Phosphinic peptide analogues as potent inhibitors of Corynebacterium rathayii bacterial collagenase

Abstract

Pseudo-substrate analogues of collagenase from Corynebacterium rathayii, in which the scissile peptide bond is replaced by a phosphinic moiety, were synthesized and evaluated as inhibitors of this enzyme. The phosphinic tetrapeptide, Z-Phe-psi(PO2CH2)-Gly-Pro-Nle (1), was found to be a potent inhibitor of collagenase with a Ki value of 8 nM. Increasing the length of the phosphinic-containing inhibitors from tetra- to hepta-peptide size further improves the potency of these compounds. The heptapeptide analogue, Z-Phe-Gly-Pro-Phe-psi(PO2CH2)-Gly-Pro-Nle-OMe, with a Ki value of 0.6 nM, is the most potent inhibitor reported to date for bacterial collagenases. A comparison between the phosphinic analogue Z-Phe-psi(PO2CH2)-Gly-Pro-Nle (1) and the phosphonamide peptide Z-Phe-psi(PO2NH)-Gly-Pro-Nle (2) shows that for bacterial collagenase the replacement of a CH2 by an NH group results only in a modest increase in affinity from Ki = 8 nM for compound 1 to Ki = 6 nM for compound 2. Most of the phosphorus-containing inhibitors of this series are slow- or slow-tight-binding inhibitors with second-order rate constants for association and dissociation varying respectively for the kon values from 1 x 10(3) to 26 x 10(3) M-1.s-1 and for the koff values from 3 x 10(-4) to 2 x 10(-5) s-1. Interestingly, the lower affinity of the molecule containing a D residue in the P1 position of the inhibitor, compared with the molecule with an L residue in this position, is mainly the consequence of a lower rate constant for association of these D stereoisomers with the enzyme. This study demonstrates that phosphinic peptide analogues are potent inhibitors of a bacterial collagenase. The development of new phosphinic peptides should lead to the discovery of potent inhibitors of other zinc metalloproteases. Details of how the analogues were synthesized are given in Supplementary Publication SUP 50176 (14 pages), which has been deposited with the British Library Document Supply Centre, Boston Spa, Wetherby, W. Yorkshire LS23 7BQ, from whom copies can be obtained on the terms indicated in Biochem. J. (1994) 297, 9.

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

“…All these phosphinic peptides, as good analogues of the substrates of zinc metalloproteases in the transition state, are expected to be potent inhibitors of this enzyme family. In fact, as demonstrated in this study, but also in previous work (15), phosphinic peptides provided that they contain the right amino acid sequence, which ensures an optimal recognition of these inhibitors by the target zinc metalloprotease, are highly potent inhibitors of this class of proteases. In this work, we have identified a phosphinic sample (Z-(L,D) Phe(PO 2 CH 2 ) (L,D) AlaArg-Met, a mixture of four distereoisomers) displaying a K i value of 70 pM.…”

“…The side chain protecting groups used were Asp(tBu), Glu(tBu), Ser(tBu), Thr(tBu), Tyr(tBu), Lys(Boc), His(Trt), Asn(Trt), Gln(Trt), and Arg(Pmc). The phosphinic pseudodipeptides Z-(L,D) Phe(PO 2 -CH 2 )GlyOH and Z-(L,D) Phe(PO 2 -CH 2 ) (L,D) AlaOH were prepared as previously described (15). The protection of the hydroxyphosphinyl group by the adamantyl group will be published elsewhere.…”

“…Peptides containing a phosphinic bond (PO 2 CH 2 ) instead of a phosphonamide bond (PO 2 NH) were selected because the former are more chemically stable than the latter. Furthermore, with bacterial collagenase, a zinc metalloprotease, we recently demonstrated that the phosphinic peptide inhibitors have nearly the same potency as the corresponding parent phosphonamide peptide inhibitors (15).…”

Phosphorus‐containing peptides as mixed inhibitors of endopeptidase 3.4.24.15 and 3.4.24.16: effect on neurotensin degradation in vitro and in vivo

“…The longitudinal smooth muscle (including the myenteric plexus) was dissected out from whole ileum according to Paton & Zar (1968) and strips 1.5-2 cm in length were set up for isometric tension recording in 3 ml organ baths containing modified Tyrode solution (mM: NaCl 136.8, KCl 2.7, MgSO4 1, NaH2PO4 0.4, NaHCO3 11.9, CaCl2 2.5, glucose 5.5) to which was added 100 nM neostygmine methylsulphate. The The synthesis of phosphorus-containing peptides has been described previously (Dive et al, 1990;Yiotakis et al, 1994).…”

RXP 407, a phosphinic peptide, is a potent inhibitor of angiotensin I converting enzyme able to differentiate between its two active sites

“…To this end, we screened different phosphinic peptide libraries, using a systematic approach previously described (14,15). Such phosphinic peptides were chosen because they are highly potent inhibitors of several zinc-metallopeptidases (14)(15)(16)(17)(18) and are good analogues of the substrate in the transition-state for this class of enzyme (19). They were screened on wild-type ACE and ACE mutants in which either the N-or the C-domain of ACE had been inactivated, as reported before (3).…”