RESUMO
The binding affinity and kinetics of target engagement are fundamental to establishing structure-activity relationships (SARs) for prospective therapeutic agents. Enhancing these binding parameters for operative targets, while minimizing binding to off-target sites, can translate to improved drug efficacy and a widened therapeutic window. Compound activity is typically assessed through modulation of an observed phenotype in cultured cells. Quantifying the corresponding binding properties under common cellular conditions can provide more meaningful interpretation of the cellular SAR analysis. Consequently, methods for assessing drug binding in living cells have advanced and are now integral to medicinal chemistry workflows. In this review, we survey key technological advancements that support quantitative assessments of target occupancy in cultured cells, emphasizing generalizable methodologies able to deliver analytical precision that heretofore required reductionist biochemical approaches.
Assuntos
Química Farmacêutica/métodos , Corantes Fluorescentes/química , Ensaios de Triagem em Larga Escala , Técnicas de Sonda Molecular , Terapia de Alvo Molecular/métodos , Técnicas de Transferência de Energia por Ressonância de Bioluminescência , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Genes Reporter , Humanos , Cinética , Imagem Óptica/métodos , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/farmacologia , Relação Estrutura-AtividadeRESUMO
Protein kinases are intensely studied mediators of cellular signaling. While traditional biochemical screens are capable of identifying compounds that modulate kinase activity, these assays are limited in their capability of predicting compound behavior in a cellular environment. Here, we aim to bridge target engagement and compound-cellular phenotypic behavior by utilizing a bioluminescence resonance energy transfer (BRET) assay to characterize target occupancy within living cells for Bruton's tyrosine kinase (BTK). Using a diverse chemical set of BTK inhibitors, we determine intracellular engagement affinity profiles and successfully correlate these measurements with BTK cellular functional readouts. In addition, we leveraged the kinetic capability of this technology to gain insight into in-cell target residence time and the duration of target engagement, and to explore a structural hypothesis.