Inhibition of Collagenase by Mycosporine-like Amino Acids from Marine Sources.

Matrix metalloproteinases play an important role in extracellular matrix remodeling. Excessive activity of these enzymes can be induced by UV light and leads to skin damage, a process known as photoaging. In this study, we investigated the collagenase inhibition potential of mycosporine-like amino acids, compounds that have been isolated from marine organisms and are known photoprotectants against UV-A and UV-B. For this purpose, the commonly used collagenase assay was optimized and for the first time validated in terms of relationships between enzyme-substrate concentrations, temperature, incubation time, and enzyme stability. Three compounds were isolated from the marine red algae Porphyra sp. and Palmaria palmata, and evaluated for their inhibitory properties against Chlostridium histolyticum collagenase. A dose-dependent, but very moderate, inhibition was observed for all substances and IC50 values of 104.0 µM for shinorine, 105.9 µM for porphyra, and 158.9 µM for palythine were determined. Additionally, computer-aided docking models suggested that the mycosporine-like amino acids binding to the active site of the enzyme is a competitive inhibition.

Engineering D-Amino Acid Containing Collagen Like Peptide at the Cleavage Site of Clostridium histolyticum Collagenase for Its Inhibition.

Collagenase is an important enzyme which plays an important role in degradation of collagen in wound healing, cancer metastasis and even in embryonic development. However, the mechanism of this degradation has not yet been completely understood. In the field of biomedical and protein engineering, the design and development of new peptide based materials is of main concern. In the present work an attempt has been made to study the effect of DAla in collagen like peptide (imino-poor region of type I collagen) on the structure and stability of peptide against enzyme hydrolysis. Effect of replacement of DAla in the collagen like peptide has been studied using circular dichroic spectroscopy (CD). Our findings suggest that, DAla substitution leads to conformational changes in the secondary structure and favours the formation of polyproline II conformation than its L-counterpart in the imino-poor region of collagen like peptides. Change in the chirality of alanine at the cleavage site of collagenase in the imino-poor region inhibits collagenolytic activity. This may find application in design of peptides and peptidomimics for enzyme-substrate interaction, specifically with reference to collagen and other extra cellular matrix proteins.

The effect of mouthwashes containing biguanides on the progression of erosion in dentin.

BACKGROUND: Dental erosion is caused by frequent exposure to acids without the involvement of microorganism. This study analyzed the effect of biguanides (polyhexamethylene biguanide - PHMB and chlorhexidine - CHX) on dentin erosion due to their possible influence on the enzymatic degradation of the demineralized organic matrix. METHOD: Sixty bovine dentin specimens were prepared. On both sides of their surface, nail varnish was applied to maintain the reference surfaces for the determination of dentin loss. Samples were cyclically de- and remineralized for 6 days. Demineralization was performed with a 0.87 M citric acid solution (6×5 min daily). Thereafter, samples were treated with distilled water (negative control), 0.12% CHX (positive control), 0.07% PHMB, Sanifill Perio Premium™ (0.07% PHMB plus 0.05% NaF), or F solution (0.05% NaF) for 1 min and then subjected to enzymatic challenge for 10 min using a bacterial collagenase (Clostridium hystoliticum, 100 µg/ml). Dentin loss was assessed using profilometry (µm) daily. Data were analyzed using 2-way repeated measures-ANOVA and Bonferroni's test (p < 0.05). RESULTS: Dentin loss progressed significantly for all groups during the 6 days. After the 3rd day, Sanifill Premium™, CHX, and PHMB significantly reduced dentin erosion compared to control. On the 6th day, the lowest mean (±SD) dentin loss was observed for Sanifill Perio Premium™ (94.4 ± 3.9 µm). PHMB and CHX led to intermediate dentin loss (129.9 ± 41.2 and 135.3 ± 33.5 µm, respectively) that was significantly lower than those found for negative control (168.2 ± 6.2 µm). F (157.4 ± 6.1 µm) did not significantly differ from negative control. CONCLUSIONS: Sanifill Perio Premium™ mouthwash has a good potential to reduce dentin loss, which might be associated with the presence of PHMB.

Protective effect of Withania somnifera and Cardiospermum halicacabum extracts against collagenolytic degradation of collagen.

The irreversible destruction of extracellular matrix (ECM) such as cartilage, tendon, and bone that comprise synovial joints is the hallmark of both rheumatoid arthritis and osteoarthritis by over-expression of matrix metalloproteinase (MMP)-collagenases. We report herein the detailed study on the inhibitory effects of Withania somnifera extract (WSE) and Cardiospermum halicacabum extract (CHE) on Clostridium histolyticum collagenase (ChC) activity against the degradation of the ECM component of bovine Achilles tendon type I collagen by hydroxyproline assay method. Interaction of WSE and CHE with ChC exhibited 71% and 88% inhibition, respectively, to the collagenolytic activity of ChC against collagen degradation, and the inhibition was found to be concentration-dependent. The inhibition kinetics of ChC by both the extracts has been deduced from the extent of hydrolysis of N-[3-(2-furyl) acryloyl]-Leu-Gly-Pro-Ala. Both WSE and CHE are provided competitive and mixed type inhibition on ChC activity, respectively. Circular dichroism studies of ChC on treatment with WSE and CHE revealed changes in the secondary structure of collagenase. These results suggest that the WSE and CHE facilitated collagen stabilization through collagenase inhibition.

QSAR analysis of some 5-amino-2-mercapto-1,3,4-thiadiazole based inhibitors of matrix metalloproteinases and bacterial collagenase.

A quantitative structure-activity relationship (QSAR) study has been performed on 5-amino-2-mercapto-1,3,4-thiadiazole based inhibitors of matrix metalloproteinases (MMPs) and a bacterial collagenase known as Clostridium histolyticum collagenase (ChC) to understand the structural features influencing the affinity of these inhibitors towards the enzyme. The compounds in the selected series were characterized by topological and fragmental descriptors calculated using QuaSAR module of molecular operating environment (MOE). An indicator variable was also assigned to account for the presence of amide function in vicinity of sulfonamide group in the parent structure. Correlations between different inhibitory activities and calculated predictor variables were established through stepwise multiple regression employing the method of least squares. The results of the study indicates that MMP inhibitory activity of 5-amino-2-mercapto-1,3,4-thiadiazoles can be successfully explained in terms of topology of the molecule. The obtained correlations also suggest that increase in the number of fluorine atoms in the aromatic ring will augment inhibitory activity of these molecules against all the MMPs probably by virtue of hydrogen bond interaction with some complementary groups in the active site of the enzymes. One prime requirement for better inhibition of MMPs (except for MMP-1) and ChC identified from the present study is the presence of amide function in vicinity of sulfonamide group in the parent structure as suggested by the presence of indicator variable in almost all correlations. While MMP-1 and ChC inhibitory activity of the compounds studied is shown to be dependent on Kier's first order carbon valence molecular connectivity index indicating that increase in branching and presence of heteroatoms in the molecule will improve the MMP-1 and ChC inhibitory potency of 5-amino-2-mercapto-1,3,4-thiadiazoles, correlations derived for other enzymes (MMP-2, MMP-8, MMP-9) are quite similar. In addition to the number of fluorine atoms and presence of indicator variable, MMP-2, MMP-8 and MMP-9 inhibitory activity of 5-amino-2-mercapto-1,3,4-thiadiazoles is found to be dependent on Kier's alpha modified index of third order in such a way that infer, terminally branched functions will increase the affinity of these molecules to the MMPs.

Quantum theoretic QSAR of benzene derivatives: some enzyme inhibitors.

Our previously developed approach to the development of QSAR equations for benzene derivatives, originally for phenylalkylamine hallucinogens, has been applied to four new systems: sulfonamide inhibitors of the enzymes carbonic anhydrase, thrombin, trypsin, and Clostridium histolyticum collagenase. The novel features involve the energies and nodal orientations of pi-like orbitals, and an allowance for the symmetry of the benzene nucleus. The resulting equations give better fits, better predictivity and are more easily interpretable than those resulting from traditional QSAR methods.

Inhibition effects of (+)-catechin-aldehyde polycondensates on proteinases causing proteolytic degradation of extracellular matrix.

Inhibition effects of (+)-catechin-aldehyde polycondensates against the activity of proteinases, Clostridium histolyticum collagenase (ChC) and human neutrophil elastase (HNE) causing proteolytic degradation of extracellular matrix (ECM), have been investigated. In normal tissues, a balance is reached between the formation and destruction of ECM, leading to a state of homeostasis. However, uncontrolled destruction of ECM contributes to tumor invasion and metastasis. In the measurement of the inhibition activity on ChC and HNE, the polycondensates exhibited superior effects compared to the catechin monomer. Kinetic assays of ChC and HNE inhibition by the polycondensate clearly showed a mixed-type inhibition. These data demonstrate that the polycondensates are a new class of proteinase inhibitors useful for a potent therapeutic agent.

A quantitative structure-activity relationship study on Clostridium histolyticum collagenase inhibitors: roles of electrotopological state indices.

A quantitative structure-activity relationship (QSAR) study has been made on eight different series of Clostridium histolyticum collegenase (ChC) inhibitors. These series are comprised of four different groups of sulfonylated amino acids and their corresponding hydroxamates. In each series, the inhibition potency of the compounds has been found to be significantly correlated with the electrotopological state (E-state) indices of nitrogen and sulfur atoms of the sulfonylated amino group in the molecules, showing the importance of the electronic characterstics of these atoms in controlling the inhibition potency of the compounds.

Protease inhibitors: synthesis of bacterial collagenase and matrix metalloproteinase inhibitors incorporating arylsulfonylureido and 5-dibenzo-suberenyl/suberyl moieties.

Novel matrix metalloproteinase (MMP)/bacterial collagenase inhibitors are reported, considering the sulfonylated amino acid hydroxamates as lead molecules. A series of compounds was prepared by reaction of arylsulfonyl isocyanates with N-(5H-dibenzo[a,d]cyclohepten-5-yl)- and N-(10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5-yl) methyl glycocolate, respectively, followed by the conversion of the COOMe to the carboxylate/hydroxamate moieties. The corresponding derivatives with methylene and ethylene spacers between the polycyclic moiety and the amino acid functionality were also obtained by related synthetic strategies. These new compounds were assayed as inhibitors of MMP-1, MMP-2, MMP-8 and MMP-9, and of the collagenase isolated from Clostridium histolyticum (ChC). Some of the new derivatives reported here proved to be powerful inhibitors of the four MMPs mentioned above and of ChC, with activities in the low nanomolar range for some of the target enzymes, depending on the substitution pattern at the sulfonylureido moiety and on the length of the spacer through which the dibenzosuberenyl/suberyl group is connected with the rest of the molecule. Several of these inhibitors also showed selectivity for the deep pocket enzymes (MMP-2, MMP-8 and MMP-9) over the shallow pocket ones MMP-1 and ChC.

Inhibition of collagenase breakdown of equine corneas by tetanus antitoxin, equine serum and acetylcysteine.

OBJECTIVE: To determine whether tetanus antitoxin, equine serum, and acetylcysteine, which are currently used in the treatment of equine corneal ulcer, inhibit the digestion of equine corneal collagen when exposed to collagenase in vitro. ANIMALS STUDIED: Corneas from 40 adult horses. PROCEDURES: Sections of equine corneas were incubated with saline, a solution of bacterial collagenase in saline, bacterial collagenase in saline plus equine tetanus antitoxin, bacterial collagenase in saline plus equine serum, or bacterial collagenase in saline plus acetylcysteine. Each one of the collagenase inhibitors was tested at different concentrations. The degree of corneal collagen digestion was determined by concentrations of hydroxyproline released into the incubation media and/or by weight loss of the cornea. RESULTS: Corneas exposed to collagenase released a significant (0.05 level) large amount of hydroxyproline (43.1 +/- 2.3 microg/mL/100 mg cornea/5 h) and decreased cornea weight by up to 89%. Blood serum (200 microL/mL), purified albumin or globulin fractions of serum, tetanus antitoxin (120 units/mL), and acetylcysteine (20 mg/mL) when used at the highest concentrations blocked collagenase digestive activity by approximately 50%. Dilution of inhibitors decreased corneal protection and linearly increased corneal weight loss. Purified equine serum albumin and globulin fractions were equally effective in protecting corneas. CONCLUSIONS: This experiment indicates that tetanus antitoxin, serum and acetylcysteine equally protected corneas from collagenase digestion, in vitro. However, a clinical trial is needed to establish relative therapeutic value.

A quantitative structure-activity relationship study on some matrix metalloproteinase and collagenase inhibitors.

A quantitative structure-activity relationship (QSAR) study is made on some hydroxamic acid-based inhibitors of matrix metalloproteinases (MMPs) and a bacterial collagenase, namely Clostridium histolyticum collagenase (ChC), that also belongs to an MMP family, M-31, using Kier's valence molecular connectivity index (1)chi(v) of the substituents and electrotopological state (E-state) indices of some atoms. The results indicate that out of the four MMPs (MMP-1, MMP-2, MMP-8, and MMP-9) studied, MMP-2 and MMP-9 can be structurally quite similar, but widely differing from MMP-1 and MMP-8 and ChC. For MMP-2 and MMP-9, the inhibition activity of compounds is shown to depend on both (1)chi(v )and E-state indices, while for MMP-1 and MMP-8 it is shown to depend only on E-state indices and for ChC only on (1)chi(v). However, in all the cases, an aromatic group like C(6)F(5) or 3-CF(3)-C(6)H(4) attached to SO(2) moiety in the compounds is indicated to be equally beneficial, due to probably the involvement of fluorine atom(s) in charge-charge interactions with the Zn(2+) ion of the enzymes or in the formation of the hydrogen bonds with some sites of the receptors.

Free fatty acids inhibit the activity of Clostridium histolyticum collagenase and human neutrophil elastase.

We investigated the ability of free fatty acids to inhibit the activity of Clostridium histolyticum collagenase (EC 3.4.24.3) and human neutrophil elastase (EC 3.4.21.37). We determined the activity of collagenase by degradation of resorufin-labeled casein fluorimetrically. The determination of the elastase activity was performed by a spectrophotometric method using a 4-nitroanilide peptide substrate. We found that most of the tested fatty acids inhibited collagenase at concentrations between 50 microM and 500 microM. For elastase we found an inhibition of the activity at concentrations between 500 nM and 50 microM. The most potent inhibitory fatty acids of both enzymes differed. Thus, as a result for collagenase we can assume that the saturated fatty acids with C(16)-C(19) were the most potent ones. For elastase the inhibition rate of unsaturated acids was much higher than the rate of the saturated ones. The highly active erucic acid with an IC(50) value of 450 nM (elastase) is remarkable.

Protease inhibitors: synthesis of matrix metalloproteinase and bacterial collagenase inhibitors incorporating 5-amino-2-mercapto-1,3,4-thiadiazole zinc binding functions.

Matrix metalloproteinase (MMP)/bacterial collagenase inhibitors incorporating 5-amino-2-mercapto-1,3,4-thiadiazole zinc binding functions are reported. A series of compounds was prepared by reaction of arylsulfonyl isocyanates or arylsulfonyl halides with phenylalanyl-alanine, followed by coupling with 5-amino-2-mercapto-1,3,4-thiadiazole in the presence of carbodiimides. These new compounds were assayed as inhibitors of human MMP-1, MMP-2, MMP-8 and MMP-9, and of the collagenase isolated from the anaerobe Clostridium histolyticum (ChC). The new derivatives proved to be powerful inhibitors of these metalloproteases, with activities in the low micromolar range for some of the target enzymes, depending on the substitution pattern at the arylsulfonyl(ureido) moieties.

Protease inhibitors: synthesis of a series of bacterial collagenase inhibitors of the sulfonyl amino acyl hydroxamate type.

A series of sulfonyl amino acyl hydroxamates incorporating alkyl/arylsulfonyl-N-2-nitrobenzyl-L-alanine was prepared. Related compounds were obtained by reaction of N-2-nitrobenzyl-L-Ala with aryl isocyanates, arylsulfonyl isocyanates, or benzoyl isothiocyanate, followed by the conversion of the COOH into the CONHOH moiety. The new compounds were assayed as inhibitors of the Clostridium histolyticum collagenase (ChC), a bacterial protease involved in the degradation of extracellular matrix. Many of the obtained hydroxamates proved to be effective bacterial collagenase inhibitors, the main contributor to activity being the substitution pattern at the sulfonamido moiety. The best ChC inhibitors were those containing pentafluorophenylsulfonyl and 3- and 4-protected-aminophenylsulfonyl P(1)(') groups among others, with affinities in the low nanomolar range. This study also proves that the 2-nitrobenzyl- moiety, similarly to the 4-nitrobenyl one previously investigated (Scozzafava, A.; Supuran, C. T. J. Med. Chem. 2000, 43, 1858-1865) is an efficient P(2)(') anchoring moiety for obtaining potent bacterial collagenase inhibitors.

Protease inhibitors: Synthesis of L-alanine hydroxamate sulfonylated derivatives as inhibitors of clostridium histolyticum collagenase.

L-alanine hydroxamate derivatives were obtained by reaction of alkyl/arylsulfonyl halides with L-alanine, followed by treatment with benzyl chloride, and conversion of the COOH moiety to the CONHOH group with hydroxylamine in the presence of carbodiimides. Other derivatives were obtained by reaction of N-benzyl-alanine with aryl isocyanates, arylsulfonyl isocyanates or benzoyl isothiocyanate, followed by a similar conversion of the COOH to the CONHOH moiety. The obtained compounds were assayed as inhibitors of Clostridium histolyticum collagenase, ChC (EC 3.4.24.3), a zinc enzyme which degrades triple helical collagen. The hydroxamate derivatives were generally 100-500 times more active than the corresponding carboxylates. In the series of synthesized derivatives, substitution patterns leading to the most potent ChC inhibitors were those involving perfluoroalkylsulfonyl- and substituted-arylsulfonyl moieties, such as pentafluorophenylsulfonyl, 3- and 4-protected-aminophenylsulfonyl-, 3- and 4-carboxy-phenylsulfonyl-, 3-trifluoromethyl-phenylsulfonyl-, or 1- and 2-naphthylsulfonyl among others. Similarly to the matrix metalloproteinase (MMP) hydroxamate inhibitors, ChC inhibitors of the type reported here must incorporate hydrophobic moieties at the P(2') and P(3') sites, in order to achieve tight binding to the enzyme.

Protease inhibitors: synthesis of potent bacterial collagenase and matrix metalloproteinase inhibitors incorporating N-4-nitrobenzylsulfonylglycine hydroxamate moieties.

A series of compounds was prepared by reaction of alkyl/arylsulfonyl halides with N-4-nitrobenzylglycine, followed by conversion of the COOH to the CONHOH group, with hydroxylamine in the presence of carbodiimides. Other structurally related compounds were obtained by reaction of N-4-nitrobenzylglycine with aryl isocyanates, arylsulfonyl isocyanates, or benzoyl isothiocyanate, followed by the similar conversion of the COOH into the CONHOH moiety. Another subseries of derivatives was prepared from sulfanilyl- or metanilyl-4-nitrobenzylglycine by reaction with arylsulfonyl isocyanates, followed by conversion of the COOH to the hydroxamate moiety. The new compounds were assayed as inhibitors of four matrix metalloproteinases (MMPs), MMP-1, MMP-2, MMP-8, and MMP-9, and of the Clostridium histolyticum collagenase (ChC). Some of the prepared hydroxamate derivatives proved to be very effective collagenase/gelatinase inhibitors, depending on the substitution pattern at the sulfonamido moiety. Substitutions leading to best inhibitors of MMP-1, a short pocket enzyme, were those involving pentafluorophenylsulfonyl or 3-trifluoromethylphenylsulfonyl moieties at P(1') (K(I)'s of 3-5 nM). For MMP-2, MMP-8, and MMP-9 (deep-pocket enzymes), best inhibitors were especially those containing long perfluoroalkylsulfonyl and substituted-arylsulfonyl moieties, such as pentafluorophenylsulfonyl, 3- and 4-protected-aminophenylsulfonyl, 3- and 4-carboxyphenylsulfonyl, arylsulfonylureido, or arylsulfonylureidosulfanilyl/metanilyl moieties, at P(1'). Bulkier groups in this position, such as 1- and 2-naphthyl, substituted-naphthyl, or quinolin-8-yl moieties among others, led to less effective MMP/ChC inhibitors. Best ChC inhibitors were again those containing pentafluorophenylsulfonyl or 3- and 4-protected-aminophenylsulfonyl P(1') anchoring groups, suggesting that this protease is also a short-pocket wider-neck one (more similar to MMP-1). This study also proves that the 4-nitrobenzyl moiety is an efficient P(2') anchoring moiety and that sulfonylureido, ureido, or carboxythioureido substitutions at P(1') are also tolerated for obtaining potent sulfonylated amino acid hydroxamate-like MMP/ChC inhibitors.

Protease inhibitors: synthesis of clostridium histolyticum collagenase inhibitors incorporating sulfonyl-L-alanine hydroxamate moieties.

A series of hydroxamates was obtained by the reaction of N-(4-nitrobenzyl)-L-alanine with alkyl/arylsulfonyl halides, followed by conversion of the COOH group into CONHOH. Structurally-related compounds were prepared similarly by using arylsulfonyl isocyanates, aryl isocyanates or arylsulfenyl halides instead of the sulfonyl halides. Many of the new compounds showed nanomolar affinity for the bacterial collagenase isolated from the pathogen Clostridium histolyticum.

Protease inhibitors - part 5. Alkyl/arylsulfonyl- and arylsulfonylureido-/arylureido- glycine hydroxamate inhibitors of Clostridium histolyticum collagenase.

Reaction of alkyl/arylsulfonyl halides with glycine afforded a series of derivatives which were first N-benzylated by treatment with benzyl chloride, and then converted to the corresponding hydroxamic acids with hydroxylamine in the presence of carbodiimide derivatives. Other derivatives were obtained by reaction of N-benzyl-glycine with aryl isocyanates, arylsulfonyl isocyanates or benzoyl isothiocyanate, followed by conversion of their COOH group into the CONHOH moiety, as mentioned above. The 90 new compounds reported here were assayed as inhibitors of the Clostridium histolyticum collagenase (EC 3.4.24.3), a zinc enzyme which degrades triple helical regions of native collagen. The prepared hydroxamate derivatives were generally 100-500 times more active than the corresponding carboxylates. In the series of synthesized hydroxamates, substitution patterns leading to the best inhibitors were those involving perfluoroalkylsulfonyl- and substituted-arylsulfonyl moieties, such as pentafluorophenylsulfonyl, 3- and 4-carboxyphenylsulfonyl-, 3-trifluoromethyl-phenylsulfonyl or 1- and 2-naphthyl among others. Thus, it seems that similarly to the matrix metalloproteinase (MMP) hydroxamate inhibitors, Clostridium histolyticum collagenase inhibitors should incorporate hydrophobic moieties at the P(1') and P(2') sites, whereas the alpha-carbon substituent may be a small and compact moiety (such as H, for the Gly derivatives reported here). Such compounds might lead to the design of collagenase inhibitor-based drugs useful as anti-cancer, anti-arthritis or anti-bacterial agents for the treatment of corneal keratitis.

Protease inhibitors. Part 8: synthesis of potent Clostridium histolyticum collagenase inhibitors incorporating sulfonylated L-alanine hydroxamate moieties.

A series of hydroxamates was prepared by reaction of alkyl/arylsulfonyl halides with N-2-chlorobenzyl-L-alanine, followed by conversion of the COOH moiety to the CONHOH group, with hydroxylamine in the presence of carbodiimides. Other structurally related compounds were obtained by reaction of N-2-chlorobenzyl-L-alanine with aryl isocyanates, arylsulfonyl isocyanates or benzoyl isothiocyanate, followed by the similar conversion of the COOH into the CONHOH moiety. The new compounds were assayed as inhibitors of the Clostridium histolyticum collagenase, ChC (EC 3.4.24.3), a bacterial zinc metallo-peptidase which degrades triple helical collagen as well as a large number of synthetic peptides. The prepared hydroxamate derivatives proved to be 100-500 times more active collagenase inhibitors than the corresponding carboxylates. Substitution patterns leading to best ChC inhibitors (both for carboxylates as well as for the hydroxamates) were those involving perfluoroalkylsulfonyl- and substituted-arylsulfonyl moieties, such as pentafluorophenylsulfonyl; 3- and 4-protected-aminophenylsulfonyl-; 3- and 4-carboxyphenylsulfonyl-; 3-trifluoromethyl-phenylsulfonyl; as well as 1- and 2-naphthyl-, quinoline-8-yl- or substituted-arylsulfonylamidocarboxyl moieties among others. Similarly to the matrix metalloproteinase (MMP) hydroxamate inhibitors, ChC inhibitors of the type reported here must incorporate hydrophobic moieties at the P2' and P3' sites, in order to achieve tight binding to the enzyme. This study also proves that the 2-chlorobenzyl moiety, investigated here for the first time, is an efficient P2' anchoring moiety for obtaining potent ChC inhibitors.

Protease inhibitors. Part 7. Inhibition of Clostridium histolyticum collagenase with sulfonylated derivatives of L-valine hydroxamate.

Sulfonylated L-valine hydroxamate derivatives were obtained by reaction of alkyl/arylsulfonyl halides with the title amino acid, followed by treatment with benzyl chloride, and conversion of the COOH moiety to the CONHOH group. Other derivatives were obtained by reaction of N-benzyl-L-valine with arylisocyanates, arylsulfonylisocyanates or benzoylisothiocyanate, followed by the similar conversion of the COOH into the CONHOH moiety, with hydroxylamine in the presence of carbodiimides. The obtained compounds were assayed as inhibitors of the Clostridium histolyticum collagenase, ChC (EC 3.4.24.3), a zinc enzyme which degrades triple helical collagen. The hydroxamate derivatives were generally 100-500 times more active than the corresponding carboxylates. In the series of synthesized derivatives, substitution patterns leading to best ChC inhibitors were those involving perfluoroalkylsulfonyl- and substituted-arylsulfonyl moieties, such as pentafluorophenylsulfonyl; 3- and 4-protected-aminophenylsulfonyl-; 3- and 4-carboxyphenylsulfonyl-; 3-trifluoromethylphenylsulfonyl; or 1- and 2-naphthyl among others. Similarly to the matrix metalloproteinase hydroxamate inhibitors, ChC inhibitors of the type reported here must incorporate hydrophobic moieties at the P(2') and P(3') subsites, in order to achieve tight binding to the enzyme. Such compounds might lead to drugs useful in the treatment corneal bacterial keratitis.