Abstract
A new systematic approach, based on combinatorial chemistry of phosphinic peptides, is proposed for rapid development of highly potent and selective inhibitors of zinc metalloproteases. This strategy first evaluates the effects on the inhibitory potency and selectivity of the following 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 identity of the residues that optimized both the potency and the selectivity. We demonstrate the efficiency of this novel approach in rapid identification of the first potent inhibitor of the mammalian zinc endopeptidase neurolysin(24-16), able to discriminate between this enzyme and the related zinc endopeptidase thimet oligopeptidase(24-15). The most potent and selective inhibitor developed in this study, Pro-LPhePsi(PO2CH2)Gly-Pro, displays a Ki value of 4 nM for 24-16 and is 2000 times less potent on 24-15. The specific recognition of such a free phosphinic tetrapeptide by 24-16, as well as the unique specificity of the 24-16 S2 and S2' subsites for proline, unveiled by this study, are discussed in terms of their possible significance for the function of this enzyme and its related zinc endopeptidase activities.
Highlights
Potent enzyme inhibitors have long been recognized as powerful tools for assessing the physiological roles of enzymes and have sometimes led to therapeutic drugs able to modulate their activities in vivo (1)
We demonstrated that the systematic substitution by 20 amino acids of the YaaЈ and ZaaЈ positions in phosphinic peptides of the general formula Z-Phe⌿(PO2CH2)XaaЈYaaЈ-ZaaЈ, with XaaЈ representing Gly or Ala, led to the identification of an extremely potent inhibitor of 24-15 (ZPhe⌿(PO2CH2)Ala-Arg-Met; Ki ϭ 15 pM), a molecule that is 3 orders of magnitude less potent toward 24-16 (8)
Description of the Approach—The novel approach developed in this study was devised in an attempt to analyze the effects on the inhibitory potency and selectivity of the size of the phosphinic peptide, the position of the phosphinic bond in the peptide sequence, and the state of the N- and C-terminal extremities of the peptides
Summary
Potent enzyme inhibitors have long been recognized as powerful tools for assessing the physiological roles of enzymes and have sometimes led to therapeutic drugs able to modulate their activities in vivo (1). This situation applies to the zinc metalloprotease family, for which a general strategy has been to synthesize short peptide sequences containing a particular functional group (thiol, carboxyalkyl, hydroxamate) able to interact with the zinc atom of the active site, while the remaining peptide sequence fits within the enzyme cavity This traditional approach has led to remarkable results in terms of potency (2), but taking into account the rapid expansion of the zinc metalloprotease family (3), we felt that it would be important to devise a more systematic approach for discovering highly potent and selective inhibitors of these enzymes.
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