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Going After Ebola

How small-molecule drugs fit into binding pockets in their targets is one of the central questions of medicinal chemistry. A new paper from a group at Oxford gives a good example of how varied that process can be – it’s looking at a number of drugs that have been shown to interfere (to some degree) with infection by the Ebola virus. Ebola has been the subject of a number of screens, which you can be sure were run by people paying very careful attention to their lab technique and protective equipment. A surprising number of approved drugs have shown some activity – no rockin’ inhibitors at the nanomolar level, unfortunately, but definitely a higher hit rate than one might have expected from an antiviral screen.

The compounds that hit certainly didn’t seem to have much in common. Among them are ibuprofen, the estrogen receptor modulator toremifene, the muscarinic antagonist benzatropine, the old calcium channel blocker bepridil, and the serotonin reuptake inhibitors Paxil (paroxetine) and Zoloft (sertraline). That’s a pretty good spread of mechanisms and structures, and no, the two SSRIs don’t seem to lead anywhere mechanistically. So what’s going on?

It turns out that all of these drugs bind to the Ebolavirus glycoprotein (EBOV GP), a key surface protein on the viral particles that is responsible for its attachment to cells and entry through the cell membrane. Not only do all these compounds bind to the same protein, they actually bind to the same pocket, albeit by forming different interactions in each case. At right is an overlay of the six structures, and you can see that they carve out a common volume, for the most part. You have to be careful with that sort of thinking, because most compounds this size can be jiggered so that they look to be carving out such a space, but in this case, though, these are structures based on X-ray data of the EPOV GP/ligand complexes, collected at the heavy-duty DIAMOND synchrotron source in the UK. There are several interactions that are suggested by the data, among them that hydrophobic subpocket near the V66/A101 residues (where a phenyl ring of bepridil fits perfectly). The X-ray structures also show some movements in the protein itself on the binding of different inhibitors, although none that seem directly related to the small molecule interactions.

But sertraline doesn’t hit either of the subpockets; it’s all out in the middle. Two molecule of benzatropine fit into the protein at the same time (one of those is shown in green in the figure above), and paroxetine (which is in grey) doesn’t seem to fill all of the common volume very well at all. Interestingly, all the interactions seen are hydrophobic ones – there’s not a hydrogen bond in sight. And none of the compounds exploit all the interactions seen across the whole set, which strongly suggests that there should be hybrid structures that do a better job.

All of these compounds are way up in the micromolar range in their binding constants,  and they all show destabilization of the EBOV GP in thermal shift assays. Those of you familiar with fragment-based drug discovery will recognize the process – low affinity, multiple molecules per site, different compounds scattered around the binding pocket, and so on. These compounds are larger than traditional fragments, but the the region where they’re binding is larger than many traditional binding sites, so you end up in the same mode.

If you’re willing to go out into these weaker interactions, you’d probably find that an awful lot of drug molecules hit an awful lot of things at (say) 100 micromolar. It’s just that most of these binding events have little or no functional relevance. And viruses have very few moving parts to them, which often makes drug discovery difficult. In the Ebola case, though, this protein is a critical, unique, Swiss-army-knife thing whose functions cannot be messed around with. Bepridil, for example, provides 100% protection from Ebola infection in a mouse model and sertraline hits 70%, which is not too bad at all for micromolar phenotypic screening hits. Even more suggestively, the antiviral efficacy correlates quite well with the thermal shift data, a strong indication that messing with the protein’s conformation is indeed the mechanism of action.

You’d have to think that an optimized EBOV GP binder could be very effective indeed, and this paper points the way to assembling such compounds. You’ve got the protein target, you’ve got a strongly relevant biophysical assay, you’ve got X-ray data from a list of compounds in the binding site, and you’ve got an animal model – that’s about as much as a med-chem team can ask for down here on Earth, and I hope that something comes of it.

25 comments on “Going After Ebola”

  1. anoano says:

    Target for Numerate and their AI platform to have another go after DOD contract few years ago

  2. Peter Kenny says:

    The article notes “The reported IC50 is 3 μM for sertraline compared to 5 μM for bepridil in Vero cells; however, bepridil provided better protection than sertraline in a mouse model”. The affinities for bepridil (0.29 mM) sertraline (0.95 mM) binding to EBOV GP would appear to be far too weak to explain the activity in the Vero cell assay.

    1. anon says:

      The compounds display basic amines. They are likely accumulating in the acidic late endosomes that are formed during EboV entry, and interfering with the virus/host fusion step as opposed to inhibiting virus entry into the cell. It is maybe the accumulation that makes these compounds appear to have a higher affinity for the glycoprotein.

      I would bet my house that they were discovered in a cell-based phenotypic screen.

    2. Calvin says:

      In general I’d say that for viruses there is a much larger disparity between the isolated protein biochemical assay versus the whole virus (without getting into tox effects, cell type variation etc). It can go in both directions so the difference can be massive or tiny. This is largely because the replication complex and all the other associated machinery of viruses can be conformationally very different in the cell versus just the isolated enzyme. And those complexes break up and move and reform in unexpected ways so the biochemical proteins can be a pretty poor representation of what is going on. So I’m not entirely surprised that there is such a disconnect here. In the end most antiviral approaches start with the biochemical assay to get a decent starting point and feel for activity and then optimise exclusively on the whole cell assay. I remember Janssen publishing some compounds in HCV where they had a disconnect between the biochemical assays and the whole virus replicon and despite tonnes of structural biology comparing many different sequences and binding of compounds they couldn’t explain the difference between the cell results and the biochemical results. All part of the fun I guess.

      1. Peter Kenny says:

        I would generally anticipate a compound being less potent in a cell-based assay than its affinity for the the isolated target would have one believe. Low permeability and/or high efflux may result in relatively low intracellular concentration and the compound may also have to compete with intracellular substrates and cofactors. The most likely explanation for a compound being more potent in the cell-based assay is that it is hitting a target other than the one we thought/hoped it was hitting. Concentration in acidic compartments and active influx would be other possible explanations. If unexpectedly high activity is only observed in vivo then it may be due to an active metabolite. I’ve linked a recent article in which this type of disconnect in assay results was discussed.

  3. anon the II says:

    I’m a little leary of getting much from this thermal shift data. Most potent drugs stabilize the protein target on binding, i.e. they raise it’s “melting point” in a thermal shift assay. This makes sense because the complex is basically a more stable beast. Invoking a possible mechanism of action based on the thermal shift data pointing in the wrong direction seems a bit hand wavy.

    If it’s not noise, this seems like the perfect data set for a drug discovery effort based on the “fragment approach”. Waiting with baited breath.

    1. Peter S. Shenkin says:

      That’s “bated breath”, unless you’ve been eating fish…. 🙂

  4. Radpharmchem says:

    “which strongly suggests that there should be hybrid structures that do a better job”.

    Do you mean like a LINKER between any two of the drugs to exploit more of the binding pockets??

    1. RM says:

      Given that it looks like the various compounds overlap in 3D space to a rather large extent (rather than bind to completely independent pockets), just slapping a linker between two of them is unlikely to work. Instead, you’re likely going to have to take a “grafting” approach, combining moieties from one compound with ones from another (and possibly some from a third), on something like a joint/hybrid scaffold.

      1. Radpharmchem says:

        Thanks for the clarification. That makes alot of sense now.

    2. Derek Lowe says:

      No, there doesn’t seem to be room for that, since they’re all sitting in roughly the same area. You’re going to have to make compounds that have the phenyl of one drug, a ring or another, a side chain of another, all in one new structure. That’s not simple, but that’s what med-chem is for. . .

      1. Radpharmchem says:

        Thanks Derek for clarifying that. I understand what you mean now.
        Yeah doesn’t sound so simple but looks like a very good idea worth the try.

  5. Wavefunction says:

    It’s important to be careful when deriving “common pharmacophores” from data like this. A lot of times the affinity comes from distributed, piecewise small interactions, and creating a common pharmacophore will get rid of these interactions.

    1. Derek Lowe says:

      That’s absolutely right – I’ve had some of these “greatest hits” structures just totally evaporate. But there’s only one way to find out!

  6. Barry says:

    Is the incidence of Ebola enough to pay for R&D of a novel anti-viral?

    displaying “the phenyl of one molecule, the sidechain of another…” is just what Bartlett’s CAVEAT is for

    1. DrOcto says:

      The last few outbreaks have hit poor populations, and were otherwise well contained by quarentene procedures. So not much opportunity for profit.

      Given that development from scratch takes decades, there is something to be said for using public funds (or an altruistic company donation) to prepare for the worst.

    2. Zune fan says:

      @Barry and DrOcto
      Funding: I seem to remember that in the last big Ebola outbreak the Gates Foundation slapped down something like $50M to aid the caregivers etc. after it was in full swing. Even half that money could fund several groups to find a viable candidate or candidates. Profits not much after, but would make financial sense for a small organization to undertake.

      1. Barry says:

        If you’re looking no further than starting a Med. Chem. project that no one will take to the clinic (real bucks) or to market, you’re wasting everyone’s time. The characteristic high fever says Ebola’s an excellent candidate for a vaccine ( although I don’t know how fast it mutates). That’s the place to put finite resources.

        1. Zune Fan says:

          If you get a real clinical candidate, you can be sure Gates would take it through development. Of course no company would do it. For Gates, it makes financial sense for them in terms of not having to throw big money at the next outbreak. Fifty million dollars for last one–could have spent that on preemptive cures. But that would require planning. Vaccine makes sense but all eggs in one basket is not usually wise.

  7. Mol Biologist says:

    Hey Barry, you are always smart. Virus like we are they know better where to put the needle. Derek always forget thatM human are more than it. We are lucky and have more options compared to viruses.

  8. Jbosch says:

    The thermal shift has 5% [DMSO] and they test the glycosylated protein, while the crystallization was performed with 10% [DMSO] and unglycosylated protein. For my taste pretty high levels of DMSO, nevertheless they succeeded in getting some nice structures.

    A shape complementarity approach with an eye on chemical Tanimoto scores could be helpful with the prior knowledge of the compounds now. I’m thinking of for example ROCS, defining an overall shape and adding chemical constraints then go wild on the 10 million compounds virtually and start testing a subset in bioassays.

  9. Barry says:

    Let’s add Ebola to the list (with novel antibiotics) of projects that probably need to be pursued but which won’t be served by the magic of the Free Market. We need a mechanism apart from “a period of market exclusivity” guaranteed by the USPTO. I for one don’t want to rely on Bill and Melinda (grateful as I am) to shape our research efforts

    1. Janex says:

      Well the world governments don’t have enough money to fund it directly (or frankly the drug/vaccine development expertise). Even Bill and Melinda can’t fund everything! But what the government can do is give something of value. One example could be FDA priority review vouchers. Those sell for roughly $100 million. So programs of this type could be rewarded with between 1-10 vouchers. At the end of phase 2, a CDC committee could assign the voucher reward based upon novelty, clinical utility for untreatable diseases etc according to pre-determined criteria. There are certainly other rewards that could be given and the pre-determined criteria and other details would need to be hammered out but something of this sort would work.

      1. Kamil Pabis says:

        Of course they have enough money. Most countries invest very little into developmental aid and this is de facto a form of developmental aid. There is no evidence that countries that invest a lot into aid suffer negative consequences (e.g. Scandinavia) so there is room to spend more. Money is rarely the issue, but political will is.

    2. Scott says:

      Get one case of Ebola back in the US (or Europe) during the next big outbreak, I guarantee there will be sufficient monetary motivation.

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