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.

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.

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.

Inhibitory effect of tea catechins on collagenase activity.

A major purpose of this study was to examine inhibitory effect of the catechin derivatives from Japanese green tea Camellia sinensis on collagenase activity. The crude tea catechins, which contain (+)-catechin (C), (-)-epicatechin (EC), (+)-gallocatechin (GC), (-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECg), and (-)-epigallocatechin gallate (EGCg), were tested for their ability to inhibit the prokaryotic and eukaryotic cell derived collagenase activities. Among the tea catechins tested, ECg and EGCg showed the most potent inhibitory effect on collagenase activity when an optimal concentration of tea catechins (100 micrograms/ml) was added to reaction mixture containing collagenase and collagen. Preincubation of collagenase with tea catechins reduced the collagenase activity as well. In contrast to ECg and EGCg, the other four tea catechins (C, EC, EGC, and GC) did not show any collagenase inhibitory effect. Our results suggest that the steric structure of 3-galloyl radical is important for the inhibition of collagenase activity. The collagenase activity in the gingival crevicular fluid from highly progressive adult periodontitis was completely inhibited by the addition of tea catechins. These results demonstrated that tea catechins containing galloyl radical possess the ability to inhibit both eukaryotic and prokaryotic cell derived collagenase.

Clostridium histolyticum collagenases: a new look at some old enzymes.

Seven collagenases denoted by the letters alpha, beta, gamma, delta, epsilon, zeta and eta have been purified to homogeneity from the culture filtrate of Clostridium histolyticum. All seven enzymes are zinc proteinases that require calcium ions for activity and have essential carboxyl, tyrosyl and lysyl residues. These enzymes can be divided into two classes on the basis of the sequence homologies in their polypeptide chains, as revealed from a comparison of their tryptic digests. This division into classes is also supported by a comparison of their specificities toward peptide substrates, their interaction with substrate-analog inhibitors, and their mode of attack of triple helical collagens. The sequence specificities of these enzymes have been studied in detail. The specificities of the two classes are similar, but complementary. Both classes exhibit both endopeptidase and tripeptidylcarboxypeptidase activities, where the latter is thought to facilitate removal of Gly-X-Y triplets from the C-terminus of collagen fragments. The mode of attack of these collagenases on triple helical type I, II and III collagens is very similar for the enzymes within each class, but different for the two classes. The class I enzymes first hydrolyze loci near the ends of the triple helical domains of these collagen molecules, while the class II enzymes make their initial cleavages in the interior. The sites of these initial cleavages are being sequenced and preliminary results indicate that they do not resemble the tissue collagenase cleavage site with respect to either their imino acid content or distribution. The kinetic parameters for the hydrolysis of type I, II and III collagens have been measured and are similar in magnitude to those for the tissue collagenases. Synthetic peptide substrate-analog inhibitors have been prepared for both classes of collagenases and shown to be transition-state-analog inhibitors.

[Studies on collagenase inhibitors. IV. Inhibitors of bacterial collagenase in Coptidis rhizoma].

A hot aqueous extract of Coptidis Rhizoma had an inhibitory effect on the bacterial collagenase from Clostridium histolyticum. Active principles were isolated by silica gel column chromatography from the CHCl3 extract. Consequently, two inhibitors obtained were identified with the chloride of berberine and coptisine. The concentrations of the berberine and coptisine in the assay mixture to give 50% inhibition (IC50) were 0.73 mM and 0.16 mM, respectively. The type of inhibition by coptisine chloride was shown to be a mixture type from Lineweaver-Burk plots. Tetrahydroberberine, a reduction product of berberine chloride, had no inhibitory effect. This result suggests that the quaternary nitrogen of the alkaloids plays an important role in inhibitory activity.

Expression of metalloproteinases and metalloproteinase inhibitor in human arthritic synovium.

The expression of messenger RNA encoding neutral metalloproteinases and the tissue inhibitor of metalloproteinases (TIMP) in human arthritic synovium was evaluated in situ, using RNA probes. Interstitial collagenase and stromelysin were expressed by synovial lining cells in patients with active rheumatoid arthritis (RA). Proteinase messenger RNA was found both in cells expressing mononuclear phagocyte antigens and in cells that were negative for the antigens. TIMP was also expressed predominantly along the synovial lining layer. In highly inflammatory RA, TIMP expression appeared less intense than that of the proteases. In osteoarthritic synovium, TIMP was expressed at easily detectable levels, whereas the expression of collagenase and stromelysin was less prominent. The balance between expression of the metalloproteinases and of the metalloproteinase inhibitor in synovium appears to be altered during inflammation. These results are consistent with the notion that synovium plays different roles in the cartilage damage of RA and of osteoarthritis.

The inhibitory effect of plant extracts on the collagenolytic activity and cytotoxicity of human gingival fibroblasts by Porphyromonas gingivalis crude enzyme.

It is well known that plant extracts inhibit some enzymatic activities. The present study examined the inhibitory effects of natural plant extracts against the collagenolytic activity of Porphyromonas gingivalis. The enzyme was isolated from a culture supernatant of P. gingivalis 381. The aqueous and 50% ethanolic extracts of Ginkgo biloba, Mosla chinensis, Salvia officinalis, Cinnamomum cassia, and a catechin extract of Camellia sinensis exhibited strong inhibitory effects on collagenolytic activity. The activities of these plant extracts were higher than that of tetracycline-HCl. They also inhibited the cytotoxicity of P. gingivalis crude enzyme against human gingival fibroblasts. C. sinensis catechin was the most effective agent in neutralizing the cytotoxicity of P. gingivalis. The aqueous and 50% ethanolic extracts of C. cassia had relatively strong anti-cytotoxic activity. Although the other samples strongly inhibited the collagenolytic activity of P. gingivalis, they were not effectively anti-cytotoxic. The present findings suggest that C. sinensis and C. cassia extracts are effective in reducing the pathogenicity of periodontopathic bacteria.

Inhibitory effects of aqueous extract of cacao bean husk on collagenase of Bacteroides gingivalis.

Natural materials containing tannin were examined to determine whether they possessed any inhibitory activity against collagenase obtained from the culture supernatant of Bacteroides gingivalis. The collagenolytic activity was determined with Collagenokit CLN-100 (Collagen Technological Co., Tokyo) using FITC-conjugated collagen type 1. Among the test samples, only an aqueous extract of cacao (Theobroma cacao) bean husk strongly inhibited the bacterial collagenase. This inhibitory potency was at the same level as that of tetracycline-HCl.

In vitro inhibition of arachidonic acid metabolism by two novel retinoid analogs.

Ro 23-6457, (all-E)-3,7-dimethyl-9-[2-(trifluoromethyl)-6-(nonyloxy)phenyl]-2, 4,6,8- nonatetraenoic acid, and Ro 23-2895, (all-E)-9-[2-(nonyloxy)phenyl]-3,7-dimethyl-2,4,6,8-nonatetraen oic acid, are two novel retinoid analogs which exhibit antiinflammatory activity in both the developing and the established rat adjuvant arthritis models [8]. Here we investigated the effect of these two compounds on the production of arachidonic acid (AA) metabolites in two in vitro test systems [i.e., Ca2+ ionophore A23187 (I)-stimulated resident rat peritoneal macrophages (MO) and cytokine-stimulated human dermal fibroblasts (HDF)]. Both compounds, Ro 23-6457 and Ro 23-2895, significantly inhibited the release of 14C-AA metabolites and the production of LTB4, PGE2, and 6-keto-PGF1 alpha in I-stimulated MO, at concentrations of 1-33 microM. Both compounds also inhibited the production of PGE2 in HDF stimulated by either rhuIL-1 alpha or huTNF alpha at concentrations of 1 x 10(-5) to 1 x 10(-7) M. Ro 23-2895 was also a potent inhibitor of IL-1-induced collagenase production in rheumatoid synovial cells (IC50 approximately 1 to 2.5 x 10(-8) M). The inhibitory profile of these novel compounds in these cell systems is therefore similar to that of other known antiinflammatory retinoids (e.g., all-trans- and 13-cis-retinoic acid). Inhibitory effects such as those described here might in part contribute to the antiinflammatory activity of these compounds in vivo.