07 March 2022

Virtual screening succeeds against the SARS-CoV-2 main protease

Today marks exactly two years since Practical Fragments first mentioned SARS-CoV-2. Since then, COVID-19 has killed more than 6 million people worldwide. Multiple effective vaccines have been developed and approved, along with a couple small-molecule drugs, but the virus is here to stay, and more drugs will be needed. This brings us to an open-access paper published in J. Am. Chem. Soc. by Jens Carlsson (Uppsala University) and a large group of international collaborators.
The so-called main protease (Mpro, or 3CLp) has been an antiviral target since the earliest days of the pandemic; the work we highlighted two years ago focused on a crystallographic screen against this enzyme. The new paper describes two virtual screening approaches.
The first started with a library of 235 million virtual compounds, mostly from Enamine’s “readily available for synthesis” (REAL) collection. Each compound was docked in thousands of different orientations against the active site of Mpro using DOCK3.7. Despite the staggering numbers (more than 223 trillion complexes!), the screen took just a day on 3500 CPU cores. The top 300,000 compounds were clustered based on similarity, and 100 molecules were synthesized. Nineteen of these showed binding by SPR, and three also inhibited the enzyme. Crystal structures were obtained for two of these, and both bound similarly to the predicted binding modes.
Compounds 1 and 3 each contain a hydantoin moiety that makes multiple hydrogen bonds to the protein, and merging elements led to low micromolar compounds such as compound 15. Further optimization ultimately delivered compound 19.

Compound 19 was potent in SPR and biochemical assays. Though it binds noncovalently, it had comparable cellular activity to nirmatrelvir, the recently approved covalent inhibitor of Mpro. Compound 19 showed nanomolar cell potency against SARS-CoV-1 and MERS-CoV and good selectivity against ten human proteases. The in vitro stability and permeability of compound 19 are also promising.
In addition to this de novo virtual screen, the researchers performed a second screen starting from one of the fragments identified crystallographically at Diamond Light Source. Of 93 molecules purchased and experimentally tested, 21 showed binding by SPR and 5 of these also inhibited the enzyme, with the most potent compound showing low micromolar activity.
There are several lessons from this paper. First, despite searching hundreds of millions of compounds, the best hits had only modest activity. This is perhaps surprising given the high fragment hit rates observed against Mpro in crystallographic and NMR screens, though it is worth noting that those fragments were even weaker binders.
Second, the hit rate from the na├»ve virtual screen was similar to that from the experimentally derived fragment screen. The researchers suggest that perhaps docking “may be more proficient in ranking diverse chemotypes rather than differentiating between closely related elaborations of the same scaffold.” In other words, virtual screens seem better at evaluating diverse starting points rather many similar molecules.
Third, despite the fact that the de novo virtual screen was not explicitly fragment-based, compound 1 does actually adhere to the rule of three. From there, addition of just six atoms improved affinity by >600-fold while also improving ligand efficiency.
Finally, this work is a testament to the utility of combining massive virtual screening with readily synthesizable compounds: the researchers note that it took less than four months to progress from compound 1 to nanomolar inhibitors.
This work relied heavily on rapid chemical synthesis done in Ukraine. Indeed, the two most popular fragment suppliers are both largely based in that country. Over the years many of us have come to know Ukrainian scientists not just as trusted colleagues but also as friends. I wish them and their families safety, and strength.

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