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

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 sPLA ₂-IIA species specifically inhibit the enzyme from which they are derived. We have now used an analogue screen of the human pentapeptide ⁷⁰FLSYK⁷⁴ in which side-chain residues were substituted, together with molecular docking approaches that modeled low-energy conformations of ⁷⁰FLSYK⁷⁴ bound to human sPLA ₂-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 an Escherichia 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.