We also employed differential scanning fluorimetry (DSF) to evaluate shifts in TMPRSS2 melting temperatures (ΔTms) for TMPRSS2 and TMPRSS2 S441A proteins in the presence of '2222, '2602, 6amidino-2-naphthol, nafamostat, and '157. All noncovalent ligands were tested at their maximum achievable solubility in aqueous buffer. The Tm values for TMPRSS2 and TMPRSS2 S441A in the absence of ligands were (52.1±0.1)°C and (49.5±0.3)°C, respectively. The noncovalent ligand 6-amidino-2-naphthol induced Tm shifts of (8.2±0.4)°C and (7.3±0.7)°C for TMPRSS2 and TMPRSS2 S441A, respectively, at a ligand concentration of 10 mM (Fig. ). The TMPRSS2 thermal stabilizations induced by 6-amidino-2naphthol were linear on a semi-logarithmic scale and did not show signs of saturation even at the highest ligand concentration (Fig. ). Nafamostat and '157 induced large maximum ΔTm values for TMPRSS2 at (23.6±0.2)°C and (8.7±0.5)°C, respectively, and demonstrated saturation binding in the TMPRSS2 thermal stabilization plot that indicated they achieved maximum ligand occupancies, likely due to covalent ligand binding (Extended Data Fig. ). In contrast, nafamostat and '157 incubated with TMPRSS2 S441A only induced small ΔTms of (1.50±0.07)°C and (2.1±0.1)°C, respectively, across the same ligand concentration ranges and did not exhibit saturation binding in the thermal stabilization plot (hollow symbols; Fig. , Extended Data Fig. ). The '2602 ligand induced a TMPRSS2 ΔTm of (3.2±0.2)°C at a ligand concentration of 2 mM, but solubility challenges prevented any higher '2602 concentrations being tested (green triangles Fig. ; Extended Data Fig. ). Across the tested '2602 concentration range, no saturation binding was observed and instead the '2602 plot resembled the TMPRSS2 thermal stabilization trends induced by 6amidino-2-naphthol. The '2602 ligand only showed a small TMPRSS2 S441A ΔTm of (0.85±0.07)°C at a ligand concentration of 4 mM, suggesting this TMPRSS2 residue was critical for '2602 binding. Thus, through two independent biophysical methods, we confirmed the results obtained in the molecular docking screens that '2222/'2602 noncovalently bind within the TMPRSS2 S1 site and rely on the TMPRSS2 Ser441 for binding.