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

Investigation and Conformational Analysis of Fluorinated Nucleoside Antibiotics Targeting Siderophore Biosynthesis

Antibiotic resistance represents one of the greatest threats to public health. The adenylation inhibitor 5′-<i>O</i>-[<i>N</i>-(salicyl)­sulfamoyl]­adenosine (SAL-AMS) is the archetype for a new class of nucleoside antibiotics that target iron acquisition in pathogenic microorganisms and is especially effective against <i>Mycobacterium tuberculosis</i>, the causative agent of tuberculosis. Strategic incorporation of fluorine at the 2′ and 3′ positions of the nucleoside was performed by direct fluorination to enhance activity and improve drug disposition properties. The resulting SAL-AMS analogues were comprehensively assessed for biochemical potency, whole-cell antitubercular activity, and in vivo pharmacokinetic parameters. Conformational analysis suggested a strong preference of fluorinated sugar rings for either a 2′-<i>endo</i>, 3′-<i>exo</i> (South), or a 3′-<i>endo</i>,2′-<i>exo</i> (North) conformation. The structure–activity relationships revealed a strong conformational bias for the C3′-<i>endo</i> conformation to maintain potent biochemical and whole-cell activity, whereas improved pharmacokinetic properties were associated with the C2′-<i>endo</i> conformation

Development of a Selective Activity-Based Probe for Adenylating Enzymes: Profiling MbtA Involved in Siderophore Biosynthesis from <i>Mycobacterium tuberculosis</i>

MbtA is an adenylating enzyme from <i>Mycobacterium tuberculosis</i> that catalyzes the first step in the biosynthesis of the mycobactins. A bisubstrate inhibitor of MbtA (Sal-AMS) was previously described that displays potent antitubercular activity under iron-replete as well as iron-deficient growth conditions. This finding is surprising since mycobactin biosynthesis is not required under iron-replete conditions and suggests off-target inhibition of additional biochemical pathways. As a first step toward a complete understanding of the mechanism of action of Sal-AMS, we have designed and validated an activity-based probe (ABP) for studying Sal-AMS inhibition in <i>M. tuberculosis</i>. This probe labels pure MbtA as well as MbtA in mycobacterial lysate, and labeling can be completely inhibited by preincubation with Sal-AMS. Furthermore, this probe provides a prototypical core scaffold for the creation of ABPs to profile any of the other 66 adenylating enzymes in <i>Mtb</i> or the multitude of adenylating enzymes in other pathogenic bacteria

Absolute Quantitative MALDI Imaging Mass Spectrometry: A Case of Rifampicin in Liver Tissues

Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) elucidates molecular distributions in thin tissue sections. Absolute pixel-to-pixel quantitation has remained a challenge, primarily lacking validation of the appropriate analytical methods. In the present work, isotopically labeled internal standards are applied to tissue sections to maximize quantitative reproducibility and yield accurate quantitative results. We have developed a tissue model for rifampicin (RIF), an antibiotic used to treat tuberculosis, and have tested different methods of applying an isotopically labeled internal standard for MALDI IMS analysis. The application of the standard and subsequently the matrix onto tissue sections resulted in quantitation that was not statistically significantly different from results obtained using HPLC-MS/MS of tissue extracts. Quantitative IMS experiments were performed on liver tissue from an animal dosed <i>in vivo</i>. Each microspot in the quantitative images measures the local concentration of RIF in the thin tissue section. Lower concentrations were detected from the blood vessels and around the portal tracts. The quantitative values obtained from these measurements were comparable (>90% similarity) to HPLC-MS/MS results obtained from extracts of the same tissue

Synthesis and Pharmacokinetic Evaluation of Siderophore Biosynthesis Inhibitors for Mycobacterium tuberculosis

MbtA catalyzes the first committed biosynthetic step of the mycobactins, which are important virulence factors associated with iron acquisition in Mycobacterium tuberculosis. MbtA is a validated therapeutic target for antitubercular drug development. 5′-<i>O</i>-[<i>N</i>-(Salicyl)­sulfamoyl]­adenosine (<b>1</b>) is a bisubstrate inhibitor of MbtA and exhibits exceptionally potent biochemical and antitubercular activity. However, <b>1</b> suffers from suboptimal drug disposition properties resulting in a short half-life (<i>t</i>_1/2), low exposure (AUC), and low bioavailability (<i>F</i>). Four strategies were pursued to address these liabilities including the synthesis of prodrugs, increasing the p<i>K</i>_a of the acyl-sulfonyl moiety, modulation of the lipophilicity, and strategic introduction of fluorine into <b>1</b>. Complete pharmacokinetic (PK) analysis of all compounds was performed. The most successful modifications involved fluorination of the nucleoside that provided substantial improvements in <i>t</i>_1/2 and AUC. Increasing the p<i>K</i>_a of the acyl-sulfonyl linker yielded incremental enhancements, while modulation of the lipophilicity and prodrug approaches led to substantially poorer PK parameters

Non-Nucleoside Inhibitors of BasE, an Adenylating Enzyme in the Siderophore Biosynthetic Pathway of the Opportunistic Pathogen <i>Acinetobacter baumannii</i>

Siderophores are small-molecule iron chelators produced by bacteria and other microorganisms for survival under iron limiting conditions such as found in a mammalian host. Siderophore biosynthesis is essential for the virulence of many important Gram-negative pathogens including <i>Acinetobacter baumannii</i>, <i>Klebsiella pneumoniae</i>, <i>Pseudomonas aeruginosa</i>, and <i>Escherichia coli.</i> We performed high-throughput screening against BasE, which is involved in siderophore biosynthesis in <i>A. baumannii</i>, and identified 6-phenyl-1-(pyridin-4-ylmethyl)-1<i>H</i>-pyrazolo­[3,4-<i>b</i>]­pyridine-4-carboxylic acid <b>15</b>. Herein we report the synthesis, biochemical, and microbiological evaluation of a systematic series of analogues of the HTS hit <b>15</b>. Analogue <b>67</b> is the most potent analogue with a <i>K</i>_D of 2 nM against BasE. Structural characterization of the inhibitors with BasE reveals that they bind in a unique orientation in the active site, occupying all three substrate binding sites, and thus can be considered as multisubstrate inhibitors. These results provide a foundation for future studies aimed at increasing both enzyme potency and antibacterial activity

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

Linking High-Throughput Screens to Identify MoAs and Novel Inhibitors of <i>Mycobacterium tuberculosis</i> Dihydrofolate Reductase

Though phenotypic and target-based high-throughput screening approaches have been employed to discover new antibiotics, the identification of promising therapeutic candidates remains challenging. Each approach provides different information, and understanding their results can provide hypotheses for a mechanism of action (MoA) and reveal actionable chemical matter. Here, we describe a framework for identifying efficacy targets of bioactive compounds. High throughput biophysical profiling against a broad range of targets coupled with machine learning was employed to identify chemical features with predicted efficacy targets for a given phenotypic screen. We validate the approach on data from a set of 55 000 compounds in 24 historical internal antibacterial phenotypic screens and 636 bacterial targets screened in high-throughput biophysical binding assays. Models were built to reveal the relationships between phenotype, target, and chemotype, which recapitulated mechanisms for known antibacterials. We also prospectively identified novel inhibitors of dihydrofolate reductase with nanomolar antibacterial efficacy against <i>Mycobacterium tuberculosis</i>. Molecular modeling provided structural insight into target–ligand interactions underlying selective killing activity toward mycobacteria over human cells

A High-Throughput Screen To Identify Inhibitors of ATP Homeostasis in Non-replicating <i>Mycobacterium tuberculosis</i>

Growing evidence suggests that the presence of a subpopulation of hypoxic non-replicating, phenotypically drug-tolerant mycobacteria is responsible for the prolonged duration of tuberculosis treatment. The discovery of new antitubercular agents active against this subpopulation may help in developing new strategies to shorten the time of tuberculosis therapy. Recently, the maintenance of a low level of bacterial respiration was shown to be a point of metabolic vulnerability in <i>Mycobacterium tuberculosis</i>. Here, we describe the development of a hypoxic model to identify compounds targeting mycobacterial respiratory functions and ATP homeostasis in whole mycobacteria. The model was adapted to 1,536-well plate format and successfully used to screen over 600,000 compounds. Approximately 800 compounds were confirmed to reduce intracellular ATP levels in a dose-dependent manner in <i>Mycobacterium bovis</i> BCG. One hundred and forty non-cytotoxic compounds with activity against hypoxic non-replicating <i>M. tuberculosis</i> were further validated. The resulting collection of compounds that disrupt ATP homeostasis in <i>M. tuberculosis</i> represents a valuable resource to decipher the biology of persistent mycobacteria