SARS-CoV-2 resistance pathways to EDP-235

SARS-CoV-2 infections continue to frequently occur, and treatment options remain limited, emphasizing the need for additional development of therapeutics. Here, we have conducted resistance profiling of EDP-235, a next-generation coronavirus 3CL^pro inhibitor in clinical development. High-throughput passaging for resistance resulted in 29 out of 180 replicates surviving at 256-fold the EC₅₀. Sequencing of these viruses identified a myriad of emergent mutations, with T21I, L50F, P132S, S144A, E166A, and P168S observed most frequently. Pathway analyses indicated that T21I was the most frequently initially selected mutation, followed by E166A then L50F. Among combinations, L50F + E166A + P168S was the most commonly observed, and cellular reporter assays found this mutant to be the most resistant 3CL^pro against EDP-235 (578-fold resistance). A large-scale approach to studying the barrier to resistance was developed and used to compare EDP-235 and nirmatrelvir, with EDP-235 demonstrating a higher barrier to resistance against SARS-CoV-2. Finally, EDP-235 resistance selection was conducted for an alphacoronavirus, HCoV-229E. The resistance-associated mutations which emerged against this coronavirus differed from those observed for SARS-CoV-2, indicating that the viral backbone affects resistance selection for coronaviruses and emphasizing the usage of appropriate viruses. These results further our understanding of SARS-CoV-2 drug resistance and support the continued development of EDP-235 and other inhibitors for COVID-19.