Lilly M. Saleena

Discovery of potent inhibitor for matrix metalloproteinase-9 by pharmacophore based modeling and dynamics simulation studies

Matrix metalloproteinase-9 (MMP-9) is an attractive target for anticancer therapy. In the present study ligand based pharmacophore modeling was performed to elucidate the structural elements for a diverse class of MMP-9 inhibitors. The pharmacophore model was validated through Güner-Henry (GH) scoring method. The final pharmacophore model consisted of three hydrogen bond acceptors (HBA), and two ring aromatic regions (RA). This model was utilized to screen the natural compound database to seek novel compounds as MMP-9 inhibitors. The identified hits were validated using molecular docking and molecular dynamics simulation studies. Finally, one compound named Hinokiflavone from Juniperus communis had high binding free energy of -26.54kJ/mol compared with the known inhibitors of MMP-9. Cytotoxicity for hinokiflavone was evaluated by MTT assay. Inhibition of MMP-9 in the presence of hinokiflavone was detected by gelatin zymography and gelatinolytic inhibition assay. Results revealed that the natural compounds derived based on the developed pharmacophore model would be useful for further design and development of MMP-9 inhibitors.

Potent inhibitors precise to S1′ loop of MMP-13, a crucial target for osteoarthritis

Matrix metalloproteinase-13 (MMP-13) is the primary MMP involved in cartilage degradation through its particular ability to cleave type-II collagen. This protein is expressed by chondrocytes and synovial cells in human osteoarthritis and rheumatoid arthritis; hence, it is an attractive target for the treatment of arthritic diseases. Currently available inhibitors lack specificity for metalloproteinase because of a common Zn binding site in MMPs; thus, there is a need to identify selective MMP-13 inhibitors for osteoarthritis therapy. Because selectivity is the major concern, both ligand-based and protein-based pharmacophore methodologies were used to identity potent and selective MMP-13 inhibitors. Different hypotheses were validated, and the best hypothesis was used to screen Zinc (natural and chemical) databases to seek novel scaffolds as MMP-13 inhibitors. The identified hits were validated using different strategies, such as Glide Standard precision, extra precision, E-model energies and receiver operating curve (ROC). In addition, potent inhibitors were selected based on two criteria: a similar binding mode as that of the active site PB3 crystal ligand and crucial amino acid interactions that are catalytically important for the function of MMP-13. The candidate potent inhibitors ZINC 02535232, ZINC 08399795, ZINC 12419118 and ZINC 00624580 nearly reproduced the H-bond interactions formed in the crystal structure of 1XUC with reasonable RMSD values exhibiting a novel interaction pattern that was not previously observed in MMP-13 inhibitors. The identified potent hits with diverse chemical scaffolds may be useful in designing new MMP-13 inhibitors.

Lead identification and optimization of novel collagenase inhibitors; pharmacophore and structure based studies.

In this study, chemical feature based pharmacophore models of MMP-1, MMP-8 and MMP-13 inhibitors have been developed with the aid of HypoGen module within Catalyst program package. In MMP-1 and MMP-13, all the compounds in the training set mapped HBA and RA, while in MMP-8, the training set mapped HBA and HY. These features revealed responsibility for the high molecular bioactivity, and this is further used as a three dimensional query to screen the knowledge based designed molecules. These pharmacophore models for collagenases picked up some potent and novel inhibitors. Subsequently, docking studies were performed for the potent molecules and novel hits were suggested for further studies based on the docking score and active site interactions in MMP-1, MMP-8 and MMP-13.

Genetic engineering of Clostridium thermocellum DSM1313 for enhanced ethanol production

BackgroundThe twin problem of shortage in fossil fuel and increase in environmental pollution can be partly addressed by blending of ethanol with transport fuel. Increasing the ethanol production for this purpose without affecting the food security of the countries would require the use of cellulosic plant materials as substrate. Clostridium thermocellum is an anaerobic thermophilic bacterium with cellulolytic property and the ability to produce ethanol. But its application as biocatalyst for ethanol production is limited because pyruvate ferredoxin oxidoreductase, which diverts pyruvate to ethanol production pathway, has low affinity to the substrate. Therefore, the present study was undertaken to genetically modify C. thermocellum for enhancing its ethanol production capacity by transferring pyruvate carboxylase (pdc) and alcohol dehydrogenase (adh) genes of the homoethanol pathway from Zymomonas mobilis.ResultsThe pdc and adh genes from Z. mobilis were cloned in pNW33N, and transformed to Clostridium thermocellum DSM 1313 by electroporation to generate recombinant CTH-pdc, CTH-adh and CTH-pdc-adh strains that carried heterologous pdc, adh, and both genes, respectively. The plasmids were stably maintained in the recombinant strains. Though both pdc and adh were functional in C. thermocellum, the presence of adh severely limited the growth of the recombinant strains, irrespective of the presence or absence of the pdc gene. The recombinant CTH-pdc strain showed two-fold increase in pyruvate carboxylase activity and ethanol production when compared with the wild type strain.ConclusionsPyruvate decarboxylase gene of the homoethanol pathway from Z mobilis was functional in recombinant C. thermocellum strain and enhanced its ability to produced ethanol. Strain improvement and bioprocess optimizations may further increase the ethanol production from this recombinant strain.

Field- and Gaussian-based 3D-QSAR studies on barbiturate analogs as MMP-9 inhibitors

Matrix metalloproteinase-9 (MMP-9) is one of the important enzymes belongs to gelatinase family and involved in multiple cellular processes including proliferation, angiogenesis, and metastasis. Various studies show that N-substituted homopiperazine barbiturates are promising and also selective for the MMP-9 inhibition. In this study, we selected and reported 49 barbiturate derivatives as inhibitors of MMP-9 and performed structure-based 3D-QSAR studies to elucidate the important structural features responsible for binding affinity. Crystal structure of MMP-9 complexed with barbiturate available in PDB was selected and performed quantum-polarized ligand docking for the reported inhibitors. Receptor-based alignment obtained from the docked poses were used for field- and Gaussian-based 3D-QSAR study. The results of both field and Gaussian models gave the good predictive correlation coefficient (test set) q2 of 0. 77 and 0.85, and the conventional correlation coefficient (training set) r2 of 0.845 and 0.928, respectively. The results of 3D-QSAR and docking studies validate each other and provided insight into the structural requirements for MMP-9 inhibition. The outcomes of the contour maps for both steric and Gaussian models results indicate that steric and hydrophobic interactions are major contributing factor for the activity and these findings will be useful to design the new inhibitors against MMP-9.

E-Pharmacophore and Molecular Dynamics Study of Flavonols and Dihydroflavonols as Inhibitors Against DiHydroOrotate DeHydrogenase

DiHydroOrotate DeHydrogenase [huDHODH] is a therapeutic target for Rheumatoid arthritis [RA]. Leflunomide [A771726] is a widely used synthetic inhibitor against huDHODH. We to find more efficient lead like compounds. A four featured E-Pharmacophore A1D4H6R7 was built based on the inhibitor A771726. This pharmacophore was validated by checking its ability to identify known highly active inhibitors of huDHODH and assigning higher fitness scores to them. A reverse validation was also performed where random 4 featured pharmacophores were built and its efficiency in identifying actives was compared with our E-Pharmacophore. Our Epharmacophore was very efficient, since it passed both validations by picking the known active molecules with high fitness scores. This validated E- pharmacophore was searched against the KEGG phytochemicals subset database. This search resulted in 18 molecules which were subjected to docking with huDHODH. The molecules with docking score greater than that of A771726 were selected. The docking results were further validated using MM/GBSA which gave similar ranking with high binding free energy values. The four molecules 6-Methoxytaxifolin, Rhamnetin, Rhamnazin and Pinoquercetin were taken for explicit 3ns simulation and it was observed that all four molecules had acceptable RMSD values and stable interactions. Thus our study, suggests four phytomolecules that might inhibit huDHODH more efficiently than A771726. Interestingly, some of the obtained hits have already been proven in vitro anti-inflammatory activity which confirms that, the developed E-pharmacophore can be used to identify novel small molecules against inflammatory target, huDHODH.

Silica Solubilization Potential of Certain Bacterial Species in the Presence of Different Silicate Minerals

Silicate solubilisng bacteria (SSB) are distributed in soil, water, aquatic sediments and in silicate minerals but their population is smaller than the total bacteria indicating their uniqueness. Pond sediment showed a higher SSB with 40×104 g−1 while sugarcane field soil recorded 31×104 g−1 dry weight. SSB population in silicate minerals varied with the highest in phyto-sil followed by muscovite but very low in quartz and illite. The silicate solubilisation potential of the six isolates tested on eight minerals varied with the isolates and mineral. Magnesium trisilicate was more easily solubilised than, quartz, or muscovite. Four of the elite isolates were characterized by 16S r RNA sequencing and were found to be Bacillus flexus, B. mucilaginosus, B. megaterium and Pseudomonas fluorescens. The GC/MS analysis of organic acids produced in the medium containing feldspar and quartz by Bacillus flexus and B. muicilaginosus showed variation with the minerals. The release of silica in solution serves as a nutrient for life forms.

Direct estimation of ethanol as a negative peak from alcoholic beverages and fermentation broths by reversed phase-HPLC

A relatively simple and straightforward technique utilizing reversed phase liquid chromatography with a PDA-UV detector was used for the detection and quantification of ethanol. Samples of alcoholic beverages and fermentation broths were injected without any prior chemical pre-treatment or structural modification. The C18 column was equilibrated with water as a mobile phase having a high UV absorbing compound, acetone, added in an optimized percentage. Ethanol when injected resulted in a negative peak within 4 minutes of runtime, and the absolute area/height of the peak was used for quantification. The method was validated for linearity with the detection ranging from 0.1–100% v/v. Intra-day and inter-day precision experimental results were within acceptable limits. The accuracy and repeatability of this method can reduce the dependency of using specific columns and detectors for the analysis of ethanol.

Molecular Docking and Dynamics Simulation Study on the Influence of Zn 2+ on the Binding Modes of Aggrecanase with its Inhibitors

Zinc plays a vital role in structural organization, regulation of function and stabilization of the folded protein, which ultimately activates or inactivates the binding sites of the protein. Its transition makes a major change in the protein and its binding affinity. The ligand binding aggrecanases can be influenced by Zn2+ ions; therefore the study focuses on checking the binding mode in the presence and absence of zinc using Docking and Molecular dynamics simulation. The crystal structure with zinc was considered as wild type (ADAMTS-4-1Zn2+, ADAMTS-5-1Zn2+) and the crystal structure without zinc was considered as the mutant type (ADAMTS-4-0Zn2+, ADAMTS-5-0Zn2+). Mutations were made manually by deleting the zinc atom. ADAMTS-4-1Zn2+ had the best Glide score of -12.66 kcal·mol−1, whereas ADAMTS-4-0Zn2+ had -11.69 kcal·mol−1. ADAMTS-4-1Zn2+ had the best glide energy of -72.29 kcal·mol−1, whereas ADAMTS-4-0Zn2+ had-68.44 kcal·mol−1. ADAMTS-4-1Zn2+ had the best glide e-model of -116.34, whereas ADAMTS-4-0Zn2+ had -104.264. The RMSD value for ADAMTS-4-1Zn2+ and ADAMTS-4-0Zn2+ was 1.9. These results suggested that the absence of zinc decreases the binding affinity of ADAMTS-4 with its inhibitor. ADAMTS-5-1Zn2+ had the best Glide score of -8.32 kcal·mol−1, whereas ADAMTS-5-0Zn2+ had -6.62 kcal·mol−1. ADAMTS-5-1Zn2+ had the best glide energy of -70.28 kcal·mol−1, whereas ADAMTS-5-0Zn2+ had -66.02 kcal·mol−1. ADAMTS-5-1Zn2+ had the best glide e-model of-108.484, whereas ADAMTS-5-0Zn2+ had -93.81. The RMSD value for ADAMTS-5-1Zn2+ and ADAMTS-5-0Zn2+ was 0.48Å. These results confirmed that the absence of zinc decreased the binding affinity of ADAMTS-5 with its inhibitor whereas the presence extended the docking energy range and strengthened the binding affinity. Per-residue interaction study, MM-GBSA and Molecular Dynamics showed that all the four complexes underwent extensive structural changes whereas the complex with zinc was stable throughout the simulation period.