Homology Modeling of Wild-type,D516V,and H526L Mycobacterium Tuberculosis RNA Polymerase and Their Molecular Docking Study with Inhibitors

Abstract

Rifamicyns (Rifs) are antibiotic widely used for the treatment of tuberculosis (TB); nevertheless, their efficacy has been limited by a high percentage of mutations, principally in the rpoB gene. In this work, the first three-dimensional molecular model of the hypothetical structures for the wild-type and D516V and H526L mutants of Mycobacterium tuberculosis (mtRNAP) were elucidated by a homology modeling method. In addition, the orientations and binding affinities of some Rifs with those new structures were investigated. Our findings could be helpful for the design of new more potent rifamycin analogs.     

RNA polymerase-rifamycin. A molecular model of inhibition

By fluorimetric titration of Rifs (E. coli B) and Rifr (E. coli rpoB255) RNA polymerases with rifamycin, the mutant polymerase was demonstrated to bind rifamycin. A comparison of spatial structures of rifamycin and dinucleotide fragment of RNA in the hybrid with DNA revealed their similarity. Taking into account this structural similarity and also the fact that two phosphodiester bonds can be formed by RNA polymerase in the presence of rifamycin, a model for the inhibition mode was proposed. According to this model, rifamycin occupies the place of two terminal nucleotides of synthesized, but not translocated pentanucleotide in the transcribing complex. Asp-516 of the wild type beta-subunit was assumed to form a hydrogen bond with the rifamycin C(23) hydroxyl group. On the base of this model, reduced "cycling" synthesis of tetra-, penta-... up to decanucleotides by the Rifr RNA polymerase, in comparison with Rifs, was predicted.     

Molecular Modeling of Mycobacterium Tuberculosis DNA Gyrase and its Molecular Docking Study with Gatifloxacin Inhibitors

Abstract

Mycobacterium tuberculosis (Mt) is a leading cause of infectious disease in the world today. This outlook is aggravated by a growing number of Mt infections in individuals who are immunocompromised as a result of HIV infections. Thus, new and more potent anti-tuberculosis agents are necessary. Therefore, DNA gyrase was selected as a target enzyme to combat Mt. In this work, the first three-dimensional molecular model of the hypothetical structures for the Mycobacterium tuberculosis DNA gyrase (mtDNAg) was elucidated by a homology modeling method. In addition, the orientations and binding affinities of some gatifloxacin analogs with those new structures were investigated. Our findings could be helpful for the design of new more potent gatifloxacin analogs.     

Synthesis of methyl 5-S-alkyl-5-thio-D-arabinofuranosides and evaluation of their antimycobacterial activity

The emergence of drug resistant tuberculosis necessitates a search for new antimycobacterial compounds. The antigen 85 (ag85) complex is a family of mycolyl transferases involved in the synthesis of trehalose-6,6'-dimycolate and the mycolated hexasaccharide motif found at the terminus of the arabinogalactan in mycobacterium. Enzymes involved in the synthesis of cell wall structures like these are potential targets for the development of new antiinfectives. To potentially inhibit the ag85 complex, methyl 5-S-alkyl-5-thio-arabinofuranoside analogues were designed based on docking studies with ag85C derived from Mycobacterium tuberculosis. The target arabinofuranosides were then synthesized and the antibacterial activity evaluated against Mycobacterium smegmatis ATCC 14468. Two of the compounds, 5-S-octyl-5-thio-alpha-d-arabinofuranoside (8) and 5-S-octyl-5-thio-beta-d-arabinofuranoside (11), showed MICs of 256 and 512microg/mL, respectively. Attempts to directly evaluate acyltransferase inhibitory activity of the arabinofuranosides against ag85C are also described. In conclusion, a new class of antimycobacterial arabinofuranosides has been discovered.     

Design, synthesis, and evaluation of novel ethambutol analogues

Ethambutol is one of the front-line agents recommended by the World Health Organization for the treatment of tuberculosis. In an effort to develop more potent therapies to treat tuberculosis, novel unsymmetrical ethambutol analogues were successfully synthesized by a new route utilizing novel building blocks synthesized using Ellman's sulfinyl chemistry. The resulting analogues were tested for anti-tuberculosis activity yielding compounds with comparable anti-tuberculosis activity to ethambutol and increased lipophilicity that may instill better tissue penetration and serum half-life.     

The progress made in determining the Mycobacterium tuberculosis structural proteome

Mycobacterium tuberculosis is a highly infectious pathogen that is still responsible for millions of deaths annually. Effectively treating this disease typically requires a course of antibiotics, most of which were developed decades ago. These drugs are, however, not effective against persistent tubercle bacilli and the emergence of drug-resistant stains threatens to make many of them obsolete. The identification of new drug targets, allowing the development of new potential drugs, is therefore imperative. Both proteomics and structural biology have important roles to play in this process, the former as a means of identifying promising drug targets and the latter allowing understanding of protein function and protein-drug interactions at atomic resolution. The determination of M. tuberculosis protein structures has been a goal of the scientific community for the last decade, who have aimed to supply a large amount of structural data that can be used in structure-based approaches for drug discovery and design. Only since the genome sequence of M. tuberculosis has been available has the determination of large numbers of tuberculosis protein structures been possible. Currently, the molecular structures of 8.5% of all the pathogen's protein-encoding ORFs have been determined. In this review, we look at the progress made in determining the M. tuberculosis structural proteome and the impact this has had on the development of potential new drugs, as well as the discovery of the function of crucial mycobaterial proteins.     

Autoluminescent Mycobacterium tuberculosis for rapid, real-time, non-invasive assessment of drug and vaccine efficacy

Preclinical efforts to discover and develop new drugs and vaccines for tuberculosis are hampered by the reliance on colony-forming unit (CFU) counts as primary outcomes for in vivo efficacy studies and the slow growth of Mycobacterium tuberculosis. The utility of bioluminescent M. tuberculosis reporter strains for real-time in vitro and ex vivo assessment of drug and vaccine activity has been demonstrated but a simple, non-invasive, real-time surrogate marker to replace CFU counts for real-time evaluation of drug and vaccine efficacy in vivo has not been described. We describe the development of a fully virulent and stable autoluminescent strain of M. tuberculosis and proof-of-concept experiments demonstrating its utility for in vivo bioluminescence imaging to assess the efficacy of new drugs and vaccines for tuberculosis in a mouse model. Relative light unit (RLU) counts paralleled CFU counts during the active phase of bacterial growth, with a lower limit of detection of approximately 10(6) CFU in live, anesthetized mice. Experiments distinguishing active from inactive anti-tuberculosis drugs and bacteriostatic drug effects from bactericidal effects were completed in less than 5 days. The ability of a recombinant BCG vaccine to limit bacterial growth was demonstrated within 3 weeks. Use of this autoluminescent reporter strain has the potential to drastically reduce the time, effort, animals and costs consumed in the evaluation of drug activity in vitro and the in vivo assessment of drug and vaccine efficacy.     

New flavanones from the leaves of Cryptocarya chinensis and their antituberculosis activity

Four new flavanones, cryptoflavanones A-D (1-4, resp.), together with eight known compounds, were isolated from the leaves of Cryptocarya chinensis. The structures of these new compounds were determined by spectral analyses. Among the isolated compounds, pinocembrin (5) and cryptocaryone (6) exhibited antituberculosis activity against Mycobacterium tuberculosis H(37) Rv strain in vitro with MIC values of 3.5 and 25.0 μg/ml, respectively.     

Reactivation of M. tuberculosis infection in trans-membrane tumour necrosis factor mice

Of those individuals who are infected with M. tuberculosis, 90% do not develop active disease and represents a large reservoir of M. tuberculosis with the potential for reactivation of infection. Sustained TNF expression is required for containment of persistent infection and TNF neutralization leads to tuberculosis reactivation. In this study, we investigated the contribution of soluble TNF (solTNF) and transmembrane TNF (Tm-TNF) in immune responses generated against reactivating tuberculosis. In a chemotherapy induced tuberculosis reactivation model, mice were challenged by aerosol inhalation infection with low dose M. tuberculosis for three weeks to establish infection followed chemotherapeutic treatment for six weeks, after which therapy was terminated and tuberculosis reactivation investigated. We demonstrate that complete absence of TNF results in host susceptibility to M. tuberculosis reactivation in the presence of established mycobacteria-specific adaptive immunity with mice displaying unrestricted bacilli growth and diffused granuloma structures compared to WT control mice. Interestingly, bacterial re-emergence is contained in Tm-TNF mice during the initial phases of tuberculosis reactivation, indicating that Tm-TNF sustains immune pressure as in WT mice. However, Tm-TNF mice show susceptibility to long term M. tuberculosis reactivation associated with uncontrolled influx of leukocytes in the lungs and reduced IL-12p70, IFNγ and IL-10, enlarged granuloma structures, and failure to contain mycobacterial replication relative to WT mice. In conclusion, we demonstrate that both solTNF and Tm-TNF are required for maintaining immune pressure to contain reactivating M. tuberculosis bacilli even after mycobacteria-specific immunity has been established.     

Isolation, Structure elucidation, and antimycobacterial properties of dimeric naphtho-gamma-pyrones from the marine-derived fungus Aspergillus carbonarius

Two new dimeric naphtho-gamma-pyrones, compounds 1 and 2, were isolated from the AcOEt extract of the fungal strain WZ-4-11 of Aspergillus carbonarius, together with eight known analogues, including 10,10'-bifonsecin B (3), 6'-O-demethylnigerone (4), nigerone (5), isonigerone (6), fonsecin (7), rubrofusarin B (8), TMC 256A1 (9), and flavasperone (10). Their structures were elucidated by means of UV, CD, IR, and 1D- and 2D-NMR spectroscopy, in combination with HR-MS analysis. The fully assigned (1)H- and (13)C-NMR data of 3, and the (13)C-NMR data of 6 are reported for the first time. Compounds 1 and 2 showed weak antimycobacterial activities against Mycobacterium tuberculosis H37Rv, with MIC values of 43.0 and 21.5 microM, resp.     

Synthesis, biological evaluation and SAR study of novel pyrazole analogues as inhibitors of Mycobacterium tuberculosis

As a continuation of our previous work that turned toward the identification of antimycobacterial compounds with innovative structures, two series of pyrazole derivatives were synthesized by parallel solution-phase synthesis and were assayed as inhibitors of Mycobacterium tuberculosis (MTB), which is the causative agent of tuberculosis. One of these compounds showed high activity against MTB (MIC = 4 microg/mL). The newly synthesized pyrazoles were also computationally investigated to analyze their fit properties to the pharmacophoric model for antitubercular compounds previously built by us and to refine structure-activity relationship analysis.     

The crystal structure of Rv0793, a hypothetical monooxygenase from <Emphasis Type="BoldItalic">M.␣tuberculosis</Emphasis>

Mycobacterium tuberculosis infects millions worldwide. The Structural Genomics Consortium for M. tuberculosis has targeted all genes from this bacterium in hopes of discovering and developing new therapeutic agents. Open reading frame Rv0793 from M. tuberculosis was annotated with an unknown function. The 3-dimensional structure of Rv0793 has been solved to 1.6 A resolution. Its structure is very similar to that of Streptomyces coelicolor ActVA-Orf6, a monooxygenase that participates in tailoring of polyketide antibiotics in the absence of a cofactor. It is also similar to the recently solved structure of YgiN, a quinol monooxygenase from Escherichia coli. In addition, the structure of Rv0793 is similar to several structures of other proteins with unknown function. These latter structures have been determined recently as a result of structural genomic projects for various bacterial species. In M. tuberculosis, Rv0793 and its homologs may represent a class of monooygenases acting as reactive oxygen species scavengers that are essential for evading host defenses. Since the most prevalent mode of attack by the host defense on M. tuberculosis is by reactive oxygen species and reactive nitrogen species, Rv0793 may provide a novel target to combat infection by M. tuberculosis.     

Novel drug target strategies against Mycobacterium tuberculosis

The resurgence of drug resistant tuberculosis (TB) is a significant global healthcare challenge. Mycobacterium tuberculosis (MTB), TB's causative agent, evades the host immune system and drug regimes by entering prolonged periods of non-proliferation or dormancy. In infected individuals, the immune system sequesters MTB into structures called granulomas where the bacterium survives by shifting into a non-replicative state. Although still not well understood, progress has been made in characterizing the genetic program of MTB, activated by DosR (DevR) signal transduction that allows adaptation to the hypoxic, nutrient limiting granuloma microenvironment. Recent work, especially the identification genes involved in regulatory networks and the Enduring Hypoxic Response (EHR), hold promise for developing new drugs targeting dormancy phase MTB.     

CXCL10/IP-10 release is induced by incubation of whole blood from tuberculosis patients with ESAT-6, CFP10 and TB7.7

IFN-gamma responses to Mycobacterium tuberculosis specific antigens are used as in vitro diagnostic tests for tuberculosis infection. The tests are sensitive and specific for latent and active tuberculosis disease, but sensitivity may be reduced during immunosuppression. The objective of the study was to explore new ways to improve the diagnosis of tuberculosis infection using CXCL10 and IL-2 as alternative markers to IFN-gamma. CXCL10, IL-2, and IFN-gamma responses to stimulation with ESAT-6/CFP10/TB7.7 were assessed in 12 Quantiferon positive, 8 Quantiferon negative tuberculosis patients and 11 Quantiferon negative controls. CXCL10 and IL-2 were determined by multiplex and IFN-gamma by the Quantiferon ELISA. The median antigen specific CXCL10, IFN-gamma, and IL-2 responses in patients with tuberculosis were 870 pg/ml (range 261-1576 pg/ml), 217 pg/ml (81-1273 pg/ml), 59 pg/ml (14-276 pg/ml) respectively, and the CXCL10 responses were significantly higher than any of the other cytokines measured (p=0.001). In 4/7 individuals with a negative (n=6) or indeterminate (n=1) Quantiferon test, antigen specific CXCL10 responses were detectable at high levels ranging from 196-532 pg/ml. In conclusion CXCL10 was strongly induced after M. tuberculosis specific stimulation and sensitivity appeared superior to the Quantiferon test. Our findings suggest that CXCL10 may serve as an alternative or additional marker for the immunodiagnosis of tuberculosis.     

The mRNA interferases, MazF-mt3 and MazF-mt7 from Mycobacterium tuberculosis target unique pentad sequences in single-stranded RNA

mRNA interferases are sequence-specific endoribonucleases encoded by toxin-antitoxin (TA) systems in bacterial genomes. Previously, we demonstrated that Mycobacterium tuberculosis contains at least seven genes encoding MazF homologues (MazF-mt1 to -mt7) and determined cleavage specificities for MazF-mt1 and MazF-mt6. Here we have developed a new general method for the determination of recognition sequences longer than three bases for mRNA interferases with the use of phage MS2 RNA as a substrate and CspA, an RNA chaperone, which prevents the formation of secondary structures in the RNA substrate. Using this method, we determined that MazF-mt3 cleaves RNA at UU˘CCU or CU˘CCU and MazF-mt7 at U˘CGCU (˘indicates the cleavage site). As pentad sequence recognition is more specific than those of previously characterized mRNA interferases, bioinformatics analysis was carried out to identify M. tuberculosis mRNAs that may be resistant to MazF-mt3 and MazF-mt7 cleavage. The pentad sequence was found to be significantly underrepresented in several genes, including members of the PE and PPE families, large families of proteins that play a role in tuberculosis immunity and pathogenesis. These data suggest that MazF-mt3 and MazF-mt7 or other mRNA interferases that target longer RNA sequences may alter protein expression through differential mRNA degradation, a regulatory mechanism that may allow adaptation to environmental conditions, including those encountered by pathogens such as M. tuberculosis during infection.     

Ornithine lipid of Mycobacterium tuberculosis: its distribution in some slow- and fast-growing mycobacteria

An ornithine-amide lipid is present in Mycobacterium tuberculosis. Its structure was established by a combination of chemical analysis and mass spectrometry. 3-Hydroxyoctadecanoic and 3-hydroxyeicosanoic acids (and homologues) were found to be linked through an amide bond to the alpha-amino group of L-ornithine, the hydroxyl group of the fatty acid being esterified mainly by tuberculostearic acid (10-methyloctadecanoic acid). This ornithine-amide lipid was detected in several other slow-growing pathogenic mycobacteria by thin layer chromatography, but not in an avirulent strain (H37 Ra) of M. tuberculosis. In each case mass spectrometry showed that all the structures were identical, thus revising an earlier reported structure for the lipid from M. bovis.     

Feasibility, Yield, and Cost of Active Tuberculosis Case Finding Linked to a Mobile HIV Service in Cape Town, South Africa: A Cross-sectional Study

Background

The World Health Organization is currently developing guidelines on screening for tuberculosis disease to inform national screening strategies. This process is complicated by significant gaps in knowledge regarding mass screening. This study aimed to assess feasibility, uptake, yield, treatment outcomes, and costs of adding an active tuberculosis case-finding program to an existing mobile HIV testing service.

Methods and Findings

The study was conducted at a mobile HIV testing service operating in deprived communities in Cape Town, South Africa. All HIV-negative individuals with symptoms suggestive of tuberculosis, and all HIV-positive individuals regardless of symptoms were eligible for participation and referred for sputum induction. Samples were examined by microscopy and culture. Active tuberculosis case finding was conducted on 181 days at 58 different sites. Of the 6,309 adults who accessed the mobile clinic, 1,385 were eligible and 1,130 (81.6%) were enrolled. The prevalence of smear-positive tuberculosis was 2.2% (95% CI 1.1–4.0), 3.3% (95% CI 1.4–6.4), and 0.4% (95% CI 1.4 015–6.4) in HIV-negative individuals, individuals newly diagnosed with HIV, and known HIV, respectively. The corresponding prevalence of culture-positive tuberculosis was 5.3% (95% CI 3.5–7.7), 7.4% (95% CI 4.5–11.5), 4.3% (95% CI 2.3–7.4), respectively. Of the 56 new tuberculosis cases detected, 42 started tuberculosis treatment and 34 (81.0%) completed treatment. The cost of the intervention was US$1,117 per tuberculosis case detected and US$2,458 per tuberculosis case cured. The generalisability of the study is limited to similar settings with comparable levels of deprivation and TB and HIV prevalence.

Conclusions

Mobile active tuberculosis case finding in deprived populations with a high burden of HIV and tuberculosis is feasible, has a high uptake, yield, and treatment success. Further work is now required to examine cost-effectiveness and affordability and whether and how the same results may be achieved at scale.     

Combined Species Identification, Genotyping, and Drug Resistance Detection of Mycobacterium tuberculosis Cultures by MLPA on a Bead-Based Array

The population structure of Mycobacterium tuberculosis is typically clonal therefore genotypic lineages can be unequivocally identified by characteristic markers such as mutations or genomic deletions. In addition, drug resistance is mainly mediated by mutations. These issues make multiplexed detection of selected mutations potentially a very powerful tool to characterise Mycobacterium tuberculosis. We used Multiplex Ligation-dependent Probe Amplification (MLPA) to screen for dispersed mutations, which can be successfully applied to Mycobacterium tuberculosis as was previously shown. Here we selected 47 discriminative and informative markers and designed MLPA probes accordingly to allow analysis with a liquid bead array and robust reader (Luminex MAGPIX technology). To validate the bead-based MLPA, we screened a panel of 88 selected strains, previously characterised by other methods with the developed multiplex assay using automated positive and negative calling. In total 3059 characteristics were screened and 3034 (99.2%) were consistent with previous molecular characterizations, of which 2056 (67.2%) were directly supported by other molecular methods, and 978 (32.0%) were consistent with but not directly supported by previous molecular characterizations. Results directly conflicting or inconsistent with previous methods, were obtained for 25 (0.8%) of the characteristics tested. Here we report the validation of the bead-based MLPA and demonstrate its potential to simultaneously identify a range of drug resistance markers, discriminate the species within the Mycobacterium tuberculosis complex, determine the genetic lineage and detect and identify the clinically most relevant non-tuberculous mycobacterial species. The detection of multiple genetic markers in clinically derived Mycobacterium tuberculosis strains with a multiplex assay could reduce the number of TB-dedicated screening methods needed for full characterization. Additionally, as a proportion of the markers screened are specific to certain Mycobacterium tuberculosis lineages each profile can be checked for internal consistency. Strain characterization can allow selection of appropriate treatment and thereby improve treatment outcome and patient management.     

Structural genomics of Mycobacterium tuberculosis: a preliminary report of progress at UCLA

The growing list of fully sequenced genomes, combined with innovations in the fields of structural biology and bioinformatics, provides a synergy for the discovery of new drug targets. With this background, the TB Structural Genomics Consortium has been formed. This international consortium is comprised of laboratories from 31 universities and institutes in 13 countries. The goal of the consortium is to determine the structures of over 400 potential drug targets from the genome of Mycobacterium tuberculosis and analyze their structures in the context of functional information. We summarize the efforts of the UCLA consortium members. Potential drug targets were selected using a variety of bioinformatics methods and screened for certain physical and species-specific properties to yield a starting group of protein targets for structure determination. Target determination methods include protein phylogenetic profiles and Rosetta Stone methods, and the use of related biochemical pathways to select genes linked to essential prokaryotic genes. Criteria imposed on target selection included potential protein solubility, protein or domain size, and targets that lack homologs in eukaryotic organisms. In addition, some protein targets were chosen that are specific to M. tuberculosis, such as PE and PPE domains. Thus far, the UCLA group has cloned 263 targets, expressed 171 proteins and purified 40 proteins, which are currently in crystallization trials. Our efforts have yielded 13 crystals and eight structures. Seven structures are summarized here. Four of the structures are secreted proteins: antigen 85B; MPT 63, which is one of the three major secreted proteins of M. tuberculosis; a thioredoxin derivative Rv2878c; and potentially secreted glutamate synthetase. We also report the structures of three proteins that are potentially essential to the survival of M. tuberculosis: a protein involved in the folate biosynthetic pathway (Rv3607c); a protein involved in the biosynthesis of vitamin B5 (Rv3602c); and a pyrophosphatase, Rv2697c. Our approach to the M. tuberculosis structural genomics project will yield information for drug design and vaccine production against tuberculosis. In addition, this study will provide further insights into the mechanisms of mycobacterial pathogenesis.