As of November 23, more than 58 million people worldwide have contracted COVID-19, and more than 1.3 million have died. Each of these numbers is roughly two orders of magnitude higher than in this post published exactly eight months ago. Progress towards vaccines and biological treatments has been stunningly fast, but small molecules could still play a role. Towards this end, Sebastian Günther, Alke Meents, and nearly 100 collaborators from the Center for Free-Electron Laser Science at DESY and multiple other institutions have just posted a preprint on bioRxiv.
The researchers were interested in drug repurposing, in which approved or clinical-stage molecules are tested against a new target. Typically this is done in some sort of biochemical or cell-based assay, but in this case the researchers chose crystallographic screening against the main protease (Mpro) from SARS-CoV-2. An independent fragment screen against the same target was published recently in Nat. Comm. (I wrote a companion Comment, and both articles are open-access.)
The current screen of 5953 compounds may be the largest crystallographic screen in history, and the first I know of that used drug-sized molecules rather than fragments. Even more impressive, all compounds were co-crystallized with Mpro, a much more tedious process than the usual soaking. The advantage of co-crystallization is that the protein is more able to change conformation in response to compound binding, but the disadvantage is that small molecules may prevent crystallization. Ultimately 3955 compounds allowed crystal formation, of which 3228 produced crystals that diffracted better than 2.5 Å, and 1196 produced usable datasets. The result? Just 37 unique binders, or 0.6%. Comparing this to the 96 fragment hits from the smaller fragment library is complicated by differences in methodologies, but it does seem likely that molecular complexity played a role in the lower hit rate: the median molecular weight of the drugs screened, 366.5 Da, comfortably exceeds fragment space.
Among the 37 binders, only 29 gave sufficiently well-resolved electron density to determine binding modes. Of these, ten bound covalently. The catalytic cysteine seems particularly reactive, as evidenced by the fact that seven structures showed maleate – a common pharmaceutical counterion – covalently bound. One of the covalent molecules, calpeptin, is a cysteine protease inhibitor that had previously been reported to be active against SARS-CoV-2, but the others are less predictable. In addition to the active site, some molecules bound to two possibly allosteric sites.
Crystallographic hits were tested for inhibition of viral replication in cells. Ten were active, and a few (calpeptin, pelitinib, and isofloxythepin) had single digit micromolar activity. Interestingly, despite being designed as a covalent kinase inhibitor, pelitinib binds noncovalently. In contrast, isofloxythepin, a non-covalent dopamine receptor antagonist, binds covalently.
In addition to the cell-based screen, many of the compounds also showed binding by native electrospray ionization mass spectrometry (ESI-MS). However, as we’ve noted previously, the correlation between affinity and ESI-MS binding can be tenuous. It would be nice to see the affinity or activity of the compounds via a more quantitative method. Indeed, the researchers note that none of the non-peptidic molecules had previously been reported as Mpro inhibitors, so they may be quite weak. Another problem is that – in contrast to the crystallographic fragment screen – none of the coordinates seem to have been released yet. Hopefully this will be rectified when the paper is formally published.
This campaign is yet more evidence that crystallography has come into its own as a primary screening methodology. The researchers note that they “now routinely measure 450 datasets per day,” with a goal of reaching 1000. Whether or not these results impact the course of COVID-19, the techniques developed will likely impact future drug discovery efforts.