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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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May 22, 2008

Killing Proteins Wholesale

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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.

Comments (6) + TrackBacks (0) | Category: Biological News | Toxicology


COMMENTS

1. Kay on May 22, 2008 7:35 AM writes...

Are you hinting that the rule of 5 might not be reliable?

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2. MTK on May 22, 2008 4:09 PM writes...

I guess I'm not that surprised with the results. If you had to rank the electrophilicity of each of the reagents, the primary sulfonate ester would certainly be the most reactive, so the fact that it reacted with more amino acid residues than the others seems consistent. Also Michael addition under these conditions, at least to maleimides, is known(assumed?) to be Cys specific as is the addition to alpha-halo amides.

The one thing as synthetic chemists that we need to remember is that these protein labeling studies are usually quite low yielding compared to what we're used to. I believe that if you fished out over 50% of Cys containing peptides from a complex mixture using a biotinylated maleimide, there'd be dancing in the streets.

This means that various methods which in theory should give similar answer sets often times do not. A good example is in quantitative proteomics where it's often stated that to get the most complete answer you might need to use three different methods, since there is a high degree of non-overlap between the answer sets derived using each method.

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3. Ian Musgrave on May 23, 2008 12:44 AM writes...

Yikes! We were doing carbonylations a few years back. In the test tube our reagent generated lysine, histidine and cyseine adducts. When added to a real cell (PC-12), we recovered lots of carbonyl adducts, but we could never show a lysine adduct. Puzzled us mightily, but at least someone else has the same issue.

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4. A-non-y-mous on May 23, 2008 5:09 AM writes...

When thinking about the reactivity of protein side chains, you gotta think about pK and protonation states, too.

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5. CMC guy on May 23, 2008 9:27 AM writes...

#4 A-non-y-mous is right on. Makes me think of Watson & Crick in the movie "Double Helix" as was a major insight to structure coordination. Don't recall in the book it was quite as a dramatic "Ah Ha" (or British equivalent) moment but still was a key theme.

Permalink to Comment

6. Javaslinger on May 26, 2008 6:03 PM writes...

British equiv. --> Blimey!

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