First-in-class inhibitors of Nsp15 endoribonuclease of SARS-CoV-2: modeling, synthesis, and enzymatic assay of thiazolidinedione and rhodanine analogs.

During infection, the coronavirus Nsp15, a uridine-specific endoribonuclease, suppresses the host cell's antiviral response. Recently, researchers have paid more attention to this relatively underexplored yet potentially viable drug target. In this study, we employed fluorescence resonance energy transfer-based screening assays to identify potent Nsp15 inhibitors. Subsequently, we used active-site in silico docking methods to design new molecules with enhanced inhibitory properties. Solution assays were used to measure the potency and determine the mechanism of these inhibitors. We identified a novel class of thiazolidinedione and rhodanine analogs that inhibit SARS-CoV-2 Nsp15. Docking these compounds into the uridine-binding site shows that most analogs form two hydrogen bonds with Ser294. The most potent inhibitors are compounds KCO237 and KCO251 (half-maximal inhibitory concentration: 0.304 μM, 0.931 μM respectively). The inhibition kinetics of KCO237 and KCO251 best align with a reversible mixed inhibition model. Mutating Ser294 did not completely abolish Nsp15 activity or the inhibitory effect of KCO237 or KCO251. These findings suggest that thiazolidinedione and rhodanine analogs likely inhibit Nsp15 by binding to the uridine active site while also implicating a possible secondary allosteric binding site. The ability of these compounds to inhibit VERO 6 cell infection with SARS-CoV-2 at subtoxic levels highlights their potential for development as novel antiviral treatments for SARS-CoV-2 and other coronavirus-related diseases.