Abstract 2764: Application of NanoBRET target engagement cellular assay for development of inhibitors against protein kinases, RAS, and epigenetic proteins in cancer therapy

Abstract

Abstract NanoBRET target engagement (TE) is the first biophysical technique that broadly enables the quantitative determination of protein inhibitor occupancy in live cells, without disruption of cellular membrane integrity. This quantitative capability is achieved in live cells via BRET with an optimized set of cell-permeable tracers, allowing the measurement of compound binding to a selected cellular target protein. Our NanoBret assay is scaled to 384-well plate format and Z’ analysis demonstrated the assay to be High Throughput Screening compatible. Protein kinases, RAS, and epigenetic proteins are very important drug targets. NanoBRET TE cellular assay has enabled the development of live-cell quantitative compound binding assays for >360 individual wild type and mutant protein kinases including serine/threonine-specific kinases, receptor tyrosine kinases, and all 20 cyclin-dependent kinases (CDKs). RAS is a well-known oncogene that is frequently mutated in most lung, pancreatic and colorectal cancers and is associated with poor disease prognosis. The recent discovery of a unique switch II binding pocket and successful inhibition of the KRAS (G12C) mutant by covalent inhibitors have led to the resurgence of interest in the design of inhibitors targeting RAS directly. Our data demonstrate that NanoBRET TE cellular assay can measure the apparent affinity of RAS inhibitors by competitive displacement of a NanoBRET RAS switch I/II pocket tracer, reversibly bound to the LgBiT- and SmBiT- KRAS, KRAS (G12C), KRAS (G12D), KRAS (G12V), KRAS (G13D), KRAS (Q61H), KRAS (Q61L), KRAS (Q61R), KRAS-G12A, KRAS-G12S, HRAS, HRAS-G12C, and HRAS-G12V fusion constructs co-transfected in live HEK293 cells. Our data also show NanoBRET cellular assay can be used to measure compound binding affinity to epigenetic drug target proteins such as BRD4, BRD4-BD1, BRD4-BD2, HDAC6, HDAC1, HDAC2, and HDAC3. Taken together, our results suggest NanoBRET TE cellular assay can serve as a great tool to facilitate new drug discovery against human cancers. Citation Format: Jianghong Wu, Yuan Wang, Shawn McGinley, Peter Gallagher, Li Liang, Alisha Di Lanni, Ashley Miles, Mohammad Haque, Yong Wan, Haiching Ma. Application of NanoBRET target engagement cellular assay for development of inhibitors against protein kinases, RAS, and epigenetic proteins in cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2764.