Inhibitors of 3&beta-Hydroxysteroid? Dehydrogenase and Metabolic? and Enzymatic Characterization of the Cholesterol? ? Metabolizing Intracellular Growth? Operon? of? <italic>Mycobacterium tuberculosis</italic>

Abstract

New drugs with novel mechanisms of action are required to meet the severe threat to human health posed by the emergence of multidrug and extensively drug resistant strains of Mycobacterium tuberculosis (M. tuberculosis). The cholesterol metabolism pathway in M. tuberculosis is a potential source of energy as well as secondary metabolite production that is important for survival of M. tuberculosis in the host macrophage. Oxidation and isomerization of 3Β-hydroxysterols to 4-en-3-ones by M. tuberculosis 3Β-hydroxysteroid dehydrogenase (3Β-HSD) is required for sterol metabolism, and inhibitors of 3Β-HSD are important for targeting the cholesterol metabolic pathway. In this work, we evaluated a series of azasteroids for inhibition of 3Β-HSD. Our structure-activity studies indicate that the 6-aza version of cholesterol is the best and tightest binding competitive inhibitor (Ki = 100 nM) of the steroid substrate and are consistent with cholesterol being the preferred substrate of M. tuberculosis 3Β-HSD. The intracellular growth (igr) operon is required for in vitro growth using cholesterol as a sole carbon source. The function of igr operon and its role in cholesterol metabolism is yet to be established. Here we describe the biosynthetic preparation of isotopically labeled 13C- [1,7,15,22,26]-cholesterol and employ it as a tool to investigate the cholesterol-derived metabolite profile of the M. tuberculosis H37Rv Δigr mutant strain by high resolution LC/MS. Culture supernatants from the Δigr mutant accumulate a cholesterol-derived metabolite not observed in H37Rv wild type or complemented strains. Multidimensional NMR and mass spectral analysis revealed the structure of this cholesterol-derived catabolite to be a late stage metabolic product: methyl 1Β-(2'-propanoate)-3aΑ-H-4Α(3'-propanoic acid)-7aΑ-methylhexahydro-5-indanone. The computationally annotated functions of the six genes of the igr operon are a lipid transfer protein (ltp2/Rv3540c), two (R)-specific enoyl-CoA hydratases (Rv3541c and Rv3542c), two acyl-CoA dehydrogenases (fadE29/Rv3543c and fadE28/Rv3544c), and a cytochrome P450 (cyp125/Rv3545c). Heterologous expression in E. coli demonstrated that FadE28 forms a heteromeric complex with FadE29, and that likewise, Rv3542c forms a heteromeric complex with Rv3541c. Biophysical characterization of each complex established they form novel Α2Β2 heterotetramers. Using synthetic substrates analogous to the metabolite identified in M. tuberculosis H37Rv Δigr mutant strain, we verified the catalytic activity of the purified, recombinant FadE28-FadE29 and Rv3541c-Rv3542c protein complexes to be dehydrogenation and hydration of the 2'-propanoate-CoA side chain. We conclude the igr operon is required for degradation of the 2'-propanoate side chain fragment during metabolism of cholesterol by M. tuberculosis.