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Covalent Fragments Yield A Pile of Information

What happens when you expose a reactive, covalent-bond-forming compound to cell extracts (or to living cells)? The answer is complicated. You might expect the compound to go around grabbing every reactive group it sees, shotgunning across the proteins it encounters and labeling without discrimination. But it turns out that you only see that with the nastiest compounds, the sorts of things that fume when you open the reagent bottle. Even things that look fairly reactive actually pick their targets more carefully than you’d think (here’s a post with a number of links to discussions on this).

There are several ways you can tune this effect. If you have a weakly active electrophile in your probe (such as an epoxide), then it’s probably only going to react when it fits just right next to a suitable nucleophile, which means that the affinity of the compound itself is going to mean more than the reactivity of the warhead. If you don’t have many binding features in your molecule and not much of a warhead, it’s probably not going to do much at all. That’s been the question behind the idea of reactive covalent-linking fragment-sized compounds, then: what’s the balance between the one-size-fits-many (to some degree of binding) behavior of fragments and the reactivity of the covalent linking group?

There have been a few examples exploring this area in the literature, and a new paper from Ben Cravatt’s group at Scripps has more to add. (Here’s a post at Practical Fragments on the paper as well, and an article from the San Diego Union Tribune). This work takes a library of 50 reasonably small electrophilic probes (mostly chloroacetamides and substituted acrylamides) and runs them across the human proteome. Careful mass spec analysis showed that these molecules labeled 758 Cys residues in 637 proteins (out of 2248 that they could quantify), and a closer look at just which residues and just which proteins showed some interesting features. Good old druggable proteins with reactive Cys residues in their active sites accounted for some of the hits, as you would have expected. But there were also plenty of unusual hits – transcription factors, scaffolding proteins, and others that don’t usually pick up on small molecule compounds, much less fragment-sized ones. Overall, only 14% of the proteins that reacted were found in the DrugBank database.

The reactivity was concentration-dependent, and even some of the more wide-ranging compounds showed selectivity if you dialed down their concentrations in the assay. And there were wide variations among the proteins when it came to which electrophiles they liked – looking at the heat map, I’d have to say that it would have been impossible to predict which of the fifty probes would show activity towards any given protein, because they’re all over the place. I get the impression that 150 probes or more would probably have given a similar result – there’s really no such thing as a Cys-reactive compound that’s just going to go in and pick them all up. (Update: well, maybe iodoacetamide, which was used as the positive control. See the comments section). You can predict whether a given protein has Cys residues that are more likely to be reactive, but not what they’re going to react with.

And there’s apparently no such thing as a single thiol that’s going to pick up all the reactive fragments, either. Interestingly, the hit rates of the fragments did not really correlate with their reactivity towards glutathione, which is a quick-and-dirty assay often used to assess reactive compounds. It may well be time to get rid of that one; there are just too many other variables, it seems, for it to tell you anything really useful.

The group also tried the experiment with 20 of the probes on intact cells. This mostly recapitulated the lysate data, but with some real outliers in both directions (which is a phenomenon that’s been seen before in protein-labeling experiments). One interesting effect was noted with caspase 8 – there were several fragments that labeled it in both experiments, but none of them seemed to be inhibitors of the enzyme when they were assayed, even at high concentrations. It turned out that they were labeling the (inactive) procaspase form of the protein, and the paper shows some further SAR development of one of the fragments into what seems like a very useful chemical probe.

Beyond chemical probes, there have been many reports (two recent ones) of covalent Cys targeting as a possible mode for drug action, and this sort of study would seem to lead right into some useful approaches for that as well. If your interests overlap at all with the topic of covalent drug mechanisms, chemical probes, cysteine reactivity, or new target discovery, the whole paper (which is a big one, once you get into the Supplementary Information) is well worth a close look.

15 comments on “Covalent Fragments Yield A Pile of Information”

  1. Isidore says:

    Biogen’s MS drug Tecfidera (dimethyl fumarate) has been shown to modify covalently oxidized cysteines on Keap 1 and not much else, in spite of its reactivity and the fact that it is, structurally, a very simple molecule.

    1. Terry Moore says:

      At the same time, there’s a recent paper that suggests that Tecfidera works independently of Nrf2 in MS, which is negatively regulated by Keap1. Nrf2 activation was thought to be a major mechanism of action of Tecfidera. http://www.pnas.org/content/113/17/4777.full.pdf

      1. Isidore says:

        Maybe so, but my point was that dimethyl fumarate, in spite of the simplicity of its structure and its reactivity likes to bind covalently to oxidized cysteines of Keap 1, as has been demonstrated by experiments similar to those carried out by the Cravatt group, even though there are quite a few proteins with oxidized cysteines that would be chemically reactive towards this compound.

  2. CMCguy says:

    Is there anything new here? Irreversible enzyme inhibitors has had a long history of interest although believe little actual drug outputs. Attaching to a protein Cys group has been a well studied approach, partly because it can show up as key part of the active enzyme locale and do find reactive enough is biologic environment to target in almost tune-able windows. Never was clear why such suicide inhibitors were so looked down upon (maybe was application of that tag?) verses reversible counterparts although promiscuity was always a concern and typically refining selectivity plus checking side reactivity other Cys enzymes was demanded where studies about the apoptotic caspases being a prime example. I do believe others have previously tried such reactive molecule cellular stew experiments then tried to pull out applicable info where maybe now with modern tools and techniques Carvatts was able to advance the approach by expanding on details however still appears to be long way from the data to a medicine.

    1. cynical1 says:

      What is a “reasonable” drug output in the area? I would argue that beta-lactam antibiotics constitute more than a little drug output all by themselves. There’s about 45 of them or so. What about omeprazole, MAO inhibitors, aspirin, naloxazone, phenoxybenzamine, or ritonavir, which irreversibly reacts with the P450 to give it’s pharmacoenhancement effect? (I’ve taken esomeprazole for years.) I won’t count alkylating agents for cancer because…..well, that’s why they’re so toxic and non-selective. Agreed there aren’t that many but certainly quite a few. I know when I worked in med chem for 30 years, management were very, very resistant to covalent inhibitors. That’s likely the real reason there aren’t more. Maybe the cited work will change some prejudice in the area. Dunno…….

      If mother nature hadn’t given us the beta-lactams, a good percentage of us wouldn’t be around.

      1. CMCguy says:

        cynical1 you are of course correct as I totally overlooked the betalactam MOA being in this classification. When thinking of the few of the others that ultimately did become drugs know they faced stronger than normal resistance and believe there were many other potential candidates which never made it to the finish for one reason or the other. Having seen the prejudiced attitudes of R&D management I wonder if discovery people in some cases gave in to the pressure and abandoned promising compounds because grew tired of the negativity they faced.

  3. Peter Kenny says:

    This certainly looks like an interesting study although it seems to be directed more at target discovery than at lead discovery. Some of the probes (e.g. 7, 61, 63) are perhaps on the large side to be considered to be fragments although this would be more a criticism of jumping on the fragment band wagon than of the approach itself. The authors seem to be equating covalency with irreversibility (which may the case for the warheads that they’re using) but this doesn’t have to be the case. That said, for what they’re doing reversible binding to cysteines (e.g with nitriles) is unlikely to work. Covalent modification of cysteines in these experiments is likely to be subject to kinetic (rather than thyermodynamic) control. The inherent reactvity of the cysteine will be a factor but so will the ability of the probe (and protein) to get the reacting atoms into a stable transition state geometry without forming bad (i.e. destabilising) contacts between probe and protein.

  4. HTSguy says:

    RE the comment about the glutathione reactivity assay: this is commonly done by incubating the compound with 5 mM glutathione (not the 125 uM used here) and then measuring the loss of compound potency in one’s assay. Since all of the compounds tested in Ext. Data Fig. 2 reacted to some extent, one might conclude that the usual assay with a higher concentration of glutathione is still useful.

  5. Curious Wavefunction says:

    I found the following statement interesting as an example of the complexities of enzyme biochemistry:
    “Several fragments targeted the catalytic cysteine nucleophile C360 of the protease caspase-8 (CASP8) in isoTOP-ABPP experiments performed in vitro and in situ. Curiously, however, these fragments exhibited marginal to no inhibition of active CASP8 using either substrate or activity-based probe (Rho-DEVD-AOMK probe) assays. This initially puzzling outcome was explained when we discovered that the electrophilic fragments selectively labelled the inactive zymogen (pro-), but not active form of CASP8.”

  6. CurtF. says:

    …there’s really no such thing as a Cys-reactive compound that’s just going to go in and pick them all up.

    This is a bit of a nitpick, but the study used iodoacetamide as a “universal” probe to react with and label all unlabeled cysteines (after an initial treatment of cells with a more structurally complex, less reactive electrophile). They are thus assuming that there is a Cys-reactive compound that’s going to go in and pick them all up…and it’s name is iodoacetamide.

    Practically speaking, their mass spec analysis *only* looks at iodoacetamide-reacted products, so if there were cysteines that were unreactive to iodoacetamide, they wouldn’t show up in this paper at all.

    1. Derek Lowe says:

      Good point! I’ve added a note to the post about this. . .

      1. Curt F. says:

        Thanks for the reply and I apologize for screwing up the italics tags so badly in my comment.

  7. Elair says:

    Cool paper. Suggestion, given the emphasis on chemical reactivity, it may be prudent to more effectively integrate involvement of some of the synthetic chemists that helped make the compounds in the biological portion of the work and write-up. This, after all, is their area of expertise, and it may help to further the insights you obtained.

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