RESUMO
It has been recently demonstrated that HIV-1 reverse transcriptase is the target of two diterpenes, (6 R)-6-hydroxydichotoma-3,14-diene-1,17-dial (compound 1) and (6 R)-6-acetoxydichotoma-3,14-diene-1,17-dial (compound 2), that inhibit HIV-1 replication in vitro. In this work, the effects of both diterpenes on the kinetic properties of the recombinant HIV-1 reverse transcriptase (RT) enzyme were evaluated. RNA-dependent DNA-polymerase (RDDP) activity assays demonstrated that both diterpenes behave as non-competitive inhibitors with respect to dTTP and uncompetitive inhibitors with respect to poly(rA).oligo(dT) template primers. The K(i) values obtained for compounds 1 and 2 were 10 and 35 microM, respectively. Neither of these diterpenes affected the DNA-dependent DNA-polymerase (DDDP) activity of the HIV-1 RT. The RDDP activities of AMV-RT and MMLV-RT enzymes were also inhibited by compounds 1 and 2. In contrast to the HIV-1 enzyme, the DDDP activities of AMV-RT and MMLV-RT enzymes were significantly reduced by compound 1. Taken together, our results demonstrate that compound 1 is a more effective inhibitor of the viral reverse transcriptases from HIV-1, AMV and MMLV than compound 2. The kinetic behavior analyses of the HIV-1 RT demonstrate that both diterpenes have similar mechanisms of inhibition of RDDP activity.
Assuntos
Fármacos Anti-HIV/farmacologia , Diterpenos/farmacologia , Transcriptase Reversa do HIV/efeitos dos fármacos , Phaeophyceae/química , Inibidores da Transcriptase Reversa/farmacologia , Fármacos Anti-HIV/química , Fármacos Anti-HIV/isolamento & purificação , Vírus da Mieloblastose Aviária/enzimologia , DNA Polimerase Dirigida por DNA/efeitos dos fármacos , Diterpenos/química , Diterpenos/isolamento & purificação , Transcriptase Reversa do HIV/genética , Vírus da Leucemia Murina de Moloney/enzimologia , DNA Polimerase Dirigida por RNA/efeitos dos fármacos , Proteínas Recombinantes/efeitos dos fármacos , Inibidores da Transcriptase Reversa/química , Inibidores da Transcriptase Reversa/isolamento & purificação , Proteínas Virais/efeitos dos fármacosRESUMO
At low concentrations, dimethyl sulfoxide (DMSO) stimulated the avian myeloblastosis virus reverse transcriptase activity. About 40% stimulation was obtained in the presence of 5% (v/v) DMSO, using activated DNA and polyriboadenylic acid (poly(rA)) as templates, and Mg2+ as divalent cation. A similar stimulation by DMSO was observed with Mn2+ for the poly(rA)-dependent reverse transcriptase activity. DMSO at concentrations higher than 15% inhibited the reverse transcriptase reactions, independent of the template-primers used. An exception was detected with the 2'-fluoro analog of poly(rA) as template, where an activation of 100% was found in the presence of 20% DMSO. The stimulation caused by DMSO could be due to a reduction of the apparent Km value for poly(rA) from 9.1 to 3.3 micrograms/ml.
Assuntos
Vírus da Mieloblastose Aviária/enzimologia , Dimetil Sulfóxido/farmacologia , DNA Polimerase Dirigida por RNA/efeitos dos fármacos , DNA Polimerase Dirigida por RNA/metabolismoRESUMO
Atlow concentrations, dimethyl sulfoxide (DMSO), stimulated the avian myeloblastosis virus reverse transcriptase activity. About 40 percent stimulation was obtained in the presence of 5 percent (v/v) DMSO, using activated DNA and polyriboadenylic acid (poly(rA)) as templates, and Mg2+ as divalent cation. A similar stimulation by DMSO was observed with Mn2+ for the poly(rA)-dependent reverse transcriptase activity. DMSO at concentrations higher than 15 percent inhibited the reverse transcriptase reactions, independent of the template-primers used. An exception was detected with the 2'-fluoro analog of poly(rA) as template, where an activation of 100 percent was found in the presence of 20 percent DMSO. The stimulation caused by DMSO could be due to a reduction of the apparent Km value for poly(rA) from 9.1 to 3.3 µg/ml
Assuntos
Animais , Dimetil Sulfóxido/farmacologia , DNA Polimerase Dirigida por RNA/metabolismo , Vírus da Mieloblastose Aviária/enzimologiaRESUMO
Several homologous polynucleotides have been tested as inhibitors on the reactions catalyzed by avian myeloblastosis virus (AMV) reverse transcriptase, in the presence of polyribonucleotides and 2'-fluorinated polynucleotides as templates. Polynucleotides differentially inhibited the reactions catalyzed by reverse transcriptase in the presence of these synthetic templates. Polyriboadenylic acid (poly(rA), poly(2'-O-methyladenylic acid) (poly(Am)), poly(2'-fluoro-2'-deoxyadenylic acid) (poly(dAfl), polyinosinic acid (poly(rI)) and polyuridylic acid poly(rU)) inhibited the polyribonucleotide-, but not the 2'-fluorinated polynucleotide-directed reverse transcriptase activity.
Assuntos
DNA/biossíntese , Polidesoxirribonucleotídeos/metabolismo , Polinucleotídeos/farmacologia , Polirribonucleotídeos/metabolismo , DNA Polimerase Dirigida por RNA/metabolismo , Vírus da Mieloblastose Aviária/enzimologia , Relação Dose-Resposta a Droga , Flúor/farmacologia , Manganês/farmacologia , Poli A/farmacologia , Poli I/farmacologia , Poli U/farmacologia , Inibidores da Transcriptase Reversa , Moldes GenéticosRESUMO
Extracts from higher plants were evaluated for inhibitory activity against avian myeloblastosis virus reverse transcriptase. The assay may serve as a useful prescreen for potential anti-HIV bioactives.
Assuntos
Antivirais/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Extratos Vegetais/farmacologia , Inibidores da Transcriptase Reversa , Vírus da Mieloblastose Aviária/enzimologia , HIV/efeitos dos fármacos , Porto RicoRESUMO
Extracts from higher plants were evaluated for inhibitory activity against avian myeloblastosis virus reverse transcriptase. The assay may serve as a useful prescreen for potential anti-HIV bioactives
Assuntos
Antivirais/farmacologia , Extratos Vegetais/farmacologia , DNA Polimerase Dirigida por RNA/antagonistas & inibidores , Avaliação Pré-Clínica de Medicamentos/métodos , HIV/efeitos dos fármacos , Porto Rico , Vírus da Mieloblastose Aviária/enzimologiaRESUMO
Nalidixic acid, a very specific inhibitor of bacterial DNA synthesis, has been studied for its action on the avian myeloblastosis virus reverse transcriptase activity. The drug inhibited the DNA synthesis reaction catalyzed by the viral enzyme in the presence of different template-primers. The inhibitory effect by nalidixic acid was higher with polyriboadenylic acid than with polyribocytidylic acid as a synthetic template. With activated DNA as a template nalidixic acid preferentially inhibited the TMP incorporation when compared with the dAMP incorporation. Both these results showed the importance of the presence of adenine in the templates for a more efficient inhibition by nalidixic acid. The inhibition for this drug was also shown in the presence of Mn2+ instead of Mg2+ as the divalent cation, and with a 2'-fluorinated analogue of polyriboadenylic acid as the template. Kinetic data showed a non-competitive inhibition by nalidixic acid in relation to polyriboadenylic acid and to TTP in the reaction catalyzed by reverse transcriptase.
Assuntos
Vírus da Mieloblastose Aviária/efeitos dos fármacos , Ácido Nalidíxico/farmacologia , Inibidores da Transcriptase Reversa , Vírus da Mieloblastose Aviária/enzimologia , Vírus da Mieloblastose Aviária/genética , DNA Viral/biossíntese , Cinética , Polinucleotídeos/síntese química , Polinucleotídeos/metabolismo , Moldes GenéticosRESUMO
The polyamine spermine can not replace Mg2+ in the reactions catalyzed by avian myeloblastosis virus reverse transcriptase. In the presence of suboptimal Mg2+ concentrations, spermine enhanced the reverse transcriptase activities with viral 70 S RNA and synthetic polyriboadenylic acid as templates, about 4-fold and 2-fold, respectively. A similar effect occurred in the polyriboadenylic acid-directed reaction using Mn2+ as divalent cation. On the other hand, no stimulation by spermine was observed with activated DNA and polydeoxyadenylic acid as template in the reactions catalyzed by reverse transcriptase.
Assuntos
Vírus da Leucose Aviária/enzimologia , Vírus da Mieloblastose Aviária/enzimologia , DNA Polimerase Dirigida por RNA/metabolismo , Espermina/farmacologia , Vírus da Mieloblastose Aviária/genética , Cátions Bivalentes , DNA/biossíntese , Magnésio/farmacologia , Poli dA-dT/metabolismo , RNA Viral/metabolismo , Moldes GenéticosRESUMO
Poly (2'-O-methylcytidylic acid) is recognized as a template in reactions catalyzed by RNA-dependent DNA polymerases in the presence of Mn2+ as divalent cation. We report that kinetic data obtained for dGTP and template under optimal experimental conditions in the reaction catalyzed by reverse transcriptase showed some similarities between the poly (2'-O-methylcytidylic acid)Mn2+ and polyribocytidylic acid/Mg2+ systems. The reaction was inhibited by the action of N-ethylmaleimide and novobiocin, and to a lesser extent by ethidium bromide and tetramethyl ethidium bromide
Assuntos
Vírus da Mieloblastose Aviária/enzimologia , Guanosina Trifosfato/metabolismo , Poli C/metabolismo , DNA Polimerase Dirigida por RNA/metabolismo , Sítios de Ligação , Magnésio/metabolismo , Manganês/metabolismoRESUMO
Poly (2'-O-methylcytidylic acid) is recognized as a template in reactions catalyzed by RNA-dependent DNA polymerases in the presence of Mn2+ as divalent cation. We report that kinetic data obtained for dGTP and template under optimal experimental conditions in the reaction catalyzed by reverse transcriptase showed some similarities between the poly (2'-O-methylcytidylic acid)/Mn2+ and polyribocytidylic acid/Mg2+ systems. The reaction was inhibited by the action of N-ethylmaleimide and novobiocin, and to a lesser extent by ethidium bromide and tetramethyl ethidium bromide.
Assuntos
Vírus da Leucose Aviária/enzimologia , Vírus da Mieloblastose Aviária/enzimologia , Guanosina Trifosfato/metabolismo , Poli C/farmacocinética , Polirribonucleotídeos/farmacocinética , DNA Polimerase Dirigida por RNA/metabolismo , Sítios de Ligação , Magnésio/metabolismo , Manganês/metabolismoRESUMO
Poly (2'-O-methyladenylic acid), poly (Am), is recognized as template in the reaction catalyzed by avian myeloblastosis virus, AMV, reverse transcriptase better in the presence of Mn than Mg as divalent cation. An apparent KM value of 2.5 x 10 M in relation to TTP and an inhibition of the reaction at poly (Am concentration higher than 5 microng/ml have been observed. Ethidium bromide, EtBr, tetramethyl ethidium bromide, TMEtBr, dideoxy TTP, and novobiocin are inhibitors of the poly (Am)-directed reverse transcriptase reaction in the presence of Mn