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.

Anthranilic amide and imidazobenzothiadiazole compounds disruptMycobacterium tuberculosismembrane potential

“…From the benzothiadiazole series, the profile of compound 2 under non-replicating conditions at both pH 4.5 and pH 6.8 has been previously reported. 13 Concentrations ranging from 10–100 μM decreased CFU in the culture >4 logs within 7 days, with all concentrations sterilizing the culture within 14 days. 13 …”

“… 13 Concentrations ranging from 10–100 μM decreased CFU in the culture >4 logs within 7 days, with all concentrations sterilizing the culture within 14 days. 13 …”

“…We previously reported a whole-cell screening effort targeted at identifying compounds that disrupt pH homeostasis in Mycobacterium tuberculosis . 13 We hypothesized that such compounds would prove bactericidal under acidic conditions. Five compound hits were selected and demonstrated to have pH-dependent bactericidal activity.…”

“…Five compound hits were selected and demonstrated to have pH-dependent bactericidal activity. 13 Of the clustered hits resulting from this screen, we selected several for further development.…”

NSC 18725, a Pyrazole Derivative Inhibits Growth of Intracellular Mycobacterium tuberculosis by Induction of Autophagy

“…Phenotypic screening addresses challenges associated with cell wall penetration, pro-drug activation and results in the identification of accessible and essential bacterial targets (Swinney, 2013;Dhiman and Singh, 2018;Yuan and Sampson, 2018). Several groups have performed modified phenotypic screening by incorporating conditions such as acidic, low oxygen, nutrient starvation, reactive nitrogen intermediates, and fatty acids as carbon source in their screening assays (Cho et al, 2007;Mak et al, 2012;Grant et al, 2013;VanderVen et al, 2015;Early et al, 2019). In addition, high-content screening has also resulted in identification of compounds that inhibit growth of intracellular M. tuberculosis (Christophe et al, 2009(Christophe et al, , 2010Brodin et al, 2010;Pethe et al, 2013;Stanley et al, 2014).…”