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
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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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+ TrackBacks (0) | Category: Biological News
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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+ TrackBacks (0) | Category: Biological News | Press Coverage
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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+ TrackBacks (0) | Category: Biological News | Drug Assays | Drug Development
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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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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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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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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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, |
