Identification of Compounds with pH-Dependent Bactericidal Activity against Mycobacterium tuberculosis

Abstract

To find new inhibitors of Mycobacterium tuberculosis that have novel mechanisms of action, we miniaturized a high throughput screen to identify compounds that disrupt pH homeostasis. We adapted and validated a 384-well format assay to determine intrabacterial pH using a ratiometric green fluorescent protein. We screened 89000 small molecules under nonreplicating conditions and confirmed 556 hits that reduced intrabacterial pH (below pH 6.5). We selected five compounds that disrupt intrabacterial pH homeostasis and also showed some activity against nonreplicating 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 bissulfone, and a thiadiazole, none of which are known antibacterial agents. All of these five compounds demonstrated bactericidal activity against nonreplicating 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 disrupters of membrane potential. These compounds are useful starting points for work to elucidate their mechanism of action and their utility for drug discovery.

Keywords: Mycobacterium tuberculosis; antibacterial; bactericidal; drug discovery; pH homeostasis; phenotypic screen.

Figure 1

High throughput screen. (A) Lysates were generated from M. tuberculosis expressing pH-sensitive green fluorescent protein in buffered solution. Fluorescence was measured at excitation/emission 400/516 nm and 485/516 nm, and the ratio was calculated. Data are average and standard deviation from 2 independent experiments. The line of best fit using the least-squares method was derived to generate a standard curve. (B) Duplicate plates for minimum signal (5 μM monensin), midpoint signal (0.5 μM monensin), and maximum signal (DMSO) were tested on 3 different days using M. tuberculosis incubated in phosphocitrate buffer at pH 4.5 for 48 h. Day 1, black; day 2, dark gray; day 3, light gray. The Z′ was >0.5 for each plate. (C) For each compound, the intrabacterial pH was calculated based on the standard curve in panel A; compounds that dropped the pH < 6.5 were considered active.

Figure 2

Kill kinetics. Compounds were tested for their ability to kill M. tuberculosis in phosphate citrate buffer at pH 6.8 (left panels) and pH 4.5 buffer (right panels): (A, B) test compounds; (C) control compounds. Data are the average and standard deviation from two independent experiments.

Figure 2

Kill kinetics. Compounds were tested for their ability to kill M. tuberculosis in phosphate citrate buffer at pH 6.8 (left panels) and pH 4.5 buffer (right panels): (A, B) test compounds; (C) control compounds. Data are the average and standard deviation from two independent experiments.