RESUMO
In order to identify potential cytochrome P-450 3A4 (drug-metabolizing enzyme) inducers at an early stage of the drug discovery process, a cell-based transactivation high-throughput luciferase reporter assay for the human pregnane X receptor (PXR) in HepG2 cells has been implemented and multiplexed with a viability end point for data interpretation, as part of a Lead Profiling portfolio of assays. As a routine part of Lead Profiling operations, assays are periodically evaluated for utility as well as for potential improvements in technology or process. We used a recent evaluation of our PXR-transactivation assay as a model for the application of Lean Thinking-based process analysis to lab-bench assay optimization and automation. This resulted in the development of a 384-well multiplexed homogeneous assay simultaneously detecting PXR transactivation and HepG2 cell cytotoxicity. In order to multiplex fluorescent and luminescent read-outs, modifications to each assay were necessary, which included optimization of multiple assay parameters such as cell density, plate type, and reagent concentrations. Subsequently, a set of compounds including known cytotoxic compounds and PXR inducers were used to validate the multiplexed assay. Results from the multiplexed assay correlate well with those from the singleplexed assay formats measuring PXR transactivation and viability separately. Implementation of the multiplexed assay for routine compound profiling provides improved data quality, sample conservation, cost savings, and resource efficiencies.
Assuntos
Citocromo P-450 CYP3A/biossíntese , Avaliação Pré-Clínica de Medicamentos/métodos , Linhagem Celular Tumoral , Criopreservação , Relação Dose-Resposta a Droga , Eficiência , Indução Enzimática/efeitos dos fármacos , Humanos , Luciferases/genética , Luciferases/metabolismo , Receptor de Pregnano X , Receptores de Esteroides/biossíntese , Receptores de Esteroides/genética , Padrões de Referência , Reprodutibilidade dos Testes , Relação Estrutura-Atividade , TransfecçãoRESUMO
We review strategic approaches taken over an eight-year period at BMS to implement new high-throughput approaches to lead discovery. Investments in compound management infrastructure and chemistry library production capability allowed significant growth in the size, diversity and quality of the BMS compound collection. Screening platforms were upgraded with robust automated technology to support miniaturized assay formats, while workflows and information handling technologies were streamlined for improved performance. These technology changes drove the need for a supporting organization in which critical engineering, informatics and scientific skills were more strongly represented. Taken together, these investments led to significant improvements in speed and productivity as well a greater impact of screening campaigns on the initiation of new drug discovery programs.
Assuntos
Desenho de Fármacos , Indústria Farmacêutica/economia , Tecnologia Farmacêutica/métodos , Gastos de Capital , Investimentos em Saúde , Setor Privado , Tecnologia Farmacêutica/economiaRESUMO
The application of parallel synthesis to lead optimization programs in drug discovery has been an ongoing challenge since the first reports of library synthesis. A number of approaches to the application of parallel array synthesis to lead optimization have been attempted over the years, ranging from widespread deployment by (and support of) individual medicinal chemists to centralization as a service by an expert core team. This manuscript describes our experience with the latter approach, which was undertaken as part of a larger initiative to optimize drug discovery. In particular, we highlight how concepts taken from the manufacturing sector can be applied to drug discovery and parallel synthesis to improve the timeliness and thus the impact of arrays on drug discovery.