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

The SARS-CoV-2 virus causes COVID-19, and several of its gene products have been successfully targeted for antiviral drug development, including the 3C-like protease (3CL^pro). The substrate-binding site of 3CL^pro exhibits significant conformational plasticity. While available X-ray crystal structures reveal substantial loop variability and molecular dynamics simulations indicate that conformational heterogeneity persists in ligand-bound complexes, the slow-timescale thermodynamic and kinetic landscape of these complexes in solution remains incompletely defined. Using ¹⁹F NMR spectroscopy, we characterize a slow-exchange conformational equilibrium in both the covalent nirmatrelvir (NMV) and noncovalent ensitrelvir (ENS) complexes of 3CL^pro. For the wild-type 3CL^pro-NMV complex at 298 K, joint dual-field line shape and exchange spectroscopy analysis resolves a millisecond-timescale exchange between two distinct states (population ratio ∼ 65:35; k_ex  ≈ 52 s^-1; ΔG^‡ ≈ 15 kcal mol^-1). Co-existing bound states also persist in both the noncovalent NMV-[C145A] mutant (minor state population, p_B  ≈ 18%) and wild-type ENS (p_B  ≈ 38%) complexes, demonstrating that this slow-exchange heterogeneity is an intrinsic property of the ligated protease rather than a consequence of covalent attachment. These dynamics are not readily explained by available crystal structures or conventional microsecond molecular dynamics simulations, suggesting that in solution the inhibited enzyme samples alternative, energetically accessible conformations that are not fully represented in the crystal lattice. This study highlights the utility of solution-state ¹⁹F NMR for quantifying low-energy conformational states relevant to drug-target energetics and inhibitor design.