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

Glucose Phosphorylation Is Required for <em>Mycobacterium tuberculosis</em> Persistence in Mice

<div><p><em>Mycobacterium tuberculosis</em> (Mtb) is thought to preferentially rely on fatty acid metabolism to both establish and maintain chronic infections. Its metabolic network, however, allows efficient co-catabolism of multiple carbon substrates. To gain insight into the importance of carbohydrate substrates for Mtb pathogenesis we evaluated the role of glucose phosphorylation, the first reaction in glycolysis. We discovered that Mtb expresses two functional glucokinases. Mtb required the polyphosphate glucokinase PPGK for normal growth on glucose, while its second glucokinase GLKA was dispensable. ¹³C-based metabolomic profiling revealed that both enzymes are capable of incorporating glucose into Mtb's central carbon metabolism, with PPGK serving as dominant glucokinase in wild type (wt) Mtb. When both glucokinase genes, <em>ppgK</em> and <em>glkA</em>, were deleted from its genome, Mtb was unable to use external glucose as substrate for growth or metabolism. Characterization of the glucokinase mutants in mouse infections demonstrated that glucose phosphorylation is dispensable for establishing infection in mice. Surprisingly, however, the glucokinase double mutant failed to persist normally in lungs, which suggests that Mtb has access to glucose <em>in vivo</em> and relies on glucose phosphorylation to survive during chronic mouse infections.</p> </div

Both PPGK and GLKA mediate glucose incorporation into central carbon metabolism.

<p>Schematic illustration of the metabolic pathways studied using carbon tracing analysis and isotopic incorporation of U¹³C-glucose into the intracellular pool of selected metabolites. Isotopic labeling is indicated on the y-axis as nmol labeled/mg protein/16 h labeling interval. Each bar represents the mean of three sample replicates and error bars indicate standard deviation from the mean. nd = not detected. * P≤0.05, ** P≤0.01, *** P≤0.001. Data are representative of two independent experiments.</p

PpgK is required for normal growth with glucose as sole carbon source.

<p>Growth of wt Mtb (A) and <i>ΔppgK</i> (B) in carbon defined media with 0.4% glycerol (squares), 0.4% glucose (circles) or no carbon (asterisks). Open circles depict growth of the complemented mutant in glucose containing medium. (C) Growth of wt and <i>ΔppgK</i> in response to increasing glucose concentration. Data represent two to three independent experiments.</p

Rv0650 (GLKA) can mediate growth on glucose.

<p>(A) Growth of <i>ΔppgK</i> and <i>ΔppgK</i> complemented with <i>ppgK</i> on an integrative plasmid or with <i>glkA</i> on an episomal plasmid in carbon defined media with 0.4% glucose. (B, C) Growth of <i>ΔglkA</i> (B) and <i>ΔppgKΔglkA</i> (C) in carbon defined media with 0.4% glycerol (squares), 0.4% glucose (circles) or no carbon (asterisks). Open circles depict growth of the <i>ΔppgKΔglkA</i> mutant complemented with <i>ppgK</i> in glucose containing medium. (D) Glucose dose response in liquid media of wt, <i>ΔglkA</i> and <i>ΔppgK ΔglkA</i>. (E, F) Growth of wt (E) and <i>ΔppgKΔglkA</i> (F) in carbon defined media with 0.2% acetate and 0.2% acetate+0.2% glucose. Data represent two to three independent experiments.</p

Gluokinases are dispensable for trehalose metabolism and survival during starvation.

<p>(A) Growth in carbon defined media with 0.4% trehalose. (B) Survival of wt, <i>ΔglkA</i>, <i>ΔppgK</i>, and <i>ΔppgKΔglkA</i> in PBS. (C) Survival of <i>ΔppgKΔglkA</i> in carbon defined media containing 0.4% glucose or no carbon.</p

The glucokinase double mutant is hypersusceptible to hydrogen peroxide, but attenuated in phagocyte oxidase deficient mice.

<p>(A) Susceptibility to low pH, reactive nitrogen intermediates and hydrogen peroxide. Strains were exposed to pH 4.5 for 6 days, to 3 mM NaNO₂ at pH 5.5 for 3 days and to 5 mM H₂O₂ for 4 hrs and bacterial survival was determined by plating CFU. Data are means ± s.d. of triplicate cultures. Hypersusceptibility of <i>ΔppgKΔglkA</i> to 5 mM H₂O₂ was demonstrated in three independent experiments. (B) Bacterial titers in lungs of gp96^phox−/− mice infected with wt and <i>ΔppgKΔglkA</i>. Data are means ± s.d. from three mice per time point per group and representative of two independent experiments.</p

Glucose phosphorylation is required for mycobacterial persistence in lungs of chronically infected mice.

<p>Bacterial titers in lungs (A) and spleens (B) from mice infected with wt, glucokinase mutants and complemented mutant. Data are means ± s.d. from four mice per time point per group and represent two independent experiments.</p

Nitazoxanide Disrupts Membrane Potential and Intrabacterial pH Homeostasis of <i>Mycobacterium tuberculosis</i>

Nitazoxanide (Alinia), a nitro-thiazolyl antiparasitic drug, kills diverse microorganisms by unknown mechanisms. Here we identified two actions of nitazoxanide against <i>Mycobacterium tuberculosis</i> (Mtb): disruption of Mtb’s membrane potential and pH homeostasis. Both actions were shared by a structurally related antimycobacterial compound, niclosamide. Reactive nitrogen intermediates were reported to synergize with nitazoxanide and its deacetylated derivative tizoxanide in killing Mtb. Herein, however, we could not attribute this to increased uptake of nitazoxanide or tizoxanide as monitored by targeted metabolomics, nor to increased impact of nitazoxanide on Mtb’s membrane potential or intrabacterial pH. Thus, further mechanisms of action of nitazoxanide or tizoxanide may await discovery. The multiple mechanisms of action may contribute to Mtb’s ultralow frequency of resistance against nitazoxanide

Fig3B_Glycine

Microsoft excel file containing raw data for Fig3B Glycine