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.     

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 comparative QSAR study on carbonic anhydrase and matrix metalloproteinase inhibition by sulfonylated amino acid hydroxamates

A quantitative structure-activity relationship (QSAR) study is made on the inhibition of a few isozymes of carbonic anhydrase (CA) and some matrix metalloproteinases (MMPs), both zinc containing families of enzymes, by sulfonylated amino acid hydroxamates. For both enzymes, the inhibition potency of the hydroxamates is found to be well correlated with Kier's first-order valence molecular connectivity index 1chi(v) of the molecule and electrotopological state indices of some atoms. From the results, it is suggested that while hydroxamate-CA binding may involve mostly polar interactions, hydroxamate-MMP and hydroxamate-ChC (ChC: Clostridium histolyticum collagenase, another zinc enzyme related to MMPs) bindings may involve some hydrophobic interactions. Both MMPs and ChC also possess some electronic sites of exactly opposite nature to the corresponding sites in CAs. A group such as C6F5 present in the sulfonyl moiety is shown to be advantageous in both CA and MMP (also ChC) inhibitions, which is supposed to be due to the interaction of this group with Zn2+ ion present in the catalytic site of both families of enzymes.     

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.     

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.     

A Novel Approach for the Synthesis of seco C-Nucleoside Analogues

Abstract

The synthesis of 4-amino-3-(D-gluco- or D-galacto-pentitol-1.yl)-5-mercapto-1,2,4-triazoles and their conversion to the respective 6-methyl-3-(1,2,3,4,5-penta-O-acetyl-pentitol-1-yl)1,2,4-triazolo[3,4-b]1,3,4-thiadiazoles have been achieved. The vicinal coupling constants were used to deduce the favored conformations.     

Inhibition of collagenase and metalloproteinases by aloins and aloe gel

The effects of Aloe barbadensis gel and aloe gel constituents on the activity of microbial and human metalloproteinases have been investigated. Clostridium histolyticum collagenase (ChC) results dose-dependently inhibited by aloe gel and the activity-guided fractionation led to an active fraction enriched in phenolics and aloins. Aloins have been shown to be able to bind and to inhibit ChC reversibly and non-competitively. Aloe gel and aloins are also effective inhibitors of stimulated granulocyte matrix metalloproteinases (MMPs). The remarkable structural resemblances between aloins and the pharmacophore structure of inhibitory tetracyclines, suggest that the inhibitory effects of aloins are via an interaction between the carbonyl group at C(9) and an adjacent hydroxyl group of anthrone (C(1) or C(8)) at the secondary binding site of enzyme, destabilizing the structure of granulocyte MMPs.     

Synthesis and biological evaluation of 2-trifluoromethyl/sulfonamido-5,6-diaryl substituted imidazo[2,1-b]-1,3,4-thiadiazoles: a novel class of cyclooxygenase-2 inhibitors

A series of 2-trifluoromethyl/sulfonamido-5,6-diarylsubstituted imidazo[2,1-b]-1,3,4-thiadiazole derivatives 15a-j have been synthesized by the reaction of 2-amino-5-trifluoromethyl/sulfonamido-1,3,4-thiadiazoles 14a-b and appropriately substituted alpha-bromo-1,2-(p-substituted)diaryl-1-ethanones 13a-h. Structures of these compounds were established by IR, (1)H NMR, (13)C NMR, Mass, and HRMS data. The selected compounds were evaluated for their preliminary in vitro cyclooxygenase inhibitory activity against COX-2 and COX-1enzymes using colorimetric method. The compounds tested showed selective inhibitory activity toward COX-2 (80.6-49.4%) over COX-1 (30.6-8.6), amongst them compounds 15f and 15j showed appreciable COX-2 selective inhibitory activity. These compounds also exhibited significant anti-inflammatory activity (70.09-42.32%), which is comparable to that of celecoxib in the carrageenan-induced rat paw edema method.     

Phage display of tissue inhibitor of metalloproteinases-2 (TIMP-2): identification of selective inhibitors of collagenase-1 (metalloproteinase 1 (MMP-1))

Tissue inhibitor of metalloproteinases-2 (TIMP-2) is a broad spectrum inhibitor of the matrix metalloproteinases (MMPs), which function in extracellular matrix catabolism. Here, phage display was used to identify variants of human TIMP-2 that are selective inhibitors of human MMP-1, a collagenase whose unregulated action is linked to cancer, arthritis, and fibrosis. Using hard randomization of residues 2, 4, 5, and 6 (L1) and soft randomization of residues 34-40 (L2) and 67-70 (L3), a library was generated containing 2 × 10(10) variants of TIMP-2. Five clones were isolated after five rounds of selection with MMP-1, using MMP-3 as a competitor. The enriched phages selectively bound MMP-1 relative to MMP-3 and contained mutations only in L1. The most selective variant (TM8) was used to generate a second library in which residues Cys(1)-Gln(9) were soft-randomized. Four additional clones, selected from this library, showed a similar affinity for MMP-1 as wild-type TIMP-2 but reduced affinity for MMP-3. Variants of the N-terminal domain of TIMP-2 (N-TIMP-2) with the sequences of the most selective clones were expressed and characterized for inhibitory activity against eight MMPs. All were effective inhibitors of MMP-1 with nanomolar K(i) values, but TM8, containing Ser(2) to Asp and Ser(4) to Ala substitutions, was the most selective having a nanomolar K(i) value for MMP-1 but no detectable inhibitory activity toward MMP-3 and MMP-14 up to 10 μM. This study suggests that phage display and selection with other MMPs may be an effective method for discovering tissue inhibitor of metalloproteinase variants that discriminate between specified MMPs as targets.     

Studies on collagen-tannic acid-collagenase ternary system: Inhibition of collagenase against collagenolytic degradation of extracellular matrix component of collagen

We report the detailed studies on the inhibitory effect of tannic acid (TA) on Clostridium histolyticum collagenase (ChC) activity against degradation of extracellular matrix component of collagen. The TA treated collagen exhibited 64% resistance against collagenolytic hydrolysis by ChC, whereas direct interaction of TA with ChC exhibited 99% inhibition against degradation of collagen and the inhibition was found to be concentration dependant. The kinetic inhibition of ChC has been deduced from the extent of hydrolysis of N-[3-(2-furyl) acryloyl]-Leu-Gly-Pro-Ala (FALGPA). This data provides a selective competitive mode of inhibition on ChC activity seems to be influenced strongly by the nature and structure of TA. TA showed inhibitor activity against the ChC by molecular docking method. This result demonstrated that TA containing digalloyl radical possess the ability to inhibit the ChC. The inhibition of ChC in gaining new insight into the mechanism of stabilization of collagen by TA is discussed.     

Studies on collagen-tannic acid-collagenase ternary system: Inhibition of collagenase against collagenolytic degradation of extracellular matrix component of collagen

We report the detailed studies on the inhibitory effect of tannic acid (TA) on Clostridium histolyticum collagenase (ChC) activity against degradation of extracellular matrix component of collagen. The TA treated collagen exhibited 64% resistance against collagenolytic hydrolysis by ChC, whereas direct interaction of TA with ChC exhibited 99% inhibition against degradation of collagen and the inhibition was found to be concentration dependant. The kinetic inhibition of ChC has been deduced from the extent of hydrolysis of N-[3-(2-furyl) acryloyl]-Leu-Gly-Pro-Ala (FALGPA). This data provides a selective competitive mode of inhibition on ChC activity seems to be influenced strongly by the nature and structure of TA. TA showed inhibitor activity against the ChC by molecular docking method. This result demonstrated that TA containing digalloyl radical possess the ability to inhibit the ChC. The inhibition of ChC in gaining new insight into the mechanism of stabilization of collagen by TA is discussed.     

Tetrahydroisoquinoline based sulfonamide hydroxamates as potent matrix metalloproteinase inhibitors

The synthesis and MMP inhibitory activity of a series of tetrahydroisoquinoline based sulfonamide hydroxamates are described. In nine MMPs tested, most of the compounds display potent inhibition activity except for MMP-7. Some subtle isozyme selectivity is observed by varying the substituents at the 6- and 7-positions and aromatic ring of arylsulfonyl groups.     

A Comparative QSAR Study on Carbonic Anhydrase and Matrix Metalloproteinase Inhibition by Sulfonylated Amino Acid Hydroxamates

A quantitative structure-activity relationship (QSAR) study is made on the inhibition of a few isozymes of carbonic anhydrase (CA) and some matrix metalloproteinases (MMPs), both zinc containing families of enzymes, by sulfonylated amino acid hydroxamates. For both enzymes, the inhibition potency of the hydroxamates is found to be well correlated with Kier's first-order valence molecular connectivity index ¹χ^v of the molecule and electrotopological state indices of some atoms. From the results, it is suggested that while hydroxamate-CA binding may involve mostly polar interactions, hydroxamate-MMP and hydroxamate-ChC (ChC: Clostridium histolyticum collagenase, another zinc enzyme related to MMPs) bindings may involve some hydrophobic interactions. Both MMPs and ChC also possess some electronic sites of exactly opposite nature to the corresponding sites in CAs. A group such as C 6 F 5 present in the sulfonyl moiety is shown to be advantageous in both CA and MMP (also ChC) inhibitions, which is supposed to be due to the interaction of this group with Zn 2+ ion present in the catalytic site of both families of enzymes.     

Identification and enzymatic characterization of two diverging murine counterparts of human interstitial collagenase (MMP-1) expressed at sites of embryo implantation

Remodeling of fibrillar collagen in mouse tissues has been widely attributed to the activity of collagenase-3 (matrix metalloproteinase-13 (MMP-13)), the main collagenase identified in this species. This proposal has been largely based on the repeatedly unproductive attempts to detect the presence in murine tissues of interstitial collagenase (MMP-1), a major collagenase in many species, including humans. In this work, we have performed an extensive screening of murine genomic and cDNA libraries using as probe the full-length cDNA for human MMP-1. We report the identification of two novel members of the MMP gene family which are contained within the cluster of MMP genes located at murine chromosome 9. The isolated cDNAs contain open reading frames of 464 and 463 amino acids and are 82% identical, displaying all structural features characteristic of archetypal MMPs. Comparison for sequence similarities revealed that the highest percentage of identities was found with human interstitial collagenase (MMP-1). The new proteins were tentatively called Mcol-A and Mcol-B (Murine collagenase-like A and B). Analysis of the enzymatic activity of the recombinant proteins revealed that both are catalytically autoactivable but only Mcol-A is able to degrade synthetic peptides and type I and II fibrillar collagen. Both Mcol-A and Mcol-B genes are located in the A1-A2 region of mouse chromosome 9, Mcol-A occupying a position syntenic to the human MMP-1 locus at 11q22. Analysis of the expression of these novel MMPs in murine tissues revealed their predominant presence during mouse embryogenesis, particularly in mouse trophoblast giant cells. According to their structural and functional characteristics, we propose that at least one of these novel members of the MMP family, Mcol-A, may play roles as interstitial collagenase in murine tissues and could represent a true orthologue of human MMP-1.     

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.     

Inhibitory effects of glycoprotein-120 (G-120) from Ulmus davidiana Nakai on cell growth and activation of matrix metalloproteinases

Excessive breakdown of extracellular matrix by metalloproteinases (MMPs) occurs in many pathological conditions. Consequently, methods for inhibiting MMP activity have therapeutic potential. In this study, we investigated the effect of G-120, a 120 kDa glycoprotein purified from the Oriental herbal plant, Ulmus davidiana Nakai (UDN), on the activity and production of several MMPs by evaluating its growth inhibitory effect on NIH 3T3 cells. Tritium uptake assays showed that proliferation of NIH 3T3 cells was strongly suppressed, and G-120-mediated inhibition of DNA synthesis proved to involve a cytostatic, rather than a cytotoxic, effect, as shown by cytotoxicity and apoptosis assays. More importantly, G-120 strongly reduced the gelatinolytic and collagenase activities of MMP proteins, as well as expression of MMP-2 and MMP-9. Electrophoretic mobility shift assays revealed that it suppressed the DNA binding activity of NF-kappaB. Collectively, our observations show that G-120 strongly inhibits the activation of MMPs and NF-kappaB.     

Synthesis of radioiodinated probes targeted toward matrix metalloproteinase-12

Matrix metalloproteinase-12 (MMP-12, macrophage elastase) is a member of the MMP family that is responsible for the degradation of extracellular matrix, and is associated with the inflammatory process of chronic obstructive pulmonary disease (COPD). COPD, characterized by progressive and irreversible airflow obstruction, is recently a major cause of mortality and morbidity worldwide. Herein, to develop radioiodinated probes for the early diagnosis of COPD, we designed and synthesized novel MMP-12-targeted dibenzofuran compounds (1–3) with a variety of linker structures (carbamate, amide, and sulfonamide). In competitive enzyme activity assays, it was revealed that the linker structures significantly affected the inhibitory activity against and selectivity for MMP-12. Compound 1, with carbamate linker, demonstrated potent MMP-12 inhibitory activity (IC₅₀?=?8.5?nM) compared to compound 2, with amide linker, and compound 3, with sulfonamide linker. Using bromo-substituted carbamate 13 as a radioiodination precursor, [¹²⁵I]1 was successfully prepared to high radiochemical purity (over 98%) and good specific radioactivity (4.1?GBq/μmol). These results suggest that radioiodinated compound 1 is potent as a novel MMP-12-targeted probe.     

Effect of heparin and related glycosaminoglycan on PDGF-induced lung fibroblast proliferation, chemotactic response and matrix metalloproteinases activity

Fibroblast migration, proliferation, extracellular matrix protein synthesis and degradation are the key events in various biological and pathological processes in pulmonary fibrosis. In addition, biopsy specimens from the lungs of patients with pulmonary fibrosis show increased numbers of mast cells which have metachromatic granules containing heparin, histamine and proteases. Little is known about how these products influence pulmonary fibrosis. In the present study, we investigated the effect of heparin and related glycosaminoglycans on PDGF-induced lung fibroblast proliferation and chemotactic response in vitro. In addition, we examined the effect of heparin on both the induction of matrix metalloproteinases (MMPs) and MMPs activity in lung fibroblasts in vitro. Heparin, de-N-sulphated heparin but not heparan sulphate inhibited PDGF-induced lung fibroblast proliferation. In contrast, only heparin inhibited PDGF-stimulated human lung fibroblast chemotaxis. Negatively charged poly-L-glutamic acid had no effect on either fibroblast proliferation or chemotaxis. Thus the negative charge alone cannot account for the ant-proliferative and anti-chemotactic effects of heparin. Furthermore, heparin and heparan sulphate also had no inhibitory effect on induction of MMPS, including MMP-1 (interstitial collagenase), MMP-2 (gelatinase A) and MMP-9 (gelatinase B). Only heparin inhibited both MMP-1 and MMP-2/MMP-9 activity. Additionally, tissue inhibitor of metalloproteinase type 1 (TIMP-1) and type 2 (TIMP-2) inhibited PDGF-stimulated human lung fibroblast chemotaxis. The ability of heparin to inhibit fibroblast chemotaxis may account for the inhibitory effect of heparin on MMP activity. The above results suggested that heparin and related glycosaminoglycans differentially regulate PDGF-induced lung fibroblast proliferation, chemotaxis and MMPs activity and further that these effects may have a key role in extracellular matrix remodeling in inflammatory lung disease.     

Double-headed acyclo C-nucleoside analogues. Functionalized 1,2-bis-(1,2,4-triazol-3-yl)ethane-1,2-diol

Reaction of L-tartaric acid with thiocarbohydcrazide afforded (1R, 2S)-1,2-bis(4-amino-5-mercapto-1,2,4-triazol-3-yl)-ethane-1,2-diol (3). The functional groups in 3 allowed the construction of fused heterocycles on the 1,2,4-triazole rings, mainly of the 1,2,4-triazolo[3,4-b][1,3,4]thiadiazine type as in 4, 5, 7, 10, 13 and 1,2,4-triazolo[3,4-b][1,3,4]thiadiazole type as in 14.