Structural Basis of Mycobacterium tuberculosis Transcription and Transcription Inhibition.

Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis, which kills 1.8 million annually. Mtb RNA polymerase (RNAP) is the target of the first-line antituberculosis drug rifampin (Rif). We report crystal structures of Mtb RNAP, alone and in complex with Rif, at 3.8-4.4 Å resolution. The results identify an Mtb-specific structural module of Mtb RNAP and establish that Rif functions by a steric-occlusion mechanism that prevents extension of RNA. We also report non-Rif-related compounds-Nα-aroyl-N-aryl-phenylalaninamides (AAPs)-that potently and selectively inhibit Mtb RNAP and Mtb growth, and we report crystal structures of Mtb RNAP in complex with AAPs. AAPs bind to a different site on Mtb RNAP than Rif, exhibit no cross-resistance with Rif, function additively when co-administered with Rif, and suppress resistance emergence when co-administered with Rif.

Development of New Tuberculosis Drugs: Translation to Regimen Composition for Drug-Sensitive and Multidrug-Resistant Tuberculosis.

Tuberculosis (TB) kills more people than any other infectious disease. Challenges for developing better treatments include the complex pathology due to within-host immune dynamics, interpatient variability in disease severity and drug pharmacokinetics-pharmacodynamics (PK-PD), and the growing emergence of resistance. Model-informed drug development using quantitative and translational pharmacology has become increasingly recognized as a method capable of drug prioritization and regimen optimization to efficiently progress compounds through TB drug development phases. In this review, we examine translational models and tools, including plasma PK scaling, site-of-disease lesion PK, host-immune and bacteria interplay, combination PK-PD models of multidrug regimens, resistance formation, and integration of data across nonclinical and clinical phases.We propose a workflow that integrates these tools with computational platforms to identify drug combinations that have the potential to accelerate sterilization, reduce relapse rates, and limit the emergence of resistance.

The Chemical Property Position of Bedaquiline Construed by a Chemical Global Positioning System-Natural Product.

Bedaquiline is a novel adenosine triphosphate synthase inhibitor anti-tuberculosis drug. Bedaquiline belongs to the class of diarylquinolines, which are antituberculosis drugs that are quite different mechanistically from quinolines and flouroquinolines. The fact that relatively similar chemical drugs produce different mechanisms of action is still not widely understood. To enhance discrimination in favor of bedaquiline, a new approach using eight-score principal component analysis (PCA), provided by a ChemGPS-NP model, is proposed. PCA scores were calculated based on 35 + 1 different physicochemical properties and demonstrated clear differences when compared with other quinolines. The ChemGPS-NP model provided an exceptional 100 compounds nearest to bedaquiline from antituberculosis screening sets (with a cumulative Euclidian distance of 196.83), compared with the different 2Dsimilarity provided by Tanimoto methods (extended connective fingerprints and the Molecular ACCess System, showing 30% and 182% increases in cumulative Euclidian distance, respectively). Potentially similar compounds from publicly available antituberculosis compounds and Maybridge sets, based on bedaquiline's eight-dimensional similarity and different filtrations, were identified too.

3-Aryl-substituted imidazo[1,2-a]pyridines as antituberculosis agents.

Novel inhibitors are needed to tackle tuberculosis. Herein, we report the 3-aryl-substituted imidazo[1,2-a]pyridines as potent antituberculosis agents. A small library of 3-aryl-substituted imidazo[1,2-a]pyridines was synthesized using direct arylation, followed by nitro reduction and finally Pd-catalyzed C-N coupling reactions. The compounds thus obtained were evaluated against Mycobacterium tuberculosis H37Rv. Compound 26 was identified as an antituberculosis lead with a minimum inhibitory concentration of 2.3 µg/ml against M. tuberculosis H37Rv. This compound showed a selectivity index of 35. The docking of 26 in the active site of the M. tuberculosis cytochrome bc1 complex cytochrome b subunit (Mtb QcrB) revealed key π-π interactions of compound 26 with the Tyr389 and Trp312 residues of Mtb QcrB.

Synthesis of novel 4,5-dihydropyrrolo[1,2-a]quinoxalines, pyrrolo[1,2-a]quinoxalin]-2-ones and their antituberculosis and anticancer activity.

A facile strategy was developed for the synthesis of biologically important 4,5-dihydropyrrolo[1,2-a]quinoxalines and pyrrolo[1,2-a]quinoxalin]-2-ones by treating 2-(1H-pyrrol-1-yl)anilines with imidazo[1,2-a]pyridine-3-carbaldehyde or isatin, using amidosulfonic acid (NH3 SO3 ) as a solid catalyst in water at room temperature. The protocol has been extended to electrophile ninhydrin. The catalyst could be recycled for six times without the loss of activity. The compounds were evaluated for their antituberculosis, antibacterial, and anticancer activities. It is worth noting that compounds 3d and 3e demonstrated a minimum inhibitory concentration value of 6.25 µM against Mycobacterium tuberculosis H37Rv, whereas compounds 3d, 3g, 5d, 5e, and 5i showed a remarkable inhibition of A549, DU145, HeLa, HepG2, MCF-7, and B16-F10 cell lines, respectively. Staphylococcus aureus was inhibited by compounds 5b, 5e, 5d, 5g, and 5l at 32 µg/ml.

Synthesis and antituberculosis activity of new acylthiosemicarbazides designed by structural modification.

Acylthiosemicarbazides 8a-n were designed by structural modification of lead Compound 7. The syntheses of 8a-n involve a five-step procedure starting from carboxylic acids. Compounds 8a-n were tested against three Mycobacterium tuberculosis strains to measure their inhibitory antituberculosis activities. These activities could be explained according to the presence or absence of the chlorine substituent in the aromatic ring of the amide joined to the thiosemicarbazide core. Thiosemicarbazide derivative 8n is a candidate for the development of novel antitubercular agents. Ongoing studies are focused on exploring the mechanism by which these compounds inhibit M. tuberculosis cell growth.

Design, synthesis, and bioevaluation of a novel class of (E)-4-oxo-crotonamide derivatives as potent antituberculosis agents.

A series of novel (E)-4-oxo-2-crotonamide derivatives were designed and synthesized to find potent antituberculosis agents. All the target compounds were evaluated for their in vitro activity against Mycobacterium tuberculosis H37Rv(MTB). Results reveal that 4-phenyl moiety at part A and short methyl group at part C were found to be favorable. Most of the derivatives displayed promising activity against MTB with MIC ranging from 0.125 to 4 µg/mL. Especially, compound IIIa16 was found to have the best activity with MIC of 0.125 µg/mL against MTB and with MIC in the range of 0.05-0.48 µg/mL against drug-resistant clinical MTB isolates.

Pharmacokinetic-pharmacodynamic and dose-response relationships of antituberculosis drugs: recommendations and standards for industry and academia.

BACKGROUND: Antimicrobial pharmacokinetic-pharmacodynamic (PK/PD) science is vital to early antibiotic drug development to enable more efficient dose-effect study designs, identification of doses that may suppress drug resistance and choice of susceptibility breakpoints. Proper conduct of such studies is essential in the field of tuberculosis. METHODS: We conducted an exhaustive review of literature on the hollow fiber system (HFS) model, murine model, and guinea pig model of tuberculosis as well as clinical studies to identify PK/PD studies that have been applied to antituberculosis therapy. Lessons learned are presented as recommendations and standards for both industry and academia in the field of antituberculosis drug development. RESULTS: PK/PD studies have been performed for both first-line and experimental antituberculosis agents. When properly designed exposure-effect and dose-fractionation studies have been performed in preclinical models, optimal drug exposures, and PK/PD parameters identified in these models have been found to be similar to clinical studies. Susceptibility breakpoints identified using these methods differed from previous concentrations in the literature but were found to be similar to those in prospective clinical studies. CONCLUSIONS: Preclinical PK/PD studies are essential value added in the development of antituberculosis agents. We provide 8 recommendations and standards for the proper conduct of such studies.

Discovery of 2,9-diaryl-6-carbamoylpurines as a novel class of antitubercular agents.

A series of novel 9-alkyl/aryl-2-aryl-6-carbamoylpurines were synthesized, and their activity against Mycobacterium tuberculosis strain H37Rv was assessed. The SAR analysis on the first set of derivatives, with an alkyl or aryl unit at N-9 and a phenolic unit at C-2, showed that the activity depends on the purine ring substituents at N-9 and C-2. A phenyl group at N-9 combined with a 3-hydroxyphenyl or 4-hydroxyphenyl at C-2 improve the activity. The most active compound of this set has a phenyl group at N-9 and a 4-hydroxyphenyl group at C-2, displaying an IC90 = 1.2 µg/mL and a selectivity index higher than 25.5. This compound served as a Hit to design the second set of derivatives. A phenyl group at N-9 was maintained, and the group at C-2 was diversified. The SAR analysis showed that the aryl unit at C-2 must have an oxygen or nitrogen atom bonded in the para position. A proton, a small alkyl or a substituted aryl group may also be bonded to the oxygen. The compound with the 4-methoxyphenyl group at C-2, 1Bd, exhibits the highest activity with an IC90 < 0.19 µg/mL. This compound is highly potent against M. tuberculosis strain H37Rv and non-toxic for VERO mammalian cells with an SI > 153.8. Compound 1Bd was also non-cytotoxic against primary macrophage cultures at IC90, 2xIC90, and 10xIC90 and significantly reduced the bacterial load in M. tuberculosis-infected macrophages at the same concentrations. Compound 1Bd showed a favorable pharmacokinetic profile when administered orally, with major lung and liver accumulation. In vivo antimycobacterial efficacy of 1Bd was tested at 25 mg/kg. At the tested regimen, a decrease in bacterial burden was observed in the liver. Optimization of the treatment regimen should be performed to fully potentiate the in vivo efficacy of our lead molecule, particularly in the lung, the main target organ of M. tuberculosis.

3,5-disubstituted pyridines with potent activity against drug-resistant Mycobacterium tuberculosis clinical isolates.

Aim: We designed and synthesized a series of compounds with a 3,5-disubstituted pyridine moiety and evaluated them against Mycobacterium tuberculosis (Mtb) and drug-resistant Mtb clinical isolates.Methodology: A library of 3,5-disubstituted pyridine was synthesized. The compounds were screened for activity against M. tuberculosis H37Rv. The optimal substitutions needed for the activity were identified through structure-activity relationship (SAR) studies.Results: From the screening studies, compounds 24 and 26 were identified as potent members of this series with Minimum Inhibitory Concentration (MIC) of 1.56 µg/ml against M. tuberculosis H37Rv. These compounds did not show any inhibition against a panel of ESKAPE pathogens at >50 µg/ml indicating their selective killing of M. tuberculosis H37Rv. Importantly, compound 24 showed a selectivity index of 54.64 against CHO-K1 and 78.26 against VERO cell lines, while compound 26 showed a selectivity index of 108.5 against CHO-K1 and 63.2 against VERO cell lines, respectively. Compound 24 formed a stable complex with the target protein DprE1 with predicted binding energy -8.73 kcal/mol and inhibited multidrug-resistant clinical isolate of M. tuberculosis at 6.25 µg/ml.Conclusion: This study identified the 3,5-disubstituted pyridine derivative 24 with potent antituberculosis activity and can be taken forward to generate new preclinical candidate.

[Box: see text].

A minimal PBPK model to accelerate preclinical development of drugs against tuberculosis.

Introduction: Understanding drug exposure at disease target sites is pivotal to profiling new drug candidates in terms of tolerability and efficacy. Such quantification is particularly tedious for anti-tuberculosis (TB) compounds as the heterogeneous pulmonary microenvironment due to the infection may alter lung permeability and affect drug disposition. Murine models have been a longstanding support in TB research so far and are here used as human surrogates to unveil the distribution of several anti-TB compounds at the site-of-action via a novel and centralized PBPK design framework. Methods: As an intermediate approach between data-driven pharmacokinetic (PK) models and whole-body physiologically based (PB) PK models, we propose a parsimonious framework for PK investigation (minimal PBPK approach) that retains key physiological processes involved in TB disease, while reducing computational costs and prior knowledge requirements. By lumping together pulmonary TB-unessential organs, our minimal PBPK model counts 9 equations compared to the 36 of published full models, accelerating the simulation more than 3-folds in Matlab 2022b. Results: The model has been successfully tested and validated against 11 anti-TB compounds-rifampicin, rifapentine, pyrazinamide, ethambutol, isoniazid, moxifloxacin, delamanid, pretomanid, bedaquiline, OPC-167832, GSK2556286 - showing robust predictability power in recapitulating PK dynamics in mice. Structural inspections on the proposed design have ensured global identifiability and listed free fraction in plasma and blood-to-plasma ratio as top sensitive parameters for PK metrics. The platform-oriented implementation allows fast comparison of the compounds in terms of exposure and target attainment. Discrepancies in plasma and lung levels for the latest BPaMZ and HPMZ regimens have been analyzed in terms of their impact on preclinical experiment design and on PK/PD indices. Conclusion: The framework we developed requires limited drug- and species-specific information to reconstruct accurate PK dynamics, delivering a unified viewpoint on anti-TB drug distribution at the site-of-action and a flexible fit-for-purpose tool to accelerate model-informed drug design pipelines and facilitate translation into the clinic.

Recent advances in oxazolidinones as antituberculosis agents.

Tuberculosis (TB) is an infectious and fatal disease caused by Mycobacterium tuberculosis (Mtb) and remains a serious public health threat; therefore, the development of new antitubercular agents is a priority for the World Health Organization's End TB strategy and the United Nations' Sustainable Development Goals to eradicate TB. Oxazolidinones are a class of synthetic antibacterial agents with a distinct mode of action developed for the treatment of Gram-positive bacterial infections. Many oxazolidinones exhibit good activity against Mtb, and some are currently in clinical trials for multidrug-resistant TB and extensively drug-resistant TB therapy. In this review, the mechanism of action, activity and toxicity of oxazolidinones and recent progress in the research and development of oxazolidinones as anti-TB agents are summarized.

(2Z)-3-Hydroxy-3-(4-R-Phenyl)-Prop-2-Enedithioic Acids as New Antituberculosis Compounds.

BACKGROUND: Tuberculosis is an infectious disease caused by the bacillus Mycobacterium tuberculosis. Compounds including a sulfur-containing scaffold have been shown to be key scaffolds in various antituberculosis agents. Interestingly, the 3-hydroxy-3-phenyl-prop-2-enedithioic acids 11a-j have, to the best of our knowledge, not been previously described as antituberculosis agents. PURPOSE: In the present study, we investigated the role of substituents attached to the phenyl ring of a 3-hydroxy-3-phenyl-prop-2-enedithioic acid scaffold (compounds 11a-j) in inhibiting the growth of M. tuberculosis strain H37Rv. METHODS: (Z)-3-hydroxy-3-(4-R-phenyl)-prop-2-enedithioic acids 11b-j, with R groups including various electron-donating or electron-withdrawing groups, were designed by structurally modifying the lead compound 11a. The syntheses of 11a-j involved each one-step procedure starting from the corresponding substituted acetophenone. Compounds 11a-j were tested against M. tuberculosis strain H37Rv to evaluate their bacterial growth inhibitory activities. ADMET profiles were predicted by employing three different methods. In addition, molecular docking studies were carried out, based on the molecular similarities of the synthesized compounds with ethionamide (5), on the active site of the M. tuberculosis H37Rv (3R)-hydroxyacyl-ACP (HadAB) dehydratase heterodimer. RESULTS: The antituberculosis activities of compounds 11a-j could be explained in terms of the presence of electron-donating or electron-withdrawing substituents on the aromatic ring of the substituted 3-hydroxy-3-phenyl)-prop-2-enedithioic acid core. The activity and selectivity index (SI) value of (Z)-3-hydroxy-3-(4-nitrophenyl)-prop-2-enedithioic acid 11e suggested that this compound could be used for the design of novel antituberculosis agents. Most of the synthesized molecules showed an acceptable ADME profile and a low probability of being toxic. Docking studies of 11d and 11e showed them forming hydrogen bonds with the ACys61 residue of the HadAB enzyme. CONCLUSION: Our results suggested that the antituberculosis compound 11e could be used for the design of novel antituberculosis agents.

Differential Expression of Resistant and Efflux Pump Genes in MDR-TB Isolates.

BACKGROUND: Numerous investigations demonstrate efflux as a worldwide bacterial mode of action which contributes to the resistance of drugs. The activity of antibiotics, which subjects to efflux, can be improved by the combined usage of efflux inhibitors. However, the efflux role to the overall levels of antibiotic resistance of clinical M. tuberculosis isolates is inadequately comprehended and is still disregarded by many. METHODS: Here, we assessed the contribution of resistant genes associated with isoniazid (INH) and rifampin (R) resistance to the levels of drug resistance in the (27) clinical isolates of MDR-TB. Additionally, the role of the resistance for six putative drug efflux pump genes to the antibiotics was investigated. The level of katG expression was down-regulated in 24/27 (88.88%) of MDR-TB isolates. Of the 27 MDR-TB isolates, inhA, oxyR-ahpC, and rpoB showed either overexpression or up-regulation in 8 (29.62%), 4 (14.81 %), and 24 (88.88%), respectively. Moreover, the efflux pump genes drrA, drrB, efpA, Rv2459, Rv1634, and Rv1250 were overexpressed under INH/RIF plus fresh pomegranate juice (FPJ) stress signifying the efflux pumps contribution to the overall levels of the resistance of MDR-TB isolates. CONCLUSION: These results displayed that the levels of drug resistance of MDR-TB clinical isolates are due to combination among drug efflux pump and the presence of mutations in target genes, a truth which is often ignored by the specialists of tuberculosis in favour of the almost undoubted significance of drug target- gene mutations for the resistance in M. tuberculosis.

Synthesis and biological evaluation of 2,4,5-trisubstituted thiazoles as antituberculosis agents effective against drug-resistant tuberculosis.

The dormant and resistant form of Mycobacterium tuberculosis presents a challenge in developing new anti-tubercular drugs. Herein, we report the synthesis and evaluation of trisubstituted thiazoles as antituberculosis agents. The SAR study has identified a requirement of hydrophobic substituent at C2, ester functionality at C4, and various groups with hydrogen bond acceptor character at C5 of thiazole scaffold. This has led to the identification of 13h and 13p as lead compounds. These compounds inhibited the dormant Mycobacterium tuberculosis H37Ra strain and M. tuberculosis H37Rv selectively. Importantly, 13h and 13p were non-toxic to CHO cells. The 13p showed activity against multidrug-resistant tuberculosis isolates.

Synthesis and biological activity of furoxan derivatives against Mycobacterium tuberculosis.

Tuberculosis (TB) remains a serious health problem responsible to cause millions of deaths annually. The scenario becomes alarming when it is evaluated that the number of new drugs does not increase proportionally to the emergence of resistance to the current therapy. Furoxan derivatives, known as nitric oxide (NO) donors, have been described to exhibit antitubercular activity. Herein, a novel series of hybrid furoxan derivatives (1,2,5-oxadiazole 2-N-oxide) (compounds 4a-c, 8a-c and 14a-c) were designed, synthesized and evaluated in vitro against Mycobacterium tuberculosis (MTB) H37Rv (ATCC 27294) and a clinical isolate MDR-TB strain. The furoxan derivatives have exhibited MIC90 values ranging from 1.03 to 62 µM (H37Rv) and 7.0-50.0 µM (MDR-TB). For the most active compounds (8c, 14a, 14b and 14c) the selectivity index ranged from 3.78 to 52.74 (MRC-5 cells) and 1.25-34.78 (J774A.1 cells). In addition, it was characterized for those compounds logPo/w values between 2.1 and 2.9. All compounds were able to release NO at levels ranging from 0.16 to 44.23%. Among the series, the phenylsulfonyl furoxan derivatives (compounds 14a-c) were the best NO-donor with the lowest MIC90 values. The most active compound (14c) was also stable at different pHs (5.0 and 7.4). In conclusion, furoxan derivatives were identified as new promising compounds useful to treat tuberculosis.

[Multidrug resistant tuberculosis among health personnel in Côte d'Ivoire]. / Tuberculose multirésistante chez le personnel de santé en Côte d'Ivoire.

UNLABELLED: Multidrug resistance tuberculosis (MDR-TB) of health workers raises the question of hospital-borne transmission of infection. OBSERVATIONS: We report 4 cases of MDR-TB confirmed at the health workers over a period of 8 years (January, 2005 to December 2012), in the 2 services of pulmonology from Abidjan to Côte d'Ivoire). It was about young grown-up patients (aged between 28 and 39 years), all HIV negatives, in a no-win situation of antituberculosis treatment (3 patients/4). The most concerned staffs were the male nurses (2/4). Two agents worked in general hospital and the only one in a pulmonology department at the time of the diagnosis. The tuberculosis was of lung seat with bilateral radiographic hurt (3/4) and multiples excavations (4/4). The case index, when it was identified (2/2), was a family case. Among 3 agents who benefited from a second line treatment, 1 died further to an extensive drug resistance and 2 are declared to be cured. The fourth died before the beginning of the treatment. These cases of cure were in touch with a premature care. CONCLUSION: Multidrug resistant tuberculosis at the health workers could have a negative impact on the antituberculosis fight imposing rigorous measures of infection control and better implication of the occupational medicine.

Differential expression of efflux pump genes of Mycobacterium tuberculosis in response to varied subinhibitory concentrations of antituberculosis agents.

Several reports have elaborated on the role of efflux pumps in drug resistance in Mycobacterium tuberculosis by analysing the mRNA expression profiles. However, there is no uniformity in the subinhibitory concentrations of drugs chosen in these studies. Some investigators studied the expression of efflux pumps under a drug concentration of 1/2 minimum inhibitory concentration (MIC), while others used 1/3, 1/4 or 1/8MIC. The present study was planned to understand the effect of different concentrations of antituberculosis drugs on the expression of efflux pump genes. Log phase culture of the laboratory strain M. tuberculosis H37Rv was exposed to rifampicin (RIF), isoniazid (INH), streptomycin (SM) and ethambutol (EMB) at different drug concentrations (1/2MIC, 1/3MIC and 1/4MIC). The expression of 10 putative efflux pump genes was studied using quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). We observed an optimal expression of efflux pumps at higher concentrations of INH; and at lower concentrations of RIF and EMB. However, in the presence of SM, a decreased expression of efflux genes with increasing concentrations of the drug was confounded by a significant reduction in Colony Forming Units (CFU).

Effects of type 2 diabetes mellitus on the population pharmacokinetics of rifampin in tuberculosis patients.

Diabetes mellitus (DM) is a well-known risk factor to develop tuberculosis (TB). Some reports indicate the serum concentrations of anti-TB drugs are lower in patients with TB and DM than those with TB only. Therefore, we developed a nonlinear mixed-effects model (NONMEM) to determine the population PK parameters of rifampin and assessed the effects of DM status in patients with TB. One-compartment linear modeling with first-order absorption was evaluated using the 206 plasma samples of rifampin from 54 patients with DM. Based on the final model, DM affected the absorption rate constant (ka) and the volume of distribution (Vd) of rifampin. The body mass index (BMI) of the patients affected rifampin clearance (CL). The ka of rifampin in patients with TB and DM was greater than that in patients with TB only. Further, the predicted Vd in patients with DM was greater than that in patients without DM. As Vd is inversely correlated with plasma concentrations, the rifampin concentrations were predicted to be lower in the patients with DM. The authors recommend administering the greater doses of rifampin for the treatment of TB in patients with DM compared with the doses for the patients without DM to prevent treatment failure.

Structure activity relationships of 4-hydroxy-2-pyridones: A novel class of antituberculosis agents.

Pyridone 1 was identified from a high-throughput cell-based phenotypic screen against Mycobacterium tuberculosis (Mtb) including multi-drug resistant tuberculosis (MDR-TB) as a novel anti-TB agent and subsequently optimized series using cell-based Mtb assay. Preliminary structure activity relationship on the isobutyl group with higher cycloalkyl groups at 6-position of pyridone ring has enabled us to significant improvement of potency against Mtb. The lead compound 30j, a dimethylcyclohexyl group on the 6-position of the pyridone, displayed desirable in vitro potency against both drug sensitive and multi-drug resistant TB clinical isolates. In addition, 30j displayed favorable oral pharmacokinetic properties and demonstrated in vivo efficacy in mouse model. These results emphasize the importance of 4-hydroxy-2-pyridones as a new chemotype and further optimization of properties to treat MDR-TB.