RESUMO
Feline immunodeficiency virus (FIV), the causative agent of an acquired immunodeficiency syndrome in cats (feline AIDS), is a ubiquitous health threat to the domestic and feral cat population, also triggering disease in wild animals. No registered antiviral compounds are currently available to treat FIV-infected cats. Several human antiviral drugs have been used experimentally in cats, but not without the development of serious adverse effects. Here we report on the treatment of six naturally FIV-infected cats, suffering from moderate to severe disease, with the antiretroviral compound (R)-9-(2-phosphonylmethoxypropyl)-2,6-diaminopurine ([R]-PMPDAP), a close analogue of tenofovir, a widely prescribed anti-HIV drug in human medicine. An improvement in the average Karnofsky score (pretreatment 33.2 ± 9.4%, post-treatment 65±12.3%), some laboratory parameters (ie, serum amyloid A and gammaglobulins) and a decrease of FIV viral load in plasma were noted in most cats. The role of concurrent medication in ameliorating the Karnofsky score, as well as the possible development of haematological side effects, are discussed. Side effects, when noted, appeared mild and reversible upon cessation of treatment. Although strong conclusions cannot be drawn owing to the small number of patients and lack of a placebo-treated control group, the activity of (R)-PMPDAP, as observed here, warrants further investigation.
Assuntos
Adenina/análogos & derivados , Antivirais/uso terapêutico , Síndrome de Imunodeficiência Adquirida Felina/tratamento farmacológico , Vírus da Imunodeficiência Felina/efeitos dos fármacos , Compostos Organofosforados/uso terapêutico , Adenina/uso terapêutico , Animais , Gatos , Síndrome de Imunodeficiência Adquirida Felina/virologia , Humanos , Injeções Subcutâneas/veterinária , Avaliação de Estado de Karnofsky , Resultado do Tratamento , Replicação Viral/efeitos dos fármacosRESUMO
New large-scale synthetic approach to antiretroviral agent 9-[2-(R)-(phosphonomethoxy)propyl]-2,6-diaminopurine, (R)-PMPDAP, was developed. Reaction of (R)-propanediol carbonate with 2,6-diaminopurine afforded exclusively (R)-9-(2-hydroxypropyl)-2,6-diaminopurine which was subsequently used for introduction of a phosphonomethyl residue using TsOCH(2)P(O)(OiPr)(2) or BrCH(2)P(O)(OiPr)(2) followed by deprotection of ester groups. All minor ingredients and by-products formed during the process were identified and further studied. The final product was obtained in high yield and its high enantiomeric purity (>99%) was confirmed by chiral capillary electrophoretic analysis using ß-cyclodextrin as a chiral selector. Antiretroviral activity data of (R)-PMPDAP and its diverse prodrugs against HIV and FIV were investigated. Akin to (R)-PMPDAP, both prodrugs inhibit FIV replication in a selective manner. Compared to the parent molecule, the amidate prodrug was 10-fold less active against FIV in cell culture, whereas the alkoxyalkyl ester prodrug was 200-fold more potent in inhibiting FIV replication in vitro.
Assuntos
Adenina/análogos & derivados , Antivirais/química , Compostos Organofosforados/química , Pró-Fármacos/química , Adenina/química , Adenina/farmacologia , Antivirais/farmacologia , HIV-1/efeitos dos fármacos , Vírus da Imunodeficiência Felina/efeitos dos fármacos , Compostos Organofosforados/farmacologia , Pró-Fármacos/farmacologia , EstereoisomerismoRESUMO
Pradimicin S (PRM-S) is a highly water-soluble, negatively charged derivative of the antibiotic pradimicin A (PRM-A) in which the terminal xylose moiety has been replaced by 3-sulfated glucose. PRM-S does not prevent human immunodeficiency virus (HIV) adsorption on CD4(+) T cells, but it blocks virus entry into its target cells. It inhibits a wide variety of HIV-1 laboratory strains and clinical isolates, HIV-2, and simian immunodeficiency virus (SIV) in various cell culture systems (50% and 90% effective concentrations [EC(50)s and EC(90)s] invariably in the lower micromolar range). PRM-S inhibits syncytium formation between persistently HIV-1- and SIV-infected cells and uninfected CD4(+) T lymphocytes, and prevents dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN)-mediated HIV-1 and SIV capture and subsequent virus transmission to CD4(+) T cells. Surface plasmon resonance (SPR) studies revealed that PRM-S strongly binds to gp120 in a Ca(2+)-dependent manner at an affinity constant (K(D)) in the higher nanomolar range. Its anti-HIV activity and HIV-1 gp120-binding properties can be dose-dependently reversed in the presence of an (alpha-1,2)mannose trimer. Dose-escalating exposure of HIV-1-infected cells to PRM-S eventually led to the isolation of mutant virus strains that had various deleted N-glycosylation sites in the envelope gp120 with a strong preference for the deletion of the high-mannose-type glycans. Genotypic resistance development occurred slowly, and significant phenotypic resistance occurred only after the sequential appearance of up to six mutations in gp120, pointing to a high genetic barrier of PRM-S. The antibiotic is nontoxic against a variety of cell lines, is not mitogenic, and does not induce cytokines and chemokines in peripheral blood mononuclear cells as determined by the Bio-Plex human cytokine 27-plex assay. It proved stable at high temperature and low pH. Therefore, PRM-S may qualify as a potential anti-HIV drug candidate for further (pre)clinical studies, including its microbicidal use.
Assuntos
Antraciclinas/farmacologia , Fármacos Anti-HIV/farmacologia , HIV-1/efeitos dos fármacos , Antraciclinas/química , Antibacterianos/química , Antibacterianos/farmacologia , Fármacos Anti-HIV/química , Anti-Infecciosos/química , Anti-Infecciosos/farmacologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/virologia , Células CACO-2 , Linhagem Celular , Quimiocinas/biossíntese , Técnicas de Cocultura , Citocinas/biossíntese , Farmacorresistência Viral/genética , Estabilidade de Medicamentos , Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/metabolismo , Proteína gp41 do Envelope de HIV/metabolismo , HIV-1/genética , HIV-1/fisiologia , HIV-2/efeitos dos fármacos , Células HeLa , Humanos , Modelos Moleculares , Vírus da Imunodeficiência Símia/efeitos dos fármacos , Solubilidade , Internalização do Vírus/efeitos dos fármacosRESUMO
Alcian Blue (AB), a phthalocyanine derivative, is able to prevent infection by a wide spectrum of human immunodeficiency virus type 1 (HIV-1), HIV-2, and simian immunodeficiency virus strains in various cell types [T cells, (co)receptor-transfected cells, and peripheral blood mononuclear cells]. With the exception of herpes simplex virus, AB is inactive against a broad variety of other (DNA and RNA) viruses. Time-of-addition studies show that AB prevents HIV-1 infection at the virus entry stage, exactly at the same time as carbohydrate-binding agents do. AB also efficiently prevents fusion between persistently HIV-1-infected HUT-78 cells and uninfected (CD4(+)) lymphocytes, DC-SIGN-directed HIV-1 capture, and subsequent transmission to uninfected (CD4(+)) T lymphocytes. Prolonged passaging of HIV-1 at dose-escalating concentrations of AB resulted in the selection of mutant virus strains in which several N-glycans of the HIV-1 gp120 envelope were deleted and in which positively charged amino acid mutations in both gp120 and gp41 appeared. A mutant virus strain in which four N-glycans were deleted showed a 10-fold decrease in sensitivity to the inhibitory effect of AB. These data suggest that AB is likely endowed with carbohydrate-binding properties and can be considered an important lead compound in the development of novel synthetic nonpeptidic antiviral drugs targeting the glycans of the envelope of HIV.
Assuntos
Azul Alciano/farmacologia , Fármacos Anti-HIV/farmacologia , Proteína gp120 do Envelope de HIV/metabolismo , Azul Alciano/química , Azul Alciano/metabolismo , Células Cultivadas , Farmacorresistência Viral , Células Gigantes/efeitos dos fármacos , Glicosilação , Proteína gp120 do Envelope de HIV/química , HIV-1/efeitos dos fármacos , Humanos , Mutação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
It was recently shown that capture of HIV-1 by DC-SIGN-expressing cells and the subsequent transmission of HIV to CD4+ T-lymphocytes can be prevented by carbohydrate-binding agents (CBAs), whereas polyanions were unable to block virus capture by DC-SIGN. In this study, we could show that a short pre-exposure of HIV-1 to both mannose- and N-acetylglucosamine (GlcNAc)-specific CBAs or polyanions dose-dependently prevented virus capture by L-SIGN-expressing 293T-REx/L-SIGN cells and subsequent syncytia formation in co-cultures of the drug-exposed HIV-1-captured 293T-REx/L-SIGN cells and uninfected C8166 CD4+ T-lymphocytes. Additionally, the inhibitory potential of the compounds against L-SIGN-mediated HIV-1 capture and transmission was more pronounced than observed for DC-SIGN expressing293T-REx/DC-SIGN cells. The excess value of CBAs and polyanions to prevent HIV-1 capture and transmission by DC-SIGN and L-SIGN-expressing cells to susceptible T-lymphocytes could be of interest for the development of new drug leads targeting HIV entry/fusion.
Assuntos
Fármacos Anti-HIV/farmacologia , Linfócitos T CD4-Positivos/virologia , Moléculas de Adesão Celular/antagonistas & inibidores , HIV-1/efeitos dos fármacos , Lectinas Tipo C/antagonistas & inibidores , Lectinas/farmacologia , Polímeros/farmacologia , Receptores de Superfície Celular/antagonistas & inibidores , Ligação Viral/efeitos dos fármacos , Linhagem Celular , Relação Dose-Resposta a Droga , HIV-1/fisiologia , Humanos , PolieletrólitosRESUMO
Reduction of intramolecular disulfides in the HIV-1 envelope protein gp120 occurs after its binding to the CD4 receptor. Protein disulfide isomerase (PDI) catalyzes the disulfide reduction in vitro and inhibition of this enzyme blocks viral entry. PDI belongs to the thioredoxin protein superfamily that also includes human glutaredoxin-1 (Grx1). Grx1 is secreted from cells and the protein has also been found within the HIV-1 virion. We show that Grx1 efficiently catalyzes gp120, and CD4 disulfide reduction in vitro, even at low plasma levels of glutathione. Grx1 catalyzes the reduction of two disulfide bridges in gp120 in a similar manner as PDI. Purified anti-Grx1 antibodies were shown to inhibit the Grx1 activity in vitro and block HIV-1 replication in cultured peripheral blood mononuclear cells. Also, the polyanion PRO2000, that was previously shown to prevent HIV entry, inhibits the Grx1- and PDI-dependent reduction of gp120 disulfides. Our findings suggest that Grx1 activity is important for HIV-1 entry and that Grx1 and the gp120 intramolecular disulfides are novel pharmacological targets for rational drug development.
Assuntos
Antígenos CD4/metabolismo , Glutarredoxinas/metabolismo , Proteína gp120 do Envelope de HIV/metabolismo , HIV-1/fisiologia , Replicação Viral/fisiologia , Animais , Catálise , Bovinos , Dissulfetos/metabolismo , Glutarredoxinas/antagonistas & inibidores , Glutarredoxinas/farmacologia , Glutationa/metabolismo , HIV-1/metabolismo , Humanos , Naftalenossulfonatos/farmacologia , Oxirredução , Polímeros/farmacologia , Isomerases de Dissulfetos de Proteínas/metabolismo , Proteínas Recombinantes/farmacologia , Internalização do Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacosRESUMO
Carbohydrate-binding agents (CBAs), such as the mannose-specific Hippeastrum hybrid agglutinin (HHA) and the GlcNAc-specific Urtica dioica agglutinin (UDA), frequently select for glycan deletions in all different domains of HIV-1 gp120, except in the V1/V2 domain. To reveal the underlying mechanisms, a broad variety of 31 different virus strains containing one or several N-glycan deletions in V1/V2 of the gp120 of the X4-tropic HIV-1(NL4.3) were constructed by chimeric virus technology. No co-receptor switch to CCR5 was observed for any of the replication-competent mutant virus strains. With a few exceptions, the more glycans were deleted in the gp120 V1/V2 domain, the more the replication capacity of the mutant viruses became compromised. None of the mutant virus strains showed a markedly decreased sensitivity to the inhibitory activity of HHA and UDA. Instead, an up to 2- to 10-fold higher sensitivity to the inhibitory activity of these CBAs was observed. Our data may provide an explanation why glycan deletions in the gp120 V1/V2 domain rarely occur under CBA pressure and confirm the important functional role of the glycans in the HIV-1 gp120 V1/V2 domain. The gp120 V1/V2 loop glycans of HIV-1 should therefore be considered as a hot spot and novel target for specific therapeutic drug intervention.
Assuntos
Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/metabolismo , HIV-1/química , HIV-1/fisiologia , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Replicação Viral , Substituição de Aminoácidos/genética , Animais , Fármacos Anti-HIV/metabolismo , Fármacos Anti-HIV/farmacologia , Linhagem Celular , Glicosilação , Proteína gp120 do Envelope de HIV/genética , HIV-1/efeitos dos fármacos , HIV-1/genética , Humanos , Concentração Inibidora 50 , Lectinas/metabolismo , Lectinas/farmacologia , Testes de Sensibilidade Microbiana , Mutagênese Sítio-Dirigida , Proteínas Mutantes/genética , Mutação de Sentido Incorreto , Ligação ProteicaRESUMO
Carbohydrate-binding agents (CBAs), such as the plant lectins Hippeastrum hybrid agglutinin (HHA) and Urtica dioica agglutinin (UDA), but also the nonpeptidic antibiotic pradimicin A (PRM-A), inhibit entry of HIV into its target cells by binding to the glycans of gp120. Given the high sequence identity and similarity between the envelope gp120 glycoproteins of HIV and simian immunodeficiency virus (SIV), the inhibitory activity of a variety of CBAs were evaluated against HIV-1, HIV-2, and SIV. There seemed to be a close correlation for the inhibitory potential of CBAs against HIV-1, HIV-2, and SIV replication in cell culture and syncytia formation in cocultures of persistently SIV-infected HUT-78 cell cultures and uninfected CEM cells. CBAs also inhibit transmission of the SIV to T lymphocytes after capture of the virus by dendritic cell-specific ICAM3-grabbing nonintegrin (DC-SIGN)-expressing cells. A total of 8 different SIV strains were isolated after prolonged HHA, UDA, and PRM-A exposure in virus-infected cell cultures. Each virus isolate consistently contained at least 2 or 3 glycan deletions in its gp120 envelope and showed decreased sensitivity to the CBAs and cross-resistance toward all CBAs. Our data revealed that CBAs afford SIV and HIV-1 inhibition in a similar manner regarding prevention of virus infection, DC-SIGN-directed virus capture-related transmission, and selection of drug-resistant mutant virus strains. Therefore, SIV(mac251)-infected monkeys might represent a relevant animal model to study the efficacy of CBAs in vivo.
Assuntos
Antivirais/metabolismo , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos/tendências , Farmacorresistência Viral/fisiologia , HIV/metabolismo , Vírus da Imunodeficiência Símia/metabolismo , Animais , Antraciclinas/metabolismo , Antraciclinas/farmacologia , Antivirais/farmacologia , Sítios de Ligação/efeitos dos fármacos , Sítios de Ligação/fisiologia , Metabolismo dos Carboidratos/efeitos dos fármacos , Metabolismo dos Carboidratos/fisiologia , Células Cultivadas , Farmacorresistência Viral/efeitos dos fármacos , HIV/efeitos dos fármacos , Humanos , Lectinas de Plantas/farmacocinética , Lectinas de Plantas/farmacologia , Vírus da Imunodeficiência Símia/efeitos dos fármacosRESUMO
The coumarins represent a unique class of non-nucleoside reverse transcriptase inhibitors (NNRTIs) that were isolated from tropical plants. (+)-Calanolide A, the most potent compound of this class, selects for the T139I resistance mutation in HIV-1 reverse transcriptase (RT). Seven RTs mutated at amino acid position 139 (Ala, Lys, Tyr, Asp, Ile, Ser, and Gln) were constructed by site-directed mutagenesis. The mutant T139Q enzyme retained full catalytic activity compared with wild-type RT, whereas the mutant T139I, T139S, and T139A RTs retained only 85 to 50% of the activity. Mutant T139K, T139D, and T139Y RTs had seriously impaired catalytic activities. The mutations in the T139I and T139D RTs were shown to destabilize the RT heterodimer. (+)-Calanolide A lost inhibitory activity (up to 20-fold) against the mutant T139Y, T139Q, T139K, and T139I enzymes. All of the mutant enzymes retained marked susceptibility toward the other NNRTIs, including nevirapine, delavirdine, efavirenz, thiocarboxanilide UC-781, quinoxaline GW867420X, TSAO [[2',5'-bis-O-(tert-butyldimethylsilyl)-beta-D-ribofuranosyl]-3'-spiro-5''-(4''-amino-1'',2''-oxathiole-2'',2''-dioxide)] derivatives, and the nucleoside inhibitor, ddGTP. The fact that the T139I RT 1) proved to be resistant to (+)-calanolide A, 2) represents a catalytically efficient enzyme, and 3) requires only a single transition point mutation (ACA-->ATA) in codon 139 seems to explain why mutant T139I RT virus strains, but not virus strains containing other amino acid changes at this position, predominantly emerge in cell cultures under (+)-calanolide A pressure.
Assuntos
Cumarínicos/farmacologia , Transcriptase Reversa do HIV/antagonistas & inibidores , Inibidores da Transcriptase Reversa/farmacologia , Treonina/fisiologia , Transcriptase Reversa do HIV/química , Transcriptase Reversa do HIV/genética , Cinética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Piranocumarinas , Treonina/químicaRESUMO
The highly conserved Asn136 is in close proximity to the nonnucleoside reverse transcriptase (RT) inhibitor (NNRTI)-specific lipophilic pocket of human immunodeficiency virus type 1 (HIV-1) RT. Site-directed mutagenesis has revealed that the catalytic activity of HIV-1 RT mutated at position Asn136 is heavily compromised. Only 0.07 to 2.1% of wild-type activity is retained, depending on the nature of the amino acid change at position 136. The detrimental effect of the mutations at position 136 occurred when the mutated amino acid was present in the p51 subunit but not in the p66 subunit of the p51/p66 RT heterodimer. All mutant enzymes could be inhibited by second-generation NNRTIs such as efavirenz. They were also markedly more sensitive to the inactivating (denaturating) effect of urea than wild-type RT, and the degree of increased urea sensitivity was highly correlated with the degree of (lower) catalytic activity of the mutant enzymes. Replacing wild-type Asn136 in HIV-1 RT with other amino acids resulted in notably increased amounts of free p51 and p66 monomers. Our findings identify a structural/functional role for Asn136 in stabilization of the RT p66/p51 dimer and provide hints for the rational design of novel NNRTIs or drugs targeting either Asn136 in the beta7-beta8 loop of p51 or its anchoring point on p66 (the peptide backbone of His96) so as to interfere with the RT dimerization process and/or with the structural support that the p51 subunit provides to the p66 subunit and which is essential for the catalytic enzyme activity.
Assuntos
Asparagina/genética , Desenho de Fármacos , Transcriptase Reversa do HIV/antagonistas & inibidores , Transcriptase Reversa do HIV/genética , Inibidores da Transcriptase Reversa/síntese química , Asparagina/química , Catálise , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Transcriptase Reversa do HIV/química , Humanos , Mutação , Estrutura Secundária de Proteína/genética , Subunidades Proteicas/antagonistas & inibidores , Subunidades Proteicas/química , Subunidades Proteicas/genética , Inibidores da Transcriptase Reversa/farmacologiaRESUMO
Amino acids N137 and P140 in the p51 subunit of HIV-1 reverse transcriptase (RT) are part of the beta7-beta8-loop that contributes to the formation of the base of the non-nucleoside RT inhibitor (NNRTI)-binding pocket and makes up a substantial part of the dimerization interface. Amino acid P95 in p66 also markedly contributes to the dimerization binding energy. Nine RT mutants at amino acid 137 were constructed bearing the mutations Y, K, T, D, A, Q, S, H or E. The prolines at amino acid positions 95 and 140 were replaced by alanine in separate enzymes. We found that all mutant RT enzymes showed a dramatically decreased RNA-dependent DNA polymerase activity. None of the mutant RT enzymes showed marked resistance against any of the clinically used NNRTIs but they surprisingly lost significant sensitivity for NRTIs such as ddGTP. The denaturation analyses of the mutant RTs by urea are suggestive for a relevant role of N137 in the stability of the RT heterodimer and support the view that the beta7-beta8 loop in p51 is a hot spot for RT dimerization and instrumental for efficient polymerase catalytic activity. Consequently, N137 and P140 in p51 and P95 in p66 should be attractive targets in the design of new structural classes of RT inhibitors aimed at compromising the optimal interaction of the beta7-beta8 loop in p51 at the p66/p51 dimerization interface.
Assuntos
Fármacos Anti-HIV/farmacologia , Asparagina/metabolismo , Desenho de Fármacos , Transcriptase Reversa do HIV/química , Transcriptase Reversa do HIV/metabolismo , HIV-1/enzimologia , Fenilalanina/metabolismo , Fármacos Anti-HIV/química , Asparagina/genética , Catálise , Estabilidade Enzimática , Transcriptase Reversa do HIV/genética , HIV-1/genética , Humanos , Concentração Inibidora 50 , Modelos Moleculares , Mutação/genética , Fenilalanina/genética , Estrutura Terciária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Inibidores da Transcriptase Reversa/química , Inibidores da Transcriptase Reversa/farmacologia , Ureia/farmacologiaRESUMO
The phenylmethylthiazolylthiourea (PETT) derivative MSK-076 shows, besides high potency against human immunodeficiency virus type 1 (HIV-1), marked activity against HIV-2 (50% effective concentration, 0.63 microM) in cell culture. Time-of-addition experiments pointed to HIV-2 reverse transcriptase (RT) as the target of action of MSK-076. Recombinant HIV-2 RT was inhibited by MSK-076 at 23 microM. As was also found for HIV-1 RT, MSK-076 inhibited HIV-2 RT in a noncompetitive manner with respect to dGTP and poly(rC).oligo(dG) as the substrate and template-primer, respectively. MSK-076 selected for A101P and G112E mutations in HIV-2 RT and for K101E, Y181C, and G190R mutations in HIV-1 RT. The selected mutated strains of HIV-2 were fully resistant to MSK-076, and the mutant HIV-2 RT enzymes into which the A101P and/or G112E mutation was introduced by site-directed mutagenesis showed more than 50-fold resistance to MSK-076. Mapping of the resistance mutations to the HIV-2 RT structure ascertained that A101P is located at a position equivalent to the nonnucleoside RT inhibitor (NNRTI)-binding site of HIV-1 RT. G112E, however, is distal to the putative NNRTI-binding site in HIV-2 RT but close to the active site, implying a novel molecular mode of action and mechanism of resistance. Our findings have important implications for the development of new NNRTIs with pronounced activity against a wider range of lentiviruses.
Assuntos
Fármacos Anti-HIV/farmacologia , Farmacorresistência Viral , HIV-2/efeitos dos fármacos , DNA Polimerase Dirigida por RNA/efeitos dos fármacos , Inibidores da Transcriptase Reversa/farmacologia , Tiazóis/farmacologia , Triazóis/farmacologia , Sequência de Aminoácidos , Células Cultivadas , Efeito Citopatogênico Viral , Transcriptase Reversa do HIV , HIV-1/efeitos dos fármacos , HIV-2/enzimologia , HIV-2/genética , Humanos , Testes de Sensibilidade Microbiana , Modelos Moleculares , Dados de Sequência Molecular , Mutação , DNA Polimerase Dirigida por RNA/química , DNA Polimerase Dirigida por RNA/genética , Tiazóis/química , Triazóis/químicaRESUMO
To map the determinants of the lack of susceptibility of feline immunodeficiency virus (FIV) reverse transcriptase (RT) to anti human immunodeficiency virus type 1 (HIV-1) non-nucleoside RT inhibitors (NNRTIs), a variety of chimeric HIV-1/FIV RTs were constructed. The majority of chimeric RTs had an affinity (Km) for their natural substrates comparable with that of the wild-type HIV-1 and FIV RTs, but their catalytic efficacy was decreased. Whereas HIV-1 RT could be made entirely insensitive to NNRTIs by exchanging the amino acid sequence 97 through 205 of FIV RT, none of the reverse FIV/HIV-1 RT chimeras gained susceptibility to NNRTIs. The amino acids that are thought to be involved in NNRTI susceptibility and that are different from those in HIV-1 RT have also been introduced in FIV RT. These mutant RTs gained virtually no susceptibility to efavirenz or capravirine. Vice versa, when these HIV-1-specific amino acids were replaced by their FIV RT counterparts in HIV-1 RT, susceptibility to the NNRTIs was lost. Thus, replacing segments or substituting relevant amino acids in FIV RT by their HIV-1 RT counterparts did not suffice to make FIV RT sensitive toward NNRTIs and was often accompanied by a decrease or even total loss of polymerase activity. It is postulated that, in contrast to the results found for HIV-1/HIV-2 RT chimeras and supported by the crystal structure of HIV-2 RT, there exist significant differences in the structure and/or flexibility of FIV RTs that may prevent NNRTIs from interacting with the FIV RT.
Assuntos
HIV-1/enzimologia , Vírus da Imunodeficiência Felina/enzimologia , DNA Polimerase Dirigida por RNA/metabolismo , Proteínas Recombinantes de Fusão/antagonistas & inibidores , Inibidores da Transcriptase Reversa/farmacologia , Sequência de Aminoácidos , Animais , Fármacos Anti-HIV/farmacologia , Catálise , Gatos , Nucleotídeos de Desoxiguanina/farmacologia , Didesoxinucleotídeos , Foscarnet/farmacologia , Transcriptase Reversa do HIV/antagonistas & inibidores , Transcriptase Reversa do HIV/química , Humanos , Imidazóis , Cinética , Dados de Sequência Molecular , Nevirapina/farmacologia , DNA Polimerase Dirigida por RNA/química , DNA Polimerase Dirigida por RNA/efeitos dos fármacos , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Compostos de EnxofreRESUMO
Non-nucleoside reverse transcriptase inhibitors (NNRTIs) are specific for human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) and do not inhibit HIV-2. Given that the amino acids lining the NNRTI-specific pocket of HIV-1 RT display higher similarity to the corresponding feline immunodeficiency virus (FIV) RT amino acids than to HIV-2 RT, the susceptibility of FIV RT and chimeric HIV-1/FIV RTs to NNRTIs and the role of the p51 subunit in the inhibitory action of NNRTIs were investigated. We found that the wild-type FIV RT and the FIVp66/HIVp51 chimeric enzyme showed no susceptibility for NNRTIs. On the other hand, the chimeric HIVp66/FIVp51 RT retained a sensitivity spectrum for NNRTIs similar to that of the wild-type HIV-1 RT. The noncompetitive nature of inhibition of HIV-1 RT by nevirapine was also observed with the HIVp66/FIVp51 chimeric enzyme. Inhibition of the chimeric RTs by nucleoside reverse transcriptase inhibitors and foscarnet was in the same range as observed for the corresponding HIVp66/HIVp51 and FIVp66/FIVp51 wild-type enzymes. The chimeric RTs had an affinity (K(m)) for their dNTP substrate and template/primer comparable with that of the wild-type HIV-1 and FIV RTs, but their catalytic efficacy (k(cat)) was markedly decreased. This decreased catalytic efficacy of the RT chimeras may suggest suboptimal interactions between p66 and p51 in the chimeric enzymes. Our results point to a minor role of the p51 subunit in the sensitivity to RT inhibitors.