Macromolecular synthesis and membrane perturbation assays for mechanisms of action studies of antimicrobial agents.

The definition and confirmation of the mechanism of action of an NCE is central to antimicrobial drug discovery. Most antibiotics currently in clinical use selectively target and block one or more bacterial macromolecular synthesis processes, e.g., DNA replication, RNA synthesis (transcription), protein synthesis (translation), cell wall (peptidoglycan) synthesis, and fatty acid (lipid) biosynthesis. This unit includes two protocols for determining the effect of test compounds on macromolecular synthesis, one in test tube format and the other in 96-well plate format. A membrane potential depolarization protocol is also provided. Disruption of cell membrane integrity may be a legitimate mechanism of action for antibacterials, but it also may be the result of nonspecific cell membrane activity, an effect that must be ruled out for mammalian cells. These assays provide useful means for verifying inhibition of an intended target pathway with investigational antimicrobial compounds. They can also be used as valuable secondary assays for lead optimization to eliminate inhibitors that display nonselective toxicity.