title: A novel approach to the design of inhibitors of human secreted phospholipase A2 based on native peptide inhibition.
abstract: Human Type IIA secreted phospholipase A(2) (sPLA(2)-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 sPLA(2)-IIA is inhibited by a pentapeptide sequence comprising residues 70-74 of the native sPLA(2)-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 (70)FLSYK(74) in which side-chain residues were substituted, together with molecular docking approaches that modeled low-energy conformations of (70)FLSYK(74) bound to human sPLA(2)-IIA, to generate inhibitors with improved potency. Importantly, the modeling studies showed a close association between the NH(2) 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 sPLA(2)-IIA as determined by surface plasmon resonance studies. Two potential peptide interaction sites were identified on sPLA(2)-IIA from the modeling studies, one in the NH(2)-terminal helix and the other in the beta-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 sPLA(2)-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.
authors: Arthritis and Inflammation Research Programme, Garvan Institute of Medical Research, University of New South Wales, St. Vincent's Hospital, Sydney, New South Wales 2010, Australia.
authors: W B
authors: WB
authors: Church
authors: A S
authors: AS
authors: Inglis
authors: A
authors: A
authors: Tseng
authors: R
authors: R
authors: Duell
authors: P W
authors: PW
authors: Lei
authors: K J
authors: KJ
authors: Bryant
authors: K F
authors: KF
authors: Scott
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D018360:Crystallography, X-Ray; D015195:Drug Design; D004791:Enzyme Inhibitors; D054501:Group II Phospholipases A2; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D009842:Oligopeptides / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology*; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D010456:Peptides, Cyclic / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D010741:Phospholipases A / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D054467:Phospholipases A2; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry; D013329:Structure-Activity Relationship; D014162:Transfection
chemical_list: D004791:Enzyme Inhibitors; D009842:Oligopeptides; D010446:Peptide Fragments; D010456:Peptides, Cyclic; D011994:Recombinant Proteins; D010741:Phospholipases A; D054501:Group II Phospholipases A2; D054467:Phospholipases A2