RESUMEN
Polymerase eta (Polη) is a translesion synthesis DNA polymerase directly linked to cancer development. It can bypass several DNA lesions thereby rescuing DNA damage-stalled replication complexes. We previously presented evidence implicating Saccharomyces cerevisiae Polη in transcription elongation, and identified its specific RNA extension and translesion RNA synthetic activities. However, RNA synthesis by Polη proved rather inefficient under conditions optimal for DNA synthesis. Searching for factors that could enhance its RNA synthetic activity, we have identified the divalent cation of manganese. Here, we show that manganese triggers drastic changes in the activity of Polη. Kinetics experiments indicate that manganese increases the efficiency of ribonucleoside incorporation into RNA by ~400-2000-fold opposite undamaged DNA, and ~3000 and ~6000-fold opposite TT dimer and 8oxoG, respectively. Importantly, preference for the correct base is maintained with manganese during RNA synthesis. In contrast, activity is strongly impaired, and base discrimination is almost lost during DNA synthesis by Polη with manganese. Moreover, Polη shows strong preference for manganese during RNA synthesis even at a 25-fold excess magnesium concentration. Based on this, we suggest that a new regulatory mechanism, selective metal cofactor utilization, modulates the specificity of Polη helping it to perform distinct activities needed for its separate functions during replication and transcription.
Asunto(s)
ADN Polimerasa Dirigida por ADN/efectos de los fármacos , ADN Polimerasa Dirigida por ADN/metabolismo , Metales/farmacología , ARN/metabolismo , Saccharomyces cerevisiae/enzimología , Catálisis/efectos de los fármacos , ADN/metabolismo , Reparación del ADN/efectos de los fármacos , Replicación del ADN/efectos de los fármacos , ARN Polimerasas Dirigidas por ADN/efectos de los fármacos , ARN Polimerasas Dirigidas por ADN/metabolismo , Activación Enzimática/efectos de los fármacos , Iones Pesados , Cinética , Metales/química , Polimerizacion/efectos de los fármacos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Especificidad por Sustrato/efectos de los fármacosRESUMEN
We compared the ability of commercial and non-commercial, phenotypic and genotypic rapid drug susceptibility tests (DSTs) to detect rifampicin resistance (RR)-conferring 'disputed' mutations frequently missed by Mycobacterium Growth Indicator Tube (MGIT), namely L430P, D435Y, L452P, and I491F. Strains with mutation S450L served as positive control while wild-types were used as negative control. Of the 38 mutant strains, 5.7% were classified as RR by MGIT, 16.2% by Trek Sensititre MYCOTB MIC plate, 19.4% by resazurin microtiter plate assay (REMA), 50.0% by nitrate reductase assay (NRA), and 62.2% by microscopic observation direct susceptibility testing (MODS). Reducing MGIT rifampicin concentration to 0.5 µg/ml, and/or increasing incubation time, enhanced detection of disputed mutations from 5.7% to at least 65.7%, particularly for mutation I491F (from 0.0 to 75.0%). Compared with MGIT at standard pre-set time with 0.25 µg/ml ECOFF as breakpoint, we found a statistically significant increase in the ability of MGIT to resolve disputed mutants and WT strains at extended incubation period of 15 and 21 days, with 0.5 µg/ml and 1 µg/ml ECOFF respectively. MODS detected 75.0% of the I491F strains and NRA 62.5%, while it was predictably missed by all molecular assays. Xpert MTB/RIF, Xpert Ultra, and GenoscholarTB-NTM + MDRTB detected all mutations within the 81 bp RR determining region. Only GenoType MTBDRplus version 2 missed mutation L430P in 2 of 11 strains. Phenotypic and genotypic DSTs varied greatly in detecting occult rifampicin resistance. None of these methods detected all disputed mutations without misclassifying wild-type strains.
Asunto(s)
Proteínas Bacterianas/genética , ARN Polimerasas Dirigidas por ADN/genética , Farmacorresistencia Bacteriana/genética , Mutación , Mycobacterium tuberculosis/genética , Rifampin/farmacología , Antituberculosos , Proteínas Bacterianas/efectos de los fármacos , ARN Polimerasas Dirigidas por ADN/efectos de los fármacos , Genes Bacterianos , Genotipo , Pruebas de Sensibilidad Microbiana , Mycobacterium tuberculosis/efectos de los fármacosRESUMEN
Current influenza antivirals have limitations with regard to their effectiveness and the potential emergence of resistance. Encouragingly, several new compounds which inhibit the polymerase of influenza viruses have recently been shown to have enhanced pre-clinical and clinical effectiveness compared to the neuraminidase inhibitors, the mainstay of influenza antiviral therapy over the last two decades. In this review we focus on four compounds which inhibit polymerase function, baloxavir marboxil, favipiravir, pimodivir and AL-794 and discuss their clinical and virological effectiveness, their propensity to select for resistance and their potential for future combination therapy with the most commonly used neuraminidase inhibitor, oseltamivir.
Asunto(s)
Antivirales/farmacología , Antivirales/uso terapéutico , ARN Polimerasas Dirigidas por ADN/efectos de los fármacos , Neuraminidasa/antagonistas & inhibidores , Orthomyxoviridae/efectos de los fármacos , Amidas/farmacología , Dibenzotiepinas , Combinación de Medicamentos , Farmacorresistencia Viral/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Humanos , Virus de la Influenza A/efectos de los fármacos , Virus de la Influenza B/efectos de los fármacos , Morfolinas , Oseltamivir/farmacología , Oxazinas , Pirazinas/farmacología , Piridinas , Piridonas , Serina Endopeptidasas/farmacología , Tiepinas , TriazinasRESUMEN
We show that trimethoprim (TMP), an antibiotic in current use, displays a strong synergistic effect on mutagenesis in Escherichia coli when paired with the base analog 2-aminopurine (2AP), resulting in a 35-fold increase in mutation frequencies in the rpoB-Rifr system. Combination therapies are often employed both as antibiotic treatments and in cancer chemotherapy. However, mutagenic effects of these combinations are rarely examined. An analysis of the mutational spectra of TMP, 2AP, and their combination indicates that together they trigger a response via an alteration in deoxynucleoside triphosphate (dNTP) ratios that neither compound alone can trigger. A similar, although less strong, response is seen with the frameshift mutagen ICR191 and 2AP. These results underscore the need for testing the effects on mutagenesis of combinations of antibiotics and chemotherapeutics.
Asunto(s)
2-Aminopurina/farmacología , Antibacterianos/farmacología , Escherichia coli/genética , Mutagénesis/efectos de los fármacos , Mutágenos/farmacología , Trimetoprim/farmacología , ARN Polimerasas Dirigidas por ADN/efectos de los fármacos , Sinergismo Farmacológico , Escherichia coli/efectos de los fármacos , Proteínas de Escherichia coli/efectos de los fármacosRESUMEN
Background: The emergence and spread of drug-resistant Tuberculosis (TB) is a major public health threat. TB resistance originates in the course of treatment due to genomic mutations in Mycobacterium tuberculosis (MTB). An increase in new cases with drug-resistant TB could be an indicator of high levels of circulating resistant strains. This study was conducted to determine the occurrence and frequency of genomic mutations that mediate Isoniazid (INH) and Rifampicin (RIF) resistance among isolates from untreated TB cases in urban Blantyre, Malawi. Methods: A cross-sectional retrospective study was conducted on a panel of 141(n=141) MTB clinical isolates recovered between June 2010 and January 2012 from >2+ Ziehl-Neelsen smear positive new pulmonary-TB patients with no history of treatment. Frozen isolates were revived using the BACTEC MGIT detection system. DNA was extracted using GenoLyse DNA extraction kit and detection of genomic mutations was carried out using the GenoType MTBDRplus Ver 2.0 assay. Results: Out of the 141 isolates studied, 3 (2.1%) were found carrying mutations in the katG gene that confer resistance to Isoniazid (INH). No mutations were detected in the inhA promoter region gene that confer weak INH resistance or in the rpoB gene that confer Rifampicin resistance. All katG mutant genes had a S315T1 single point mutation, a genomic alteration that mediates high INH resistance. Conclusion: The katG mutant gene conferring resistance to INH was the only genomic mutation observed among the isolates studied and the frequency of occurrence was low. Our findings suggest low levels of circulating drug-resistant MTB strains in urban Blantyre, Malawi.
Asunto(s)
Antibióticos Antituberculosos/farmacología , Proteínas Bacterianas/genética , Isoniazida/farmacología , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/genética , Rifampin/farmacología , Tuberculosis Resistente a Múltiples Medicamentos/genética , Proteínas Bacterianas/efectos de los fármacos , Catalasa/efectos de los fármacos , Estudios Transversales , ARN Polimerasas Dirigidas por ADN/efectos de los fármacos , Humanos , Malaui , Técnicas de Diagnóstico Molecular , Mutación , Mycobacterium tuberculosis/aislamiento & purificación , Oxidorreductasas/efectos de los fármacos , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas/genética , Estudios RetrospectivosRESUMEN
RNA polymerase containing the stress response regulator σS subunit (RpoS) plays a key role in bacterial survival in hostile environments in nature and during infection. Here we devise and validate a simple cell-based high throughput luminescence assay for this holoenzyme suitable for screening large chemical libraries in a robotic platform.
Asunto(s)
Proteínas Bacterianas/genética , ARN Polimerasas Dirigidas por ADN/genética , Evaluación Preclínica de Medicamentos/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Salmonella enterica/genética , Factor sigma/genética , Antibacterianos/farmacología , Proteínas Bacterianas/efectos de los fármacos , ARN Polimerasas Dirigidas por ADN/efectos de los fármacos , Regulación Bacteriana de la Expresión Génica , Mediciones Luminiscentes , Robótica , Salmonella enterica/efectos de los fármacos , Factor sigma/efectos de los fármacosRESUMEN
Objective: To comprehensively identify compensatory mutations in rpoA, rpoB and rpoC genes of rifampicin-resistant Mycobacterium tuberculosis (RIF-r MTB) and to evaluate the effect of rifampicin-resistant mutation type and lineage background on occurrence of compensatory mutations. Methods: Published MTB whole genome sequencing data were searched and downloaded. RIF-r MTB was identified through known rifampicin-resistant mutations. Based on parallel evolutionary patterns, we identified putative compensatory mutations in the phylogenetic tree and calculated proportions of accumulating compensatory mutations in each rifampicin-resistant mutations' type and lineage background of RIF-r MTB. Statistic significance was analyzed by chi-square test. Results: A total of 8 453 global MTB whole genome sequencing data were downloaded form ENA (covering 12 countries), including 1 749 RIF-r MTB. Based on phylogenetic analysis, we totally identified 60 putative compensatory mutations (6 in rpoA gene, 16 in rpoB gene and 38 in rpoC gene), 11 of which were newly reported. RIF-R strains carrying rpoB S450L (41.7%, 279/669) had a significant higher chance to accumulate compensatory mutations than strains with other rpoB mutations (8.0%, 31/388, χ(2)=378.5, P<0.000 1). In addition, RIF-R strains from lineage 2 (34.0%, 223/656) had a significant higher chance to accumulate compensatory mutations than strains from other lineages [lineage1: 4.7%(2/43), 2/43, lineage3: 12.5%(4/32), 4/32, lineage4: 15.1%(78/517), 78/517; χ(2)=238.5, P<0.000 1]. Conclusions: Our study comprehensively identified putative rifampicin-resistant compensatory mutations of rifampicin resistance. RIF-R strains carrying rpoB S450L mutation or from lineage 2 had a significantly higher chance to accumulate compensatory mutations than strains either with other rpoB mutations or from other lineages.
Asunto(s)
ARN Polimerasas Dirigidas por ADN/genética , Mycobacterium tuberculosis/genética , Rifampin/farmacología , Proteínas Bacterianas/efectos de los fármacos , Proteínas Bacterianas/genética , ARN Polimerasas Dirigidas por ADN/efectos de los fármacos , Humanos , Pruebas de Sensibilidad Microbiana , Mutación , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/crecimiento & desarrollo , FilogeniaRESUMEN
Recent cases of severe toxicity during clinical trials have been associated with antiviral ribonucleoside analogs (e.g. INX-08189 and balapiravir). Some have hypothesized that the active metabolites of toxic ribonucleoside analogs, the triphosphate forms, inadvertently target human mitochondrial RNA polymerase (POLRMT), thus inhibiting mitochondrial RNA transcription and protein synthesis. Others have proposed that the prodrug moiety released from the ribonucleoside analogs might instead cause toxicity. Here, we report the mitochondrial effects of several clinically relevant and structurally diverse ribonucleoside analogs including NITD-008, T-705 (favipiravir), R1479 (parent nucleoside of balapiravir), PSI-7851 (sofosbuvir), and INX-08189 (BMS-986094). We found that efficient substrates and chain terminators of POLRMT, such as the nucleoside triphosphate forms of R1479, NITD-008, and INX-08189, are likely to cause mitochondrial toxicity in cells, while weaker chain terminators and inhibitors of POLRMT such as T-705 ribonucleoside triphosphate do not elicit strong in vitro mitochondrial effects. Within a fixed 3'-deoxy or 2'-C-methyl ribose scaffold, changing the base moiety of nucleotides did not strongly affect their inhibition constant (Ki) against POLRMT. By swapping the nucleoside and prodrug moieties of PSI-7851 and INX-08189, we demonstrated that the cell-based toxicity of INX-08189 is mainly caused by the nucleoside component of the molecule. Taken together, these results show that diverse 2' or 4' mono-substituted ribonucleoside scaffolds cause mitochondrial toxicity. Given the unpredictable structure-activity relationship of this ribonucleoside liability, we propose a rapid and systematic in vitro screen combining cell-based and biochemical assays to identify the early potential for mitochondrial toxicity.
Asunto(s)
Antivirales/toxicidad , Mitocondrias/efectos de los fármacos , Ribonucleósidos/química , Ribonucleósidos/toxicidad , Adenosina/análogos & derivados , Amidas/toxicidad , Línea Celular/efectos de los fármacos , Citidina/análogos & derivados , Citidina/toxicidad , ARN Polimerasas Dirigidas por ADN/efectos de los fármacos , Guanosina Monofosfato/análogos & derivados , Guanosina Monofosfato/toxicidad , Humanos , Concentración 50 Inhibidora , Proteínas Mitocondriales/metabolismo , Nucleósidos/toxicidad , Profármacos/farmacología , Biosíntesis de Proteínas/efectos de los fármacos , Pirazinas/toxicidad , ARN/metabolismo , ARN Mitocondrial , Sofosbuvir/toxicidad , Relación Estructura-Actividad , Sitio de Iniciación de la Transcripción/efectos de los fármacos , Transcripción Genética/efectos de los fármacosRESUMEN
Influenza virus infection is a global public health issue. The efficacy of antiviral agents for influenza virus has been limited by the emergence of drug-resistant virus strains. Thus, there is an urgent need to identify novel antiviral therapies. Our previous studies have found that Cryptoporus volvatus extract can potently inhibit influenza virus replication in vitro and in vivo. However, the effective component of Cryptoporus volvatus, which mediates the antiviral activity, hasn't been identified. Here, we identified a novel anti-influenza virus molecule, Cryptoporic acid E (CAE), from Cryptoporus volvatus. Our results showed that CAE had broad-spectrum anti-influenza activity against 2009 pandemic strain A/Beijing/07/2009 (H1N1/09pdm), seasonal strain A/Beijing/CAS0001/2007(H3N2), mouse adapted strains A/WSN/33 (H1N1), and A/PR8/34 (H1N1). We further investigated the mode of CAE action. Time-course-analysis indicated that CAE exerted its inhibition mainly at the middle stages of the replication cycle of influenza virus. Subsequently, we confirmed that CAE inhibited influenza virus RNA polymerase activity and blocked virus RNA replication and transcription in MDCK cells. In addition, we found that CAE also impaired influenza virus infectivity by directly targeting virus particles. Our data suggest that CAE is a major effective component of Cryptoporus volvatus.
Asunto(s)
Antivirales/farmacología , Virus de la Influenza A/efectos de los fármacos , Polyporaceae/metabolismo , Sesquiterpenos/antagonistas & inhibidores , Replicación Viral/efectos de los fármacos , Animales , Supervivencia Celular/efectos de los fármacos , ARN Polimerasas Dirigidas por ADN/efectos de los fármacos , Perros , Humanos , Técnicas In Vitro , Subtipo H1N1 del Virus de la Influenza A/efectos de los fármacos , Subtipo H3N2 del Virus de la Influenza A/efectos de los fármacos , Virus de la Influenza A/enzimología , Gripe Humana/tratamiento farmacológico , Concentración 50 Inhibidora , Pulmón/virología , Células de Riñón Canino Madin Darby , Ratones , Infecciones por Orthomyxoviridae/tratamiento farmacológico , Infecciones por Orthomyxoviridae/virología , ARN Viral/efectos de los fármacos , Ribavirina/farmacología , Sesquiterpenos/química , Virión/efectos de los fármacosRESUMEN
Since 1967, Rifampin (RMP, a Rifamycin) has been used as a first line antibiotic treatment for tuberculosis (TB), and it remains the cornerstone of current short-term TB treatment. Increased occurrence of Rifamycin-resistant (RIFR ) TB, â¼41% of which results from the RpoB S531L mutation in RNA polymerase (RNAP), has become a growing problem worldwide. In this study, we determined the X-ray crystal structures of the Escherichia coli RNAPs containing the most clinically important S531L mutation and two other frequently observed RIFR mutants, RpoB D516V and RpoB H526Y. The structures reveal that the S531L mutation imparts subtle if any structural or functional impact on RNAP in the absence of RIF. However, upon RMP binding, the S531L mutant exhibits a disordering of the RIF binding interface, which effectively reduces the RMP affinity. In contrast, the H526Y mutation reshapes the RIF binding pocket, generating significant steric conflicts that essentially prevent any RIF binding. While the D516V mutant does not exhibit any such gross structural changes, certainly the electrostatic surface of the RIF binding pocket is dramatically changed, likely resulting in the decreased affinity for RIFs. Analysis of interactions of RMP with three common RIFR mutant RNAPs suggests that modifications to RMP may recover its efficacy against RIFR TB.
Asunto(s)
Antibióticos Antituberculosos/farmacología , Proteínas Bacterianas/genética , ARN Polimerasas Dirigidas por ADN/ultraestructura , Mycobacterium tuberculosis/efectos de los fármacos , Rifampin/farmacología , Tuberculosis Pulmonar/tratamiento farmacológico , Sitios de Unión/genética , Cristalografía por Rayos X , ARN Polimerasas Dirigidas por ADN/efectos de los fármacos , ARN Polimerasas Dirigidas por ADN/genética , Farmacorresistencia Bacteriana/genética , Escherichia coli/genética , Humanos , Mutación/genética , Mycobacterium tuberculosis/genética , Conformación Proteica , ARN Bacteriano , Tuberculosis Pulmonar/microbiologíaRESUMEN
Worldwide, respiratory syncytial virus (RSV) causes severe disease in infants, the elderly, and immunocompromised people. No vaccine or effective antiviral treatment is available. RSV is a member of the non-segmented, negative-strand (NNS) group of RNA viruses and relies on its RNA-dependent RNA polymerase to transcribe and replicate its genome. Because of its essential nature and unique properties, the RSV polymerase has proven to be a good target for antiviral drugs, with one compound, ALS-8176, having already achieved clinical proof-of-concept efficacy in a human challenge study. In this article, we first provide an overview of the role of the RSV polymerase in viral mRNA transcription and genome replication. We then review past and current approaches to inhibiting the RSV polymerase, including use of nucleoside analogs and non-nucleoside inhibitors. Finally, we consider polymerase inhibitors that hold promise for treating infections with other NNS RNA viruses, including measles and Ebola.
Asunto(s)
Antivirales/uso terapéutico , ARN Polimerasas Dirigidas por ADN/antagonistas & inhibidores , Mononegavirales/efectos de los fármacos , Virus Sincitial Respiratorio Humano/efectos de los fármacos , Antivirales/farmacología , Ensayos Clínicos como Asunto , ARN Polimerasas Dirigidas por ADN/efectos de los fármacos , ARN Polimerasas Dirigidas por ADN/metabolismo , Desoxicitidina/análogos & derivados , Desoxicitidina/uso terapéutico , Fiebre Hemorrágica Ebola/tratamiento farmacológico , Humanos , Sarampión/tratamiento farmacológico , Mononegavirales/enzimología , Mononegavirales/genética , Nucleósidos/agonistas , ARN Mensajero , ARN Polimerasa Dependiente del ARN/efectos de los fármacos , Infecciones por Virus Sincitial Respiratorio/tratamiento farmacológico , Virus Sincitial Respiratorio Humano/enzimología , Virus Sincitial Respiratorio Humano/genética , Transcripción Genética , Replicación Viral/efectos de los fármacosRESUMEN
Toxicity has emerged during the clinical development of many but not all nucleotide inhibitors (NI) of hepatitis C virus (HCV). To better understand the mechanism for adverse events, clinically relevant HCV NI were characterized in biochemical and cellular assays, including assays of decreased viability in multiple cell lines and primary cells, interaction with human DNA and RNA polymerases, and inhibition of mitochondrial protein synthesis and respiration. NI that were incorporated by the mitochondrial RNA polymerase (PolRMT) inhibited mitochondrial protein synthesis and showed a corresponding decrease in mitochondrial oxygen consumption in cells. The nucleoside released by the prodrug balapiravir (R1626), 4'-azido cytidine, was a highly selective inhibitor of mitochondrial RNA transcription. The nucleotide prodrug of 2'-C-methyl guanosine, BMS-986094, showed a primary effect on mitochondrial function at submicromolar concentrations, followed by general cytotoxicity. In contrast, NI containing multiple ribose modifications, including the active forms of mericitabine and sofosbuvir, were poor substrates for PolRMT and did not show mitochondrial toxicity in cells. In general, these studies identified the prostate cell line PC-3 as more than an order of magnitude more sensitive to mitochondrial toxicity than the commonly used HepG2 cells. In conclusion, analogous to the role of mitochondrial DNA polymerase gamma in toxicity caused by some 2'-deoxynucleotide analogs, there is an association between HCV NI that interact with PolRMT and the observation of adverse events. More broadly applied, the sensitive methods for detecting mitochondrial toxicity described here may help in the identification of mitochondrial toxicity prior to clinical testing.
Asunto(s)
Antivirales/farmacología , ARN Polimerasas Dirigidas por ADN/efectos de los fármacos , Hepacivirus/efectos de los fármacos , Hepatitis C Crónica/tratamiento farmacológico , Mitocondrias/efectos de los fármacos , Línea Celular , ADN Polimerasa gamma , ADN Polimerasa Dirigida por ADN/genética , ARN Polimerasas Dirigidas por ADN/genética , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacología , Guanosina Monofosfato/análogos & derivados , Guanosina Monofosfato/farmacología , Humanos , Mitocondrias/genética , Mitocondrias/metabolismo , Nucleósidos/farmacología , Consumo de Oxígeno/efectos de los fármacos , Biosíntesis de Proteínas/efectos de los fármacos , ARN/genética , ARN Mitocondrial , Sofosbuvir/farmacología , Transcripción Genética/efectos de los fármacos , Transcripción Genética/genética , Replicación Viral/efectos de los fármacosRESUMEN
The RNA polymerase of Influenza A virus (IAV), which is comprised of three units PA, PB1 and PB2, is involved in transcription and replication of the influenza virus. In order to develop effective treatment for IAV, researchers have focused on designing drugs targeting IAV polymerase. Currently, crystal structures of the IAV polymerase PA-PB1, PB1-PB2 complexes and the PA subunit have been obtained by several groups, providing useful information regarding potential binding sites in drug design. However, to gain full understanding of the molecular mechanism of IAV polymerase in viral transcription and replication, thereby aiding drug development, a complete atomistic structure of the RNA polymerase is required. In this paper, we employed computer-aided drug design tools to describe the complete structure of the RNA polymerase and proposed a putative mechanism. We predict that the combination of Vancomycin and Oseltamivir will be an effective drug to universally treat IAVs with no resultant drug resistance if this putative mechanism is true.
Asunto(s)
Antivirales/farmacología , Diseño Asistido por Computadora , Diseño de Fármacos , Terapia Molecular Dirigida , Antivirales/administración & dosificación , Sitios de Unión , ARN Polimerasas Dirigidas por ADN/química , ARN Polimerasas Dirigidas por ADN/efectos de los fármacos , ARN Polimerasas Dirigidas por ADN/metabolismo , Farmacorresistencia Viral , Quimioterapia Combinada , Humanos , Virus de la Influenza A/efectos de los fármacos , Virus de la Influenza A/enzimología , Gripe Humana/tratamiento farmacológico , Gripe Humana/virología , Oseltamivir/administración & dosificación , Oseltamivir/farmacología , Vancomicina/administración & dosificación , Vancomicina/farmacología , Replicación Viral/efectos de los fármacosRESUMEN
Mutations in rpoB (RNA polymerase ß-subunit) can cause high-level resistance to rifampicin, an important first-line drug against tuberculosis. Most rifampicin-resistant (Rif(R)) mutants selected in vitro have reduced fitness, and resistant clinical isolates of M. tuberculosis frequently carry multiple mutations in RNA polymerase genes. This supports a role for compensatory evolution in global epidemics of drug-resistant tuberculosis but the significance of secondary mutations outside rpoB has not been demonstrated or quantified. Using Salmonella as a model organism, and a previously characterized Rif(R) mutation (rpoB R529C) as a starting point, independent lineages were evolved with selection for improved growth in the presence and absence of rifampicin. Compensatory mutations were identified in every lineage and were distributed between rpoA, rpoB and rpoC. Resistance was maintained in all strains showing that increased fitness by compensatory mutation was more likely than reversion. Genetic reconstructions demonstrated that the secondary mutations were responsible for increasing growth rate. Many of the compensatory mutations in rpoA and rpoC individually caused small but significant reductions in susceptibility to rifampicin, and some compensatory mutations in rpoB individually caused high-level resistance. These findings show that mutations in different components of RNA polymerase are responsible for fitness compensation of a Rif(R) mutant.
Asunto(s)
Antibióticos Antituberculosos/farmacología , ARN Polimerasas Dirigidas por ADN/genética , Aptitud Genética , Rifampin/farmacología , Salmonella typhimurium/genética , Proteínas Bacterianas/efectos de los fármacos , Proteínas Bacterianas/genética , ARN Polimerasas Dirigidas por ADN/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Mutación , Fenotipo , Estructura Terciaria de Proteína , Salmonella typhimurium/efectos de los fármacos , Salmonella typhimurium/crecimiento & desarrolloRESUMEN
OBJECTIVE: To observe the effect of curcumin on the expression of Abeta42 and its degrading enzyme NEP in APP/PS1 double transgenic mice. METHOD: 3-month old APP/PS1 dtg mice were randomly divided into model group, positive control group and curcumin large, medium and small dose group. After 3 months, Morris water maze, immunohistochemistry, Western blot were applied to detect learning and memory ability of animal. RESULT: Behavior detection, compared with the model group, treatment group showed varying degrees of difference in place navigation test and space exploration experiments (P < 0.01 or P < 0.05). The expression of Abeta42 and its degrading enzyme NEP, Abeta42-positive cells of hippocampus CA1 region significantly increased in model mice as compared with normal control group (P < 0.01). CONCLUSION: Curcumin can improve learning and memory ability of APP/PS1 double transgenic mice through increasing the expression of Abeta-degrading enzyme NEP and decreasing the expression of Abeta42.
Asunto(s)
Péptidos beta-Amiloides/efectos de los fármacos , Péptidos beta-Amiloides/metabolismo , Región CA1 Hipocampal/metabolismo , Curcumina/farmacología , Aprendizaje por Laberinto/efectos de los fármacos , Fragmentos de Péptidos/efectos de los fármacos , Fragmentos de Péptidos/metabolismo , Animales , Región CA1 Hipocampal/efectos de los fármacos , ARN Polimerasas Dirigidas por ADN/efectos de los fármacos , ARN Polimerasas Dirigidas por ADN/metabolismo , Memoria/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ratones TransgénicosRESUMEN
Mycobacterium tuberculosis (MTB) infects over 9 million people globally and claims approximately 2 million lives annually. Rifampin (Rif) is one of the first-line anti-tuberculosis drugs that inhibits transcription by binding to the ß subunit (encoded by the rpoB gene) of the prokaryotic RNA polymerase (RNAP). A highly conserved 81 base pair core region among the ß subunit of prokaryotes harbors most of the point mutations leading to rifamycin-resistant (RifR) mutations, where the majority of the clinically relevant MTB RifR mutations result from amino acid substitutions of one of the following three amino acids: ßAsp435, ßHis445, and ßSer450 (MTB numbering). In this study, to determine the direct effect of rifamycins on the MTB RNAP, co-overexpression vectors were constructed to co-express the core subunits of wild-type and RifR mutants of MTB RNAP. The three aforementioned amino acids were each mutated to the most prevalent substitution found in the MTB clinical isolates (Asp435Val, His445Tyr, Ser450Leu) in the rpoB gene via site-directed mutagenesis. After purification via two-step column chromatography, the in vitro activity of the wild-type and RifR mutant MTB RNAPs was assessed via rolling circle transcription assay. The apparent IC(50) values for three key rifamycins (rifampin (Rif), rifabutin (Rbn), and rifaximin (Rfx)) were determined and these results indicate that the mutant RNAPs demonstrate approximately 10(3)-fold or greater loss of affinities for rifamycins relative to wild-type MTB RNAP. Along with the MTB RNAPs, rifamycin inhibition of the Escherichia coli RNAP counterparts was also assessed. Previously, it has been reported that Gram-positive bacteria (particularly mycobacteria) are more sensitive to rifamycins than Gram-negative bacteria. Under our experimental conditions, the rifamycin IC(50)s for wild-type and RifR mutants of MTB and E. coli RNAPs (wild-type and corresponding mutants) were very similar; therefore, the difference in sensitivity toward rifamycins does not reside in the RNAP itself. The correlation between the sensitivity of rifamycins and permeability into cells was evaluated using the wild-type E. coli strains (TG2 and DH5α) and a mutant E. coli strain with efflux pump defects (EC2880, tolC(-)/imp(-)). The MICs were drastically lower in the EC2880 strain, consistent with previous reports that the differential sensitivity of MTB and E. coli to rifamycins is not related to the RNAP, but rather has to do with efflux pumps in E. coli. Future work will focus on the elucidation of the molecular interaction of these MTB RifR mutants with rifamycins to provide insight to the design of novel rifamycins.
Asunto(s)
Antibacterianos/farmacología , ARN Polimerasas Dirigidas por ADN/efectos de los fármacos , Farmacorresistencia Bacteriana , Escherichia coli/efectos de los fármacos , Mutación , Mycobacterium tuberculosis/efectos de los fármacos , Rifamicinas/farmacología , Tuberculosis/genética , Secuencia de Bases , Análisis por Conglomerados , Diseño de Fármacos , Farmacorresistencia Bacteriana/genética , Escherichia coli/enzimología , Escherichia coli/genética , Predisposición Genética a la Enfermedad/genética , Humanos , Pruebas de Sensibilidad Microbiana , Mutación/efectos de los fármacos , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Polimorfismo de Nucleótido Simple , Tuberculosis/tratamiento farmacológico , Regulación hacia ArribaRESUMEN
The hepatitis C virus (HCV) RNA polymerase (RdRp) may be a target of the drug ribavirin, and it is an object of drug development. Independent isolates of any HCV subtype differ genetically by approximately 10%, but the effects of this variation on enzymatic activity and drug sensitivity are poorly understood. We proposed that nucleotide use profiles (G/U ratio) among subtype 1b RdRps may reflect their use of ribavirin. Here, we characterized how subtype 1b genetic variation affects RNA polymerase activity and evaluated the G/U ratio as a surrogate for ribavirin use during pegylated interferon α and ribavirin therapy. Genetic and biochemical variation in the RdRp was compared between responders who would be largely sensitive to ribavirin and relapsers who would be mostly resistant. There were no consistent genetic differences between responder and relapser RdRps. RNA polymerization, RNA binding and primer usage varied widely among the RdRps, but these parameters did not differ significantly between the response groups. The G/U ratio among a set of subtype 1a RdRps increased rather than decreased following failed therapy, as would be expected if it reflected ribavirin use. Finally, RdRp activity was significantly associated with ALT levels. These data indicate that (i) current genetic approaches cannot predict RNA polymerase behaviour, (ii) the G/U ratio is not a surrogate for ribavirin use, (iii) RdRp activity may contribute to liver disease by modulating viral mRNA and antigen levels, and (iv) drug candidates should be tested against multiple patient-derived enzymes to ensure widespread efficacy even within a viral subtype.
Asunto(s)
Antivirales/uso terapéutico , ARN Polimerasas Dirigidas por ADN/efectos de los fármacos , Hepacivirus/efectos de los fármacos , Hepatitis C/tratamiento farmacológico , Proteínas no Estructurales Virales/metabolismo , Antivirales/administración & dosificación , Antivirales/farmacología , ARN Polimerasas Dirigidas por ADN/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Quimioterapia Combinada , Variación Genética/genética , Hepacivirus/enzimología , Hepacivirus/genética , Hepatitis C/virología , Humanos , Interferón-alfa/administración & dosificación , Interferón-alfa/farmacología , Interferón-alfa/uso terapéutico , ARN Viral/genética , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Recurrencia , Ribavirina/administración & dosificación , Ribavirina/farmacología , Ribavirina/uso terapéutico , Proteínas no Estructurales Virales/genéticaRESUMEN
The synthetic methods for 4'-C-modified nucleosides as well as structure activity relationship of obtained compounds towards hepatitis C virus are reviewed.
Asunto(s)
Nucleósidos de Pirimidina/química , Nucleósidos de Pirimidina/síntesis química , Relación Estructura-Actividad , ARN Polimerasas Dirigidas por ADN/efectos de los fármacos , Hepacivirus/efectos de los fármacos , Humanos , Estructura Molecular , Nucleósidos de Pirimidina/farmacología , ARN Helicasas/efectos de los fármacosRESUMEN
OBJECTIVE: Growth of white adipose tissue takes place in normal development and in obesity. A pool of adipose progenitors is responsible for the formation of new adipocytes and for the potential of this tissue to expand in response to chronic energy overload. However, factors controlling self-renewal of human adipose progenitors are largely unknown. We investigated the expression profile and the role of activin A in this process. RESEARCH DESIGN AND METHODS: Expression of INHBA/activin A was investigated in three types of human adipose progenitors. We then analyzed at the molecular level the function of activin A during human adipogenesis. We finally investigated the status of activin A in adipose tissues of lean and obese subjects and analyzed macrophage-induced regulation of its expression. RESULTS: INHBA/activin A is expressed by adipose progenitors from various fat depots, and its expression dramatically decreases as progenitors differentiate into adipocytes. Activin A regulates the number of undifferentiated progenitors. Sustained activation or inhibition of the activin A pathway impairs or promotes, respectively, adipocyte differentiation via the C/EBPß-LAP and Smad2 pathway in an autocrine/paracrine manner. Activin A is expressed at higher levels in adipose tissue of obese patients compared with the expression levels in lean subjects. Indeed, activin A levels in adipose progenitors are dramatically increased by factors secreted by macrophages derived from obese adipose tissue. CONCLUSIONS: Altogether, our data show that activin A plays a significant role in human adipogenesis. We propose a model in which macrophages that are located in adipose tissue regulate adipose progenitor self-renewal through activin A.
Asunto(s)
Activinas/fisiología , Tejido Adiposo/citología , Glucosafosfato Deshidrogenasa/genética , Obesidad Mórbida/patología , Células Madre/citología , Delgadez/patología , Activinas/genética , Activinas/farmacología , Tejido Adiposo/efectos de los fármacos , Tejido Adiposo/patología , Adulto , Diferenciación Celular , División Celular , ARN Polimerasas Dirigidas por ADN/efectos de los fármacos , ARN Polimerasas Dirigidas por ADN/genética , Dexametasona/farmacología , Regulación de la Expresión Génica , Glucosafosfato Deshidrogenasa/efectos de los fármacos , Humanos , Obesidad Mórbida/genética , Obesidad Mórbida/prevención & control , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Madre/efectos de los fármacos , Células Madre/patología , Proteína de Unión a TATA-Box/efectos de los fármacos , Proteína de Unión a TATA-Box/genéticaRESUMEN
Facile evaluation of mixed-salt effect on the strongly salt-dependent thermodynamic and kinetic parameters of protein-DNA complexes is of importance for relevant biochemical and biophysical studies. In pursuit of this aim, binding isotherms for open transcription complex (RPo) of Escherichia coli RNA polymerase (R) at lambdaP(R) promoter DNA (P) were determined as a function of salt concentration in pure NaCl and Tris/HCl solutions, and as a function of [NaCl] in the presence of fixed concentrations of MgCl(2) and Tris/HCl. A concept of equivalent salt concentrations, i.e. concentrations at which the binding equilibrium constant is the same, was introduced and applied for prediction of binding isotherms in mixed salt solutions. Full coincidence between the experimental and predicted isotherms indicated that individual contributions of salts to the global salt-effect are additive in a broad range of salt concentrations. A generalized formula for calculation of salt equivalents characteristic for any of the thermodynamic or kinetic parameters of a complex (e.g., free energy, binding equilibrium and association/dissociation kinetic rate constants) is presented and its applicability to a number of protein-DNA complexes and dsDNA melting demonstrated using authors' own and literature data.