Synthesis and antimycobacterial activity of 4-[5-(substituted phenyl)-4, 5-dihydro-3-isoxazolyl]-2-methylphenols

Compounds based on the isoxazoline moiety were screened for their antimycobacterial activity in vitro against Mycobacterium tuberculosis H37R (MTB), and INH (isoniazid) resistant Mycobacterium tuberculosis (INHR-MTB) using the agar dilution method and bactec 460. Among the synthesized compounds, 4-[5-(4-bromophenyl)-4,5-dihydro-3-isoxazolyl]-2-methylphenol (4l) was found to be the most active agent against MTB and INHR-MTB with minimum inhibitory concentration of 0.62 μM. When compared to INH, compound (4l) was 1.12 fold and 3.0 fold more active against MTB and INHR-MTB, respectively.     

结核分枝杆菌对宿主巨噬细胞死亡方式的调控

结核分枝杆菌（Mycobacterium tuberculosis,Mtb）感染后能抑制宿主巨噬细胞（M西）的免疫反应,并在其中生存、复制。研究表明Mtb减毒株感染主要诱导宿主Mφ凋亡,凋亡能抑制胞内Mtb的活力;而Mtb毒力株感染能抑制凋亡的完成,诱导Mφ坏死,最终导致Mtb扩散、感染临近细胞。通过对Mtb感染诱导宿主Mφ不同死亡方式的讨论,进一步认识Mtb的致病机制。     

Tuberculosis vaccine design: influence of the completed genome sequence

Tuberculosis continues to be a major health problem, with more adults dying from Mycobacterium tuberculosis than any other pathogen world-wide.With the onset of the HIV epidemic and an increase in drug-resistant M. tuberculosis strains, the need for an improved vaccine has become an international priority.The recent completion of the genome sequences for two M. tuberculosis strains provides a wealth of information that can be used to design new strategies for vaccine development. The challenge comes in making rational choices from among the 4,000 genes of the most probable candidate immunogens or virulence genes.Thus, a well-designed screen is needed to reduce the number of candidates that must be tested. Presently, the most valuable role that bioinformatics can play is to provide such a screen.     

Tuberculosis patients co-infected with Mycobacterium bovis and Mycobacterium tuberculosis in an urban area of Brazil

In this cross-sectional study, mycobacteria specimens from 189 tuberculosis (TB) patients living in an urban area in Brazil were characterised from 2008-2010 using phenotypic and molecular speciation methods (pncA gene and oxyR pseudogene analysis). Of these samples, 174 isolates simultaneously grew on Löwenstein-Jensen (LJ) and Stonebrink (SB)-containing media and presented phenotypic and molecular profiles of Mycobacterium tuberculosis, whereas 12 had molecular profiles of M. tuberculosis based on the DNA analysis of formalin-fixed paraffin wax-embedded tissue samples (paraffin blocks). One patient produced two sputum isolates, the first of which simultaneously grew on LJ and SB media and presented phenotypic and molecular profiles of M. tuberculosis, and the second of which only grew on SB media and presented phenotypic profiles of Mycobacterium bovis. One patient provided a bronchial lavage isolate, which simultaneously grew on LJ and SB media and presented phenotypic and molecular profiles of M. tuberculosis, but had molecular profiles of M. bovis from paraffin block DNA analysis, and one sample had molecular profiles of M. tuberculosis and M. bovis identified from two distinct paraffin blocks. Moreover, we found a low prevalence (1.6%) of M. bovis among these isolates, which suggests that local health service procedures likely underestimate its real frequency and that it deserves more attention from public health officials.     

Antimycobacterial activity: synthesis of novel 3-(substituted phenyl)-6,7-dimethoxy-3a,4-dihydro-3H-indeno[1,2-c]isoxazole analogues

In this study, a series of novel 3-(substituted phenyl)-6,7-dimethoxy-3a,4-dihydro-3H-indeno[1,2-c]isoxazole analogues were synthesized and evaluated for antimycobacterial activity against Mycobacterium tuberculosis (MTB) H₃₇Rv and isoniazid resistant M. tuberculosis (INHR-MTB). All the newly synthesized compounds were showing moderate to high inhibitory activities. The compound 6,7-dimethoxy-3-(4-chloro phenyl)-4H-indeno[1,2-c]isoxazole (4b) was found to be the most promising compound, active against MTB H₃₇Rv and INHR-MTB with minimum inhibitory concentrations of 0.22 and 0.34 μM.     

Cloning and characterization of aspartate-beta-semialdehyde dehydrogenase from Mycobacterium tuberculosis H37 Rv

AIMS: To clone and characterize the aspartate-beta-semialdehyde dehydrogenase of Mycobacterium tuberculosis H37Rv. METHODS AND RESULTS: The asd gene of M. tuberculosis H37Rv was cloned in pGEM-T Easy vector, subcloned in expression vector pQE30 having a T5 promoter, and overexpressed in Escherichia coli. The ASD enzyme was expressed to levels of 40% but was found to be inactive. Functional ASD was obtained by altering induction and growth conditions and the enzyme was purified to near homogeneity using nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography. The K(m) and V(max) values for the three substrates L-ASA, NADP and Pi, the turnover number and specific activity of the enzyme were determined. CONCLUSIONS: Functional ASD enzyme of M. tuberculosis was obtained by gene cloning and protein purification using affinity chromatography. The K(cat) and specific activity of the enzyme were 8.49 s(-1) and 13.4 micromol min(-1) microg(-1) respectively. Significance and Impact of the Study: The ASD enzyme is a validated drug target. We characterized this enzyme from M. tuberculosis and future work would focus on deducing the three-dimensional structure of the enzyme and design of inhibitors, which could be used as drugs against TB.     

结核病诊断技术新进展

结核病疫情亟需快速、有效的诊断方法,但长久以来其诊断一直依靠传统的结核分枝杆菌抗酸染色涂片和培养技术,并不能满足快速诊断的需要。为解决这个全球性的问题,近年来出现了多种结核病新的诊断技术和方法,如新的影像学检查及计算机辅助技术、显微镜学诊断技术、结核分枝杆菌快速培养技术、免疫学诊断技术,以及结核分枝杆菌特异性核酸扩增技术。本文主要介绍几种最有代表性的技术和方法,其中部分技术已获得世界卫生组织(World Health Organization,WHO)的推荐,部分正在进行大规模临床研究或验证,反映了近年来结核病诊断发展的新方向。     

Experimental studies on the growth and usnic acid production in "lichen"Usnea ghattensis in vitro

The study was aimed to optimize the culture conditions for the production of usnic acid in the cultured cell aggregates composed of symbionts in lichen Usnea ghattensis in vitro. The cultured lichen tissue composed of symbionts appeared after about 2-3 weeks of inoculation in water-agar and malt-yeast extract (MYE) media and shown the production of usnic acid after 2-3 months of inoculation. However, the growth of symbionts was strongly affected by different culture conditions. The addition of excess carbon and nitrogen sources in the media has significantly enhanced the growth as well as usnic acid content. The cultured symbionts in MYE medium having 4% sucrose, 4% polyethyl glycol (PEG) gave 7.63 g dry biomass with 3.9 microg usnic acid/g dry biomass. In water-agar medium having 4% sucrose and 4% PEG gave 3.08 g dry biomass with 1.11 microg usnic acid/g dry biomass. The positive effects of medium on the growth of symbionts and the production of usnic acid are seemed to be due to nutritional factors.     

Extensively drug-resistant tuberculosis: current challenges and threats

Extensively drug-resistant tuberculosis (XDR-TB) is defined as tuberculosis caused by a Mycobacterium tuberculosis strain that is resistant to at least rifampicin and isoniazid among the first-line antitubercular drugs (multidrug-resistant tuberculosis; MDR-TB) in addition to resistance to any fluroquinolones and at least one of three injectable second-line drugs, namely amikacin, kanamycin and/or capreomycin. Recent studies have described XDR-TB strains from all continents. Worldwide prevalence of XDR-TB is estimated to be c. 6.6% in all the studied countries among multidrug-resistant M. tuberculosis strains. The emergence of XDR-TB strains is a reflection of poor tuberculosis management, and controlling its emergence constitutes an urgent global health reality and a challenge to tuberculosis control activities in all parts of the world, especially in developing countries and those lacking resources and as well as in countries with increasing prevalence of HIV/AIDS.     

Characterization of Mycobacterium tuberculosis diaminopimelic acid epimerase: paired cysteine residues are crucial for racemization

Recently, the overproduction of Mycobacterium tuberculosis diaminopimelic acid (DAP) epimerase MtDapF in Escherichia coli using a novel codon alteration cloning strategy and the characterization of the purified enzyme was reported. In the present study, the effect of sulphydryl alkylating agents on the in vitro activity of M. tuberculosis DapF was tested. The complete inhibition of the enzyme by 2-nitro-5-thiocyanatobenzoate, 5,5'-dithio-bis(2-nitrobenzoic acid) and 1,2-benzisothiazolidine-3-one at nanomolar concentrations suggested that these sulphydryl alkylating agents modify functionally significant cysteine residues at or near the active site of the epimerase. Consequently, the authors extended the characterization of MtDapF by studying the role of the two strictly conserved cysteine residues. The putative catalytic residues Cys87 and Cys226 of MtDapF were replaced individually with both serine and alanine. Residual epimerase activity was detected for both the serine replacement mutants C87S and C226S in vitro. Kinetic analyses revealed that, despite a decrease in the K(M) value of the C87S mutant for DAP that presumably indicates an increase in nonproductive substrate binding, the catalytic efficiency of both serine substitution mutants was severely compromised. When either C87 or C226 were substituted with alanine, epimerase activity was not detected emphasizing the importance of both of these cysteine residues in catalysis.     

Mycobacterial surface moieties are released from infected macrophages by a constitutive exocytic event

Bacterial cell wall constituents are released from mycobacterial phagosomes and actively traffic within infected macrophages. Colocalization of fluorescently tagged bacterial moieties with endocytic tracers revealed the dynamic movement of released mycobacterial constituents into the endocytic network with accumulation in tubular lysosomal-like compartments. The released bacterial constituents not only penetrated the infected host cell but were also present in an extracellular microvesicular fraction. To identify the intracellular source of these exocytic compartments, released vesicular material was isolated from culture supernatants by differential ultracentrifugation and characterized by Western blot and electron microscopy analyses. The presence of lysosomal membrane proteins and lysosomal proteases suggested that labeled mycobacterial cell wall constituents access a constitutive lysosomal exocytic pathway. An abundance of multilamellar extracellular compartments morphologically reminiscent of MHC class II-enriched compartments (MIIC) implicated a MHC class II transport pathway in the extracellular release of bacterial constituents. Increases in intracellular free calcium have previously been shown to trigger lysosomal exocytosis by inducing fusion of lysosomes with the plasma membrane. To test if an increase in calcium would stimulate exocytosis with release of mycobacterial constituents, infected macrophages were exposed to the calcium ionophore A23187. The ionophore triggered the release of a microvesicular fraction containing labeled bacterial moieties, implicating calcium-regulated lysosomal exocytosis as a trafficking pathway by which mycobacterial products are released from infected macrophages.     

Use of a codon alteration strategy in a novel approach to cloning the Mycobacterium tuberculosis diaminopimelic acid epimerase

Previous attempts to express the diaminopimelate epimerase gene dapF of Mycobacterium tuberculosis in Escherichia coli resulted in undetectable enzyme yields. We used silent mutation of the first 10 codons of the recombinant ORF in an attempt to reduce the formation of secondary structures that might occur near the 5' end of the mRNA and inhibit translation. This significantly increased the yield of the enzyme, which was purified and characterized biochemically. This strategy could be generally applied to other mycobacterial genes that are difficult to express hetero-specifically and here provided pure M. tuberculosis DapF, a good foundation for future research in antimycobacterial agents.     

Synthesis of various 3-nitropropionamides as Mycobacterium tuberculosis isocitrate lyase inhibitor

Various 3-nitropropionamides were synthesized and evaluated for in vitro activities against log and starved phase culture of two mycobacterial species and Mycobacterium tuberculosis (MTB) isocitrate lyase (ICL) enzyme inhibition studies. Among 22 compounds, 1-cyclopropyl-7-(3,5-dimethyl-4-(3-nitropropanoyl)piperazin-1-yl)-6-fluoro-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (22) was found to be the most active compound in vitro with MICs of 0.16 and 0.04 μM against log- and starved-phase culture of MTB. Compound 22 also showed good enzyme inhibition of MTB ICL with IC(50) of 0.10 ± 0.01 μM. The docking studies also confirmed the binding potential of the compounds at the ICL active site.