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

Conformationally Constrained Cinnolinone Nucleoside Analogues as Siderophore Biosynthesis Inhibitors for Tuberculosis

5′-<i>O</i>-[<i>N</i>-(Salicyl)­sulfamoyl]­adenosine (Sal-AMS, <b>1</b>) is a nucleoside antibiotic that inhibits incorporation of salicylate into siderophores required for bacterial iron acquisition and has potent activity against <i>Mycobacterium tuberculosis</i> (<i>Mtb</i>). Cinnolone analogues exemplified by <b>5</b> were designed to replace the acidic acyl-sulfamate functional group of <b>1</b> (p<i>K</i>_a = 3) by a more stable sulfonamide linkage (p<i>K</i>_a = 6.0) in an attempt to address potential metabolic liabilities and improve membrane permeability. We showed <b>5</b> potently inhibited the mycobacterial salicylate ligase MbtA (apparent <i>K</i>_i = 12 nM), blocked production of the salicylate-capped siderophores in whole-cell <i>Mtb</i>, and exhibited excellent antimycobacterial activity under iron-deficient conditions (minimum inhibitor concentration, MIC = 2.3 μM). To provide additional confirmation of the mechanism of action, we demonstrated the whole-cell activity of <b>5</b> could be fully antagonized by the addition of exogenous salicylate to the growth medium. Although the total polar surface area (tPSA) of <b>5</b> still exceeds the nominal threshold value (140 Å) typically required for oral bioavailability, we were pleasantly surprised to observe introduction of the less acidic and conformationally constrained cinnolone moiety conferred improved drug disposition properties as evidenced by the 7-fold increase in volume of distribution in Sprague–Dawley rats

Validation of CoaBC as a Bactericidal Target in the Coenzyme A Pathway of Mycobacterium tuberculosis

Mycobacterium tuberculosis relies on its own ability to biosynthesize coenzyme A to meet the needs of the myriad enzymatic reactions that depend on this cofactor for activity. As such, the essential pantothenate and coenzyme A biosynthesis pathways have attracted attention as targets for tuberculosis drug development. To identify the optimal step for coenzyme A pathway disruption in M. tuberculosis, we constructed and characterized a panel of conditional knockdown mutants in coenzyme A pathway genes. Here, we report that silencing of <i>coaBC</i> was bactericidal in vitro, whereas silencing of <i>panB</i>, <i>panC</i>, or <i>coaE</i> was bacteriostatic over the same time course. Silencing of <i>coaBC</i> was likewise bactericidal in vivo, whether initiated at infection or during either the acute or chronic stages of infection, confirming that CoaBC is required for M. tuberculosis to grow and persist in mice and arguing against significant CoaBC bypass via transport and assimilation of host-derived pantetheine in this animal model. These results provide convincing genetic validation of CoaBC as a new bactericidal drug target

Expanding Benzoxazole-Based Inosine 5′-Monophosphate Dehydrogenase (IMPDH) Inhibitor Structure–Activity As Potential Antituberculosis Agents

New drugs and molecular targets are urgently needed to address the emergence and spread of drug-resistant tuberculosis. <i>Mycobacterium tuberculosis</i> (<i>Mtb</i>) inosine 5′-monophosphate dehydrogenase 2 (<i>Mtb</i>IMPDH2) is a promising yet controversial potential target. The inhibition of <i>Mtb</i>IMPDH2 blocks the biosynthesis of guanine nucleotides, but high concentrations of guanine can potentially rescue the bacteria. Herein we describe an expansion of the structure–activity relationship (SAR) for the benzoxazole series of <i>Mtb</i>IMPDH2 inhibitors and demonstrate that minimum inhibitory concentrations (MIC) of ≤1 μM can be achieved. The antibacterial activity of the most promising compound, <b>17b</b> (<b>Q151</b>), is derived from the inhibition of <i>Mtb</i>IMPDH2 as demonstrated by conditional knockdown and resistant strains. Importantly, guanine does not change the MIC of <b>17b</b>, alleviating the concern that guanine salvage can protect <i>Mtb</i> in vivo. These findings suggest that <i>Mtb</i>IMPDH2 is a vulnerable target for tuberculosis

Transcriptional responses to drug treatment reveal that a single gene representing logically associated gene clusters is sufficient for MoA diagnosis in <i>Mycobacterium tuberculosis</i>.

<p>Groups of drugs clustered separately based on the known mechanism of action, while SAR series of compounds with novel MoA (cyclomarin) showed a fingerprint distinct from any of the compounds tested. Periods following drug names represent duplicates. X-axis represents the 90 genes that were chosen from the microarray as representative of 90 clusters, y-axis lists the different drug treatments. i, ii, and iii, represent 3 clusters namely, cell wall inhibitor, RNA polymerase inhibitor and Cyclomarin series, respectively.</p

Pathway enrichment of functional classes of genes on PCR array.

<p>Pathway enrichment of functional classes of genes on PCR array.</p

List of investigational and FDA approved anti-tubercular drugs and their mode of action.

<p>MIC₅₀ listed for anti-tubercular investigational and SAR compounds, when available.</p

Transcriptional responses of <i>M. bovis</i> BCG to anti-tubercular drugs profiled using qPCR.

<p>A compendium of qPCR results can show differences in the MOA of anti-tubercular drugs. (A) Spatial grouping of drug expression profiles on a dendrogram after qPCR profiling using hierarchical clustering. From the list of drugs tested, two major clusters emerge cell wall inhibitors, and those that inhibit DNA/protein synthesis. (B) Similarity matrix of expression profiles for chemical inhibitors using qPCR profiling. The blue-red color scale shows the degree of correlation of drugs expression profiles ranging from −1 to 1 respectively. ×- depicts transcriptional responses at 2× or 0.5× the MIC₅₀, while those that have no × designation were done 1× the MIC₅₀.</p

Hierarchical clustering and correlations of anti-tubercular drugs.

<p>Transcriptional responses data obtained from microfluidic experiment are used for the analysis. The detailed descriptions for genes represented in this figure are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069191#pone.0069191.s003" target="_blank">Table S1</a>. (A) Hierarchical clustering via average linkage of Pearson correlations for FDA approved drugs. The individual genes are represented in y-axis and the compound treatment is in the x-axis. (B) Pearson correlations for 23 anti-tubercular drugs. Correlations were calculated between 2 independent microfluidic experiments, following median normalization to account for plate effects.</p

Expanding Benzoxazole-Based Inosine 5′-Monophosphate Dehydrogenase (IMPDH) Inhibitor Structure–Activity As Potential Antituberculosis Agents

New drugs and molecular targets are urgently needed to address the emergence and spread of drug-resistant tuberculosis. <i>Mycobacterium tuberculosis</i> (<i>Mtb</i>) inosine 5′-monophosphate dehydrogenase 2 (<i>Mtb</i>IMPDH2) is a promising yet controversial potential target. The inhibition of <i>Mtb</i>IMPDH2 blocks the biosynthesis of guanine nucleotides, but high concentrations of guanine can potentially rescue the bacteria. Herein we describe an expansion of the structure–activity relationship (SAR) for the benzoxazole series of <i>Mtb</i>IMPDH2 inhibitors and demonstrate that minimum inhibitory concentrations (MIC) of ≤1 μM can be achieved. The antibacterial activity of the most promising compound, <b>17b</b> (<b>Q151</b>), is derived from the inhibition of <i>Mtb</i>IMPDH2 as demonstrated by conditional knockdown and resistant strains. Importantly, guanine does not change the MIC of <b>17b</b>, alleviating the concern that guanine salvage can protect <i>Mtb</i> in vivo. These findings suggest that <i>Mtb</i>IMPDH2 is a vulnerable target for tuberculosis