Computer-assisted screening of mycobacterial growth inhibitors: Exclusion of frequent hitters with the assistance of the multiple target screening method.

Background: The emergence of frequent hitters (FHs) remains a challenge in drug discovery. We have previously used in silico structure-based drug screening (SBDS) to identify antimycobacterial candidates. However, excluding FHs has not been integrated into the SBDS system. Methods: A dataset comprising 15,000 docking score (protein-compound affinity matrix) was constructed by multiple target screening (MTS): DOCK-GOLD two-step docking simulations with 154,118 compounds versus the 30 target proteins essential for mycobacterial survival. After extraction of 141 compounds from the protein-compound affinity matrix, compounds determined to be FHs or false positives were excluded. Antimycobacterial properties of the top nine compounds selected through SBDS were experimentally evaluated. Results: Nine compounds designated KS1-KS9 were selected for experimental evaluation. Among the selected compounds, KS3, identified as adenosylhomocysteinase inhibitor, showed a potent inhibitory effect on antimycobacterial growth (inhibitory concentration [IC]50 = 1.2 M). However, the compound also showed potent cytotoxicity. Conclusion: The MTS method is applicable in SBDS for the identification of enzyme-specific inhibitors.

Effect of methotrexate exposure at middle gestation on the inner plate of the ocular cup and lens in the rat fetus.

Pregnant rats were treated intraperitoneally with a single dose of methotrexate (MTX) 90 mg/kg on gestation day (GD) 13, and fetal eyeballs were examined time-dependently from GD 13.5 to 15.5. Throughout the experimental period, the inner plate of the ocular cup in the MTX group was significantly thinner than that in the control group. In the inner plate of the ocular cup on GD 15 and 15.5, whereas a developed ganglion cell layer was observed in the control group, the ganglion cell layer in the MTX group was undeveloped and indistinguishable. Disturbance of the arrangement of lens fiber cells, narrowing of the hyaloid cavity of the optic cup, and hypoplasia of optic nerve fibers were observed in the MTX group on GD 15 and 15.5. Increase of pyknosis and decrease of mitosis were induced in the optic cup and the lens epithelium of the MTX group. In the inner plate of the optic cup and the lens epithelium of the MTX group, the cleaved caspase-3- and TUNEL-positive rates increased significantly throughout the experimental period. The phospho-histone H3-positive rate in the inner plate of the optic cup decreased significantly from GD 13.5 to 14.5, and it recovered on GD 15. On the other hand, the phospho-histone H3-positive rate in the lens epithelium decreased significantly throughout the experimental period. These results suggested that optic tissue on GD 13 in rats was sensitive to MTX.

Identification of novel potential antibiotics for tuberculosis by in silico structure-based drug screening.

The enoyl-acyl carrier protein reductase of Mycobacterium tuberculosis (MTB) is a key enzyme of the type II fatty acid synthesis system. It is involved in the production of mycolic acid and is a known target for isoniazid, an effective antibiotic for tuberculosis treatment. The increasing prevalence of tuberculosis in many areas of the world, which is associated with the rise of drug-resistant MTB strains, presents a major global health threat. In this study, we attempted to identify novel antibiotics specifically targeting the MTB enoyl-acyl carrier protein reductase. We performed in silico structure-based drug screening using the crystal structure data for the MTB enoyl-acyl carrier protein reductase (PDB code; 2H7I) and a virtual compound library, which includes 152,102 chemicals. By a two-step screening method using DOCK (first screening) and GOLD (second screening), we identified 5 chemical compounds expected to have high binding affinity to the active center of the MTB enoyl-acyl carrier protein reductase. Moreover, we examined the antibiotic effects of these chemical compounds on model bacterial strains by in vitro experiments. We found that a chemical compound, which has a basic skeleton comprised of dibenzofuran, acetoamide, trizol, furyl and methylphenyl groups, completely inhibited the growth of Mycobacterium vanbaalenii and had no toxic effects on enterobacteria and cultured mammalian cells. Therefore, the chemical compound is likely to be useful in the research and development of new antibiotics for tuberculosis.