Sequence Analysis of the Gene Region Encoding ESAT-6, Ag85B,and Ag85 C Proteins from Clinical Isolates of Mycobacterium tuberculosis

Mycobacterium tuberculosis secreted proteins in culture filtrate and early phase of infection, such as early secretory antigen target 6 (ESAT-6), culture filtrate protein 10 (CFP-10), and antigen 85 complex i.e. Ag85A, Ag85B, and Ag85C which played roles in adherence, invasion, cytolysis, and evading cytosol of macrophage, were virulence factors that determined the immune responses important on pathogenesis of Tuberculosis (TB), including granuloma formation or tissue that determine the degree of disease. The purpose of this research was to analyze the gene region sequence encoding ESAT-6, Ag85B, and Ag85C of Mycobacterium tuberculosis. Mycobacterium tuberculosis strain analyzed was taken from sputum of pulmonary TB patients in East Java, Indonesia. Sequenced DNA analyzed using GENETYX Ver.10. There were no SNPs both inside and outside epitope region of gene encoding ESAT-6, Ag85B, and Ag85C from clinical isolates of Mycobacterium tuberculosis. From this study, it could be concluded that the highly conserved gene region encoding ESAT-6, Ag85B, and Ag85C revealed no sequence polymorphism SNPs in epitope regions among Mycobacterium tuberculosis clinical isolates from sputum specimens of pulmonary TB patients.     

Arabinofuranose disaccharide analogs as inhibitors of Mycobacterium tuberculosis

Several octyl 5-O-(α-d-arabinofuranosyl)-α-d-arabinofuranoside disaccharide analogs substituted at the 5-position of the non-reducing end sugar were synthesized and tested in vitro against Mycobacterium tuberculosis (M.tb.), Mycobacterium avium complex (MAC) as well as in a cell free assay system for arabinosyltransferase acceptor/inhibitor activity. A few compounds showed interesting inhibitory activity in the cell free assay as well as against the whole microorganism in vitro.     

A highly atom economic, chemo-, regio- and stereoselective synthesis, and discovery of spiro-pyrido-pyrrolizines and pyrrolidines as antimycobacterial agents

The 1,3-dipolar cycloaddition of azomethine ylides derived from acenaphthenequinone and α-amino acids viz. proline, phenylglycine and sarcosine to a series of 1-methyl-3,5-bis[(E)-arylmethylidene]tetrahydro-4(1H)-pyridinones afforded novel spiro-pyrido-pyrrolizines and pyrrolidines chemo-, regio- and stereoselectively in quantitative yields. These compounds were screened for their in vitro activity against Mycobacterium tuberculosis H37Rv (MTB), multi-drug resistant Mycobacterium tuberculosis (MDR-TB) and Mycobacterium smegmatis using agar dilution method. Among the synthesized compounds, spiro-[2.2″]acenaphthene-1″-one-spiro[3.3′]-5′-(2-chlorophenylmethylidene)-1′-methyltetrahydro-4′(1H)-pyridinone-4-(2-chlorophenyl)hexahydro-1H-pyrrolizine (3e) was found to be the most active with a minimum inhibitory concentration (MIC) of 0.4 μg/mL against MTB and MDR-TB.     

Hit Compounds and Associated Targets in Intracellular Mycobacterium tuberculosis

Mycobacterium tuberculosis (Mtb), the etiological agent of tuberculosis, is one of the most devastating infectious agents in the world. Chemical-genetic characterization through in vitro evolution combined with whole genome sequencing analysis was used identify novel drug targets and drug resistance genes in Mtb associated with its intracellular growth in human macrophages. We performed a genome analysis of 53 Mtb mutants resistant to 15 different hit compounds. We found nonsynonymous mutations/indels in 30 genes that may be associated with drug resistance acquisitions. Beyond confirming previously identified drug resistance mechanisms such as rpoB and lead targets reported in novel anti-tuberculosis drug screenings such as mmpL3, ethA, and mbtA, we have discovered several unrecognized candidate drug targets including prrB. The exploration of the Mtb chemical mutant genomes could help novel drug discovery and the structural biology of compounds and associated mechanisms of action relevant to tuberculosis treatment.     

The synthesis of a single enantiomer of a major α-mycolic acid of M. tuberculosis

We report a synthesis of a single enantiomer of a mycolic acid from Mycobacterium tuberculosis containing a di-cis-cyclopropane. The method can be simply varied to modify the chain lengths or the absolute stereochemistry of either cyclopropane.     

Efficient synthesis of pyruvate ketals of carbohydrates

An efficient method for the preparation of pyruvate ketals of carbohydrates has been developed using N-iodosuccinimide or 1,3-dibromo-5,5-dimethylhydantoin as activator and methyl 2,2-di(ethylthio)propionate. The method has been applied for the preparation of a pyruvate ketal-containing trisaccharide present in the bacterial cell-wall of Mycobacterium smegmatis.     

Virtual screening and repositioning of inconclusive molecules of beta-lactamase Bioassays—A data mining approach

This study focuses on the best possible way forward in utilizing inconclusive molecules of PubChem bioassays AID 1332, AID 434987 and AID 434955, which are related to beta-lactamase inhibitors of Mycobacterium tuberculosis (Mtb). The inadequacy in the experimental methods that were observed during the invitro screening resulted in an inconclusive dataset. This could be due to certain moieties present within the molecules. In order to reconsider such molecules, insilico methods can be suggested in place of invitro methods For instance, datamining and medicinal chemistrymethods: have been adopted to prioritise the inconclusive dataset into active or inactive molecules. These include the Random Forest algorithm for dataminning, Lilly MedChem rules for virtually screening out the promiscuity, and Self Organizing Maps (SOM) for clustering the active molecules and enlisting them for repositioning through the use of artificial neural networks. These repositioned molecules could then be prioritized for downstream drug discovery analysis.     

Purification,Characterization, and Structural Studies of a Sulfatase from Pedobacter yulinensis

Sulfatases are ubiquitous enzymes that hydrolyze sulfate from sulfated organic substrates such as carbohydrates, steroids, and flavones. These enzymes can be exploited in the field of biotechnology to analyze sulfated metabolites in humans, such as steroids and drugs of abuse. Because genomic data far outstrip biochemical characterization, the analysis of sulfatases from published sequences can lead to the discovery of new and unique activities advantageous for biotechnological applications. We expressed and characterized a putative sulfatase (PyuS) from the bacterium Pedobacter yulinensis. PyuS contains the (C/S)XPXR sulfatase motif, where the Cys or Ser is post-translationally converted into a formylglycine residue (FGly). His-tagged PyuS was co-expressed in Escherichia coli with a formylglycine-generating enzyme (FGE) from Mycobacterium tuberculosis and purified. We obtained several crystal structures of PyuS, and the FGly modification was detected at the active site. The enzyme has sulfatase activity on aromatic sulfated substrates as well as phosphatase activity on some aromatic phosphates; however, PyuS did not have detectable activity on 17α-estradiol sulfate, cortisol 21-sulfate, or boldenone sulfate.     

The synthesis of (3,4,5,6-tetrahydro-4-oxo-2h-1,3-thiazin-2-yl)-alkanoic acids,their derivatives and some related compounds

5,6-Dihydro-2H-1,3-thiazin-4(3H)-ones 2–10 related to the natural antituberculytic Mycobacidine 1, have been synthesised. Some of the compounds of types 2,3 and 5 proved highly active in vitro against Mycobacterium tuberculosis typ. humanus (H₃₇R_v), but the in vitro activities are diminished or suspended in the presence of biotine.     

Synthesis and in vitro activity of 6-amino-2,9-diarylpurines for Mycobacterium tuberculosis

A mild method was developed to synthesize 6-amino-2-phenolic-9-alkyl and 9-arylpurines. The new compounds were screened for antibacterial activity against Mycobacterium tuberculosis strain H₃₇Rv. The 9-tolyl derivatives with a dimethylamino or a piperidine substituent in C6 and the 3-hydroxyphenyl or 4-hydroxyphenyl substituent in C2 were highly active against the bacilli.     

New metabolites with antibacterial activity from the marine angiosperm Cymodocea nodosa

Four new metabolites (1-4) have been isolated from the organic extract of the seagrass Cymodocea nodosa, collected at the coastal area of Porto Germeno, in Attica Greece. Compounds 1 and 2 belong to the structural class of diarylheptanoids, which have been found only once before in marine organisms [Kontiza, I.; Vagias, C.; Jakupovic, J.; Moreau, D.; Roussakis, C.; Roussis, V. Tetrahedron Lett.2005, 46, 2845-2847]. Compound 3 is a new meroterpenoid, while compound 4, to the best of our knowledge, is the first briarane diterpene isolated from seaweeds, and only the second analog of this class with a tricyclic skeleton. Furthermore metabolite 4 is the first brominated briarane diterpene. The structures and the relative stereochemistry of the new natural products were established by spectral data analyses. The new metabolites were submitted for evaluation of their antibacterial activity against multidrug-resistant (MDR) pathogens including methicillin-resistant (MRSA) strains of Staphylococcus aureus, as well as the rapidly growing mycobacteria, Mycobacterium phlei, Mycobacterium smegmatis, and Mycobacterium fortuitum.     

Oligoglycol carbonate prodrugs of 5-modified 2'-deoxyuridines: synthesis and antibacterial activity

In order to develop a new generation of antibacterial nucleosides, a representative set of novel 3'- and 5'-tri- or tetraethylene glycol prodrug forms of 5-alkyloxymethyl-2'- deoxyuridines was synthesized. These compounds were at least two orders of magnitude more soluble than the parent nucleosides, possessed significant inhibitory activity against a set of bacteria including resistant strains of Staphylococcus aureus and Mycobacterium smegmatis, and showed low cytotoxicity. The obtained data indicate that glycol carbonates are convenient and prospective for usage in prodrugs of nucleoside derivatives with antibacterial activity.     

Chromatographic and serologic identity of the specific phenolglycolipids PGL-K1 from Mycobacterium kansasii and PGL-G1 from M. Gastri

Mycobacterium kansasii and M. gastri formed chromatographically and serologically identical phenolglycolipid antigens, designated respectively PGL-K₁ and PGL-G₁, in ELISA. Antisera prepared in rabbits did not bind the PGL-1 from M. leprae nor the crude extracts of 20 other mycobacterial species.     

Relative stereochemical assignment of C-33 and C-35 in the antibiotic gladiolin

Gladiolin is a macrolide antibiotic isolated from Burkholderia gladioli BCC0238 with promising activity against Mycobacterium tuberculosis, including several multidrug resistant strains. The configuration of all but one of the stereogenic centers of gladiolin has previously been elucidated using a combination of NOESY NMR experiments and predictive sequence analysis of the polyketide synthase responsible for its assembly. However, it was not possible to assign the configuration of the C-35 methyl group using such methods. Here we report the synthesis of C-33/C-35-syn and C-33/C-35-anti mimics of the C-30 to C-38 fragment of gladiolin from (R) and (S)-citronellol, respectively. Comparison of HSQC NMR data for the mimics and the natural product showed that the C-35 methyl is anti to the C-33 hydroxyl group, indicating that gladiolin has the 35S configuration.     

Asymmetric whole cell biotransformations in biphasic ionic liquid/water-systems by use of recombinant Escherichia coli with intracellular cofactor regeneration

Ionic liquids such as [BMIM][PF₆] and [BMIM][NTF] are already known as good alternatives to organic solvents in biphasic biotransformation. Herein, we report about a systematic procedure based on physical properties to identify more commercially available ionic liquids exhibiting the potential to improve the efficiency of whole cell biocatalyses. This approach resulted in the identification of seven other water immiscible ionic liquids. These ionic liquids were rated by their biocompatibility, their substrate- and product-specific distribution coefficients and by for example performed asymmetric reductions of several prochiral ketones. With the use of a recombinant Escherichia coli as biocatalyst, overproducing a Lactobacillus brevis alcohol dehydrogenase and a Mycobacterium vaccae N10 formate dehydrogenase for cofactor regeneration, the great potential of asymmetric whole cell biotransformations in biphasic ionic liquid/water-systems were demonstrated in simple batch processes.     

Reduction of tellurite and deesterification of fluorescein diacetate are not well correlated with the viability of mycobacteria

Both Mycobacterium leprae and M. lepraemurium (MLM) were capable of reducing tellurium as tellurite ion (Te⁴⁺) to elemental tellurium (Te), seen by electron microscopy as fine crystals within the bacterial cells. There appeared to be close correspondence between the capacity to reduce tellurite, bright green fluorescence after staining with fluorescein diacetate (FDA) and the ability of M. smegmatis to multiply in culture. Likewise, there appeared to be correspondence between tellurite reduction and fluorescence after FDA staining for MLM subjected to prolonged storage in the cold or to heating at 70°C. However, correspondence with tellurite-reduction or fluorescence after FDA staining was not observed when death of MLM occured     

Synthesis and biological evaluation of 4-thiazolidinone derivatives as antitubercular and antimicrobial agents

New series of N-[2-{2-(substitutedphenyl)-4-oxo-5-(substitutedbenzylidene)-1,3-thiazolidine}-iminoethyl]-2-amino-5-nitrothiazole, 5(a–m) have been synthesized from 2-amino-5-nitrothiazole as a starting material by conventional as well as microwave methods. All the synthesized compounds 4(a–m) were screened for their antibacterial and antifungal activities against some selected bacteria and fungi and antitubercular activity screened against Mycobacterium tuberculosis. The structure of all the synthesized compounds were confirmed by chemical and spectral analyses such as IR, ¹H NMR, ¹³C NMR and FAB-Mass.     

The first syntheses of single enantiomers of the major methoxymycolic acid of Mycobacterium tuberculosis

The synthesis of three stereoisomers of a major homologue of the methoxymycolic acids present in Mycobacterium tuberculosis is described.     

Hirsutellones A-E, antimycobacterial alkaloids from the insect pathogenic fungus Hirsutella nivea BCC 2594

Five new alkaloids, hirsutellones A-E, were isolated from the insect pathogenic fungus Hirsutella nivea BCC 2594. Their structures were elucidated by spectroscopic analysis and X-ray crystallography. Hirsutellones displayed significant growth inhibitory activity against Mycobacterium tuberculosis H₃₇Ra.     

Bipinnapterolide B, a bioactive oxapolycyclic diterpene from the Colombian gorgonian coral Pseudopterogorgia bipinnata

A unique oxapolycyclic diterpenoid, named bipinnapterolide B, has been isolated from the Colombian gorgonian octocoral Pseudopterogorgia bipinnata, and its structure was deduced from spectral and X-ray diffraction studies. Bipinnapterolide B (3) inhibits the growth of Mycobacterium tuberculosis.