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.