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

Fragment-Based Approach to Targeting Inosine-5′-monophosphate Dehydrogenase (IMPDH) from <i>Mycobacterium tuberculosis</i>

Tuberculosis (TB) remains a major cause of mortality worldwide, and improved treatments are needed to combat emergence of drug resistance. Inosine 5′-monophosphate dehydrogenase (IMPDH), a crucial enzyme required for <i>de novo</i> synthesis of guanine nucleotides, is an attractive TB drug target. Herein, we describe the identification of potent IMPDH inhibitors using fragment-based screening and structure-based design techniques. Screening of a fragment library for <i>Mycobacterium thermoresistible</i> (<i>Mth</i>) IMPDH ΔCBS inhibitors identified a low affinity phenylimidazole derivative. X-ray crystallography of the <i>Mth</i> IMPDH ΔCBS–IMP–inhibitor complex revealed that two molecules of the fragment were bound in the NAD binding pocket of IMPDH. Linking the two molecules of the fragment afforded compounds with more than 1000-fold improvement in IMPDH affinity over the initial fragment hit

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

Design and implementation of HTS campaign.

<p><b>A</b>) Plate layout for 320-array with four sets of controls (n = 16 for each) in columns 1, 2, 23, and 24. <b>B</b>) Results from Blank Plate Validation using DMSO alone in all 320 assay wells.</p

Screening results.

<p><b>A</b>) Activity of compounds from LISSP4 (grey) and Diversity (black) libraries shown as percent inhibition against PanC_MTB. <b>B</b>) Concentration response curves (CRCs) for two representative hits and nafronyl oxalate. <b>C</b>) Structures of compound <b>1</b> and <b>2</b>.</p

The resuscitation-promoting factors of are required for virulence and resuscitation from dormancy but are collectively dispensable for growth -2

<p><b>Copyright information:</b></p><p>Taken from "The resuscitation-promoting factors of are required for virulence and resuscitation from dormancy but are collectively dispensable for growth "</p><p></p><p>Molecular Microbiology 2007;67(3):672-684.</p><p>Published online 21 Dec 2007</p><p>PMCID:PMC2229633.</p><p>© 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd</p

Single-time point fluorescence assay for PanC.

<p><b>A</b>) Reaction schematic. Upper panel shows the reaction catalyzed by PanC and the enzyme cascade that is initiated by the reaction product AMP resulting in βNADH oxidation. Lower panel shows the final βNADH dependent fluorescence generating reaction that is coupled to the PanC-initiated enzyme cascade. <b>B</b>) Low-throughput assay: kinetic reaction monitoring the rate of βNADH oxidation. <b>C</b>) High-throughput assay: fluorescent resorufin signal generated by residual βNADH following the PanC initiated enzyme cascade. <b>D</b>) Fluorescence generated with varied βNADH in solution using the same conditions as in (B).</p

Biochemical characterization of Class 1 compounds.

<p>Michaelis-Menten plots with varied concentrations of pantoate and <b>A</b>) compound <b>1</b> and <b>B</b>) compound <b>2</b>. Graphpad Prism was used to fit the data to nonlinear regressions.</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

Chemical validation of Mycobacterium tuberculosis phosphopantetheine adenylyltransferase using fragment linking and CRISPR interference.

The coenzyme A (CoA) biosynthesis pathway has attracted attention as a potential target for much-needed novel antimicrobial drugs, including for the treatment of tuberculosis (TB), the lethal disease caused by Mycobacterium tuberculosis (Mtb). Seeking to identify inhibitors of Mtb phosphopantetheine adenylyltransferase (MtbPPAT), the enzyme that catalyses the penultimate step in CoA biosynthesis, we performed a fragment screen. In doing so, we discovered three series of fragments that occupy distinct regions of the MtbPPAT active site, presenting a unique opportunity for fragment linking. Here we show how, guided by X-ray crystal structures, we could link weakly-binding fragments to produce an active site binder with a KD < 20 µM and on-target anti-Mtb activity, as demonstrated using CRISPR interference. This study represents a big step toward validating MtbPPAT as a potential drug target and designing a MtbPPAT-targeting anti-TB drug