Structural Determinants of Indole-2-carboxamides: Identification of Lead Acetamides with Pan Antimycobacterial Activity.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (M.tb), is one of the leading causes of death in developing countries. Non-tuberculous mycobacteria (NTM) infections are rising and prey upon patients with structural lung diseases such as chronic obstructive pulmonary disease (COPD) and cystic fibrosis. All mycobacterial infections require lengthy treatment regimens with undesirable side effects. Therefore, new antimycobacterial compounds with novel mechanisms of action are urgently needed. Published indole-2-carboxamides (IC) with suggested inhibition of the essential transporter MmpL3 showed good potency against whole-cell M.tb, yet had poor aqueous solubility. This project focused on retaining the required MmpL3 inhibitory pharmacophore and increasing the molecular heteroatom percentage by reducing lipophilic atoms. We evaluated pyrrole, mandelic acid, imidazole, and acetamide functional groups coupled to lipophilic head groups, where lead acetamide-based compounds maintained high potency against mycobacterial pathogens, had improved in vitro ADME profiles over their indole-2-carboxamide analogs, were non-cytotoxic, and were determined to be MmpL3 inhibitors.

Ethanol metabolism results in a G2/M cell-cycle arrest in recombinant Hep G2 cells.

Previous studies using the Hep G2-based VA cells showed that ethanol metabolism resulted in both cytotoxicity and impaired DNA synthesis, causing reduced accumulation of cells in culture. To further characterize the ethanol oxidation-mediated impairment of DNA synthesis we analyzed the cell-cycle progression of VA cells. These studies showed approximately a 6-fold increase in the percentage of cells in the G2/M phase of the cell cycle after 4 days of ethanol exposure. The G2/M transition requires activity of the cyclin-dependent kinase, Cdc2. Cdc2 is positively regulated by association with cyclin B1, and negatively regulated by phosphorylation of amino acids Thr14 and Tyr15. Immunoblot analysis revealed that ethanol metabolism had little affect on total Cdc2 content in these cells, but resulted in the accumulation of up to 20 times the amount of cyclin B1, indicating that cyclin B1 was available for formation of Cdc2/cyclin B1 complexes. Co-immunoprecipitation revealed that 6 times more Cdc2/cyclin B1 complexes were present in the ethanol-treated cells compared with the controls. Investigation of the phosphorylation state of Cdc2 revealed that ethanol oxidation increased the amount of the phosphorylated inactive form of Cdc2 by approximately 3-fold. Thus, the impairment in cell-cycle progression could not be explained by a lack of cyclin B1, or the ability of Cdc2 and cyclin B1 to associate, but instead resulted, at least in part, from impaired Cdc2 activity. In conclusion, ethanol oxidation by VA cells results in a G2/M cell-cycle arrest, mediated by accumulation of the phosphorylated inactive form of Cdc2.