A new proteinase 3 substrate with improved selectivity over human neutrophil elastase

We report the synthesis and enzymatic studies on a new proteinase 3 intermolecular quenched substrate with enhanced selectivity over neutrophil elastase. Using combinatorial chemistry methods, we were able to synthesize the hexapeptide library with the general formula ABZ-Tyr-Tyr-Abu-X₁′-X₂′-X₃′-Tyr(3-NO₂)-NH₂ using the mix and split method. The iterative deconvolution of such a library allowed us to obtain the sequence ABZ-Tyr-Tyr-Abu-Asn-Glu-Pro-Tyr(3-NO₂)-NH₂ with a high specificity constant (k_cat/K_M = 1534 × 10³ M⁻¹ s⁻¹) and superior selectivity over neutrophil elastase and other neutrophil-derived serine proteases. Moreover, using the obtained substrate, we were able to detect a picomolar concentration of proteinase 3 (PR3). Incubation of the above-mentioned substrate with neutrophil lysate resulted in a strong fluorescent signal that was significantly reduced in the presence of a PR3 selective inhibitor.