RESUMEN
The novel virucidal protein cyanovirin-N (CV-N) binds with equally high affinity to soluble forms of either H9 cell-produced or recombinant glycosylated HIV-1 gp120 (sgp120) or gp160 (sgp160). Fluorescence polarization studies showed that CV-N is also capable of binding to the glycosylated ectodomain of the HIV-envelope protein gp41 (sgp41) (as well as SIV glycoprotein 32), albeit with considerably lower affinity than the sgp120/CV-N interaction. Pretreatment of CV-N with either sgp120 or sgp41 abrogated the neutralizing activity of CV-N against intact, infectious HIV-1 virions. Isothermal calorimetry and optical biosensor binding studies showed that CV-N bound to recombinant sgp120 with a K(d) value ranging from 2 to 45 nM and to sgp41 with a K(d) value of 606 nM; furthermore, they indicated an approximate 5:1 stoichiometry for CV-N binding to sgp120 and a 1:1 stoichiometry for CV-N binding to sgp41. Circular dichroism studies additionally illuminated the binding of CV-N with both sgp120 and sgp41, providing the first direct evidence that conformational changes are a consequence of CV-N interactions with both HIV-1 envelope glycoproteins.
Asunto(s)
Fármacos Anti-VIH/farmacología , Proteínas Bacterianas , Proteínas Portadoras/farmacología , Proteína gp120 de Envoltorio del VIH/metabolismo , Proteína gp41 de Envoltorio del VIH/metabolismo , VIH/efectos de los fármacos , Fármacos Anti-VIH/antagonistas & inhibidores , Unión Competitiva , Técnicas Biosensibles , Calorimetría , Proteínas Portadoras/antagonistas & inhibidores , Dicroismo Circular , Polarización de Fluorescencia , Proteína gp120 de Envoltorio del VIH/farmacología , Proteína gp41 de Envoltorio del VIH/farmacología , Proteínas Recombinantes/metabolismo , Espectrometría de Fluorescencia , Factores de TiempoRESUMEN
Aqueous extracts from the African plant Myrianthus holstii potently inhibited the infection of the T-lymphoblastoid cell line, CEM-SS, by human immunodeficiency virus-1(RF) (HIV-1(RF)). The active constituent, M. holstii lectin (MHL), was purified by LH-20 column chromatography and reversed phase HPLC. MHL, a 9284-Da cysteine-rich protein, was characterized by amino acid analysis, N-terminal sequencing, ESIMS, and matrix-assisted laser-desorption ionization-time-of-flight mass spectrometry. Pure MHL had anti-HIV activity, with an EC(50) value of 150 nM. Delaying the addition of MHL for up to 8 h after initial exposure of CEM-SS cells to virus did not result in loss of the antiviral activity; however, if addition of the compound was delayed for 16 h or more, there was a marked decrease in the antiviral activity. MHL bound to a virus-free, soluble form of the viral envelope protein gp120 but did not inhibit the subsequent binding to a cell-free, soluble form of the cellular receptor CD4.
Asunto(s)
VIH-1/efectos de los fármacos , Lectinas/aislamiento & purificación , Raíces de Plantas/química , Plantas/química , Secuencia de Aminoácidos , Antígenos CD4/química , Agregación Celular/efectos de los fármacos , Células Cultivadas , Quitinasas/análisis , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Electroforesis en Gel de Poliacrilamida , Cromatografía de Gases y Espectrometría de Masas , Proteína gp120 de Envoltorio del VIH/química , Infecciones por VIH/tratamiento farmacológico , Pruebas de Hemaglutinación , Humanos , Lectinas/química , Lectinas/farmacología , Datos de Secuencia Molecular , Lectinas de Plantas , Alineación de Secuencia , Análisis de Secuencia de Proteína , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , TanzaníaRESUMEN
We have isolated and sequenced a novel 11-kDa virucidal protein, named cyanovirin-N (CV-N), from cultures of the cyanobacterium (blue-green alga) Nostoc ellipsosporum. We also have produced CV-N recombinantly by expression of a corresponding DNA sequence in Escherichia coli. Low nanomolar concentrations of either natural or recombinant CV-N irreversibly inactivate diverse laboratory strains and primary isolates of human immunodeficiency virus (HIV) type 1 as well as strains of HIV type 2 and simian immunodeficiency virus. In addition, CV-N aborts cell-to-cell fusion and transmission of HIV-1 infection. Continuous, 2-day exposures of uninfected CEM-SS cells or peripheral blood lymphocytes to high concentrations (e.g., 9,000 nM) of CV-N were not lethal to these representative host cell types. The antiviral activity of CV-N is due, at least in part, to unique, high-affinity interactions of CV-N with the viral surface envelope glycoprotein gp120. The biological activity of CV-N is highly resistant to physicochemical denaturation, further enhancing its potential as an anti-HIV microbicide.
Asunto(s)
Fármacos Anti-VIH/aislamiento & purificación , Proteínas Bacterianas , Proteínas Portadoras/aislamiento & purificación , Proteína gp120 de Envoltorio del VIH/metabolismo , Síndrome de Inmunodeficiencia Adquirida/transmisión , Secuencia de Aminoácidos , Fármacos Anti-VIH/química , Fármacos Anti-VIH/metabolismo , Secuencia de Bases , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Fusión Celular , Supervivencia Celular/efectos de los fármacos , Ensayo de Inmunoadsorción Enzimática , Humanos , Datos de Secuencia Molecular , Peso Molecular , Unión Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Volumetría , UltrafiltraciónRESUMEN
Calanolide A, recently discovered in extracts from the tropical rainforest tree, Calophyllum lanigerum, is a novel inhibitor of the human immunodeficiency virus (HIV) type 1. The compound is essentially inactive against strains of the less common HIV type 2. The present study focused on the further characterization of the selective antiviral activity and mechanism of action of calanolide A. The compound inhibited a wide variety of laboratory strains of HIV type 1, with EC50 values ranging from 0.10 to 0.17 microM. The compound similarly inhibited promonocytotropic and lymphocytotropic isolates from patients in various stages of HIV disease, as well as drug-resistant strains. Viral life-cycle studies indicated that calanolide A acted early in the infection process, similar to the known HIV reverse transcriptase (RT) inhibitor 2', 3'-dideoxycytidine. In enzyme inhibition assays, calanolide A potently and selectively inhibited recombinant HIV type 1 RT but not cellular DNA polymerases or HIV type 2 RT within the concentration range tested. Serial passage of the virus in host cells exposed to increasing concentrations of calanolide A yielded a calanolide A resistant virus strain. RT from the resistant virus was not inhibited by calanolide A but retained sensitivity to other nonnucleoside as well as nucleoside RT inhibitors, including 3'-azido-2',3'-dideoxythymidine triphosphate and nevirapine. The study substantially supports the conclusion that calanolide A represents a novel subclass of nonnucleoside RT inhibitor which merits consideration for anti-HIV drug development.
Asunto(s)
Fármacos Anti-VIH/farmacología , Cumarinas/farmacología , VIH-1/efectos de los fármacos , Inhibidores de la Transcriptasa Inversa/farmacología , Línea Celular , Humanos , Inhibidores de la Síntesis del Ácido Nucleico , PiranocumarinasRESUMEN
Calanolide A, first isolated from the tropical rain forest tree Calophyllum lanigerum, is a potent human immunodeficiency virus type-1 (HIV-1) specific reverse transcriptase (RT) inhibitor, broadly active against diverse HIV-1 strains, including nucleoside and nonnucleoside-resistant variants. We examined the biochemical mechanism of inhibition of HIV-1 RT by calanolide A. Two template/primer systems were examined: ribosomal RNA and homopolymeric rA-dT 12-18. Calanolide A inhibited HIV-1 RT by a complex mechanism involving two calanolide A binding sites. With respect to either deoxynucleotide triphosphate (dNTP) or template/primer binding, one site was competitive and the other was uncompetitive. The data indicated that calanolide A bound near the active site of the enzyme and interfered with dNTP binding. Calanolide A inhibited HIV-1 RT in a synergistic fashion with nevirapine, further distinguishing it from the general class of nonnucleoside RT inhibitors. At certain concentrations, calanolide A bound HIV-1 RT in a mutually exclusive fashion with respect to both the pyrophosphate analog, phosphonoformic acid and the acyclic nucleoside analog 1-ethoxymethyl-5-ethyl-6-phenylthio-2-thiouracil. This indicates that calanolide A shares some binding domains with both phosphonoformic acid and 1-ethoxymethyl-5-ethyl-6-phenylthio-2-thiouracil, presumably reflecting that it interacts with RT near both the pyrophosphate binding site and the active site of the enzyme.
Asunto(s)
Fármacos Anti-VIH/farmacología , Cumarinas/farmacología , Transcriptasa Inversa del VIH/antagonistas & inhibidores , Inhibidores de la Transcriptasa Inversa/farmacología , Sitios de Unión , Humanos , Cinética , PiranocumarinasRESUMEN
UC 38, a simple analog of oxathiin carboxanilide, UC 84, lacking the oxathiin ring, was found to be a potent inhibitor of human immunodeficiency virus (HIV)-1-induced cell killing and HIV replication in a variety of human cell lines, as well as in human peripheral blood lymphocytes and macrophages. UC 38 was active against a wide range of biologically diverse laboratory and clinical strains of HIV-1. However, UC 38 was inactive against HIV-2 and both nevirapine- and pyridinone-resistant strains of HIV-1. UC 38 selectively inhibited HIV-1 reverse transcriptase (RT), but not HIV-2 RT. Combination of UC 38 with 3'-azido-3'-deoxythymidine synergistically inhibited HIV-induced cell killing. An HIV-1 isolate resistant to UC 38 was selected in cell culture, and the mutations in the RT nucleotide sequences were determined. Comparison with the wild-type RT sequence revealed an amino acid change at position 181 (Tyr to Cys). The UC 38-resistant virus was found to be cross-resistant to a variety of structurally diverse non-nucleoside RT inhibitors. UC 38 was susceptible to rapid degradation in vitro and in vivo; yet, nontoxic in vivo concentrations of UC 38 many-fold in excess of the in vitro effective concentrations could be achieved and maintained after s.c. or p.o. administration in hamsters. These results establish UC 38 as a new chemotype within the general class of HIV-1-specific RT inhibitors. The favorable physical characteristics, lack of toxicity, potency and bioavailability of UC 38 may make it a candidate for combination chemotherapy of acquired immune deficiency syndrome.
Asunto(s)
Antivirales/farmacología , Benzoatos/farmacología , VIH-1/efectos de los fármacos , ADN Polimerasa Dirigida por ARN/efectos de los fármacos , Inhibidores de la Transcriptasa Inversa/farmacología , Tiocarbamatos/farmacología , Animales , Antivirales/farmacocinética , Benzoatos/farmacocinética , Disponibilidad Biológica , Carboxina/análogos & derivados , Carboxina/farmacocinética , Carboxina/farmacología , Cricetinae , Análisis Mutacional de ADN , ADN Viral/análisis , ADN Viral/genética , Esquema de Medicación , Farmacorresistencia Microbiana , Estabilidad de Medicamentos , Sinergismo Farmacológico , Transcriptasa Inversa del VIH , VIH-1/enzimología , Humanos , Masculino , Ratones , Pruebas de Sensibilidad Microbiana , Ratas , Inhibidores de la Transcriptasa Inversa/farmacocinética , Tiocarbamatos/farmacocinética , Zidovudina/farmacologíaRESUMEN
Anti-HIV activity and the inhibition of phorbol ester receptor binding activity in two species of Maprounea were traced to small amounts of highly potent phorbol esters of the daphnane type. The triterpenes previously isolated from this genus were found to be devoid of biological activity when scrupulously purified. Four new triterpene esters were elucidated; two [3,4] were found in M. africana, while three [4,6,7] were found in M. membranacea. Nmr assignments have also been made for two previously known compounds [2,5] in this group.
Asunto(s)
VIH/efectos de los fármacos , Plantas Medicinales/química , Triterpenos/química , Triterpenos/farmacología , Acetilación , Animales , Unión Competitiva/efectos de los fármacos , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Cromatografía Líquida de Alta Presión , Cromatografía de Gases y Espectrometría de Masas , Hidrólisis , Técnicas In Vitro , Espectroscopía de Resonancia Magnética , Forbol 12,13-Dibutirato/farmacología , Ratas , Inhibidores de la Transcriptasa Inversa , Especificidad de la Especie , Triterpenos/aislamiento & purificaciónRESUMEN
We have biologically and biochemically evaluated a structurally diverse group of HIV-1-specific reverse transcriptase (RT) inhibitors and determined that the members of this class share many common properties. These include reproducible and selective antiviral activity against a panel of biologically distinct laboratory and clinical strains of HIV-1, activity against HIV-1 in a wide variety of cultured and fresh human cells, and potent inhibition of HIV-1 RT when evaluated using a heteropolymeric ribosomal RNA template assay. Each of the HIV-1-specific compounds was capable of inhibiting HIV replication when challenged at high m.o.i., further distinguishing them from the nucleoside analogs 3'-azido-3'-deoxythymidine (AZT) and 2',3'-dideoxycytidine (ddC). When tested in combination with AZT, each of the HIV-1-specific compounds synergistically inhibited the replication of HIV-1. HIV-1 isolates resistant to different HIV-1-specific inhibitors exhibited heterogeneous patterns of cross-resistance to other members of this pharmacologic class. Four distinct phenotypic classes have been defined through the use of drug-resistant virus isolates which derive from distinct mutations in the RT. These results indicate that the various subgroups of HIV-1-specific inhibitors interact differently with HIV-1 RT, suggesting important potential implications for drug combination therapeutic strategies.
Asunto(s)
Antivirales/farmacología , VIH-1/efectos de los fármacos , Inhibidores de la Transcriptasa Inversa , Línea Celular , Evaluación de Medicamentos , Interacciones Farmacológicas , Farmacorresistencia Microbiana , Transcriptasa Inversa del VIH , Humanos , Fenotipo , Reproducibilidad de los Resultados , Zidovudina/farmacologíaRESUMEN
Studies of the mechanism of action of michellamine B, a novel anti-human immunodeficiency virus (HIV) alkaloid from the tropical plant Ancistrocladus korupensis, have revealed that the compound acts at two distinct stages of the HIV life cycle. The compound had no direct effect on HIV virions and did not block the initial binding of HIV to target cells. Postinfection time course studies revealed that the agent partially inhibited HIV-induced cell killing and syncytium formation when added up to 48 h following acute infection; however, viral reproduction was fully inhibited only when the compound was added immediately after infection. Time-limited treatments of HIV-infected cells revealed that michellamine B had to be present continuously to provide maximum antiviral protection. HIV replication in cells in which infection was already fully established or in chronically infected cells was unaffected by michellamine B. Biochemical studies showed that michellamine B inhibited the enzymatic activities of reverse transcriptases (RTs) from both HIV type 1 and HIV type 2 as well as two different nonnucleoside drug-resistant RTs with specific amino acid substitutions. In addition, human DNA polymerases alpha and beta were inhibited by the alkaloid. Michellamine B exerted a potent dose-dependent inhibition of cell fusion in two independent cell-based fusion assays. Thus, michellamine B acts both at an early stage of the HIV life cycle by inhibiting RT as well as at later stages by inhibiting cellular fusion and syncytium formation.
Asunto(s)
Antivirales/farmacología , VIH/efectos de los fármacos , Isoquinolinas/farmacología , Naftalenos/farmacología , ADN Polimerasa Dirigida por ARN , Fusión Celular/efectos de los fármacos , Línea Celular , Transcriptasa Inversa del VIH , Células HeLa , Humanos , Inhibidores de la Transcriptasa InversaRESUMEN
We have studied the effects of four nonnucleoside inhibitors, including the novel natural product inhibitor calanolide A, on molecular chimeras containing complementary segments of human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2) reverse transcriptases (RTs). All four compounds specifically inhibited the DNA polymerase activity of HIV-1 RT but had no apparent effect on the RNase H activity of this enzyme or on the DNA polymerase or RNase H activity of HIV-2 RT. Three of these compounds showed the generally expected patterns of resistance and susceptibility with the various chimeric RTs. However, the inhibition patterns of the chimeric RTs by calanolide A provided evidence that there is a segment between residues 94 and 157 in HIV-1 RT that is critical for inhibition. However, the data also suggest that there may be a second segment located between amino acids 225 and 427 in HIV-1 RT that is also important for specifying susceptibility to the drug.
Asunto(s)
Antivirales/farmacología , Cumarinas/farmacología , VIH-1/enzimología , VIH-2/enzimología , Proteínas Recombinantes de Fusión/antagonistas & inhibidores , Inhibidores de la Transcriptasa Inversa , Transcriptasa Inversa del VIH , VIH-1/efectos de los fármacos , VIH-2/efectos de los fármacos , Mutagénesis Insercional , Piranocumarinas , ADN Polimerasa Dirigida por ARN/genética , Proteínas Recombinantes de Fusión/genéticaRESUMEN
A number of chemically distinct nonnucleoside inhibitors of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) have been reported. Several lines of evidence, including the isolation of RT mutants that show cross resistance, suggest that, despite their structural diversity, many of these inhibitors bind to a common site on HIV-1 RT. We have recently reported that, on the basis of analyses of HIV-1/HIV-2 chimeras, the natural product calanolide A may interact with a different site or sites in HIV-1 RT. We have used BspMI cassette mutagenesis to prepare a collection of HIV-1 RT mutants that show resistance to the known members of the general class of nonnucleoside inhibitors. This collection of mutants can be used to determine whether a new drug will show cross resistance with known inhibitors and to define amino acid positions critical for the action of the drugs. The mutants were used to analyze calanolide A, 1H,3H-thiazolo[3,4-a]benzimidazole(4i), and the acyclic nucleoside analog 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine. These analyses suggest that all three drugs interact with HIV-1 RT within the previously defined common binding site for nonnucleoside inhibitors. However, the drugs respond differently to the panel of drug-resistant HIV-1 RTs, indicating that while the binding sites of the drugs overlap they are not identical.
Asunto(s)
Antivirales/química , Bencimidazoles/farmacología , Cumarinas/farmacología , VIH/efectos de los fármacos , Inhibidores de la Transcriptasa Inversa , Tiazoles/farmacología , Timina/análogos & derivados , Secuencia de Aminoácidos , Farmacorresistencia Microbiana , VIH/enzimología , Transcriptasa Inversa del VIH , Piranocumarinas , Relación Estructura-Actividad , Timina/farmacologíaRESUMEN
Eight new coumarin compounds (1-8) were isolated by anti-HIV bioassay-guided fractionation of an extract of Calophyllum lanigerum. The structures of calanolide A (1), 12-acetoxycalanolide A (2), 12-methoxycalanolide A (3), calanolide B (4), 12-methoxycalanolide B (5), calanolide C (6) and related derivatives 7 and 8 were solved by extensive spectroscopic analyses, particularly HMQC, HMBC, and difference NOE NMR experiments. The absolute stereochemistry of calanolide A (1) and calanolide B (4) was established by a modified Mosher's method. Calanolides A (1) and B (4) were completely protective against HIV-1 replication and cytopathicity (EC50 values of 0.1 microM and 0.4 microM, respectively), but were inactive against HIV-2. Some of the related compounds also showed evidence of anti-HIV-1 activity. Studies with purified bacterial recombinant reverse transcriptases (RT) revealed that the calanolides are HIV-1 specific RT inhibitors. Moreover, calanolide A was active not only against the AZT-resistant G-9106 strain of HIV-1 but also against the pyridinone-resistant A17 strain. This was of particular interest since the A17 virus is highly resistant to previously known HIV-1 specific, non-nucleoside RT inhibitors (e.g., TIBO; BI-RG-587; L693,593) which comprise a structurally diverse but apparently common pharmacologic class. The calanolides represent a substantial departure from the known class and therefore provide a novel new anti-HIV chemotype for drug development.
Asunto(s)
Antivirales/farmacología , Cumarinas/farmacología , VIH-1/efectos de los fármacos , Árboles/química , Antivirales/química , Antivirales/aislamiento & purificación , Cromatografía Liquida , Cumarinas/química , Cumarinas/aislamiento & purificación , Efecto Citopatogénico Viral/efectos de los fármacos , Farmacorresistencia Microbiana , Transcriptasa Inversa del VIH , VIH-1/enzimología , VIH-1/fisiología , VIH-2/efectos de los fármacos , VIH-2/fisiología , Humanos , Hidrólisis , Espectroscopía de Resonancia Magnética , Piranocumarinas , Inhibidores de la Transcriptasa Inversa , Relación Estructura-Actividad , Células Tumorales Cultivadas , Replicación Viral/efectos de los fármacosRESUMEN
The inosinate dehydrogenase (IMPD) inhibitors ribavirin, tiazofurin and mycophenolic acid were found to stimulate by as much as 20-fold the anabolism of the anti-HIV agent 2' ,3'dideoxyguanosine to its 5'-diphosphate (ddGDP) in a human T-cell culture system (Molt-4 cells). Stimulation of the further conversion to ddGTP (the active form of the drug) was lesser in magnitude but still highly significant (up to 4-fold at appropriate concentrations of ribavirin or tiazofurin). In parallel with these increases, the inhibitors also produced increases of up to 35-fold in IMP levels. These results support the proposal that the initial phosphorylation of ddGuo is catalyzed by a phosphotransferase (5'-nucleotidase) which utilizes IMP as its phosphate donor (Johnson and Fridland, [1989] Molec. Pharmacol. 36, 291-295). Concomitant with this increase in 5'-phosphorylation of ddGuo, an increase in its anti-HIV activity of up to 6.5-fold was observed when this agent was combined with ribavirin (5 microM) in the H9 [corrected] cell assay system.
Asunto(s)
Antivirales/metabolismo , Didesoxinucleósidos/metabolismo , IMP Deshidrogenasa/antagonistas & inhibidores , Ácido Micofenólico/farmacología , Ribavirina/análogos & derivados , Ribavirina/farmacología , Línea Celular , Didesoxinucleósidos/farmacología , VIH-1/efectos de los fármacos , Humanos , Cinética , Nucleótidos/aislamiento & purificación , FosforilaciónRESUMEN
Monocyte/macrophages (M/M) are an important target cell for human immunodeficiency virus (HIV) infection in the body. The study of HIV infection in these cells, however, is rather complicated because they represent a variable population, and because HIV entry and replication in M/M may be markedly influenced by a number of factors. These must be considered in therapeutic approaches to HIV infection. In the present set of experiments, we studied the interaction between certain agents which increase the infection of monocyte/macrophages (M/M) by HIV and two groups of anti-HIV agents: dideoxynucleosides and specific inhibitors of gp120-CD4 binding. We found that the cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF), which markedly enhances HIV replication in M/M, does not affect the activity of recombinant soluble CD4 (sCD4) or OKT4A, two agents which block gp120-CD4 binding. However, it had varying effects on different dideoxynucleosides: GM-CSF increased the net anti-HIV activity of 3'-azido2',3'-dideoxythymidine (AZT), while at the same time it reduced the activity of 2',3'-dideoxycytidine (ddC) and 2',3'-dideoxyinosine (ddI). These effects probably represent an interplay between varying effects of GM-CSF on drug entry and phosphorylation. In additional experiments, we showed that very low concentrations of anti-HIV antibodies could enhance HIV infection of the U937 monocytoid cell line. Interestingly, while this effect has been hypothesized to occur through a CD4-independent mechanism, we found that the anti-HIV activities of both sCD4 and OKT4A were unchanged under conditions of enhancement.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Antivirales/farmacología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Anticuerpos Anti-VIH/inmunología , Infecciones por VIH/tratamiento farmacológico , VIH/efectos de los fármacos , Macrófagos/microbiología , Monocitos/microbiología , Antígenos CD4/metabolismo , Línea Celular , VIH/crecimiento & desarrollo , VIH/inmunología , Proteína gp120 de Envoltorio del VIH/metabolismo , Humanos , Macrófagos/efectos de los fármacos , Monocitos/efectos de los fármacos , Replicación Viral/efectos de los fármacosRESUMEN
We tested the in vitro inhibitory activities of three 2',3'-dideoxynucleosides and two inhibitors of viral binding in combinations against the infectivity and cytopathic effect of human immunodeficiency virus type 1. 3'-Azido-2',3'-dideoxythymidine, 2',3'-dideoxyinosine, or 2',3'-dideoxycytidine, combined with recombinant soluble CD4 (sCD4), brought about synergistic antiretroviral activity without toxicity at clinically achievable concentrations. Combinations of 2',3'-dideoxynucleosides plus dextran sulfate exerted similar synergistic antiviral effects without concomitant increases in toxicities. When sCD4 and dextran sulfate were combined, apparent antagonism was observed. We confirmed that no combination of sCD4 plus 3'-azido-2',3'-dideoxythymidine, 2',3'-dideoxyinosine, or 2',3'-dideoxycytidine significantly increased the inhibitory effect on colony formation of human myeloid-monocytic bone marrow cells in vitro at the concentrations used in this study. These data might have clinical relevance for the treatment of patients infected with human immunodeficiency virus.
Asunto(s)
Antivirales/farmacología , Antígenos CD4/farmacología , Didesoxinucleósidos/farmacología , VIH-1/efectos de los fármacos , Replicación Viral/efectos de los fármacos , Antivirales/toxicidad , Enfermedades de la Médula Ósea/inducido químicamente , Efecto Citopatogénico Viral/efectos de los fármacos , Sulfato de Dextran , Dextranos/farmacología , Didanosina/farmacología , Didesoxinucleósidos/toxicidad , Sinergismo Farmacológico , VIH-1/metabolismo , VIH-1/fisiología , Humanos , Proteínas Recombinantes/farmacologíaRESUMEN
We have previously described the application of an automated microculture tetrazolium assay (MTA) involving dimethyl sulfoxide solubilization of cellular-generated 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-formazan to the in vitro assessment of drug effects on cell growth (M.C. Alley et al., Proc. Am. Assoc. Cancer Res., 27:389, 1986; M.C. Alley et al., Cancer Res. 48:589-601, 1988). There are several inherent disadvantages of this assay, including the safety hazard of personnel exposure to large quantities of dimethyl sulfoxide, the deleterious effects of this solvent on laboratory equipment, and the inefficient metabolism of MTT by some human cell lines. Recognition of these limitations prompted development of possible alternative MTAs utilizing a different tetrazolium reagent, 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl] -2H- tetrazolium hydroxide (XTT), which is metabolically reduced in viable cells to a water-soluble formazan product. This reagent allows direct absorbance readings, therefore eliminating a solubilization step and shortening the microculture growth assay procedure. Most human tumor cell lines examined metabolized XTT less efficiently than MTT; however, the addition of phenazine methosulfate (PMS) markedly enhanced cellular reduction of XTT. In the presence of PMS, the XTT reagent yielded usable absorbance values for growth and drug sensitivity evaluations with a variety of cell lines. Depending on the metabolic reductive capacity of a given cell line, the optimal conditions for a 4-h XTT incubation assay were 50 micrograms of XTT and 0.15 to 0.4 microgram of PMS per well. Drug profiles obtained with representative human tumor cell lines for several standard compounds utilizing the XTT-PMS methodology were similar to the profiles obtained with MTT. Addition of PMS appeared to have little effect on the metabolism of MTT. The new XTT reagent thus provides for a simplified, in vitro cell growth assay with possible applicability to a variety of problems in cellular pharmacology and biology. However, the MTA using the XTT reagent still shares many of the limitations and potential pitfalls of MTT or other tetrazolium-based assays.