RESUMEN
Camel single-domain antibody fragments or Nanobodies, are practical in a wide range of applications. Their unique biochemical and biophysical properties permit an intracellular expression and antigen targeting. The availability of an efficient intracellular selection step would immediately identify the best intracellularly performing functional antibody fragments. Therefore, we assessed a bacterial-two-hybrid system to retrieve such Nanobodies. With GFP as an antigen we demonstrate that antigen-specific Nanobodies of sub-micromolar affinity and stability above 30 kJ/mol, at a titer of 10(-4) can be retrieved in a single-step selection. This was further proven practically by the successful recovery from an 'immune' library of multiple stable, antigen-specific Nanobodies of good affinity for HIV-1 integrase or nucleoside hydrolase. The sequence diversity, intrinsic domain stability, antigen-specificity and affinity of these binders compare favorably to those that were retrieved in parallel by phage display pannings.
Asunto(s)
Camelus/inmunología , Clonación Molecular/métodos , Fragmentos de Inmunoglobulinas/genética , Fragmentos de Inmunoglobulinas/inmunología , Secuencia de Aminoácidos , Animales , Afinidad de Anticuerpos , Camelus/genética , Línea Celular , Escherichia coli/genética , Expresión Génica , Proteínas Fluorescentes Verdes/análisis , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/inmunología , Integrasa de VIH/inmunología , VIH-1/enzimología , Fragmentos de Inmunoglobulinas/química , Fragmentos de Inmunoglobulinas/aislamiento & purificación , Datos de Secuencia Molecular , N-Glicosil Hidrolasas/inmunología , Biblioteca de Péptidos , Estabilidad Proteica , Trypanosoma vivax/enzimologíaRESUMEN
Raltegravir is the first integrase strand-transfer inhibitor (INSTI) approved for use in highly active antiretroviral therapy (HAART) for the management of HIV infection. Resistance to antiretrovirals can compromise the efficacy of HAART regimens. Therefore it is important to understand the emergence of resistance to RAL and cross-resistance to other INSTIs including potential second-generation INSTIs such as MK-2048. We have now studied the question of whether in vitro resistance selection (IVRS) with RAL initiated with viruses derived from clinical isolates would result in selection of resistance mutations consistent with those arising during treatment regimens with HAART containing RAL. Some correlation was observed between the primary mutations selected in vitro and during therapy, initiated with viruses with identical IN sequences. Additionally, phenotypic cross-resistance conferred by specific mutations to RAL and MK-2048 was quantified. N155H, a RAL-associated primary resistance mutation, was selected after IVRS with MK-2048, suggesting similar mechanisms of resistance to RAL and MK-2048. This was confirmed by phenotypic analysis of 766 clonal viruses harboring IN sequences isolated at the point of virological failure from 106 patients on HAART (including RAL), where mutation Q148H/K/R together with additional secondary mutations conferred reduced susceptibility to both RAL and MK-2048. A homology model of full length HIV-1 integrase complexed with viral DNA and RAL or MK-2048, based on an X-ray structure of the prototype foamy virus integrase-DNA complex, was used to explain resistance to RAL and cross-resistance to MK-2048. These findings will be important for the further discovery and profiling of next-generation INSTIs.
Asunto(s)
Farmacorresistencia Viral , Inhibidores de Integrasa VIH/farmacología , VIH-1/efectos de los fármacos , Integrasas/genética , Pirrolidinonas/farmacología , Terapia Antirretroviral Altamente Activa , Línea Celular , Codón/genética , Genotipo , Inhibidores de Integrasa VIH/química , VIH-1/genética , VIH-1/aislamiento & purificación , VIH-1/patogenicidad , Humanos , Integrasas/metabolismo , Pruebas de Sensibilidad Microbiana/métodos , Modelos Moleculares , Estructura Molecular , Mutación , Fenotipo , Plasma/virología , Pirrolidinonas/química , Quinolonas/química , Quinolonas/farmacología , Raltegravir Potásico , TransfecciónRESUMEN
Emergence of resistance to raltegravir reduces its treatment efficacy in HIV-1-infected patients. To delineate the effect of resistance mutations on viral susceptibility to integrase inhibitors, in vitro resistance selections with raltegravir and with MK-2048, an integrase inhibitor with a second-generation-like resistance profile, were performed. Mutation Q148R arose in four out of six raltegravir-selected resistant viruses. In addition, mutations Q148K and N155H were selected. In the same time frame, no mutations were selected with MK-2048. Q148H/K/R and N155H conferred resistance to raltegravir, but only minor changes in susceptibility to MK-2048. V54I, a previously unreported mutation, selected with raltegravir, was identified as a possible compensation mutation. Mechanisms by which N155H, Q148H/K/R, Y143R and E92Q confer resistance are proposed based on a structural model of integrase. These data improve the understanding of resistance against raltegravir and cross-resistance to MK-2048 and other integrase inhibitors, which will aid in the discovery of second-generation integrase inhibitors.
Asunto(s)
Fármacos Anti-VIH/farmacología , Farmacorresistencia Viral , Inhibidores de Integrasa VIH/farmacología , Integrasa de VIH/genética , VIH-1/efectos de los fármacos , Mutación Missense , Pirrolidinonas/farmacología , Sustitución de Aminoácidos/genética , Análisis Mutacional de ADN , Integrasa de VIH/química , VIH-1/genética , Humanos , Modelos Moleculares , Estructura Terciaria de Proteína , Raltegravir PotásicoRESUMEN
Selective delivery of antiretrovirals to human immunodeficiency virus (HIV) infected cells may reduce toxicities associated with long-term highly active antiretroviral therapy (HAART), may improve therapeutic compliance and delay the emergence of resistance. We developed sterically stabilized pegylated liposomes coated with targeting ligands derived from the Fab' fragment of HIV-gp120-directed monoclonal antibody F105, and evaluated these liposomes as vehicles for targeted delivery of a novel HIV-1 protease inhibitor. We demonstrated that the immunoliposomes were selectively taken up by HIV-1-infected cells and localized intracellularly, enabling the establishment of a cytoplasmic reservoir of protease inhibitor. In antiviral experiments, the drug delivered by the immunoliposomes showed greater and longer antiviral activity than comparable concentrations of free drug or drug encapsulated in non-targeted liposomes. In conclusion, by combining a targeting moiety with drug-loaded liposomes, efficient and specific uptake by non-phagocytic HIV-infected cells was facilitated, resulting in drug delivery to infected cells. This approach to targeted delivery of antiretroviral compounds may enable the design of drug regimens for patients that allow increased therapeutic adherence and less toxic treatment of HIV infection.
Asunto(s)
Fármacos Anti-VIH/farmacología , Proteína gp120 de Envoltorio del VIH/metabolismo , Inhibidores de la Proteasa del VIH/farmacología , VIH-1/efectos de los fármacos , Liposomas/metabolismo , Liposomas/farmacología , Replicación Viral/efectos de los fármacos , Línea Celular , Portadores de Fármacos/farmacología , Infecciones por VIH/tratamiento farmacológico , Inhibidores de la Proteasa del VIH/síntesis química , Inhibidores de la Proteasa del VIH/química , VIH-1/metabolismo , VIH-1/fisiología , Humanos , Liposomas/química , Polietilenglicoles/química , Polietilenglicoles/farmacología , Linfocitos T/virologíaRESUMEN
A series of C-5 methyl substituted 4-arylthio- and 4-aryloxy-3-iodopyridin-2(1H)-ones has been synthesized as new pyridinone analogues for their evaluation as anti-HIV inhibitors. The optimization at the 5-position was developed through an efficient use of the key intermediates 5-ethoxycarbonyl- and 5-cyano-pyridin-2(1H)-ones (14 and 15). Biological studies revealed that several compounds show potent HIV-1 reverse transcriptase inhibitory properties, for example, compounds 93 and 99 are active at 0.6-50 nM against wild type HIV-1 and a panel of major simple/double HIV mutant strains.
Asunto(s)
Fármacos Anti-VIH/síntesis química , Fármacos Anti-VIH/farmacología , VIH/efectos de los fármacos , Yodopiridonas/síntesis química , Yodopiridonas/farmacología , Fármacos Anti-VIH/química , Línea Celular , VIH/enzimología , VIH/genética , Transcriptasa Inversa del VIH/antagonistas & inhibidores , Transcriptasa Inversa del VIH/genética , Humanos , Concentración 50 Inhibidora , Yodopiridonas/química , Mutación , Bibliotecas de Moléculas Pequeñas/síntesis química , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacologíaRESUMEN
Integration of viral DNA into the host chromosome is an essential step in the life cycle of retroviruses and is facilitated by the viral integrase enzyme. The first generation of integrase inhibitors recently approved or currently in late-stage clinical trials shows great promise for the treatment of human immunodeficiency virus (HIV) infection, but virus is expected to develop resistance to these drugs. Therefore, we used a novel resistance selection protocol to follow the emergence of resistant HIV in the presence of the integrase inhibitor elvitegravir (GS-9137). We find the primary resistance-conferring mutations to be Q148R, E92Q, and T66I and demonstrate that they confer a reduction in susceptibility not only to elvitegravir but also to raltegravir (MK-0518) and other integrase inhibitors. The locations of the mutations are highlighted in the catalytic sites of integrase, and we correlate the mutations with expected drug-protein contacts. In addition, mutations that do not confer reduced susceptibility when present alone (H114Y, L74M, R20K, A128T, E138K, and S230R) are also discussed in relation to their position in the catalytic core domain and their proximity to known structural features of integrase. These data broaden the understanding of antiviral resistance against integrase inhibitors and may give insight facilitating the discovery of second-generation compounds.
Asunto(s)
Farmacorresistencia Viral , Integrasa de VIH/genética , VIH-1/efectos de los fármacos , VIH-1/genética , Inhibidores de Integrasa/farmacología , Mutación Missense , Quinolonas/farmacología , Dominio Catalítico , Análisis Mutacional de ADN , Integrasa de VIH/química , Humanos , Modelos Moleculares , Estructura MolecularRESUMEN
The development of antiviral assays using an ATP/luminescence-based readout to profile antiviral compounds against the positive-strand RNA viruses: yellow fever virus (YFV), West Nile virus (WNV), Sindbis virus, and Coxsackie B virus, representing three virus families, is described. This assay readout is based upon the bioluminescent measurement of ATP in metabolically active cells. Antiviral efficacy was determined by measuring the ATP level in cells that were protected from the viral cytopathic effect (CPE) by the presence of antiviral compounds. The antiviral assay parameters were optimized and the assays were validated using a panel of different reference compounds to determine the intra- and inter-assay reproducibility. The signal-to-noise ratios for the yellow fever virus and West Nile virus assays were 7.5 and 36, respectively, comparing favorably with a signal-to-noise ratio of only 1.5 in the yellow fever virus neutral red dye uptake assay, an alternative readout for CPE inhibition. For Coxsackie B and Sindbis viruses, the signal-to-noise ratios were 40 and 50, respectively. These assays are robust, high-throughput, reproducible, and give much improved signal-to-noise ratios than those of dye uptake assays.
Asunto(s)
Adenosina Trifosfato/metabolismo , Antivirales/farmacología , Efecto Citopatogénico Viral/efectos de los fármacos , Luminiscencia , Virus ARN/efectos de los fármacos , Virus Sindbis/efectos de los fármacos , Animales , Chlorocebus aethiops , Enterovirus Humano B/efectos de los fármacos , Humanos , Pruebas de Sensibilidad Microbiana/métodos , Virus ARN/clasificación , Reproducibilidad de los Resultados , Células Vero , Virus del Nilo Occidental/efectos de los fármacos , Virus de la Fiebre Amarilla/efectos de los fármacosRESUMEN
The high incidence of cross-resistance between human immunodeficiency virus type 1 (HIV-1) protease inhibitors (PIs) limits their sequential use. This necessitates the development of PIs with a high genetic barrier and a broad spectrum of activity against PI-resistant HIV, such as tipranavir and darunavir (TMC114). We performed a surface plasmon resonance-based kinetic study to investigate the impact of PI resistance-associated mutations on the protease binding of five PIs used clinically: amprenavir, atazanavir, darunavir, lopinavir, and tipranavir. With wild-type protease, the binding affinity of darunavir was more than 100-fold higher than with the other PIs, due to a very slow dissociation rate. Consequently, the dissociative half-life of darunavir was much higher (>240 h) than that of the other PIs, including darunavir's structural analogue amprenavir. The influence of protease mutations on the binding kinetics was tested with five multidrug-resistant (MDR) proteases derived from clinical isolates harboring 10 to 14 PI resistance-associated mutations with a decreased susceptibility to various PIs. In general, all PIs bound to the MDR proteases with lower binding affinities, caused mainly by a faster dissociation rate. For amprenavir, atazanavir, lopinavir, and tipranavir, the decrease in affinity with MDR proteases resulted in reduced antiviral activity. For darunavir, however, a nearly 1,000-fold decrease in binding affinity did not translate into a weaker antiviral activity; a further decrease in affinity was required for the reduced antiviral effect. These observations provide a mechanistic explanation for darunavir's potent antiviral activity and high genetic barrier to the development of resistance.
Asunto(s)
Inhibidores de la Proteasa del VIH/metabolismo , Proteasa del VIH/genética , Proteasa del VIH/metabolismo , VIH-1/efectos de los fármacos , Sulfonamidas/metabolismo , Secuencia de Aminoácidos , Darunavir , Farmacorresistencia Viral Múltiple , Farmacorresistencia Viral , Inhibidores de la Proteasa del VIH/farmacología , VIH-1/enzimología , VIH-1/genética , Humanos , Cinética , Pruebas de Sensibilidad Microbiana/métodos , Datos de Secuencia Molecular , Mutación , Sulfonamidas/farmacologíaRESUMEN
Fusion of host cell and human immunodeficiency virus type 1 (HIV-1) membranes is mediated by the 2 "heptad-repeat" regions of the viral gp41 protein. The collapse of the C-terminal heptad-repeat regions into the hydrophobic grooves of a coiled-coil formed by the corresponding homotrimeric N-terminal heptad-repeat regions generates a stable 6-helix bundle. This brings viral and cell membranes together for membrane fusion, facilitating viral entry. The authors developed an assay based on soluble peptides derived from the gp41 N-terminal heptad-repeat region (IQN36) as well as from the C-terminal region (C34). Both peptides were labeled with fluorophores, IQN36 with allophycocyanin (APC) and C34 with the lanthanide europium (Eu3+). Formation of the 6-helix bundle brings both fluorophores in close proximity needed for Förster resonance energy transfer (FRET). Compounds that interfere with binding of C34-Eu with IQN36-APC suppress the FRET signal. The assay was validated with various peptides and small molecules, and quenching issues were addressed. Evaluation of a diversified compound collection in a high-throughput screening campaign enabled identification of small molecules with different chemical scaffolds that inhibit this crucial intermediate in the HIV-1 entry process. This study's observations substantiate the expediency of time-resolved FRET-based assays to identify small-molecule inhibitors of protein-protein interactions.
Asunto(s)
Antivirales/análisis , Antivirales/farmacología , Transferencia Resonante de Energía de Fluorescencia/métodos , VIH-1/efectos de los fármacos , Pruebas de Sensibilidad Microbiana/métodos , Internalización del Virus/efectos de los fármacos , Secuencia de Aminoácidos , Unión Competitiva , Proteína gp41 de Envoltorio del VIH/química , Modelos Moleculares , Datos de Secuencia Molecular , Fragmentos de Péptidos/análisis , Fragmentos de Péptidos/farmacología , Homología de Secuencia de AminoácidoRESUMEN
Novel diarylpyrimidines (DAPY), which represent next generation of non-nucleoside reverse transcriptase inhibitors (NNRTIs), were synthesized and their activities against human immunodeficiency virus type I (HIV-1) assessed. Modulations at positions 2 and 6 of the left phenyl ring generated interesting derivatives of TMC278 displaying high potency against wild-type and mutant viruses compared to nevirapine and efavirenz. The pharmacokinetic profile of the best newly synthesized DAPY was evaluated and compared with TMC278 now in phase II clinical trials.
Asunto(s)
VIH-1/efectos de los fármacos , Nitrilos/síntesis química , Nitrilos/farmacología , Pirimidinas/química , Inhibidores de la Transcriptasa Inversa/síntesis química , Inhibidores de la Transcriptasa Inversa/farmacología , Animales , Cromatografía Líquida de Alta Presión , Perros , Femenino , Espectroscopía de Resonancia Magnética , Masculino , Espectrometría de Masas , Pirimidinas/síntesis química , Pirimidinas/farmacología , Ratas , Ratas Wistar , Rilpivirina , Espectrofotometría UltravioletaRESUMEN
The use of targeting moieties is a new and exciting field of scientific research for facilitating the specific delivery of therapeutic agents in HIV-infected patients. The interaction of a potential targeting moiety with its ligand is a crucial factor in the evaluation of a targeted approach for chemotherapeutic intervention. Therefore, we have further characterized the interaction between a potential targeting agent, the monoclonal human antibody F105, and its ligand gp120, a glycoprotein expressed on the surface of HIV-1 infected cells. We demonstrate the specificity of binding and entry of F105 to infected cells. F105 was rapidly taken up into the cell and accumulated in the Golgi apparatus. Kinetic analysis of the F105-gp120 interaction revealed an equilibrium dissociation constant (K(D)) of 0.62 nM, compared with the gp120-CD4 interaction where the K(D) was determined at 35 nM. Consequently, F105 displayed a higher gp120 affinity. This was due to a slower dissociation as compared with the natural ligand. These data further underline the potential of monoclonal antibodies as targeting agents, and offer new insights into the possibility of F105 as a targeting moiety for the delivery of antiretroviral drugs to HIV-1 infected cells.
Asunto(s)
Síndrome de Inmunodeficiencia Adquirida/terapia , Anticuerpos Monoclonales/metabolismo , Proteína gp120 de Envoltorio del VIH/inmunología , VIH-1 , Inmunoglobulina G/metabolismo , Cadenas kappa de Inmunoglobulina/metabolismo , Anticuerpos Monoclonales/uso terapéutico , HumanosRESUMEN
BACKGROUND: Resistance to antiretroviral (ARV) drugs constitutes one of the greatest limitations to effective long-term therapy for human immunodeficiency virus (HIV) infection--a problem alleviated, but by no means overcome, by the application of carefully selected, sequential combination regimens. The rational development of novel therapeutics with the ability to suppress viraemia effectively and also address the complex problems of resistance offers a useful way forward for HIV therapy. The investigational drug TMC114 (darunavir) was designed to be active against both wild-type HIV and strains that are resistant to currently available protease inhibitors. SCOPE: This review describes the challenges posed by HIV drug resistance and the novel approach taken in the design and selection of TMC114. Articles were identified by searching MEDLINE in September 2005 (search limits: 1995-2006) using the terms: TMC114, darunavir, resistance, screening, selection, ARV therapy, HIV and HAART. Additional data included bibliographies from identified articles. FINDINGS: With the continuing problem of resistance, ARV drugs must be designed with broad-spectrum activity forming a central part of their development and screening. Drugs must not only be able to treat existing resistant strains, but must also possess more intrinsic resilience to the development of resistance. TMC114 was designed and selected with strong emphasis on potent activity across a range of both wild-type and resistant HIV strains and high binding affinity to HIV protease. CONCLUSION: This review illustrates that future treatment strategies should include screening against multiple-resistant strains to optimize the identification of novel therapeutic agents for the treatment of HIV.
Asunto(s)
Fármacos Anti-VIH/uso terapéutico , Farmacorresistencia Viral , Infecciones por VIH/tratamiento farmacológico , Darunavir , Farmacorresistencia Viral Múltiple , Inhibidores de la Proteasa del VIH/uso terapéutico , Humanos , Sulfonamidas/uso terapéuticoRESUMEN
We have discovered a novel class of human immunodeficiency virus (HIV) reverse transcriptase (RT) inhibitors that block the polymerization reaction in a mode distinct from those of the nucleoside or nucleotide RT inhibitors (NRTIs) and nonnucleoside RT inhibitors (NNRTIs). For this class of indolopyridone compounds, steady-state kinetics revealed competitive inhibition with respect to the nucleotide substrate. Despite substantial structural differences with classical chain terminators or natural nucleotides, these data suggest that the nucleotide binding site of HIV RT may accommodate this novel class of RT inhibitors. To test this hypothesis, we have studied the mechanism of action of the prototype compound indolopyridone-1 (INDOPY-1) using a variety of complementary biochemical tools. Time course experiments with heteropolymeric templates showed "hot spots" for inhibition following the incorporation of pyrimidines (T>C). Moreover, binding studies and site-specific footprinting experiments revealed that INDOPY-1 traps the complex in the posttranslocational state, preventing binding and incorporation of the next complementary nucleotide. The novel mode of action translates into a unique resistance profile. While INDOPY-1 susceptibility is unaffected by mutations associated with NNRTI or multidrug NRTI resistance, mutations M184V and Y115F are associated with decreased susceptibility, and mutation K65R confers hypersusceptibility to INDOPY-1. This resistance profile provides additional evidence for active site binding. In conclusion, this class of indolopyridones can occupy the nucleotide binding site of HIV RT by forming a stable ternary complex whose stability is mainly dependent on the nature of the primer 3' end.
Asunto(s)
Replicación del ADN/efectos de los fármacos , Transcriptasa Inversa del VIH/antagonistas & inhibidores , VIH-1/enzimología , Indoles/farmacología , Nitrilos/farmacología , Piridonas/farmacología , Inhibidores de la Transcriptasa Inversa/farmacología , Cartilla de ADN , Ensayo de Cambio de Movilidad Electroforética , Transcriptasa Inversa del VIH/genética , Indoles/síntesis química , Indoles/química , Cinética , Nitrilos/síntesis química , Nitrilos/química , Piridonas/síntesis química , Piridonas/química , Análisis de Secuencia de ADNRESUMEN
The severity and global spread of the 2003 outbreak of the severe acute respiratory syndrome-associated coronavirus (SARS-CoV) highlighted the risks to human health posed by emerging viral diseases and emphasized the need for specific therapeutic agents instead of relying on existing broadly active antiviral compounds. The development of rapid screening assays is essential for antiviral drug discovery. Thus, a screening system for anti-SARS-CoV agents was developed, which evaluated compound potency, specificity and cytotoxicity at the initial screening phase. Cell lines were engineered to constitutively express an enhanced green fluorescent protein (EGFP) and used to detect (1) antiviral potency in SARS-CoV infection tests; (2) antiviral specificity in tests using the porcine coronavirus transmissible gastroenteritis virus (TGEV); and (3) cytotoxicity in the same assays without virus challenge. The assay system involves minimal manipulation after assay set-up, facilitates automated read-out and minimizes risks associated with hazardous viruses. The suitability of this assay system in drug discovery was demonstrated by screening of 3388 small molecule compounds. The results show that these assays can be applied to high-throughput screening for identification of inhibitors selectively active against SARS-CoV.
Asunto(s)
Antivirales/análisis , Antivirales/farmacología , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/efectos de los fármacos , Antivirales/química , Antivirales/metabolismo , Línea Celular , Evaluación Preclínica de Medicamentos , Proteínas Fluorescentes Verdes , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/fisiología , Replicación Viral/efectos de los fármacosRESUMEN
Human immunodeficiency virus type-1 integrase is an essential enzyme for effective viral replication and hence a valid target for the design of inhibitors. We report here on the design and synthesis of a novel series of phthalimide analogues as integrase inhibitors. The short synthetic pathway enabled us to synthesize a series of analogues with a defined structure diversity. The presence of a single carbonyl-hydroxy-aromatic nitrogen motif was shown to be essential for the enzymatic activity and this was confirmed by molecular docking studies. The enzymatically most active compound from this series is 7-(3,4-dichlorobenzyl)-5,9-dihydroxypyrrolo[3,4-g]quinoxaline-6,8-dione (15l) with an IC(50) value of 112 nM on the HIV-1 integrase enzyme, while ((7-(4-chlorobenzyl)-5,9-dihydroxy-pyrrolo[3,4-g]quinoxaline-6,8-dione (15k)) showed an EC(50) of 270 nM against HIV-1 in a cell-based assay.
Asunto(s)
Fármacos Anti-VIH/síntesis química , Inhibidores de Integrasa VIH/síntesis química , Integrasa de VIH/metabolismo , Compuestos Heterocíclicos con 3 Anillos/síntesis química , Ftalimidas/síntesis química , Pirroles/síntesis química , Quinoxalinas/síntesis química , Fármacos Anti-VIH/química , Fármacos Anti-VIH/farmacología , Sitios de Unión , Línea Celular , Integrasa de VIH/química , Inhibidores de Integrasa VIH/química , Inhibidores de Integrasa VIH/farmacología , VIH-1/efectos de los fármacos , Compuestos Heterocíclicos con 3 Anillos/química , Compuestos Heterocíclicos con 3 Anillos/farmacología , Humanos , Modelos Moleculares , Ftalimidas/química , Ftalimidas/farmacología , Unión Proteica , Pirroles/química , Pirroles/farmacología , Quinoxalinas/química , Quinoxalinas/farmacología , Relación Estructura-ActividadRESUMEN
This paper reports the synthesis and the antiviral properties of new diarylpyrimidine (DAPY) compounds as nonnucleoside reverse transcriptase inhibitors (NNRTIs). The synthesis program around this new DAPY series was further optimized to produce compounds displaying improved activity against a panel of eight clinically relevant single and double mutant strains of human immunodeficiency virus type 1 (HIV-1).
Asunto(s)
Fármacos Anti-VIH/síntesis química , Transcriptasa Inversa del VIH/metabolismo , VIH-1/efectos de los fármacos , Pirimidinas/síntesis química , Inhibidores de la Transcriptasa Inversa/síntesis química , Fármacos Anti-VIH/farmacología , Fármacos Anti-VIH/toxicidad , Línea Celular , Farmacorresistencia Viral , Transcriptasa Inversa del VIH/genética , VIH-1/genética , Humanos , Mutación , Pirimidinas/farmacología , Pirimidinas/toxicidad , Inhibidores de la Transcriptasa Inversa/farmacología , Inhibidores de la Transcriptasa Inversa/toxicidad , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
BACKGROUND: Naturally-occurring polymorphisms at the human immunodeficiency virus type 1 (HIV-1) protease which have been associated to resistance to protease inhibitors (PIs) in clade B viruses are frequently found in non-B subtypes, with unknown clinical significance. OBJECTIVE: To assess the susceptibility of non-B viruses to different PIs. STUDY DESIGN: Plasma samples from 58 drug-naive individuals infected with HIV-1 non-B subtypes (2A, 22C, 2D, 1F, 29G and 2J) defined by phylogenetic analyses of the protease gene were tested using a phenotypic assay (PhenoSense, ViroLogic, South San Francisco, CA, USA). Twenty of them were further analyzed with another assay (Antivirogram, Virco, Mechelen, Belgium). All 58 non-B viruses harbored amino acid substitutions associated with reduced PI susceptibility in clade B (positions 10, 20, 36, 63, 70, 77 and 82). RESULTS: Using PhenoSense-HIV assay, all but two individuals harbored viruses completely susceptible to all six PIs tested (indinavir (IDV), ritonavir (RTV), saquinavir (SQV), nelfinavir (NFV), amprenavir (APV), lopinavir (LPV)). The two viruses with reduced susceptibility belonged to clade G. The first virus, which had K20I, M36I and V82I, showed 2.9-fold decreased susceptibility to APV, while the second virus showed 3.9-fold decreased susceptibility to both NFV and RTV, with amino acid substitutions K20I, M36I, L63P and V82I. Of note, several other viruses displayed the same constellation of mutations but without showing any reduced susceptibility, suggesting that these polymorphisms per se do not affect PI susceptibility. CONCLUSION: PI susceptibility in HIV-1 non-B viruses seems to be preserved despite the presence of polymorphic changes which have been associated to PI resistance in clade B viruses.
Asunto(s)
Inhibidores de la Proteasa del VIH/farmacología , Proteasa del VIH/genética , VIH-1/efectos de los fármacos , VIH-1/enzimología , Polimorfismo Genético , Farmacorresistencia Viral , VIH-1/clasificación , VIH-1/genética , Humanos , Masculino , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Mutación , Fenotipo , Juego de Reactivos para Diagnóstico , Análisis de Secuencia de ADNRESUMEN
The long-term efficacy of making resistance testing routinely available to clinicians has not been established. We conducted a clinical trial at 6 US military hospitals in which volunteers infected with human immunodeficiency virus type-1 were randomized to have routine access to phenotype resistance testing (PT arm), access to genotype resistance testing (GT arm), or no access to either test (VB arm). The primary outcome measure was time to persistent treatment failure despite change(s) in antiretroviral therapy (ART) regimen. Overall, routine access to resistance testing did not significantly increase the time to end point. Time to end point was significantly prolonged in the PT arm for subjects with a history of treatment with > or =4 different ART regimens or a history of treatment with nonnucleoside reverse-transcriptase inhibitors before the study, compared with that in the VB arm. These results suggest that routine access to resistance testing can improve long-term virologic outcomes in HIV-infected patients who are treatment experienced but may not impact outcome in patients who are naive to or have had limited experience with ART.
Asunto(s)
Fármacos Anti-VIH/uso terapéutico , Farmacorresistencia Viral , Infecciones por VIH/tratamiento farmacológico , VIH-1/efectos de los fármacos , Adulto , Terapia Antirretroviral Altamente Activa , Femenino , Humanos , Masculino , Pruebas de Sensibilidad Microbiana , Factores de Tiempo , Insuficiencia del TratamientoRESUMEN
OBJECTIVE: To assess the prevalence of modest (< 10-fold) decreases in baseline non-nucleoside reverse transcriptase inhibitor (NNRTI) susceptibility and their impact on virological response to NNRTI-containing triple therapy in drug-naive individuals. METHODS: Baseline HIV resistance phenotype, genotype and response to therapy were examined retrospectively for all antiretroviral-naive individuals initiating therapy with two nucleoside analogues and an NNRTI in British Columbia, Canada, between 05/1997 and 08/1999 (n = 279), followed until July 31 2001. Time to viral suppression (first of at least two consecutive plasma viral loads < 400 copies HIV RNA copies/ml) and viral rebound (to > or = 400 copies/ml after first pVL < 400 copies HIV RNA copies/ml), were estimated by Kaplan-Meier methods. Multivariate analyses were performed using Cox proportional hazards regression. RESULTS: Nevirapine was the most commonly prescribed NNRTI (96%). Four- to 10-fold decreased susceptibility to NNRTIs was observed in > 30% of untreated individuals at baseline, an observation strongly driven by decreased susceptibility to delavirdine (22.4%). A > 10-fold decrease in susceptibility to any NNRTI was observed only rarely (< 2%). There was no association between four- and 10-fold decreased baseline susceptibility to NNRTIs and virological outcome (P > 0.05). In multivariate analyses, the strongest predictors of poor virological response to NNRTI-based therapy were baseline plasma viral load and the proportion of time on therapy in the first year of follow-up. There was no relationship between the presence of previously reported mutations associated with decreased NNRTI susceptibility (at codons 135 and 283 in HIV reverse transcriptase) and virological response. CONCLUSIONS: These data suggest that the clinically significant level of resistance to NNRTIs, particularly nevirapine, in drug-naive individuals is likely greater than four- to 10-fold.
Asunto(s)
Fármacos Anti-VIH/uso terapéutico , Farmacorresistencia Viral , Infecciones por VIH/tratamiento farmacológico , VIH-1/efectos de los fármacos , Inhibidores de la Transcriptasa Inversa/uso terapéutico , Adulto , Fármacos Anti-VIH/farmacología , Farmacorresistencia Viral/genética , Quimioterapia Combinada , Femenino , Infecciones por VIH/virología , Transcriptasa Inversa del VIH/genética , VIH-1/fisiología , Humanos , Masculino , Pruebas de Sensibilidad Microbiana , Persona de Mediana Edad , Nevirapina/farmacología , Nevirapina/uso terapéutico , ARN Viral/sangre , Inhibidores de la Transcriptasa Inversa/farmacología , Resultado del Tratamiento , Carga ViralRESUMEN
Insertions in the protease gene of HIV-1 were rarely found and are not associated with reduced effectiveness of protease inhibitors although they are thought to be selected by protease inhibitor therapy. This is the first report of a 6-basepair insertion in the protease gene prior to protease inhibitor therapy.