RESUMO
PSI-7977, a prodrug of 2'-F-2'-C-methyluridine monophosphate, is the purified diastereoisomer of PSI-7851 and is currently being investigated in phase 3 clinical trials for the treatment of hepatitis C. In this study, we profiled the activity of PSI-7977 and its ability to select for resistance using a number of different replicon cells. Results showed that PSI-7977 was active against genotype (GT) 1a, 1b, and 2a (strain JFH-1) replicons and chimeric replicons containing GT 2a (strain J6), 2b, and 3a NS5B polymerase. Cross-resistance studies using GT 1b replicons confirmed that the S282T change conferred resistance to PSI-7977. Subsequently, we evaluated the ability of PSI-7977 to select for resistance using GT 1a, 1b, and 2a (JFH-1) replicon cells. S282T was the common mutation selected among all three genotypes, but while it conferred resistance to PSI-7977 in GT 1a and 1b, JFH-1 GT 2a S282T showed only a very modest shift in 50% effective concentration (EC(50)) for PSI-7977. Sequence analysis of the JFH-1 NS5B region indicated that additional amino acid changes were selected both prior to and after the emergence of S282T. These include T179A, M289L, I293L, M434T, and H479P. Residues 179, 289, and 293 are located within the finger and palm domains, while 434 and 479 are located on the surface of the thumb domain. Data from the JFH-1 replicon variants showed that amino acid changes within the finger and palm domains together with S282T were required to confer resistance to PSI-7977, while the mutations on the thumb domain serve to enhance the replication capacity of the S282T replicons.
Assuntos
Antivirais/farmacologia , Hepacivirus/efeitos dos fármacos , Hepacivirus/genética , Uridina Monofosfato/análogos & derivados , Linhagem Celular , Genótipo , Humanos , Replicon/efeitos dos fármacos , Replicon/genética , Sofosbuvir , Uridina Monofosfato/farmacologia , Replicação Viral/efeitos dos fármacosRESUMO
BACKGROUND: Nucleoside reverse transcriptase inhibitors (NRTIs) are an effective class of agents that has played a vital role in the treatment of HIV infections. (-)-ß-D-(2R,4R)-dioxolane-thymine (DOT) is a thymidine analogue that is active against wild-type and NRTI-resistant HIV-1 mutants. It has been shown that the anti-HIV activity of DOT is limited due to poor monophosphorylation. METHODS: To further enhance the anti-HIV activity of DOT, an extensive structure-activity relationship analysis of phosphoramidate prodrugs of DOT monophosphate was undertaken. These prodrugs were evaluated for anti-HIV activity using Hela CD4 ß-gal reporter cells (P4-CCR5 luc cells). RESULTS: Among the synthesized prodrugs, the 4-bromophenyl benzyloxy l-alanyl phosphate derivative of DOT was the most potent, with a 50% effective concentration of 0.089 µM corresponding to a 75-fold increase in activity relative to the parent nucleoside DOT with no increased cytotoxicity. The metabolic stability of a selected number of potent DOT phosphoramidates was also evaluated in simulated gastric fluid, simulated intestinal fluid, human plasma and liver S9 fractions. CONCLUSIONS: A series of new phosphoramidate prodrugs of DOT were prepared and evaluated as inhibitors of HIV replication in vitro. Metabolic stability studies indicated that these DOT phosphoramidate derivatives have the potential to show acceptable stability in the gastrointestinal tract, but they metabolize rapidly in the liver.
Assuntos
Amidas/farmacologia , Fármacos Anti-HIV/farmacologia , Dioxolanos/farmacologia , Ácidos Fosfóricos/farmacologia , Pró-Fármacos/farmacologia , Timina/análogos & derivados , Cromatografia Líquida de Alta Pressão , Dioxolanos/química , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Espectrofotometria Ultravioleta , Timina/química , Timina/farmacologiaRESUMO
PSI-352938, a cyclic phosphate nucleotide, and PSI-353661, a phosphoramidate nucleotide, are prodrugs of ß-D-2'-deoxy-2'-α-fluoro-2'-ß-C-methylguanosine-5'-monophosphate. Both compounds are metabolized to the same active 5'-triphosphate, PSI-352666, which serves as an alternative substrate inhibitor of the NS5B RNA-dependent RNA polymerase during HCV replication. PSI-352938 and PSI-353661 retained full activity against replicons containing the S282T substitution, which confers resistance to certain 2'-substituted nucleoside/nucleotide analogs. PSI-352666 was also similarly active against both wild-type and S282T NS5B polymerases. In order to identify mutations that confer resistance to these compounds, in vitro selection studies were performed using HCV replicon cells. While no resistant genotype 1a or 1b replicons could be selected, cells containing genotype 2a JFH-1 replicons cultured in the presence of PSI-352938 or PSI-353661 developed resistance to both compounds. Sequencing of the NS5B region identified a number of amino acid changes, including S15G, R222Q, C223Y/H, L320I, and V321I. Phenotypic evaluation of these mutations indicated that single amino acid changes were not sufficient to significantly reduce the activity of PSI-352938 and PSI-353661. Instead, a combination of three amino acid changes, S15G/C223H/V321I, was required to confer a high level of resistance. No cross-resistance exists between the 2'-F-2'-C-methylguanosine prodrugs and other classes of HCV inhibitors, including 2'-modified nucleoside/-tide analogs such as PSI-6130, PSI-7977, INX-08189, and IDX-184. Finally, we determined that in genotype 1b replicons, the C223Y/H mutation failed to support replication, and although the A15G/C223H/V321I triple mutation did confer resistance to PSI-352938 and PSI-353661, this mutant replicated at only about 10% efficiency compared to the wild type.
Assuntos
Óxidos P-Cíclicos/farmacologia , Farmacorresistência Viral , Guanosina Monofosfato/análogos & derivados , Hepacivirus/efeitos dos fármacos , Hepacivirus/genética , Mutação/genética , Nucleosídeos/farmacologia , RNA Viral/genética , Replicon/efeitos dos fármacos , Antivirais/farmacologia , Guanosina Monofosfato/farmacologia , Hepatite C/tratamento farmacológico , Hepatite C/genética , Hepatite C/virologia , Humanos , Fenótipo , Pró-Fármacos/farmacologia , Conformação Proteica , RNA Polimerase Dependente de RNA/antagonistas & inibidores , RNA Polimerase Dependente de RNA/metabolismo , Replicon/genética , Replicação Viral/efeitos dos fármacos , Replicação Viral/genéticaRESUMO
PSI-353661, a phosphoramidate prodrug of 2'-deoxy-2'-fluoro-2'-C-methylguanosine-5'-monophosphate, is a highly active inhibitor of genotype 1a, 1b, and 2a HCV RNA replication in the replicon assay and of genotype 1a and 2a infectious virus replication. PSI-353661 is active against replicons harboring the NS5B S282T or S96T/N142T amino acid alterations that confer decreased susceptibility to nucleoside/tide analogs as well as mutations that confer resistance to non-nucleoside inhibitors of NS5B. Replicon clearance studies show that PSI-353661 was able to clear cells of HCV replicon RNA and prevent a rebound in replicon RNA. PSI-353661 showed no toxicity toward bone marrow stem cells or mitochondrial toxicity. The metabolism to the active 5'-triphosphate involves hydrolysis of the carboxyl ester by cathepsin A (Cat A) and carboxylesterase 1 (CES1) followed by a putative nucleophilic attack on the phosphorus by the carboxyl group resulting in the elimination of phenol and the alaninyl phosphate metabolite, PSI-353131. Histidine triad nucleotide-binding protein 1 (Hint 1) then removes the amino acid moiety, which is followed by hydrolysis of the methoxyl group at the O(6)-position of the guanine base by adenosine deaminase-like protein 1 (ADAL1) to give 2'-deoxy-2'-fluoro-2'-C-methylguanosine-5'-monophosphate. The monophosphate is phosphorylated to the diphosphate by guanylate kinase. Nucleoside diphosphate kinase is the primary enzyme involved in phosphorylation of the diphosphate to the active triphosphate, PSI-352666. PSI-352666 is equally active against wild-type NS5B and NS5B containing the S282T amino acid alteration.
Assuntos
Antivirais/farmacologia , Guanosina Monofosfato/análogos & derivados , Hepacivirus/efeitos dos fármacos , Pró-Fármacos/farmacologia , Replicação Viral/efeitos dos fármacos , Biotransformação , Catepsina A/metabolismo , Cromatografia Líquida de Alta Pressão , Clonagem Molecular , Avaliação Pré-Clínica de Medicamentos , Guanosina Monofosfato/antagonistas & inibidores , Guanosina Monofosfato/farmacologia , Guanilato Quinases/metabolismo , Células Hep G2 , Hepacivirus/genética , Hepacivirus/fisiologia , Hepatócitos/efeitos dos fármacos , Humanos , Ácido Láctico/metabolismo , Luciferases/metabolismo , Testes de Sensibilidade Microbiana , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mutação , Proteínas do Tecido Nervoso/metabolismo , Fenol/metabolismo , Fosforilação , Pró-Fármacos/química , Replicon , Proteínas não Estruturais Virais/antagonistas & inibidoresRESUMO
PSI-352938 is a novel cyclic phosphate prodrug of ß-D-2'-deoxy-2'-α-fluoro-2'-ß-C-methylguanosine 5'-monophosphate that has potent activity against hepatitis C virus (HCV) in vitro. The studies described here characterize the in vitro anti-HCV activity of PSI-352938, alone and in combination with other inhibitors of HCV, and the cross-resistance profile of PSI-352938. The effective concentration required to achieve 50% inhibition for PSI-352938, determined using genotype 1a-, 1b-, and 2a-derived replicons stably expressed in the Lunet cell line, were 0.20, 0.13, and 0.14 µM, respectively. The active 5'-triphosphate metabolite, PSI-352666, inhibited recombinant NS5B polymerase from genotypes 1 to 4 with comparable 50% inhibitory concentrations. In contrast, PSI-352938 did not inhibit the replication of hepatitis B virus or human immunodeficiency virus in vitro. PSI-352666 did not significantly affect the activity of human DNA and RNA polymerases. PSI-352938 and its cyclic phosphate metabolites did not affect the cyclic GMP-mediated activation of protein kinase G. Clearance studies using replicon cells demonstrated that PSI-352938 cleared cells of HCV replicon RNA and prevented replicon rebound. An additive to synergistic effect was observed when PSI-352938 was combined with other classes of HCV inhibitors, including alpha interferon, ribavirin, NS3/4A inhibitors, an NS5A inhibitor, and nucleoside/nucleotide and nonnucleoside inhibitors. Cross-resistance studies showed that PSI-352938 remained fully active against replicons containing the S282T or the S96T/N142T amino acid alteration. Replicons that contain mutations conferring resistance to various classes of nonnucleoside inhibitors also remained sensitive to inhibition by PSI-352938. PSI-352938 is currently being evaluated in a phase I clinical study in genotype 1-infected individuals.
Assuntos
Antivirais/farmacologia , Óxidos P-Cíclicos/farmacologia , Desoxiguanosina/análogos & derivados , Hepacivirus/efeitos dos fármacos , Nucleosídeos/farmacologia , Pró-Fármacos/farmacologia , RNA Viral/biossíntese , Replicon/efeitos dos fármacos , Desoxiguanosina/farmacologia , Farmacorresistência Viral , HumanosRESUMO
The hepatitis C virus (HCV) NS5B RNA polymerase facilitates the RNA synthesis step during the HCV replication cycle. Nucleoside analogs targeting the NS5B provide an attractive approach to treating HCV infections because of their high barrier to resistance and pan-genotype activity. PSI-7851, a pronucleotide of beta-D-2'-deoxy-2'-fluoro-2'-C-methyluridine-5'-monophosphate, is a highly active nucleotide analog inhibitor of HCV for which a phase 1b multiple ascending dose study of genotype 1-infected individuals was recently completed (M. Rodriguez-Torres, E. Lawitz, S. Flach, J. M. Denning, E. Albanis, W. T. Symonds, and M. M. Berry, Abstr. 60th Annu. Meet. Am. Assoc. Study Liver Dis., abstr. LB17, 2009). The studies described here characterize the in vitro antiviral activity and cytotoxicity profile of PSI-7851. The 50% effective concentration for PSI-7851 against the genotype 1b replicon was determined to be 0.075+/-0.050 microM (mean+/-standard deviation). PSI-7851 was similarly effective against replicons derived from genotypes 1a, 1b, and 2a and the genotype 1a and 2a infectious virus systems. The active triphosphate, PSI-7409, inhibited recombinant NS5B polymerases from genotypes 1 to 4 with comparable 50% inhibitory concentrations. PSI-7851 is a specific HCV inhibitor, as it lacks antiviral activity against other closely related and unrelated viruses. PSI-7409 also lacked any significant activity against cellular DNA and RNA polymerases. No cytotoxicity, mitochondrial toxicity, or bone marrow toxicity was associated with PSI-7851 at the highest concentration tested (100 microM). Cross-resistance studies using replicon mutants conferring resistance to modified nucleoside analogs showed that PSI-7851 was less active against the S282T replicon mutant, whereas cells expressing a replicon containing the S96T/N142T mutation remained fully susceptible to PSI-7851. Clearance studies using replicon cells demonstrated that PSI-7851 was able to clear cells of HCV replicon RNA and prevent viral rebound.
Assuntos
Antivirais/farmacologia , Nucleotídeos de Desoxiuracil/farmacologia , Inibidores Enzimáticos/farmacologia , Hepacivirus/efeitos dos fármacos , Pró-Fármacos/farmacologia , Replicação Viral/efeitos dos fármacos , Amidas/química , Amidas/farmacologia , Antivirais/química , Linhagem Celular Tumoral , Nucleotídeos de Desoxiuracil/química , Inibidores Enzimáticos/química , Genótipo , Hepacivirus/classificação , Hepacivirus/enzimologia , Humanos , Ácidos Fosfóricos/química , Ácidos Fosfóricos/farmacologia , Pró-Fármacos/química , RNA Viral/genética , RNA Viral/metabolismo , RNA Polimerase Dependente de RNA/antagonistas & inibidores , Replicon/efeitos dos fármacos , Proteínas não Estruturais Virais/antagonistas & inibidoresRESUMO
APOBEC3G and APOBEC3F exhibit antiretroviral activity primarily as a consequence of their ability to deaminate cytidines in retroviral DNA. Here, we compare the properties of APOBEC3F and APOBEC3G from human, macaque, and African green monkey (AGM). While all APOBEC proteins tested exhibited anti-HIV-1 activity, human APOBEC3F was, surprisingly, 10- to 50-fold less potent than human APOBEC3G. However, similar discrepancies in antiviral potency were not found when pairs of proteins from macaque and AGM were compared. Intrinsic differences in the ability of each APOBEC protein to induce hypermutation, rather than differences in packaging efficiency, partially accounted for variable antiretroviral activity. Each of four primate lentivirus Vif proteins reduced human and AGM APOBEC3F expression and antiviral activity, but all were only partially effective and species-specific effects were relatively minor. Overall, highly efficient and species-specific neutralization of APOBEC3G, and less efficient neutralization of APOBEC3F, appears to be a general property of Vif proteins.
Assuntos
Fármacos Anti-HIV , Citidina Desaminase/fisiologia , Desaminase APOBEC-3G , Sequência de Aminoácidos , Animais , Fármacos Anti-HIV/antagonistas & inibidores , Chlorocebus aethiops , Citidina Desaminase/antagonistas & inibidores , Citosina Desaminase/antagonistas & inibidores , Citosina Desaminase/química , Citosina Desaminase/fisiologia , Regulação da Expressão Gênica , Produtos do Gene vif/fisiologia , HIV-1/efeitos dos fármacos , HIV-1/crescimento & desenvolvimento , Humanos , Macaca mulatta , Mutação , Nucleosídeo Desaminases/antagonistas & inibidores , Nucleosídeo Desaminases/química , Nucleosídeo Desaminases/fisiologia , RNA Viral/genética , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/química , Proteínas Repressoras/fisiologia , Homologia de Sequência de Aminoácidos , Montagem de Vírus , Inativação de Vírus , Produtos do Gene vif do Vírus da Imunodeficiência HumanaRESUMO
The HIV-1 Vif protein counteracts the antiviral activity exhibited by the host cytidine deaminases APOBEC3G and APOBEC3F. Here, we show that defective vif alleles can readily be found in HIV-1 isolates and infected patients. Single residue changes in the Vif protein sequence are sufficient to cause the loss of Vif-induced APOBEC3 neutralization. Interestingly, not all the detected defects lead to a complete inactivation of Vif function since some mutants retained selective neutralizing activity against APOBEC3F but not APOBEC3G or vice versa. Concordantly, independently hypermutated proviruses with distinguishable patterns of G-to-A substitution attributable to cytidine deamination induced by APOBEC3G, APOBEC3F, or both enzymes were present in individuals carrying proviruses with completely or partly defective Vif variants. Natural variation in Vif function may result in selective and partial neutralization of cytidine deaminases and thereby promote viral sequence diversification within HIV-1 infected individuals.
RESUMO
APOBEC3G is promiscuous with respect to its antiretroviral effect, requiring that it be packaged into diverse retrovirus particles. Here, we show that most virally encoded human immunodeficiency virus type 1 particle components are dispensable for APOPEC3G incorporation. However, replacement of the nucleocapsid (NC) Gag domain with a leucine zipper abolished APOBEC3G incorporation. Moreover, coprecipitation analysis showed that APOBEC3G-Gag interaction requires NC and nonspecific RNA. These observations suggest that APOBEC3G exploits an essential property of retroviruses, namely, RNA packaging, to infiltrate particles. Because it is, therefore, difficult to evolve specific sequences that confer escape from APOBEC3G, these findings may explain why lentiviruses evolved an activity that induces its destruction.
Assuntos
HIV-1/metabolismo , Proteínas/metabolismo , Vírion/metabolismo , Desaminase APOBEC-3G , Citidina Desaminase , Produtos do Gene gag/fisiologia , Nucleosídeo Desaminases , RNA Viral/fisiologia , Proteínas Repressoras , Montagem de VírusRESUMO
Ex vivo gene therapy is emerging as a promising approach for the treatment of neurodegenerative diseases and central nervous system (CNS) trauma. We have shown previously that human adult astrocytes can be expanded in vitro and can express various therapeutic transgenes (Ridet et al. [1999] Hum. Gene Ther. 10:271-280; Serguera et al. [ 2001] Mol. Ther. 3:875-881). Here, we grafted normal and lentivirally-modified human adult astrocytes into the striatum and spinal cord of nude mice to test whether they are suitable candidates for ex vivo CNS gene therapy. Transplanted cells survived for at least 2 months (longest time analyzed) and sustained transgene expression. Importantly, the absence of proliferating cell nuclear antigen (PCNA) staining, a hallmark of cell division, ascertains the safety of these cells. Thus, adult human astrocytes are a promising tool for human CNS repair; they may make autologous ex vivo gene transfer feasible, thereby avoiding the problems of immunological rejection and the side effects of immunosuppressors.
Assuntos
Astrócitos/transplante , Transplante de Tecido Encefálico/métodos , Terapia Genética/métodos , Transplantes , Adulto , Animais , Apoptose/fisiologia , Astrócitos/química , Transplante de Tecido Encefálico/efeitos adversos , Transplante de Tecido Encefálico/ética , Técnicas de Cultura de Células/ética , Técnicas de Cultura de Células/métodos , Córtex Cerebral/química , Córtex Cerebral/transplante , Feminino , Terapia Genética/efeitos adversos , Terapia Genética/ética , Humanos , Camundongos , Camundongos Nus , Transplante Autólogo/ética , Transplante Autólogo/métodos , Transplantes/éticaRESUMO
Mucopolysaccharidosis type VII (MPS VII) is an inherited disease caused by beta-glucuronidase (beta-glu) deficiency. This deficiency results in the lysosomal accumulation of glycosaminoglycans in all tissues and affects a wide range of organs, including the central nervous system (CNS). Gene transfer is a promising approach to therapy for MPS VII because it allows extensive delivery of the enzyme to the affected tissues. We studied neurotransplantation of primary human cells to supply beta-glucuronidase to the CNS. Human neural progenitor cells (HNPC) were amplified and cotransduced with two lentiviral vectors, one encoding the green fluorescent protein and the other the human beta-glu. We show that these cells strongly expressed both transgenes in culture. When grafted into the mouse striatum, HNPC differentiated into neurons and astrocytes and expressed the two transgenes for at least 6 months. This study therefore paves the way for the treatment of MPS VII by long-term delivery of the appropriate enzyme.
Assuntos
Terapia Genética , Glucuronidase/genética , Mucopolissacaridose VII/terapia , Neurônios/transplante , Transplante de Células-Tronco , Fatores Etários , Animais , Transplante de Tecido Encefálico , Corpo Estriado/cirurgia , Feminino , Transplante de Tecido Fetal , Expressão Gênica , Proteínas de Fluorescência Verde , Humanos , Indicadores e Reagentes/metabolismo , Lentivirus/genética , Proteínas Luminescentes/genética , Camundongos , Camundongos Nus , Neurônios/fisiologia , Células-Tronco/fisiologia , Transgenes/genéticaRESUMO
BACKGROUND: A central triple-stranded DNA structure created during HIV-1 reverse transcription, the central flap, acts as a cis-active nuclear import determinant of the HIV-1 DNA genome. Insertion of the sequences responsible for formation of the central DNA flap into an HIV-1-derived vector strongly enhances its transduction efficiency. METHODS: HIV-1 vectors with or without inclusion of the DNA flap and encoding the same melanoma polyepitope were constructed to transduce dendritic cells (DCs) and to evaluate their capacity for induction of melanoma-specific cytotoxic T-lymphocyte (CTL) responses ex vivo and in vivo. RESULTS: HIV-1 vectors including the DNA flap transduced up to 100% of immature mouse and human DCs. Inoculation of HLA-A2.1 transgenic mice with this flap vector elicited vigorous and multi-specific long-term anti-melanoma CTL responses, whereas the parental vector lacking the flap sequence was less efficient. Furthermore, human DCs transduced ex vivo with the recombinant DNA flap vector displayed efficient multi-specific primary human CTL responses against melanoma. CONCLUSION: Lentiviral vectors including the DNA flap should be powerful tools both for active immunization and for the ex vivo priming of CTL for tumor immunotherapy.