Fragment-based screening targeting an open form of the SARS-CoV-2 main protease binding pocket

To identify starting points for therapeutics targeting SARS-CoV-2, the Paul Scherrer Institute and Idorsia decided to collaboratively perform an X-ray crystallographic fragment screen against its main protease. Fragment-based screening was carried out using crystals with a pronounced open conformation of the substrate binding pocket. Of 631 fragments soaked, a total of 29 hits bound either in the active site (24 hits), a remote binding pocket (2 hits) or at crystal packing interfaces (3 hits). Notably, two fragments with a pose sterically incompatible with a more occluded crystal form were identified. Two isatin-based electrophilic fragments bound covalently to the catalytic cysteine residue. Our structures also revealed a surprisingly strong influence of the crystal form on the binding pose of three published fragments used as positive controls, with implications for fragment screening by crystallography. Synopsis An X-ray crystallographic screen on SARS-CoV-2 3CL protease resulted in 29 fragment hits, including two isatin-based reversible covalent binders, and revealed a strong influence of the crystal form used for fragment soaking on the bound conformation of three additional reference fragments.