Cyclic Peptides with a Phosphinic Bond as Potent Inhibitors of a Zinc Bacterial Collagenase

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-LPhe⌿(PO 2 CH 2 )Gly-Pro, displays a K i 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 S 2 and S 2 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.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). However, to be valuable tools, such inhibitors should also be as selective as possible and not interfere with all of the members of a particular enzyme family. This problem is of particular significance for the inhibitors designed based on enzyme mechanism considerations. 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. For this purpose, we elected to synthesize, by combinatorial synthesis methods, libraries of phosphinic peptides and then screened them with the proteases of interest to find potent and selective inhibitors. Such peptides have been chosen because they were proven to be highly potent inhibitors of several zinc metalloproteases, as expected for good transition state analogues of these enzymes (4 -7). We recently tested this novel strategy for developing potent and specific inhibitors of two closely related mammalian zinc endopeptidases, the endopep...

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“…The L (R configuration) or D (S configuration) stereochemistry of the pseudophenylalanine residue was assigned as described previously (21).…”

Section: Enzyme Assays and Inhibition Studiesmentioning

confidence: 99%

Development of the First Potent and Selective Inhibitor of the Zinc Endopeptidase Neurolysin Using a Systematic Approach Based on Combinatorial Chemistry of Phosphinic Peptides

Jiráček

Yiotakis

Vincent

et al. 1996

Journal of Biological Chemistry

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“…Peptide purities were checked by TLC, analytical HPLC (Vydac 218TP104 column), and mass spectrometry. Attribution of the L (R configuration) or D (S configuration) stereochemistry of the pseudophenylalanine residue was determined according to the procedure previously described (24).…”

Section: Enzyme Assays and Inhibition Studiesmentioning

confidence: 99%

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

Jiráček

Yiotakis

Vincent

et al. 1995

Journal of Biological Chemistry

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113

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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.The endopeptidase 24-15 (EC 3.4.24.15) 1 belongs to the zinc metalloprotease family (1) and resembles a peptidase previously purified from rabbit brain by Camargo et al. (2). Later, endopeptidase 24-15 was named thimet oligopeptidase with respect to the thiol and metal dependence of its catalytic activity (3-5). Molecular cloning of the cDNA of endopeptidase 24-15 revealed a HEXXH motif, which characterizes peptidases belonging to this family, and a cysteine residue, lying on the C-terminal side of the second histidine of the zinc binding motif (6). This cysteine residue has been proposed to be responsible for activation of the enzyme by 2-mercaptoethanol or dithiothreitol, as well as for inhibition of the enzyme activity by thiol reactive reagents (6).24-15 displays several biochemical and physicochemical properties (for a review, see Ref. 7) in common with another zinc-containing metallopeptidase, endopeptidase . Interestingly, these two peptidases have the ability to hydrolyze numerous bioactive or synthetic peptides at the same cleavage site, suggesting that they have a closely related active site (7-9).We previously reported that phosphodiepryl03, a phosphonamide peptide, acts as a potent mixed inhibitor of 24-15 and 24-16, with K i values in the nanomolar range (10). This inhibitor was shown to be unable to block the activity of several other zinc peptidases such as endopeptidase 24-11, angiotensin-converting enzyme, aminopeptidase M, leucine aminopeptidase, and carboxypeptidases A and B. We particularly studied the effect of this inhibitor in vivo on the neurotensin catabolism, since we previously established that 24-15 and 24-16 participated in vitro in the inactivation of this neuropeptide (11,12). We established that phosphodiepryl03 prevented neurotensin degradation in vivo in vascularly perfused dog ileum (13). More recently, several phosphodiepryl03 analogues were also proved to be potent but still acting as mixed inhibitors of

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“…[36] By coordinating to zinc, the oxy-anion of phosphinic acids mimics the unstable tetrahedral transition state of the peptide bond cleavage site, a feature which is crucial to their function as inhibitors. [27,36] The phosphinic moiety has been utilized for the construction of many inhibitors of a variety of enzymes such as MMP-3, [28,35] collagenases, [25,37,38] other zinc metalloproteases, [22, 26, 27, 39±41] HIV-protease, [42,43] other aspartic proteases, [32,44,45] ligases, [23,24,46,47] and yet other enzymes.…”

Section: Introductionmentioning

confidence: 99%

“…[48±50] Phosphinic peptides have been prepared both in solution [27,28,37,44] and more recently on the solid phase. [22,39,40, 51±53] The solid-phase synthesis technique [54] has a number of advantages in the synthesis of sequential oligomeric molecules.…”

Section: Introductionmentioning

confidence: 99%

Phosphinic Peptide Matrix Metalloproteinase-9 Inhibitors by Solid-Phase Synthesis Using a Building Block Approach

et al. 1999

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The solid-phase synthesis of an array of different pseudopeptides containing a phosphinic glycine ± leucine moiety (-GY{P(O)OH-CH 2 }L-) [1] is described. The resulting pseudopeptides were shown to act as matrix metalloproteinase-9 (MMP-9) inhibitors. Starting from available materials, the protected amino acid isosters benzyloxycarbonyl aminomethylphosphinic acid (glycine analogue) and ethyl a-isobutylacrylate (leucine analogue) were synthesized and coupled with the bis(trimethylsilyl)phosphonite. Protective group interchange yielded a protected phosphinic dipeptide building block 1 ready for use in solid-phase peptide synthesis. Solid-phase peptide synthesis was performed with 9-fluorenylmethyloxycarbonyl (Fmoc) chemistry on a polyethylene glycol polyamide (PEGA) support and the coupling of 1 (1.5 equiv) was carried out with standard activation. The P 4 ± P 2 and P 2 ' ± P 4 ' positions of the pseudopeptides were designed by analogy of the cleavage sequences of different natural extracellular matrix protein substrates with a synthetic peptide substrate of MMP-9. The crude peptides were obtained in high yield and purity as determined by RP-HPLC, and were characterized by electrospray mass spectrometry and amino acid analysis after purification. Enzyme kinetic investigations with MMP-9 of the purified peptide inhibitors showed K i values in the range from mm to nm.

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Cyclic Peptides with a Phosphinic Bond as Potent Inhibitors of a Zinc Bacterial Collagenase

Cited by 24 publications

References 12 publications

Development of the First Potent and Selective Inhibitor of the Zinc Endopeptidase Neurolysin Using a Systematic Approach Based on Combinatorial Chemistry of Phosphinic Peptides

Development of the First Potent and Selective Inhibitor of the Zinc Endopeptidase Neurolysin Using a Systematic Approach Based on Combinatorial Chemistry of Phosphinic Peptides

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

Phosphinic Peptide Matrix Metalloproteinase-9 Inhibitors by Solid-Phase Synthesis Using a Building Block Approach

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