Brahm S. Srivastava

Synthesis and bioevaluation of glycosyl ureas as α-glucosidase inhibitors and their effect on mycobacterium

Glycosyl amino esters (2-13) on reaction with different isocyanates resulted in quantitative conversion to glycosyl ureas (14--32). Few of the selected ureas (15-20, 22-28, 30 and 32) on cyclative amidation with DBU/TBAB/4 A MS gave respective dihydropyrimidinones in fair to good yields (33-47). The compounds were screened for alpha-glucosidase inhibitory activity and two (19 and 23) of them showed strong inhibition against rat intestinal alpha-glucosidase. The compounds were also screened against Mycobacterium aurum, however, only one (19) of them exhibited marginal antitubercular activity.

Synthesis of glycosylated β-amino acids as new class of antitubercular agents

Abstract A series of glycosylated β-amino acids was prepared and evaluated against Mycobacterium tuberculosis, M. avium, M. fortuitum and M. smegmatis. The compounds were designed to mimic the enzyme d -alanine racemase and glycosyl transferase involved in the biosynthesis of essential cell wall peptidoglycan and arabinogalactan. Though most of the compounds exhibited little activity, however, one showed significant activity against all the strains in cell culture and activity was confirmed by B actec method.

Comparative expression analysis of rpf-like genes of Mycobacterium tuberculosis H37Rv under different physiological stress and growth conditions.

Mycobacterium tuberculosis H37Rv possesses five resuscitation-promoting factors, RpfA-E, which are required for the resuscitation of dormancy in mycobacteria induced by prolonged incubation of the culture in stationary phase. This study explores the transcriptional profile of all the rpf-like genes of M. tuberculosis H37Rv in the exponential phase, stationary phase, non-culturable phase and Rpf-mediated resuscitation phase. The relative expression was also monitored under acid stress, nutrient starvation and low-oxygen (hypoxia) conditions by real-time quantitative PCR. We show differential relative expression of the rpf genes during different stages of growth and under stress. During early resuscitation, all rpf genes were expressed with maximal expression ratios for rpfA and rpfD. rpfC was consistently expressed during all stages of growth and nutrient starvation. Acid stress induced higher relative expression of rpfD and rpfE and hypoxia of rpfC and rpfE. These results therefore provide further evidence that the rpf genes perform distinct roles during cell growth and cell survival under different physiological stresses, and are consistent with the rpf-like genes being differentially regulated.

Identification of an antimicrobial entity from the cyanobacterium Fischerella sp. isolated from bark of Azadirachta indica (Neem) tree

The active principle in a methanolic extract of the laboratory-grown cyanobacterium, Fischerella sp. isolated from Neem (Azadirachta indica) tree bark was active against Mycobacterium tuberculosis, Enterobacter aerogenes, Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella typhi, Escherichia coli as well as three multi-drug resistant E. coli strains in in vitro assays. Based on MS, UV, IR 1H NMR analyses the active principle is proposed to be hapalindole T having the empirical formula C21H23N2ClSO and a molecular weight of 386 with the melting point range 179–182 °C. The estimated production of Hapalindole T from the cyanobacterium is 1.25 mg g−1 lyophilized biomass. It is suggested that cyanobacteria colonizing specialized niches such as tree bark could be an antibacterial drug resource.

Synthesis and antimycobacterial activity of 3,5-disubstituted thiadiazine thiones☆

A series of 3,5-disubstituted thiadiazine thiones (4-24) have been synthesized by reaction of primary amines with carbon disulphide followed by cyclocondensation of the resulting intermediate with formaldehyde and primary amines or amino acids. The compounds were screened for antitubercular activity in vitro against Mycobacterium tuberculosis H37Rv. Three compounds 4, 12 and 18 showed antimycobacterial activity with MIC 12.5 microg/mL. Compound 4, was tested in vitro against five multidrug resistant (MDR) strains of M. tuberculosis and was found to be active. Compound 4 also exhibited activity in vivo. While all the mice died in the untreated group, the mean survival time (MST) of the compound treated mice was enhanced, 33% mice were surviving in treated group and the load of bacilli in the lung was considerably less in the compound treated group than in the untreated control group.

Genetic rearrangements leading to disruption of heterologous gene expression in mycobacteria : An observation with Escherichia coli β-galactosidase in Mycobacterium smegmatis and its implication in vaccine development

Different mycobacteria carrying cloned genes for heterologous protective antigens have been proposed as vaccine vehicles. In this study, the stability of the expression of beta-galactosidase was studied in Mycobacterium smegmatis using integrative (pMV361::lacZ) and replicative (pMV261::lacZ) vectors. Recombinant M. smegmatis forms blue colonies on X-gal plates. Occasional white mutants encountered while plating on X-gal plates were genetically analysed. The loss of lacZ phenotype was due to insertion of an IS element in lacZ gene of integrative vector whereas in case of replicative vectors, loss of lacZ phenotype was due to deletions of different sizes in the lacZ gene and the Phsp60 promoter region. The frequency of such events was rare, 1.7 x 10(-5) in integrative vector and 2 x 10(-3) in the case of replicative vector. The integrative vector seemed more stable in terms of expression of foreign genes in mycobacteria. Hence, the rearrangements reported in the present study warrant serious consideration before implementing mycobacteria as recombinant vaccines.

Baylis–Hillman Reaction: Convenient Ascending Syntheses and Biological Evaluation of Acyclic Deoxy Monosaccharides as Potential Antimycobacterial Agents

A series of acyclic deoxy carbohydrate derivatives from easily available carbohydrate enals 1, 2, 3 or 5 were prepared involving the Baylis-Hillman reaction. These newly formed carbohydrate based Baylis-Hillman adducts and their amino derivatives were evaluated for their antimycobacterial activity against Mycobacterium tuberculosis H(37)R(v). Among the compounds evaluated for their antimycobacterial activity, compound (10) showed the desired activity in the range of 3.125 microg/mL.

Fast-growing, non-infectious and intracellularly surviving drug-resistant Mycobacterium aurum: a model for high-throughput antituberculosis drug screening

OBJECTIVES Enoyl acyl-carrier-protein reductase (InhA), the primary endogenous target for isoniazid and ethionamide, is crucial to type-II fatty acid biosynthesis (FAS-II). The objectives of this study were first to generate InhA mutants of Mycobacterium aurum, secondly to characterize InhA-mediated isoniazid and ethionamide resistance mechanisms across those mutants and finally to investigate the interaction of InhA with enzymes in the FAS-II pathway in M. aurum. METHODS Spontaneous mutants were generated by isoniazid overdose and limited broth dilution, while for genetically modified mutants sense-antisense DNA technology was used. Southern hybridization and immunoprecipitation were both used to identify the InhA homologue in M. aurum. The latter method was further used to compare the level of InhA expression in M. aurum with that in corresponding mutants. Isoniazid/ethionamide susceptibility modulation was examined in vitro and ex vivo using a resazurin assay as well as by cfu counting. In addition, circular dichroism and the bacterial two-hybrid system were exploited to investigate the interaction of InhA with other enzymes of the FAS-II pathway. RESULTS A Mycobacterium tuberculosis InhA homologue was detected in M. aurum. Susceptibility to isoniazid/ethionamide was significantly altered in genetically modified mutants and simultaneously InhA was overexpressed in both spontaneous and genetically modified mutants. InhA interacts with other FAS-II enzymes of M. aurum in vivo. CONCLUSION Close resemblance of isoniazid/ethionamide action on InhA between M. tuberculosis and M. aurum further supports the use of fast-growing and intracellularly surviving drug-resistant M. aurum to substitute for highly virulent, extremely slow-growing M. tuberculosis strains in the early stage of antituberculosis inhibitor screening.

In vivo activity of thiophene-containing trisubstituted methanes against acute and persistent infection of non-tubercular Mycobacterium fortuitum in a murine infection model

OBJECTIVES Mycobacterium fortuitum causes opportunist non-tubercular infection in humans. Chronic infection of M. fortuitum has been clinically documented and requires prolonged chemotherapy. The objectives of this study were to characterize acute and persistent infection of M. fortuitum in a murine infection model and to screen thiophene-containing trisubstituted methanes active against both acute and persistent infection. METHODS A murine infection model of M. fortuitum was used. Bacillary count, bioluminescence, disease symptoms, host immune response, drug susceptibility and mortality were measured. Reactivation of persistent bacilli was induced by dexamethasone. Trisubstituted methanes containing thiophene rings were synthesized and screened in vitro by agar dilution and BACTEC assay and in mice. Cytotoxicity was tested with Vero monkey kidney cells using a resazurin assay. RESULTS The acute infection in mice was marked by a 3 log rise in viable counts, the appearance of disease symptoms and a rise in the Th1 immune response. Bacilli were susceptible to fluoroquinolones. This was followed by persistent infection, in which disappearance of disease symptoms, a decline in Th1 response and non-susceptibility to fluoroquinolones was observed. When the mice were immunocompromised on day 40 post-infection (persistent state) by dexamethasone, a rise in viable counts, symptoms and susceptibility to fluoroquinolones and a prominent Th1 response reappeared. Two lead compounds were found that cleared the mice of bacilli in acute infection and caused a 2.29-2.99 log reduction in cfu of persistent bacilli. CONCLUSIONS The study established acute and persistent infection in mice and identified two promising anti-M. fortuitum compounds with a selectivity index >10.

Comparative efficacy of biodegradable liposomes and microspheres as carriers for delivery of Vibrio cholerae antigens in the intestine

The effect of the encapsulated antigens of Vibrio cholerae and their route of administration in induction of immune response was studied in experimental cholera. The antigenic proteins of V. cholerae El Tor strain KB207 were obtained by fractionation of cell-free lysate by high-performance liquid chromatography. The antigenic proteins were pooled and encapsulated in biodegradable liposomes and poly(D,L) lactic co-glycolic acid microspheres. Rabbits were immunized with free as well as encapsulated antigens by different routes. Liposome-encapsulated antigens delivered intraintestinally offered maximum protection. Orally or intraintestinally delivered antigens in microspheres failed to elicit a significant immune response, although as a carrier microspheres were comparable to liposomes when judged by the subcutaneous route. The results suggested that liposomes and microspheres could be used as carriers of protective antigens of V. cholerae for effective immunization.