RESUMEN
HIV-1 Vpu targets the host cell proteins CD4 and BST-2/Tetherin for degradation, ultimately resulting in enhanced virus spread and host immune evasion. The discovery and characterization of small molecules that antagonize Vpu would further elucidate the contribution of Vpu to pathogenesis and lay the foundation for the study of a new class of novel HIV-1 therapeutics. To identify novel compounds that block Vpu activity, we have developed a cell-based 'gain of function' assay that produces a positive signal in response to Vpu inhibition. To develop this assay, we took advantage of the viral glycoprotein, GaLV Env. In the presence of Vpu, GaLV Env is not incorporated into viral particles, resulting in non-infectious virions. Vpu inhibition restores infectious particle production. Using this assay, a high throughput screen of >650,000 compounds was performed to identify inhibitors that block the biological activity of Vpu. From this screen, we identified several positive hits but focused on two compounds from one structural family, SRI-41897 and SRI-42371. We developed independent counter-screens for off target interactions of the compounds and found no off target interactions. Additionally, these compounds block Vpu-mediated modulation of CD4, BST-2/Tetherin and antibody dependent cell-mediated toxicity (ADCC). Unfortunately, both SRI-41897 and SRI-42371 were shown to be specific to the N-terminal region of NL4-3 Vpu and did not function against other, more clinically relevant, strains of Vpu; however, this assay may be slightly modified to include more significant Vpu strains in the future.
Asunto(s)
Fármacos Anti-VIH , VIH-1 , Proteínas del Virus de la Inmunodeficiencia Humana , Proteínas Reguladoras y Accesorias Virales , Proteínas Viroporinas , Fármacos Anti-VIH/química , Antígeno 2 del Estroma de la Médula Ósea/metabolismo , Proteínas Ligadas a GPI/metabolismo , VIH-1/metabolismo , Proteínas del Virus de la Inmunodeficiencia Humana/antagonistas & inhibidores , Proteínas del Virus de la Inmunodeficiencia Humana/metabolismo , Virus de la Leucemia del Gibón/metabolismo , Bibliotecas de Moléculas Pequeñas , Proteínas Reguladoras y Accesorias Virales/antagonistas & inhibidores , Proteínas Reguladoras y Accesorias Virales/metabolismo , Proteínas Viroporinas/antagonistas & inhibidoresRESUMEN
Bone marrow stromal cell antigen 2 (BST-2), also known as CD317 or tetherin, has been identified as a host restriction factor that suppresses the release of enveloped viruses from host cells by physically tethering viral particles to the cell surface; however, this host defense can be subverted by multiple viruses. For example, human immunodeficiency virus (HIV)-1 encodes a specific accessory protein, viral protein U (Vpu), to counteract BST-2 by binding to it and directing its lysosomal degradation. Thus, blocking the interaction between Vpu and BST-2 will provide a promising strategy for anti-HIV therapy. Here, we report a NanoLuc Binary Technology (NanoBiT)-based high-throughput screening assay to detect inhibitors that disrupt the Vpu-BST-2 interaction. Out of more than 1000 compounds screened, four inhibitors were identified with strong activity at nontoxic concentrations. In subsequent cell-based BST-2 degradation assays, inhibitor Y-39983 HCl restored the cell-surface and total cellular level of BST-2 in the presence of Vpu. Furthermore, the Vpu-mediated enhancement of pesudotyped viral particle production was inhibited by Y-39983 HCl. Our findings indicate that our newly developed assay can be used for the discovery of potential antiviral molecules with novel mechanisms of action.
Asunto(s)
Fármacos Anti-VIH/farmacología , Infecciones por VIH/tratamiento farmacológico , VIH-1/efectos de los fármacos , Ensayos Analíticos de Alto Rendimiento/métodos , Proteínas del Virus de la Inmunodeficiencia Humana/antagonistas & inhibidores , Dominios y Motivos de Interacción de Proteínas/efectos de los fármacos , Proteínas Reguladoras y Accesorias Virales/antagonistas & inhibidores , Antígenos CD/metabolismo , Proteínas Ligadas a GPI/antagonistas & inhibidores , Proteínas Ligadas a GPI/metabolismo , Infecciones por VIH/metabolismo , Infecciones por VIH/virología , Células HeLa , Proteínas del Virus de la Inmunodeficiencia Humana/metabolismo , Humanos , Nanotecnología/métodos , Proteínas Reguladoras y Accesorias Virales/metabolismo , Replicación ViralRESUMEN
BIT225 is a first-in-class inhibitor of human immunodeficiency virus (HIV) type 1 Vpu. A phase II trial enrolled 36 HIV-1-infected, treatment-naive participants in Thailand to receive standard-of-care antiretroviral therapy (ART), tenofovir disoproxil fumarate/emtricitabine/efavirenz (Atripla), with 100 or 200 mg of BIT225 or placebo (daily) for 12 weeks. Combined treatment with BIT225 and ART was found to be generally safe and well tolerated, with antiviral efficacy comparable to that of ART alone. The secondary end point-soluble CD163, a marker of monocyte/macrophage inflammation-was noted to be significantly decreased in the BIT225 arm. Plasma-derived activated CD4+ and CD8+ T cells, natural killer cells, and interleukin 21 were increased in those treated with BIT225. These findings are consistent with inhibition of the known effects of HIV Vpu and may reflect clinically important modulation of inflammatory and immune function. Further clinical study is planned to both confirm and extend these important findings in treatment-naive, and treatment-experienced individuals. Clinical Trials Registration. Australian New Zealand Clinical Trials Registry (Universal Trial Number U1111-1191-2194).
Asunto(s)
Fármacos Anti-VIH , Combinación Efavirenz, Emtricitabina y Fumarato de Tenofovir Disoproxil , Guanidinas/uso terapéutico , Infecciones por VIH , Pirazoles/uso terapéutico , Fármacos Anti-VIH/uso terapéutico , Australia , Linfocitos T CD4-Positivos , Linfocitos T CD8-positivos , Quimioterapia Combinada , Combinación Efavirenz, Emtricitabina y Fumarato de Tenofovir Disoproxil/uso terapéutico , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/inmunología , VIH-1 , Proteínas del Virus de la Inmunodeficiencia Humana/antagonistas & inhibidores , Humanos , Inflamación/tratamiento farmacológico , Tailandia , Proteínas Reguladoras y Accesorias Virales/antagonistas & inhibidores , Proteínas Viroporinas/antagonistas & inhibidoresRESUMEN
The importance of new effective treatment methodologies for human immunodeficiency virus (HIV) is undeniable for the medical society. Viral protein U (Vpu), one of the disparaged accessory proteins of HIV, is responsible for the dissemination of viral particles, and HIV mutants lacking Vpu protein have remarkably reduced pathogenicity. Here, we explored the marine natural products to find the leading structures which can potentially inhibit the activity of Vpu in silico. To fulfill this goal, we set up a virtual screening based on molecular docking to evaluate the binding capacity of different marine products to Vpu. For validation, we used molecular dynamics simulation and monitored the root mean square deviation value and binding interactions. The results were intriguing when we realized that the hit compounds (phlorotannins) had previously been identified as reverse transcriptase and HIV protease inhibitors. This research inaugurates a new road to combat HIV by multifaceted mode of action of these marine natural products without putting the normal cells in jeopardy (with their safe toxicological profile).
Asunto(s)
Antirretrovirales/química , Organismos Acuáticos/química , Productos Biológicos/química , VIH-1/química , Proteínas del Virus de la Inmunodeficiencia Humana , Simulación del Acoplamiento Molecular , Proteínas Reguladoras y Accesorias Virales , Proteínas del Virus de la Inmunodeficiencia Humana/antagonistas & inhibidores , Proteínas del Virus de la Inmunodeficiencia Humana/química , Humanos , Proteínas Reguladoras y Accesorias Virales/antagonistas & inhibidores , Proteínas Reguladoras y Accesorias Virales/químicaRESUMEN
Novel ethyl 2-(5-aryl-1H-imidazol-1-yl)-acetates 17 and propionates 18, together with their acetic acid 19 and acetohydrazide 20 derivatives, were designed and synthesized using TosMIC chemistry. Biological evaluation of these newly synthesized scaffolds in the HIV-1 Vpu- Host BST-2 ELISA assay identified seven hits (17a, 17b, 17c, 17g, 18a, 20f and 20g) with greater than 50% inhibitory activity. These hits were validated in the HIV-1 Vpu- Host BST-2 AlphaScreen™ and six of the seven compounds were found to have comparable percentage inhibitory activities to those of the ELISA assay. Compounds 17b and 20g, with consistent percentage inhibitory activities across the two assays, had IC50 values of 11.6 ± 1.1 µM and 17.6 ± 0.9 µM in a dose response AlphaScreen™ assay. In a cell-based HIV-1 antiviral assay, compound 17b exhibited an EC50 = 6.3 ± 0.7 µM at non-toxic concentrations (CC50 = 184.5 ± 0.8 µM), whereas compound 20g displayed antiviral activity roughly equivalent to its toxicity (CC50 = 159.5 ± 0.9 µM). This data suggests that compound 17b, active in both cell-based and biochemical assays, provides a good starting point for the design of possible lead compounds for prevention of HIV-1 Vpu and host BST-2 protein binding in new anti-HIV therapeutics.
Asunto(s)
Fármacos Anti-VIH/farmacología , VIH-1/efectos de los fármacos , Proteínas del Virus de la Inmunodeficiencia Humana/antagonistas & inhibidores , Imidazoles/farmacología , Multimerización de Proteína/efectos de los fármacos , Proteínas Reguladoras y Accesorias Virales/antagonistas & inhibidores , Fármacos Anti-VIH/síntesis química , Antígenos CD , Línea Celular , Diseño de Fármacos , Proteínas Ligadas a GPI/antagonistas & inhibidores , VIH-1/química , Humanos , Imidazoles/síntesis química , Pruebas de Sensibilidad Microbiana , Replicación Viral/efectos de los fármacosRESUMEN
Protein-protein interaction (PPI) inhibitors are a rapidly expanding class of therapeutics. Recent advances in our understanding of PPIs and success of early examples of PPI inhibitors demonstrate the feasibility of targeting PPIs. This review summarizes the techniques used for the discovery and optimization of a diverse set PPI inhibitors, focusing on the development of PPI inhibitors as new antibacterial and antiviral agents. We close with a summary of the advances responsible for making PPI inhibitors realistic targets for therapeutic intervention and brief outlook of the field.
Asunto(s)
Antibacterianos/farmacología , Antivirales/farmacología , Enfermedades Transmisibles/tratamiento farmacológico , Inhibidores de Fusión de VIH/farmacología , Inhibidores de Integrasa VIH/farmacología , Proteínas del Virus de la Inmunodeficiencia Humana/antagonistas & inhibidores , Antibacterianos/química , Antivirales/química , Bacterias/efectos de los fármacos , Enfermedades Transmisibles/microbiología , Enfermedades Transmisibles/virología , Inhibidores de Fusión de VIH/química , Inhibidores de Integrasa VIH/química , Interacciones Microbiota-Huesped/efectos de los fármacos , Proteínas del Virus de la Inmunodeficiencia Humana/metabolismo , HumanosRESUMEN
Infections by the human immunodeficiency virus type 1 (HIV-1), the causative agent of the acquired immunodeficiency syndrome (AIDS), are still totaling an appalling 36.7 millions worldwide, with 1.1 million AIDS deaths/year and a similar number of yearly new infections. All this, in spite of the discovery of HIV-1 as the AIDS etiological agent more than 30 years ago and the introduction of an effective combinatorial antiretroviral therapy (cART), able to control disease progression, more than 20 years ago. Although very effective, current cART is plagued by the emergence of drug-resistant viral variants and most of the efforts in the development of novel direct-acting antiviral agents (DAAs) against HIV-1 have been devoted toward the fighting of resistance. In this review, rather than providing a detailed listing of all the drugs and the corresponding resistance mutations, we aim, through relevant examples, at presenting to the general reader the conceptual shift in the approaches that are being taken to overcome the viral resistance hurdle. From the classic 'running faster' strategy, based on the development of novel DAAs active against the mutant viruses selected by the previous drugs and/or presenting to the virus a high genetic barrier toward the development of resilience, to a 'jumping higher' approach, which looks at the cell, rather than the virus, as a source of valuable drug targets, in order to make the cellular environment non-permissive toward the replication of both wild-type and mutated viruses.
Asunto(s)
Fármacos Anti-VIH/uso terapéutico , Diseño de Fármacos , Farmacorresistencia Viral Múltiple , Quimioterapia Combinada , Infecciones por VIH/tratamiento farmacológico , VIH-1/efectos de los fármacos , Modelos Biológicos , Animales , Fármacos Anti-VIH/efectos adversos , Fármacos Anti-VIH/química , Fármacos Anti-VIH/farmacología , Terapia Antirretroviral Altamente Activa/efectos adversos , Antagonistas de los Receptores CCR5/química , Antagonistas de los Receptores CCR5/farmacología , Antagonistas de los Receptores CCR5/uso terapéutico , ARN Helicasas DEAD-box/antagonistas & inhibidores , ARN Helicasas DEAD-box/química , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , Quimioterapia Combinada/efectos adversos , Infecciones por VIH/metabolismo , Infecciones por VIH/virología , Inhibidores de la Proteasa del VIH/efectos adversos , Inhibidores de la Proteasa del VIH/química , Inhibidores de la Proteasa del VIH/farmacología , Inhibidores de la Proteasa del VIH/uso terapéutico , VIH-1/genética , VIH-1/crecimiento & desarrollo , VIH-1/fisiología , Interacciones Huésped-Patógeno/efectos de los fármacos , Proteínas del Virus de la Inmunodeficiencia Humana/antagonistas & inhibidores , Proteínas del Virus de la Inmunodeficiencia Humana/química , Proteínas del Virus de la Inmunodeficiencia Humana/genética , Proteínas del Virus de la Inmunodeficiencia Humana/metabolismo , Humanos , Estructura Molecular , Terapia Molecular Dirigida , Mutación , Conformación Proteica , Inhibidores de la Transcriptasa Inversa/química , Inhibidores de la Transcriptasa Inversa/farmacología , Inhibidores de la Transcriptasa Inversa/uso terapéutico , Fenómenos Fisiológicos de los Virus/efectos de los fármacos , Replicación Viral/efectos de los fármacosRESUMEN
Protein-protein interactions (PPI) are essential in every step of the HIV replication cycle. Mapping the interactions between viral and host proteins is a fundamental target for the design and development of new therapeutics. In this review, we focus on rational development of anti-HIV-1 peptides based on mapping viral-host and viral-viral protein interactions all across the HIV-1 replication cycle. We also discuss the mechanism of action, specificity and stability of these peptides, which are designed to inhibit PPI. Some of these peptides are excellent tools to study the mechanisms of PPI in HIV-1 replication cycle and for the development of anti-HIV-1 drug leads that modulate PPI.
Asunto(s)
Fármacos Anti-VIH/farmacología , Proteínas del Virus de la Inmunodeficiencia Humana/antagonistas & inhibidores , Péptidos/metabolismo , Fármacos Anti-VIH/síntesis química , Fármacos Anti-VIH/química , Proteínas del Virus de la Inmunodeficiencia Humana/metabolismo , Humanos , Unión Proteica/efectos de los fármacos , Replicación Viral/efectos de los fármacosRESUMEN
The continued burden of HIV in resource-limited regions such as parts of sub-Saharan Africa, combined with adverse effects and potential risks of resistance to existing antiretroviral therapies, emphasize the need to identify new HIV inhibitors. Here we performed a virtual screen of molecules from the pan-African Natural Product Library, the largest collection of medicinal plant-derived pure compounds on the African continent. We identified eight molecules with structural similarity to reported interactors of Vpu, an HIV-1 accessory protein with reported ion channel activity. Using in vitro HIV-1 replication assays with a CD4+ T cell line and peripheral blood mononuclear cells, we confirmed antiviral activity and minimal cytotoxicity for two compounds, ixoratannin A-2 and boldine. Notably, ixoratannin A-2 retained inhibitory activity against recombinant HIV-1 strains encoding patient-derived mutations that confer resistance to protease, non-nucleoside reverse transcriptase, or integrase inhibitors. Moreover, ixoratannin A-2 was less effective at inhibiting replication of HIV-1 lacking Vpu, supporting this protein as a possible direct or indirect target. In contrast, boldine was less effective against a protease inhibitor-resistant HIV-1 strain. Both ixoratannin A-2 and boldine also inhibited in vitro replication of hepatitis C virus (HCV). However, BIT-225, a previously-reported Vpu inhibitor, demonstrated antiviral activity but also cytotoxicity in HIV-1 and HCV replication assays. Our work identifies pure compounds derived from African plants with potential novel activities against viruses that disproportionately afflict resource-limited regions of the world.
Asunto(s)
Aporfinas/farmacología , Productos Biológicos/química , VIH-1/efectos de los fármacos , Proantocianidinas/farmacología , Aporfinas/química , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/virología , Línea Celular , Farmacorresistencia Viral , Guanidinas/farmacología , VIH-1/fisiología , Hepacivirus/efectos de los fármacos , Hepacivirus/fisiología , Proteínas del Virus de la Inmunodeficiencia Humana/antagonistas & inhibidores , Proteínas del Virus de la Inmunodeficiencia Humana/metabolismo , Humanos , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/virología , Simulación del Acoplamiento Molecular , Proantocianidinas/química , Pirazoles/farmacología , Proteínas Reguladoras y Accesorias Virales/antagonistas & inhibidores , Proteínas Reguladoras y Accesorias Virales/metabolismo , Replicación Viral/efectos de los fármacosRESUMEN
Rational design of proteins with novel binding specificities and increased affinity is one of the major goals of computational protein design. Epitope-scaffolds are a new class of antigens engineered by transplanting viral epitopes of predefined structure to protein scaffolds, or by building protein scaffolds around such epitopes. Epitope-scaffolds are of interest as vaccine components to attempt to elicit neutralizing antibodies targeting the specified epitope. In this study we developed a new computational protocol, MultiGraft Interface, that transplants epitopes but also designs additional scaffold features outside the epitope to enhance antibody-binding specificity and potentially influence the specificity of elicited antibodies. We employed MultiGraft Interface to engineer novel epitope-scaffolds that display the known epitope of human immunodeficiency virus 1 (HIV-1) neutralizing antibody 2F5 and that also interact with the functionally important CDR H3 antibody loop. MultiGraft Interface generated an epitope-scaffold that bound 2F5 with subnanomolar affinity (K(D) = 400 pM) and that interacted with the antibody CDR H3 loop through computationally designed contacts. Substantial structural modifications were necessary to engineer this antigen, with the 2F5 epitope replacing a helix in the native scaffold and with 15% of the native scaffold sequence being modified in the design stage. This epitope-scaffold represents a successful example of rational protein backbone engineering and protein-protein interface design and could prove useful in the field of HIV vaccine design. MultiGraft Interface can be generally applied to engineer novel binding partners with altered specificity and optimized affinity.
Asunto(s)
Anticuerpos Monoclonales/metabolismo , Anticuerpos Neutralizantes/metabolismo , Anticuerpos Antivirales/metabolismo , Antígenos/química , Regiones Determinantes de Complementariedad/química , Proteínas del Virus de la Inmunodeficiencia Humana/antagonistas & inhibidores , Modelos Moleculares , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/genética , Anticuerpos Neutralizantes/química , Anticuerpos Neutralizantes/genética , Anticuerpos Antivirales/química , Anticuerpos Antivirales/genética , Afinidad de Anticuerpos , Especificidad de Anticuerpos , Antígenos/genética , Antígenos/metabolismo , Anticuerpos ampliamente neutralizantes , Regiones Determinantes de Complementariedad/genética , Regiones Determinantes de Complementariedad/metabolismo , Biología Computacional , Epítopos/química , Epítopos/metabolismo , Anticuerpos Anti-VIH , Proteínas del Virus de la Inmunodeficiencia Humana/química , Proteínas del Virus de la Inmunodeficiencia Humana/genética , Proteínas del Virus de la Inmunodeficiencia Humana/metabolismo , Internet , Datos de Secuencia Molecular , Biblioteca de Péptidos , Conformación Proteica , Ingeniería de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Programas InformáticosRESUMEN
Various drugs found in Chinese herbs are well known for their antiviral potency. We have tested several flavonoids with respect to their potency to block the viral protein U of the human immunodeficiency type 1 virus, which is believed to form a cation-permeable ion channel in the infected cell. We used Xenopus oocytes with heterologously expressed viral protein U as model system to test the efficacy of the drugs in voltage-clamp experiments. This method had been demonstrated in the past as a useful tool to screen drugs for their potency in inhibition of ion channel activity. The viral protein U-mediated current could be inhibited by Ba(2+) with a K1/2 value of 1.6 mM. Therefore, we determined viral protein U-mediated current as current component blocked by 10 mM Ba(2+). We screened several flavonoids with respect to their effects on this current. The flavonols quercetin and kaempferol, and the flavanols (-)epigallochatechin and (-)epichatechin were ineffective. The flavanone naringenin showed at 20 µM slight (about 10%) inhibition. The most potent drug was the isoflavon genistein which exhibited at 20 µM significant inhibition of about 40% with a K1/2 value of 81 ± 4 µM. We suggest that viral ion channels, in general, may be a good target for development of antiviral agents, and that, in particular, isoflavons may be candidates for development of drugs targeting viral protein U.
Asunto(s)
Antivirales/farmacología , Flavanonas/farmacología , Flavonoides/farmacología , Genisteína/farmacología , Infecciones por VIH/tratamiento farmacológico , VIH-1/efectos de los fármacos , Animales , Antivirales/química , Femenino , Flavanonas/química , Flavonoides/química , Expresión Génica , Genisteína/química , Proteínas del Virus de la Inmunodeficiencia Humana/antagonistas & inhibidores , Humanos , Canales Iónicos/efectos de los fármacos , Técnicas de Placa-Clamp , ARN Complementario/genética , Transgenes , Proteínas Reguladoras y Accesorias Virales/antagonistas & inhibidores , Liberación del Virus/efectos de los fármacos , Xenopus laevisRESUMEN
The cellular protein tetherin tethers the HIV-1 viral particles on the cellular membrane to inhibit the replication of HIV-1. However, the HIV-1 accessory protein Vpu counteracts the antiviral function of tetherin. In this study, two retroviral vector plasmids were constructed. One inhibited the vpu gene expression; the other one over-expressed the tetherin. Both retroviral vector plasmids could be packaged in the packaging cell line PT67 to obtain the corresponding retroviruses. The retroviral vector plasmids' functions of tetherin over-expression or vpu-RNAi were detected at the cell level. Retroviral vector plasmids were transfected to PT67 cells at different ratios from 0T3V to 3T0V, and then mixed retroviruses were harvested. The antiviral functions of mixed retroviruses were detected in HIV-1 infected TZM-bl cells. The results showed that packaged mixed retroviruses could repress the replication of HIV-1 in TZM-bl cells.
Asunto(s)
VIH-1/fisiología , Ensamble de Virus , Replicación Viral , Antígenos CD/biosíntesis , Antígenos CD/genética , Línea Celular , Células Epiteliales/virología , Proteínas Ligadas a GPI/biosíntesis , Proteínas Ligadas a GPI/genética , Expresión Génica , Proteínas del Virus de la Inmunodeficiencia Humana/antagonistas & inhibidores , Proteínas del Virus de la Inmunodeficiencia Humana/genética , Humanos , Plásmidos , Proteínas Reguladoras y Accesorias Virales/antagonistas & inhibidores , Proteínas Reguladoras y Accesorias Virales/genéticaRESUMEN
Bone marrow stromal cell antigen 2 (BST-2) inhibits the release of enveloped viruses from the cell surface. Various viral counter measures have been discovered, which allow viruses to escape BST-2 restriction. Human immunodeficiency virus type 1 (HIV-1) encodes viral protein U (Vpu) that interacts with BST-2 through their transmembrane domains and causes the downregulation of cell surface BST-2. In this study, we used a computer modeling method to establish a molecular model to investigate the binding interface of the transmembrane domains of BST-2 and Vpu. The model predicts that the interface is composed of Vpu residues I6, A10, A14, A18, V25, and W22 and BST-2 residues L23, I26, V30, I34, V35, L41, I42, and T45. Introduction of mutations that have been previously reported to disrupt the Vpu-BST-2 interaction led to a calculated higher binding free energy (MMGBSA), which supports our molecular model. A pharmacophore was also generated on the basis of this model. Our results provide a precise model that predicts the detailed interaction occurring between the transmembrane domains of Vpu and BST-2 and should facilitate the design of anti-HIV agents that are able to disrupt this interaction.
Asunto(s)
Antígenos CD/química , Células de la Médula Ósea/química , Técnicas Químicas Combinatorias/métodos , VIH-1/química , Proteínas del Virus de la Inmunodeficiencia Humana/química , Modelos Moleculares , Complejos Multiproteicos/química , Proteínas Reguladoras y Accesorias Virales/química , Antígenos CD/genética , Membrana Celular/química , Membrana Celular/genética , Proteínas Ligadas a GPI/antagonistas & inhibidores , Proteínas Ligadas a GPI/química , Proteínas Ligadas a GPI/genética , Proteínas del Virus de la Inmunodeficiencia Humana/antagonistas & inhibidores , Proteínas del Virus de la Inmunodeficiencia Humana/genética , Humanos , Simulación de Dinámica Molecular , Complejos Multiproteicos/genética , Valor Predictivo de las Pruebas , Unión Proteica/genética , Mapeo de Interacción de Proteínas , Multimerización de Proteína/genética , Estructura Secundaria de Proteína/genética , Células del Estroma/química , Proteínas Reguladoras y Accesorias Virales/antagonistas & inhibidores , Proteínas Reguladoras y Accesorias Virales/genéticaRESUMEN
We examined the antiviral activity of ADAR1 against HIV-1. Our results indicated that ADAR1 in a transfection system inhibited production of viral proteins and infectious HIV-1 in various cell lines including 293T, HeLa, Jurkat T and primary CD4+ T cells, and was active against a number of X4 and R5 HIV-1 of different clades. Further analysis showed that ADAR1 inhibited viral protein synthesis without any effect on viral RNA synthesis. Mutational analysis showed that ADAR1 introduced most of the A-to-G mutations in the rev RNA, in the region of RNA encoding for Rev Response Element (RRE) binding domain and in env RNA. These mutations inhibited the binding of rev to the RRE and inhibited transport of primary transcripts like gag, pol and env from nucleus to cytoplasm resulting in inhibition of viral protein synthesis without any effect on viral RNA synthesis. Furthermore, ADAR1 induced mutations in the env gene inhibited viral infectivity.
Asunto(s)
Adenosina Desaminasa/farmacología , Fármacos Anti-VIH/farmacología , VIH-1/efectos de los fármacos , Proteínas de Unión al ARN/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Secuencia de Bases , Células Cultivadas , Regulación Viral de la Expresión Génica/efectos de los fármacos , Genes env/efectos de los fármacos , Genes env/genética , VIH-1/genética , VIH-1/fisiología , Proteínas del Virus de la Inmunodeficiencia Humana/antagonistas & inhibidores , Proteínas del Virus de la Inmunodeficiencia Humana/biosíntesis , Proteínas del Virus de la Inmunodeficiencia Humana/efectos de los fármacos , Humanos , Datos de Secuencia Molecular , Mutación , Estructura Terciaria de Proteína , ARN Viral/biosíntesis , ARN Viral/efectos de los fármacos , ARN Viral/genética , Proteínas de Unión al ARN/genéticaRESUMEN
Persistence of a reservoir of latently infected memory T cells provides a barrier to HIV eradication in treated patients. Several reports have implicated the involvement of SWI/SNF chromatin remodeling complexes in restricting early steps in HIV infection, in coupling the processes of integration and remodeling, and in promoter/LTR transcription activation and repression. However, the mechanism behind the seemingly contradictory involvement of SWI/SNF in the HIV life cycle remains unclear. Here we addressed the role of SWI/SNF in regulation of the latent HIV LTR before and after transcriptional activation. We determined the predicted nucleosome affinity of the LTR sequence and found a striking reverse correlation when compared to the strictly positioned in vivo LTR nucleosomal structure; sequences encompassing the DNase hypersensitive regions displayed the highest nucleosome affinity, while the strictly positioned nucleosomes displayed lower affinity for nucleosome formation. To examine the mechanism behind this reverse correlation, we used a combinatorial approach to determine DNA accessibility, histone occupancy, and the unique recruitment and requirement of BAF and PBAF, two functionally distinct subclasses of SWI/SNF at the LTR of HIV-infected cells before and after activation. We find that establishment and maintenance of HIV latency requires BAF, which removes a preferred nucleosome from DHS1 to position the repressive nucleosome-1 over energetically sub-optimal sequences. Depletion of BAF resulted in de-repression of HIV latency concomitant with a dramatic alteration in the LTR nucleosome profile as determined by high resolution MNase nucleosomal mapping. Upon activation, BAF was lost from the HIV promoter, while PBAF was selectively recruited by acetylated Tat to facilitate LTR transcription. Thus BAF and PBAF, recruited during different stages of the HIV life cycle, display opposing function on the HIV promoter. Our data point to the ATP-dependent BRG1 component of BAF as a putative therapeutic target to deplete the latent reservoir in patients.
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Proteínas Cromosómicas no Histona/fisiología , Duplicado del Terminal Largo de VIH/genética , VIH-1/fisiología , Proteínas del Virus de la Inmunodeficiencia Humana/fisiología , Nucleosomas/fisiología , Latencia del Virus , Acetilación , Ensamble y Desensamble de Cromatina , Proteínas Cromosómicas no Histona/antagonistas & inhibidores , Proteínas Cromosómicas no Histona/genética , Regulación Viral de la Expresión Génica , VIH-1/genética , Proteínas del Virus de la Inmunodeficiencia Humana/antagonistas & inhibidores , Proteínas del Virus de la Inmunodeficiencia Humana/genética , Humanos , Células Jurkat , Modelos Genéticos , Regiones Promotoras Genéticas , Linfocitos T/virología , Activación Transcripcional , Activación Viral/genética , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismo , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/fisiologíaRESUMEN
Among its many roles, the HIV-1 accessory protein Vpu performs a viroporin function and also antagonizes the host cell restriction factor tetherin through its transmembrane domain. BIT225 is a small molecule inhibitor that specifically targets the Vpu viroporin function, which, in macrophages, resulted in late stage inhibition of virus release and decreased infectivity of released virus, a phenotype similar to tetherin-mediated restriction. Here, we investigated whether BIT225 might mediate its antiviral function, at least in part, via inhibition of Vpu-mediated tetherin antagonism. Using T-cell lines inducible for tetherin expression, we found that BIT225 does not exert its antiviral function by inhibiting Vpu-mediated tetherin downmodulation from the cell surface, the main site of action of tetherin activity. In addition, results from a bioluminescence resonance energy transfer (BRET) assay showed that the Vpu-tetherin interaction was not affected by BIT225. Our data provide support for the concept that tetherin antagonism and viroporin function are separable on the Vpu transmembrane and that viroporin function might be cell-type dependent. Further, this work contributes to the characterization of BIT225 as an inhibitor that specifically targets the viroporin function of Vpu.
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Fármacos Anti-VIH/farmacología , Guanidina/farmacología , Guanidinas/farmacología , VIH-1/fisiología , Proteínas del Virus de la Inmunodeficiencia Humana/antagonistas & inhibidores , Proteínas del Virus de la Inmunodeficiencia Humana/farmacología , Pirazoles/farmacología , Proteínas Reguladoras y Accesorias Virales/antagonistas & inhibidores , Proteínas Reguladoras y Accesorias Virales/farmacología , Antígenos CD/metabolismo , Línea Celular , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Proteínas Ligadas a GPI/antagonistas & inhibidores , Proteínas Ligadas a GPI/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , VIH-1/efectos de los fármacos , VIH-1/metabolismo , Proteínas del Virus de la Inmunodeficiencia Humana/química , Humanos , Estructura Terciaria de Proteína , Proteínas Reguladoras y Accesorias Virales/química , Liberación del Virus/efectos de los fármacosRESUMEN
Bone marrow stromal cell antigen 2 (BST-2, also known as Tetherin) inhibits HIV-1 release and thereby severely impairs viral replication. HIV-1 accessory protein Vpu induces the down-regulation of cell surface BST-2, and counteracts the antiviral function of BST-2. Blocking Vpu-mediated down-regulation of cell surface BST-2 is viewed as a new opportunity for developing anti-HIV drugs. In this study, we have developed a high-throughput cell-based ELISA to identify small molecules that antagonize HIV-1 Vpu function and consequently inhibit HIV-1 replication through rescuing the antiviral activity of host BST-2. This cell-ELISA shows an excellent correlation with results obtained by flow cytometry (FACS). Under optimal conditions, a Z' factor of 0.605 was achieved in a 96-well format. Together, these results demonstrate that this assay can be used to quantify the cell surface level of BST-2 and be adapted to a high-throughput screening for novel anti-HIV compounds.
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Fármacos Anti-VIH/farmacología , Antígenos CD/metabolismo , VIH-1/efectos de los fármacos , Ensayos Analíticos de Alto Rendimiento , Proteínas del Virus de la Inmunodeficiencia Humana , Unión Proteica/efectos de los fármacos , ARN Interferente Pequeño/farmacología , Células del Estroma/efectos de los fármacos , Proteínas Reguladoras y Accesorias Virales , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Membrana Celular/virología , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Proteínas Ligadas a GPI/agonistas , Proteínas Ligadas a GPI/metabolismo , Silenciador del Gen/efectos de los fármacos , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/metabolismo , Infecciones por VIH/patología , Infecciones por VIH/virología , VIH-1/genética , VIH-1/metabolismo , Células HeLa , Proteínas del Virus de la Inmunodeficiencia Humana/antagonistas & inhibidores , Proteínas del Virus de la Inmunodeficiencia Humana/genética , Proteínas del Virus de la Inmunodeficiencia Humana/metabolismo , Humanos , Plásmidos , Reacción en Cadena en Tiempo Real de la Polimerasa , Células del Estroma/citología , Células del Estroma/metabolismo , Transfección , Proteínas Reguladoras y Accesorias Virales/antagonistas & inhibidores , Proteínas Reguladoras y Accesorias Virales/genética , Proteínas Reguladoras y Accesorias Virales/metabolismo , Acoplamiento Viral/efectos de los fármacos , Replicación Viral/efectos de los fármacosRESUMEN
BACKGROUND: Vicriviroc is a next-generation antiretroviral compound that blocks HIV from entering uninfected cells by binding to the virus's cellular co-receptor chemokine receptor 5 (CCR5). A potent inhibitor of HIV infection of human cells both in vitro and in vivo, vicriviroc is in development for use in treatment-naïve HIV-1-infected individuals. These patients often receive antiretroviral therapy regimens that include a ritonavir-enhanced protease inhibitor. Such regimens have a high potential for drug-drug interactions because many of the antiretroviral agents inhibit or induce elements of drug elimination pathways, such as the hepatic cytochromes, which may alter drug concentrations and affect both safety and efficacy. The aim of this set of studies was to determine what, if any, dose adjustments or monitoring would be required to use vicriviroc in regimens containing the most common antiretroviral agents. METHODS: Drug-drug interactions between vicriviroc and 11 other antiretroviral compounds were investigated in fixed-sequence or parallel-group clinical trials lasting 12-35 days. Fixed-sequence studies were conducted with the protease inhibitors atazanavir, darunavir, fosamprenavir, indinavir, nelfinavir, saquinavir and tipranavir. In these studies vicriviroc was administered with ritonavir for a fixed duration, followed by administration of vicriviroc with ritonavir plus the protease inhibitor. Parallel-group studies conducted with lopinavir, zidovudine/lamivudine and tenofovir disoproxil fumarate randomized subjects to receive vicriviroc with or without the study drug. All subjects enrolled in the studies were healthy male and female adults. STATISTICAL METHODS: The log-transformed data for vicriviroc primary pharmacokinetic parameters on appropriate days were statistically analysed using a one-way analysis of variance (ANOVA) model extracting the effects due to treatment. Steady state was evaluated by an ANOVA model on trough concentrations using day and subject as class variables. RESULTS: Vicriviroc exposure was not affected by the concurrently administered antiretroviral drugs in any clinically relevant manner, nor did vicriviroc have a clinically relevant effect on the exposure of other drugs. The drug combinations studied were safe and well tolerated, with most adverse events reported as mild to moderate. Aside from the known toxicities of the other antiretroviral drugs, no clinically relevant changes in blood chemistry, haematological parameters, ECGs or vital signs were associated with either vicriviroc or combination treatment. CONCLUSIONS: No dose modification or monitoring of vicriviroc concentrations is necessary when vicriviroc is co-administered with any of the antiretroviral agents reviewed here. The lack of drug-drug interactions suggests that it will be possible to add vicriviroc at the single clinically prescribed dose level to various background regimens that include a boosted protease inhibitor, with all other drugs also prescribed at their standard doses.
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Antirretrovirales/farmacocinética , Antagonistas de los Receptores CCR5 , Infecciones por VIH/tratamiento farmacológico , Proteínas del Virus de la Inmunodeficiencia Humana/antagonistas & inhibidores , Piperazinas/farmacocinética , Pirimidinas/farmacocinética , Adolescente , Adulto , Antirretrovirales/efectos adversos , Antirretrovirales/sangre , Antirretrovirales/uso terapéutico , Disponibilidad Biológica , Interacciones Farmacológicas , Quimioterapia Combinada/efectos adversos , Femenino , Inhibidores de la Proteasa del VIH/efectos adversos , Inhibidores de la Proteasa del VIH/sangre , Inhibidores de la Proteasa del VIH/farmacocinética , Inhibidores de la Proteasa del VIH/uso terapéutico , Semivida , Humanos , Masculino , Persona de Mediana Edad , Piperazinas/efectos adversos , Piperazinas/sangre , Piperazinas/uso terapéutico , Pirimidinas/efectos adversos , Pirimidinas/sangre , Pirimidinas/uso terapéutico , Ritonavir/farmacología , Adulto JovenRESUMEN
The human immunodeficiency virus (HIV), the causative agent of acquired immunodeficiency syndrome (AIDS), relies heavily on protein-protein interactions in almost every step of its lifecycle. Targeting these interactions, especially those between virus and host proteins, is increasingly viewed as an ideal avenue for the design and development of new therapeutics. In this tutorial review, we outline the lifecycle of HIV and describe some of the protein-protein interactions that control and regulate each step of this process, also detailing efforts to develop therapies that target these interactions.
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VIH/metabolismo , Proteínas del Virus de la Inmunodeficiencia Humana/metabolismo , VIH/enzimología , Proteína gp120 de Envoltorio del VIH/química , Integrasa de VIH/química , Proteasa del VIH/química , Proteasa del VIH/genética , Transcriptasa Inversa del VIH/química , Proteínas del Virus de la Inmunodeficiencia Humana/antagonistas & inhibidores , Humanos , Unión Proteica , Replicación ViralRESUMEN
Ion channels are membrane proteins that are found in a number of viruses and which are of crucial physiological importance in the viral life cycle. They have one common feature in that their action mode involves a change of electrochemical or proton gradient across the bilayer lipid membrane which modulates viral or cellular activity. We will discuss a group of viral channel proteins that belong to the viroproin family, and which participate in a number of viral functions including promoting the release of viral particles from cells. Blocking these channel-forming proteins may be "lethal", which can be a suitable and potential therapeutic strategy. In this review we discuss seven ion channels of viruses which can lead serious infections in human beings: M2 of influenza A, NB and BM2 of influenza B, CM2 of influenza C, Vpu of HIV-1, p7 of HCV and 2B of picornaviruses.