Applications of Ensemble Docking in Potential Inhibitor Screening for Mycobacterium tuberculosis Isocitrate Lyase Using a Local Plant Database.

Isocitrate lyase (ICL) is a persistent factor for the survival of dormant stage Mycobacterium tuberculosis (MTB), thus a potential drug target for tuberculosis treatment. In this work, ensemble docking approach was used to screen for potential inhibitors of ICL. The ensemble conformations of ICL active site were obtained from molecular dynamics simulation on three dimer form systems, namely the apo ICL, ICL in complex with metabolites (glyoxylate and succinate), and ICL in complex with substrate (isocitrate). Together with the ensemble conformations and the X-ray crystal structures, 22 structures were used for the screening against Malaysian Natural Compound Database (NADI). The top 10 compounds for each ensemble conformation were selected. The number of compounds was then further narrowed down to 22 compounds that were within the Lipinski's Rule of Five for drug-likeliness and were also docked into more than one ensemble conformation. Theses 22 compounds were furthered evaluate using whole cell assay. Some compounds were not commercially available; therefore, plant crude extracts were used for the whole cell assay. Compared to itaconate (the known inhibitor of ICL), crude extracts from Manilkara zapota, Morinda citrifolia, Vitex negundo, and Momordica charantia showed some inhibition activity. The MIC/MBC value were 12.5/25, 12.5/25, 0.78/1.6, and 0.39/1.6 mg/mL, respectively. This work could serve as a preliminary study in order to narrow the scope for high throughput screening in the future.

Lactobacillus Strains Alleviated Aging Symptoms and Aging-Induced Metabolic Disorders in Aged Rats.

Aging is an inevitable and ubiquitous progress that affects all living organisms. A total of 18 strains of lactic acid bacteria (LAB) were evaluated on the activation of adenosine monophosphate-activated protein kinase (AMPK), an intracellular energy sensor mediating lifespan extension. The cell-free supernatant (CFS) of Lactobacillus fermentum DR9 (LF-DR9), Lactobacillus paracasei OFS 0291 (LP-0291), and Lactobacillus helveticus OFS 1515 (LH-1515) showed the highest activation of AMPK and was further evaluated. The phosphorylation of AMPK by these three LAB strains was more evident in U2OS and C2C12 cells, compared to the other cell lines and control (P < .05). Using premature senescent Sprague-Dawley rats induced by D-galactose (D-gal), the administration of LAB (10 log CFU/rat/day) for 12 weeks prevented the shortening of telomere length in D-gal-treated rats compared to the untreated control (P < .05). LF-DR9 lowered gene expression of p53, a known senescent biomarker, in gastrocnemius muscle and tibia compared to the control. The selected LAB strains also enhanced lipid, renal, and liver profile of rats, suggesting added potential of the strains in preventing aging-induced metabolic diseases. Strain LP-0291 and LH-1515 showed ability to adhere to mucin, no antibiotic resistance, tolerated and proliferated under gastric and intestinal simulated conditions, and inhibited the growth of pathogens Escherichia coli, Staphylococcus aureus, and Staphylococcus epidermidis, comparable to commercial probiotic LF-DR9 and Lactobacillus sakei Probio 65. This study provided an insight into the potential of LAB for exhibiting antisenescence effects, with potentials as new medicinal foods for targeted antiaging therapies.

Eurycomanone, the major quassinoid in Eurycoma longifolia root extract increases spermatogenesis by inhibiting the activity of phosphodiesterase and aromatase in steroidogenesis.

ETHNOPHARMACOLOGICAL RELEVANCE: Eurycoma longifolia Jack (Simaroubaceae family), known locally as 'Tongkat Ali' by the ethnic population, is popularly taken as a traditional remedy to improve the male libido, sexual prowess and fertility. Presently, many tea, coffee and carbonated beverages, pre-mixed with the root extract are available commercially for the improvement of general health and labido. Eurycomanone, the highest concentrated quassinoid in the root extract of E. longifolia improved fertility by increasing testosterone and spermatogenesis of rats through the hypothalamus-pituitary-gonadal axis, but the mechanisms underlying the effects are not totally clear. AIM OF THE STUDY: To provide evidences on the plant ethnopharmacological use and the involvement of eurycomanone, the major indigenous plant quassinoid in testosterone steroidogenesis and spermatogenesis increase. MATERIAL AND METHODS: The rat testicular Leydig cell-rich interstitial cells were isolated and incubated in the culture medium M199. The viability of the cells was determined with trypan blue staining and the concentration of the viable cells was counted with a haemocytometer. The 3ß-hydroxysteroid dehydrogenase (HSD) staining method was used to measure the abundance of Leydig cells in the preparation. Eurycomanone and the standard steroidogenesis inhibitors were incubated with 1.0 × 10(5) cells, and after 2h, the testosterone and the oestrogen concentrations were determined by the ELISA method. Computational molecular docking was performed to determine the binding affinity of the compound at the respective steroidogenesis enzymes. RESULTS: Eurycomanone (EN) significantly increased testosterone production dose-dependently at 0.1, 1.0 and 10.0 µM (P<0.05), but the two lower doses when combined with 3-isobutyl-1-methylxanthine (IBMX), the phosphodiesterase inhibitor were not significantly higher than EN or IBMX alone, except at a higher concentration. The molecular docking studies indicated EN and IBMX were binding at different sites of the enzyme. EN has no reversal of inhibition by aminoglutethimide, ketoconazole or nifedipine at the respective steroidogenesis enzyme. The quassinoid was also non-responsive to the inhibition of oestrogen receptor by tamoxifen, but displayed improved formestane inhibition of aromatase in reducing oestrogen production. The molecular docking studies further supported that EN and formestane bound to aromatase with similar orientations and free energy binding values. CONCLUSION: Eurycomanone enhanced testosterone steroidogenesis at the Leydig cells by inhibiting aromatase conversion of testosterone to oestrogen, and at a high concentration may also involve phosphodiesterase inhibition. The quassinoid may be worthy for further development as a phytomedicine to treat testosterone-deficient idiopathic male infertility and sterility.

Discovery of potential M2 channel inhibitors based on the amantadine scaffold via virtual screening and pharmacophore modeling.

The M2 channel protein on the influenza A virus membrane has become the main target of the anti-flu drugs amantadine and rimantadine. The structure of the M2 channel proteins of the H3N2 (PDB code 2RLF) and 2009-H1N1 (Genbank accession number GQ385383) viruses may help researchers to solve the drug-resistant problem of these two adamantane-based drugs and develop more powerful new drugs against influenza A virus. In the present study, we searched for new M2 channel inhibitors through a combination of different computational methodologies, including virtual screening with docking and pharmacophore modeling. Virtual screening was performed to calculate the free energies of binding between receptor M2 channel proteins and 200 new designed ligands. After that, pharmacophore analysis was used to identify the important M2 protein-inhibitor interactions and common features of top binding compounds with M2 channel proteins. Finally, the two most potential compounds were determined as novel leads to inhibit M2 channel proteins in both H3N2 and 2009-H1N1 influenza A virus.