The critical platforms of PROTAC validation

In recent years, proteolysis targeting chimeric (PROTAC) technology has become an effective endogenous protein degradation tool in drug discovery. As a class of heterobifunctional small molecules, PROTACs recruit an E3 ubiquitin ligase to a given substrate protein, resulting in its targeted degradation through the endogenous ubiquitin-proteasome system (UPS). PROTAC consists of three chemical elements: a ligand that binds to a target protein, a ligand that binds to an E3 ubiquitin ligase, and a linker that connects the two ligands. Compared with traditional drug development, PROTAC technology possesses unique advantages including overcoming drug resistance and targeting the “undruggable”.

Here we generated a PROTAC target, like KRAS, BRD4, ESR1, associated cell lines to accelerate the discovery of new protein degradation drugs and the target protein degradation therapeutics by an optimized insertion system. Initially, we developed a system of different tag targeted cell lines, which are amenable to introduce HiBiT, Flag, GFP and also Halo Tag to target protein by CRISPaint or CRISPR Cas9 mediated HDR system. We also developed an arbitrary point mutation system based on the optimized PE3 mutation system, which can randomly modify any point mutation to simulate disease models in vitro. During this process, we have also optimized a series of validation systems, like Targeted Protein Degradation (TPD), High-throughput flow cytometry (HTFC) and In-Cell Western (ICW) to shorten the detection time and improve detection stability. We also developed a HiBiT and GFP dual-signal system, which can readily validate target protein degradation and cell viability. This detection system holds the promise to speed up the development of new drugs and contribute to anti-tumor therapy.

11 PROTAC platform_5307_Ruifeng


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