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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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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 ┬ÁM, 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


COMMENTS

1. Mac on July 22, 2014 9:14 AM writes...

Interesting take, but what's really excited me here is that this type of library could be used for phenotypic screening (even by academic labs or on contract) with a very strong chance of effective target ID. I hope he'll start a company to synthesize 50k of these.

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2. Molecular Geek on July 22, 2014 9:19 AM writes...

you've got an unclosed italics tag in there somewhere, Derek.

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3. luysii on July 22, 2014 10:10 AM writes...

[ Proc. Natl. Acad. Sci. vol. 110 pp. 9344 - 9349 '13 ] The binding pockets of naturally occuring proteins fall into about 400 classes. The same is true for single domain proteins which were computationally generated to be compact. So pockets are a spandrel -- a byproduct of compact protein structure (which can't be fully compact). Natural selection can then act on these pockets to tune them for specific ligands.

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4. anonymouscb on July 22, 2014 10:15 AM writes...

Everything is a scaffolding protein until we figure out what it does. Sometimes they are and other times they are things like p300/CBP

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5. Anon on July 22, 2014 1:47 PM writes...

I don't have access to the paper but have a question.
Did they take two of their library compounds (with different handles/tags, add a first, watch what happens, and then add the second to see if the first set of binding molecules was altered?
Sort of a quick control to see if this library technique alters the cellular system?

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6. NoDrugsNoJobs on July 22, 2014 1:56 PM writes...

#5 - Interesting idea

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7. entropyGain on July 23, 2014 10:08 AM writes...

Once again Derek is the goto expert to for perspective on science news

http://www.utsandiego.com/news/2014/jul/22/cravatt-small-molecule-protein/

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