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About this Author
DBL%20Hendrix%20small.png College chemistry, 1983

Derek Lowe The 2002 Model

Dbl%20new%20portrait%20B%26W.png After 10 years of blogging. . .

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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August 14, 2014

Proteins Grazing Against Proteins

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Posted by Derek

A huge amount of what's actually going on inside living cells involves protein-protein interactions. Drug discovery, for obvious reasons, focuses on the processes that depend on small molecules and their binding sites (thus the preponderance of receptor ligands and enzyme inhibitors), but small molecules are only part of the story in there.

And we've learned a fair amount about all this protein-protein deal-making, but there's clearly a lot that we don't understand at all. If we did, perhaps we'd have more compounds that can target them. Here's a very basic topic about which we know very little: how tight are the affinities between all these interacting proteins? What's the usual level, and what's the range? What does the variation in binding constants say about the signaling pathways involved, and the sorts of binding surfaces that are being presented? How long do these protein complexes last? How weak can one of these interactions be, and still be physiologically important?

A new paper has something to say about that last part. The authors have found a bacterial system where protein phosphorylation takes place effectively although the affinity between the two partners (KD) is only around 25 millimolar. That's very weak indeed - for those outside of drug discovery, small-molecule drug affinities are typically well over a million times that level. We don't know how common or important such weak interactions are, but this work suggests that we're going to have to look pretty far up the scale in order to understand things, and that's probably going to require new technologies to quantify such things. Unless we figure out that huge, multipartner protein dance that's going on, with all its moves and time signatures, we're not going to understand biochemistry. The Labanotation for a cell would be something to see. . .

Comments (4) + TrackBacks (0) | Category: Biological News | Chemical Biology

July 22, 2014

Put Them in Cells and Find Out

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Posted by Derek

So, when you put some diverse small molecules into cellular assays, how many proteins are they really hitting? You may know a primary target or two that they're likely to interact with, or (if you're doing phenotypic screening), you may not have any idea at all. But how many proteins (or other targets) are there that bind small molecules at all?

This is a question that many people are interested in, but hard data to answer it are not easily obtained. There have been theoretical estimates via several techniques, but (understandably) not too much experimental evidence. Now comes this paper from Ben Cravatt's group, and it's one of the best attempts yet.

What they've done is to produce a library of compounds, via Ugi chemistry, containing both a photoaffinity handle and an alkyne (for later "click" tagging). They'd done something similar before, but the photoaffinity group in that case was a benzophenone, which is rather hefty. This time they used a diazirine, which is both small and the precursor to a very reactive carbene once it's irradiated. (My impression is that the diazirine is the first thing to try if you're doing photoaffinity work, for just those reasons). They made a small set of fairly diverse compounds (about 60), with no particular structural biases in mind, and set out to see what these things would label.

They treated PC-3 cells (human prostate-cancer derived) with each member of the library at 10 µM, then hit them with UV to do the photoaffinity reaction, labeled with a fluorescent tag via the alkyne, and fished for proteins. What they found was a pretty wide variety, all right, but not in the nonselective shotgun style. Most compounds showed distinct patterns of protein labeling, and most proteins picked out distinct SAR from the compound set. They picked out six members of the library for close study, and found that these labeled about 24 proteins (one compound only picked up one target, while the most promiscuous compound labeled nine). What's really interesting is that only about half of these were known to have any small-molecule ligands at all. There were proteins from a number of different classes, and some (9 out of 24) weren't even enzymes, but rather scaffolding and signaling proteins (which wouldn't be expected to have many small-molecule binding possibilities).

A closer look at non-labeled versions of the probe compounds versus more highly purified proteins confirmed that the compounds really are binding as expected (in some cases, a bit better than the non-photoaffinity versions, in some cases worse). So even as small a probe as a diazirine is not silent, which is just what medicinal chemists would have anticipated. (Heck, even a single methyl or fluoro isn't always silent, and a good thing, too). But overall, what this study suggests is that most small molecules are going to hit a number of proteins (1 up to a dozen?) in any given cell with pretty good affinity. It also (encouragingly) suggests that there are more small-molecule binding sites than you'd think, with proteins that have not evolved for ligand responses still showing the ability to pick things up.

There was another interesting thing that turned up: while none of the Ugi compounds was a nonselective grab-everything compound, some of the proteins were. A subset of proteins tended to pick up a wide variety of the non-clickable probe compounds, and appear to be strong, promiscuous binders. Medicinal chemists already know a few of these things - CYP metabolizing enzymes, serum albumin, and so on. This post has some other suggestions. But there are plenty more of them out there, unguessable ones that we don't know about yet (in this case, PTGR and VDAC subtypes, along with NAMPT). There's a lot to find out.

Comments (7) + TrackBacks (0) | Category: Chemical Biology | Drug Assays

July 16, 2014

An Easy Way to Make Cyclic Peptides

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Posted by Derek

If you ever find yourself needing to make large cyclic peptides, you now have a new option. This paper in Organic Letters describes a particularly clean way to do it: let glutathione-S-transferase (GST) do the work for you. Bradley Pentelute's group at MIT reports that if your protein has a glutathione attached at one end, and a pentafluoroaryl Cys at the other, that GST will step in and promote the nucleophilic aromatic substitution reaction to close the two ends together.
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This is an application of their earlier work on the uncatalyzed reaction and on the use of GST for ligation.. Remarkably, the GST method seems to product very high yields of cyclic peptides up to at least 40 residues, and at reasonable concentration (10 mM) of the starting material, under aqueous conditions. Cyclic peptides themselves are interesting beasts, often showing unusual properties compared to the regular variety, and this method look as it will provide plenty more of them for study.

Comments (7) + TrackBacks (0) | Category: Chemical Biology | Chemical News

July 14, 2014

Modifying Red Blood Cells As Carriers

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Posted by Derek

What's the best carrier to take some sort of therapeutic agent into the bloodstream? That's often a tricky question to work out in animal models or in the clinic - there are a lot of possibilities. But what about using red blood cells themselves?

That idea has been in the works for a few years now, but there's a recent paper in PNAS reporting on more progress (here's a press release). Many drug discovery scientists will have encountered the occasional compound that partitions into erythrocytes all by itself (those are usually spotted by their oddly long half-lives after in vivo dosing, mimicking the effect of plasma protein binding). One of the early ways that people have attempted to try this deliberately was forcing a compound into the cells, but this tends to damage them and make them quite a bit less useful. A potentially more controllable method would be to modify the surfaces of the RBCs themselves to serve as drug carriers, but that's quite a bit more complex, too. Antibodies have been tried for this, but with mixed success.

That's what this latest paper addresses. The authors (the Lodish and Ploegh groups at Whitehead/MIT) introduce modified surface proteins (such as glycophorin A) that are substrates for Ploegh's sortase technology (two recent overview papers), which allows for a wide variety of labeling.

Experiments using modified fetal cells in irradiated mice gave animals that had up to 50% of their RBCs modified in this way. Sortase modification of these was about 85% effective, so plenty of label can be introduced. The labeling process doesn't appear to affect the viability of the cells very much as compared to wild-type - the cells were shown to circulate for weeks, which certainly breaks the records held by the other modified-RBC methods.

The team attached either biotin tags and specific antibodies to both mouse and human RBCs, which would appear to clear the way for a variety of very interesting experiments. (They also showed that simultaneous C- and N-terminal labeling is feasible, to put on two different tags at once). Here's the "coming attractions" section of the paper:

he approach presented here has many other possible applications; the wide variety of possible payloads, ranging from proteins and peptides to synthetic compounds and fluorescent probes, may serve as a guide. We have conjugated a single-domain antibody to the RBC surface with full retention of binding specificity, thus enabling the modified RBCs to be targeted to a specific cell type. We envision that sortase-engineered cells could be combined with established protocols of small-molecule encapsulation. In this scenario, engineered RBCs loaded with a therapeutic agent in the cytosol and modified on the surface with a cell type-specific recognition module could be used to deliver payloads to a precise tissue or location in the body. We also have demonstrated the attachment of two different functional probes to the surface of RBCs, exploiting the subtly different recognition specificities of two distinct sortases. Therefore it should be possible to attach both a therapeutic moiety and a targeting module to the RBC surface and thus direct the engineered RBCs to tumors or other diseased cells. Conjugation of an imaging probe (i.e., a radioisotope), together with such a targeting moiety also could be used for diagnostic purposes.

This will be worth keeping an eye on, for sure, both as a new delivery method for small (and not-so-small) molecules, fof biologics, and for its application to all the immunological work going on now in oncology. This should keep everyone involved busy for some time to come!

Comments (7) + TrackBacks (0) | Category: Biological News | Chemical Biology | Pharmacokinetics

June 2, 2014

Single-Cell Compound Measurements - Now In A Real Animal

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Posted by Derek

parp%20cells%20copy.jpg
Last year I mentioned an interesting paper that managed to do single-cell pharmacokinetics on olaparib, a poly(ADP) ribose polymerase 1 (PARP1) inhibitor. A fluorescently-tagged version of the drug could be spotted moving into cells and even accumulating in the nucleus. The usual warnings apply: adding a fluorescent tag can disturb the various molecular properties that you're trying to study in the first place. But the paper did a good set of control experiments to try to get around that problem, and this is still the only way known (for now) to get such data.

The authors are back with a follow-up paper that provides even more detail. They're using fluorescence polarization/fluorescence anisotropy microscopy. That can be a tricky technique, but done right, it provides a lot of information. The idea (as the assay-development people in the audience well know) is that when fluorescent molecules are excited by polarized light, their emission is affected by how fast they're rotating. If the rotation is slowed down to below the fluorescence lifetime of the molecules (as happens when they're bound to a protein), then you see more polarization in the emitted light, but if the molecules are tumbling around freely, that's mostly lost. There are numerous complications - you need to standardize each new system according to how much things change in increasingly viscous solutions, the fluorophores can't get too close together, you have to be careful with the field of view in your imaging system to avoid artifacts - but that's the short form.

In this case, they're using near-IR light to do the excitation, because those wavelengths are well known to penetrate living cells well. Their system also needs two photons to excite each molecule, which improves signal-to-noise and the two-photon dye is a BODIPY compound. These things have been used in fluorescence studies with wild abandon for the past few years - at one point, I was beginning to think that the acronym was a requirement to get a paper published in Chem. Comm. They have a lot of qualities (cell penetration, fluorescence lifetime, etc.) that make them excellent candidates for this kind of work.

This is the same olaparib/BODIPY hybrid used in the paper last year, and you see the results. The green fluorescence is nonspecific binding, while the red is localized to the nuclei, and doesn't wash out. If you soak the cells with unlabeled olaparib beforehand, though, you don't see this effect at all, which also argues for the PARP1-bound interpretation of these results. This paper takes things even further, though - after validating this in cultured cells, they moved on to live mice, using an implanted window chamber over a xenograft.

And they saw the same pattern: quick cellular uptake of the labeled drug on infusion into the mice, followed by rapid binding to nuclear PARP1. The intracellular fluorescence then cleared out over a half-hour period, but the nuclear-bound compound remained, and could be observed with good signal/noise. This is the first time I've seen an experiment like this. Although it's admittedly a special case (which takes advantage of a well-behaved fluorescently labeled drug conjugate, to name one big hurdle), it's a well-realized proof of concept. Anything that increases the chances of understanding what's going on with small molecules in real living systems is worth paying attention to. It's interesting to note, by the way, that the olaparib/PARP1 system was also studied in that recent whole-cell thermal shift assay technique, which does not need modified compounds. Bring on the comparisons! These two techniques can be used to validate each other, and we'll all be better off.

Comments (4) + TrackBacks (0) | Category: Biological News | Chemical Biology | Pharmacokinetics

May 30, 2014

Covalent Fragments

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Posted by Derek

Many drug discovery researchers now have an idea of what to expect when a fragment library is screened against a new target. And some have had the experience of screening covalent, irreversible inhibitor structures against targets (a hot topic in recent years). But can you screen with a library of irreversibly-binding fragments?

This intersection has occurred to more than one group, but this paper marks the first published example that I know of. The authors, Alexander Statsyuk and co-workers at Northwestern, took what seems like a very sound approach. They were looking for compounds that would modify the active-site residues of cysteine proteases, which are the most likely targets in the proteome. But balancing the properties of a fragment collection with those of a covalent collection is tricky. Red-hot functional groups will certainly label your proteins, but they'll label the first things they see, which isn't too useful. If you go all the way in the other direction, epoxides are probably the least reactive covalent modifier, but they're so tame that unless they fit into a binding site perfectly, they might not do anything at all - and what are the chances that a fragment-sized molecule will bind that well? How much room is there in the middle?

That's what this paper is trying to find out. The team first surveyed a range of reactive functional groups against a test thiol, N-acetylcysteine. They attached an assortment of structures to each reactive end, and they were looking for two things: absolute reactivity of each covalent modifier, and how much it mattered as their structures varied. Acrylamides dropped out as a class because their more reactive examples were just too hot - their reactivity varied up to 2000x across a short range of examples. Vinylsulfonamides varied 8-fold, but acrylates and vinylsulfones were much less sensitive to structural variation. They picked acrylates as the less reactive of the two.

A small library of 100 diverse acrylates were then prepared (whose members still only varied about twofold in reactivity), and these were screened (100 micromolar) against papain as a prototype cysteine protease. They'd picked their fragments so that everything had a distinct molecular weight, so whole-protein mass spec could be used as a readout. Screening ten sets of ten mixtures showed that the enzyme picked out three distinct fragments from the entire set, a very encouraging result. Pretreatment of the enzyme with a known active-site labeling inhibitor shut down any reaction with the three hits, as it should have.

Keep in mind that this also means that 97 reasonably-sized acrylates were unable to label the very reactive Cys in the active site of papain, and that they did not label any surface residues. This suggests that the compounds that did make it in did so because of some structure-driven binding selectivity, which is just the territory that you want to be in. Adding an excess of glutathione to the labeling experiments did not shut things down, which also suggests that these are not-very-reactive acrylates whose structures are giving them an edge. Screen another enzyme, and you should pick up a different set of hits.

And that's exactly what they did next. Screening a rhinovirus cysteine protease (HRV3C) gave three totally new hits - not as powerful against that target as the other three were against papain, but real hits. Two other screens, against USP08 and UbcH7, did not yield any hits at all (except a couple of very weak ones against the former when the concentration was pushed hard). A larger reactive fragment library would seem to be the answer here; 100 compounds really isn't very much, even for fragment space, when you get down to it.

So this paper demonstrates that you can, in fact, find an overlap between fragment space and covalent inhibition, if you proceed carefully. Now here's a question that I'm not sure has ever been answered: if you find such a covalent fragment, and optimize it to be a much more potent binder, can you then pull the bait-and-switch by removing the covalent warhead, and still retain enough potency? Or is that too much to ask?

Comments (10) + TrackBacks (0) | Category: Chemical Biology | Chemical News | Drug Assays

May 28, 2014

The Science Chemogenomics Paper is Revised

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Posted by Derek

The Science paper on chemogenomic signatures that I went on about at great length has been revised. Figure 2, which drove me and every other chemist who saw it up the wall, has been completely reworked:

To improve clarity, the authors revised Fig. 2 by (i) illustrating the substitution sites of fragments; (ii) labeling fragments numerically for reference to supplementary materials containing details about their derivation; and (iii) representing the dominant tautomers of signature compounds. The authors also discovered an error in their fragment generation software that, when corrected, resulted in slightly fewer enriched fragments being identified. In the revised Fig. 2, they removed redundant substructures and, where applicable, illustrated larger substructures containing the enriched fragment common among signature compounds.

Looking it over in the revised version, it is indeed much improved. The chemical structures now look like chemical structures, and some of the more offensive "pharmacophores" (like tetrahydrofuran) have now disappeared. Several figures and tables have been added to the supplementary material to highlight where these fragments are in the active compounds (Figure S25, an especially large addition), and to cross-index things more thoroughly.

So the most teeth-gritting parts of the paper have been reworked, and that's a good thing. I definitely appreciate the work that the authors have put into making the work more accurate and interpretable, although these things really should have been caught earlier in the process.

Looking over the new Figure S25, though, you can still see what I think are the underlying problems with the entire study. That's the one where "Fragments that are significantly enriched in specific sets of signature compounds (FDR ≤ 0.1 and signature compounds fraction ≥ 0.2) are highlighted in blue within the relevant signature compounds. . .". It's a good idea to put something like that in there, but the annotations are a bit odd. For example, the compounds flagged as "6_cell wall" have their common pyridines highlighted, even though there's a common heterocyclic core that that all but one those pyridines are attached to (it only varies by alkyl substitutents). That single outlier compound seems to be the reason that the whole heterocycle isn't colored in - but there are plenty of other monosubstituted pyridines on the list that have completely different signatures, so it's not like "monosubstituted pyridine" carries much weight. Meanwhile, the next set ("7_cell wall") has more of the exact same series of heterocycles, but in this case, it's just the core heterocycle that's shaded in. That seems to be because one of them is a 2-substituted isomer, while the others are all 3-substituted, so the software just ignores them in favor of coloring in the central ring.

The same thing happens with "8_ubiquinone biosynthesis and proteosome". What gets shaded in is an adamantane ring, even though every single one of the compounds is also a Schiff base imine (which is a lot more likely to be doing something than the adamantane). But that functional group gets no recognition from the software, because some of the aryl substitution patterns are different. One could just as easily have colored in the imine, though, which is what happens with the next category ("9_ubiquinone biosynthesis and proteosome"), where many of the same compounds show up again.

I won't go into more detail; the whole thing is like this. Just one more example: "12_iron homeostasis" features more monosubstituted pyridines being highlighted as the active fragment. But look at the list: there's are 3-aminopyridine pieces, 4-aminomethylpyridines, 3-carboxylpyridines, all of them substituted with all kinds of stuff. The only common thread, according to the annotation software, is "pyridine", but those are, believe me, all sorts of different pyridines. (And as the above example shows, it's not like pyridines form some sort of unique category in this data set, anyway).

So although the most eye-rolling features of this work have been cleaned up, the underlying medicinal chemistry i