Tetrahydropyrazolo[1,5-a]Pyrimidine-3-Carboxamide and N-Benzyl-6′,7′-Dihydrospiro[Piperidine-4,4′-Thieno[3,2-c]Pyran] analogues with bactericidal efficacy against Mycobacterium tuberculosis targeting MmpL3

Mycobacterium tuberculosis is a major human pathogen and the causative agent for the pulmonary disease, tuberculosis (TB). Current treatment programs to combat TB are under threat due to the emergence of multi-drug and extensively-drug resistant TB. As part of our efforts towards the discovery of new anti-tubercular leads, a number of potent tetrahydropyrazolo[1,5-a]pyrimidine-3-ca​rboxamide(THPP) and N-benzyl-6′,7′-dihydrospiro[piperidine-4,​4′-thieno[3,2-c]pyran](Spiro) analogues were recently identified against Mycobacterium tuberculosis and Mycobacterium bovis BCG through a high-throughput whole-cell screening campaign. Herein, we describe the attractive in vitro and in vivo anti-tubercular profiles of both lead series. The generation of M. tuberculosis spontaneous mutants and subsequent whole genome sequencing of several resistant mutants identified single mutations in the essential mmpL3 gene. This ‘genetic phenotype’ was further confirmed by a ‘chemical phenotype’, whereby M. bovis BCG treated with both the THPP and Spiro series resulted in the accumulation of trehalose monomycolate. In vivo efficacy evaluation of two optimized THPP and Spiro leads showed how the compounds were able to reduce >2 logs bacterial cfu counts in the lungs of infected mice

Novel insight into the reaction of nitro, nitroso and hydroxylamino benzothiazinones and of benzoxacinones with Mycobacterium tuberculosis DprE1

Abstract Nitro-substituted 1,3-benzothiazinones (nitro-BTZs) are mechanism-based covalent inhibitors of Mycobacterium tuberculosis decaprenylphosphoryl-β-D-ribose-2′-oxidase (DprE1) with strong antimycobacterial properties. We prepared a number of oxidized and reduced forms of nitro-BTZs to probe the mechanism of inactivation of the enzyme and to identify opportunities for further chemistry. The kinetics of inactivation of DprE1 was examined using an enzymatic assay that monitored reaction progress up to 100 min, permitting compound ranking according to k inact/K i values. The side-chain at the 2-position and heteroatom identity at the 1-position of the BTZs were found to be important for inhibitory activity. We obtained crystal structures with several compounds covalently bound. The data suggest that steps upstream from the covalent end-points are likely the key determinants of potency and reactivity. The results of protein mass spectrometry using a 7-chloro-nitro-BTZ suggest that nucleophilic reactions at the 7-position do not operate and support a previously proposed mechanism in which BTZ activation by a reduced flavin intermediate is required. Unexpectedly, a hydroxylamino-BTZ showed time-dependent inhibition and mass spectrometry corroborated that this hydroxylamino-BTZ is a mechanism-based suicide inhibitor of DprE1. With this BTZ derivative, we propose a new covalent mechanism of inhibition of DprE1 that takes advantage of the oxidation cycle of the enzyme

High-Content Screening Technology Combined with a Human Granuloma Model as a New Approach To Evaluate the Activities of Drugs against Mycobacterium tuberculosis

International audienceTuberculosis remains a major health problem due to the emergence of drug-resistant strains of Mycobacterium tuberculosis. Some models have provided valuable information about drug resistance and efficacy; however, the translation of these results into effective human treatments has mostly proven unsuccessful. In this study, we adapted high-content screening (HCS) technology to investigate the activities of antitubercular compounds in the context of an in vitro granuloma model. We observed significant shifts in the MIC 50 s between the activities of the compounds under extracellular and granuloma conditions. T uberculosis (TB) is a respiratory disease that is one of the major causes of mortality and morbidity worldwide. Almost 20 people develop TB and four patients die from the disease every minute somewhere in the world (1). The disease can be cured by drug treatment involving a regimen of several drugs. The World Health Organization (WHO) estimates that there were around 0.5 million new cases of multidrug-resistant TB (MDR-TB) in 2012 (2, 3). A recent report confirmed that MDR-TB is a continuing worldwide health problem, even in high-income countries with a low incidence of TB (4). The immune system undoubtedly plays a major role in TB control (5). Signaling events of the immune system lead to the formation of a granuloma, the hallmark of TB. Granulomas are an immune microenvironment in which the infection can be controlled but also a niche in which bacilli can thrive and modulate immune responses to ensure their survival for long periods without causing damage (6, 7). Granulomas are cell aggregates that form tridimensional and heterogeneous structures (8, 9). We previously described an in vitro granuloma model involving human blood cells either infected with mycobacteria or incubated with mycobacterial antigens (8, 10). Either of these models resulted in the formation of a typical epithelioid granuloma with morphological characteristics and properties of cellular differentiation similar to those of natural granulomas (8, 10, 11). This model may help bridge the gap between the in vitro evaluation of MICs and costly in vivo efficacy studies in guinea pigs. In particular, compounds that are effective in the in vitro granuloma model could be prioritized for guinea pig studies. High-content screening (HCS) technology has been used to identify anti-TB compounds (12–14). This technology has several advantages over traditional phenotypic screening approaches. This technology has mostly been used in single-cell experiments, because it is adapted to identify and analyze images (12, 13); however , in this paper, we focus on the use of granulomas instead of single cells. In this report, we describe the development of a novel HCS method to evaluate the activities of reference compounds against a green fluorescent protein (GFP)-expressing H37Rv strain of Mycobacterium tuberculosis (MTB-GFP) within 5 days, and we compared the MIC 50 values with those of classical liquid cultures (extracellular), intracellular growth (single-cell assay), and within granulomas, all using the same bacterial strain. MTB-GFP strains (13) were grown for 3 weeks at 37°C until the log phase was reached. The test inoculum was prepared by diluting the culture with phosphate-buffered saline (PBS) medium or complete RPMI (RPMIc) medium (containing RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum [FBS], with 1% glutamine, 1% HEPES, 1% nonessential amino acids) to a final bacterial concentration of 1 ϫ 10 6 CFU/ml. In vitro human granuloma formation was performed by incu-bating peripheral blood mononuclear cells (PBMC) of healthy volunteers; the cells were purified by gradient density sedimenta-tion (15) with MTB-GFP using an appropriate multiplicity of infection in RPMIc medium at 37°C with low shaking (125 rpm) for 1 h; they were then washed with PBS containing 2% FBS and suspended in RPMIc medium at a concentration of 3.5 ϫ 10 5 cells per well in 384-well plates (Greiner). The cells were then incu-bated for 3 days at 37°C in a 5% CO 2 atmosphere to allow the formation of granulomas. Intragranuloma bacterial quantification was performed by fixing granulomas with 4% paraformaldehyde (Electron Microscopy Sciences) in PBS and staining with Hoechst (Sigma); each step consisted of a 30-min incubation period at room temperature

A new piperidinol derivative targeting mycolic acid transport in Mycobacterium abscessus

International audienceThe natural resistance of Mycobacterium abscessus to most commonly available antibiotics seriously limits chemotherapeutic treatment options, which is particularly challenging for cystic fibrosis patients infected with this rapid-growing mycobacterium. New drugs with novel molecular targets are urgently needed against this emerging pathogen. However, the discovery of such new chemotypes has not been appropriately performed. Here, we demonstrate the utility of a phenotypic screen for bactericidal compounds against M. abscessus using a library of compounds previously validated for activity against M. tuberculosis. We identified a new piperidinol-based molecule, PIPD1, exhibiting potent activity against clinical M. abscessus strains in vitro and in infected macrophages. Treatment of infected zebrafish with PIPD1 correlated with increased embryo survival and decreased bacterial burden. Whole genome analysis of M. abscessus strains resistant to PIPD1 identified several mutations in MAB_4508, encoding a protein homologous to MmpL3. Biochemical analyses demonstrated that while de novo mycolic acid synthesis was unaffected, PIPD1 strongly inhibited the transport of trehalose monomycolate, thereby abrogating mycolylation of arabinogalactan. Mapping the mutations conferring resistance to PIPD1 on a MAB_4508 tridimensional homology model defined a potential PIPD1-binding pocket. Our data emphasize a yet unexploited chemical structure class against M. abscessus infections with promising translational development possibilities

Design, synthesis and structure-activity relationship study of wollamide B; a new potential anti TB agent

<div><p>Wollamide B is a cationic antimycobacterial cyclohexapeptide that exhibits activity against <i>Mycobacterium bovis (M</i>. <i>bovis)</i> (IC₅₀ of 3.1 μM). Aiming to define its structural activity relationship (SAR), optimizing potency and pharmacokinetic properties, libraries of analogues were synthesized following a standard Fmoc-based solid phase peptide synthesis approach. The antimycobacterial activities of wollamide B and all the synthesized analogues were tested against <i>Mycobacterium tuberculosis</i> (<i>Mtb</i>) H37Rv. Parallely, <i>in vitro</i> drug metabolism and pharmacokinetic (ADME) profiling was done for the synthesized compounds to evaluate their drug likeness. Among the 25 synthesized wollamides five of them showed potent activities with MICs ≤ 3.1 μM and found to be nontoxic against human HepG2 cells up to 100 μM. The results of the <i>in vitro</i> ADME profiling revealed the remarkable plasma stability and very good aqueous solubility of the class in general while the metabolic stability was found to be moderate to low. Of particular note, compounds <b>7c</b> (MIC = 1.1 μM) and <b>13c</b> (0.6 μM) that exhibited good balance of antimycobacterial activity vs. optimal pharmacokinetic properties could be used as a new lead for further development.</p></div

Accelerating early antituberculosis drug discovery by creating mycobacterial indicator strains that predict mode of action

Due to the rise of drug-resistant forms of tuberculosis, there is an urgent need for novel antibiotics to effectively combat these cases and shorten treatment regimens. Recently, drug screens using whole-cell analyses have been shown to be successful. However, current high-throughput screens focus mostly on stricto sensu life/death screening that give little qualitative information. In doing so, promising compound scaffolds or nonoptimized compounds that fail to reach inhibitory concentrations are missed. To accelerate early tuberculosis (TB) drug discovery, we performed RNA sequencing on Mycobacterium tuberculosis and Mycobacterium marinum to map the stress responses that follow upon exposure to subinhibitory concentrations of antibiotics with known targets, ciprofloxacin, ethambutol, isoniazid, streptomycin, and rifampin. The resulting data set comprises the first overview of transcriptional stress responses of mycobacteria to different antibiotics. We show that antibiotics can be distinguished based on their specific transcriptional stress fingerprint. Notably, this fingerprint was more distinctive in M. marinum. We decided to use this to our advantage and continue with this model organism. A selection of diverse antibiotic stress genes was used to construct stress reporters. In total, three functional reporters were constructed to respond to DNA damage, cell wall damage, and ribosomal inhibition. Subsequently, these reporter strains were used to screen a small anti-TB compound library to predict the mode of action. In doing so, we identified the putative modes of action for three novel compounds, which confirms the utility of our approach

Microsomal stabilities of wollamide B analogues.

<p>Microsomal stabilities of wollamide B analogues.</p