The neutrophil serine proteases proteinase 3 (PR3) and human neutrophil elastase (HNE) are specific regulators of the immune response and targets for several chronic inflammatory diseases including chronic obstructive pulmonary disease (COPD). Despite sharing a high sequence similarity, the two enzymes have different substrate specificities and functions. While a plethora of HNE inhibitors exist, both peptide-based and non-peptide, PR3 specific inhibitors are still in their infancy. Based on the detailed map of the ligand binding sites of PR3 that we drew earlier [1], we designed substrates specific for PR3 [2] using in silico design, docking and molecular dynamics simulations. In molecular dynamics simulations of the enzyme-substrate complexes, we could observe that the N-terminal fluorophore 2-aminobenzoic acid (Abz) and the C-terminal quencher N-(2,4-dinitrophenyl) ethylenediamine (EDDnp) groups of some of the substrates interact with the substrate recognition sites of the enzyme and contribute significantly to the binding free energy. We confirm this with enzymatic assays where Abz and EDDnp were found to significantly contribute to substrate hydrolysis by PR3. We next designed ketomethylene-based peptidomimetic inhibitors based on the substrates. As for the substrates, the presence of Abz and EDDnp in the peptidomimetic inhibitors is also important for securing high inhibitory potency towards PR3. The best inhibitor (Abz-VADnV[Ψ](COCH2)ADYQ-EDDnp) shows a low micromolar IC50 value for PR3 and is selective for PR3 compared to HNE. It displays a competitive and reversible inhibition mechanism. FRET peptides containing Abz and EDDnp as fluorophore and quencher groups, respectively, have been widely used to probe proteases specificity. We would therefore recommend a cautious approach while using Abz/EDDnp substrates in fluorescence assays for mapping substrate binding sites. [1] E.Hajjar, B.Korkmaz, B.O.Brandsdal, V.Witko-Sarsat and N.Reuter. J. Med. Chem. (2006), 49(4):1248-60 [2] S.Narawane, A.Budnjo, C.Grauffel, B.E.Haug, N.Reuter. J. Med. Chem. (2014) 57(3):1111-1115

In silico design, synthesis, and assays of specific substrates and peptidomimetic inhibitors for proteinase 3

249th National Meeting (2015)

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.