A Novel Approach to the Design of Inhibitors of Human Secreted Phospholipase A2 Based on Native Peptide Inhibition* 210

Human Type IIA secreted phospholipase A2 (sPLA2-IIA) is an important modulator of cytokine-dependent inflammatory responses and a member of a growing superfamily of structurally related phospholipases. We have previously shown that sPLA2-IIA is inhibited by a pentapeptide sequence comprising residues 70–74 of the native sPLA2-IIA protein and that peptides derived from the equivalent region of different sPLA2-IIA species specifically inhibit the enzyme from which they are derived. We have now used an analogue screen of the human pentapeptide70FLSYK74 in which side-chain residues were substituted, together with molecular docking approaches that modeled low-energy conformations of 70FLSYK74 bound to human sPLA2-IIA, to generate inhibitors with improved potency. Importantly, the modeling studies showed a close association between the NH2 and COOH termini of the peptide, predicting significant enhancement of the potency of inhibition by cyclization. Cyclic compounds were synthesized and indeed showed 5–50-fold increased potency over the linear peptide in anEscherichia coli membrane assay. Furthermore, the potency of inhibition correlated with steady-state binding of the cyclic peptides to sPLA2-IIA as determined by surface plasmon resonance studies. Two potential peptide interaction sites were identified on sPLA2-IIA from the modeling studies, one in the NH2-terminal helix and the other in the β-wing region, and in vitro association assays support the potential for interaction of the peptides with these sites. The inhibitors were effective at nanomolar concentrations in blocking sPLA2-IIA-mediated amplification of cytokine-induced prostaglandin synthesis in human rheumatoid synoviocytes in culture. These studies provide an example where native peptide sequences can be used for the development of potent and selective inhibitors of enzyme function.