A binding-depth-dependent continuum of ADAM17/TACE inhibition revealed by punicalagin-derived polyphenols

The metalloprotease ADAM17/TACE is a critical therapeutic target, yet selective inhibition remains difficult due to its conserved catalytic site. This study investigates the inhibitory mechanisms of punicalagin (PNG) and gallic acid (GAA) in comparison with marimastat (MRM) using integrated enzyme kinetics, molecular docking, molecular dynamics (MD), quantum mechanical (QM), and UV-visible analyses. Kinetic and dose-response analyses suggest that PNG is consistent with non-competitive inhibition (IC₅₀ ≈ 0.18 μM; Ki ≈ 2.3-4.9 μM), characterized by strong suppression of catalytic turnover; GAA displays behavior consistent with mixed-type inhibition (∼68% maximal inhibition; Ki ≈ 2.3-6.4 μM); and MRM shows competitive-like inhibition (IC₅₀ = 23-49 nM; Ki ≈ 8.6 nM), likely reflecting direct Zn²⁺ catalytic-site engagement. These mechanistic assignments are based on convergent multi-method evidence rather than kinetic fitting alone, given the limited inhibitor concentration range available. Structural and MD analyses revealed distinct binding regions, from distal regulatory sites (PNG) to the Zn²⁺ catalytic pocket (MRM), with interaction energies correlating with binding depth. QM and spectroscopic results provided qualitative support for ligand-dependent electronic redistribution. These findings suggest a binding-depth-dependent inhibition continuum as a plausible mechanistic framework, highlighting ligand positioning as a potentially important determinant of selective metalloprotease inhibition; however, definitive mechanistic characterization will require expanded kinetic datasets and orthogonal binding validation. Quantum mechanical calculations and UV-visible spectroscopy confirm that differential ligand binding depths induce distinct electronic redistribution profiles in enzyme-ligand complexes, with distal exosite binding more likely to induce conformational-level electronic perturbations than catalytic-site engagement.