An Investigation of Molecular Targeting of MMP-9 for Endometriosis Using Algal Bioactive Molecules

Endometriosis is a chronic inflammatory disease that occurs due to the presence of endometrial tissue outside the uterine cavity. It affects from 5% to 10% of women of reproductive age. High levels of matrix metalloproteinase (especially MMP-9) have been observed in women suffering from endometriosis. Thus, the aim of this study was to investigate the naturally anti-inflammatory compounds available from an algal source that can target the MMP-9 by various in silico approaches. The target 1L6J (Crystal structure of human matrix metalloproteinase MMP-9) structure was retrieved from the PDB database. Five compounds such as Eckol, Sargafuran, Vitamin E, Docosahexaenoic acid, Fucoidan and Elagolix were selected based on ‘Lipinski’s rule of five’ using the PubChem database. The pharmacokinetics, ADMET properties and biological activity of these compounds were predicted computationally using databases such as PreADME, SWISS-ADME, pkCSM and PASS. Comparative analysis of the bioactive compounds with the target was performed by AutoDock 4.2.6. Using LigPlot v.2.2, the target residues interacting with the compounds were visualised in a 2D manner. Based on the results, Eckol exhibited the highest binding energy value of −7.82 kcal/mol, whereas the Elagolix (control drug) showed a binding energy of −4.88 kcal. We conclude that Eckol can be a potent inhibitor of target MMP-9 with least side effects when compared to the control drug. Hence, this compound can be effectively explored by further in vitro and in vivo studies to develop more effective treatments for Endometriosis.     

Expression and biochemical characterization of two novel feruloyl esterases derived from fecal samples of Rusa unicolor and Equus burchelli

Two novel genes (tvms10a, tvmz2a) were identified in the metagenomic DNA of Rusa unicolor and Equus burchelli fecal samples. The amplified PCR product of tvms10a is composed of 917bp and the gene was found to encode a protein containing 165 amino acids, while the tvmz2a PCR product was 1053bp long encoding 298 amino acid proteins. The gene has 72% primary sequence identity with Clostridiales sp. These amplified PCR products which can encode FAE were cloned into pGEMT Easy TA cloning vector and then sub-cloned into the EcoRI site of pET32a expression vector to generate pET32-tvms10a and pET32-tvmz2a, which was then transformed into Escherichia coli BL21. The recombinants were grown in LB medium and gene expression was induced with IPTG for 6h. Purified recombinant Tvms10a and Tvmz2a proteins showed molecular masses of 18.6 and 31.2kDa respectively, and displayed hydrolytic activity towards substrate ethyl ferulate. The activities of Tvms10a and Tvmz2a produced in E. coli were 15 and 9U/min respectively, and their specific activities 16.6 and 10.4U/mg protein respectively. The optimal pH is between 5.0 and 8.0 and the optimal temperature is 37°C for enzyme reaction. Unusually, these proteins were found to be capable of releasing ferulic acid (FA) and diferulic acid (diFA) from untreated crude plant cell wall materials. The substrate utilization preferences and sequence similarity of these clones place it in the type-D sub-class of FAE.     

Molecular cloning, expression and characterization of a novel feruloyl esterase enzyme from the symbionts of termite(Coptotermes formosanus) gut

Termites play an important role in the degradation of dead plant materials and have acquired endogenous and symbiotic cellulose digestion capabilities. The feruloyl esterase enzyme (FAE) gene amplified from the metagenomic DNA of Coptotermes formosanus gut was cloned in the TA cloning vector and subcloned into a pET32a expression vector. The Ft3-7 gene has 84% sequence identity with Clostridium saccharolyticum and shows amino acid sequence identity with predicted xylanase/chitin deacetylase and endo-1,4-beta-xylanase. The sequence analysis reveals that probably Ft3-7 could be a new gene and that its molecular mass was 18.5 kDa. The activity of the recombinant enzyme (Ft3-7) produced in Escherichia coli (E.coli) was 21.4 U with substrate ethyl ferulate and its specific activity was 24.6 U/mg protein. The optimum pH and temperature for enzyme activity were 7.0 and 37oC, respectively. The substrate utilization preferences and sequence similarity of the Ft3-7 place it in the type-D sub-class of FAE.     

Larvicidal and insect growth regulator effect of α-amyrin acetate from Catharanthus Roseus Linn against the malaria vector Anopheles Stephensi Liston (Diptera: Culicidae)

Vector control is a serious concern in developing countries. Over the past two decades, phytochemicals have received progressively more attention as insecticide alternatives, and they have recently become the focus in the concept of integrated vector control. α-Amyrin acetate, the n-hexane fraction of acetone extract from the leaves of Catharanthus roseus , was evaluated for its larvicidal, pupicidal and fecundity effects as well as insect growth regulator activity against the malaria vector Anopheles stephensi Liston. The highest concentration of 1 p.p.m. produced 100% mortality in first to second instars and 94% mortality in third and fourth instars. In addition, the duration of larval instars and the total developmental time were prolonged, while female longevity and fecundity were markedly decreased. The suppression of pupation and adult emergence was probably due to its action similar to juvenile hormone analogs in combination with growth regulator activity and toxicity, which reduced the overall performance of the malaria vector An. stephensi .