RESUMEN
Recent advances in targeted protein degradation have enabled chemical hijacking of the ubiquitin-proteasome system to treat disease. The catalytic rate of cereblon (CRBN)-dependent bifunctional degradation activating compounds (BiDAC), which recruit CRBN to a chosen target protein, resulting in its ubiquitination and proteasomal degradation, is an important parameter to consider during the drug discovery process. In this work, an in vitro system was developed to measure the kinetics of BRD4 bromodomain 1 (BD1) ubiquitination by fitting an essential activator kinetic model to these data. The affinities between BiDACs, BD1, and CRBN in the binary complex, ternary complex, and full ubiquitination complex were characterized. Together, this work provides a new tool for understanding and optimizing the catalytic and thermodynamic properties of BiDACs.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Bioensayo , Proteínas de Ciclo Celular/metabolismo , Oxindoles/farmacología , Ftalimidas/farmacología , Procesamiento Proteico-Postraduccional , Factores de Transcripción/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Sistema Libre de Células/química , Sistema Libre de Células/metabolismo , Células HeLa , Humanos , Cinética , Oxindoles/síntesis química , Ftalimidas/síntesis química , Complejo de la Endopetidasa Proteasomal/efectos de los fármacos , Unión Proteica , Dominios Proteicos , Proteolisis/efectos de los fármacos , Termodinámica , Factores de Transcripción/química , Factores de Transcripción/genética , Ubiquitina-Proteína Ligasas/genética , Ubiquitinación/efectos de los fármacosRESUMEN
Despite decades of effort, the sensitivity of patient tumors to individual drugs is often not predictable on the basis of molecular markers alone. Therefore, unbiased, high-throughput approaches to match patient tumors to effective drugs, without requiring a priori molecular hypotheses, are critically needed. Here, we improved upon a method that we previously reported and developed called high-throughput dynamic BH3 profiling (HT-DBP). HT-DBP is a microscopy-based, single-cell resolution assay that enables chemical screens of hundreds to thousands of candidate drugs on freshly isolated tumor cells. The method identifies chemical inducers of mitochondrial apoptotic signaling, a mechanism of cell death. HT-DBP requires only 24 hours of ex vivo culture, which enables a more immediate study of fresh primary tumor cells and minimizes adaptive changes that occur with prolonged ex vivo culture. Effective compounds identified by HT-DBP induced tumor regression in genetically engineered and patient-derived xenograft (PDX) models of breast cancer. We additionally found that chemical vulnerabilities changed as cancer cells expanded ex vivo. Furthermore, using PDX models of colon cancer and resected tumors from colon cancer patients, our data demonstrated that HT-DBP could be used to generate personalized pharmacotypes. Thus, HT-DBP appears to be an ex vivo functional method with sufficient scale to simultaneously function as a companion diagnostic, therapeutic personalization, and discovery tool.