately printed off a copy to study more closely. Now I’ll see if I can interest anyone who wasn’t already intruiged! First off, some discussion of protein tagging, so if you’re into that stuff already, you may want to skip ahead.
So, let’s say you have a molecule that has some interesting biological effect, but you’re not sure how it works. You have suspicions that it’s binding to some protein and altering its effects (always a good guess), but which protein? Protein folks love fluorescent assays, so if you could hang some fluorescent molecule off one end of yours, perhaps you could start the hunt: expose your cells to the tagged molecule, break them open, look for the proteins that glow. There are complications, though. You’d have to staple the fluorescent part on in a way that didn’t totally mess up that biological activity you care about, which isn’t always easy (or even possible). The fact that most of the good fluorescent tags are rather large and ugly doesn’t help. But there’s more trouble: even if you manage to do that, what’s to keep your molecule from drifting right back off of the protein while you’re cleaning things up for a look at the system? Odds are it will, unless it has a really amazing binding constant, and that’s not the way to bet.
One way around that problem is sticking yet another appendage on to the molecule, a so-called photoaffinity label. These groups turn into highly reactive species on exposure to particular wavelengths of light, ready to form a bond with the first thing they see. If your molecule is carrying one when it’s bound to your mystery protein, shining light on the system will likely cause a permanent bond to form between the two. Then you can do all your purifications and separations, and look at your leisure for which proteins fluoresce.
This is “activity-based protein profiling”, and it’s a hot field. There are a lot of different photoaffinity labels, and a lot of ways to attach them, and likewise with the fluorescent groups. The big problem, as mentioned above, is that it’s very hard to get both of those on your molecule of interest and still keep its biological activity – that’s an awful lot of tinsel to carry around. One slick solution is to use a small placeholder for the big fluorescent part. This, ideally, would be some little group that will hide out innocently during the whole protein-binding and photoaffinity-labeling steps, then react with a suitably decorated fluorescent partner once everything’s in place. This assembles your glowing tag after the fact.
A favorite way to do that step is through an azide-acetylene cycloaddition reaction, the favorite of Barry Sharpless’s “click” reactions. Acetylenes are small and relatively unreactive, and at the end of the process, after you’ve lysed the cells and released all their proteins, you can flood your system with azide-substituted fluorescent reagent. The two groups react irreversibly under mild catalytic conditions to make a triazole ring linker, which is a nearly ideal solution that’s getting a lot of use these days (more on this another day).
So, now to this paper. What this group did was label a known compound (from Ron Breslow's group at Columbia) that targets histone deacetylase (HDAC) enzymes, SAHA, now on the market as Vorinostat. There are a lot of different subtypes of HDAC, and they do a lot of important but obscure things that haven’t been worked out yet. It’s a good field to discover protein function in.
When they modified SAHA in just the way described above, with an acetylene and a photoaffinity group, it maintained its activity on the known enzymes, so things looked good. They then exposed it to cell lysate, the whole protein soup, and found that while it did label HDAC enzymes, it seemed to label a lot of other things in the background. That kind of nonspecific activity can kill an assay, but they tried the label out on living cells anyway, just to see what would happen.
Very much to their surprise, that experiment led to much cleaner and more specific labeling of HDACs. The living system was much nicer than the surrogate, which (believe me) is not how things generally go. Some HDACs were labeled much more than others, though, and my first thought on reading that was “Well, yeah, sure, your molecule is a more potent binder to some of them”.
But that wasn’t the case, either. When they profiled their probe molecule’s activity versus a panel of HDAC enzymes, they did indeed find different levels of binding – but those didn’t match up with which ones were labeled more in the cells. (One explanation might be that the photoaffinity label found some of the proteins easier to react with than others, perhaps due to what was nearby in each case when the reactive species formed).
Their next step was to make a series of modified SAHA scaffolds and rig them up with the whole probe apparatus. Exposing these to cell lysate showed that many of them performed fine, labeling HDAC subtypes as they should, and with different selectivities than the original. But when they put these into cells, none of them worked as well as the plain SAHA probe – again, rather to their surprise. (A lot of work went into making and profiling those variations, so I suspect that this wasn’t exactly the result the team had hoped for - my sympathies to Cravatt and especially to his co-author Cleo Salisbury). The paper sums the situation up dryly: "These results demonstrate that in vitro labeling is not necessarily predictive of in situ labeling for activity-based protein profiling probes".
And that matches up perfectly with my own prejudices, so it must be right. I've come to think, over the years, that the way to go is to run your ideas against the most complex system you think that they can stand up to - in fact, maybe one step beyond that, because you may have underestimated them. A strict reductionist might have stopped after the cell lysate experiments in this case - clearly, this probe was too nonspecific, no need to waste time on the real system, eh? But the real system, the living cell, is real in complex ways that we don't understand well at all, and that makes this inference invalid.
The same goes for medicinal chemistry and drug development. If you say "in vitro", I say "whole cells". If you've got it working in cells, I'll call for mice. Then I'll see your mice and raise you some dogs. Get your compounds as close to reality as you can before you pass judgment on them.
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January 8, 2008
Posted by Derek
I came across a neat article in Nature from a group working on a new technique in neuroscience imaging. They expressed an array of four differently colored fluorescent proteins in developing neurons in vivo, and placed them so that recombination events would scramble the relative expression of the multiple transgenes as the cell population expands. That leads to what they’re calling a “brainbow”: a striking array of about a hundred different shades of fluorescent neurons, tangled into what looks like a close-up of a Seurat painting.
The good part is that the entire neuron fluoresces, not just a particular structure inside it. Being able to see all those axons opens up the possibility of tracking how the cells interact in the developing brain – where synapses form and when. That should keep everyone in this research group occupied for a good long while.
What I particularly enjoyed, though, was the attitude of the lab head, Jeff Lichtman of Harvard. He states that he doesn’t really know exactly what they’re looking for, but that this technique will allow them to just sit back and see what there is to see. That’s a scientific mode with a long history, basically good old Francis-Bacon style induction, but we don’t actually get a chance to do it as much as you’d think.
That varies by the area being under investigation. In general, the more complex and poorly understood the object of study, the more appropriate it is to sit back and take notes, rather than go in trying to prove some particular hypothesis. (Neuroscience, then, is a natural!) In a chemistry setting, though, I wouldn’t recommend setting up five thousand sulfonamide formations just to see what happens, because we already have a pretty good idea of what’ll happen. But if you’re working on new metal-catalyzed reactions, a big screen of every variety of metal complex you can find might not be such a bad idea, if you’ve got the time and material. There’s a lot that we don’t know about those things, and you could come across an interesting lead.
Some people get uncomfortable with “fishing expedition” work like this, though. In the med-chem labs, I’ve seen some fishy glances directed at people who just made a bunch of compounds in a series because no one else had made them and they just wanted to see what would happen. While I agree that you don’t want to run a whole project like that, I think that the suspicion is often misplaced, considering how many projects start from high-throughput screening. We don’t, a priori, usually have any good idea of what molecules should bind to a new drug target. Going in with an advanced hypothesis-driven approach often isn’t as productive as just saying “OK, let’s run everything we’ve got past the thing, see what sticks, and take it from there”.
But the feeling seems to be that a drug project (and its team members) should somehow outgrow the random approach as more knowledge comes in. Ideally, that would be the case. I’m not convinced, though, that enough med-chem projects generate enough detailed knowledge about what will work and what won’t to be able to do that. (There’s no percentage in beating against structural trends that you have evidence for, but trying out things that no one’s tried yet is another story). It’s true that a project has to narrow down in order to deliver a lead compound to the clinic, but getting to the narrowing-down stage doesn’t have to be (and usually isn’t) a very orderly process.
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+ TrackBacks (0) | Category: Biological News | Drug Development | The Central Nervous System | Who Discovers and Why
December 5, 2007
Posted by Derek
How many hits can a drug – or a whole class of drugs – take? Avandia (rosiglitazone) has been the subject of much wrangling about cardiovascular risk in its patient population of Type II diabetics. But there have also been scattered reports of increases in fractures among people taking it or Actos (pioglitazone), the other drug with the same mechanism of action.
Now Ron Evans and his co-workers at Salk, who know about as much PPAR-gamma biology as there is to know, have completed a difficult series of experiments that provides some worrying data about what might be going on. Studying PPAR-gamma’s function in mice is tricky, since you can’t just step in and knock it out (that’s embryonic lethal), and its function varies depending on the tissue where it’s expressed. (That latter effect is seen across many other nuclear receptors, which is just one of the things that make their biology so nightmarishly complex).
So tissue-specific knockouts are the way to go, but the bones are an interesting organ. The body is constantly laying down new bone tissue and reabsorbing the old. Evans and his team managed to knock out the system in osteoclasts (the bone-destroying cells), but not osteoblasts (the bone-forming ones). It’s been known for years that PPAR-gamma has effects on the development of the latter cells, which makes sense, because it also affects adipocytes (fat cells), and those two come from the same lineage. But no one’s been able to get a handle on what it does in osteoclasts, until now.
It turns out that without PPAR-gamma, the bones of the mice turned out larger and much more dense than in wild-type mice. (That’s called osteopetrosis, a word that you don’t hear very much compared to its opposite). Examining the tissue confirmed that there seemed to be normal numbers of osteoblasts, but far fewer osteoclasts to reabsorb the bone that was being produced. Does PPAR stimulation do the opposite? Unfortunately, yes – there had already been concern about possible effects on bone formation because of the known effects on osteoblasts, but it turned out that dosing rosiglitazone in mice actually stimulates their osteoclasts. This double mode of action, which was unexpected, speeds up the destruction of bone and at the same time slow down its formation. Not a good combination.
So there’s a real possibility that long-term PPAR-gamma agonist use might lead to osteoporosis in humans. If this is confirmed by studies of human osteoclast activity, that may be it for the glitazones. They seem to have real benefit in the treatment of diabetes, but not with these consequences. Suspicion of cardiovascular trouble, evidence of osteoporosis – diabetic patients have enough problems already.
As I’ve mentioned here before, I think that PPAR biology is a clear example of something that has turned out to be (thus far) too complex for us to deal with. (Want a taste? Try this on for size, and let me assure that this is a painfully oversimplified diagram). We don’t understand enough of the biology to know what to target, how to target it, and what else might happen when we do. And we've just proven that again. I spent several years working in this field, and I have to say, I feel safer watching it from a distance.
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+ TrackBacks (0) | Category: Biological News | Diabetes and Obesity | Toxicology
November 11, 2007
Posted by Derek
As you root through genomic sequences - and there are more and more of them to root through these days - you come across some stretches of DNA that hardly seem to vary at all. The hard-core "ultraconserved" parts, first identified in 2004, are absolutely identical between mice, rats, and humans. Our last common ancestor was rather a long time ago (I know, I know - everyone works with some people who seem to be exceptions, but bear with me), so these things are rather well-preserved.
Even important enzyme sequences vary a bit among the three species, so what could these pristine stretches (some of which are hundreds of base pairs long) be used for? The assumption, naturally, has been that whatever it is, it must be mighty important, but if we're going to be scientists, we can't just go around assuming that what we think must be right. A team at Lawrence Berkeley and the DOE put things to the test recently by identifying four of the ultraconserved elements that all seem to be located next to critical genes - and deleting them.
The knockout mice turned out to do something very surprising indeed. They were born normally, but then they grew up normally. When they reached adulthood, though, they were completely normal. Exhaustive biochemical and behavioral tests finally uncovered the truth: they're basically indistinguishable from the wild type. Hey, I told you it was surprising. This must have been the last thing that the researchers expected.
Reaction to these results has been a series of raised eyebrows and furrowed foreheads. Deleting any of the known genes near the ultraconserved sequences confirms that they, anyway, are as important as they're billed to be. And these genes show the usual level of difference that you see among the three species. So what's this unchanged, untouchable, but apparently disposable stuff in there with them?
No one knows. And it's a real puzzle, the answer to which is going to be tangled up with a lot of our basic ideas about genes and evolution. To a good first approximation, it's hard to see how (or why) something like this should be going on. So what, exactly, are we missing? Something important? And if so, what else have we missed, too?
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October 29, 2007
Posted by Derek
There was an intriguing paper published earlier this month from Manfred Reetz and co-workers at the Max Planck Institute. It's not only an interesting finding, but a good example of making lemonade from lemons.
They were looking at an enzyme called tHisF, a thermostable beast from a marine microorganism that's normally involved in histamine synthesis. It has an acid/base catalytic site, so Reetz's group, which has long been involved in pushing enzymes to do more than they usually do, was interested in seeing if this one would act as an esterase/hydrolase.
And so it did - not as efficiently as a real esterase, but not too shabby when given some generic nitrophenyl esters to chew on. There was some structure-activity trend at work: the larger the alkyl portion of the ester, the less the enzyme liked it. Given a racemic starting material, it did a good job of resolution, spitting out the R alcohol well over the S isomer. All just the sort of thing you'd expect from a normal enzyme.
Next, they used the crystal structure of the protein and previous work on the active site to see which amino acids were important for the esterase activity. And here's where the wheels came off. They did a series of amputations to all the active side chains, hacking aspartic acids and cysteines down to plain old alanine. And none of it did a thing. To what was no doubt a room full of shocked expressions, the enzyme kept rolling along exactly as before, even with what were supposed to be its key parts missing.
Further experiments confirmed that the active site actually seems to have nothing at all to do with the hydrolase activity. So what's doing it? They're not sure, but there must be some other non-obvious site that's capable of acting like a completely different enzyme. I'm sure that they're actively searching for it now, probably by doing a list of likely point mutations until they finally hit something that stops the thing.
So how often does this sort of thing happen? Are there other enzymes with "active sites" that no one's ever recognized? If so, do these have any physiological relevance? No one knows yet, but a whole new area of enzymology may have been opened up. I look forward to seeing more publications on this, and I'll enjoy them all the more knowing that they came from a series of frustrating, head-scratching "failed" experiments. Instead of pouring things into the waste can, Reetz and his co-workers stayed the course, and my hat's off to them.
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October 15, 2007
Posted by Derek
The news of a possible diagnostic test for Alzheimer’s disease is very interesting, although there’s always room to wonder about the utility of a diagnosis of a disease for which there is little effective therapy. The sample size for this study is smaller than I’d like to see, but the protein markers that they’re finding seem pretty plausible, and I’m sure that many of them will turn out to have some association with the disease.
But let’s run some numbers. The test was 91% accurate when run on stored blood samples of people who were later checked for development of Alzheimer’s, which compared to the existing techniques is pretty good. Is it good enough for a diagnostic test, though? We’ll concentrate on the younger elderly, who would be most in the market for this test.The NIH estimates that about 5% of people from 65 to 74 have AD. According to the Census Bureau (pdf), we had 17.3 million people between those ages in 2000, and that’s expected to grow to almost 38 million in 2030. Let’s call it 20 million as a nice round number.
What if all 20 million had been tested with this new method? We’ll break that down into the two groups – the 1 million who are really going to get the disease and the 19 million who aren’t. When that latter group gets their results back, 17,290,000 people are going to be told, correctly, that they don’t seem to be on track to get Alzheimer’s. Unfortunately, because of that 91% accuracy rate, 1,710,000 people are going to be told, incorrectly, that they are. You can guess what this will do for their peace of mind. Note, also, that almost twice as many people have just been wrongly told that they’re getting Alzheimer’s than the total number of people who really will.
Meanwhile, the million people who really are in trouble are opening their envelopes, and 910,000 of them are getting the bad news. But 90,000 of them are being told, incorrectly, that they’re in good shape, and are in for a cruel time of it in the coming years.
The people who got the hard news are likely to want to know if that’s real or not, and many of them will take the test again just to be sure. But that’s not going to help; in fact, it’ll confuse things even more. If that whole cohort of 1.7 million people who were wrongly diagnosed as being at risk get re-tested, about 1.556 million of them will get a clean test this time. Now they have a dilemma – they’ve got one up and one down, and which one do you believe? Meanwhile, nearly 154,000 of them will get a second wrong diagnosis, and will be more sure than ever that they’re on the list for Alzheimer’s.
Meanwhile, if that list of 910,000 people who were correctly diagnosed as being at risk get re-tested, 828 thousand of them will hear the bad news again and will (correctly) assume that they’re in trouble. But we’ve just added to the mixed-diagnosis crowd, because almost 82,000 people will be incorrectly given a clean result and won’t know what to believe.
I’ll assume that the people who got the clean test the first time will not be motivated to check again. So after two rounds of testing, we have 17.3 million people who’ve been correctly given a clean ticket, and 828,000 who’ve been correctly been given the red flag. But we also have 154,000 people who aren’t going to get the disease but have been told twice that they will, 90,000 people who are going to get it but have been told that they aren’t, and over 1.6 million people who have been through a blender and don’t know anything more than when they started.
Sad but true: 91% is just not good enough for a diagnostic test. And getting back to that key point in the first paragraph, would 100% be enough for a disease that we can't do anything about? Wait for an effective therapy, is my advice, and for a better test.
Update: See the comments for more, because there's more to it than this. For one thing, are the false positive and false negative rates for this test the same? (That'll naturally make a big difference). And how about differential diagnosis, using other tests to rule out similar conditions? On the should-you-know question, what about the financial and estate planning implications of a positive test - shouldn't those be worth something? (And there's another topic that no one's brought up yet: suicide, which you'd have to think would be statistically noticeable. . .)
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+ TrackBacks (0) | Category: Alzheimer's Disease | Biological News
October 11, 2007
Posted by Derek
Now we open the sedate, learned pages of Nature Methods, a fine journal that specializes in new techniques in molecular and chemical biology. In the August issue, the correspondence section features. . .well, a testy response to a paper that appeared last year in Nature Methods.
“Experimental challenge to a ‘rigorous’ BRET analysis of GPCR oligimerization” is the title. If you don’t know the acronyms, never mind – journals like this have acronyms like leopards have spots. The people doing the complaining, Ali Salahpour and Bernard Masri of Duke, are taking issue with a paper from Oxford by John James, Simon Davis, and co-workers. The original paper described a bioluminescence energy transfer (BRET) method to see if G-protein coupled receptors (GPCRs) were associating with each other on cell surfaces. (GPCRs are hugely important signaling systems and drug targets – think serotonin, dopamine, opiates, adrenaline – and it’s become clear in recent years that they can possibly hook up in various unsuspected combinations on the surfaces of cells in vivo).
Salahpour and Masri take strong exception to the Oxford paper’s self-characterization:
“Although the development of new approaches for BRET analysis is commendable, part of the authors’ methodological approach falls short of being ‘rigorous’. . .Some of the pitfalls of their type-1 and type-2 experiments have already been discussed elsewhere (footnote to another complaint about the same work, which also appeared earlier this year in the same journal - DBL). Here we focus on the type-2 experiments and report experimental data to refute some of the results and conclusions presented by James et al.”
That’s about an 8 out of 10 on the scale of nasty scientific language, translating as “You mean well but are lamentably incompetent.” The only way to ratchet things up further is to accuse someone of bad faith or fraud. I won’t go into the technical details of Salahpour and Masri’s complaints; they have to do with the mechanism of BRET, the effect on it of how much GPCR protein is expressed in the cells being studied, and the way James et al. interpreted their results versus standards. The language of these complaints, though, is openly exasperated, full of wording like “unfortunately”, “It seems unlikely”, “we can assume, at best” “(does) not permit rigorous conclusions to be drawn”, “might be erroneous”, “inappropriate and a misinterpretation”, “This could explain why”, “careful examination also (raises) some concerns”, and so on. After the bandilleros and picadors have done their work in the preceding paragraphs, the communication finishes up with another flash of the sword:
In summary, we agree with James and colleagues that type-2 experiments are useful and informative. . .Unfortunately, the experimental design proposed in James et al. to perform type-2 experiments seems incorrect and cannot be interpreted. . .”
James and Davis don’t take this with a smile, naturally. The journal gave them a space to reply to the criticisms, as is standard practice, and as they did for the earlier criticism. (At least the editors know that people are reading the papers they accept. . .) They take on many of the Salahpour/Masri points, claiming that their refutations were done under completely inappropriate conditions, among other things. And they finish up with a flourish, too:
"As we have emphasized, we were not the first to attempt quantitative analysis of BRET data. Previously, however, resonance energy transfer theory was misinterpreted (for example, ref. 4) or applied incorrectly (for example, ref. 5). (Note - reference 4 is to a paper by the first people to question their paper earlier this year, and reference 5 is to the work of Salahpour himself, a nice touch - DBL). The only truly novel aspect of our experiments is that we verified our particular implementation of the theory by analyzing a set of very well-characterized. . .control proteins. (Note - "as opposed to you people" - DBL). . . .In this context, the technical concerns of Salahpour and Masri do not seem relevant."
It's probably safe to say that the air has not yet been cleared. I'm not enough of a BRET hand to say who's right here, but it looks like we're all going to have some more chances to make up our minds (and to appreciate the invective along the way).
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+ TrackBacks (0) | Category: Biological News | Drug Assays | The Scientific Literature
September 6, 2007
Posted by Derek
It’s useful to be reminded every so often of how much you don’t know. There’s a new paper in PNAS that’ll do that for a number of its readers. The authors report a new protein, one of the iron-sulfur binding ones. There are quite a few of these known already, so this wouldn’t be big news by itself. But this one is the first of its kind to be found in the outer mitochondrial membrane, which makes it a bit more interesting.
It also has a very odd structure – well, odd to us humans anyway, for all we know things like this are all over the place and we haven’t stumbled across one until now. There’s a protein fold here which not only has never been seen in the 650 or iron-sulfur proteins with solved structures, it’s never been seen in any protein at all. That’s worth a good publication, for sure.
The part that’ll really throw people, though, is that this protein (named mitoNEET, for the amino acids that make up its weird fold) binds a known drug whose target we all thought we already knew. Actos (pioglitazone) turns out to associate with it, which is a very interesting surprise. We already knew the glitazones as PPAR-gamma ligands. We didn’t understand them as PPAR ligands (no one understands them very well, despite many years and many, many scores of millions of dollars), but that was generally accepted as their site of action.
And now there’s another one, which is going to make the pioglitazone story even more complex. Reading between the lines of the paper, I get the strong impression that the authors were fishing for another pioglitazone binding site, using modified versions of the drug to label proteins, and hit the jackpot with this one. (And good for them - that's a hard technique to get to work). There’s been some speculation that the compound might have effects on mitochondria that wouldn’t necessarily be PPAR-mediated, and this is strong circumstantial evidence for it.
What’s more, I can’t think of any other iron-sulfur proteins that are targets of small molecules. Just last week, I was talking about the diversity of binding sites and interactions that we haven’t explored in medicinal chemistry, and here’s an example for you.
This paper raises a pile of questions: what does mitoNEET do? Shuttle iron-sulfur complexes around? (If so, to where, and to what purpose?) Is it involved in diabetes, or other diseases of metabolism? Does pioglitazone modify its activity in vivo, whatever that activity is? How well does it bind the drug, anyway, and what does the structure of that complex look like? Does Avandia (rosiglitazone) bind, too, and if not, why not? Are there other proteins in this family, and do they also have drug interactions that we don’t know about? Ah, we’ll all be employed forever in this business, for as long as people can stand it.
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+ TrackBacks (0) | Category: Biological News | Diabetes and Obesity
July 17, 2007
Posted by Derek
A commentor to my Proteomics 101 post the other day brought up an important point: that before you can have a chance to figure out what a protein is doing, you have to know that it exists. Finding the darn things is no small job, since you're digging through piles of chemically similar stuff to unearth them. What's more, we can't just ignore 'em: some of the low-concentration proteins are also correspondingly important and powerful.
Nasty arguments can erupt over whether a given protein and its proposed functions even exist. Crockery is flying over one of those right now, an insulin-like protein hormone dubbed "visfatin" by its discoverers in Osaka a couple of years ago. Well, in this case the protein probably exists, but does it do what it's advertised to do? An insulin mimic secreted by fat cells would be worth knowing about, but there doesn't seem to be enough of it present in the blood to do much of anything, given how well it binds to its putative targets. There are also reports that some of that data in the Osaka paper are hard to reproduce.
Complicating things even more is the (apparently well-founded) contention that visfatin is a re-discovery of a protein already known as PBEF, which is identical to another protein named Nampt. (Each "discovering" group assigned their own name, a situation that happens so often in biology that people don't even notice it any more).
The whipped topping on the whole thing is a accusation of misconduct by someone in Japan, which led to an investigation by Osaka University, which has now recommended that the original paper be retracted. Its lead author, Iichiro Shimomura, does not agree, as you might well imagine. The points of contention are many: whether the misconduct was real at all, or whether it describes real events that don't rise to the level of misconduct, or whether the conclusions of the paper are invalidated or not by them, and so on.
An early solution appears unlikely. And we still don't know what exactly visfatin/PBEF/Nampt is doing. Next time you wonder how things are going over in the proteome, consider this one.
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+ TrackBacks (0) | Category: Biological News | Diabetes and Obesity | The Dark Side
November 6, 2006
Posted by Derek
One of the things I like most about science is that you really don't know what's going to happen next. That's especially true in the areas where things have just barely settled down. Before that, when a field is new, no one knows what to expect, so in a way there aren't really any surprising results: everything's a surprise. A much more settled area, by contrast, is far less likely to produce surprises, although when one shows up it really stands out. But a field where people are just starting to exhale and think that maybe they've finally figured out what's going on - that has the best combination of high contrast and a real likelihood for craziness.
Here's a perfect example, since I was just expressing some doubts about the immediate commercial potentials of RNA interference the other day. In a paper coming out in PNAS, a group at UCSF was investigating the use of some small double-stranded RNAs, just the sort of thing that can be used for RNAi experiments. But they found (to their great surprise) that their experiments were stimulating the transcription of their targeted genes, rather than shutting them down. Needless to say, this was not what anyone expected, and I'll bet the folks involved repeated these things many, many times before they could trust their own eyes. There are plenty of other people who won't believe it until they've seen it with theirs.
On a molecular biology level, it's hard to say just what's going on. The authors, according to this news item from Science (probably subscriber-only) say that they've found some rules about which genes will be susceptible to the technique and which won't, which will be released soon. (Translation: as soon as they can be reasonably sure that they won't make fools of themselves - this paper took enough nerve as it is).
The Science article includes a good deal of if-this-holds-up language, which is appropriate for such a weird discovery. (Are the editors there wondering why they didn't get a chance to publish the article themselves, or did they have the chance and turn it down?) At any rate, if-it-holds-up this effect will simultaneously complicate the RNAi field a great deal (it was gnarly enough already, thanks) and also open a door to some really unusual experiments. Upregulating genes isn't very easy, and there are no doubt many ideas that have been waiting on a way to do it. There are therapeutic possibilities, too, naturally - but they'll have to wait on the same difficulties as the other RNA therapies.
Anyway, I'm happy to see this. It opens up some completely new biology, and it opens a door to a potential Nobel for the discoverers should everything work out. And it always cheers me up when something totally unexpected flies down like this and lands on the lawn.
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October 18, 2006
Posted by Derek
There's a curious paper (subscriber-only link) in the latest Nature that's getting some attention, titled "A linguistic model for the rational design of antimicrobial peptides". For non-subscribers, here's a synopsis of the work from the magazine's news site.
A group at MIT headed by Gregory Stephanopolous has been studying various antimicrobial peptides, which are secreted by all kinds of organisms as antibiotics. Taking the amino acid sequences of several hundred of these and feeding them into a linguistic pattern-analysing program suggested some common features, which they then used to synthesize 42 new unnatural candidates. The hit rate for these was about 50%, which is far, far more than you'd expect if you weren't tuning in to some sort of useful rules.
It's the concept of "peptide grammar" that seems to be the news hook here. But I'm quite puzzled by all the fuss, because looking for homology among protein sequences is one of the basic bioinformatics tools. I have to wonder what the MIT group found with their linguistics program that they wouldn't have found with biology software. What they're doing is good old structure-activity relationship work, the lifeblood of every medicinal chemist. Well, it's perhaps better described as sequence-activity relationships, but sequence is just a code for structure. There's nothing here that any drug company's bioinformatics people wouldn't be able to do for you, as far as I can see.
So why haven't they? Well, despite the article's mention of a potential 50,000 further peptides of this type, the reason is probably because not many people care. After all, we're talking about small peptides here, of the sort that are typically just awful candidates for real-world drugs. And I'm not just babbling theory here - many people have actually tried for many years now to commercialize various antimicrobial peptides and landed flat on their faces.
You won't see a mention of that history in the Nature news story, unfortunately. They do, to their credit, mention (albeit in the fourth paragraph from the end) that peptides are troublesome development candidates. That's where it also says that there are reports that bacteria can become resistant even to these proteins, which prompts me to remind everyone that bacteria can become resistant to everything short of freshly extruded magma. It's in the very last paragraph of the story, though, that Robert Hancock of UBC in Vancouver says just what I was thinking when I started reading:
(Hancock) questions how different the linguistics technique is from other computational methods used to find similarities between protein sequences. "What's new is the catchy title," he says.
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+ TrackBacks (0) | Category: Biological News | Drug Development | Infectious Diseases
March 14, 2006
Posted by Derek
I received (some time ago) an answer from Miguel Cizin and the folks at Docoop, makers of Neowater. (If you haven't seen the first parts of this story, they're here and here). In that last post, I had a number of look-under-the-hood physical chemistry questions about the stuff, in an attempt to figure out if there's anything to it or not. Here they are in order, with the provided answers:
1. How much of Neowater's characteristics can be explained under the usual framework of colligative properties? That is, by how much is the boiling point of Neowater elevated, and by how much is its freezing point depressed?
The company provided some differential scanning and isothermal titration calorimetry data in response to this, which I appreciate. I'm no expert in this area, but to my eye the ITC plots look broadly similar, but with a noticeably longer half-life to thermal equilibrium in the Neowater runs. (It's not noted what substance was being injected in these experiments).
2. Similarly, what's its vapor pressure at STP? Does it show a negative deviation from Raoult's Law (as you'd expect from the descriptions in the patent of Neowater's structure), and is this deviation much greater than expected given the low levels of particulate matter contained? The literature on the DoCoop web site, I should note, mentions that Neowater evaporates more slowly than regular water.
DoCoop replies: "Neowater indeed evaporates more slowly than regular water, since the water molecules are less available as they are attracted to the charged nanoparticles, hence it takes more energy to dislodge them. The difference in the vapor pressure is one of the mechanisms of action that we use to alter the dynamics of reactions to benefit our customers. You are right, there is a difference in the vapor pressure indeed. We will not enter here into the metrics or actual values, since it is proprietary for use by customers, so we focused our answer on the claim itself only, rather than the detail, and hope you understand us." This isn't as complete an answer as I'd wish for - in fact, it doesn't add anything at all to what we've already been told, and I have a hard time believing that a deviation from Raoult's Law is proprietary information. But we'll let that go for now.
3. In the same vein, what's the surface tension of Neowater as compared to the water it's produced from? I could imagine it going either way - if large clusters of water are occupied around the nanoparticles, the surface layer of water may not form in as ordered a fashion, leading to lower surface tension. On the other hand, if Neowater is better thought of as a collection of larger polar "balls" of hydrated particles, perhaps the value could end up higher.
Answer: "Exactly as you stated above. This is another mechanism of action in Neowater that we use to the benefit of our customers for the enhancement of their reaction. In Neowater, the dynamic range of surface tension is +15% to -15% around 72 dyn."
Actually, I think that should be dyn/cm, and that value is smack on top of the normal values for water (between 72 and 73). We're left to wonder what could cause it to vary higher and lower, though, and to wonder which of my explanations were correct. The DoCoop website has a picture of the stuff on a hydrophilic surface, showing a higher surface tension. I should note that if you want lower values, a drop of detergent will do the job nicely.
4. What's the conductivity of Neowater as compared to its untreated form? How does it change in the presence of small amounts of electrolytes as compared to regular water?
Answer: "Neowater's conductivity is like that in RO or distilled water. Neowater has no ions. It will change if (they're added). We are in the process of starting a research project with a NJ-based University on this application for batteries."
5. Have the rates of standard nucleophilic displacement reactions and/or cycloadditions been measured in Neowater? The presence or absence of a polar transition state and the resultant effect on reaction rate would make an interesting test of its properties. (Neowater is stated to be a "more hydrophobic" form of the liquid). Which reminds me: have Neowater's dipole moment and dielectric constant been determined?
Answer: " Neowater is an irregular media from the point of view of nucleophillic and cycloadditions. We did not find the right method to characterize this irregularity. We are open to suggestions because one of our business opportunities is in crystallization of proteins, where this issue is central. We do see irregularities of the nucleophilic behavior in Neowater with our university partners that are developing this application at the Weizman Institute in Israel. Regarding the dielectric constant measurement, there is a change in it in Neowater vs. regular water. We could not conclude yet the correlation b/w the shift in the structure of the "spinnor network" within Neowater if this is what you are trying to understand."
I would think that if you have a system that shows that Neowater is an "irregular" medium, then you'd have a method to begin characterizing it right there. But I'll wait to see if something comes out of the Weizmann work. For cycloadditions, I'd suggest looking at some of the aqueous Diels-Alder work from the 1980s.
And as for my question #6, about whether deuterated Neowater had been prepared, the company indicates that it hasn't done anything in that direction yet, although they are looking into the idea of using Neowater as an MRI contrast agent.
So, where does this leave us? While I appreciate the company taking time to answer my queries, I can't say that I'm all that much more informed compared to what I'd been able to find out from their press releases. That's clearly the way that they'd like to keep it, which is naturally their right from a business standpoint.
But from the scientific end, I have trouble buying into this "It's all proprietary for the use of our customers and the enhancement of shareholder value" explanation. Because if Neowater were really the sort of breakthrough that DoCoop's material makes it sound like, it would be worth a slew of research papers which would give it more scientific credibility. And since the company has already worked to secure its patent rights, such papers would certainly be feasible - desirable, even, considering the publicity that would follow.
And besides, if you want to know about the effects of nanoparticles in water, you can turn to the people who actually do publish their results. Perhaps any rate enhancement in PCR runs with Neowater is due to enhanced thermal conductivity - after all, temperature cycling is an essential part of the technique. How did I come to this conclusion? By reading this paper on the effects of aqueous nanoparticles on PCR reactions. It's a perfectly reasonable paper, and contains, as far as I can see, more data than DoCoop has ever released.
While we're on that subject, here's a site that will tell you so much about the effect of nanoparticles on thermal conductivity that you'll wish you'd never asked. Similarly, if you'd like to know more about the effect that nanoparticles have on water's surface tension, you could go here. If you wanted to learn more about the properties of water confined to nanoscale environments, you'd get a lot more out of this guy or this one than you would out of DoCoop's literature and patent filings, not that that would be very difficult.
So, all in all, I continue to be not very impressed. If Neowater were the kind of wild breakthrough that the company claims it to be, it would be worth more than its current use as a sort of STP-oil-treatment for PCR reactions. The company can, of course, have the last laugh on me over the next few years, and I wish them luck in doing so. But I'm betting that any breakthroughs in the aqueous nanoparticle area will find their way into the scientific literature in a more convincing fashion.
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February 2, 2006
Posted by Derek
Genetic Engineering News is sort of an odd publication. Primarily a vehicle for big, glossy color ads, it publishes some articles of its own: guest editorials, roundups of news from conferences and trade shows, that sort of thing. And it also publishes plenty of things that are (that have to be) slightly rewritten press releases - the sort of articles that start off:
"InterCap Corp. and SynaDynaGen say that their research collaboration on biosecurity proteomics through RNA interference and four-dimensional mass spectrometry, now with the great taste of fish, is yielding results that will make customers roll over on their backs and pant. Speaking at the Weaseltech Investor's Conference, company spokescreatures vowed to. . ."
One of these in the December issue, though, is weird enough that you can hear the editorial staff wrestling with their better selves. Phrases like "The company claims. . ." and "Company spokesmen maintain. . ." keep running through the whole article. It's titled "Water-Based Nanotech for the Life Sciences", and profiles a small Israeli company called (oddly) DoCoop. What DoCoop is selling is water.
But not just any water. . .Neowater! (Trademarked, natch). This is "a stable system of highly hydrated, inert nanoparticles", which supposedly have thousands of ordered hydration shells around them. This, the company says, modifies the bulk properties of the water. And what does that buy you?
Well, according to the company (there, I'm doing it, too), it will do pretty much everything except change the cat's litter box for you. It makes reactions run faster, at lower concentrations. It improves all biochemical assays and molecular biology techniques - PCR, RNA interference, ELISAs, you name it. Brief mentions are made of delivering molecules directly into cells with the stuff. It has applications in diagnostic kits, in drug delivery, in protein purification, and Cthulu only knows what else.
Some of these claims would seem to directly clash with each other. In the space of a few paragraphs, we hear that Neowater behaves "like a strong detergent", but somehow accelerates the growth of bacteria in culture. But at the same time it also prevents the formation of biofilms. And it increases the potency of antibiotics against bacteria, too. How it manages to do these things simultaneously is left, apparently, as an exercise for the reader.
The company claims that it has plenty of customers, and that it's working with several pharmaceutical companies to develop some of these applications. A search through the literature turned up one European molecular biology paper that mentioned using their PCR enhancing kit, so they've sold some Neowater for sure. But I'd like to turn this one over to the readers: have any of you seen this stuff? Know anyone who uses it?
And is everyone else's crank radar pinging as loudly as mine is? The thing is, unless a superior variety has up and evolved on us, cranks don't usually go out and form their own molecular biology reagent companies and place press releases in Genetic Engineering News. I'm profoundly sceptical of the claims this company makes, but I have the feeling that they're sincere in making them. Very odd, very odd indeed.
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January 9, 2006
Posted by Derek
Update: Since the site was down most of Tuesday, I'm leaving this post up another day. Things have only worsened since I put it up, though. . .
I've been withholding my comments on the South Korean stem cell controversy, waiting to see how the story finally settled out. Well, it's good and settled now: the entire enterprise was a fraud. Here's a timeline of the whole sorry business, for people who need a recap. Start at the bottom of that page to experience it in the most painfully realistic way.
My first impulse, in the manner of anyone belonging to a group (biomedical researchers) whose reputations have been dented by such a case, is to point out that, yes, "the system worked". The fraudulent research was discovered and rooted out, papers were retracted, funding lost, brows slapped, all of it. And it hasn't taken that long, either. It's useful to point these things out to people who would like to throw mud on the whole enterprise of science.
See, for example, this blog review of a recent book on scientific fraud. Contrary to its repeated assertions, scientists do indeed realize that fraud happens, because every working scientist has seen it. For starters, most large academic departments have tales of grad students or post-docs whose work could never be trusted. And all of us in research have run into papers in the literature whose techniques won't reproduce, no matter what, and the suspicions naturally grow that they were the product of exaggeration or wishful thinking. The number of possible publications sins alone is huge: yields of chemical reactions get padded, claims of novelty and generality get inflated, invalidating research from other labs doesn't get cited.
It's painful for me to admit it, but this kind of thing goes on all the time. And as long as the labs are staffed with humans, we're not going to be able to get rid of it. The best we can do is discourage it and correct it when we can.
But takes me to the second standard impulse that strikes in these situations, which is to ask what in the world these people were thinking. That's what's always puzzled me about major scientific fraud. The more interesting your work is, the more fame you stand to gain from your results, the more certain you are to be found out if you fake it. There are obscure areas that you could forge and fake around in for years, and journals in which you could publish your phony results without anyone ever being the wiser. Of course, by definition those won't do you much good - heck, you might as well do real work by that point.
But faking the big ones, the worldwide-headline national-hero stuff - you can't get away with that for long, and Professor Hwang didn't. The closest parallels I can think of are the recent Jan Hendrik Schoen case and the thirty-year-old Summerlin mouse scandal. (These and several other infamous cases are summarized here and here. I honestly find it hard to believe that there are others of that magnitude that anyone got away with.
I've never been able to imagine the state of mind of someone involved in this kind of thing. There you are, famous for something you've completely made up. In front of you are the cameras and reporters, while behind you, off in the distance, are hundreds of other scientists around the world busily trying to reproduce your amazing results. Every minute, they get closer to finding you out. How can anyone smile for the television crews under such conditions?
It's tempting to speculate about the state of the Korean scientific establishment and the role of Korea culture itself in this latest blowup. But such things have happened everywhere. The Korean factor certainly led to Hwang being an instant national figure with his face on every magazine and a dozen microphones trained on him wherever he turned. But it's not a Korean failing that did him in, it's a human one.
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December 6, 2005
Posted by Derek
The medical-blog roundup known as Grand Rounds is up today at Dr. Charles, with a wide selection of good reading.
And this is a good time to announce that I'm going to be hosting the next installment a week from now. Please feel free to send along links to any good blog posts on medical topics - your own, or ones you've come across when you're supposed to be working.
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November 17, 2004
Posted by Derek
A notable feature of 21st century molecular biology (so far!) is the emphasis on RNA. I've written before about RNA interference, a hugely popular (and hugely researched) way to silence the expression of proteins in living cells. Wide swaths of academia and industry are now devoted to figuring out all the details of these pathways, key parts of which are built into the cellular machinery. They turn out to regulate gene expression in ways that weren't even thought of before the late 1990s, and I've said for several years now that this field is the most obvious handful of tickets to Stockholm that I've ever seen. (Naturally, there are some worries that the whole field has perhaps been a bit over-promoted. . .)
Shutting off the production of targeted proteins is a wonderful thing, both from the basic research viewpoint and the clinical one. The more control you can have over the process, the better, and RNAi has been extremely promising. But as we're learning more about the system, complications are creeping in. Don't they always. . .
It turns out that the small interfering RNAs that are used, and are supposed to be the most efficacious and the most specific, aren't always what they seem. A disturbing recent study used one targeting luciferase, a firefly protein with no close relatives in the human genome. But applying it to the human-derived HeLa cell line showed effects on over 1800 genes - some of which only showed up at high concentrations, true, but none of these would have shown up at all in the ideal world we might have been living in for a while. There have also been experiments with RNAs that have deliberately made with slight mismatches for their intended target, and some of them work rather too well.
Finally, as I mentioned about a year ago, there are reports that these small RNAs can set off an interferon response, suggesting that the technique can cause cells to respond as if they're under infectious attack. As you'd imagine, this can also complicate the interpretation of an experiment, especially if you're already targeting something that might interact with any of these pathways (and plenty of things do.)
None of these yellow flags are particularly large, but there are several of them now and probably more waiting to be noticed. (A good brief roundup of the situation can be found in the November issue of Trends in Genetics, for those with access.) Perhaps as we learn more we'll find ways to obviate these problems. If there's one thing for sure, it's that we haven't figured out all the tricks that RNA is capable of. But the companies that are racing to get RNAi therapies into the clinic are watching all this a bit nervously, hoping that they're not going to be those fools that you always hear about rushing in.
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August 17, 2004
Posted by Derek
I'm going to take off from another comment, this one from Ron, who asks (in reference to the post two days ago): "would it not be fair to say that cellular biochemistry gets even more complicated the more we learn about it?
It would indeed be fair. I think that as a scientific field matures it goes through several stages. Brute-force collection of facts and observations comes early on, as you'd figure. Then the theorizing starts, with better and better theories being honed by more targeted experiments. This phase can be mighty lengthy, depending on the depth of the field and the number of outstanding problems it contains. A zillion inconsistent semi-trivialities can take a long time to sort out (think of the mathematical proof of the Four-Color Theorem), as can a smaller number of profound headscratchers (like, say, a reconciliation of quantum mechanics with relativity as they deal with gravity.)
If the general principles discovered are powerful enough, things can get simpler to understand. Think of the host of problems that early 20th-century physics had, many of which resolved themselves as applications of quantum mechanics. Earlier, chemistry went through something similar earlier, on a smaller scale, with the adoption of the stereochemical principles of van't Hoff. Suddenly, what seemed to be several separate problems turned out to be facets of one explanation: that atoms had regular three-dimensional patterns of bonding to other atoms. (If that sounds too obvious for such emphasis, keep in mind that this notion was fiercely ridiculed at resisted at the time.)
Cell biology is up to its pith helmet in hypotheses, and is nowhere near out of the swamps of fact collection. As in all molecular biology, the sheer number of different systems is making for a real fiesta. Your average cell is a morass of interlocking positive and negative feedback loops, many of which only show up fleetingly, under certain conditions, and in very defined locations. Some general principles have been established, but the number of things that have to be dealt with is still increasing, and I'm not sure when it's going to level out.
For example, the other day a group at Sugen (now Pfizer) published a paper establishing just how many genes there are in mice that code for protein kinase enzymes. Through adding phosphoryl groups, these enzymes are extremely important actors in the activation, transport, and modulation of the activities of thousands upon thousands of other proteins, and it turns out that there are exactly 540 of them. (Doubtless there are some variations as they get turned into proteins, but that's how many genes there are.) And that's that.
Now, that earlier discovery of protein phosphorylation as a signaling mechanism was a huge advance, and it has been appropriately rewarded. And knowing just how many different kinase enzymes there are is a step forward, too. But figuring out all the proteins they interact with, and when, and where, and what happens when they do - well, that's first cousin to hard work.
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April 22, 2004
Posted by Derek
I mentioned the other day that not everything in that Stuart Schreiber interview sounded sane to me, (although more of it does than I'd expected). The interviewer, Joanna Owens, asks him to expand on a statement he made about ten years ago: famously (in some circles, at any rate) Schreiber said that he wanted to - and thought that eventually he could - produce a small-molecule partner for every human gene.
A worthy goal, to be sure, but a honking big one, too. To his credit, though, Schreiber isn't making light of it:
". . .that challenge understates what we really want to do, which is to use small molecules to modulate the individual function(s) of multifunctional proteins, activating or inactivating individual functions as necessary. This is one of the differences between small molecules, for example, and the knockout of knowckdown technologies, where you inactivate everything to do with the protein of interest."
Note how things have appropriately expanded. There are a lot more proteins than there are genes (a lot more, given the surprisingly lowball figure for the total size of the human genome), and the number of protein activities is several times larger than that. He's absolutely right that this figure is the real bottom line. But here comes that Muhammed Ali side of his personality:
"Small molecules allow you to gain control rapidly, and can be delivered simply but, most importantly, we've shown that we can discover molecules that only modulate one of several functions of a single protein. . .(the scientific community has) identified 5000 out of the required 500,000 small molecules, which is similar to where the Human Genome Project was in year two of its 12-year journey. That might be a useful calibration - optimistically, we're ten years away."
Midway through that paragraph is where I start pulling back on a set of imaginary reins. Whoa up, there, Schreibster! Let's take the assumptions in order:
Small molecules allow you to gain control rapidly. . . Compared to transcription-level technology, this is largely correct. But the effects of small-molecule treatment often take a while to make themselves known, for a variety of reasons that we don't fully understand. The problem's particularly acute in larger systems - look at how long it takes for many CNS drugs to have any meaningful clinical effect. And these complex systems have other weird aspects, which make the phrase "gain control" seem a bit too confident. U-shaped dose-response relationships are common. Look at what you find in toxicology, where you see threshold effects and even hormesis, with large and small doses of the same substance showing opposite effects.
. . .and can be delivered simply. . . Well, when they can be delivered at all, I guess. But there more of them that come bouncing back at us than we'd like. In every drug research program I've been involved with, there are plenty of reasonable-looking compounds that hit the molecular target hard, but then don't perform in the cellular assay. You can come up with a lot of hand-waving rationales: perhaps the main series of compounds is riding in on some sort of active transport and these outliers can't, or they're getting actively pumped back out of the cell, or they hit some other sinkhole binding site that the others escape, and so on. Figuring out what's going on is an entire research project in itself, and rarely undertaken. Every time someone tells me that drug delivery is simple, I can feel my hair begin to frizz.
. . .we've shown that we can discover molecules that only modulate one of several functions of a single protein. . . True enough, and a very interesting accomplishment. But the generality of it is, to put the matter gently, unproven. It would not surprise me at all if there turn out to be many proteins whose functions can't be independently inhibited. The act of binding a small molecule to alter one of the functions would cause the other ones to change. And a bigger problem will be distinguishing these effects from the consequences of actually taking out that first function cleanly: how will you know when you've altered the system?
. . .which is similar to where the Human Genome Project was in year two. . . True, but that and forty dollars will get you an Aldrich Chemical can opener. The comparison isn't just optimistic - it's crazy. The problems that the genome sequencers faced were engineering problems - difficult, tricky, infuriating ones, but with solutions that were absolutely within the realm of possibility. Faster machines were made, with more computing power, and new techniques were applied to make use of them.
But as I've been saying, I'm not sure that the Maximum Inhibitor Library that Schreiber's talking about is even possible at all. Don't get me wrong - I hope that it is. We'll learn so much biochemistry that our heads will hurt. But its feasibility is very much open to question, to many questions, and we won't even begin to know the answers until we've put in a lot more work.
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April 13, 2004
Posted by Derek
You've probably heard of the hypothesis that a reasonable amount of dirt is good for you, especially in childhood. (My kids are certainly taking no chances.) The idea is that the immune system needs a certain amount of challenge to develop properly, so trying to live too antiseptic a life is a mistake. I think that this is very likely correct, and it turns out that it's especially correct if you're a zebrafish.
Not many of my readers are zebrafish, at least as far as I can tell from my referral logs, but they're an influential demographic. Danio rerio isn't as well known outside biology as say, the fruit fly, but it's a workhorse model organism for vertebrate development. Zebrafish are small, fast-growing, and the embryos are nearly transparent in their earlier stages. (Xenopus frogs share these characteristics, and have their partisans, too.)
The March 30th issue of the Proceedings of the National Academy of Sciences, with a Warholian zebrafish cover, features a study from Washington U. where the fish were raised under strictly aseptic (gnotobiotic) conditions. That's not easy to do, but if you make absolutely sure that no bacteria are present, it turns out that the embryos don't even develop properly. The defects are in the gut, which makes a lot of sense.
It turns out that colonization by normal intestinal flora is vital - zebrafish and their bacteria have become evolutionarily entangled. The bacteria actually induce some crucial gene expression by their presence, and the developmental program just doesn't have an aseptic default setting. There hasn't been an aseptic zebrafish since the beginning of biological time.
OK, these guys swim around in tropical pools, floating in a bacterial soup. But we're floating in one, too, just at a slightly lower density. Every part of a human body that can be easily (benignly) colonized by bacteria already is. Are there similar developmental effects in man? It wouldn't surprise me at all. No one's going to be running that exact embryo experiment, needless to say, but there are probably ways to sneak up on the answer using cell cultures. There's never been an aseptic human baby, either. . .although this is enough to make a person wonder about situations where a pregnant mother has had to take a long course of powerful antibiotics.
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January 19, 2004
Posted by Derek
Signaling between cells is weirder than we used to think it was. There's a hardy perennial, all right - that sentence could have been written whenever you like for the past fifty years or so. But the surprises keep on coming. Some of the most intense communication needs are between neurons, as you'd expect, and it looks like nature has taken advantage of all kinds of things to achieve greater bandwidth.
Everyone now learns about nitric oxide and its effects when they study physiology. The thought of a toxic gas as a neurotransmitter was a tough one to deal with at first, but the evidence was overwhelming. Then in the 1990s, two more oddities were proposed in the same category, and they're even more poisonous: hydrogen sulfide and carbon monoxide. Comments of the "You have to be kidding" sort greeted the initial work in this field, but the nitric oxide work had opened the door. It now appears that these two are, indeed, important signaling molecules in the brain.
Hydogen sulfide has a number of physiological effects, other than poisoning you (or, in lower concentrations, making you choke on its delightful aroma.) It seems to act on smooth muscle along with nitric oxide (now, there's a combination I would go out of my way to avoid breathing), and it also seems to have a role in laying down long-term memory. Carbon monoxide also seems to have a number of different functions - it's vasoactive, like its gaseous partners, but also seems to be involved in the immune response and in cellular protection and repair.
Taking up in the same way as the nitric oxide research, which has stimulated a huge amount of drug discovery work over the years, people are now trying similar tricks with these new gases. Look for more and more work on these in the drug industry as their mechanisms get fleshed out.
What's next? Well, it's not impossible that some other small-molecule gases have their own pathways, too. These things have properties that aren't shared by any other molecules, and perhaps they're being put to use. Ammonia, sulfur dioxide, and nitrous oxide have all been proposed as candidates. Thinking along those lines, I have to wonder about the small alkyl derivatives like methylamine and dimethyl sufide, too. Why not? But if someone gets around to claiming chlorine or the other halogens, I'm going to start to wonder. And if there turns out to be a physiological role for the noble gases, I'd start to suspect that Einstein was wrong: maybe God's approach to scientific laws is malicious after all. It would explain a lot, now that I think about it. . .
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January 18, 2004
Posted by Derek
Remember the genomics gold rush? Back about five or six years ago? Sure you do! People were lining up to throw money at companies that could deliver human gene sequences, as part of the never-ending search for new drug targets. (OK, it's not quite never-ending, but for the time horizon we have in the industry, we'll stick with that adjective.) Well, a good part of the reasoning behind all those sequencing deals may have just taken another hit.
Even while the genomic craze was at its peak, there were doubters. Gene sequences should, in principle, read out directly into protein sequences. But there are already some complications, since DNA and RNA sequences can, at various points, be spliced and recombined. We think we know the signs of that happening, but it's still another thing to worry about.
But even if there's no funny business, it's not easy getting useful information from a raw sequence. We still can't predict protein structures de novo, not to the degree that medicinal chemistry needs. You can learn a few things - homology statistics can place your unknown protein into a known family, or (failing that) at least tell you stuff like whether it's likely to be membrane-bound. But knowing that something's, say, a G-protein-coupled receptor sure isn't enough to tell you what it does in vivo, or if it's a valid drug target (and if so, for what disease.)
And there's always been a lot more to the cohort of proteins than just the corresponding genomic sequences. That's where the doubting voices got louder. Proteins get modified in all kinds of ways. They get phosphorylated and glycosylated around their outsides, for example, which can profoundly change their function. And they can get sliced up into smaller proteins, too. Happens all the time - plenty of bioactive proteins are produced from a larger percursor, carved off as needed like sandwich meat at the deli counter. (The enzymes that do the carving can be very good drug targets indeed.)
Enter the latest craziness, from J. C. Yang's lab at the National Cancer Institute. There's an exhilarating (or alarming, depending on your point of view) paper in the ">latest issue of Nature (427, 252), whose authors have seen something that no one had ever seen in higher organisms. They've shown that not only can proteins be chopped up in the cell, but that the various fragments can be spliced back together in new combinations. In their case, they showed that cells could produce a nine-amino-acid peptide from a 49-amino-acid precursor. The middle 40 got snipped out, and the two ends were spliced together to make the nine-mer. You're never going to be able to read off the sequence for that one, now, are you?
This sort of thing goes on all the time in single-celled creatures, and is known all the way up to, oh, bean plants. But it had sure never been seen in mammalian cells. How does this process happen, and how important is it? Who knows! It might turn out to be a rare curiosity, or it might turn out to be something really important that we've completely missed seeing all these years. To be sure, no one's reporting coming across a lot of important proteins whose sequences couldn't be matched in the genome somewhere. But there are an awful lot of proteins whose sequence we don't know, so the upper and lower bounds of this new phenomenon are fuzzy.
This paper, you can bet, has already set off a flurry of research. Perhaps there are some unexplained proteomic problems out there which this will turn out to answer. And here's a prediction: it wouldn't surprise me if protein splicing had already been seen by someone else, who looked at the data, thought about it, and said "Naaaah. That can't happen. Must have messed something up somewhere. . ." If this turns out to be physiologically important, it's Nobel material for sure. Listen closely, and you may hear the sound of someone kicking themselves.
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January 11, 2004
Posted by Derek
In my industry, you hear a lot of talk about drug targets and their relative chances of success. Targets fall into several broad classes, and when you take a close look, there are clearly some that are easier to hit than others. The G-protein coupled receptors (GPCRs) are one of those (antihistamines and beta-blockers are classic examples), and various hydrolytic enzymes are another (ACE inhibitors, HIV protease inhibitors, PDE inhibitors like Viagra, etc.)
But there are some other categories that are severely under-represented. "Interaction" targets is what I'd call a broad group of these. The ligands for the easier enzymes and GPCRs fit into defined binding pockets, which have evolved for small molecules, It's the old lock-and-key picture. But trying to affect the binding between two proteins, or of a protein with a stretch of DNA/RNA - now, that's something else again. There's no single binding pocket there, at least not on the scale of a drug-sized molecule. Instead of fitting different-shaped keys into existing locks, we're faced with trying to wedge something in between a door and its frame.
It's hard to get in there, and our molecules are often too small to have much effect. But the number of drug targets in this class is huge; we're going to have to come to terms with them eventually. . But for now, one of the best ways is to carefully study the various high-value targets and see if there are some that look more likely to work, given what we already know how to do. That's what a group at Roche has been up to recently, and they've reported their success in an online preprint in Science.
They're after a protein called MDM2, which acts as a brake on the activity of a more famous protein from the p53 tumor-suppression gene. In many cancers, it would be good to block this interaction and get the p53 system as back to being revved-up as possible. (Of course, in many other cancers, this gene has already been taken out of action by one mutation or another, which is probably a key step in their formation. Those won't be candidates for MDM2 blocking therapies.)
In 1996, a group at Sloan-Kettering published an X-ray crystal structure of the two proteins, which showed that there was a fairly clear pocket that seemed responsible for a lot of the binding. It looked like a possible candidate for a small molecule, but this is the first report of real success in targeting it (although others are hard at work.) The Roche group found some polyaryl imidazoline structures through high-throughput screening that seem to do the job. One of them is even orally active in a rodent tumor model, which is quite an accomplishment. And as proof of the mechanism, the compounds are inactive against those cancer cell lines that have already lost their p53 gene.
This is good news, since we can always use another route to cancer therapy. But I'm not sure how broadly applicable this is going to be. I'm sure that there will be talk of new interest in protein-protein drug targets, but this one is (unfortunately) an anomaly. That type of small, reasonably well-defined pocket that plays a role here doesn't show up that often, and it's not like people haven't been looking. News that these things can succeed will stimulate more work in the area, true. But that's where a lot of the effort was going already, because other protein-protein targets have seemed destined to fail.
My mental picture of those targets is of two oil tankers slowly coming together, brought closer as dozens of small grappling hooks whiz out and clang onto different parts of their decks. With a small molecule, we're trying to interfere with that by sticking a fishing boat in between them. Not easy, but we're going to have to figure it out eventually. Protein-protein interactions are a hot topic these days (go off and Google "proteomics", but stand well clear while you do it!) so we're bound to learn a lot more in the next few years.
For now, congratulations to Roche as they move forward toward the clinic. They'll be the first to find out what blocking MDM2 binding is going to do to animals - how well it'll treat those with cancer, and what side effects it might have on those without. I hope there's daylight in between those two groups!
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December 9, 2002
Posted by Derek
I'll bet that this is the only hit for a Google search for that word! I typed it out as I was thinking about how some major classes of biomolecules - protein, carbohydrate, lipid, and nucleic acid - are perceived. If you look at the number of papers published, and the number of details worked out in their field, you'd think that proteins are the single most important constituent of a living cell, followed by DNA / RNA. Are they?
I think this is partly an artifact of how easy things are to work with. There are only five purine / pyrimidine bases used in all DNA and RNA, and only two sugars (ribose or deoxyribose.) That level of simplicity is what's allowed sequencing techniques to become so automated so quickly. I'm not saying that there isn't plenty of complexity in the area - you get all sorts of hard-to-sequence hairpins and the like - but having only a few building blocks has helped enormously.
Proteins are the next step up. There are twenty-odd amino acids that you have to worry about, which gets pretty combinatorially complicated. (If you wanted to make, say 100 milligrams for your compound files of every 20-amino-acid protein combination there is, you'd run into a severe problem having to do with the amount of available carbon on earth.) Direct protein sequencing can be done, but it's nowhere near as easy as it is for DNA. Proteins have the advantage of being much easier to handle than nucleic acids, though, and many of them are robust enough to stand all kinds of mistreatment. That helped biochemists get a good start on enzymes before any other aspect of molecular biology got on its feet at all.
So, how about lipids? Here's where things start to get ugly. There are a *lot* more than 20 or 30 kinds of lipid molecules in a living system - all sorts of chain lengths, unsaturations, cis/trans isomers, mono-di-and-triglycerides and so on.(I won't even get into phosphorylation, since that's a big variable in the protein world, too.) And what about steroids, prostaglandins, and all the other lipid-derived stuff? All of these things are a real a pain to work with, too, since they're often found transiently or at very small concentration and their solubility is almost always awful by definition. It takes some really good techniques to separate the various lipid constituents out of the greasy mess.
And carbohydrates? I worked a lot with smaller ones in my graduate school days, and people still look at me funny for it. Sugars are as bad as they come for complexity - there are plenty of them, and they can be connected any number of ways to make macromolecules. By contrast, proteins are basically linear front-to-back chains (curled up, twisted, fractal-dimension space-filling chains, but chains nonetheless.) Complex carbohydates branch out all over the place, and they'll really make your life miserable. Despite years of work, there's not a general way (yet) to automatically sequence one, although the situation is getting better. But if we had to depend on carbohydrate sequencing to read the genetic code, we'd be up the creek for sure. Their physical properties can be quite squirreley, too, making them very little fun to purify.
So there are at least two important classes of biomolecules that probably don't get their due, because they're a lot more hostile to work with. And that should tell you how well we can handle the mixes between them - glycosylated proteins, nucleic-acid protein complexes, lipid conjugates. Pretty poorly, is how. It's a mess out there.
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October 14, 2002
Posted by Derek
There's been a flurry of news about gene therapy, a high-risk high-reward area of research from the very beginning. The biggest success stories came recently in the treatment of X-linked severe combined immunodeficiency (SCID,) the so-called "bubble boy" disease. But the course of true therapy never did run smooth, and there have been potentially dire complications.
SCID is fortunately rare, because it's a bad-news condition. Patients are essentially left without a functioning immune system, which makes everyone in that position die early from opportunistic infections. The sort of thing that would give a healthy individual a nasty cough for a few days is a fatal illness if you don't have T-cells and their partners. The most common genetic defect that lead to this condition is a loss of the enzyme adenosine deaminase, but there are several others that will put you in the same boat. The recent good news/bad news incidents concen SCID which was mediated by a loss of a protein called gc (for gamma-chain,) which is involved in cytokine signaling. There are some significant differences in trying to treat these two varieties, but gc-loss is probably easier to treat (a relative judgement if ever there was one; they're both tough.)
The standard therapy is bone marrow transplantation. This uses tissue from a matched healthy donor, usually after some level of intentional destruction of the existing marrow. When things really are matched identically, the prognosis is excellent, but the problem is that finding such a tissue match isn't always easy. A lesser degree of similarity, HLA-haploidentical tissue matching, is the next option. Survival rates in those cases are lower, although still around 75%, which most surely beats an early and certain death. But these patients don't usually get the full range of their immune response back. Specifically, B cells and NK cells aren't restored to normal levels, and even T-cell counts can start falling with time.
So there's room for improvement, and if you're a patient for whom no good tissue match exists, there's room for a lot of improvement. Thus gene therapy. The basic idea is similar to using bone marrow from a donor, only you donate your own marrow, newly refurbished, to yourself. The original marrow cells are replaced with genetically altered cells which have had the proper gene spliced into them.
Which sounds reasonably simple, but getting the gene into the cells is the voodoo part of the whole sequence. There are any number of ways of doing that, each with their known advantages and disadvantages, and each with plenty of unknown things waiting to emerge. Much of the progress in gene therapy has come from refining the vectors used to introduce the genes, but it's still a pretty crude process. In the standard method a crippled form of a retrovirus is used, one without RNA sequences for some key proteins that it would need to reproduce itself.
The problem is, these retroviruses go around jamming in genetic material all over the place. Sometimes it'll end up in a place where it can get transcribed into active protein, and sometimes it won't. If it inserts right into the middle of some key cellular gene that has to be read off later, the cell will probably die when it tries to do that. You just incubate as many stem cells as you can get, and hope for the best.
In several of the patients, that's what they got. They seem to have completely restored immune systems, a first for non-tissue-matching SCID patients. But in one case, the gene appears to have inserted itself into precisely the wrong place, making nonsense out of a gene that codes for a known growth-checking protein called LMO-2. This could have happened in only one cell out of the entire transplant, but one cell is enough. Loss of this protein has sent it into full-tilt reproduction and growth, which is another word for cancer. A new man-made form of leukemia was the result.
Analysis of the proliferating T-cells showed that, indeed, the necessary protein had the viral sequences wedged into it. The boy involved has a family history of a higher incidence of tumors, and he had a chicken-pox infection after his transplant (which must have been a scary test of its efficacy.) Either of these could have made the situation worse. He's receiving chemotherapy now, and as of last report the prognosis is cautiously optimism that the rogue cells can be brought under control.
So, does this stop the gene therapy world in its tracks? Not at all, as it turns out. In what I think is a very realistic risk/reward appraisal, an FDA advisory committee met last week and decided to press on with such experiments in the US. After all, it's the only chance these patients have. And a pediatric oncologist for the National Cancer Institute put it well: "If we threw out every therapy in cancer that causes cancer," she said, "we would get rid of some of our most effective ones." For better or worse, that's the state of the art. Good luck to all involved.
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