RESUMEN
BACKGROUND: Human respiratory syncytial virus (hRSV) is a highly contagious pathogen and is the most common cause of bronchiolitis and pneumonia for infants and children under one year of age. Worldwide, greater than 33 million children under five years of age are affected by hRSV resulting in three million hospitalizations and 200,000 deaths. However, severe lower respiratory tract disease may occur at any age, especially among the elderly or those with compromised cardiac, pulmonary, or immune systems. There is no vaccine commercially available. Existing therapies for the acute infection are ribavirin and the prophylactic humanized monoclonal antibody (Synagis® from MedImmune) that is limited to use in high risk pediatric patients. Thus, the discovery of new inhibitors for hRSV would be clinically beneficial. RESULTS: We have developed and validated a 384-well cell-based, high-throughput assay that measures the cytopathic effect of hRSV (strain Long) in HEp-2 cells using a luminescent-based detection system for signal endpoint (Cell Titer Glo®). The assay is sensitive and robust, with Z factors greater than 0.8, signal to background greater than 35, and signal to noise greater than 24. Utilizing this assay, 313,816 compounds from the Molecular Libraries Small Molecule Repository were screened at 10 µM. We identified 7,583 compounds that showed greater than 22% CPE inhibition in the primary screen. The top 2,500 compounds were selected for confirmation screening and 409 compounds showed at least 50% inhibition of CPE and were considered active. We selected fifty-one compounds, based on potency, selectivity and chemical tractability, for further evaluation in dose response and secondary assays Several compounds had SI50 values greater than 3, while the most active compound displayed an SI50 value of 58.9. CONCLUSIONS: A robust automated luminescent-based high throughput screen that measures the inhibition of hRSV-induced cytopathic effect in HEp-2 cells for the rapid identification of potential inhibitors from large compound libraries has been developed, optimized and validated. The active compounds identified in the screen represent different classes of molecules, including aryl sulfonylpyrrolidines which have not been previously identified as having anti-hRSV activity.
Asunto(s)
Antivirales/aislamiento & purificación , Descubrimiento de Drogas/métodos , Ensayos Analíticos de Alto Rendimiento , Virus Sincitial Respiratorio Humano/efectos de los fármacos , Automatización de Laboratorios/métodos , Efecto Citopatogénico Viral/efectos de los fármacos , Células Hep G2 , Hepatocitos/virología , Humanos , Mediciones Luminiscentes , PotexvirusRESUMEN
A Rh/tetramethylcyclopentadienyl complex containing a tethered functionality has been demonstrated to give excellent results in the asymmetric transfer hydrogenation of ketones in both aqueous and formic acid/triethylamine media.
RESUMEN
[reaction: see text] A rhodium(III) catalyst for asymmetric transfer hydrogenation of ketones has been designed. The incorporation of a tethering group between the diamino group and the cyclopentadienyl unit provides extra stereochemical rigidity. The catalyst is capable of enantioselective reduction of a range of ketones in excellent ee using formic acid/triethylamine as both the solvent and the reducing agent.
RESUMEN
Protein-protein interactions are generally challenging to target by small molecules. To address the challenge, we have used a multidisciplinary approach to identify small-molecule disruptors of protein-protein interactions that are mediated by SUMO (small ubiquitin-like modifier) proteins. SUMO modifications have emerged as a target with importance in treating cancer, neurodegenerative disorders, and viral infections. It has been shown that inhibiting SUMO-mediated protein-protein interactions can sensitize cancer cells to chemotherapy and radiation. We have developed highly sensitive assays using time-resolved fluorescence resonance energy transfer (TR-FRET) and fluorescence polarization (FP) that were used for high-throughput screening (HTS) to identify inhibitors for SUMO-dependent protein-protein interactions. Using these assays, we have identified a nonpeptidomimetic small molecule chemotype that binds to SUMO1 but not SUMO2 or 3. NMR chemical shift perturbation studies have shown that the compounds of this chemotype bind to the SUMO1 surface required for protein-protein interaction, despite the high sequence similarity of SUMO1 and SUMO2 and 3 at this surface.
Asunto(s)
Transferencia Resonante de Energía de Fluorescencia , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/química , Secuencias de Aminoácidos , Sitios de Unión , Ligandos , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Unión Proteica , Conformación ProteicaRESUMEN
A quinazolinedione-derived screening hit 2 was discovered with cellular antiviral activity against respiratory syncytial virus (CPE EC50 = 2.1 µM), moderate efficacy in reducing viral progeny (4.2 log at 10 µM), and marginal cytotoxic liability (selectivity index, SI â¼ 24). Scaffold optimization delivered analogs with improved potency and selectivity profiles. Most notable were compounds 15 and 19 (EC50 = 300-500 nM, CC50 > 50 µM, SI > 100), which significantly reduced viral titer (>400,000-fold), and several analogs were shown to block the activity of the RNA-dependent RNA-polymerase complex of RSV.
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Antivirales/farmacología , Benzamidas/farmacología , Proliferación Celular/efectos de los fármacos , Quinazolinonas/farmacología , ARN Polimerasa Dependiente del ARN/antagonistas & inhibidores , Infecciones por Virus Sincitial Respiratorio/tratamiento farmacológico , Virus Sincitiales Respiratorios/efectos de los fármacos , Replicación Viral/efectos de los fármacos , Antivirales/síntesis química , Benzamidas/síntesis química , Diseño de Fármacos , Células Hep G2 , Humanos , Concentración 50 Inhibidora , Modelos Moleculares , Estructura Molecular , Quinazolinonas/síntesis química , Quinazolinonas/química , ARN Polimerasa Dependiente del ARN/metabolismo , Infecciones por Virus Sincitial Respiratorio/virología , Relación Estructura-ActividadRESUMEN
The authors conducted a high-throughput screening campaign for inhibitors of SV40 large T antigen ATPase activity to identify candidate antivirals that target the replication of polyomaviruses. The primary assay was adapted to 1536-well microplates and used to screen the National Institutes of Health Molecular Libraries Probe Centers Network library of 306 015 compounds. The primary screen had an Z value of ~0.68, signal/background = 3, and a high (5%) DMSO tolerance. Two counterscreens and two secondary assays were used to prioritize hits by EC(50), cytotoxicity, target specificity, and off-target effects. Hits that inhibited ATPase activity by >44% in the primary screen were tested in dose-response efficacy and eukaryotic cytotoxicity assays. After evaluation of hit cytotoxicity, drug likeness, promiscuity, and target specificity, three compounds were chosen for chemical optimization. Chemical optimization identified a class of bisphenols as the most effective biochemical inhibitors. Bisphenol A inhibited SV40 large T antigen ATPase activity with an IC(50) of 41 µM in the primary assay and 6.2 µM in a cytoprotection assay. This compound class is suitable as probes for biochemical investigation of large T antigen ATPase activity, but because of their cytotoxicity, further optimization is necessary for their use in studying polyomavirus replication in vivo.
Asunto(s)
Adenosina Trifosfatasas/antagonistas & inhibidores , Antígenos Transformadores de Poliomavirus/metabolismo , Ensayos Analíticos de Alto Rendimiento/métodos , Fenoles/farmacología , Animales , Antivirales/farmacología , Compuestos de Bencidrilo , Línea Celular , Chlorocebus aethiops , Perros , Evaluación Preclínica de Medicamentos , Inhibidores Enzimáticos/farmacología , Poliomavirus/enzimología , Bibliotecas de Moléculas Pequeñas/análisisRESUMEN
A high-throughput, cell-based screen was used to identify chemotypes as inhibitors for human respiratory syncytial virus (hRSV). Optimization of a sulfonylpyrrolidine scaffold resulted in compound 5o that inhibited a virus-induced cytopathic effect in the entry stage of infection (EC50 = 2.3 ± 0.8 µM) with marginal cytotoxicity (CC50 = 30.9 ± 1.1 µM) and reduced viral titer by 100-fold. Compared to ribavirin, sulfonylpyrrolidine 5o demonstrated an improved in vitro potency and selectivity index.
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Antivirales/síntesis química , Pirrolidinas/síntesis química , Quinolinas/síntesis química , Virus Sincitiales Respiratorios/efectos de los fármacos , Sulfonamidas/síntesis química , Sulfonas/síntesis química , Antivirales/química , Antivirales/farmacología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Efecto Citopatogénico Viral/efectos de los fármacos , Ensayos Analíticos de Alto Rendimiento , Humanos , Pirrolidinas/química , Pirrolidinas/farmacología , Quinolinas/química , Quinolinas/farmacología , Virus Sincitiales Respiratorios/fisiología , Ribavirina/farmacología , Estereoisomerismo , Relación Estructura-Actividad , Sulfonamidas/química , Sulfonamidas/farmacología , Sulfonas/química , Sulfonas/farmacología , Carga Viral/efectos de los fármacos , Internalización del Virus/efectos de los fármacos , Replicación Viral/efectos de los fármacosRESUMEN
Rh(III) catalysts containing a tetramethylcyclopentadienyl group linked by a 'tether' to a tosylated diamine ligand have previously been reported by our group for the asymmetric transfer hydrogenation (ATH) of ketones. The extension of these catalysts to the asymmetric reduction of imines, as well as to more highly functionalized substrates is reported. In some cases, the catalysts give better ee values than other methods for these transformations at lower catalyst loadings. The introduction of a methoxy group into the tethering aryl ring does not negate the performance of the catalyst, thus opening up a route to supported derivatives.