The Mce3R Regulon Is Required for 6-Azasteroid Potentiation of Anti-TB Drug Activity

Abstract

Mycobacterium tuberculosis (Mtb) infects humans and can live dormant within macrophages for decades, residing in a granuloma structure that arises from the host immune response and cholesterol is important for Mtb persistence in the host. Disruption of cholesterol-regulated genes results in reduced intracellular Mtb survival in animal models of infection. We phenotypically screened approximately 50 6-azasteroids against Mtb. We selected this steroid scaffold for its advantageous biophysical and pharmacokinetic properties. We found that at low micromolar concentrations a subset of 6-azasteroids sensitizes Mtb to the TB drug isoniazid and reduces the isoniazid MIC 15-30-fold. Two analogs selected for further study analogously sensitize Mtb to bedaquiline, and they improve the bactericidal activity of bedaquiline and isoniazid. Both 6-azasteroids improve the efficacy of isoniazid and bedaquiline under conditions of low oxygen, and demonstrate high synergy with bedaquiline (FIC index = 0.21, MIC BDQ = 16 nM at 1 µM 6-azasteroid). The rate of spontaneous resistance to 6-azasteroid is approximately 10 -7 and the 6-azasteroid resistance mutants retain their sensitivity to isoniazid and bedaquiline. Intriguingly, genes in the cholesterol-regulated Mce3R regulon are required for 6-azasteroid activity and genes in the cholesterol catabolism pathway are not. The Mce3R regulon has been implicated in stress resistance, and is not present in saprophytic strains of mycobacteria. We conclude that the Mce3R regulon encodes a cholesterol-dependent pathway important for pathogenesis that contributes to drug tolerance. Small molecule strategies to target the Mce3R regulon present an attractive avenue for further development of co-drugs that can improve existing tuberculosis chemotherapies. SIGNIFICANCEOne third of the world's population carries the infectious agent Mycobacterium tuberculosis (Mtb) that causes tuberculosis (TB), and every 17 seconds someone dies of TB worldwide. TB is the number one cause of death from a bacterial infectious disease. Current treatments require drug regimens of long duration to effectively cure TB disease and have many associated toxicities that are compounded by the high doses of long duration required. We have identified small molecules that increase Mtb susceptibility to existing TB drugs. The target of these small molecules is a pathway important for resistance of Mtb to stress. Development of a co-drug therapy has the potential to shorten treatment times and reduce toxicities associated with treatment of TB.