New thiol inhibitor of Achromobacter iophagus collagenase

Abstract

New synthetic mercaptotripeptides (HS-CH2-CH2-CO-Pro-Yaa) which inhibit Achromobacter iophagus collagenase were produced in order to obtain more powerful bacterial collagenase inhibitors than currently available, and to investigate the specificity of the S3' subsite of the enzyme.Since similar bindmg constants were found for inhibitors carrying uncharged residues of various sizes in the P3' position (Yaa = Ala, Leu, Phe, Pro, Hyp) steric hindrance at the collagenase S3' appears relatively limited.The compound (HS-CH2-CH2-CO-Pro-Arg), which carries an arginine residue in the position P3' and had the highest inhibition constant of the series tested (Ki = 0.5 bM), proved to be the strongest inhibitor so far reported in the literature. The weakest in the present series was the compound (HS-CH2-CH2-CO-Pro-Asp) which carries an aspartic residue in position P3' and had a Ki = 70 pM.The present work revealed that the charged groups in the P3' position play a key role in the interaction of the inhibitors with the enzyme.The collagenase produced by Achromobacter iophagus (EC 3.4.24.8.) is a zinc protease which cleaves native collagen and synthetic substrates at the Xaa-Gly bond in the Xaa-GlyPro-Yaa sequences [I]. A relationship between the chemical structure of a series of synthetic substrates and their capacity to be hydrolysed was shown in previous kinetic studies [2]. The synthesis of specific collagenase inhibitors should also permit the definition of the structural and dynamic properties of the ligand (i.e. the substrate or inhbitor) endowing it with high affinity for the enzyme and allow better understanding of the specificity of collagenase.The approach of Cushman and Ondetti [3] to design inhibitors of zinc proteases led to the production of strong inhibitors, but similar experiments on bacterial collagenases failed to yield potent inhibitors [4, 51. In previous studies, we showed that a tripeptide is the optimal size for obtaining good collagenase inhibitors [6]. Such inhibitors of the general structure HS-CH2-CH2-CO-Pro-Yaa must carry a modified glycine residue at position P1' and a proline residue at position P2' (nomenclature of Schechter and Berger [7]).A series of mercaptotripeptides substituted in position Yaa were synthesized in an attempt to obtain more potent inhibitors and to elucidate the P3' subsite specificity of this bacterial collagenase. The P3' position of these inhibitors (HS-CH2-CH2-CO-Pro-Yaa) corresponds to the Yaa position in the collagen sequences (Gly-Pro-Yaa),. For this reason, the position P3' was substituted with residues most frequently encountered in the Yaa position in collagen (Hyp, Ala, Arg) [8]. Furthermore, in order to investigate the impact of charge, hydrophobicity and steric hindrance on the affinity of these inhibitors for collagenase, we chose also aspartic, proline, phenylalanine and leucine residues for substitution at P3'.Since in a previous study we observed that the affinity of this enzyme for the free tripeptide inhibitor HS-CH2-CH2-CO-Pro-Ala was 50 times higher th...

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

“…So, in order to rule out this possibility, for these cyclic peptides we have checked that their levels of inhibition, as well as their &on values, are not influenced, under our experimental conditions, by the concentration of the enzyme (e.g., by a catalytic process). Furthermore, for enzyme-inhibitor solutions yielding 50% of inhibition, we have also checked that the level of inhibition observed is the same for different times of enzymeinhibitor preincubation (12,24,36, and 48 h, respectively). In addition to the above arguments, the data reported below also support an inhibition mechanism of the bacterial collagenase by these cyclic peptides, which is independent of an hydrolytic process.…”

Phosphinic peptide analogues as potent inhibitors of Corynebacterium rathayii bacterial collagenase

“…The temperature was maintained by water circulating from a Haake P2 bath. Injection of [5][6][7][8][9][10][11][12][13][14][15] ,ul of enzyme initiated the reaction. Absorbance was continuously measured, digitized and stored in a Macintosh FX computer, equipped with a MacADIOS II Jr data-acquisition system (GW Instruments).…”

“…For several years, our laboratory has been involved in the development of inhibitors of bacterial collagenases, enzymes that also belong to the zinc metalloprotease family [7][8][9][10]. The most intensively studied collagenases have been those from Clostridium histolyticum which hydrolyse collagen, gelatin and peptides containing proline at the Xaa-Gly bond in Xaa-Gly-Pro-Yaa sequences [11,12].…”

Ketone-substrate analogues of Clostridium histolyticum collagenases: tight-binding transition-state analogue inhibitors

“…There have been few reports of synthetic inhibitors of bacterial collagenases (Yagisawa et al, 1965;Siffert & Pasquet, 1977; Galardy & Grobelny, 1983;Yiotakis et al, 1984; Grobelny & Galardy, 1985; Vencill et al, 1985; Yiotakis & Dive, 1986), and most of these have indeed been patterned after inhibitors of other metalloproteinases. For the clostridial collagenases, for example, Vencill and associates (Vencill et al, 1985) have synthesized peptide inhibitors containing hydroxamic acid, carboxymethyl groups, and thiol groups placed so that they could interact with the active site zinc atom, a strategy known to work for other zinc proteinases.…”