RESUMEN
HCV NS3/4a protease inhibitors are proven therapeutic agents against chronic hepatitis C virus infection, with boceprevir and telaprevir having recently received regulatory approval as add-on therapy to pegylated interferon/ribavirin for patients harboring genotype 1 infections. Overcoming antiviral resistance, broad genotype coverage, and a convenient dosing regimen are important attributes for future agents to be used in combinations without interferon. In this communication, we report the preclinical profile of MK-5172, a novel P2-P4 quinoxaline macrocyclic NS3/4a protease inhibitor currently in clinical development. The compound demonstrates subnanomolar activity against a broad enzyme panel encompassing major hepatitis C virus (HCV) genotypes as well as variants resistant to earlier protease inhibitors. In replicon selections, MK-5172 exerted high selective pressure, which yielded few resistant colonies. In both rat and dog, MK-5172 demonstrates good plasma and liver exposures, with 24-h liver levels suggestive of once-daily dosing. When administered to HCV-infected chimpanzees harboring chronic gt1a or gt1b infections, MK-5172 suppressed viral load between 4 to 5 logs at a dose of 1 mg/kg of body weight twice daily (b.i.d.) for 7 days. Based on its preclinical profile, MK-5172 is anticipated to be broadly active against multiple HCV genotypes and clinically important resistance variants and highly suited for incorporation into newer all-oral regimens.
Asunto(s)
Hepacivirus/efectos de los fármacos , Inhibidores de Proteasas/farmacología , Quinoxalinas/farmacología , Quinoxalinas/farmacocinética , Proteínas no Estructurales Virales/antagonistas & inhibidores , Amidas , Animales , Antivirales/farmacología , Carbamatos , Ciclopropanos , Perros , Farmacorresistencia Viral , Genotipo , Hepacivirus/enzimología , Hepacivirus/genética , Hepatitis C Crónica/tratamiento farmacológico , Hepatitis C Crónica/virología , Hígado/efectos de los fármacos , Pan troglodytes , Quinoxalinas/metabolismo , Ratas , Sulfonamidas , Carga Viral/efectos de los fármacosRESUMEN
A series of macrocyclic compounds containing a cyclic constraint in the P2-P4 linker region have been discovered and shown to exhibit excellent HCV NS3/4a genotype 3a and genotype 1b R155K, A156T, A156V, and D168V mutant activity while maintaining high rat liver exposure. The effect of the constraint is most dramatic against gt 1b A156 mutants where ~20-fold improvements in potency are achieved by introduction of a variety of ring systems into the P2-P4 linker.
Asunto(s)
Proteínas Portadoras/antagonistas & inhibidores , Hepacivirus/enzimología , Compuestos Macrocíclicos/química , Inhibidores de Proteasas/química , Proteínas no Estructurales Virales/antagonistas & inhibidores , Animales , Sitios de Unión , Proteínas Portadoras/metabolismo , Dominio Catalítico , Ciclización , Genotipo , Semivida , Hepacivirus/genética , Péptidos y Proteínas de Señalización Intracelular , Cinética , Hígado/metabolismo , Compuestos Macrocíclicos/síntesis química , Compuestos Macrocíclicos/farmacocinética , Simulación del Acoplamiento Molecular , Mutación , Inhibidores de Proteasas/síntesis química , Inhibidores de Proteasas/farmacocinética , Ratas , Relación Estructura-Actividad , Proteínas no Estructurales Virales/metabolismoRESUMEN
A series of macrocyclic compounds containing 2-substituted-quinoline moieties have been discovered and shown to exhibit excellent HCV NS3/4a genotype 3a and genotype 1b R155K mutant activity while maintaining the high rat liver exposure. Cyclization of the 2-substituted quinoline substituent led to a series of tricyclic P2 compounds which also display superb gt3a potency.
Asunto(s)
Proteínas Portadoras/antagonistas & inhibidores , Hepacivirus/enzimología , Compuestos Macrocíclicos/química , Inhibidores de Proteasas/química , Proteínas no Estructurales Virales/antagonistas & inhibidores , Animales , Proteínas Portadoras/metabolismo , Ciclización , Genotipo , Semivida , Hepacivirus/genética , Péptidos y Proteínas de Señalización Intracelular , Cinética , Hígado/metabolismo , Compuestos Macrocíclicos/síntesis química , Compuestos Macrocíclicos/farmacocinética , Inhibidores de Proteasas/síntesis química , Inhibidores de Proteasas/farmacocinética , Quinolinas/química , Ratas , Relación Estructura-Actividad , Proteínas no Estructurales Virales/metabolismoRESUMEN
BACKGROUND AND PURPOSE: Diabetes self-management education and support (DSMES) equips patients with diabetes with the knowledge needed for appropriate management. The purpose of this study was to compare perceptions of student confidence/aptitude held by students, pharmacy faculty preceptors, and patients regarding student teaching of a DSMES class. EDUCATIONAL ACTIVITY AND SETTING: The study was a prospective assessment of fourth-year ambulatory care advanced pharmacy practice experience students. Students taught a single DSMES class and evaluated their confidence using a 14-item survey before and after the class. Patients who participated in the class and a pharmacy faculty observer completed the same instrument. Responses were compared using Friedman and Wilcoxon signed-rank tests, as appropriate. FINDINGS: Twenty-six students completed the survey. Overall, students' self-perceived confidence scores significantly increased for all questions after teaching the DSMES class (P < .001 for all questions). Confidence scores among students and faculty preceptors were similar with no significant differences in perceived confidence. There were some significant differences found among student and patient scores, with patients assessing student's perceived self-confidence higher than the students on three items (P < .05). SUMMARY: Student pharmacists' perceptions of their own confidence and abilities improved from before to after teaching a DSMES class. Student and faculty preceptor confidence scores were similar. There were few differences between student and patient confidence scores, with patients rating students highly on their perceived confidence in teaching a DSMES class. Patient feedback is important to consider when evaluating student confidence and abilities.
Asunto(s)
Diabetes Mellitus , Automanejo , Estudiantes de Farmacia , Docentes de Farmacia , Humanos , Percepción , Estudios ProspectivosRESUMEN
Silencing of the integrated human immunodeficiency virus type 1 (HIV-1) genome in resting CD4(+) T cells is a significant contributor to the persistence of infection, allowing the virus to evade both immune detection and pharmaceutical attack. Nonselective histone deacetylase (HDAC) inhibitors are capable of inducing expression of quiescent HIV-1 in latently infected cells. However, potent global HDAC inhibition can induce host toxicity. To determine the specific HDACs that regulate HIV-1 transcription, we evaluated HDAC1 to HDAC11 RNA expression and protein expression and compartmentalization in the resting CD4(+) T cells of HIV-1-positive, aviremic patients. HDAC1, -3, and -7 had the highest mRNA expression levels in these cells. Although all HDACs were detected in resting CD4(+) T cells by Western blot analysis, HDAC5, -8, and -11 were primarily sequestered in the cytoplasm. Using chromatin immunoprecipitation assays, we detected HDAC1, -2, and -3 at the HIV-1 promoter in Jurkat J89GFP cells. Targeted inhibition of HDACs by small interfering RNA demonstrated that HDAC2 and HDAC3 contribute to repression of HIV-1 long terminal repeat expression in the HeLa P4/R5 cell line model of latency. Together, these results suggest that HDAC inhibitors specific for a limited number of class I HDACs may offer a targeted approach to the disruption of persistent HIV-1 infection.
Asunto(s)
Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/virología , Infecciones por VIH/virología , VIH-1/fisiología , Histona Desacetilasas/metabolismo , Citoplasma/metabolismo , Citoplasma/virología , Regulación Viral de la Expresión Génica , Duplicado del Terminal Largo de VIH , VIH-1/genética , Células HeLa , Humanos , Células Jurkat , Regiones Promotoras Genéticas , ARN Mensajero/metabolismo , Transcripción Genética , Latencia del VirusRESUMEN
RNA interference (RNAi) is a modality in which small double-stranded RNA molecules (siRNAs) designed to lead to the degradation of specific mRNAs are introduced into cells or organisms. siRNA libraries have been developed in which siRNAs targeting virtually every gene in the human genome are designed, synthesized and are presented for introduction into cells by transfection in a microtiter plate array. These siRNAs can then be transfected into cells using high-throughput screening (HTS) methodologies. The goal of RNAi HTS is to identify a set of siRNAs that inhibit or activate defined cellular phenotypes. The commonly used analysis methods including median +/- kMAD have issues about error rates in multiple hypothesis testing and plate-wise versus experiment-wise analysis. We propose a methodology based on a Bayesian framework to address these issues. Our approach allows for sharing of information across plates in a plate-wise analysis, which obviates the need for choosing either a plate-wise or experimental-wise analysis. The proposed approach incorporates information from reliable controls to achieve a higher power and a balance between the contribution from the samples and control wells. Our approach provides false discovery rate (FDR) control to address multiple testing issues and it is robust to outliers.
Asunto(s)
Genómica/métodos , Interferencia de ARN , Teorema de Bayes , Biología Computacional/métodos , Simulación por Computador , Genoma Viral , VIH/genética , Células HeLa , Hepacivirus/genética , Humanos , Modelos Genéticos , ARN Interferente Pequeño/análisis , Curva ROCRESUMEN
beta-Site amyloid precursor protein (APP)-cleaving enzyme (BACE) 1 cleavage of amyloid precursor protein is an essential step in the generation of the potentially neurotoxic and amyloidogenic A beta 42 peptides in Alzheimer's disease. Although previous mouse studies have shown brain A beta lowering after BACE1 inhibition, extension of such studies to nonhuman primates or man was precluded by poor potency, brain penetration, and pharmacokinetics of available inhibitors. In this study, a novel tertiary carbinamine BACE1 inhibitor, tertiary carbinamine (TC)-1, was assessed in a unique cisterna magna ported rhesus monkey model, where the temporal dynamics of A beta in cerebrospinal fluid (CSF) and plasma could be evaluated. TC-1, a potent inhibitor (IC(50) approximately 0.4 nM), has excellent passive membrane permeability, low susceptibility to P-glycoprotein transport, and lowered brain A beta levels in a mouse model. Intravenous infusion of TC-1 led to a significant but transient lowering of CSF and plasma A beta levels in conscious rhesus monkeys because it underwent CYP3A4-mediated metabolism. Oral codosing of TC-1 with ritonavir, a potent CYP3A4 inhibitor, twice daily over 3.5 days in rhesus monkeys led to sustained plasma TC-1 exposure and a significant and sustained reduction in CSF sAPP beta, A beta 40, A beta 42, and plasma A beta 40 levels. CSF A beta 42 lowering showed an EC(50) of approximately 20 nM with respect to the CSF [TC-1] levels, demonstrating excellent concordance with its potency in a cell-based assay. These results demonstrate the first in vivo proof of concept of CSF A beta lowering after oral administration of a BACE1 inhibitor in a nonhuman primate.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Precursor de Proteína beta-Amiloide/líquido cefalorraquídeo , Precursor de Proteína beta-Amiloide/antagonistas & inhibidores , Animales , Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Inhibidores Enzimáticos/administración & dosificación , Inhibidores Enzimáticos/farmacología , Humanos , Infusiones Intravenosas , Macaca mulatta , Ratones , Ratones Transgénicos , TransfecciónRESUMEN
Modification of the benzo rings of 3-(1,1-dioxo-2H-(1,2,4)-benzothiadiazin-3-yl)-4-hydroxy-2(1H)-quinolinones into heteroaromatic systems was investigated to enhance physicochemical properties and potency profile of this class of inhibitors. The synthesis and biological activity of the derived compounds is discussed.
Asunto(s)
Química Farmacéutica/métodos , Quinolonas/síntesis química , Proteínas no Estructurales Virales/antagonistas & inhibidores , Antivirales/síntesis química , Antivirales/farmacología , Diseño de Fármacos , Genotipo , Hepacivirus/metabolismo , Humanos , Concentración 50 Inhibidora , Modelos Químicos , Estructura Molecular , Unión Proteica , Quinolonas/farmacología , Relación Estructura-ActividadRESUMEN
The synthesis and optimisation of HCV NS5B polymerase inhibitors with improved potency versus the existing compound 1 is described. Substitution in the benzothiadiazine portion of the molecule, furnishing improvement in potency in the high protein Replicon assay, is highlighted, culminating in the discovery of 12h, a highly potent oxyacetamide derivative.
Asunto(s)
Antivirales/síntesis química , Benzotiadiazinas/química , Química Farmacéutica/métodos , Hepacivirus/enzimología , Proteínas no Estructurales Virales/antagonistas & inhibidores , Administración Oral , Animales , Antivirales/farmacología , Benzotiadiazinas/farmacología , Diseño de Fármacos , Humanos , Concentración 50 Inhibidora , Modelos Químicos , Conformación Molecular , Estructura Molecular , Ratas , Relación Estructura-ActividadRESUMEN
RNA interference (RNAi) not only plays an important role in drug discovery but can also be developed directly into drugs. RNAi high-throughput screening (HTS) biotechnology allows us to conduct genome-wide RNAi research. A central challenge in genome-wide RNAi research is to integrate both experimental and computational approaches to obtain high quality RNAi HTS assays. Based on our daily practice in RNAi HTS experiments, we propose the implementation of 3 experimental and analytic processes to improve the quality of data from RNAi HTS biotechnology: (1) select effective biological controls; (2) adopt appropriate plate designs to display and/or adjust for systematic errors of measurement; and (3) use effective analytic metrics to assess data quality. The applications in 5 real RNAi HTS experiments demonstrate the effectiveness of integrating these processes to improve data quality. Due to the effectiveness in improving data quality in RNAi HTS experiments, the methods and guidelines contained in the 3 experimental and analytic processes are likely to have broad utility in genome-wide RNAi research.
Asunto(s)
Biotecnología/métodos , Genoma , Interferencia de ARN , Apolipoproteína A-I/genética , Biotecnología/normas , Hepacivirus/genética , Control de Calidad , Proyectos de Investigación/normasRESUMEN
Tenascin-C (TN-C) is an extracellular matrix (ECM) protein expressed within remodeling systemic and pulmonary arteries (PAs), where it supports vascular smooth muscle cell (SMC) proliferation. Previously, we showed that A10 SMCs cultivated on native type I collagen possess a spindle-shaped morphology and do not express TN-C, whereas those on denatured collagen possess a well-defined F-actin stress fiber network, a spread morphology, and they do express TN-C. To determine whether changes in cytoskeletal architecture control TN-C, SMCs on denatured collagen were treated with cytochalasin D, which decreased SMC spreading and activation of extracellular signal-regulated kinase 1/2 (ERK1/2), signaling effectors required for TN-C transcription. Next, to determine whether cell shape, dictated by the F-actin cytoskeleton, regulates TN-C, different geometries of SMCs (ranging from spread to round) were engineered on denatured collagen: as SMCs progressively rounded, ERK1/2 activity and TN-C transcription declined. Because RhoA and Rho kinase (ROCK) regulate cell morphology by controlling cytoskeletal architecture, we reasoned that these factors might also regulate TN-C. Indeed, SMCs on denatured collagen possessed higher levels of RhoA activity than those on native collagen, and blocking RhoA or ROCK activities attenuated SMC spreading, ERK1/2 activity, and TN-C expression in SMCs on denatured collagen. Thus, ROCK controls the configuration of the F-actin cytoskeleton and SMC shape in a manner that is permissive for ERK1/2-dependent production of TN-C. Finally, we showed that inhibition of ROCK activity suppresses SMC TN-C expression and disease progression in hypertensive rat PAs. Thus, in addition to its role in regulating vasoconstriction, ROCK also controls matrix production.
Asunto(s)
Matriz Extracelular/metabolismo , Péptidos y Proteínas de Señalización Intracelular/fisiología , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Proteínas Serina-Treonina Quinasas/fisiología , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/farmacología , Actinas/fisiología , Animales , Vasos Sanguíneos/fisiología , Adhesión Celular/fisiología , Forma de la Célula/fisiología , Células Cultivadas , Citoesqueleto/fisiología , Citoesqueleto/ultraestructura , Progresión de la Enfermedad , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Hipertensión/inducido químicamente , Hipertensión/metabolismo , Hipertensión/fisiopatología , Técnicas In Vitro , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Monocrotalina , Músculo Liso Vascular/citología , Músculo Liso Vascular/fisiología , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/fisiología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Arteria Pulmonar/metabolismo , Arteria Pulmonar/fisiopatología , Ratas , Estrés Mecánico , Tenascina/antagonistas & inhibidores , Tenascina/biosíntesis , Tenascina/genética , Tenascina/metabolismo , Transcripción Genética/fisiología , Vasoconstricción/fisiología , Quinasas Asociadas a rho , Proteína de Unión al GTP rhoA/fisiologíaRESUMEN
Recently, we disclosed a new class of HCV polymerase inhibitors discovered through high-throughput screening (HTS) of the GlaxoSmithKline proprietary compound collection. This interesting class of 3-(1,1-dioxo-2H-1,2,4-benzothiadiazin-3-yl)-4-hydroxy-2(1H)-quinolinones potently inhibits HCV polymerase enzymatic activity and inhibits the ability of the subgenomic HCV replicon to replicate in Huh-7 cells. This report will focus on the structure-activity relationships (SAR) of substituents on the quinolinone ring, culminating in the discovery of 1-(2-cyclopropylethyl)-3-(1,1-dioxo-2H-1,2,4-benzothiadiazin-3-yl)-6-fluoro-4-hydroxy-2(1H)-quinolinone (130), an inhibitor with excellent potency in biochemical and cellular assays possessing attractive molecular properties for advancement as a clinical candidate. The potential for development and safety assessment profile of compound 130 will also be discussed.
Asunto(s)
Antivirales/síntesis química , Benzotiadiazinas/síntesis química , Hepacivirus/enzimología , Quinolonas/síntesis química , ARN Polimerasa Dependiente del ARN/antagonistas & inhibidores , Tiadiazinas/síntesis química , Animales , Antivirales/química , Antivirales/farmacología , Benzotiadiazinas/química , Benzotiadiazinas/farmacología , Disponibilidad Biológica , Proteínas Sanguíneas/metabolismo , Línea Celular , Cristalografía por Rayos X , Perros , Genotipo , Semivida , Hepacivirus/genética , Macaca fascicularis , Modelos Moleculares , Estructura Molecular , Mutación , Unión Proteica , Quinolonas/química , Quinolonas/farmacología , ARN Polimerasa Dependiente del ARN/química , Ratas , Ratas Sprague-Dawley , Relación Estructura-Actividad , Tiadiazinas/química , Tiadiazinas/farmacologíaRESUMEN
RNA interference (RNAi) high-throughput screening (HTS) experiments carried out using large (>5000 short interfering [si]RNA) libraries generate a huge amount of data. In order to use these data to identify the most effective siRNAs tested, it is critical to adopt and develop appropriate statistical methods. To address the questions in hit selection of RNAi HTS, we proposed a quartile-based method which is robust to outliers, true hits and nonsymmetrical data. We compared it with the more traditional tests, mean +/- k standard deviation (SD) and median +/- 3 median of absolute deviation (MAD). The results suggested that the quartile-based method selected more hits than mean +/- k SD under the same preset error rate. The number of hits selected by median +/- k MAD was close to that by the quartile-based method. Further analysis suggested that the quartile-based method had the greatest power in detecting true hits, especially weak or moderate true hits. Our investigation also suggested that platewise analysis (determining effective siRNAs on a plate-by-plate basis) can adjust for systematic errors in different plates, while an experimentwise analysis, in which effective siRNAs are identified in an analysis of the entire experiment, cannot. However, experimentwise analysis may detect a cluster of true positive hits placed together in one or several plates, while platewise analysis may not. To display hit selection results, we designed a specific figure called a plate-well series plot. We thus suggest the following strategy for hit selection in RNAi HTS experiments. First, choose the quartile-based method, or median +/- k MAD, for identifying effective siRNAs. Second, perform the chosen method experimentwise on transformed/normalized data, such as percentage inhibition, to check the possibility of hit clusters. If a cluster of selected hits are observed, repeat the analysis based on untransformed data to determine whether the cluster is due to an artifact in the data. If no clusters of hits are observed, select hits by performing platewise analysis on transformed data. Third, adopt the plate-well series plot to visualize both the data and the hit selection results, as well as to check for artifacts.
Asunto(s)
Interferencia de ARN/fisiología , Análisis por Conglomerados , Interpretación Estadística de Datos , Evaluación Preclínica de Medicamentos/métodos , Reacciones Falso Positivas , Biblioteca de Genes , HumanosRESUMEN
The nonstructural protein 3 (NS3) of hepatitis C virus contains a protease domain at its amino terminus and RNA helicase domain at its carboxyl terminus. To identify optimal NS3 protein for developing screening assays, we expressed full-length NS3 protease/helicase and helicase domains from both HCV type 1a (H77 strain) and 1b (Con1 strain), using either E. coli or baculovirus expression systems. Our studies showed that the full-length NS3 proteins, either with or without the presence of the NS4A domain, from either strains were at least 10-fold more efficient than the corresponding helicase domains in unwinding partial duplex RNA substrates. These findings provide a rationale for the use of full-length NS3 in high throughput screening assays to identify potent small molecule inhibitors of this important target of HCV.
Asunto(s)
ARN Helicasas/metabolismo , Serina Endopeptidasas/metabolismo , Proteínas no Estructurales Virales/metabolismo , Baculoviridae/genética , Cromatografía en Gel , Escherichia coli/genética , Estructura Terciaria de Proteína , ARN Helicasas/química , Proteínas Recombinantes/biosíntesis , Proteínas no Estructurales Virales/biosíntesis , Proteínas no Estructurales Virales/químicaRESUMEN
With the goal of identifying inhibitors of hepatitis C virus (HCV) NS3/4a protease that are potent against a wide range of genotypes and clinically relevant mutant viruses, several subseries of macrocycles were investigated based on observations made during the discovery of MK-5172. Quinazolinone-containing macrocycles were identified as promising leads, and optimization for superior cross-genotype and mutant enzyme potency as well as rat liver and plasma concentrations following oral dosing, led to the development of MK-2748. Additional investigation of a series of bis-macrocycles containing a fused 18- and 15-membered ring system were also optimized for the same properties, leading to the discovery of MK-6325. Both compounds display the broad genotype and mutant potency necessary for clinical development as next-generation HCV NS3/4a protease inhibitors.
Asunto(s)
Antivirales/farmacología , Hepacivirus/enzimología , Compuestos Macrocíclicos/farmacología , Quinazolinonas/farmacología , Sulfonas/farmacología , Proteínas no Estructurales Virales/antagonistas & inhibidores , Animales , Antivirales/química , Antivirales/farmacocinética , Cristalografía por Rayos X , Descubrimiento de Drogas , Hepacivirus/efectos de los fármacos , Hepacivirus/genética , Hepatitis C/tratamiento farmacológico , Hepatitis C/virología , Humanos , Compuestos Macrocíclicos/química , Compuestos Macrocíclicos/farmacocinética , Modelos Moleculares , Mutación , Quinazolinonas/química , Quinazolinonas/farmacocinética , Ratas , Sulfonas/farmacocinética , Proteínas no Estructurales Virales/genéticaRESUMEN
The hepatitis C virus (HCV) 3'nontranslated region (3'NTR) is important for virus infection and replicon replication. Here, we constructed a panel of chimera replicons containing non-structural (NS) and 3'NTR sequences from different HCV strains or types, and examined the requirements for stable replication. A subgenomic replicon chimera comprising the polymerase and 3'NTR from HCV strain Con1, and other non-structural genes from type 1a strain H77, supported stable colony formation and replication in Huh7 cells. However, extending the type 1a sequence to include 132 amino acids of NS5B resulted in a defective HCV replicon. In contrast, a similar chimera containing HCV strain J4 sequences linked in cis to Con1 NS5B and 3'NTR supported stable replication suggesting that the interaction between the NS proteins and the 3'NTR may represent a critical determinant. Lastly, the type 1a 3'NTR from pCV-J4L6S was unable to confer replication when paired with non-structural coding sequences from BB7 or J4 and the 3'NTR from Con1 was unable to confer replication when paired with J4 or H77 sequences. These results highlighted the importance of sequence specific interaction among 3'NTR and two distinct subdomains of the NS coding region as a determinant in supporting stable replication of subgenomic replicons. The results underscore the importance of directly cloning 3'NTR sequences from relevant clinical samples.
Asunto(s)
Hepacivirus/genética , Replicón/genética , Regiones no Traducidas 3'/genética , Secuencia de Bases , Carcinoma Hepatocelular , Línea Celular Tumoral , Quimera/genética , Cartilla de ADN , Electroporación , Genoma Viral , Hepacivirus/fisiología , Humanos , Neoplasias Hepáticas , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Reacción en Cadena de la Polimerasa/métodos , ARN Viral/genética , Reproducibilidad de los Resultados , Replicación Viral/genéticaRESUMEN
Human immunodeficiency virus (HIV)-1 depends on the host cell machinery to support its replication. To discover cellular factors associated with HIV-1 replication, we conducted a genome-scale siRNA screen, revealing more than 311 host factors, including 267 that were not previously linked to HIV. Surprisingly, there was little overlap between these genes and the HIV dependency factors described recently. However, an analysis of the genes identified in both screens revealed overlaps in several of the associated pathways or protein complexes, including the SP1/mediator complex and the NF-kappaB signaling pathway. cDNAs for a subset of the identified genes were used to rescue HIV replication following knockdown of the cellular mRNA providing strong evidence that the following six genes are previously uncharacterized host factors for HIV: AKT1, PRKAA1, CD97, NEIL3, BMP2K, and SERPINB6. This study highlights both the power and shortcomings of large scale loss-of-function screens in discovering host-pathogen interactions.
Asunto(s)
Genoma , Infecciones por VIH/genética , VIH-1/fisiología , Interacciones Huésped-Patógeno , Interferencia de ARN , Replicación Viral , Infecciones por VIH/virología , VIH-1/genética , Células HeLa , HumanosRESUMEN
Proteolysis of beta-amyloid precursor protein (APP) into amyloid beta peptide (Abeta) by beta- and gamma-secretases is a critical step in the pathogenesis of Alzheimer's Disease (AD), but the pathways regulating secretases are not fully characterized. Ubiquitinylation, which is dysregulated in AD, may affect APP processing. Here, we describe a screen for APP processing modulators using an siRNA library targeting 532 predicted ubiquitin ligases. Seven siRNA pools diminished Abeta production. Of these, siRNAs targeting PPIL2 (hCyp-60) suppressed beta-site cleavage. Knockdown of PPIL2 mRNA decreased BACE1 mRNA, while overexpression of PPIL2 cDNA enhanced BACE1 mRNA levels. Microarray analysis of PPIL2 or BACE1 knockdown indicated that genes affected by BACE1 knockdown are a subset of those dependent upon PPIL2; suggesting that BACE1 expression is downstream of PPIL2. The association of PPIL2 with BACE expression and its requirement for Abeta production suggests new approaches to discover disease modifying agents for AD.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Ácido Aspártico Endopeptidasas/metabolismo , Genoma/fisiología , Ubiquitina-Proteína Ligasas/metabolismo , Secretasas de la Proteína Precursora del Amiloide/genética , Ácido Aspártico Endopeptidasas/genética , Línea Celular , Ciclofilinas/genética , Ciclofilinas/metabolismo , Expresión Génica/fisiología , Humanos , Luciferasas/metabolismo , Análisis por Micromatrices/métodos , ARN Mensajero/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transfección/métodos , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Rare familial forms of Alzheimer's disease (AD) are thought to be caused by elevated proteolytic production of the Abeta42 peptide from the beta-amyloid-precursor protein (APP). Although the pathogenesis of the more common late-onset AD (LOAD) is not understood, BACE1, the protease that cleaves APP to generate the N terminus of Abeta42, is more active in patients with LOAD, suggesting that increased amyloid production processing might also contribute to the sporadic disease. Using high-throughput siRNA screening technology, we assessed 15,200 genes for their role in Abeta42 secretion and identified leucine-rich repeat transmembrane 3 (LRRTM3) as a neuronal gene that promotes APP processing by BACE1. siRNAs targeting LRRTM3 inhibit the secretion of Abeta40, Abeta42, and sAPPbeta, the N-terminal APP fragment produced by BACE1 cleavage, from cultured cells and primary neurons by up to 60%, whereas overexpression increases Abeta secretion. LRRTM3 is expressed nearly exclusively in the nervous system, including regions affected during AD, such as the dentate gyrus. Furthermore, LRRTM3 maps to a region of chromosome 10 linked to both LOAD and elevated plasma Abeta42, and is structurally similar to a family of neuronal receptors that includes the NOGO receptor, an inhibitor of neuronal regeneration and APP processing. Thus, LRRTM3 is a functional and positional candidate gene for AD, and, given its receptor-like structure and restricted expression, a potential therapeutic target.
Asunto(s)
Enfermedad de Alzheimer , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Ácido Aspártico Endopeptidasas/metabolismo , Proteínas de la Membrana/genética , Proteínas del Tejido Nervioso/genética , Proteínas , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Secretasas de la Proteína Precursora del Amiloide/genética , Péptidos beta-Amiloides/metabolismo , Animales , Ácido Aspártico Endopeptidasas/genética , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Células Cultivadas , Cromosomas Humanos Par 10 , Activación Enzimática , Humanos , Proteínas Repetidas Ricas en Leucina , Proteínas de la Membrana/metabolismo , Ratones , Proteínas del Tejido Nervioso/metabolismo , Neuronas/citología , Neuronas/metabolismo , Proteínas Nucleares , Fragmentos de Péptidos/metabolismo , Proteínas/genética , Proteínas/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismoRESUMEN
Recently, cell-based replicon systems for hepatitis C virus (HCV), in which the nonstructural proteins stably replicate subgenomic viral RNA in Huh7 cells, were developed. To date, one limitation of using these replicon systems to advance drug discovery is the inability of other genotypic derivatives, beyond those of two distinct strains of genotype 1b (HCV-N and Con1), to stably replicate in Huh7 cells. In this report, we evaluated a series of replicon genotype 1a-1b chimeras, as well as a complete genotype 1a replicon clone. A subgenomic replicon construct containing only type 1a sequences failed to generate stable colonies in Huh7 cells even after repeated attempts. Furthermore, addition of an NS5A adaptive mutation (S2204I) which enhances type 1b replicon efficiency was insufficient to confer replication to the wild-type 1a replicon. This subgenomic replicon was subsequently found to be inefficiently translated in Huh7 cells compared to a type 1b replicon, and the attenuation of translation mapped to the N-terminal region of NS3. Therefore, to ensure efficient translation and thereby support replication of the 1a genome, the coding sequence for first 75 residues from type 1a were replaced with the type 1b (strain Con 1) NS3 coding sequence. Although nonstructural proteins were expressed at lower levels with this replicon than with type 1b and although the amount of viral RNA was also severalfold lower (150 copies of positive-strand RNA per cell), the replicon stably replicated in Huh7 cells. Notwithstanding this difference, the ratio of positive- to negative-strand RNA of 26 was similar to that found with the type 1b replicon. Similar results were found for a 1b replicon expressing the type 1a RNA-dependent RNA polymerase. These 1a hybrid replicons maintained sensitivity to alpha interferon (IFN-alpha), albeit with an eightfold-higher 50% inhibitory concentration than type 1b replicons. Evidence is provided herein to confirm that this differential response to IFN-alpha may be attributed directly to the type 1a polymerase.