DEVELOPMENT OF HIGHLY POTENT AND SELECTIVE PHOSPHINIC PEPTIDE INHIBITORS OF ZINC ENDOPEPTIDASE-24-15 USING COMBINATORIAL CHEMISTRY

Several hundred phosphinic peptides having the general formula Z-((L,D ))Phe psi(PO2CH2)((L,D))Xaa'-Yaa'-Zaa', where Xaa' = Gly or Ala and Ya a' and Zaa' represent 20 different amino acids, have been synthesized by the combinatorial chemistry approach. Peptide mixtures or individua l peptides were evaluated for their ability to inhibit the rat brain z inc endopeptidases 24-15 and 24-16. Numerous phosphinic peptides of th is series act as potent (K-i in the nanomolar range) mixed inhibitors of these two peptidases. However, our systematic and comparative strat egy led us to delineate the residues located in P-2' and P-3' position s of the inhibitors that are preferred by these two peptidases. Thus, endopeptidase 24-15 exhibits a marked preference for inhibitors contai ning a basic residue (Arg or Lys) in the P-2' position, while 24-16 pr efers a proline in this position. The P-3' position has less influence on the inhibitory potency and selectivity, both peptidases preferring a hydrophobic residue at this position. On the basis of these observa tions, we have prepared highly potent and selective inhibitors of endo peptidase 24-15. The Z-((L,D))Phe psi(PO2-CH2)((L,D))Ala-Arg-Met compo und (mixture of the four diastereoisomers) displays a K-i value of 70 pM for endopeptidase 24-15. The most selective inhibitor of endopeptid ase 24-15 in this series, Z-((L,D))Phe psi(PO2CH2)((L,D))Ala-Arg-Phe, exhibits a K-i value of 0.160 nM and is more than 3 orders of magnitud e less potent toward endopeptidase 24-16 (K-i = 530 nM). Furthermore, at 1 mu M this selective inhibitor is unable to affect the activity of several other zinc peptidases, namely endopeptidase 24-11, angiotensi n-converting enzyme, aminopeptidase M, leucine aminopeptidase, and car boxypeptidases A and B. Therefore, Z-((L,D))Phe psi(PO2CH2)((L,D))Ala- Arg-Phe can be considered as the most potent and specific inhibitor of endopeptidase 24-15 developed to date, This new inhibitor should be u seful in assessing the contribution of this proteolytic activity in th e physiological inactivation of neuropeptides known to be hydrolyzed, at least in vitro, by endopeptidase 24-15, Our study also demonstrates that the combinatorial chemistry approach leading to the development of phosphinic peptide libraries is a powerful strategy for discovering highly potent and selective inhibitors of zinc metalloproteases and s hould find a broader application in studies of this important class of enzymes.