2-Mercapto-Quinazolinones as Inhibitors of Type II NADH Dehydrogenase and Mycobacterium tuberculosis:Structure-Activity Relationships, Mechanism of Action and Absorption, Distribution, Metabolism, and Excretion Characterization

<i>Mycobacterium tuberculosis</i> (<i>MTb</i>) possesses two nonproton pumping type II NADH dehydrogenase (NDH-2) enzymes which are predicted to be jointly essential for respiratory metabolism. Furthermore, the structure of a closely related bacterial NDH-2 has been reported recently, allowing for the structure-based design of small-molecule inhibitors. Herein, we disclose <i>MTb</i> whole-cell structure–activity relationships (SARs) for a series of 2-mercapto-quinazolinones which target the <i>ndh</i> encoded NDH-2 with nanomolar potencies. The compounds were inactivated by glutathione-dependent adduct formation as well as quinazolinone oxidation in microsomes. Pharmacokinetic studies demonstrated modest bioavailability and compound exposures. Resistance to the compounds in <i>MTb</i> was conferred by promoter mutations in the alternative nonessential NDH-2 encoded by <i>ndhA</i> in <i>MTb</i>. Bioenergetic analyses revealed a decrease in oxygen consumption rates in response to inhibitor in cells in which membrane potential was uncoupled from ATP production, while inverted membrane vesicles showed mercapto-quinazolinone-dependent inhibition of ATP production when NADH was the electron donor to the respiratory chain. Enzyme kinetic studies further demonstrated noncompetitive inhibition, suggesting binding of this scaffold to an allosteric site. In summary, while the initial <i>MTb</i> SAR showed limited improvement in potency, these results, combined with structural information on the bacterial protein, will aid in the future discovery of new and improved NDH-2 inhibitors

<i>Mycobacterium tuberculosis</i> arrests host cycle at the G₁/S transition to establish long term infection

<div><p>Signals modulating the production of <i>Mycobacterium tuberculosis (Mtb</i>) virulence factors essential for establishing long-term persistent infection are unknown. The WhiB3 redox regulator is known to regulate the production of <i>Mtb</i> virulence factors, however the mechanisms of this modulation are unknown. To advance our understanding of the mechanisms involved in WhiB3 regulation, we performed <i>Mtb in vitro</i>, intraphagosomal and infected host expression analyses. Our <i>Mtb</i> expression analyses in conjunction with extracellular flux analyses demonstrated that WhiB3 maintains bioenergetic homeostasis in response to available carbon sources found <i>in vivo</i> to establish <i>Mtb</i> infection. Our infected host expression analysis indicated that WhiB3 is involved in regulation of the host cell cycle. Detailed cell-cycle analysis revealed that <i>Mtb</i> infection inhibited the macrophage G₁/S transition, and polyketides under WhiB3 control arrested the macrophages in the G₀-G₁ phase. Notably, infection with the <i>Mtb whiB3</i> mutant or polyketide mutants had little effect on the macrophage cell cycle and emulated the uninfected cells. This suggests that polyketides regulated by <i>Mtb</i> WhiB3 are responsible for the cell cycle arrest observed in macrophages infected with the wild type <i>Mtb</i>. Thus, our findings demonstrate that <i>Mtb</i> WhiB3 maintains bioenergetic homeostasis to produce polyketide and lipid cyclomodulins that target the host cell cycle. This is a new mechanism whereby <i>Mtb</i> modulates the immune system by altering the host cell cycle to promote long-term persistence. This new knowledge could serve as the foundation for new host-directed therapeutic discovery efforts that target the host cell cycle.</p></div

Model whereby <i>Mtb</i> arrests the macrophage cell cycle to establish long term infection.

<p><i>Mtb</i> WhiB3 is a redox sensor that senses NO and low levels of oxygen (hypoxia) along with <i>in vivo</i> carbon sources available and accordingly modulates bioenergetic metabolism in response to the immediate environment. Bioenergetic homeostasis is essential for the transcription and production of polyketides under WhiB3 control. Lipids and polyketides are released from <i>Mtb</i> into the infected macrophage, where they arrest the host’s cell cycle and modulate the immune response to establish a persistent infection.</p

WhiB3 regulates transcription of respiration, lipid and intermediary metabolism in <i>Mtb</i>.

<p>Circos plot of fold change in gene expression in <i>MtbΔwhiB3</i> relative to that in wt <i>Mtb</i> H37Rv, p<0.05, expression ratio >1.52 or <0.66. p values were calculated with a 2-way ANOVA. Fold changes were calculated from the means of three experiments performed in triplicate. Functional group categories are depicted by the colored segments and annotated on the outer rim of the plot. Sub-categorization is annotated on the inside of the plot. Of note is that WhiB3 controls the expression of 50 genes involved in intermediary metabolism and respiration and 19 genes implicated in lipid metabolism. See <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006389#ppat.1006389.s004" target="_blank">S1</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006389#ppat.1006389.s005" target="_blank">S2</a> Tables.</p

Pathway mapping of <i>Mtb</i> infected macrophages reveals that <i>Mtb</i> WhiB3 regulates the host cell cycle.

<p><b>(A)</b> Top ten host pathways with the most significant differential regulation in <i>MtbΔwhiB3</i> infected RAW264.7 macrophages relative to wt <i>Mtb</i> H37Rv infected macrophages (p values were calculated with <i>t</i> tests). See <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006389#ppat.1006389.s003" target="_blank">S2 Fig</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006389#ppat.1006389.s008" target="_blank">S5</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006389#ppat.1006389.s009" target="_blank">S6</a> Tables. <b>(B)</b> LC-MS/MS identification and quantification of cytoplasmic proteins with more than 2-fold differential expression (p<0.03, ANOVA) in <i>MtbΔwhiB3</i> infected RAW264.7 macrophages relative to wt <i>Mtb</i> H37Rv infected macrophages. Fold changes were calculated from the means of samples in triplicate. See <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006389#ppat.1006389.s010" target="_blank">S7 Table</a>. <b>(C)</b> DNA synthesis in RAW264.7 macrophages infected with wt <i>Mtb</i> H37Rv, <i>MtbΔwhiB3</i> and the complemented (comp) strain (MOI 5) for 24 h. Cells were stained with DAPI (blue) to identify the nuclei and BrdU was added to the cells and incorporated into newly synthesized DNA. <b>(D)</b> Representative dot plots of BrdU incorporation versus surface staining with anti-CD3⁺ (lymphocyte marker), anti-CD11b⁺ and anti-CD11c⁺ (myeloid lineage markers) of lung cells isolated from mice infected with wt <i>Mtb</i>, <i>MtbΔwhiB3</i> or the complemented strain for 6 weeks. CD11b⁺CD11c⁺ double positive cells were also gated and evaluated for BrdU incorporation. <b>(E)</b> Percentage of CD3⁺, CD11b⁺, CD11c⁺ or CD11b⁺CD11c⁺ cells isolated from mouse lungs infected with wt <i>Mtb</i>, <i>MtbΔwhiB3</i> or the complemented strain that are positive for BrdU incorporation. (<b>F</b>) Percentage of total CD3⁺, CD11b⁺, CD11c⁺ or CD11b⁺CD11c⁺ cells isolated from mouse lungs infected with wt <i>Mtb</i>, <i>MtbΔwhiB3</i> or the complemented strain. Data are representative of two independent experiments with three mice per group and were analyzed using the unpaired Student’s <i>t</i> test. For the comparison of <i>MtbΔwhiB3</i> infected mice versus any of the other mouse groups: * p<0.01.</p

Infection with <i>Mtb</i> polyketide mutants do not alter the macrophage cell cycle.

<p><b>(A)</b> Representative dot plots of BrdU and PI incorporation into RAW264.7 macrophages infected with wt <i>Mtb</i>, <i>Mtb Tn</i>:<i>pks2</i>, <i>Mtb Tn</i>:<i>mas</i> or <i>Mtb Tn</i>:<i>ppsA</i> at MOI 5 for the indicated time points. <b>(B-D)</b> Percentage of RAW264.7 cells infected with wt <i>Mtb</i> CDC1551, <i>Mtb Tn</i>:<i>pks2</i>, <i>Mtb Tn</i>:<i>mas</i> or <i>Mtb Tn</i>:<i>ppsA</i>, at MOI 5 for the indicated time points in the <b>(B)</b> G₀-G₁ phase, <b>(C)</b> S phase and <b>(D)</b> G₂-M phases of the macrophage cell cycle. Error bars represent SD from the mean of triplicate experiments. Data are representative of two independent experiments. Unpaired Student’s <i>t</i> Test was used to calculate the p value. #, p≤0.0001; **, p≤0.001; *, p≤0.05; symbols above the bars indicates comparison to macrophages infected with wt <i>Mtb</i>.</p

<i>Mtb</i> polyketides and cell wall surface molecules under WhiB3 control modulate the host cell cycle.

<p>RAW264.7 macrophages <b>(M</b>; untreated) were treated in triplicate with <b>(B)</b> MAME, <b>(C)</b> PDIM, <b>(E)</b> PIM 1,2, <b>(F)</b> SL-1, <b>(H)</b> <i>Mtb</i> total lipid extract, or <b>(I)</b> TDM at the concentrations indicated for 24 h prior to cell cycle analysis. RAW264.7 cells treated with the solvents in which the lipids were dissolved <b>(A, D, G)</b> were used as controls. <b>(J)</b> Percentage of RAW264.7 cells in the sub-G₀, G₀-G₁, S and G₂-M phases of the cell cycle following treatment with 1 μg/ml SL-1, PDIM, PIM 1,2, MAME, TDM, total lipids or vehicle (0.1% DMSO) for 24 h, <b>(K, N)</b> combinations of SL-1 and PDIM at the concentrations indicated for 24 h and <b>(O, L)</b> combinations of PIM 1,2 and PDIM at the indicated concentrations for 24 h. Error bars represent SD of the mean of triplicate experiments. Data are representative of two independent experiments. Unpaired Student’s t-Test was used to calculate the p value. #, p<0.0001; **, p≤0.005 and *, p≤0.05.</p

<i>Mtb</i> WhiB3 modulates the host cell cycle.

<p><b>(A)</b> Schematic diagram of the host cell cycle. <b>(B)</b> Schematic diagram of the dot plot of BrdU versus PI indicating the gating strategy. <b>(C)</b> Representative dot plots of BrdU and PI incorporation into RAW264.7 macrophages infected with wt <i>Mtb</i> H37Rv, <i>MtbΔwhiB3</i> or the complemented strain at MOI 5 for the indicated time periods. <b>(D-F)</b> Percentage of RAW264.7 cells infected with wt <i>Mtb</i>, <i>MtbΔwhiB3</i> or the complemented strain at MOI 5 for the indicated time points in the <b>(D)</b> G₀-G₁ phase, <b>(E)</b> S phase and <b>(F)</b> G₂-M phases of the macrophage cell cycle. Error bars represent SD of the mean of triplicate experiments. Data are representative of two independent experiments. Unpaired Student’s t-Test was used to calculate the p value. #, p<0.0001; **, p≤0.001 and * p≤0.05.</p

Role of WhiB3 in establishment of mycobacterial infection in macrophages.

<p>Circos plot of WhiB3-dependent responses to intraphagosomal survival, p<0.05 (One way Anova) (>1.2x fold change) in BMDM. The analysis made a direct comparison of transcript levels of intracellular <i>MtbΔwhiB3</i> relative to intracellular wt <i>Mtb</i>. Functional group categories are depicted by the colored segments and annotated on the outer rim of the plot. Sub-categorization is annotated on the inside of the plot. See <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006389#ppat.1006389.s007" target="_blank">S4 Table</a>.</p

image_5_Ferritin H Deficiency in Myeloid Compartments Dysregulates Host Energy Metabolism and Increases Susceptibility to Mycobacterium tuberculosis Infection.tif

<p>Iron is an essential factor for the growth and virulence of Mycobacterium tuberculosis (Mtb). However, little is known about the mechanisms by which the host controls iron availability during infection. Since ferritin heavy chain (FtH) is a major intracellular source of reserve iron in the host, we hypothesized that the lack of FtH would cause dysregulated iron homeostasis to exacerbate TB disease. Therefore, we used knockout mice lacking FtH in myeloid-derived cell populations to study Mtb disease progression. We found that FtH plays a critical role in protecting mice against Mtb, as evidenced by increased organ burden, extrapulmonary dissemination, and decreased survival in Fth^−/− mice. Flow cytometry analysis showed that reduced levels of FtH contribute to an excessive inflammatory response to exacerbate disease. Extracellular flux analysis showed that FtH is essential for maintaining bioenergetic homeostasis through oxidative phosphorylation. In support of these findings, RNAseq and mass spectrometry analyses demonstrated an essential role for FtH in mitochondrial function and maintenance of central intermediary metabolism in vivo. Further, we show that FtH deficiency leads to iron dysregulation through the hepcidin–ferroportin axis during infection. To assess the clinical significance of our animal studies, we performed a clinicopathological analysis of iron distribution within human TB lung tissue and showed that Mtb severely disrupts iron homeostasis in distinct microanatomic locations of the human lung. We identified hemorrhage as a major source of metabolically inert iron deposition. Importantly, we observed increased iron levels in human TB lung tissue compared to healthy tissue. Overall, these findings advance our understanding of the link between iron-dependent energy metabolism and immunity and provide new insight into iron distribution within the spectrum of human pulmonary TB. These metabolic mechanisms could serve as the foundation for novel host-directed strategies.</p