RESUMEN
Recent advancements in science and engineering are revolutionizing our understanding of an individual's disease, and with this knowledge we are gaining an increasingly sophisticated understanding of how discovery can be transformed to deliver personalized medicines. To reach this future state, we must reengineer our approach to enable the use of more relevant human cellular models earlier in the drug discovery process. Stem cells and primary human cells represent more disease-relevant models than immortalized cell lines; however, due to both availability and cost, their use is limited in lead generation activities. Miniaturization of cellular assays below microtiter plate volumes will enable the use of more relevant cells in screening, but this would require a change in how test molecules are introduced to the biology. With these shifting paradigms, Discovery Supply teams at GlaxoSmithKline (GSK) are modernizing our sample handling approaches. Various emerging technologies such as microarrays, nanowells, and microfluidic devices could bring fundamental changes in conventional sample handling support as we transition from microtiter plates to well-less platforms. The discussion here is exploratory in nature and reviews ongoing proof-of-concept experiments. Our ultimate goal is to industrialize the sample management platforms to support future miniaturized biological assay systems.
Asunto(s)
Técnicas Citológicas/métodos , Descubrimiento de Drogas/métodos , Evaluación Preclínica de Medicamentos/métodos , Células Cultivadas , Ensayos Analíticos de Alto Rendimiento/métodos , Humanos , Técnicas Analíticas Microfluídicas/métodos , Células MadreRESUMEN
Sickle cell anemia (SCA) is a genetic disorder of the ß-globin gene. SCA results in chronic ischemia with pain and tissue injury. The extent of SCA symptoms can be ameliorated by treatment with drugs, which result in increasing the levels of γ-globin in patient red blood cells. Hydroxyurea (HU) is a Food and Drug Administration-approved drug for SCA, but it has dose-limiting toxicity, and patients exhibit highly variable treatment responses. To identify compounds that may lead to the development of better and safer medicines, we have established a method using primary human bone marrow day 7 erythroid progenitor cells (EPCs) to screen for compounds that induce γ-globin production. First, human marrow CD34(+) cells were cultured and expanded for 7 days and characterized for the expression of erythroid differentiation markers (CD71, CD36, and CD235a). Second, fresh or cryopreserved EPCs were treated with compounds for 3 days in 384-well plates followed by γ-globin quantification by an enzyme-linked immunosorbent assay (ELISA), which was validated using HU and decitabine. From the 7408 compounds screened, we identified at least one new compound with confirmed γ-globin-inducing activity. Hits are undergoing analysis in secondary assays. In this article, we describe the method of generating fit-for-purpose EPCs; the development, optimization, and validation of the ELISA and secondary assays for γ-globin detection; and screening results.
Asunto(s)
Evaluación Preclínica de Medicamentos/métodos , Células Precursoras Eritroides/metabolismo , Activación Transcripcional/efectos de los fármacos , gamma-Globinas/genética , Anemia de Células Falciformes/tratamiento farmacológico , Azacitidina/análogos & derivados , Azacitidina/farmacología , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/metabolismo , Ácido Butírico/farmacología , Supervivencia Celular , Metilasas de Modificación del ADN/antagonistas & inhibidores , Metilasas de Modificación del ADN/metabolismo , Decitabina , Ensayo de Inmunoadsorción Enzimática , Epigénesis Genética/efectos de los fármacos , Células Precursoras Eritroides/efectos de los fármacos , Inhibidores de Histona Desacetilasas/farmacología , Humanos , Cultivo Primario de Células , gamma-Globinas/metabolismoRESUMEN
In this study, the development of an image-based high-content screening (HCS) binding assay for the seven-transmembrane (7TM) receptor Smoothened (Smo) is described. Using BacMam-based gene delivery of Smo, BODIPY-cyclopamine as a fluorescent probe, and a confocal imaging system, a robust 384-well assay that could be used for high-throughput compound profiling activities was developed. The statistically robust HCS binding assay was developed through optimization of multiple parameters, including cell transduction conditions, Smo expression levels, the image analysis algorithm, and staining procedures. Evaluation of structurally diverse compounds, including functional Smo activators, inhibitors, and related analogs, demonstrated good compound potency correlations between high-content imaging binding, membrane fluorescence polarization binding, and gene reporter assays. Statistical analysis of data from a screening test set of compounds at a single 10-µM concentration suggested that the high-content imaging Smo binding assay is amenable for use in hit identification. The 384-well HCS assay was rapidly developed and met statistical assay performance targets, thus demonstrating its utility as a fluorescent whole-cell binding assay suitable for compound screening and profiling.
Asunto(s)
Benzamidas/metabolismo , Bencimidazoles/metabolismo , Ciclohexilaminas/metabolismo , Evaluación Preclínica de Medicamentos/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Receptores Acoplados a Proteínas G/metabolismo , Tiofenos/metabolismo , Algoritmos , Baculoviridae/genética , Benzamidas/química , Benzamidas/farmacología , Bencimidazoles/química , Bencimidazoles/farmacología , Línea Celular , Ciclohexilaminas/química , Ciclohexilaminas/farmacología , Colorantes Fluorescentes , Genes Reporteros , Células HEK293 , Humanos , Morfolinas/química , Morfolinas/metabolismo , Morfolinas/farmacología , Piperazinas/química , Piperazinas/metabolismo , Piperazinas/farmacología , Unión Proteica , Purinas/química , Purinas/metabolismo , Purinas/farmacología , Pirazoles/química , Pirazoles/metabolismo , Pirazoles/farmacología , Receptor Smoothened , Tiofenos/química , Tiofenos/farmacologíaRESUMEN
High throughput screening and subsequent optimization led to the discovery of novel quaternary ammonium salts as highly potent muscarinic acetylcholine receptor antagonists with excellent selectivity. Compounds 8a, 13a, and 13b showed excellent inhibitory activity and long duration of action in bronchoconstriction in vivo models in two species via intranasal or intratracheal administration. The novel inhaled muscarinic receptor antagonists are potentially useful therapeutic agents for the treatment of chronic obstructive pulmonary disease and other bronchoconstriction disorders.