Functional and Mechanistic Characterization of SARS-CoV-2 NSP15 Reveals Metal-Dependent Regulation and Inhibition by Direct-Binding Small Molecules

The SARS-CoV-2 non-structural protein 15 (NSP15) is a uridine-specific endoribonuclease involved in viral RNA processing and immune evasion, thereby contributing to viral pathogenesis. Although NSP15 has been structurally characterized by several studies, which report its endoribonuclease activity and regulatory features, important functional and regulatory gaps remain unaddressed, including quantitative stability, metal-ion regulation, and inhibitor-binding kinetics. To address these gaps, we used an integrated approach to investigate NSP15 regulation and inhibition. Thermochemical stability studies indicated moderate NSP15 stability, with cooperative unfolding characterized by a Cm of 1.68 M urea and a Tm of ~70 °C. Urea PAGE activity measurements revealed a strong metal-ion dependence, with Mn²⁺ as the optimal activator and marked stimulation compared to Ca2+, Mg2+, and Zn2+. Biophysical experiments using SPR and fluorescence quenching confirmed a direct protein-ligand interaction with dissociation constants of ~1–4 µM. Consistent with these results, the compounds inhibited endoribonuclease activity with IC50 values of ~8-30 µM. Alanine substitution of catalytic residues H235A and H250A completely abolished cleavage, while S294A within the B1 pocket, a Uridine-specificity determinant, reduced activity by 51–67% (10–50 nM), markedly weakened inhibitor binding, and increased inhibition susceptibility. Importantly, docking and simulation studies revealed stabilized active-site dynamics driven by strong polar interactions, as evidenced by SASA, hydrogen-bonding analysis, RMSD, RMSF, and radius of gyration (Rg) analyses, underscoring the coordinated contribution of polar contacts and hydrophobic interactions in the inhibitor-bound active-site conformation. Collectively, this work provides quantitative, mutational, and functional characterization, forming a foundation for designing NSP15-specific antivirals.