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1.
Antimicrob Agents Chemother ; 58(8): 4431-42, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24867983

RESUMO

The hepatitis C virus (HCV) nonstructural 5A (NS5A) protein is a clinically validated target for drugs designed to treat chronic HCV infection. This study evaluated the in vitro activity, selectivity, and resistance profile of a novel anti-HCV compound, samatasvir (IDX719), alone and in combination with other antiviral agents. Samatasvir was effective and selective against infectious HCV and replicons, with 50% effective concentrations (EC50s) falling within a tight range of 2 to 24 pM in genotype 1 through 5 replicons and with a 10-fold EC50 shift in the presence of 40% human serum in the genotype 1b replicon. The EC90/EC50 ratio was low (2.6). A 50% cytotoxic concentration (CC50) of >100 µM provided a selectivity index of >5 × 10(7). Resistance selection experiments (with genotype 1a replicons) and testing against replicons bearing site-directed mutations (with genotype 1a and 1b replicons) identified NS5A amino acids 28, 30, 31, 32, and 93 as potential resistance loci, suggesting that samatasvir affects NS5A function. Samatasvir demonstrated an overall additive effect when combined with interferon alfa (IFN-α), ribavirin, representative HCV protease, and nonnucleoside polymerase inhibitors or the nucleotide prodrug IDX184. Samatasvir retained full activity in the presence of HIV and hepatitis B virus (HBV) antivirals and was not cross-resistant with HCV protease, nucleotide, and nonnucleoside polymerase inhibitor classes. Thus, samatasvir is a selective low-picomolar inhibitor of HCV replication in vitro and is a promising candidate for future combination therapies with other direct-acting antiviral drugs in HCV-infected patients.


Assuntos
Antivirais/farmacologia , Benzimidazóis/farmacologia , Carbamatos/farmacologia , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Hepacivirus/efeitos dos fármacos , Proteínas não Estruturais Virais/antagonistas & inibidores , Replicação Viral/efeitos dos fármacos , Linhagem Celular , Relação Dose-Resposta a Droga , Farmacorresistência Viral/efeitos dos fármacos , Farmacorresistência Viral/genética , Sinergismo Farmacológico , Genótipo , Guanosina Monofosfato/análogos & derivados , Guanosina Monofosfato/farmacologia , Células Hep G2 , Hepacivirus/genética , Hepacivirus/crescimento & desenvolvimento , Humanos , Interferon-alfa/farmacologia , Mutação , Replicon , Ribavirina/farmacologia , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo
2.
Artigo em Inglês | MEDLINE | ID: mdl-16248033

RESUMO

In order to improve the oral bioavailability of 2-C-methylcytidine, a potent anti-HCV agent, the corresponding 3'-O-L-valinyl ester derivative (NM 283) has been synthesized Based on its ease of synthesis and its physicochemical properties, NM 283 has emerged as a promising antiviral drug for treatment of chronic HCV infection.


Assuntos
Antivirais/farmacologia , Química Farmacêutica/métodos , Desoxicitidina/química , Desoxicitidina/farmacologia , Hepacivirus/genética , Hepatite C/tratamento farmacológico , Hepatite C/virologia , Pró-Fármacos/farmacologia , Nucleosídeos de Pirimidina/síntese química , Nucleosídeos de Pirimidina/farmacologia , Animais , Antivirais/síntese química , Desenho de Fármacos , Humanos , Modelos Químicos , Pró-Fármacos/química
3.
Artigo em Inglês | MEDLINE | ID: mdl-14565259

RESUMO

In order to evaluate their antiviral properties, a series of 4'-C-methyl-beta-D-ribofuranosyl purine and pyrimidine nucleosides has been prepared. Unfortunately, none of these 4'-branched nucleosides showed any antiviral activity or cytotoxcity when tested against HIV, HBV, and Yellow Fever virus.


Assuntos
Nucleosídeos de Purina/síntese química , Nucleosídeos de Pirimidina/síntese química , Antivirais/síntese química , Desenho de Fármacos , Indicadores e Reagentes , Metilação , Nucleosídeos de Purina/química , Nucleosídeos de Pirimidina/química , Ribose
4.
Artigo em Inglês | MEDLINE | ID: mdl-14565331

RESUMO

In order to improve the oral bioavailability of LdC, valinyl esters were prepared as prodrugs. We report here the syntheses of the 3'-mono-, 5'-mono, and 3',5'-di-O-valinyl esters of LdC. The comparison of their ease of synthesis, their physicochemical properties, as well as their pharmacokinetic parameters in cynomologus monkeys has revealed 3'-mono-O-valinyl derivative as the most promising of the studied prodrugs. This compound is being developed as a new anti-HBV agent.


Assuntos
Antivirais/síntese química , Desoxicitidina/análogos & derivados , Desoxicitidina/síntese química , Desoxicitidina/farmacologia , Vírus da Hepatite B/efeitos dos fármacos , Pró-Fármacos/síntese química , Administração Oral , Animais , Antivirais/química , Antivirais/farmacocinética , Antivirais/farmacologia , Disponibilidade Biológica , Desoxicitidina/farmacocinética , Humanos , Macaca fascicularis , Conformação Molecular , Estrutura Molecular , Pró-Fármacos/química , Pró-Fármacos/farmacocinética , Pró-Fármacos/farmacologia
5.
Antimicrob Agents Chemother ; 46(6): 1728-33, 2002 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-12019082

RESUMO

beta-L-Thymidine (L-dT) and beta-L-2'-deoxycytidine (L-dC) are potent and highly specific inhibitors of hepatitis B virus (HBV) replication both in vivo and in vitro (50% effective concentrations, 0.19 to 0.24 microM in 2.2.15 cells). The intracellular metabolisms of L-dT and L-dC were investigated in HepG2 cells and primary cultured human hepatocytes. L-dT and L-dC were extensively phosphorylated in both cell types, with the 5'-triphosphate derivative being the predominant metabolite. In HepG2 cells, the 5'-triphosphate levels were 27.7 +/- 12.1 and 72.4 +/- 1.8 pmol/10(6) cells for L-dT and L-dC, respectively. In primary human hepatocytes, the 5'-triphosphate levels were 16.5 +/- 9.8 and 90.1 +/- 36.4 pmol/10(6) cells for L-dT and L-dC, respectively. Furthermore, a choline derivative of L-dCDP was detected at concentrations of 15.8 +/- 1.8 and 25.6 +/- 0.1 pmol/10(6) cells in human hepatocytes and HepG2 cells, respectively. In HepG2 cells exposed to L-dC, the 5'-monophosphate and 5'-triphosphate derivatives of beta-L-2'-deoxyuridine (L-dUMP and L-dUTP, respectively) were also observed, reaching intracellular concentrations of 6.7 +/- 0.4 and 18.2 +/- 1.0 pmol/10(6) cells, respectively. In human hepatocytes, L-dUMP and L-dUTP were detected at concentrations of 5.7 +/- 2.4 and 43.5 +/- 26.8 pmol/10(6) cells, respectively. It is likely that deamination of L-dCMP by deoxycytidylate deaminase leads to the formation of L-dUMP, as the parent compound, L-dC, was not a substrate for deoxycytidine deaminase. The intracellular half-lives of L-dTTP, L-dCTP, and L-dUTP were at least 15 h, with intracellular concentrations of each metabolite remaining above their respective 50% inhibitory concentrations for the woodchuck hepatitis virus DNA polymerase for as long as 24 h after removal of the drug from cell cultures. Exposure of HepG2 cells to L-dT in combination with L-dC led to concentrations of the activated metabolites similar to those achieved with either agent alone. These results suggest that the potent anti-HBV activities of L-dT and L-dC are associated with their extensive phosphorylation.


Assuntos
Antivirais/farmacologia , Carcinoma Hepatocelular/metabolismo , Desoxicitidina/farmacologia , Vírus da Hepatite B/efeitos dos fármacos , Hepatite B/tratamento farmacológico , Hepatócitos/efeitos dos fármacos , Neoplasias Hepáticas/metabolismo , Timidina/farmacologia , Cromatografia Líquida de Alta Pressão , Desoxicitidina/metabolismo , Meia-Vida , Hepatite B/virologia , Humanos , Fosforilação , Timidina/metabolismo , Células Tumorais Cultivadas
6.
Antivir Chem Chemother ; 12 Suppl 1: 119-29, 2001.
Artigo em Inglês | MEDLINE | ID: mdl-11594678

RESUMO

Three simple, related nucleosides, beta-L-2'-deoxycytidine (LdC), beta-Lthymidine (LdT), and beta-L-2'-deoxyadenosine (LdA), have been discovered to be potent, specific and selective inhibitors of the replication hepatitis B virus (HBV), as well as the closely related duck and woodchuck hepatitis viruses (WHV). Structure-activity relationship analysis indicates that the 3'-OH group of the beta-L-2'-deoxyribose of the beta-L-2'-deoxynucleoside confers specific anti-hepadnavirus activity. The simple nucleosides had no effect on the replication of 15 other RNA and DNA viruses, and did not inhibit human DNA polymerases (alpha, beta and gamma) or compromise mitochondrial function. The nucleosides are efficiently converted intracellularly into active triphosphate metabolites that have a long half-life. Once-daily oral administration of these compounds in the woodchuck efficacy model of chronic HBV infection reduced viral load by as much as 10(8) genome equivalents/ml serum and there was no drug-related toxicity. In addition, a decline in WHV surface antigen (WHsAg) paralleled the decrease in viral load. This class of nucleosides displays an excellent overall safety profile. The first compound, LdT, has already entered clinical trials and LdC, currently being developed as a prodrug, is expected to enter the clinic in the near future. These compounds have the potential for use in combination therapy with the goal of achieving superior viral suppression and diminishing the onset of resistance.


Assuntos
Antivirais/uso terapêutico , Hepatite B/tratamento farmacológico , Nucleosídeos/uso terapêutico , Animais , Antivirais/farmacocinética , Modelos Animais de Doenças , Humanos , Testes de Sensibilidade Microbiana , Nucleosídeos/farmacocinética
7.
Nucleosides Nucleotides Nucleic Acids ; 20(4-7): 597-607, 2001.
Artigo em Inglês | MEDLINE | ID: mdl-11563077

RESUMO

A unique series of simple unnatural L-nucleosides that specifically inhibit hepatitis B virus (HBV) replication has been discovered. These molecules have in common a hydroxyl group in the 3'-position (3'-OH) of the beta-L-2'-deoxyribose sugar that confers antiviral activity specifically against hepadnaviruses. Replacement of the 3'-OH broadens activity to other viruses. Substitution in the base decreases antiviral potency and selectivity. Human DNA polymerases and mitochondrial function are not effected. Plasma viremia is reduced up to 8 logs in a woodchuck model of chronic HBV infection. These investigational drugs, used alone or in combination, are expected to offer new therapeutic options for patients with chronic HBV infection.


Assuntos
Antivirais/farmacologia , Desoxirribonucleosídeos/farmacologia , Vírus da Hepatite B/efeitos dos fármacos , Animais , Antivirais/química , Desoxiadenosinas/química , Desoxiadenosinas/farmacologia , Desoxicitidina/química , Desoxicitidina/farmacologia , Desoxirribonucleosídeos/química , Vírus da Hepatite B da Marmota/efeitos dos fármacos , Vírus da Hepatite B da Marmota/fisiologia , Vírus da Hepatite B/fisiologia , Hepatite B Crônica/tratamento farmacológico , Humanos , Relação Estrutura-Atividade , Especificidade por Substrato , Timidina/química , Timidina/farmacologia , Replicação Viral/efeitos dos fármacos
8.
Antivir Chem Chemother ; 12(2): 99-108, 2001 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-11527047

RESUMO

The beta-L-nucleoside analogue beta-L-2',3'-dideoxy adenosine (beta-L-ddA) has been shown to exhibit limited antiviral activities. This was attributed to its rapid catabolism through cleavage of the glycosidic bond and poor phosphorylation to the nucleotide beta-L-2',3'-dideoxyadenosine-5'-mono phosphate (beta-L-ddAMP) (Placidi et al., 2000). However, the nucleotide beta-L-2',3'-dideoxyadenosine-5'-triphosphate (beta-L-ddATP) inhibited the activity of both HIV-1 reverse transcriptase (RT) and viral DNA polymerase isolated from woodchuck hepatitis virus-infected serum (a model of hepatitis B) with an inhibitory concentration (IC50) of 2.0 microM without inhibiting human DNA polymerases alpha, beta, or gamma up to a concentration of 100 microM. These results suggested that prodrugs of beta-L-ddAMP may bypass the poor metabolic activation of beta-L-ddA and lead to more potent and selective antiviral activity. Therefore, the mononucleoside phosphotriester derivative of beta-L-ddAMP incorporating the S-pivaloyl-2-thioethyl (tButylSATE) groups, beta-L-ddAMP-bis(tButylSATE) was synthesized. Beta-L-ddAMP-bis(tButylSATE) inhibited HIV replication in human peripheral blood mononuclear cells (PBMCs) and HBV replication in 2.2.15 cells with effective concentrations (EC50s) of 2 and 80 nM, respectively. Intracellular metabolism of beta-L-ddAMP-bis(tButylSATE) demonstrated that beta-L-ddATP was the predominant intracellular metabolite in PBMC and liver cells. The intracellular half-life of beta-L-ddATP was 5.4 and 9.2 h in HepG2 and PBMCs, respectively. The intracellular concentrations of beta-L-ddATP were maintained above the EC50 for the inhibition of HIV RT and hepatitis B virus (HBV) for as long as 24 h after removal of the drug.


Assuntos
Antivirais/metabolismo , Antivirais/farmacologia , Didesoxiadenosina/farmacologia , HIV/efeitos dos fármacos , Vírus da Hepatite B/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Animais , Fármacos Anti-HIV/metabolismo , Fármacos Anti-HIV/farmacologia , Cromatografia Líquida de Alta Pressão , DNA Polimerase Dirigida por DNA/metabolismo , Didesoxiadenosina/análogos & derivados , Didesoxiadenosina/metabolismo , Didesoxinucleotídeos , HIV/enzimologia , HIV/fisiologia , Meia-Vida , Células-Tronco Hematopoéticas/efeitos dos fármacos , Vírus da Hepatite B/enzimologia , Vírus da Hepatite B/fisiologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Concentração Inibidora 50 , Lamivudina/farmacologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/virologia , Marmota/sangue , Marmota/virologia , Inibidores da Síntese de Ácido Nucleico , DNA Polimerase Dirigida por RNA/metabolismo , Inibidores da Transcriptase Reversa/farmacologia , Células Tumorais Cultivadas
9.
Antimicrob Agents Chemother ; 45(1): 229-35, 2001 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-11120971

RESUMO

A unique series of simple "unnatural" nucleosides has been discovered to inhibit hepatitis B virus (HBV) replication. Through structure-activity analysis it was found that the 3'-OH group of the beta-L-2'-deoxyribose of the beta-L-2'-deoxynucleoside confers specific antihepadnavirus activity. The unsubstituted nucleosides beta-L-2'-deoxycytidine, beta-L-thymidine, and beta-L-2'-deoxyadenosine had the most potent, selective, and specific antiviral activity against HBV replication. Human DNA polymerases (alpha, beta, and gamma) and mitochondrial function were not affected. In the woodchuck model of chronic HBV infection, viral load was reduced by as much as 10(8) genome equivalents/ml of serum and there was no drug-related toxicity. In addition, the decline in woodchuck hepatitis virus surface antigen paralleled the decrease in viral load. These investigational drugs, used alone or in combination, are expected to offer new therapeutic options for patients with chronic HBV infection.


Assuntos
Antivirais/farmacologia , Vírus da Hepatite B/efeitos dos fármacos , Hepatite B/tratamento farmacológico , Nucleosídeos/farmacologia , Animais , Fármacos Anti-HIV/farmacologia , Antivirais/uso terapêutico , Células da Medula Óssea/efeitos dos fármacos , Linhagem Celular , DNA Viral/biossíntese , DNA Polimerase Dirigida por DNA/metabolismo , Desoxiadenosinas/farmacologia , Desoxiadenosinas/uso terapêutico , Desoxicitidina/farmacologia , Desoxicitidina/uso terapêutico , Feminino , HIV-1/efeitos dos fármacos , Hepatite B/virologia , Humanos , Masculino , Marmota , Nucleosídeos/uso terapêutico , Células-Tronco/efeitos dos fármacos , Timidina/farmacologia , Timidina/uso terapêutico , Replicação Viral/efeitos dos fármacos
10.
J Med Chem ; 43(21): 3906-12, 2000 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-11052795

RESUMO

As dioxolane and oxathiolane nucleosides have exhibited promising antiviral and anticancer activities, it was of interest to synthesize isoelectronically substituted oxaselenolane nucleosides, in which the 3'-CH(2) is replaced by a selenium atom. To study structure-activity relationships, various pyrimidine and purine oxaselenolane nucleosides were synthesized from the key intermediate, (+/-)-2-benzoyloxymethyl-1,2-oxaselenolane 5-acetate (6). Among the synthesized racemic nucleosides, cytosine and 5-fluorocytosine analogues exhibited potent anti-HIV and anti-HBV activities. It was of interest to obtain the enantiomerically pure isomers to determine if they have differential antiviral activities. However, due to the difficult and time-consuming nature of enantiomeric synthesis, a chiral HPLC separation was performed to obtain optical isomers from the corresponding racemic mixtures. Each pair of enantiomers of Se-ddC and Se-FddC was separated by an amylose chiral column using a mobile phase of 100% 2-propanol. The results indicate that most of the anti-HIV activity of both cytosine and fluorocytosine nucleosides resides with the (-)-isomers.


Assuntos
Antivirais/síntese química , Citosina/análogos & derivados , Citosina/síntese química , Nucleosídeos/síntese química , Compostos Organometálicos/síntese química , Selênio , Fármacos Anti-HIV/síntese química , Fármacos Anti-HIV/química , Fármacos Anti-HIV/farmacologia , Antivirais/química , Antivirais/farmacologia , Linhagem Celular , Cromatografia Líquida de Alta Pressão , Citosina/química , Citosina/farmacologia , HIV-1/efeitos dos fármacos , Vírus da Hepatite B/efeitos dos fármacos , Humanos , Nucleosídeos/química , Nucleosídeos/farmacologia , Compostos Organometálicos/química , Compostos Organometálicos/farmacologia , Estereoisomerismo , Relação Estrutura-Atividade
11.
Artigo em Inglês | MEDLINE | ID: mdl-10772713

RESUMO

Cyclopropyl carbocyclic nucleosides have been synthesized from the key intermediate 2 which was converted to the mesylated cyclopropyl methyl alcohol 3. Condensation of compound 3 with various purine and pyrimidine bases gave the desired nucleosides. All synthesized nucleosides were evaluated for antiviral activity and cellular toxicity. Among them adenine 22 and guanine 23 derivatives showed moderate antiviral activity against HIV-1 and HBV. None of the other compounds showed any significant antiviral activities against HIV-1, HBV, HSV-1 and HSV-2 in vitro up to 100 microM.


Assuntos
Antivirais/síntese química , Ciclopropanos/síntese química , Nucleosídeos/síntese química , Fármacos Anti-HIV/síntese química , Fármacos Anti-HIV/química , Fármacos Anti-HIV/farmacologia , Antivirais/química , Antivirais/farmacologia , Ciclopropanos/química , Ciclopropanos/farmacologia , HIV-1/efeitos dos fármacos , Vírus da Hepatite B/efeitos dos fármacos , Herpesvirus Humano 1/efeitos dos fármacos , Herpesvirus Humano 2/efeitos dos fármacos , Humanos , Nucleosídeos/química , Nucleosídeos/farmacologia , Estereoisomerismo , Relação Estrutura-Atividade
12.
Antiviral Res ; 45(3): 169-83, 2000 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-10771081

RESUMO

The synthesis and in vitro anti human immunodeficiency virus (HIV) and anti-hepatitis B virus (HBV) activities of some unnatural beta-L-nucleoside enantiomers related to the anti-HIV compound 2', 3'-dideoxy-3'-fluoro-5-chlorouridine (beta-D-3'Fdd5ClU) are reported. In contrast to beta-D-3'Fdd5ClU, beta-L-3'Fdd5ClU and the other L-congeners were devoid of significant anti-HIV effects, but beta-L-2',3'-dideoxy-5-chlorocytidine (beta-L-dd5ClC) and beta-L-2', 3'-dideoxy-3'-fluoro-cytidine (beta-L-3'FddC) showed a distinct anti-HBV activity. Three mononucleoside phosphotriester derivatives with S-pivaloyl-2-thioethyl (t-BuSATE) groups as biolabile phosphate protective groups were also synthesized. The bis(t-BuSATE) derivative of beta-D-3'Fdd5ClU retained anti-HIV activity in thymidine kinase deficient (TK(-)) CEM cells.


Assuntos
Antivirais/farmacologia , Didesoxinucleosídeos/farmacologia , HIV/efeitos dos fármacos , Vírus da Hepatite B/efeitos dos fármacos , Antivirais/síntese química , Linhagem Celular , Didesoxinucleosídeos/síntese química , HIV-1/efeitos dos fármacos , HIV-2/efeitos dos fármacos , Humanos , Estrutura Molecular , Estereoisomerismo
13.
Antimicrob Agents Chemother ; 44(4): 853-8, 2000 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-10722481

RESUMO

The intracellular metabolism of the beta-L- enantiomer of 2', 3'-dideoxyadenosine (beta-L-ddA) was investigated in HepG2 cells, human peripheral blood mononuclear cells (PBMC), and primary cultured human hepatocytes in an effort to understand the metabolic basis of its limited activity on the replication of human immunodeficiency virus and hepatitis B virus. Incubation of cells with 10 microM [2',3',8-(3)H]-beta-L-ddA resulted in an increased intracellular concentration of beta-L-ddA with time, demonstrating that these cells were able to transport beta-L-ddA. However, it did not result in the phosphorylation of beta-L-ddA to its pharmacologically active 5'-triphosphate (beta-L-ddATP). Five other intracellular metabolites were detected and identified as beta-L-2', 3'-dideoxyribonolactone, hypoxanthine, inosine, ADP, and ATP, with the last being the predominant metabolite, reaching levels as high as 5.14 +/- 0.95, 8.15 +/- 2.64, and 15.60 +/- 1.74 pmol/10(6) cells at 8, 4, and 2 h in HepG2 cells, PBMC, and hepatocytes, respectively. In addition, a beta-glucuronic derivative of beta-L-ddA was detected in cultured hepatocytes, accounting for 12.5% of the total metabolite pool. Coincubation of hepatocytes in primary culture with beta-L-ddA in the presence of increasing concentrations of 5'-methylthioadenosine resulted in decreased phosphorolysis of beta-L-ddA and formation of associated metabolites. These results indicate that the limited antiviral activity of beta-L-ddA is the result of its inadequate phosphorylation to the nucleotide level due to phosphorolysis and catabolism of beta-L-ddA by methylthioadenosine phosphorylase (EC 2.4.2.28).


Assuntos
Antivirais/metabolismo , Didesoxiadenosina/metabolismo , Adenosina/análogos & derivados , Adenosina/farmacologia , Antivirais/farmacologia , Linhagem Celular , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Criopreservação , Didesoxiadenosina/farmacologia , HIV/efeitos dos fármacos , Vírus da Hepatite B/efeitos dos fármacos , Humanos , Fígado/citologia , Fígado/metabolismo , Monócitos/metabolismo , Tionucleosídeos/farmacologia
14.
J Biol Chem ; 274(49): 34735-41, 1999 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-10574941

RESUMO

beta-L-2',3'-Dideoxyadenosine-5'-triphosphate (beta-L-2', 3'-dd-5'-ATP) was prepared enzymatically from the corresponding monophosphate by the use of adenylate kinase, creatine phosphate, and creatine kinase in a single step. The beta-(32)P-labeled analog was prepared similarly, but in a two step reaction. beta-L-2', 3'-dd-5'-ATP inhibited adenylyl cyclase from rat brain competitively with respect to substrate (5'-ATP.Mn(2+)) and exhibited an IC(50) approximately 24 nM. The labeled ligand was used in the development of a reversible binding assay for adenylyl cyclases. Binding of beta-L-2',3'-dd-[beta-(32)P]5'-ATP was saturable with increasing concentrations of ligand and increased in proportion to membrane protein, and was enhanced by Mn(2+) to a greater extent than by Mg(2+). Binding was displaced with adenine nucleotides known to be either competitive or noncompetitive inhibitors but not by agents known not to act on the cyclase, or by 3-isobutyl-1-methylxanthine, creatine phosphate, or creatine kinase. Binding was rapid, with a half-time for the on-rate <1.8 min and for the off-rate <0.8 min. The potency and mechanism of the inhibition of this ligand and the pattern of agents that displace binding suggest an interaction with adenylyl cyclase per se and to a configuration of the enzyme consistent with an interaction at the catalytic active site. The data suggest that this is a pretransition state inhibitor and contrasts with the equipotent 2',5'-dd-3'ATP, a post-transition state noncompetitive inhibitor.


Assuntos
Trifosfato de Adenosina/síntese química , Trifosfato de Adenosina/farmacologia , Inibidores de Adenilil Ciclases , Trifosfato de Adenosina/metabolismo , Adenilil Ciclases/metabolismo , Animais , Encéfalo/enzimologia , Catálise , Cromatografia Líquida de Alta Pressão , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Concentração Inibidora 50 , Isomerismo , Cinética , Radioisótopos de Fósforo , Ligação Proteica , Ratos
16.
Nucleosides Nucleotides ; 18(4-5): 987-8, 1999.
Artigo em Inglês | MEDLINE | ID: mdl-10432726

RESUMO

beta-L-ddAMP-bis(tbutylSATE) is a potent inhibitor of HBV replication with an EC50 = 0.1 microM. Following a 0- to 72-hrs exposure of human hepatocytes to a 10 microM [2',3'-3H] beta-L-ddAMP-bis(tbutylSATE), the pharmacologically active beta-L-ddATP was the predominant metabolite attaining a concentration of 268.53 +/- 107.97 pmoles/10(6) cells at 2 hrs. In Hep-G2 cell, beta-L-ddATP accounted for 146.8 +/- 29.8 pmoles/10(6) cells at 2 hrs with an half life of approximately 5.4 hrs. This study reveals that extensive intracellular concentrations of beta-L-ddATP after incubation of cells to the parent drug is accounting for its potent antiviral activity.


Assuntos
Antivirais/metabolismo , Nucleotídeos de Desoxiadenina/metabolismo , Vírus da Hepatite B/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Antivirais/farmacologia , Linhagem Celular , Nucleotídeos de Desoxiadenina/farmacologia , Meia-Vida , Vírus da Hepatite B/fisiologia , Humanos
17.
Nucleosides Nucleotides ; 18(4-5): 1005-6, 1999.
Artigo em Inglês | MEDLINE | ID: mdl-10432731

RESUMO

We have investigated the effects of several beta-D-ddA 5'-monophospate (beta-D-ddAMP), and their corresponding beta-L-enantiomers prodrugs against HBV replication. All ddAMP prodrugs inhibited HBV replication in a dose-dependent manner.


Assuntos
Antivirais/farmacologia , Nucleotídeos de Desoxiadenina/farmacologia , Vírus da Hepatite B/efeitos dos fármacos , Lamivudina/farmacologia , Linhagem Celular , Didesoxinucleotídeos , Vírus da Hepatite B/fisiologia , Testes de Sensibilidade Microbiana , Estereoisomerismo , Replicação Viral/efeitos dos fármacos
18.
J Med Chem ; 40(24): 3969-73, 1997 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-9397178

RESUMO

The beta-L-enantiomers of 2',3'-dideoxyadenosine and 2',3'-didehydro-2',3'-dideoxyadenosine have been stereospecifically synthesized. In an attempt to explain the previously reported antiviral activities of these compounds, their enzymatic properties were studied with respect to adenosine kinase, deoxycytidine kinase, adenosine deaminase, and purine nucleoside phosphorylase. Adenosine deaminase was strictly enantioselective and favored beta-D-ddA and beta-D-d4A, whereas adenosine kinase and purine nucleoside phosphorylase had no apparent substrate properties for the D- or L-enantiomers of beta-ddA or beta-d4A. Human deoxycytidine kinase showed a remarkable inversion of the expected enantioselectivity, with beta-L-ddA and beta-L-d4A having better substrate efficiencies than their corresponding beta-D-enantiomers. Our results demonstrate the potential of beta-L-adenosine analogues as antiviral agents and suggest that deoxycytidine kinase has a strategic importance in their cellular activation.


Assuntos
Fármacos Anti-HIV/metabolismo , Antivirais/metabolismo , Didesoxiadenosina/análogos & derivados , Didesoxiadenosina/metabolismo , Adenosina Desaminase/metabolismo , Adenosina Quinase/metabolismo , Animais , Fármacos Anti-HIV/síntese química , Fármacos Anti-HIV/farmacologia , Antivirais/síntese química , Antivirais/farmacologia , Bovinos , Desoxicitidina Quinase/metabolismo , Didesoxiadenosina/síntese química , Didesoxiadenosina/farmacologia , Estabilidade de Medicamentos , HIV/efeitos dos fármacos , Vírus da Hepatite B/efeitos dos fármacos , Humanos , Purina-Núcleosídeo Fosforilase/metabolismo , Estereoisomerismo , Relação Estrutura-Atividade
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