DEVELOPMENT OF THE FIRST POTENT AND SELECTIVE INHIBITOR OF THE ZINC ENDOPEPTIDASE NEUROLYSIN USING A SYSTEMATIC-APPROACH BASED ON COMBINATORIAL CHEMISTRY OF PHOSPHINIC PEPTIDES

A mew systematic approach, based on combinatorial chemistry of phosphi nic peptides, is proposed for rapid development of highly potent and s elective inhibitors of zinc metalloproteases. This strategy first eval uates the effects on the inhibitory potency and selectivity of the fol lowing parameters: 1) size of the phosphinic peptides, 2) position of the phosphinic bond in the sequence, and 3) the state (free or blocked ) of the peptide extremities. After this selection step, the influence of the inhibitor sequence is analyzed in order to determine the ident ity of the residues that optimized both the potency and the selectivit y, We demonstrate the efficiency of this novel approach in rapid ident ification of the first potent inhibitor of the mammalian zinc endopept idase neurolysin (24-16), able to discriminate between this enzyme and the related zinc endopeptidase thimet oligopeptidase (24-15). The mos t potent and selective inhibitor developed in this study, Pro-LPhe Psi (PO2CH2)Gly-Pro, displays a K-i value of 4 nM for 24-16 and is 2000 ti mes less potent on 24-15. The specific recognition of such a free phos phinic tetrapeptide by 24-16, as well as the unique specificity of the 24-16 S-2 and S-2' subsites for proline, unveiled by this study, are discussed ill terms of their possible significance for the function of this enzyme and its related zinc endopeptidase activities.