Structural and Functional Characterization of a Ketosteroid Transcriptional Regulator of Mycobacterium tuberculosis

Crowe, Adam M.; Stogios, P.J.; Casabon, Israël; Evdokimova, E.; Savchenko, Alexei; Eltis, Lindsay D.

doi:10.1074/jbc.m114.607481

Cited by 37 publications

(35 citation statements)

References 47 publications

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“…Curiously, neither 4-BNC nor 3OChA induced DNA release despite the previous report that the latter is able to bind to M. smegmatis KstR (19). Moreover, neither CoASH nor HIP-CoA, a downstream metabolite in which the side chain and ring A are completely degraded (15,20), acted as KstR effectors. Finally, 3-HSBNC-CoA, the equivalent of 4-BNC-CoA with a cleaved ring B, also did not act as an effector (Fig.…”

Section: Resultsmentioning

confidence: 68%

“…3ChO-CoA was quantified by recording its spectrum in water and assuming the same extinction coefficient as for 4-BNC-CoA (⑀ 248 ϭ 17,200 M Ϫ1 cm Ϫ1 (21)). 3a␣-H-4␣(3Ј-propanoyl-CoA)-7a␤-methylhexahydro-1,5-indanedione (HIP-CoA) was prepared as reported previously (15,20).…”

Section: Methodsmentioning

confidence: 99%

“…Electrophoretic Mobility Shift Assay (EMSA)-A dsDNA probe of the M. tuberculosis KstR operator sequence in the intergenic region between Rv3573c and Rv3574 (Table 1) was prepared by heating complementary single-stranded DNA oligomers to 95°C and annealing at room temperature in 20 mM Tris-HCl, 10 mM MgCl 2 , pH 8.0, 75 mM NaCl as described previously (20). Binding assays were performed in 20 mM HEPES, pH 8.0, 10 mM MgCl 2 , 75 mM NaCl, 10% glycerol and contained 6 pmol of DNA probe, 12 pmol of M. tuberculosis KstR, and 0.2 mol of each potential inducer.…”

Section: Methodsmentioning

confidence: 99%

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The Structure of the Transcriptional Repressor KstR in Complex with CoA Thioester Cholesterol Metabolites Sheds Light on the Regulation of Cholesterol Catabolism in Mycobacterium tuberculosis

Dawes

Crowe³

et al. 2016

Journal of Biological Chemistry

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Cholesterol can be a major carbon source for Mycobacterium tuberculosis during infection, both at an early stage in the macrophage phagosome and later within the necrotic granuloma. KstR is a highly conserved TetR family transcriptional repressor that regulates a large set of genes responsible for cholesterol catabolism. Many genes in this regulon, including kstR, are either induced during infection or are essential for survival of M. tuberculosis in vivo. In this study, we identified two ligands for KstR, both of which are CoA thioester cholesterol metabolites with four intact steroid rings. A metabolite in which one of the rings was cleaved was not a ligand. We confirmed the ligand-protein interactions using intrinsic tryptophan fluorescence and showed that ligand binding strongly inhibited KstR-DNA binding using surface plasmon resonance (IC 50 for Tuberculosis remains a global threat to human health due to the emergence of extremely drug-resistant forms of Mycobacterium tuberculosis and co-infection with HIV (1). Elucidation of metabolic networks essential to the pathogenesis of M. tuberculosis is an important part of the quest to identify new drug target candidates that can be exploited to tackle extremely drug-resistant tuberculosis. In particular, M. tuberculosis possesses an unusual ability to metabolize lipids, and its genome is heavily orientated toward this task with the involvement of a remarkable number of genes, ϳ250, constituting around 6% of its genome (2).Cholesterol, the dominant lipid accumulated in M. tuberculosis-induced foamy macrophages, is a key growth substrate for M. tuberculosis in the intraphagosomal environment and is also important to the bacterium in necrotizing granulomas in late stage infection (3-5). M. tuberculosis is capable of utilizing cholesterol as a sole carbon source for both energy synthesis and assimilation into membrane lipids (3, 6). Many genes in the cholesterol degradation pathway are up-regulated or are essential for M. tuberculosis infection in various models of disease (3,4,(7)(8)(9)(10)(11). Disruption of genes encoding cholesterol catabolic enzymes, either through genetic manipulation or chemically, inhibits growth of the bacterium in both macrophage and animal models, and in vitro experiments suggest that this growth inhibition may be due to the toxicity of accumulated intermediates (4, 9, 12).The cholesterol degradation pathway is regulated by two TFRs, 7 KstR and KstR2 (13,14). These regulate two distinct regulons and negatively autoregulate their own expression. KstR regulates the expression of the genes involved in the trans-

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Section: Resultsmentioning

confidence: 68%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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The Structure of the Transcriptional Repressor KstR in Complex with CoA Thioester Cholesterol Metabolites Sheds Light on the Regulation of Cholesterol Catabolism in Mycobacterium tuberculosis

Dawes

Crowe³

et al. 2016

Journal of Biological Chemistry

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“…KstR2 regulates the transcription of genes encoding C/D ring degrading enzymes. The regulators are derepressed by CoA metabolites in the pathway (12,13). We found that none of these genes are required for potentiating INH activity by 4a against Mtb when grown with either cholesterol or glycerol as the carbon source (Table 2).…”

Section: -Azasteroids Are Synergistic With Inh or Bdqmentioning

confidence: 91%

“…At least 52 cholesterolregulated genes are clustered within the "Cho-region" of the Mtb genome and encode the requisite enzymes needed for catabolism (2,8). Two TetR family regulators: KstR1 and KstR2 control transcription of the majority of these catabolism genes (10,11) and are derepressed by coenzyme-A (CoA) metabolites of the catabolism pathway (12,13).…”

Section: Introductionmentioning

confidence: 99%

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

Yang¹,

Yuan²,

Ma³

et al. 2018

Preprint

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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.

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Synthesis of DOHNAA, a Mycobacterium tuberculosis Cholesterol CD Ring Catabolite and FadD3 Substrate

et al. 2015

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DOHNAA (2) is a key catabolite in the Mycobacterium tuberculosis (Mtb) cholesterol degradation pathway. The CoA ester of 2 has been implicated in regulation of gene transcription that is ultimately responsible for degradation of the C and D rings of cholesterol. A synthetic route to 2 is reported here, by a key DMSO‐mediated Morita–Bayliss–Hillman‐type alkylation of the Hajos–Parrish dione. As DOHNAA has to date only been available from microbial sources in very small quantities, the synthesis described will enable further studies of the enzymes involved in cholesterol degradation in Mtb and facilitate ongoing structure–activity studies based on this compound scaffold.

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Structural and Functional Characterization of a Ketosteroid Transcriptional Regulator of Mycobacterium tuberculosis

Cited by 37 publications

References 47 publications

The Structure of the Transcriptional Repressor KstR in Complex with CoA Thioester Cholesterol Metabolites Sheds Light on the Regulation of Cholesterol Catabolism in Mycobacterium tuberculosis

The Structure of the Transcriptional Repressor KstR in Complex with CoA Thioester Cholesterol Metabolites Sheds Light on the Regulation of Cholesterol Catabolism in Mycobacterium tuberculosis

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

Synthesis of DOHNAA, a Mycobacterium tuberculosis Cholesterol CD Ring Catabolite and FadD3 Substrate

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