Development of Highly Potent and Selective Phosphinic Peptide Inhibitors of Zinc Endopeptidase 24-15 Using Combinatorial Chemistry

Abstract

Several hundred phosphinic peptides having the general formula Z-(L,D)Pheψ(PO2CH2)(L,D)Xaa‘-Yaa‘-Zaa‘, where Xaa‘ = Gly or Ala and Yaa‘ and Zaa‘ represent 20 different amino acids, have been synthesized by the combinatorial chemistry approach. Peptide mixtures or individual peptides were evaluated for their ability to inhibit the rat brain zinc endopeptidases 24-15 and 24-16. Numerous phosphinic peptides of this series act as potent (Ki in the nanomolar range) mixed inhibitors of these two peptidases. However, our systematic and comparative strategy led us to delineate the residues located in P2′ and P3′ positions of the inhibitors that are preferred by these two peptidases. Thus, endopeptidase 24-15 exhibits a marked preference for inhibitors containing a basic residue (Arg or Lys) in the P2′ position, while 24-16 prefers a proline in this position. The P3′ position has less influence on the inhibitory potency and selectivity, both peptidases preferring a hydrophobic residue at this position. On the basis of these observations, we have prepared highly potent and selective inhibitors of endopeptidase 24-15. The Z-(L,D)Pheψ(PO2CH2)(L,D)Ala-Arg-Met compound (mixture of the four diastereoisomers) displays a Ki value of 70 pM for endopeptidase 24-15. The most selective inhibitor of endopeptidase 24-15 in this series, Z-(L,D)Pheψ(PO2CH2)(L,D)Ala-Arg-Phe, exhibits a Ki value of 0.160 nM and is more than 3 orders of magnitude less potent toward endopeptidase 24-16 (Ki = 530 nM). Furthermore, at 1 μM this selective inhibitor is unable to affect the activity of several other zinc peptidases, namely endopeptidase 24-11, angiotensin-converting enzyme, aminopeptidase M, leucine aminopeptidase, and carboxypeptidases A and B. Therefore, Z-(L,D)Pheψ(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 useful in assessing the contribution of this proteolytic activity in the 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 should find a broader application in studies of this important class of enzymes. Several hundred phosphinic peptides having the general formula Z-(L,D)Pheψ(PO2CH2)(L,D)Xaa‘-Yaa‘-Zaa‘, where Xaa‘ = Gly or Ala and Yaa‘ and Zaa‘ represent 20 different amino acids, have been synthesized by the combinatorial chemistry approach. Peptide mixtures or individual peptides were evaluated for their ability to inhibit the rat brain zinc endopeptidases 24-15 and 24-16. Numerous phosphinic peptides of this series act as potent (Ki in the nanomolar range) mixed inhibitors of these two peptidases. However, our systematic and comparative strategy led us to delineate the residues located in P2′ and P3′ positions of the inhibitors that are preferred by these two peptidases. Thus, endopeptidase 24-15 exhibits a marked preference for inhibitors containing a basic residue (Arg or Lys) in the P2′ position, while 24-16 prefers a proline in this position. The P3′ position has less influence on the inhibitory potency and selectivity, both peptidases preferring a hydrophobic residue at this position. On the basis of these observations, we have prepared highly potent and selective inhibitors of endopeptidase 24-15. The Z-(L,D)Pheψ(PO2CH2)(L,D)Ala-Arg-Met compound (mixture of the four diastereoisomers) displays a Ki value of 70 pM for endopeptidase 24-15. The most selective inhibitor of endopeptidase 24-15 in this series, Z-(L,D)Pheψ(PO2CH2)(L,D)Ala-Arg-Phe, exhibits a Ki value of 0.160 nM and is more than 3 orders of magnitude less potent toward endopeptidase 24-16 (Ki = 530 nM). Furthermore, at 1 μM this selective inhibitor is unable to affect the activity of several other zinc peptidases, namely endopeptidase 24-11, angiotensin-converting enzyme, aminopeptidase M, leucine aminopeptidase, and carboxypeptidases A and B. Therefore, Z-(L,D)Pheψ(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 useful in assessing the contribution of this proteolytic activity in the 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 should find a broader application in studies of this important class of enzymes.