New Selective Peptidyl Di(chlorophenyl)‐phosphonate Esters to Visualize and Block Neutrophil Proteinase 3 in Human Diseases

Abstract

The function of neutrophil protease 3 (PR3) is poorly understood despite of its role in autoimmune vasculitides and its possible involvement in cell apoptosis. This makes it different from its structural homologue neutrophil elastase (HNE). Endogenous inhibitors of human neutrophil serine proteases preferentially inhibit HNE and to a lesser extent PR3. We developed selective phosphonate inhibitors with the structure: Ac‐peptidyl‐P(O‐C6H4‐4‐Cl)2. The peptidyl moiety was deduced from molecular modeling and kinetic studies comparing the substrate specificities of PR3 with a single residue PR3 mutant and HNE. The P4 and P2 positions were essential to discriminate between PR3 and HNE. We then synthesized N‐terminally biotinylated peptidyl phosphonates to identify PR3 in complex biological samples. These inhibitors resisted proteolytic degradation and rapidly inactivated PR3 in biological fluids such as inflammatory lung secretions and the urine of patients with bladder cancer. One of these inhibitors revealed intracellular PR3 in permeabilized neutrophils and on the surface of activated cells. They hardly inhibited PR3 bound to the surface of stimulated neutrophils, despite their low molecular mass, suggesting that the conformation and reactivity of membrane‐bound PR3 is altered. This finding is relevant for autoantibody binding and the subsequent activation of neutrophils in granulomatosis with polyangiitis (formerly Wegener disease).