The development of SARS-CoV-2 main protease (M^pro) inhibitors for the treatment of COVID-19 has mostly benefitted from X-ray structures and preexisting knowledge of inhibitors; however, an efficient method to generate M^pro inhibitors, which circumvents such information would be advantageous. As an alternative approach, we show here that DNA-encoded chemistry technology (DEC-Tec) can be used to discover inhibitors of M^pro. An affinity selection of a 4-billion-membered DNA-encoded chemical library (DECL) using M^pro as bait produces novel non-covalent and non-peptide-based small molecule inhibitors of M^pro with low nanomolar K_i values. Furthermore, these compounds demonstrate efficacy against mutant forms of M^pro that have shown resistance to the standard-of-care drug nirmatrelvir. Overall, this work demonstrates that DEC-Tec can efficiently generate novel and potent inhibitors without preliminary chemical or structural information.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has killed more than 4 million humans globally, but there is no bona fide Food and Drug Administration-approved drug-like molecule to impede the COVID-19 pandemic. The sluggish pace of traditional therapeutic discovery is poorly suited to producing targeted treatments against rapidly evolving viruses. Here, we used an affinity-based screen of 4 billion DNA-encoded molecules en masse to identify a potent class of virus-specific inhibitors of the SARS-CoV-2 main protease (M^pro) without extensive and time-consuming medicinal chemistry. CDD-1714, the initial three-building-block screening hit (molecular weight [MW] = 542.5 g/mol), was a potent inhibitor (inhibition constant [K_i] = 20 nM). CDD-1713, a smaller two-building-block analog (MW = 353.3 g/mol) of CDD-1714, is a reversible covalent inhibitor of M^pro (K_i = 45 nM) that binds in the protease pocket, has specificity over human proteases, and shows in vitro efficacy in a SARS-CoV-2 infectivity model. Subsequently, key regions of CDD-1713 that were necessary for inhibitory activity were identified and a potent (K_i = 37 nM), smaller (MW = 323.4 g/mol), and metabolically more stable analog (CDD-1976) was generated. Thus, screening of DNA-encoded chemical libraries can accelerate the discovery of efficacious drug-like inhibitors of emerging viral disease targets.