<p>One-third of the world's population carries <i>Mycobacterium tuberculosis </i>(<i>Mtb</i>), the infectious agent that causes tuberculosis (TB), and every 17 seconds someone dies of TB. After infection, <i>Mtb</i>can live dormant within macrophages for decades in a granuloma structure arising from the host immune response; and cholesterol is important for this persistence of <i>Mtb</i>. Current treatments require long-duration drug regimens with many associated toxicities, which are

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... which are compounded by the high doses required. We phenotypically screened 35 6-azasteroid analogues against <i>Mtb</i>and found that at low micromolar concentrations, a subset of the <a>analogues sensitized <i>Mtb</i></a>to multiple TB drugs. Two analogues were selected for further study to characterize the bactericidal activity of bedaquiline and isoniazid under normoxic and low-oxygen conditions. These two 6-azasteroids <a>showed strong synergy with bedaquiline</a>(fractional inhibitory concentration index = 0.21, bedaquiline minimal inhibitory concentration = 16 nM at 1 μM 6-azasteroid). The rate at which spontaneous resistance to one of the 6-azasteroids arose in the presence of bedaquiline was approximately 10⁻⁹, and the 6-azasteroid-resistant mutants retained their isoniazid and bedaquiline sensitivity. Genes in the cholesterol-regulated Mce3R regulon were required for 6-azasteroid activity, whereas genes in the cholesterol catabolism pathway were not. Expression of a subset of Mce3R genes was down-regulated upon 6-azasteroid treatment. The Mce3R regulon is implicated in stress resistance and is absent in saprophytic mycobacteria. This regulon encodes a cholesterol-regulated stress-resistance pathway that we conclude is important for pathogenesis and contributes to drug tolerance, and that this pathway is vulnerable to small-molecule targeting in live mycobacteria.</p>