Cyclic AMP-Mediated Inhibition of Cholesterol Catabolism in Mycobacterium tuberculosis by the Novel Drug Candidate GSK2556286

Abstract

Despite the deployment of combination tuberculosis (TB) chemotherapy, efforts to identify shorter, nonrelapsing treatments have resulted in limited success. Recent evidence indicates that GSK2556286 (GSK286), which acts via Rv1625c, a membrane-bound adenylyl cyclase in Mycobacterium tuberculosis, shortens treatment in rodents relative to standard of care drugs. Moreover, GSK286 can replace linezolid in the three-drug, Nix-TB regimen. Given its therapeutic potential, we sought to better understand the mechanism of action of GSK286. The compound blocked growth of M. tuberculosis in cholesterol media and increased intracellular cAMP levels ~50-fold. GSK286 did not inhibit growth of an rv1625c transposon mutant in cholesterol media and did not induce cyclic AMP (cAMP) production in this mutant, suggesting that the compound acts on this adenylyl cyclase. GSK286 also induced cAMP production in Rhodococcus jostii RHA1, a cholesterol-catabolizing actinobacterium, when Rv1625c was heterologously expressed. However, these elevated levels of cAMP did not inhibit growth of R. jostii RHA1 in cholesterol medium. Mutations in rv1625c conferred cross-resistance to GSK286 and the known Rv1625c agonist, mCLB073. Metabolic profiling of M. tuberculosis cells revealed that elevated cAMP levels, induced using either an agonist or a genetic tool, did not significantly affect pools of steroid metabolites in cholesterol-incubated cells. Finally, the inhibitory effect of agonists was not dependent on the N-acetyltransferase MtPat. Together, these data establish that GSK286 is an Rv1625c agonist and sheds light on how cAMP signaling can be manipulated as a novel antibiotic strategy to shorten TB treatments. Nevertheless, the detailed mechanism of action of these compounds remains to be elucidated.

Keywords: TB; adenylyl cyclase; antibiotic; antitubercular.

FIG 1

cAMP production by M. tuberculosis and Rhodococcus jostii RHA1 expressing Rv1625c treated with GSK286 (A) LC-MS total ion chromatogram from M. tuberculosis lysates following GSK286 treatment. 8-Br-cAMP (8-bromoadenosine 3′,5′-cyclic monophosphate) was supplied to M. tuberculosis as a control and run as a standard for LC-MS/MS analysis. (B) WT and an rv1625c transposon mutant of M. tuberculosis CDC1551 were grown in 7H9 medium supplemented with 0.2% glycerol, concentrated, and then incubated in 7H9 medium supplemented with 0.5 mM cholesterol with or without 5 μM GSK286. Cells were harvested after 24 h, and cell lysates were analyzed by HPLC. (C) R. jostii RHA1 carrying pTip1625 or empty pTip plasmid was grown in M9 medium supplemented with 0.5 mM cholesterol with or without 5 μM GSK286. Cell lysates were analyzed by HPLC. Data represent the means of three biological replicates.

FIG 2

GSK286 treatment reduces cholesterol-derived propionyl-CoA pools in M. tuberculosis. (A) GSK286 blocks the degradative release of ¹⁴CO₂ from ¹⁴C-cholesterol in wild-type M. tuberculosis during growth in cholesterol-acetate media. (B) The GFP signal from the prpD′::GFP reporter decreases in wild-type M. tuberculosis in an Rv1625c-dependent manner following treatment with GSK286 during growth in cholesterol-acetate media. (C) Representative images of macrophages infected with wild-type M. tuberculosis carrying the prpD′::GFP reporter (green), the constitutive M. tuberculosis mCherry signal (red), and DAPI stain (blue). DMSO-treated (top panel) and GSK286-treated (bottom panel) samples are shown. (D) Flow cytometric quantification of the prpD′::GFP reporter signal M. tuberculosis isolated from infected macrophages. The median GFP signal was quantified from 10,000 bacteria and calculated relative to the DMSO control for each strain of bacteria. Bars represent means ± the standard deviations (SD; n = 4) from two independent replicates with two technical replicates each. (A) **, P < 0.05 (Student t test); (B and D) **, P < 0.05 (ordinary one-way ANOVA with Dunnett’s multiple-comparison test).

FIG 3

Rv1625c is required for GSK286-dependent growth inhibition and block of cholesterol metabolism in M. tuberculosis. (A) MIC curves for GSK286 in M. tuberculosis grown in cholesterol media. Inhibition was calculated using alamarBlue fluorescence with 100% inhibition established using rifampicin treatment (10 μM). All of the inhibition values are normalized to this value. The x-axis values are logarithms of concentrations expressed as a molar unit. (B) GFP fluorescence inhibition curves for GSK286 using the prpD′::GFP reporter in cholesterol-acetate media. The strains included WT (wild-type M. tuberculosis), 2×Rv1625c (WT strain carrying a plasmid expressing rv1625c from an hsp60 promoter), and TnRv1625c (a transposon-disrupted mutant with a nonfunctional Rv1625c). Each point in the fluorescence inhibition curve was obtained by quantifying the GFP signal from 10,000 bacteria, and error bars indicate means ± the SD (n = 4) from two independent replicates with two technical replicates for each.

FIG 4

Spontaneous resistance mutations confer cross-resistance to the Rv1625c agonists. (A) GSK286 MIC assay in cholesterol media with resistant mutants raised against GSK286 on cholesterol. (B) mCLB073 MIC assay in cholesterol media with resistant mutants raised against GSK286 on cholesterol. (C) GSK286 MIC assay in cholesterol media with resistant mutants raised against mCLB073 on cholesterol. The data are from one experiment with two technical replicates (n = 2). Symbols indicate means, and curves display the nonlinear fit of dose response. Inhibition was calculated using alamarBlue fluorescence with 100% inhibition established using rifampicin treatment (10 μM); all inhibition values are normalized to this value. The x-axis values are logarithms of concentrations expressed as a molar unit. (D) Mutations mapped onto the structure of Rv1625c. The overall fold (PDB ID 7YZK) is shown as a cartoon representation with the transmembrane, helical, and catalytic domains shown as different shades of gray with the approximate boundaries of the membrane drawn as gray lines. The location of missense mutations arising from GSK286 treatment in this study (Table 1) are highlighted as magenta sticks. The locations of cross-resistant missense mutations arising from mCLB073 treatment are shown as cyan sticks. The location of all other mutations, including nonsense, insertion, and frameshift mutations conferring resistance to GSK286, are highlighted as yellow sticks.

FIG 5

M. tuberculosis lacking Rv1625c has no fitness defect in a mouse model. (A and B) Bacterial burdens in the lungs (A) and spleens (B) of BALB/c mice infected with ~200 CFU of WT, ΔRv1625c, or complemented strain by the aerosol route. The data are from one experiment with five mice per group for each time point. The data are means ± the SD. **, P < 0.05 (ordinary one-way ANOVA with Dunnett’s multiple-comparison test).