Identification of compounds with pH-dependent bactericidal activity against Mycobacterium tuberculosis.

To find new inhibitors of Mycobacterium tuberculosis that have novel mechanisms of action, we miniaturized a high throughput screen to identify compounds which disrupt pH homeostasis. We adapted and validated a 384-well format assay to determine intrabacterial pH using a ratiometric green fluorescent protein. We screened 89k small molecules under non-replicating conditions and confirmed 556 hits that reduced intrabacterial pH (below pH 6.5). We selected five compounds that were active in the screen and also showed some activity against non-replicating bacteria in a 4-stress model, but with no (or greatly reduced) activity against replicating bacteria. The compounds selected were two benzamide sulfonamides, a benzothiadiazole, a bis sulphone and a thiadiazole, none of which are known antibacterial agents. All of these five compounds demonstrated bactericidal activity against non-replicating bacteria in buffer. Four of the five compounds demonstrated increased activity under low pH conditions. None of the five compounds acted as ionophores or as general disruptors of membrane potential. These compounds are useful starting points for work to elucidate their mechanism of action and their utility for drug discovery.