RESUMO
We previously developed a panel of one-step real-time quantitative reverse transcription PCR (one-step qRT-PCR; hereafter referred to as qRT-PCR) assays to assess compound efficacy. However, these high-cost, conventional qRT-PCR manual assays are not amenable to high-throughput screen (HTS) analysis in a time-sensitive and complex drug discovery process. Here, we report the establishment of an automated gene expression platform using in-house lysis conditions that allows the study of various cell lines, including primary T cells. This process innovation provides the opportunity to perform genotypic profiling in both immunology and oncology therapeutic areas with quantitative studies as part of routine drug discovery program support. This newly instituted platform also enables a panel screening strategy to efficiently connect HTS, lead identification, and lead optimization in parallel.
Assuntos
Automação Laboratorial/normas , Perfilação da Expressão Gênica/normas , Ensaios de Triagem em Larga Escala/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/imunologia , Automação Laboratorial/instrumentação , Proteína 3 com Repetições IAP de Baculovírus/genética , Proteína 3 com Repetições IAP de Baculovírus/imunologia , Linhagem Celular Tumoral , Quimiocina CCL3/genética , Quimiocina CCL3/imunologia , Descoberta de Drogas/métodos , Perfilação da Expressão Gênica/instrumentação , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica , Células HCT116 , Ensaios de Triagem em Larga Escala/instrumentação , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/imunologia , Osteoblastos/citologia , Osteoblastos/metabolismo , Cultura Primária de Células , Reação em Cadeia da Polimerase em Tempo Real/normas , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/imunologia , Linfócitos T/citologia , Linfócitos T/metabolismoRESUMO
High-throughput screening (HTS) is a widespread method in early drug discovery for identifying promising chemical matter that modulates a target or phenotype of interest. Because HTS campaigns involve screening millions of compounds, it is often desirable to initiate screening with a subset of the full collection. Subsequently, virtual screening methods prioritize likely active compounds in the remaining collection in an iterative process. With this approach, orthogonal virtual screening methods are often applied, necessitating the prioritization of hits from different approaches. Here, we introduce a novel method of fusing these prioritizations and benchmark it prospectively on 17 screening campaigns using virtual screening methods in three descriptor spaces. We found that the fusion approach retrieves 15% to 65% more active chemical series than any single machine-learning method and that appropriately weighting contributions of similarity and machine-learning scoring techniques can increase enrichment by 1% to 19%. We also use fusion scoring to evaluate the tradeoff between screening more chemical matter initially in lieu of replicate samples to prevent false-positives and find that the former option leads to the retrieval of more active chemical series. These results represent guidelines that can increase the rate of identification of promising active compounds in future iterative screens.
Assuntos
Avaliação Pré-Clínica de Medicamentos , Heurística , Interface Usuário-Computador , Aprendizado de MáquinaRESUMO
Combination therapies that enhance efficacy or permit reduced dosages to be administered have seen great success in a variety of therapeutic applications. More fundamentally, the discovery of epistatic pathway interactions not only informs pharmacologic intervention but can be used to better understand the underlying biological system. There is, however, no systematic and efficient method to identify interacting activities as candidates for combination therapy and, in particular, to identify those with synergistic activities. We devised a pooled, self-deconvoluting screening paradigm for the efficient comprehensive interrogation of all pairs of compounds in 1000-compound libraries. We demonstrate the power of the method to recover established synergistic interactions between compounds. We then applied this approach to a cell-based screen for anti-inflammatory activities using an assay for lipopolysaccharide/interferon-induced acute phase response of a monocytic cell line. The described method, which is >20 times as efficient as a naïve approach, was used to test all pairs of 1027 bioactive compounds for interleukin-6 suppression, yielding 11 pairs of compounds that show synergy. These 11 pairs all represent the same two activities: ß-adrenergic receptor agonists and phosphodiesterase-4 inhibitors. These activities both act through cyclic AMP elevation and are known to be anti-inflammatory alone and to synergize in combination. Thus we show proof of concept for a robust, efficient technique for the identification of synergistic combinations. Such a tool can enable qualitatively new scales of pharmacological research and chemical genetics.