Synthesis and Antitubercular Evaluation of Some Novel 1,2,3,6-tetrahydropyrimidine-5-carbonitrile

         In an attempt to find a new class of antitubercular agents, a series of 1,2,3,6-tetrahydropyrimidine-5-carbonitrile were prepared via the reaction  of ethyl N-ethoxycarbonylbenzimidate 2a-b with cyanoacetanilide derivatives 1a-c. These compounds were screened for their antitubercular activity against M. tuberculosis. Several analogues, such as 2,6-dioxo-1-phenyl-4-p-tolyl-1,2,3,6-tetrahydropyrimidine-5-carbonitrile 3a, 1-benzyl-2, 6-dioxo-4-p-tolyl-1,2,3,6-tetrahydropyrimidine-5-carbonitrile 3c and 1-benzyl-2, 6-dioxo-4-phenyl-1,2,3,6-tetrahydropyrimidine-5-carbonitrile 3d exhibited a potent antitubercular activity with an MIC values ranging from 10-35 µg/ml. Structures of the newly synthesized compounds were established by spectral data and HRMS

Attenuation of Mycobacterium species through direct and macrophage mediated pathway by unsymmetrical diaryl urea

Tuberculosis is a major threat for mankind and the emergence of resistance strain of Mycobacterium tuberculosis (Mtb) against first line antibiotics makes it lethal for human civilization. In this study, we have synthesized different diaryl urea derivatives targeting the inhibition of mycolic acid biosynthesis. Among the 39 synthesized molecules, compounds 46, 57, 58 and 86 showed MIC values ≤ 10 μg/ml against H37Rv and mc26030 strains. The best molecule with a methyl at ortho position of the first aromatic ring and prenyl group at the meta position of the second aromatic ring showed the MIC value of 5.2 μg/ml and 1 μg/ml against H37Rv and mc26030 respectively, with mammalian cytotoxicity of 163.4 μg/ml. The effective compounds showed selective inhibitory effect on mycolic acid (epoxy mycolate) biosynthesis in14C-radiolabelled assay. At the same time these molecules also executed their potent immunomodulatory activity by up-regulation of IFN-γ and IL-12 and down-regulation of IL-10.Fil: Velappan, Anand Babu. Sastra University; IndiaFil: Charan Raja, Mamilla R.. Sastra University; IndiaFil: Datta, Dhrubajyoti. Indian Institute of Science Education and Research Pune; IndiaFil: Tsai, Yi Ting. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Halloum, Iman. Université de Montpellier; Francia. Centre National de la Recherche Scientifique; FranciaFil: Wan, Baojie. University of Illinois; Estados UnidosFil: Kremer, Laurent. Université de Montpellier; Francia. Inserm; Francia. Centre National de la Recherche Scientifique; FranciaFil: Gramajo, Hugo Cesar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Franzblau, Scott G.. University of Illinois; Estados UnidosFil: Kar Mahapatra, Santanu. Sastra University; IndiaFil: Debnath, Joy. Sastra University; Indi

Resistance to thiacetazone derivatives active against Mycobacterium abscessus involves mutations in the MmpL5 transcriptional repressor MAB_4384

Available chemotherapeutic options are very limited against Mycobacterium abscessus, which imparts a particular challenge in the treatment of cystic fibrosis (CF) patients infected with this rapid-growing mycobacterium. New drugs are urgently needed against this emerging pathogen, but the discovery of active chemotypes has not been performed intensively. Interestingly, however, the repurposing of thiacetazone (TAC), a drug once used to treat tuberculosis, has increased following the deciphering of its mechanism of action and the detection of significantly more potent analogues. We, therefore, report studies performed on a library of 38 TAC-related derivatives, previously evaluated for their antitubercular activity. Several compounds, including D6, D15 and D17, were found to exhibit potent activity in vitro against M. abscessus, Mycobacterium massiliense and Mycobacterium bolletii clinical isolates from CF and non-CF patients. Similarly to TAC in M. tuberculosis, the three analogues act as pro-drugs in M. abscessus, requiring bioactivation by the EthA enzyme, MAB_0985. Importantly, mutations in the transcriptional TetR repressor MAB_4384, with concomitant upregulation of the divergently oriented adjacent genes encoding an MmpS5/MmpL5 efflux pump system, accounted for high cross-resistance levels among all three compounds. Overall, this study uncovered a new mechanism of drug resistance in M. abscessus and demonstrated that simple structural optimization of the TAC scaffold can lead to the development of new drug candidates against M. abscessus infections

Mycobacterium abscessus-Induced Granuloma Formation Is Strictly Dependent on TNF Signaling and Neutrophil Trafficking

Mycobacterium abscessus is considered the most common respiratory pathogen among the rapidly growing non-tuberculous mycobacteria. Infections with M. abscessus are increasingly found in patients with chronic lung diseases, especially cystic fibrosis, and are often refractory to antibiotic therapy. M. abscessus has two morphotypes with distinct effects on host cells and biological responses. The smooth (S) variant is recognized as the initial airway colonizer while the rough (R) is known to be a potent inflammatory inducer associated with invasive disease, but the underlying immunopathological mechanisms of the infection remain unsolved. We conducted a comparative stepwise dissection of the inflammatory response in S and R pathogenesis by monitoring infected transparent zebrafish embryos. Loss of TNFR1 function resulted in increased mortality with both variants, and was associated with unrestricted intramacrophage bacterial growth and decreased bactericidal activity. The use of transgenic zebrafish lines harboring fluorescent macrophages and neutrophils revealed that neutrophils, like macrophages, interact with M. abscessus at the initial infection sites. Impaired TNF signaling disrupted the IL8-dependent neutrophil mobilization, and the defect in neutrophil trafficking led to the formation of aberrant granulomas, extensive mycobacterial cording, unrestricted bacterial growth and subsequent larval death. Our findings emphasize the central role of neutrophils for the establishment and maintenance of the protective M. abscessus granulomas. These results also suggest that the TNF/IL8 inflammatory axis is necessary for protective immunity against M. abscessus and may be of clinical relevance to explain why immunosuppressive TNF therapy leads to the exacerbation of M. abscessus infections

Les déshydratases des acides mycoliques chez mycobacterium abscessus : contribution à la pathogénicité et cibles thérapeutiques potentielles

Mycobacterium abscessus est une mycobactérie à croissance rapide qui a récemment émergé en tant que pathogène opportuniste, en particulier chez les patients atteints de mucoviscidose. Elle est naturellement résistante aux antibiotiques les plus couramment disponibles, limitant sérieusement les options thérapeutiques chez les patients infectés. Il apparaît donc urgent d’identifier et de caractériser de nouvelles cibles d’intérêt thérapeutique dans la lutte contre ce pathogène. Contrairement au variant lisse (S) de M. abscessus, caractérisé par une forte production de glycopeptidolipides (GPL), le variant rugueux (R) associé à une faible production de GPL est responsable des manifestations cliniques les plus sévères. Chez l’embryon de poisson zèbre, le variant R s’accompagne d’une virulence accrue avec formation de cordes mycobactériennes extracellulaires et d’abcès, engendrant une mortalité rapide des larves infectées. Les mécanismes moléculaires de la pathogénicité de M. abscessus demeurent toutefois très peu connus.Dans cette étude, nous avons identifié le gène MAB_4780, codant une déshydratase distincte du complexe HadABC impliqué dans la biosynthèse des acides mycoliques. Le gène MAB_4780, tout comme son homologue chez M. smegmatis, MSMEG_6754, ont été identifiés comme responsables de la résistance innée de ces deux espèces au thiacetazone (TAC), un agent antituberculeux de seconde intention. Nous avons inactivé le gène MAB_4780 dans le variant R de M. abscessus, ce qui a entraîné une modification de la composition en acides mycoliques, un défaut de formation des cordes mycobactériennes ainsi qu'un phénotype extrêmement atténué chez les embryons de poisson zèbre. L'atténuation in vivo du mutant MAB_4780 qui résulte très vraisemblablement de l’incapacité i) à former des cordes et ii) à inhiber la fusion phagolysosomale, s’accompagne d’une croissance intracellulaire diminuée. En outre, le mutant MAB_4780, tout comme le mutant de M. smegmatis dépourvu de MSMEG_6754, présente une croissance altérée dans l’amibe, qui représente un réservoir pour les mycobactéries environnementales. Ces résultats reflètent le rôle crucial de cette nouvelle déshydratase dans la survie de M. abscessus dans son environnement et dans l'établissement d'infections aiguës et létales. Par conséquent, cibler MAB_4780 pourrait représenter une stratégie particulièrement prometteuse pour contrôler les infections à M. abscessus. De futures études seront consacrées à l'identification d’inhibiteurs spécifiques de MAB_4780, grâce au développement d’un test d’activité et d’une structure cristalline de la protéine obtenue à très haute résolution. Nous avons également criblé contre M. abscessus une chimiothèque d’analogues structuraux du TAC, préalablement validés pour leur activité inhibitrice de la déshydratase HadABC de Mycobacterium tuberculosis. Trois d’entre eux ont montré une efficacité accrue d’un facteur 50 par rapport à la molécule parentale. La surexpression d’EthA, l’activateur du TAC, augmente la susceptibilité de M. abscessus à ces trois analogues. Ces résultats suggèrent que leur mode d'activation est similaire à celle de la TAC et indiquent que l'optimisation d’analogues du TAC pourrait conduire à une nouvelle génération de composés plus efficaces pour le traitement de M. abscessus. Des études de relations structure/activité sont envisagées afin d’améliorer l’efficacité et les propriétés pharmacologiques des analogues du TAC.Mycobacterium abscessus, a rapidly-growing mycobacterium (RGM), has emerged in recent years as an important opportunistic pathogen especially in cystic fibrosis (CF) patients. M. abscessus is naturally resistant to most commonly available antibiotics, which seriously limits the treatment options, emphasizing the urgent need for more efficient drugs and innovative therapeutic strategies to combat M. abscessus infections. The M. abscessus rough (R) low-glycopeptidolipids (GPL) producer is responsible for more severe clinical infections than the smooth (S) high-GPL producer, and is associated with increased virulence in zebrafish, including the formation of massive serpentine cords, abscesses, and rapid larval death. However, the molecular mechanisms responsible for the pathogenicity of the R strains remain elusive.Herein, we identified a novel gene, MAB_4780, encoding a dehydratase distinct from the HadABC complex, known to participate in mycolic acid biosynthesis. Both MAB_4780 and its homologue in Mycobacterium smegmatis, MSMEG_6754, are responsible for the innate resistance in these species to thiacetazone (TAC), a second-line antitubercular drug. The successful deletion of MABS_4780 in the R variant of M. abscessus resulted in an altered mycolic acid composition, a pronounced defect in cording, and an extremely attenuated phenotype in zebrafish embryos. The in vivo attenuation of the MAB_4780 mutant results from both the deficiency in cord formation and the impaired intracellular growth, presumably due to limited inhibition of the phagolysosomal fusion events. In addition, similarly to the MSMEG_6754 deletion mutant, the MAB_4780 mutant showed impaired growth in amoeba, which represents a possible reservoir for environmental mycobacteria. These results reflect the critical role of this new dehydratase in the survival of M. abscessus in its environmental hosts and its importance in establishing acute and lethal infections. Therefore, targeting MAB_4780 may represent a promising strategy to control M. abscessus infections. Future work will focus on identifying specific inhibitors targeting MAB_4780, which could greatly benefit from the combination of our dehydratase assay and our high-resolution crystal structure of the protein.We have also screened against M. abscessus a library of TAC analogues, previously validated for their inhibitory effects against the Mycobacterium tuberculosis HadABC dehydratase. Among these compounds, three exhibited a 50-fold increased potency as compared to TAC. Overexpression of EthA, known as the activator of TAC, increased the susceptibility of M. abscesuss to the three analogues, suggesting that that their mode of activation is similar to that of TAC. Overall, these data indicate that optimizing the TAC scaffold may lead to more efficient compounds against M. abscessus. Additional structure/activity relationship studies are required to further improve the efficacy and pharmacological properties of TAC analogues

Identification of inhibitors targeting Mycobacterium tuberculosis cell wall biosynthesis via dynamic combinatorial chemistry

In this study, we report a dynamic combinatorial approach along with highly efficient in situ screening to identify inhibitors of UDP-galactopyranose mutase (UGM), an essential enzyme involved in mycobacterial cell wall biosynthesis.</p

Recruitment of neutrophils is crucial for granuloma development.

<p>(A and B) WT or <i>tnfr</i> morphants were <i>iv</i> infected with R <i>Mabs</i> (tdTomato, ≈150 CFU). Kinetics of granuloma formation (A) and mean ± SEM number of granuloma per infected embryos (B) in (A); (n = 30–45, three independent experiments). The top panel shows confocal images of representative granuloma. Scale bars, 100 μm. (C) Confocal images showing a representative 3 dpi granulomas in WT <i>versus tnfr1</i> morphant <i>Tg(mpeg</i>:<i>mcherry-F)</i> embryos <i>iv</i> infected with S expressing Wasabi. Arrows indicate extracellular aggregates. Scale bars, 15 μm. (D) Confocal microscopy showing representative granulomas in WT and <i>tnfr1</i> morphant <i>Tg(mpx</i>:<i>eGFP)</i> embryos <i>iv</i> infected with R expressing tdTomato at 4 dpi. Arrows indicate extracellular cords. Scale bars, 30 μm. (E-F) Confocal microscopy monitored WT or <i>tnfr</i> morphant <i>Tg(mpx</i>:<i>eGFP)</i> embryos that were <i>iv</i> infected with R (tdTomato, ≈150 CFU). (E) Representative kinetics of neutrophil mobilization during granuloma formation. Scale bars, 20 μm. (F) Number of neutrophils recruited to WT (up) or TNFR1-depleted (down) granuloma as a function of granuloma volume. Statistical significance was determined by Fisher’s exact test of a contingency table (A), one-tailed unpaired Student’s t test (B) or Pearson correlation (F). See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005986#ppat.1005986.s017" target="_blank">S6</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005986#ppat.1005986.s018" target="_blank">S7</a> Movies.</p

<i>M</i>. <i>abscessus</i> infections are associated with a massive neutrophil mobilization.

<p>(A-D) Confocal live imaging of <i>Tg(mpx</i>:<i>eGFP)</i> larvae after <i>iv</i> (A and D) or intramuscular (B and C) infection with ≈100 <i>Mabs</i> R (tdTomato). (A-B) Time-lapse of neutrophils recruitment in caudal vein (A), monitored from 20 mpi to 110 mpi, or into muscle (B) monitored from 10 mpi to 150 mpi. Scale bars, 20 μm. (C) Recruited neutrophils phagocytizing individual mycobacteria. Scale bar, 5 μm. (D) Neutrophils (arrows) and presumptive macrophages (*), containing mycobacteria. Scale bar, 20 μm. (E) Number of infected neutrophils and macrophages counted in CHT at 4 hrs post intravenous infection with both <i>Mabs</i> variants (≈150 CFU, three independent experiments, horizontal lines indicate mean values). M, macrophage; N, neutrophils. Statistical significance was determined by one-tailed unpaired Student’s t test. (F) Confocal imaging of neutrophil-bacteria interactions in the context of R-abscess. Scale bars, 20 μm. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005986#ppat.1005986.s012" target="_blank">S1 Movie</a>.</p

Expression of TNF by macrophages during <i>Mabs</i> infection.

<p>(A) Measurement of relative <i>tnf-α</i> expression by qRT-PCR using <i>ef1α</i> as a reference gene in whole embryo over PBS. Around 150 <i>Mabs</i> R or S variants were <i>iv</i> injected and assessed at 2 and 5 dpi. Mean log₁₀ value of three independent experiments with error bars representing the standard error of the mean (SEM). (B-D) <i>Tg</i>(<i>tnf-α</i>:<i>eGFP-F</i>) larvae were injected in the otic cavity with ≈100 <i>Mabs</i> R or S variants (tdTomato) or with PBS. (B) Bright-field and fluorescence overlay microscopy showing the representative expression of <i>tnf</i> close to the injection site at 2 hpi. Scale bars, 100 μm. (C-D) Mean proportion of infected larvae with <i>tnf</i>⁺ cells (n = 10) (C) and quantification of <i>tnf</i>⁺ cells per infected larvae (each symbol represents individual embryos and horizontal lines indicate the mean values) (D) in (C) after 2 hpi. The data are representative of two experiments. (E) Distribution of <i>tnfα</i>-expressing cells revealed by the <i>tnf-α</i>:<i>eGFP-F</i> reporter transgene (arrows) in whole <i>Tg</i>(<i>tnf-α</i>:<i>eGFP-F</i>) larvae imaged live at the indicated time points post <i>iv</i> injection of PBS, <i>Mabs</i> R or S (tdTomato, ≈150 colony forming units (CFU)). The yolk (*) is auto-fluorescent. Scale bars, 200 μm. (F-H) Confocal images showing the representative <i>tnf</i> expression in a 3 dpi-granuloma (F) (scale bar, 20 μm), in a 5 dpi-brain abscess (G) (scale bar, 50 μm) or close to a 3 dpi-cord (H) (scale bars, 20 μm) in <i>Tg(tnf-α</i>:<i>eGFP-F)</i> embryos <i>iv</i> infected with <i>Mabs</i> R (tdTomato). (I-J) Confocal microscopy of a 3 dpi-granuloma showing the <i>tnf</i> expression in <i>Tg</i>(<i>tnf-α</i>:<i>eGFP-F/mpeg1</i>:<i>mCherryF</i>) (I) or <i>Tg</i>(<i>tnf-α</i>:<i>eGFP-F/LysC</i>:<i>DsRed</i>) (J) double transgenic embryos <i>iv</i> infected with <i>Mabs</i> R (E2-Crimson). Scale bars, 50 μm. Statistical significance was determined by Kruskal-Wallis test with Dunns post-test (A), Fisher’s exact test of a contingency table (C) or one-tailed unpaired Student’s t test (D).</p

<i>tnfr</i> morphants are highly susceptible to <i>Mabs</i> infections.

<p>(A-H) WT or <i>tnfr</i> morphants were <i>iv</i> infected with either R (A-H) or S (A-D) variants of <i>Mabs</i> (tdTomato, ≈150 CFU). (A) Survival of infected embryos <i>versus</i> PBS-injected embryos (n = 90, average of three independent experiments). (B-C) Representative fluorescence images of: (B) Bacterial loads (FPC, two independent experiments, horizontal lines indicate the median values); (C) 3 dpi embryos infected by either R or S variants. Scale bars, 200 μm. (D) Proportion of larvae with abscesses after 13 dpi expressed as mean ± SEM from two independent experiments (n = 40–50). (E) Kinetic of R-cord formation in whole infected embryos. Mean ± SEM from three independent experiments (n = 30). (F) Fluorescence microscopy of <i>tnfr</i> morphants exhibiting widespread R-cording in the vasculature and in the CNS at 3 dpi. Scale bar, 200μm. (G-H) Proportion of embryos containing <5 or >5 cords (G) and localization of cords (H) in (E). Error bars represent the SEM. Statistical significance was determined by log-rank test (A), one-tailed Mann-Whitney’s t test (C) or Fisher’s exact test of a contingency table (D-E and G-H).</p