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Digging Through the Proteins, Covalently

I blogged here last year about some really interesting work from the Cravatt group at Scripps. It’s sort of an intersection between fragment-based screening and screening in cells, which is an intersection that I’d previously never thought existed. That’s because fragment screening typically involves biophysical methods (NMR, SPR, DSF and others) that can pick up relatively weak binding events, ones that would probably not drive much (or any) functional readout in a cell assay. And functional readout is what screening in cells is all about: you fire in the compounds and look for what happens (ideally, a well-controlled group of effects that allow you to distinguish what could be useful compounds from things that are banging on the big piano with both hands, as it were).

So the two worlds typically don’t overlap much. When you’re screening fragments, you have no idea if they (or the compounds derived from them) are going to have the effects you want in cells, because you’re way back at the beginning, just observing binding to a purified protein, a defined target. Cell activity will come later, maybe much later, and it’s going to be up to you to look for it and try to optimize it. And when you’re doing cell screening, you have no assurances about what target or targets you might be hitting. If you’re screening as part of a larger program where you already have lots of defined selectivity data on proteins, you can be pretty sure of your readout (although never 100%), but on the other hand, if you’re phenotypically screening and just hunting for the readout you like, who knows? Target elucidation is up to you, too.

What the Cravatt paper did was to build a particular library of fragments, with two key characteristics. Every one of them had a diazirene attached to it on a side chain, which is a group that chemical biologists know and love, since under strong light it turns into a reactive species that tends to form a covalent bond with whatever might be next to it. And each fragment also had an acetylene, also beloved ever since the Sharpless group unleashed click chemistry on the world, since that gives you a handle to later attach a fluorescent group or whatever other tag you like in order to find what proteins the covalent group attached to. This library of diazirene/acetylene fragments was turned loose on cells to see what they’d label – if this sounds interesting, go back and read the blog post, and most definitely read the paper it’s referencing, because it’s a real tour de force.

Near the end of it came a further development, though – a move out of fragment space and into larger, more drug-like chemical matter. That takes us into an interplay of affinity and reactivity: a red-hot covalent labeling group has a better chance of just sticking onto everything it sees, as you’d figure. On the other end of the scale, a really unreactive group is only going to stir itself when everything lines up perfectly: relatively tight binding that brings the reacting partner group in just so, right distance, right angle, room to maneuver, and so on. In general, that means that covalent fragment screening will probably call for somewhat more reactive groups, since their affinities will seldom be very high. But as you get up to more complex chemical matter, some of these things could have really significant binding, and you could well see less reactive groups in play. (This effect has been documented many times in the literature).

Of course, the odds of finding fragment-level binding are a lot better than the odds of finding this more complex binding, which is why people screen fragment libraries in the first place (and why they use the hit rates from them as proxies for “druggability” in general”). But if you’re not aiming at a particular target, but are instead just shotgunning away through the cells looking for something interesting, that’s not as much of a problem. Odds are small that you would hit one particular target with a small-to-medium library of covalent/phtotoaffinity tagged drug-like molecules, but the odds that you’ll hit something are pretty good. And if you keep profiling with diverse chemical matter, you could end up with a collection of compounds that (1) selectively hit a lot of known disease-relevant proteins and (2) hit a lot of others that are less well characterized and might lead to new biology or target ideas entirely. In both cases, you end up with a covalent probe compound ready to take you further.

And that’s what this latest paper follows up on, calling the whole process “Inverse Drug Discovery”. It goes further by bringing in compounds with aryl fluorosulfates, a functional group recently popularized by (again!) the Sharpless and Fokin groups. This is a good example of a weakly reactive covalent warhead – to get these things to react, not only do you need a nucleophile that’s able to come in at the correct angle to the sulfur-fluorine bond, but you also need an environment on the other side that will stabilize the transition state where that fluorine is in the act of leaving and give that developing fluorine anion a good home. Otherwise the energy of that TS will be too high for the reaction to ever get over the hump, and no covalent labeling will take place.

Such a functional group would be expected to label an unusual suite of proteins, and so it proved. Incubating either live cells or cell lysates (which, as you’d expect, gave more protein hits) with three different fluorosulfates gave several labeled proteins, and the mechanism was confirmed by competition with the same probe molecules minus the alkyne group (so they wouldn’t be picked up by the eventual labeling). The group narrowed down on 12 proteins for which structures are available and whose labeling seemed robust, and 11 of them also labeled when the experiment was tried on recombinant protein alone. (The outlier just points out that there could be differences in the in vivo protein – post-translational modifications? – that made it different from the pure-protein case). These 11 proteins were labeled at specific sites, mostly tyrosines but with some lysines as well.

Some of them (such as biliverdin reductase A) were labeled in their active site, shutting the enzyme down completely, while others still labeled near the binding pocket (thiopurine S-methyltransferase). There were also typically cationic residues present nearby in these pockets, which could be expected to both make the side chains involved more nucleophilic and to possibly stabilize the departing fluoride ions. Several of the enzymes (NME1, CRABP2, e.g.) are of potential drug development interest, and several of them have never been picked up in covalent screens before, either.

This is just a glance into a whole landscape of drug-compound interactions that we’ve hardly begun to explore. It’s especially interesting to consider the less-explore functional groups, as was done here, and there are plenty of these that have never been investigated at all. Such compounds could potentially turn into drug candidates (after optimization) with their covalent warheads still as part of their mechanism, or one could try to pick up enough binding energy during that optimization to dispense with the covalent binding later on. (That second idea is theoretically possible, but I’ve never seen an example of it so far, and would be very interested in one). New territory!

14 comments on “Digging Through the Proteins, Covalently”

  1. NJBiologist says:

    “The group narrowed down on 12 proteins for which structures are available and whose labeling seemed robust, and 11 of them also labeled when the experiment was tried on recombinant protein alone. (The outlier just points out that there could be differences in the in vivo protein – post-translational modifications? – that made it different from the pure-protein case).”

    I’m surprised by this… to reproduce the labeling, these proteins’ binding sites have to not depend on chaperones, funky glycosylation or any of the myriad other things that cells do to put the finishing touches on a protein. 11/12 sounds like staggering good luck.

  2. JSR says:

    And Celgene anted up $101M to Vividion, so they must have found something more interesting than biliverdin reductase A.

  3. Imaging guy says:

    I have a got a question for you, medicinal chemistry guys. Can you determine whether a small molecule will bind covalently to a protein or proteins just from its structure? Do you have to do extensive washing to determine whether the binding is covalent or not? If washing is to be done, which is to be used as the washing agent, water or methanol? One article says this, “irreversible binding of [3H] beta FNA (funaltrexamine) was defined experimentally as that remaining following a 30-min wash with 2 changes of buffer (50 mM Tris-HCl) was added to this buffer at room temp. The washing procedure we routinely used was 5 washes at 1, 5, 20, 20, and 20 min each at room temp. Under the same washing protocol, specific [3H] naloxone binding to brain sections completely disappeared (unpublished observation). Note that the washing procedure usually employed for reversible binding is (4) 30 s washes at 0 °C [1]. In another study methanol was used as washing agent. “After vortexing and centrifugation, the precipitated protein was washed with 80% (v/v) aqueous methanol containing 10% (w/v) trichloroacetic acid, diethyl ether-methanol [1:1 (v/v)], and 80% (v/v) aqueous methanol (twice for each solvent)”. [2]
    1) Autoradiographic Study of Irreversible Binding of [H-3] Beta-Funaltrexamine to Opioid Receptors in the Rat Forebrain (PMID: 1645609)
    2) Covalent Binding and Tissue Distribution/Retention Assessment of Drugs Associated with Idiosyncratic Drug Toxicity (PMID: 18508880)

    1. Derek Lowe says:

      You can only tell in the broadest, least useful terms. There are some functional groups that are known to have affinity for Cys residues, for example, and more recent ones that seem to pick out Lys groups more often. But predicting which ones they will label is another matter. There are “supernucleophilic” Cys groups that have been mapped (by Cravatt’s team and others), and I Lys, Tyr, His, and Ser residues can be modulated similarly.

      But that just takes you as far as “If something’s going to be labeled on this protein, I’ll bet it’s going to be one of these Cys residues, and if one of those gets labeled by a compound in our collection, I’ll bet it’s going to be from one of these two or three chemical classes”. Not so useful, and plenty of things label outside those guesses (or fail to). So there’s really no substitute for experimentation, not at all.

      1. Curious Wavefunction says:

        There are a select few cases where the prediction can be better (although still unrealiable). For instance in the case of boronate inhibitors like Velcade, if you have a serine in the binding site that also happens to be close to a basic group like a lysine, there can be enough of the deprotonated form to bind to the boron. In that case your inhibitor is at least definitely worth testing.

        One of the minor but still important points this example makes is regarding the problems with computational techniques: a computational algorithm designed to predict this kind of binding would have to correctly calculate or predict the pKa s of residues in the binding site. This is still a very challenging problem.

    2. Barry says:

      and there are naturally-ocurring covalent modifiers (calicheamycin, neocarzinostatin…) that definitely took us by surprise

      1. Derek Lowe says:

        I remember when the esperamycin/calicheamycin structures and mechanism came out when I was in grad school – I was just amazed at how weird those compounds were and how they worked. Still am!

    3. Barry says:

      and there are set-up cases (e.g. peptide aldehyde or peptide nitrile or alpha-ketoamide) that will covalently (reversibly) bind the active site of e.g. a cysteine protease.
      But you still want to see the x-ray diffraction co-crystal structure to confirm what’s going on.

  4. Cellular Chemistry guy says:

    A previous paper showed stability and potential use of fluorosulfate as a binding driven warhead. See here work from Pfizer:

  5. Uncle Al says:

    Traditional pharma is equilibrium binding. Given selectivity, make that default suicide binding…plus a packet of equally costly inert pills to space the dose with target turnover. Smaller production waste streams save the Earth – a more organic pharmacopoeia. “8^>)

  6. Layne Simian says:

    Has anyone here gotten click chemistry to work even half as well as it’s reported by the Cravatt group?

  7. cranky biologist says:

    Maybe instead of Inverse Drug Discovery we could just call it Discovery. I guarantee this experiment would never be funded under NSF/NIH as described, falling under the “no fishing expeditions” rule.

  8. Picky Peter says:

    I assume “unleased” should read as “unleashed”?

    1. Derek Lowe says:

      Err, yes, it should – fixed!

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