Expedited SARS‐CoV‐2 main protease inhibitor discovery through modular ‘direct‐to‐biology’ screening

Reactive fragment (RF) screening has emerged as an efficient method for ligand discovery across the proteome, irrespective of a target's perceived tractability. To date, however, the efficiency of subsequent optimisation campaigns has largely been low‐throughput, constrained by the need for synthesis and purification of target compounds. We report an efficient platform for ‘direct‐to‐biology’ (D2B) screening of cysteine‐targeting chloroacetamide RFs, wherein synthesis is performed in 384‐well plates allowing direct assessment in downstream biological assays without purification. Here, the developed platform was used to optimise inhibitors of SARS‐CoV‐2 main protease (MPro), an established drug target for the treatment of COVID‐19. An initial RF hit was developed into a series of potent inhibitors, and further exploration using D2B screening enabled a ‘switch’ to a reversible inhibitor series. This example of ligand discovery for MPro illustrates the acceleration that D2B chemistry can offer for optimising RFs towards covalent inhibitor candidates, as well as providing future impetus to explore the evolution of RFs into non‐covalent ligands.