Divergent Redox-Resilience of Alvelestat and Sivelestat: A Mechanistic Hypothesis for Inhibitor Performance Under Oxidative Stress

Human neutrophil elastase (HNE) is a key driver of inflammatory lung disorders, pro-moting extracellular matrix degradation and tissue damage. While inhibitors such as Sivelestat and Alvelestat are clinically relevant, their performance within the aggressive oxidative microenvironment of diseased lungs remains poorly understood. Here, we employed an integrated in vitro and in silico approach to investigate their behavior under physiological and oxidative conditions and to provide a molecular-level interpretation. Under physiological conditions, enzymatic assays and steady-state kinetics confirmed that both compounds act as competitive inhibitors, with Sivelestat displaying higher baseline potency. Under oxidative stress, however, Sivelestat exhibited a near-complete loss of activity, whereas Alvelestat retained its inhibitory efficacy. Molecular modeling and molecular dynamics simulations of native and oxidized HNE variants provided a structural rationale for this divergence. Alvelestat, as a non-covalent inhibitor, maintains stable binding despite increased flexibility of the active site induced by oxidative modifications. In contrast, Sivelestat, which acts through a reversible covalent mechanism, requires a precise pre-acylation geometry. Oxidation-induced remodeling of the S1 pocket disrupts the near-attack configuration required for covalent bond formation, thereby im-pairing inhibition. Overall, these findings indicate that oxidative stress selectively compromises covalent inhibition while preserving enzymatic activity, highlighting redox re-silience as a key parameter in the design of next-generation HNE inhibitors.