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.     

A continuous spectrophotometric assay for Clostridium histolyticum collagenase

A continuously recording spectrophotometric assay has been developed for Clostridium histolyticum collagenase based on the hydrolysis of 2-furanacryloyl-l-leucylglycyl-l-prolyl-l-alanine (FALGPA). The hydrolysis of this peptide by collagenase obeys Michaelis-Menten kinetics with V = 1.8 × 10⁵μkatal/kg and K_m = 0.5 mm. FALGPA is hydrolyzed more rapidly by collagenase than any other commonly used synthetic substrate, but is not cleaved by any of the well-known proteinases such as trypsin, thermolysin, or elastase. The assay itself is rapid, convenient, and sensitive, and should greatly facilitate detailed kinetic studies of collagenase.     

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.     

A quantitative structure-activity relationship study on some series of anthranilic acid-based matrix metalloproteinase inhibitors

A quantitative structure-activity relationship (QSAR) study has been made on four different series of anthranilic acid-based matrix metalloproteinase (MMP) inhibitors, in which two substituted aryl rings, one bearing the hydroxamic acid moiety that binds with the zinc atom of MMPs, are joined through a bridge group of sulfonamide. The QSAR results indicate that the sulfonamide group plays a very important role in the inhibition activity of the inhibitors and that the effectiveness of this sulfonamide group can be increased by the presence at the aryl rings or at the sulfonamide nitrogen itself of nitrogen-containing or some such substituents that can increase the electronic character of the sulfonamide group. The hydrophobic character of the molecules is not found to be of any advantage; rather in most of the cases it is shown to have detrimental effect, suggesting that MMPs provide little opportunity to the inhibitors to have a any hydrophobic interactions with them. On the other hand, polarizability of the molecules has been found to be conducive to activity in some cases. Thus the inhibition mechanism seems to predominantly involve the electronic interactions between the inhibitors and the enzymes.     

New thiol inhibitors of Clostridium histolyticum collagenase. Importance of the P3' position

An extensive series of synthetic mercaptotripeptides (HS-CH2-CH2-CO-Pro-Yaa) was prepared, and the inhibitory constants were determined on the Clostridium histolyticum collagenase. Among the factors which control the optimal binding of these inhibitors, we found that the presence of a free C-terminal carboxylate group in the position P3' of the compounds is of primary importance. In general, the esterification of this carboxylate group decreased the potency of the inhibitors by two orders of magnitude. We observed also that the enzyme favored the inhibitors having a long linear apolar or basic side-chain at the position P3'. The present data suggest a large S3' subsite of the C. histolyticum collagenase. The compound which contains a homoarginine residue at the P3' position with a Ki of 0.2 microM proved to be the most potent synthetic inhibitor known to date for the C. histolyticum collagenase.     

A quantitative structure-activity relationship study on matrix metalloproteinase inhibitors: Piperidine sulfonamide aryl hydroxamic acid analogs

A quantitative structure-activity relationship (QSAR) study has been made on a series of piperidine sulfonamide aryl hydroxamic acid analogs acting as matrix metalloproteinase (MMP) inhibitors. The inhibitory potencies of the compounds against two MMPs, MMP-2 and MMP-13, are found to be significantly correlated with the hydrophobic properties of the molecules, suggesting that in both enzymes the hydrophobic interaction is playing a dominant role.     

A quantitative structure-activity relationship study on matrix metalloproteinase inhibitors: piperidine sulfonamide aryl hydroxamic acid analogs

A quantitative structure-activity relationship (QSAR) study has been made on a series of piperidine sulfonamide aryl hydroxamic acid analogs acting as matrix metalloproteinase (MMP) inhibitors. The inhibitory potencies of the compounds against two MMPs, MMP-2 and MMP-13, are found to be significantly correlated with the hydrophobic properties of the molecules, suggesting that in both enzymes the hydrophobic interaction is playing a dominant role.     

Anacollagenase by the action of amitriptyline on Clostridium histolyticum collagenase]

Intraperitoneal injection of a mixture of collagenase (300 U) and amitriptyline (Laroxyl*, 3 mg) induce no lesions in contrast with the severe effects of collagenase alone. Also, a complete resistance to intraperitoneal collagenase injection is observed when preceded by 3 intramuscular injections of the same mixture (associated with Freund's incomplete adjuvant). This is due to the development of collagenase antibodies, as demonstrated by nephelometry and immunodiffusion. These facts show that amitriptyline neutralizes the enzymatic properties of collagenase, without alterring its antigenicity. We propose to call this new substance anacollagenase. Such a phenomenon has never been observed with a drug. However we got identical results with other tricyclic depressants (clomipramine, imipramine, doxepine, iprindole). The mechanism of the transformation of collagenase into anacollagenase is not yet explained.     

Inhibitory action of methysergide bimaleate on the collagenase of Clostridium histolyticum

Inhibitory effects of methysergide bimaleate on the collagenase of the Clostridium histolyticum have been established. Having previously shown the inhibitory action of serotonin on this bacterial collagenase, the authors have tested methysergide bimaleate as another inhibitor. After injection in the peritoneum of the rats of methysergide bimaleate and collagenase together, lesions are minimal or absent, in contrast with the dramatic effects of collagenase alone. This shows the antagonist role of methysergide bimaleate in regard to collagenase and suggest that methysergide bimaleate reduce the collagenolysis and may elucidate the possible occurrence of fibrotic lesions after treatment of migraine by methysergide bimaleate in man.     

A quantitative structure-activity relationship study on a series of Na+, K+-ATPase inhibitors

A quantitative structure-activity relationship (QSAR) study has been made on a new series of digitalis-like Na+,K+-ATPase inhibitors in which the guanylhydrazone group has been replaced by an aminoalkyloxime group. The correlations obtained have shown that the oxime moiety, primary amine group, overall size, and polarizability of the new type of substituents are higly beneficial to the Na+,K+-ATPase inhibition potency of the compounds and that their effect can be quantitatively assessed. The study also showed that the inotropic activity of the compounds is very well correlated with their Na+,K+-ATPase inhibition potency.     

The effects of Clostridium histolyticum collagenase on amino sugar-containing compound-collagen complexes

Summary Clostridium histolyticum collagenase has been used on fetal cartilage and bone in an effort to determine its effects on amino sugar-containing-compound collagen complexes. After enzyme treatment it has been found that the staining for acid glycosaminoglycans and glycoproteins in cartilage was abolished only after previous hyaluromdase digestion. The interaction dye-substrate in bone was, instead, readily suppressed after collagenase treatment. These findings suggest a complex formation between some amino sugar-containing compounds and collagen.Supported by Research Grant DE-01952 (04) of the National Institutes of Public Health, Bethesda, Md.     

Effect of divalent metal ions on collagenase from Clostridium histolyticum.

The collagenase from Clostridium histolyticum (EC 3.4.24.3) degrades type IV collagen with Km 32 nM, indicating a high affinity for this substrate. Ferrous and ferric ions can inhibit Clostridium collagenase. Inhibition by Fe++ was of the mixed, non-competitive type, with Ki 90 microM. The inhibitory effect of Fe++ may be due to Zn++ displacement from the intrinsic functional center of this metalloprotease, since in the presence of excess amounts of Zn++ enzyme activity is retained. This inhibitory effect of Fe++ may be common for all types of collagenases, since this ion can also inhibit type IV collagenase purified from Walker 256 carcinoma, with IC50 80 microM. Cu++ can only partially inhibit Clostridium collagenase, while other divalent metal ions such as Cd++, Co++, Hg++, Mg++, Ni++ or Zn++ are devoid of any inhibitory effect on the enzyme.     

A quantitative structure-activity relationship study on some HIV-1 protease inhibitors using molecular connectivity index

A quantitative structure-activity relationship (QSAR) study has been made on two different series of tetrahydropyrimidinones acting as HIV-1 protease inhibitors. A structural parameter, the first order valence molecular connectivity index ((1)chi(v)), has been used to account for the variation in the activity. The protease inhibition activity as well as the antiviral potency of the compounds are found to be significantly correlated with (1)chi(v) of P(2)/P(2') substituents attached to the two nitrogens N1 and N3, suggesting that substituents containing less electronegative and more saturated atoms, meaning thereby the less polar or more hydrophobic substituents, will be more advantageous. Further, if P(2) and P(2') are dissimilar, the former is found to be more effective than the latter. This difference is attributed to a conformational change in the enzyme that may be more favorable to P(2) binding than to P(2') binding.     

Preparation of crude Clostridium histolyticum collagenase for therapeutic purposes.

The production of a freeze-dried enzymatic preparation from the category of crude collagenases has been described. The method is based on the utilization of a highly proteolytic Clostridium histolyticum strain whose products have more advantageous properties for therapeutic purposes than the products of the strain commonly used as yet.     

Identification of metal ligands in the Clostridium histolyticum ColH collagenase

A Clostridium histolyticum 116-kDa collagenase has an H415EXXH motif but not the third zinc ligand, as found in already characterized zinc metalloproteinases. To identify its catalytic site, we mutated the codons corresponding to the three conserved residues in the motif to other amino acid residues. The mutation affecting His415 or His419 abolished catalytic activity and zinc binding, while that affecting Glu416 did the former but not the latter. These results suggest that the motif forms the catalytic site. We also mutated the codons corresponding to other amino acid residues that are likely zinc ligands. The mutation affecting Glu447 decreased markedly both the enzymatic activity and the zinc content, while that affecting Glu446 or Glu451 had smaller effects on activity and zinc binding. These mutations caused a decrease in kcat but no significant change in Km. These results are consistent with the hypothesis that Glu447 is the third zinc ligand. The spacing of the three zinc ligands is the same in all known clostridial collagenases but not in other known gluzincins, indicating that they form a new gluzincin subfamily. The effects of mutations affecting Glu446 and Glu451 suggest that the two residues are also involved in catalysis, possibly through an interaction with the two zinc-binding histidine residues.     

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.     

Clostridium histolyticum collagenase: development of new thio ester, fluorogenic, and depsipeptide substrates and new inhibitors

A new series of thio ester, depsipeptide, and peptide substrates have been synthesized for the bacterial enzyme Clostridium histolyticum collagenase. The hydrolysis of the depsipeptide substrate was followed on a pH stat, and thio ester hydrolysis was measured by inclusion of the chromogenic thiol reagent 4,4'-dithiopyridine in the assay mixture. The best thio ester substrate, Boc-Abz-Gly-Pro-Leu-SCH2CO-Pro-Nba, had a kcat/KM of 63 000 M-1 s-1, while several shorter thio ester sequences were inactive as substrates. In general, the peptide analogues of all the reactive thio ester substrates were shown to be hydrolyzed 5-10 times faster by collagenase. In one case (Z-Gly-Pro-Leu-Gly-Pro-NH2) where a comparison was made, the peptide substrate was respectively 8- and 106-fold more readily hydrolyzed than the corresponding thio ester and ester substrates. Cleavages of the two fluorescence-quench substrates Abz-Gly-Pro-Leu-Gly-Pro-Nba and Abz-Gly-Pro-Leu-SCH2CO-Pro-Nba could be easily followed fluorogenically since a 5-10-fold increase in fluorescence occurred upon hydrolysis. The fluorescent peptide substrate is the best synthetic substrate known for C. histolyticum collagenase with a kcat/KM value of 490 000 M-1 s-1. A series of new reversible inhibitors were developed by the attachment of zinc ligating groups (hydroxamic acid, carboxymethyl, and thiol) to various peptide sequences specific for C. histolyticum collagenase. The shorter peptides designed to bind to either the P3-P1 or P1'-P3' subsites were poor to moderate inhibitors. The thiol HSCH2CH2CO-Pro-Nba had the lowest K1 (0.02 mM).(ABSTRACT TRUNCATED AT 250 WORDS)     

A quantitative structure-activity relationship study of hydroxamate matrix metalloproteinase inhibitors derived from functionalized 4-aminoprolines

A quantitative structure-activity relationship (QSAR) study has been made on the inhibitions of some matrix metalloproteinases (MMPs) by functionalized 4-aminoproline based hydroxamates. Attempts have been made to correlate the inhibition potencies of these hydroxamates with Kier's first-order valence molecular connectivity index ((1)chi(v)) of substituents and electrotopological state (E-state) indices of some atoms. The correlations obtained for the inhibitions of all the enzymes studied, i.e. MMP-1, MMP-2, MMP-3, MMP-7, and MMP-13, were not so uniform, but suggested that in almost all the cases the substituents at the amide nitrogen may be conducive to the activity, though the whole amide group may be sterically unfavourable. Similarly, in most of the cases, the substituens at the phenyl moiety have been found to be beneficial to the inhibition potency and in many cases an electronic role of SO(2) group of the sulfonylphenyl moiety has been indicated.