H37Ra Strain Research Articles

Anti tubercular minimum inhibitory concentration (MIC) and chemical characterization of ethnobotanical mixture used in the treatment of tuberculosis

Introduction: Tuberculosis (TB) is a national health priority and with failing current treatment regimens, uses of ethnobotanical therapies are being more vigorously explored. Treatment of TB using oral and inhalational administration (hawan) of a mixture of herbs (hawan samagri, HS) has been practiced in India since ages. In vitro antitubercular effect of extract of HS against H37Ra strain of Mycobacterium tuberculosis (MTB) was recently reported in an ICMR funded research project.1 Hence this study aims to document scientific data on antitubercular effect of this HS extract against clinical MTB strains. Materials and Methods: Minimum inhibitory concentration (MIC) against clinical MTB strains of two different ethnobotanical mixtures (HS1 and HS2) used traditionally in India for the indigenous treatment of TB was compared. Extraction of essential oil (EO) from the mixture was done by hydro distillation and chemical characterization was done by Gas Chromatography and mass Spectroscopy (GCMS). Resazurin Microtiter Assay (REMA) was done to study antitubercular MIC. Result and Discussion: Crude essential oil (EO) extract of HS showed significant antitubercular effect in MIC range 50 - 400 µg/ml. HS1 showed lower MIC values compared to HS2. GCMS analysis of EO showed an array of bioactive compounds known to have antitubercular and other beneficial effects. Conclusion: Scientific validation of ethnobotanical use of HS extract for antitubercular activity was obtained in this work. HS crude EO seems to be a promising natural product with potential for new drug development in the treatment of TB. Keywords: Ethnobotanical, Antitubercular activity, Essential oil, Multi drug resistant (MDR), Resazurin microtiter assay (REMA), Gas Chromatography and mass spectroscopy (GCMS), Phytochemical, Terpenoids, Phenolics, Hawan samagri (HS).

Synthesis and antimycobacterial evaluation of new 5-(1-benzyl-1H-1,2,3-triazol-4-yl)-4-methyl-2-arylthiazole derivatives

A new series of 5-(1-benzyl-1H-1,2,3-triazol-4-yl)-4-methyl-2-arylthiazole derivatives, 6a−w have been synthesized by click reaction of substituted benzylazide, 5a−d with 5-ethynyl-4-methyl-2-substituted phenylthiazole, 4a−f. The starting compounds 4-ethynyl-2-substituted phenylthiazole (4a−f) were synthesized from the corresponding thiazole aldehyde by using the Ohira−Bestmann reagent. The structure of the synthesized compounds was determined by spectral analysis. All the synthesized compounds were screened for their preliminary antitubercular activity against Mycobacterium tuberculosis H37Ra (MTB, ATCC 25177). Most of the synthesized compounds reported good activity against M. tuberculosis H37Ra strain with IC50 range of 0.58−8.23 µg/mL. Compounds 6g and 6k reported good antitubercular activity with MIC90 values of 4.71 and 2.22 µg/mL, respectively. Potential antimycobacterial activity suggested that these compounds could serve as good lead compounds for further optimization and development of a newer antitubercular candidate.

Characterisation of genes differentially expressed in macrophages by virulent and attenuated Mycobacterium tuberculosisthrough RNA-Seq analysis

Tuberculosis (TB) remains a global healthcare issue. Understanding the host-pathogen interactions in TB is vital to develop strategies and therapeutic tools for the control of Mycobacterium tuberculosis (Mtb). In this study, transcriptome analyses of macrophages infected with either the virulent Mtb strain H37Rv (Rv) or the avirulent Mtb strain H37Ra (Ra) were carried out and 750 differentially expressed genes (DEGs) were identified. As expected, the DEGs were mainly involved in the induction of innate immune responses against mycobacterial infections. Among the DEGs, solute carrier family 7 member 2 (Slc7a2) was more strongly expressed in Ra-infected macrophages. Induction of SLC7A2 was important for macrophages to control the intracellular survival of Mtb. Our results imply that SLC7A2 plays an important role in macrophages during Mtb infection. Our findings could prove useful for the development of new therapeutic strategies to control TB infection.

Design and Synthesis of 2‐Amino‐thiophene‐proline‐conjugates and Their Anti‐tubercular Activity against Mycobacterium Tuberculosis H37Ra

AbstractThe emergence of extensively drug resistant tuberculosis (XDR‐TB) and multi‐drug resistant tuberculosis (MDR‐TB) has necessitated the development of new drugs with short chemotherapy treatment regime and cost effectiveness. To overcome these challenges, we are reporting the synthesis of a series of 2‐amino‐thiophene‐proline‐conjugates which show potent in‐vitro and ex‐vivo anti‐tubercular (anti‐TB) activity against mycobacterium tuberculosis (mtb) H37Ra. The synthesis of these 2‐amino‐thiophene‐proline‐conjugates was carried out via solution phase peptide coupling reactions using methyl‐2‐aminothiophene‐3‐carboxylate 8 as an intermediate obtained by modified gewald reaction. Intermediate 8 was coupled with different amino acids to obtain dipeptides 3, 4, 5, 6 a and 7. Priliminary anti‐TB assay data encoureaged us to synthesize modified proline derivatives 6 b‐6 k via formation of a benzoxazinone intermediate 16. Most of these conjugates are active against mtb H37Ra in both active (A) and dormant (D) strains. They are also active against drug resistant mtb H37Ra strains. A trifluoroethyl ester analog, 6 i was the most potent among the series [MIC 1 μg/mL] along with 6 f and 6 g [MIC 2–6 μg/mL]. Cytotoxicity studies suggested that, these compounds are less cytotoxic to human cell lines HeLa, MCF‐7, HUVEC and hence possess high selectivity index (SI). Docking studies revealed that the binding mode of most active compounds 6 i, 6 g and 6 f is in accordance with their bioactivity studies having docking score −8.969, −8.446 and −7.865, respectively. Moreover, in silico ADME properties suggest that all the compounds possess drug like properties.

1-(Piperidin-3-yl)thymine amides as inhibitors of M. tuberculosis thymidylate kinase

A series of readily accessible 1-(piperidin-3-yl)thymine amides was designed, synthesised and evaluated as Mycobacterium tuberculosis TMPK (MtbTMPK) inhibitors. In line with the modelling results, most inhibitors showed reasonable MtbTMPK inhibitory activity. Compounds 4b and 4i were slightly more potent than the parent compound 3. Moreover, contrary to the latter, amide analogue 4g was active against the avirulent M. tuberculosis H37Ra strain (MIC50=35 µM). This finding opens avenues for future modifications.

Design, Synthesis and Biological Screening of Novel 1,3,4‐Oxadiazoles as Antitubercular Agents

AbstractA series of novel 2,5‐disubstitued 1,3,4‐oxadiazole derivatives bearing 2,2‐dimethyl‐2,3‐dihydrobenzofuran scaffold has been synthesized and screened for antitubercular activity. All the synthesized compounds were characterized by IR, 1H NMR, 13C‐NMR and Mass spectral study. The in vitro antitubercular activity of the synthesized compounds was evaluated against Mycobacterium tuberculosis H37Ra(ATCC 25177) strain. Among the synthesized compounds, four compound displayed good antitubercular activity IC50 values in low micro‐gram range (<10 μg/mL). The antitubercular data suggested that growth inhibition MTB can be imparted by the introduction of a 4‐ trifluoromethyl phenyl acetylene substituent. Specificity of these compounds was checked by screening them for their anti‐bacterial activity against four bacterial strains (Gram‐negative strains: E. coli, S. aureus; Gram‐positive strains: P. aeruginosa and B. subtilis). None of the compound displayed antibacterial activity against any of the seleted strain. Molecular docking studies were carried out on InhA (FabI/ENR) which shows that the synthesized compounds bind at the catalytic site in a most favourable manner suggesting their potential as anti‐mycobacterial agents. The research presented here was found to be adventitious for the development of new therapeutic agents against Mycobacterium infection.

Interplay of PhoP and DevR response regulators defines expression of the dormancy regulon in virulent Mycobacterium tuberculosis

The DevR response regulator of Mycobacterium tuberculosis is an established regulator of the dormancy response in mycobacteria and can also be activated during aerobic growth conditions in avirulent strains, suggesting a complex regulatory system. Previously, we reported culture medium-specific aerobic induction of the DevR regulon genes in avirulent M. tuberculosis H37Ra that was absent in the virulent H37Rv strain. To understand the underlying basis of this differential response, we have investigated aerobic expression of the Rv3134c-devR-devS operon using M. tuberculosis H37Ra and H37Rv devR overexpression strains, designated as LIX48 and LIX50, respectively. Overexpression of DevR led to the up-regulation of a large number of DevR regulon genes in aerobic cultures of LIX48, but not in LIX50. To ascertain the involvement of PhoP response regulator, also known to co-regulate a subset of DevR regulon genes, we complemented the naturally occurring mutant phoPRa gene of LIX48 with the WT phoPRv gene. PhoPRv dampened the induced expression of the DevR regulon by >70-80%, implicating PhoP in the negative regulation of devR expression. Electrophoretic mobility shift assays confirmed phosphorylation-independent binding of PhoPRv to the Rv3134c promoter and further revealed that DevR and PhoPRv proteins exhibit differential DNA binding properties to the target DNA. Through co-incubations with DNA, ELISA, and protein complementation assays, we demonstrate that DevR forms a heterodimer with PhoPRv but not with the mutant PhoPRa protein. The study puts forward a new possible mechanism for coordinated expression of the dormancy regulon, having implications in growth adaptations critical for development of latency.

Platinum(II)-azoimidazole drugs against TB and cancer: Structural studies, cytotoxicity and anti-mycobacterial activity

1-Alkyl-2-(arylazo)imidazoles (Haai-C4H9 (1), Haai-C10H21 (2)) and their Pt(II) complexes, [Pt(Haai-C4H9)Cl2] (3) and [Pt(Haai-C10H21)Cl2] (4) were synthesized and characterized by different spectroscopic studies and structural confirmation has been achieved in the case of complex 3 by single crystal X-ray diffraction analysis. Complexes 3 and 4 were evaluated for their in vitro anti-mycobacterial activity against Mycobacterium tuberculosis H37Ra (ATCC 25177) and H37Rv (ATCC 25618) strains, as well as against two clinical strains. Their cytotoxic effects on three cancer cell lines, A549 (human lung carcinoma), MCF-7 (human breast cancer) and Caco-2 (colorectal adenocarcinoma line), and one normal cell line, 3T3 (mouse normal fibroblast) were examined. The DNA interaction of the complexes shows that the intrinsic binding constant (Kb) decreases with the increasing molecular dimension and the chain length of the 1-alkyl group. The longer 1-alkyl chain substituted complex, [Pt(Haai-C10H21)Cl2] (4), is less efficient due to hydrophobic interactions than the lower homologue [Pt(Haai-C4H9)Cl2] (3) (Kb(3), 5.9(2) × 106 M−1 and molar volume, 276.03(10) cm3 mol−1; [Pt(Haai-C10H21)Cl2], Kb(4), 5.86(2) × 105 M−1 and molar volume, 397.56(8) cm3 mol−1).

Synthesis and biological evaluation of novel triazole-biscoumarin conjugates as potential antitubercular and anti-oxidant agents

The synthesis of a new series of triazole-biscoumarin conjugates by using a molecular hybridization approach is described. The newly synthesized compounds 6a–k were evaluated for their in vitro antitubercular activity against active and dormant Mtb H37Ra and anti-oxidant activity against DPPH radical scavenging. Molecular docking simulations for the antitubercular activity showed that the conjugates 6a–k bind in the pocket of the DprE1 enzyme. Most of the conjugates displayed good antitubercular activity against both the active and dormant Mtb H37Ra strain. The compound 6h displayed very good antitubercular activity against dormant Mtb H37Ra with an IC50 value of 1.44 μg/mL. Most of the synthesized conjugates exhibit excellent anti-oxidant activity with an IC50 of less than the standard BHT. Compound 6b is the most active among all the conjugates with an IC50 value of 08.17 ± 0.11 μg/mL. The molecular docking study shows good agreement between the observed antitubercular activity and the binding affinity.

Effect of inhaled air pollution particulate matter in alveolar macrophages on local pro-inflammatory cytokine and peripheral interferon γ production in response to Mycobacterium tuberculosis

Abstract Background Human alveolar macrophages are first-line cellular responders to inhaled particulate matter (PM) and microbial pathogens in the lungs. We examined whether PM exposure affects human bronchoalveolar and peripheral blood mononuclear cell responses to Mycobacterium tuberculosis . Methods Bronchoalveolar and peripheral blood mononuclear cells were obtained from 30 healthy adult volunteers living in Mexico City, Mexico. Proportions of alveolar macrophages with PM and areas of cytoplasm occupied by inhaled PM within alveolar macrophages were evaluated in 50 bronchoalveolar cells from each donor by ImageJ (version 1.49) on 1000× digital bright field microscopy cytospin images. Ambient PM 2.5 (aerodynamic diameters M tuberculosis (strain H37Ra at multiplicities of infection [MOI] of 1 and 10), lipopolysaccharide, 6 kDa early secretory antigenic target (ESAT-6), purified protein derivative, and PM 2.5 were enumerated by ELISPOT assays. Findings While 26–60% of bronchoalveolar cells harboured inhaled PM, percentages of alveolar macrophage areas covered by PM were between 0·9% (±0·16) and 5·49% (±0·78). In unstimulated bronchoalveolar cells and bronchoalveolar cells exposed in vitro to PM (PM 2.5 ; 0·1, 1·0, 10 μg/mL), frequencies of interleukin-1β and TNF-α-producing cells decreased with higher percentages of alveolar macrophages containing inhaled PM (r=–0·78, p=0·0078 and r=–0·63, p=0·038, respectively). By contrast, frequencies of interferon-γ-producing cells increased in bronchoalveolar cells with alveolar macrophages containing greater areas of inhaled PM (r=0·59, p=0·049). In bronchoalveolar cells infected in vitro with M tuberculosis , frequencies of cells producing interleukin 1β significantly decreased in volunteers with higher percentages of alveolar macrophages containing inhaled PM. Frequencies of interferon-γ-producing cells were significantly decreased in peripheral blood mononuclear cells stimulated with purified protein derivative from volunteers with higher percentages of inhaled PM-containing alveolar macrophages (r=–0·7167, p=0·0184). Interpretation The presence of inhaled PM in alveolar macrophages significantly decreases interleukin 1β and TNF-α production in bronchoalveolar cells in response to M tuberculosis infection as well as interferon-γ-production in peripheral blood mononuclear cells in response to purified protein derivative. These findings provide important insights into the potential mechanisms underlying the epidemiological observations of increased risk of tuberculosis development in air pollution exposure settings and indicate that air pollution may have an adverse impact on global tuberculosis control efforts. Funding NIEHS 5RO1ES020382-05.

Novel red fluorescence protein based microplate assay for drug screening against dormant Mycobacterium tuberculosis by using paraffin

The hypoxia model of dormancy is widely used in drug screening programs to identify novel inhibitors against latent Mycobacterium tuberculosis disease. In earlier reported microplate assays, hypoxia was maintained by either sealing the microplate or shifting in an anaerobic chamber to develop dormant phenotype. In these assays, inhibitors were added during inoculation, which mainly represents the active stage inhibitors instead of the dormant ones. Herein, the culture was covered with paraffin to develop hypoxia condition and consequently providing the advantage of adding compounds at any stage during incubation of 96-well plate. The stable expression of the red fluorescent protein in the bacilli under both actively growing as well as dormant conditions also facilitate the reliable estimation of growth and inhibition kinetics of bacilli in medium. Furthermore, S/N ratio and Z′ factor of this assay were found to be > 27 and 0.91–0.94 respectively, which confirm the robustness of the protocol. This newly developed drug-screening assay offers an easy, inexpensive, safe and high throughput-screening tool to search novel antitubercular inhibitors against both active and dormant bacilli. The red fluorescent H37Ra strain is a suitable surrogate for the more virulent H37Rv strain, and thus this effort will help in combating latent tuberculosis.

Nigella damascena L. Essential Oil-A Valuable Source of β-Elemene for Antimicrobial Testing.

The most commonly used plant source of β-elemene is Curcuma wenyujin Y. H. Chen & C. Ling (syn. of Curcuma aromatic Salisb.) with its content in supercritical CO2 extract up to 27.83%. However, the other rich source of this compound is Nigella damascena L. essential oil, in which β-elemene accounts for 47%. In this work, the effective protocol for preparative isolation of β-elemene from a new source—N. damascena essential oil—using high performance counter-current chromatography HPCCC was elaborated. Furthermore, since sesquiterpens are known as potent antimicrobials, the need for finding new agents designed to combat multi-drug resistant strains was addressed and the purified target compound and the essential oil were tested for its activity against a panel of Gram-positive and Gram-negative bacteria, fungi, and mycobacterial strains. The application of the mixture of petroleum ether, acetonitrile, and acetone in the ratio 2:1.5:0.5 (v/v) in the reversed phase mode yielded β-elemene with high purity in 70 min. The results obtained for antimicrobial assay clearly indicated that N. damascena essential oil and isolated β-elemene exert action against Mycobacterium tuberculosis strain H37Ra.

The Effect of Combining Natural Terpenes and Antituberculous Agents against Reference and Clinical Mycobacterium tuberculosis Strains.

Background: On account of emergence of multi- and extensively drug-resistant Mycobacterium tuberculosis (Mtb) strains, combinations of drugs with natural compounds were tested to search for antibiotic activity enhancers. In this work we studied terpenes (α-pinene, bisabolol, β-elemene, (R)-limonene, (S)-limonene, myrcene, sabinene), which are the main constituents of essential oil obtained from Mutellina purpurea L., a plant with described antitubercular activity, to investigate their interactions with antibiotics against reference Mtb strains and multidrug-resistant clinical isolates. Methods: The serial dilution method was used to evaluate the minimal inhibitory concentration (MIC) of tested compounds, while the fractional inhibitory concentration index (FICI) was calculated for characterization of interactions. Moreover, IC50 values of tested compounds were determined using monkey kidney epithelial cell line (GMK). Results: The combinations of all studied terpenes with ethambutol or rifampicin resulted in a synergistic interaction. Bisabolol and (R)-limonene decreased the MIC for rifampicin at least two-fold for all tested strains, however no synergistic action was observed against virulent strains. The tested terpenes showed slight (bisabolol) or no cytotoxic effect against normal eukaryotic cells in vitro. Conclusions: The obtained enhanced activity (FICI < 0.5) of ethambutol and rifampicin against H37Ra strain under the influence of the studied terpenes may be correlated to the capability of essential oil constituents to modify bacterial resistance mechanisms in general. The observed differences in avirulent and virulent bacteria susceptibility to terpenes tested separately and in combinations with antibiotics can be correlated with the differences in the cell wall structure between H37Ra mutant and all virulent strains.

Mycobacterium tuberculosis Strains H37ra and H37rv have equivalent minimum inhibitory concentrations to most antituberculosis drugs.

Mycobacterium tuberculosis (Mtb) strains H37Ra and H37Rv are commonly used to study new and re-evaluate old antituberculous agents with respect to their pharmacodynamic effects in vitro. The differences in membrane proteins and, in particular, differences in carrier proteins between Mtb H37Ra and Mtb H37Rv may have an impact on antibiotic potency. The question of whether H37Ra can be used as a reliable surrogate for H37Rv and clinical strains has not been addressed sufficiently. The purpose of this study is to provide a full comparison of susceptibility data of the most common antituberculosis (TB) agents against both Mtb strains. In addition to a literature review, in vitro checkerboard susceptibility study was conducted comparing the in vitro minimum inhibitory concentration (MIC) of 16 common antituberculous drugs against H37Ra and H37Rv. Heifets-Sanchez TB agar drug susceptibility plates were utilized. Half of the antibiotics demonstrated similar growth inhibition against both strains, while slightly differing MIC values were found for 7 of 16 drugs. With the exception of rifampicin, no marked difference in MIC against H37Ra and H37Rv was observed. While neither the attenuated (H37Ra) nor the virulent strain (H37Rv) is a clinical strain, both strains predicted MICs of clinical isolates equally well, when comparing the current in vitro results to clinical susceptibility data in the literature. H37Ra comes with the benefits of lower experimental costs and less administrative barriers including the requirement of a biosafety Level III environment.

Design, synthesis, and biological evaluation of m-amidophenol derivatives as a new class of antitubercular agents.

A series of m-amidophenol derivatives (6a-6l, 7a-7q, 9a, 9b, 12a-12c, 14 and 15) were designed and synthesized. Their antitubercular activities were evaluated in vitro against M. tuberculosis strains H37Ra and H37Rv and clinically isolated multidrug-resistant M. tuberculosis strains. Ten compounds displayed minimal inhibitory concentrations (MICs) against M. tuberculosis H37Ra below 2.5 μg mL-1 and 6g was the most active compound (MIC = 0.625 μg mL-1). Compounds 6g and 7a also showed potent inhibitory activity against M. tuberculosis H37Rv (MIC = 0.39 μg mL-1) and several clinically isolated multidrug-resistant M. tuberculosis strains (MIC = 0.39-3.125 μg mL-1). The compounds did not show inhibitory activity against normal Gram-positive and Gram-negative bacteria. They exhibited low cytotoxicity against HepG2 and RAW264.7 cell lines. The results demonstrated m-amidophenol as an attractive scaffold for the development of new antitubercular agents.

Microbial sensor for drug susceptibility testing of Mycobacterium tuberculosis.

Drug susceptibility testing (DST) of clinical isolates of Mycobacterium tuberculosis is critical in treating tuberculosis. We demonstrate the possibility of using a microbial sensor to perform DST of M. tuberculosis and shorten the time required for DST. The sensor is made of an oxygen electrode with M. tuberculosis cells attached to its surface. This sensor monitors the residual oxygen consumption of M. tuberculosis cells after treatment with anti-TB drugs with glycerine as a carbon source. In principle, after drug pretreatment for 4-5days, the response differences between the sensors made of drug-sensitive isolates are distinguishable from the sensors made of drug-resistant isolates. The susceptibility of the M. tuberculosis H37Ra strain, its mutants and 35 clinical isolates to six common anti-TB drugs: rifampicin, isoniazid, streptomycin, ethambutol, levofloxacin and para-aminosalicylic acid were tested using the proposed method. The results agreed well with the gold standard method (LJ) and were determined in significantly less time. The whole procedure takes approximately 11days and therefore has the potential to inform clinical decisions. To our knowledge, this is the first study that demonstrates the possible application of a dissolved oxygen electrode-based microbial sensor in M. tuberculosis drug resistance testing. This study used the microbial sensor to perform DST of M. tuberculosis and shorten the time required for DST. The overall detection result of the microbial sensor agreed well with that of the conventional LJ proportion method and takes less time than the existing phenotypic methods. In future studies, we will build an O2 electrode array microbial sensor reactor to enable a high-throughput drug resistance analysis.

Graph Theoretical Analysis, In Silico Modeling, Synthesis, Anti-Microbial and Anti-TB Evaluation of Novel Quinoxaline Derivatives.

We designed to synthesize a number of 2-(2-(substituted benzylidene) hydrazinyl)-N-(4-((3-(phenyl imino)-3,4-dihydro quinoxalin-2(1 H)-ylidene)amino) phenyl) acetamide S1-S13: with the hope to obtain more active and less toxic anti-microbial and anti-TB agents. A series of novel quinoxaline Schiff bases S1-S13: were synthesized from o-phenylenediamine and oxalic acid by a multistep synthesis. In present work, we are introducing graph theoretical analysis to identify drug target. In the connection of graph theoretical analysis, we utilised KEGG database and Cytoscape software. All the title compounds were evaluated for their in-vitro anti-microbial activity by using agar well diffusion method at three different concentration levels (50, 100 and 150 µg/ml). The MIC of the compounds was also determined by agar streak dilution method. The identified study report through graph theoretical analysis were highlights that the key virulence factor for pathogenic mycobacteria is a eukaryotic-like serine/threonine protein kinase, termed PknG. All compounds were found to display significant activity against entire tested bacteria and fungi. In addition the synthesized scaffolds were screened for their in vitro antituberculosis (anti-TB) activity against Mycobacterium tuberculosis (Mtb) strain H37Ra using standard drug Rifampicin. A number of analogs found markedly potent anti-microbial and anti-TB activity. The relationship between the functional group variation and the biological activity of the evaluated compounds were well discussed. The observed study report was showing that the compound S6: (4-nitro substitution) exhibited most potent effective anti-microbial and anti-TB activity out of various tested compounds.

Quantitative proteomic analysis of host responses triggered by Mycobacterium tuberculosis infection in human macrophage cells.

Macrophages are primary host of Mycobacterium tuberculosis (M.tb) and the central effector of in vivo innate immune responses against bacteria. Though the interaction between macrophages and mycobacteria has been widely investigated, the molecular mechanisms of M.tb pathogenesis in macrophages are still not clear. In this work, we investigated the altered protein expression profiles of macrophages after virulent H37Rv strain and avirulent H37Ra strain infection by tandem mass tag-based quantitative proteomics. Among 6762 identified proteins of macrophages, the expression levels of 235 proteins were significantly altered, which is supposed to be related to the infection of different strains. By bioinformatics analysis at systems level, we found that these proteins are mainly involved in the biological process of apoptosis, blood coagulation, oxidative phosphorylation, and others. The enormous variation in protein profiles between macrophages infected with H37Ra and H37Rv suggests the existence of four different immunity mechanisms that decide the fates of macrophages and M.tb. These data may provide a better understanding of M.tb pathogenesis within the host, which contributes to the prevention and clinical treatment of tuberculosis.

2-N-Arylthiazole inhibitors of Mycobacterium tuberculosis

To develop agents for the treatment of infections caused by Mycobacterium tuberculosis, a novel phenotypic screen was undertaken that identified a series of 2-N-aryl thiazole-based inhibitors of intracellular Mycobacterium tuberculosis. Analogs were optimized to improve potency against an attenuated BSL2 H37Ra laboratory strain cultivated in human macrophage cells in vitro. The insertion of a carboxylic acid functionality resulted in compounds that retained potency and greatly improved microsomal stability. However, the strong potency trends we observed in the attenuated H37Ra strain were inconsistent with the potency observed for virulent strains in vitro and in vivo.

Synthesis, biological evaluation, and molecular docking studies of novel 3-aryl-5-(alkyl-thio)-1H-1,2,4-triazoles derivatives targeting Mycobacterium tuberculosis.

A small library of new 3-aryl-5-(alkyl-thio)-1H-1,2,4-triazoles was synthesized and screened for the antimycobacterial potency against Mycobacterium tuberculosis H37 Ra strain and Mycobacterium bovis BCG both in active and dormant stage. Among the synthesized library, 25 compounds exhibited promising anti-TB activity in the range of IC50 0.03-5.88μg/ml for dormant stage and 20 compounds in the range of 0.03-6.96μg/ml for active stage. Their lower toxicity (>100μg/ml) and higher selectivity (SI=>10) against all cancer cell lines screened make them interesting compounds with potential antimycobacterial effects. Furthermore, to rationalize the observed biological activity data and to establish a structural basis for inhibition of M.tuberculosis, the molecular docking study was carried out against a potential target MTB CYP121 which revealed a significant correlation between the binding score and biological activity for these compounds. Cytotoxicity and in vivo pharmacokinetic studies suggested that 1,2,4-triazole analogues have an acceptable safety index, in vivo stability and bio-availability.