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

Bisubstrate Inhibitors of Biotin Protein Ligase in <i>Mycobacterium tuberculosis</i> Resistant to Cyclonucleoside Formation

<i>Mycobacterium tuberculosis</i> (<i>Mtb</i>), the etiological agent of tuberculosis, is the leading cause of bacterial infectious disease mortality. Biotin protein ligase (BirA) globally regulates lipid metabolism in <i>Mtb</i> through the posttranslational biotinylation of acyl coenzyme A carboxylases (ACCs) involved in lipid biosynthesis and is essential for <i>Mtb</i> survival. We previously developed a rationally designed bisubstrate inhibitor of BirA that displays potent enzyme inhibition and whole-cell activity against multidrug resistant and extensively drug resistant <i>Mtb</i> strains. Here we present the design, synthesis, and evaluation of a focused series of inhibitors, which are resistant to cyclonucleoside formation, a key decomposition pathway of our initial analogue. Improved chemical stability is realized through replacement of the adenosyl N-3 nitrogen and C-5′ oxygen atom with carbon as well as incorporation of a bulky group on the nucleobase to prevent the required <i>syn</i>-conformation necessary for proper alignment of N-3 with C-5′

Design and characterization of leaky TrxB2-TetON mutants.

<p>(A) Repression of luciferase activity of leaky tet promoters by TetR in <i>M</i>. <i>smegmatis</i>. The x-axis specifies the promoter that was used to express luciferase and its tetO. Mutated nucleotides are shown in red. The <i>kanR</i> luciferase and <i>hygR</i> TetR plasmids were integrated into the <i>M</i>. <i>smegmatis</i> chromosome at the att-L5 and att-Tweety sites, respectively. Integers on the right indicate fold change in RLUs between bacteria without (gray bars) and with TetR (white bars). Data are means ± SD of eight replicates from at least two independent experiments. (B) Growth of H37Rv and TrxB2-TetON-tetO mutants in 7H9 medium in the presence or absence of atc. (C) Kinetics of TrxB2 depletion in TrxB2-TetON-tetO mutants in the absence of atc. TrxB2 in TrxB2-TetON-tetO mutants is of increased molecular weight due to the C-terminal DAS tag. Results in (B) and (C) are representative of three independent experiments.</p

TrxB2 is essential for <i>Mtb</i> to establish and maintain infection in mice.

<p>(A and B) Quantification of bacterial loads in lungs (A) and spleens (B) of C56BL/6 mice infected with TrxB2-DUC. Mice received doxy-containing food starting at the indicated time points or not at all. Data are means ± SD of four mice per group. The limit of detection was 4 CFU per organ and is indicated by the dashed line. (C) Gross pathology of lungs from infected mice receiving doxy-containing food starting on day 35 or not at all. Lungs were isolated on day 56, 119 and 160 post infection. (D) Haematoxylin/eosin-stained lung tissue sections from mice infected with TrxB2-DUC not treated or treated with doxy-containing food starting on day 35 post infection. Images are representative of the histopathology of the four mice from each group. Scale bar, 1.0 mm. Results are representative of two independent experiments.</p

Impact of partial TrxB2 depletion on susceptibility of <i>Mtb</i> to antimicrobial compounds.

<p>(A and B) Impact of partial TrxB2 depletion on susceptibility of <i>Mtb</i> to TrxB2 inhibitors ebselen (A) and auranofin (B). TrxB2-TetON-tetO-1C was washed and suspended in 7H9 medium without atc, then cultured for 3 days to decrease TrxB2 expression before treatment with ebselen or auranofin. OD₅₈₀ was recorded and normalized to the corresponding strain without drug treatment. (C) Survival of strains after exposure to 0.65 μg/ml or 1.5 μg/ml auranofin. (D and E) Impact of partial TrxB2 depletion on susceptibility of <i>Mtb</i> to vancomycin (D) and rifampicin (E). (F) Heat-map representation of MIC₉₀ shift of partially TrxB2 depleted <i>Mtb</i> to antimicrobial compounds. The MIC₉₀ shifts are shown as the ratio of the MIC₉₀ for H37Rv to the MIC₉₀ for TrxB2-TetON-tetO-1C in the absence of atc. Data in (A) to (E) are means ± SD of three replicates and are representative of three independent experiments. Data shown in (F) are representative of at least two independent experiments.</p

TrxB2 depletion perturbs growth-essential pathways.

<p>(A) Heat-map representation of selected genes with mean expression fold changes >2 in TrxB2-DUC at 6 h post atc treatment (adjusted p<0.02 by one-way ANOVA). (B) Heat-map representation of selected genes with mean expression fold change >3 in TrxB2-DUC at 24 h post atc treatment (adjusted p<0.02 by one-way ANOVA). (C) Impact of extracellular cysteine and methionine on TrxB2 depletion-induced death. Atc-treated TrxB2-DUC cultures were supplemented with 2 mM cysteine, 2 mM methionine or both. CFU were determined at the indicated time points. (D) Impact of partial TrxB2 depletion on susceptibility of <i>Mtb</i> to mitomycin C. TrxB2-TetON-tetO-1C was cultured in 7H9 medium without atc for 3 days to decrease TrxB2 expression before treatment with mitomycin C. Data in (C) and (D) are means ± SD (n = 3 per group) and are representative of three independent experiments.</p

TrxB2 is essential for <i>Mtb</i> survival in replicating and non-replicating conditions.

<p>(A-C) Impact of TrxB2 depletion on replicating <i>Mtb</i>. (A) Immunoblot of protein extracts from H37Rv and TrxB2-DUC grown with and without atc. Blot was probed with TrxB2-specific and Eno-specific (loading control) antisera. TrxB2 in the TrxB2-DUC mutant is of increased molecular weight due to the C-terminal DAS tag. (B) Growth of individual <i>Mtb</i> strains quantified by optical density in nutrient-rich medium with or without atc. Starting density of the cultures was OD₅₈₀ ~ 0.01. (C) Survival of <i>Mtb</i> strains quantified by CFU in 7H9 medium with or without atc (n = 6 per group). (D and E) Impact of TrxB2 depletion on non-replicating <i>Mtb</i>. (D) Immunoblot of protein extracts from <i>Mtb</i> cultures during starvation in PBS with or without atc. H37Rv and TrxB2-DUC were suspended in PBS for 10 days to obtain a non-replicating state. Where indicated, atc was added to the cultures on day 10. (E) Quantification of CFU from cultures in (D) at the indicated time points (n = 3 per group). (F) Appearance of the atc-treated TrxB2-DUC culture in 7H9 medium on day 4 and on day12. (G) Growth of TrxB2-DUC in 7H9 medium, treated with atc or meropenem–clavulanate (MCA). Starting density of the cultures was OD₅₈₀ ~ 0.1. (H) Immunoblot analysis of dihydrolipoamide acyltransferase (DlaT), enolase (Eno), proteasome beta subunit (PrcB) and secreted protein antigen 85B (Ag85B) from culture supernatants in (G) at the indicated times. (I) Quantification of cell length by microscopy of indicated <i>Mtb</i> strains treated or not with atc for 4 days. Mean cell lengths (n = 100) are indicated. **** p<0.0001 by one-way ANOVA. All results are representative of at least three independent experiments. Data in (C) and (E) are means ± SD. In some panels, error bars are too small to be seen.</p

TrxB2 protects <i>Mtb</i> from thiol-specific oxidizing stress and contributes less to defense against oxidative and nitrosative stress.

<p>(A and B) Susceptibility of partially TrxB2-depleted <i>Mtb</i> to plumbagin (A) and H₂O₂ (B). TrxB2-TetON-tetO-1C was cultured in 7H9 medium without atc for 3 days to decrease TrxB2 expression before treatment with plumbagin and H₂O₂. OD₅₈₀ was recorded and normalized to the corresponding strains without drug treatment. (C) Survival of <i>Mtb</i> strains after 4 days exposure to increasing concentrations of NaNO₂ at pH 5.5. Data are means ± SD (n = 3 per group) and are representative of two independent experiments. (* p<0.05, one way ANOVA was used for group comparison). (D) Susceptibility of partially TrxB2-depleted <i>Mtb</i> to diamide. (E) Impact of dithiothreitol (DTT) on TrxB2 depletion-induced death in the TrxB2-DUC strain. *p<0.05, **p<0.01 and ***p<0.001 by unpaired Student’s t test. (F) Impact of extracellular glutathione (GSH) and catalase on TrxB2 depletion-induced death in the TrxB2-DUC strain. Data shown means ± SD (n = 3 per group) and are representative of two to three independent experiments.</p

Fragment-Based Exploration of Binding Site Flexibility in Mycobacterium tuberculosis BioA

The PLP-dependent transaminase (BioA) of Mycobacterium tuberculosis and other pathogens that catalyzes the second step of biotin biosynthesis is a now well-validated target for antibacterial development. Fragment screening by differential scanning fluorimetry has been performed to discover new chemical scaffolds and promote optimization of existing inhibitors. Calorimetry confirms binding of six molecules with high ligand efficiency. Thermodynamic data identifies which molecules bind with the enthalpy driven stabilization preferred in compounds that represent attractive starting points for future optimization. Crystallographic characterization of complexes with these molecules reveals the dynamic nature of the BioA active site. Different side chain conformational states are stabilized in response to binding by different molecules. A detailed analysis of conformational diversity in available BioA structures is presented, resulting in the identification of two states that might be targeted with molecular scaffolds incorporating well-defined conformational attributes. This new structural data can be used as part of a scaffold hopping strategy to further optimize existing inhibitors or create new small molecules with improved therapeutic potential

<i>Mtb</i> lacking FBA requires a balanced carbon diet for growth.

<p>Growth of WT and Δ<i>fba</i> was in media containing (<b>A</b>) 0.2% glucose with varying concentrations of butyrate and (<b>B</b>) 0.1% butyrate with varying concentrations of glucose. Bacteria were cultured in 96-well plates and absorbance was measured at the indicated time points. Data are means of triplicate cultures +/− SEM and representative of two independent experiments.</p