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.
Highlights
Tuberculosis (TB), caused by Mycobacterium tuberculosis, is the leading cause of death among infectious diseases.[1]
Activated macrophages can mount an effective response to M. tuberculosis infection, and if acidification of the compartment occupied by M. tuberculosis occurs, bacterial killing is observed.[5]
The pH of phagosomes occupied by M. tuberculosis ranges from 4.5 to 6.2, which is dependent on the state of activation of the macrophage.[6−11]
Summary
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. We selected five compounds for further study based on their properties in vitro (Table 2); in particular, these five confirmed activity from the screen by exhibiting a MAC6.5 less than 10 μM, showed activity against M. tuberculosis in the multistress model, and were less effective against aerobically grown, actively replicating bacteria. Bacteria, suggesting their bactericidal activity may be specific for nonreplicating organisms In some cases this activity was pronounced; for example, compound IDR-0040669 was rapidly bactericidal, resulting in sterilization after 3 days at a concentration as low as 10 μM (Figure 2B). We saw that RIF and INH were slightly more active in the acidic conditions (Figure 2C) This has not been noted before, but we see this reproducibly, suggesting that mechanisms other than disrupting pH homeostasis could lead to the identification of compounds with condition-dependent, or condition-enhanced activity. Four of the compounds were stable in buffers at both pH values (50% remaining after 5 days), while compound IDR-0054790 appeared to degrade to an unknown species in the low pH buffer (
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