RESUMEN
BACKGROUND: Our goals were to describe azithromycin (AZI) pharmacokinetics in maternal plasma (MP), fetal plasma (FP), and amniotic fluid (AF) following intra-amniotic infection (IAI) with Ureaplasma in pregnant rhesus monkeys and to explore concentration-response relationships. METHODS: Following intra-amniotic inoculation of Ureaplasma parvum, rhesus monkeys received AZI (12.5 mg/kg every 12 hours intravenously for 10 days; n = 10). Intensive pharmacokinetic sampling of MP, FP, and AF was scheduled following the first (ie, single) dose and the last (ie, multiple) dose. Noncompartmental and pharmacokinetic modeling methods were used. RESULTS: The AF area under the concentration-time curve at 12 hours was 0.22 µg×h/mL following a single dose and 6.3 µg×h/mL at day 10. MP and AF accumulation indices were 8.4 and 19, respectively. AZI AF half-life following the single dose and multiple dose were 156 and 129 hours, respectively. The median MP:FP ratio in concomitantly drawn samples was 3.2 (range, 1.3-9.6; n = 9). Eradication of U. parvum occurred at 6.6 days, with a 95% effective concentration (EC95) of 39 ng/mL for the maximum AZI AF concentration. CONCLUSIONS: Our study demonstrates that a maternal multiple-dose AZI regimen is effective in eradicating U. parvum IAI by virtue of intra-amniotic accumulation and suggests that antenatal therapy has the potential to mitigate complications associated with U. parvum infection in pregnancy, such as preterm labor and fetal sequelae.
Asunto(s)
Antibacterianos/farmacocinética , Azitromicina/farmacocinética , Corioamnionitis/tratamiento farmacológico , Complicaciones Infecciosas del Embarazo/tratamiento farmacológico , Infecciones por Ureaplasma/tratamiento farmacológico , Administración Intravenosa , Líquido Amniótico/metabolismo , Líquido Amniótico/microbiología , Animales , Antibacterianos/sangre , Antibacterianos/uso terapéutico , Azitromicina/administración & dosificación , Azitromicina/sangre , Azitromicina/uso terapéutico , Corioamnionitis/metabolismo , Modelos Animales de Enfermedad , Femenino , Sangre Fetal/metabolismo , Sangre Fetal/microbiología , Macaca mulatta , Embarazo , Complicaciones Infecciosas del Embarazo/metabolismo , Infecciones por Ureaplasma/metabolismoRESUMEN
BACKGROUND: Immune mediated changes in circulating α-1-acid glycoprotein (AAG), a type 1 acute phase protein, which binds protease inhibitors (PI), may alter protein binding and contribute to PI's pharmacokinetic (PK) variability. METHODS: In a prospective, 2-phase intensive PK study on antiretroviral naive human immunodeficiency virus (HIV)-infected subjects treated with a lopinavir-/ritonavir-based regimen, steady state PK sampling and AAG assays were performed at weeks 2 and 16 of treatment. RESULTS: Median entry age was 43 years (n = 16). Median plasma log(10) HIV-1 RNA, CD4 T-cell counts, and AAG were 5.16 copies/mL, 28 cells/µL, and 143 mg/dL, respectively.The total lopinavir area under the concentration time curve (AUC(12_total)) and maximum concentration (C(max_total)) changed linearly with AAG at mean rates of 16±7 mg*hr/L (slope ± SE); P = .04, and 1.6 ± 0.6 mg/L, P = .02, per 100 mg/dL increase in AAG levels, respectively (n = 15).A 29% drop in AAG levels between week 2 and week 16 was associated with 14% (geometric mean ratio [GMR] = 0.86; 90% confidence interval [CI] = 0.74-0.98) and 13% (GMR = 0.87; 90% CI = 0.79-0.95) reduction in AUC(12_total) and C(max_total), respectively. Neither free lopinavir PK parameters nor antiviral activity (HIV-1 RNA average AUC minus baseline) was affected by change in plasma AAG. CONCLUSIONS: Changes in plasma AAG levels alter total lopinavir concentrations, but not the free lopinavir exposure or antiviral activity. This observation may have implications in therapeutic drug monitoring.
Asunto(s)
Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/inmunología , Inhibidores de la Proteasa del VIH/sangre , Lopinavir/sangre , Orosomucoide/inmunología , Adulto , Fármacos Anti-VIH/sangre , Fármacos Anti-VIH/farmacocinética , Fármacos Anti-VIH/uso terapéutico , Área Bajo la Curva , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Femenino , VIH , Infecciones por VIH/sangre , Infecciones por VIH/metabolismo , Inhibidores de la Proteasa del VIH/farmacocinética , Inhibidores de la Proteasa del VIH/uso terapéutico , Humanos , Lopinavir/farmacocinética , Lopinavir/uso terapéutico , Masculino , Persona de Mediana Edad , Orosomucoide/metabolismo , Estudios Prospectivos , Unión Proteica , ARN Viral/sangre , Ritonavir/uso terapéuticoRESUMEN
The biggest challenge facing highly antiretroviral-experienced patients and their caregivers is the diminishing number of therapeutic options available that sustain activity despite increasing numbers of drug-resistance mutations. New options in antiretroviral treatment have been introduced: two new members of traditional antiretroviral classes (darunavir and etravirine) and two drugs with novel mechanisms of action (raltegravir and maraviroc). Each was approved for use in treatment-experienced patients. A fifth drug-containing efavirenz, tenofovir, and emtricitabine (Atripla; Bristol-Myers Squibb, New York, NY, and Gilead Sciences, Foster City, CA)-is a novel coformulation of existing drugs from two different classes, simplifying administration with the intent of increasing adherence. Because successful management of HIV infection requires the simultaneous use of three or more drugs, understanding the pharmacologic aspects of coadministration is critical. This review summarizes the pharmacokinetic properties affecting the administration of these recently approved drugs in light of highly active antiretroviral treatment guidelines.
Asunto(s)
Adenina/análogos & derivados , Fármacos Anti-VIH , Desoxicitidina/análogos & derivados , Infecciones por VIH/tratamiento farmacológico , Organofosfonatos , Oxazinas , Inhibidores de la Transcriptasa Inversa , Adenina/administración & dosificación , Adenina/farmacocinética , Fármacos Anti-VIH/administración & dosificación , Fármacos Anti-VIH/clasificación , Fármacos Anti-VIH/farmacocinética , Terapia Antirretroviral Altamente Activa , Darunavir , Desoxicitidina/administración & dosificación , Desoxicitidina/farmacocinética , Combinación de Medicamentos , Quimioterapia Combinada , Combinación Efavirenz, Emtricitabina y Fumarato de Tenofovir Disoproxil , Infecciones por VIH/virología , Humanos , Nitrilos , Organofosfonatos/administración & dosificación , Organofosfonatos/farmacocinética , Oxazinas/administración & dosificación , Oxazinas/farmacocinética , Guías de Práctica Clínica como Asunto , Piridazinas/administración & dosificación , Piridazinas/farmacocinética , Pirimidinas , Inhibidores de la Transcriptasa Inversa/administración & dosificación , Inhibidores de la Transcriptasa Inversa/farmacocinética , Sulfonamidas/administración & dosificación , Sulfonamidas/farmacocinéticaRESUMEN
The biggest challenge facing highly antiretroviral-experienced patients and their caregivers is the diminishing number of therapeutic options available that sustain activity despite increasing numbers of drug-resistance mutations. New options in antiretroviral treatment have been introduced: two new members of traditional antiretroviral classes (darunavir and etravirine) and two drugs with novel mechanisms of action (raltegravir and maraviroc). Each was approved for use in treatment-experienced patients. A fifth drug-containing efavirenz, tenofovir, and emtricitabine (Atripla; Bristol-Myers Squibb, New York, NY, and Gilead Sciences, Foster City, CA)-is a novel coformulation of existing drugs from two different classes, simplifying administration with the intent of increasing adherence. Because successful management of HIV infection requires the simultaneous use of three or more drugs, understanding the pharmacologic aspects of coadministration is critical. This review summarizes the pharmacokinetic properties affecting the administration of these recently approved drugs in light of highly active antiretroviral treatment guidelines.
RESUMEN
This work describes an assay system that has been developed to quantify raltegravir concentrations in human plasma using a liquid-liquid extraction technique paired with HPLC separation and MS-MS detection. The dynamic range of this assay extends from 1 to 3000 ng/mL, with a coefficient of determination (r(2), mean+/-SD) of 0.9992+/-0.0002. The mean precision values for calibration standards ranged from 0.6% to 3.0%, while accuracy values were 96.5-104.3%. This procedure is an accurate, precise, and sensitive method for raltegravir quantitation and was successfully validated using external proficiency testing.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Inhibidores de Integrasa VIH/sangre , Pirrolidinonas/sangre , Espectrometría de Masas en Tándem/métodos , Calibración , Humanos , Control de Calidad , Raltegravir Potásico , Sensibilidad y EspecificidadRESUMEN
Adenosine kinase (Ado kinase) from Mycobacterium tuberculosis is structurally and biochemically unique from other known Ado kinases. This purine salvage enzyme catalyzes the first step in the conversion of the adenosine analog, 2-methyl-Ado (methyl-Ado), into a metabolite with antitubercular activity. Methyl-Ado has provided proof of concept that the purine salvage pathway from M. tuberculosis may be utilized for the development of antitubercular compounds with novel mechanisms of action. In order to utilize this enzyme, it is necessary to understand the topography of the active site to rationally design compounds that are more potent and selective substrates for Ado kinase. A previous structure-activity relationship identified modifications to the base moiety of adenosine (Ado) that result in substrate and inhibitor activity. In an extension of that work, 62 Ado analogs with modifications to the ribofuranosyl moiety, modifications to the base and ribofuranosyl moiety, or modifications to the glycosidic bond position have been analyzed as substrates and inhibitors of M. tuberculosis Ado kinase. A subset of these compounds was further analyzed in human Ado kinase for the sake of comparison. Although no modifications to the ribose moiety resulted in compounds as active as Ado, the best substrates identified were carbocyclic-Ado, 8-aza-carbocyclic-Ado, and 9-[alpha-l-lyxofuranosyl]-adenine with 38%, 4.3%, and 3.8% of the activity of Ado, respectively. The most potent inhibitor identified, 5'-amino-5'-deoxy-Ado, had a K(i)=0.8muM and a competitive mode of inhibition. MIC studies demonstrated that poor substrates could still have potent antitubercular activity.
Asunto(s)
Adenosina Quinasa/química , Adenosina/análogos & derivados , Adenosina/química , Mycobacterium tuberculosis/enzimología , Humanos , Modelos Moleculares , Estructura Molecular , Ribosa/química , Relación Estructura-ActividadRESUMEN
BACKGROUND: Intracellular nucleoside reverse transcriptase inhibitor triphosphate (NRTI-TP) concentrations are crucial in suppressing HIV replication. Little is known about how commonly used dual-NRTI regimens affect the intracellular levels of NRTI-TPs, the active form of these drugs. This study investigates the effect of dual-NRTI therapy in intracellular NRTI-TP levels. METHODS: NRTI and NRTI-TP concentrations were evaluated in HIV-infected patients receiving either lamivudine (3TC) and stavudine (d4T) or lamivudine with zidovudine (ZDV); NRTI and NRTI-TP concentrations were determined using a validated HPLC/MS/MS method. Plasma HIV-1 RNA levels were determined at baseline and monthly to examine the relationship between NRTI-TP concentrations and plasma HIV-1 RNA. RESULTS: Forty-one subjects completed the study. 3TC-TP significantly increased between day 1 and week 28 from 1.48 to 5.00 pmol/10(6) peripheral blood mononuclear cells (PBMC; P < 0.0001). NRTI-TP concentrations for d4T and ZDV did not significantly increase, with values at week 28 of 0.011 and 0.02 pmol/10(6) PBMC, respectively. Mean NRTI-TP/plasma ratios were 3%, 0.007% and 0.05% for 3TC, d4T and ZDV, respectively. Linear relationships were observed between ZDV- and 3TC-TP and changes in plasma HIV-1 RNA. CONCLUSION: Of the three drugs studied, only 3TC-TP levels increased significantly between day 1 and week 28. ZDV-TP and 3TC-TP levels were unaffected by dual-NRTI therapy relative to monotherapy, regardless of the combination (3TC-ZDV or 3TC-d4T). Intracellular levels of d4T-TP were similar to previous reports for dual-NRTI therapy; however, in the case of d4T, these values appear lower than those achieved with d4T monotherapy.
Asunto(s)
Fármacos Anti-VIH/uso terapéutico , Citidina Trifosfato/análogos & derivados , Didesoxinucleótidos/sangre , Infecciones por VIH/tratamiento farmacológico , Lamivudine/análogos & derivados , Inhibidores de la Transcriptasa Inversa/uso terapéutico , Nucleótidos de Timina/sangre , Zidovudina/análogos & derivados , Fármacos Anti-VIH/sangre , Citidina Trifosfato/sangre , Quimioterapia Combinada , Femenino , Infecciones por VIH/sangre , Infecciones por VIH/virología , Humanos , Lamivudine/sangre , Lamivudine/uso terapéutico , Leucocitos Mononucleares/metabolismo , Masculino , Inhibidores de la Transcriptasa Inversa/sangre , Estavudina/sangre , Estavudina/uso terapéutico , Zidovudina/sangre , Zidovudina/uso terapéuticoRESUMEN
A sensitive and specific method for the quantitation of tenofovir (TFV) in human plasma by liquid chromatography/electrospray ionization mass spectrometry was developed and validated. Plasma samples were prepared by solid-phase extraction performed on Waters Oasis cation-exchange cartridges (30 mg). Chromatographic separation was performed isocratically on a reversed-phase Waters Atlantis dC18 column (2.0x100 mm, 3 microm). The mobile phase consisted of a hydroxylamine/acetic acid buffer (pH 6.75) and methanol (93:7, v/v). The acquisition was performed in selected ion monitoring mode for the protonated molecular ions [M+H]+ of m/z 288.2 for TFV and 212.2 for the internal standard, zalcitibine. The method was fully validated to determine its specificity, recovery, linearity and sensitivity, accuracy and precision. The analytical range was set at 1-750 ng/mL using a 200 microL plasma sample, with a mean coefficient of determination (r2) of 0.9969. The mean accuracies for the calibration standards ranged from -5.0 to 4.3%, while the precisions were within 1.2 and 6.4%. Intra-assay and inter-assay mean accuracies for three quality control concentrations (2, 60, and 600 ng/mL) ranged from -6.1 to 10.7%, while the precisions were within 1.3 and 9.1%. TFV was shown to be stable under normal storage and assay conditions; no degradation was seen when stored at -20 degrees C or -80 degrees C for up to 6 months, and after 16 h at room temperature in the injection matrix. The present method provides an accurate, precise, and sensitive tool for TFV quantitation and was successfully applied to an external proficiency-testing program and pharmacokinetic analysis.
Asunto(s)
Adenina/análogos & derivados , Cromatografía Liquida/métodos , Organofosfonatos/sangre , Inhibidores de la Transcriptasa Inversa/sangre , Espectrometría de Masa por Ionización de Electrospray/métodos , Adenina/sangre , Adenina/química , Adenina/farmacocinética , Calibración , Estabilidad de Medicamentos , Congelación , Humanos , Estructura Molecular , Organofosfonatos/química , Organofosfonatos/farmacocinética , Estándares de Referencia , Reproducibilidad de los Resultados , Inhibidores de la Transcriptasa Inversa/química , Inhibidores de la Transcriptasa Inversa/farmacocinética , Sensibilidad y Especificidad , Temperatura , Tenofovir , Factores de Tiempo , Zalcitabina/químicaRESUMEN
Tuberculosis remains a serious health problem throughout the world, and new drugs are needed to help control this disease. We have identified several purine nucleoside analogs that exhibit selective activity against Mycobacterium tuberculosis. The lead compound in this series is 2-methyl-adenosine (methyl-Ado), which is active against proliferating and nonproliferating bacteria due to its ability to inhibit protein synthesis. Methyl-Ado is activated by adenosine kinase that is expressed in M. tuberculosis cells. The primary intracellular metabolite is 2-methyl-AMP, although some methyl-ATP was also produced in the cells. Adenosine kinase has been purified from M. tuberculosis cells and its biochemical activity has been characterized and compared to that of the human homolog. The gene for adenosine kinase has been determined to be Rv2202c, which had been putatively identified as a sugar kinase. Because very little is known about purine metabolism in M. tuberculosis, we have initiated studies to characterize the enzymes that are involved in salvage of purine nucleosides. We believe that enhanced knowledge of the characteristics of the enzymes involved in purine salvage in M. tuberculosis should aid in the rational design of more potent purine analogs that can selectively inhibit M. tuberculosis replication. Compounds in this class should be active against strains of M. tuberculosis that are resistant to current agents used to treat this disease and may also target latent disease.
Asunto(s)
Antituberculosos/administración & dosificación , Antituberculosos/síntesis química , Sistemas de Liberación de Medicamentos/métodos , Mycobacterium tuberculosis/metabolismo , Nucleósidos de Purina/metabolismo , Tuberculosis/metabolismo , Tuberculosis/microbiología , Animales , Humanos , Mycobacterium tuberculosis/efectos de los fármacos , Filogenia , Tuberculosis/tratamiento farmacológicoRESUMEN
OBJECTIVES: Analyse a series of halogenated 3-deaza-adenosine analogues for efficacy against Mycobacterium tuberculosis H37Ra and determine if adenosine (Ado) kinase plays a role in the mechanism of action of these compounds. METHODS: The MIC as determined by microdilution broth assay provided a measure of antitubercular efficacy. MIC values were measured in M. tuberculosis strains H37Ra, SRICK1 (an Ado kinase-deficient strain of M. tuberculosis derived from H37Ra) and SRICK1 complemented with adoK, the gene which codes for Ado kinase in M. tuberculosis, in order to determine if Ado kinase played a role in the mechanism of action of these compounds. Furthermore, each compound was analysed as both a substrate and inhibitor for purified Ado kinases from M. tuberculosis and human sources. RESULTS: 2-Fluoro-3-deaza-adenosine, 3-fluoro-3-deaza-adenosine and 2,3-difluoro-3-deaza-adenosine exhibited antitubercular activity that was Ado kinase-dependent. Furthermore, these compounds were at least 10-fold better substrates for M. tuberculosis Ado kinase than the human homologue. CONCLUSIONS: The Ado kinase-dependent antitubercular activity exhibited by several of the halogenated 3-deaza-adenosine analogues investigated in this study warrants further investigation of these compounds as antitubercular agents. Furthermore, substrate and inhibition studies provided insight into the Ado-binding domain of Ado kinase, indicating that steric hindrance may limit the size of exocyclic modifications at the 3-position of Ado.
Asunto(s)
Adenosina Quinasa/fisiología , Antituberculosos/farmacología , Mycobacterium tuberculosis/efectos de los fármacos , Tubercidina/farmacología , Humanos , Ligandos , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
Adenosine kinase (Ado kinase, EC 2.7.1.20) is a purine salvage enzyme that phosphorylates adenosine (Ado) to AMP. Ado kinase from Mycobacterium tuberculosis also catalyzes an essential step in the conversion of 2-methyl-Ado to a compound with selective antimycobacterial activity. In order to aid in the design of more potent and selective Ado analogs, eighty nucleoside analogs with modifications to the adenine (Ade) moiety of Ado were evaluated as both substrates and inhibitors of Ado kinase from M. tuberculosis, and a subset was further tested with human Ado kinase for the sake of comparison. The best substrates were 2-aza-Ado, 8-aza-9-deaza-Ado, and 2-fluoro-Ado and the most potent inhibitors were N1-benzyl-Ado (Ki=0.19 microM), 2-fluoro-Ado (Ki=0.5 microM), 6-cyclopentyloxy-purine riboside (Ki=0.15 microM), and 7-iodo-7-deaza-Ado (Ki=0.21 microM). These studies revealed the presence of a hydrophobic pocket near the N6- and N1-positions that can accommodate substitutions at least as large as a benzyl group. The ability to fit into this pocket increased the likelihood that a compound would be an inhibitor and not a substrate. The 2-position was able to accommodate exocyclic substitutions as large as a methoxy group, although substrate activity was low. Similarly, the 7-position could bind an exocyclic group as large as a carboxamido moiety. However, all of the compounds tested with modifications at the 7-position were much better inhibitors than substrates. MIC studies performed with selected compounds have yielded several Ado analogs with promising antitubercular activity. Future studies will utilize this information for the design of new analogs that may be selective antitubercular agents.
Asunto(s)
Adenina/química , Adenosina Quinasa/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Mycobacterium tuberculosis/enzimología , Adenosina Quinasa/química , Adenosina Quinasa/metabolismo , Humanos , Pruebas de Sensibilidad Microbiana , Relación Estructura-ActividadRESUMEN
Adenosine kinase from Mycobacterium tuberculosis is the only prokaryotic adenosine kinase that has been isolated and characterized. The enzyme catalyzes the phosphorylation of adenosine to adenosine monophosphate and is involved in the activation of 2-methyladenosine, a compound that has demonstrated selective activity against M. tuberculosis. The mechanism of action of 2-methyladenosine is likely to be different from those of current tuberculosis treatments and this compound (or other adenosine analogs) may prove to be a novel therapeutic intervention for this disease. The M. tuberculosis adenosine kinase was overexpressed in Escherichia coli and the enzyme was purified with activity comparable to that reported previously. The protein was crystallized in the presence of adenosine using the vapour-diffusion method. The crystals diffracted X-rays to high resolution and a complete data set was collected to 2.2 A using synchrotron radiation. The crystal belonged to space group P3(1)21, with unit-cell parameters a = 70.2, c = 111.6 A, and contained a single protein molecule in the asymmetric unit. An initial structural model of the protein was obtained by the molecular-replacement method, which revealed a dimeric structure. The monomers of the dimer were related by twofold crystallographic symmetry. An understanding of how the M. tuberculosis adenosine kinase differs from the human homolog should aid in the design of more potent and selective antimycobacterial agents that are selectively activated by this enzyme.
Asunto(s)
Adenosina Quinasa/química , Mycobacterium tuberculosis/enzimología , Adenosina Quinasa/genética , Adenosina Quinasa/aislamiento & purificación , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Clonación Molecular , Cristalización/métodos , Dimerización , Escherichia coli/genética , Volatilización , Difracción de Rayos XRESUMEN
2-Methyladenosine (methyl-Ado) has selective activity against Mycobacterium tuberculosis (M. tuberculosis). In an effort to better understand its mechanism of action, we have characterized its metabolism in M. tuberculosis cells. The primary intracellular metabolite of methyl-Ado was 2-methyl-adenylate (methyl-AMP). Very little of the methyl-AMP was metabolized further. A M. tuberculosis strain that was resistant to methyl-Ado did not express adenosine kinase and did not convert methyl-Ado to methyl-AMP in intact cells. In contrast to these results, the primary intracellular metabolite of adenosine in M. tuberculosis cells was ATP, which was readily incorporated into RNA. The rate of metabolism of methyl-Ado to methyl-AMP was similar to the rate of metabolism of adenosine to ATP. Treatment of M. tuberculosis with methyl-Ado did not affect intracellular ATP levels. Methyl-Ado and Ado were also cleaved to 2-methyladenine and adenine, respectively, which accumulated in the medium outside the cells. These studies suggested that methyl-AMP was the active metabolite responsible for the cytotoxicity of this agent. Furthermore, because methyl-Ado was poorly metabolized in human cells, these studies indicated that the selective activity of methyl-Ado was due to its selective activation by M. tuberculosis. These studies have identified two enzyme reactions (Ado kinase and Ado cleavage) in M. tuberculosis that could be exploited for the rational design of new and selective anti-M. tuberculosis agents.
Asunto(s)
Adenosina/análogos & derivados , Adenosina/metabolismo , Mycobacterium tuberculosis/metabolismo , Adenosina Trifosfato/metabolismo , Técnicas BacteriológicasRESUMEN
Adenosine kinase (AK) is a purine salvage enzyme that catalyzes the phosphorylation of adenosine to AMP. In Mycobacterium tuberculosis, AK can also catalyze the phosphorylation of the adenosine analog 2-methyladenosine (methyl-Ado), the first step in the metabolism of this compound to an active form. Purification of AK from M. tuberculosis yielded a 35-kDa protein that existed as a dimer in its native form. Adenosine (Ado) was preferred as a substrate at least 30-fold (Km = 0.8 +/- 0.08 microM) over other natural nucleosides, and substrate inhibition was observed when Ado concentrations exceeded 5 micro M. M. tuberculosis and human AKs exhibited different affinities for methyl-Ado, with Km values of 79 and 960 microM, respectively, indicating that differences exist between the substrate binding sites of these enzymes. ATP was a good phosphate donor (Km = 1100 +/- 140 microM); however, the activity levels observed with dGTP and GTP were 4.7 and 2.5 times the levels observed with ATP, respectively. M. tuberculosis AK activity was dependent on Mg2+, and activity was stimulated by potassium, as reflected by a decrease in the Km and an increase in Vmax for both Ado and methyl-Ado. The N-terminal amino acid sequence of the purified enzyme revealed complete identity with Rv2202c, a protein currently classified as a hypothetical sugar kinase. When an AK-deficient strain of M. tuberculosis (SRICK1) was transformed with this gene, it exhibited a 5,000-fold increase in AK activity compared to extracts from the original mutants. These results verified that the protein that we identified as AK was coded for by Rv2202c. AK is not commonly found in bacteria, and to the best of our knowledge, M. tuberculosis AK is the first bacterial AK to be characterized. The enzyme shows greater sequence homology with ribokinase and fructokinase than it does with other AKs. The multiple differences that exist between M. tuberculosis and human AKs may provide the molecular basis for the development of nucleoside analog compounds with selective activity against M. tuberculosis.
Asunto(s)
Adenosina Quinasa , Adenosina/análogos & derivados , Mycobacterium tuberculosis/enzimología , Adenosina/metabolismo , Adenosina/farmacología , Adenosina Quinasa/química , Adenosina Quinasa/genética , Adenosina Quinasa/aislamiento & purificación , Adenosina Quinasa/metabolismo , Secuencia de Aminoácidos , Línea Celular , Clonación Molecular , Medios de Cultivo , Humanos , Datos de Secuencia Molecular , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/genética , Filogenia , Especificidad por SustratoRESUMEN
Epstein-Barr virus (EBV), human herpesvirus type 6 (HHV-6), and human herpesvirus type 8 (HHV-8) comprise a group of lymphotropic herpesviruses which are responsible for a wide range of diseases, including lymphoproliferative disorders and tumors. We have developed several flow cytometric assay (FACS) systems to evaluate antiviral efficacy against EBV, HHV-6 and HHV-8. Assays using either EBV or HHV-8, members of the gammaherpesvirus subfamily, have shown that while EBV responds well to acyclovir (ACV), HHV-8 was most sensitive to cidofovir (CDV). Since HHV-6 strains are divided into two sub-groups, A and B, we evaluated antiviral efficacy for strains from each group. The group A strain, HHV-6(GS), was inhibited by foscarnet (PFA), CDV and ganciclovir (GCV) in both Sup-T1 and HSB-2 cell lines. HHV-6(Z-29), a representative group B virus, was inhibited by GCV and CDV but not by PFA. Our findings indicate that flow cytometry can be utilized to efficiently evaluate new antiviral agents against lymphotropic herpesviruses and that the results are comparable to those obtained by other methods such as immunofluorescence.