Slowly exchanging bound states of SARS-CoV-2 3CLpro-inhibitor complexes revealed by 19F NMR

The SARS-CoV-2 3C-like protease (3CL^pro) is an important antiviral drug target whose substrate-binding site exhibits substantial conformational plasticity. Although X-ray crystallography and molecular dynamics simulations have revealed structural variability in 3CL^pro, the thermodynamic and kinetic landscape of inhibitor-bound conformations in solution remains incompletely characterized. Here, we use solution-state ¹⁹F NMR spectroscopy to investigate conformational heterogeneity and dynamics of 3CL^pro bound to the covalent inhibitor nirmatrelvir (NMV) and the noncovalent inhibitor ensitrelvir (ENS), using their intrinsic fluorinated groups as sensitive probes of the local protein environment. ¹⁹F NMR titration of NMV with wild-type 3CL^pro reveals two distinct bound-state resonances in slow exchange, with binding saturation a 1:1 ligand-to-protomer ratio, at 298 K. The relative populations of these states remain invariant throughout the titration, supporting an intrinsic conformational equilibrium within the inhibitor-bound active site rather than multiple binding sites or oligomeric states. Variable-temperature and ¹⁹F–¹⁹F exchange spectroscopy (EXSY), combined with dual-field Bloch–McConnell line-shape analysis, resolve millisecond-timescale exchange between the two distinct inhibitor-bound conformational states at 298 K (population ratio ~65:35; k_ex ~52 s⁻¹; ΔG^‡ ~15 kcal mol⁻¹). Importantly, two bound states also persist when NMV binds noncovalently to the catalytically inactive C145A mutant (p_B ~18%) and when wild-type 3CL^pro binds the chemically distinct noncovalent inhibitor ENS (p_B ~38%; ΔG° ~+0.3 kcal mol⁻¹). Thus, slow-exchange heterogeneity does not require covalent attachment and appears to reflect an underlying property of the inhibitor-bound protease. Despite variability in active-site loops and inhibitor poses across available crystal structures, these structures do not provide an obvious structural basis for the slow exchange detected by ¹⁹F NMR. Moreover, loop motions observed in conventional microsecond molecular dynamics simulations occur on substantially faster timescales. Together, these findings indicate that inhibited 3CL^pro samples low-energy, slowly interconverting conformational states in solution that are not fully represented by static structures or conventional simulations. Solution-state ¹⁹F NMR therefore provides a powerful approach for quantifying otherwise difficult-to-access conformational states, kinetics, and energetics relevant to drug–target interactions and inhibitor design.