Abstract 6842: LgBiT-HiBiT engineered cell lines for protein degradation detection: Comprehensive PROTACs evaluation platform with endpoint and real-time kinetic analysis

Abstract Small molecule drugs have long been integral to disease treatment, but but their limitations have become increasingly apparent over time. The advent of proteolysis-targeting chimeras (PROTACs) offers a promising solution to some of these challenges. PROTACs, also known as hetero-bifunctional compounds, represent a new therapeutic class that includes a specific ligand to bind the target protein, a suitable linker, and an E3 ubiquitin ligase substrate. Upon binding to target proteins, PROTACs recruit an E3 ligase and induce the degradation of target proteins through the ubiquitin-proteasome system. This mechanism allows PROTACs to address protein targets with small-molecule binding ligands that exhibit limited efficacy, broadening the scope of druggable proteins. Additionally, unlike traditional small molecules that merely inhibit protein activity, PROTACs eliminate both enzymatic and non-enzymatic functions of target proteins, reducing the likelihood of drug resistance. Currently, Western Blot (WB) technology is widely used to validate target protein degradation and evaluate the efficacy of PROTACs. While effective for detecting significant protein level changes, WB is time-consuming, lacks precise quantification, and is impractical for monitoring protein degradation at multiple time points due to its high cost and labor intensity. To efficiently and accurately evaluate PROTACs, Kyinno have developed a series of cell models based on the LgBiT-HiBiT technology. LgBiT and HiBiT are high-affinity protein subunits that together constitute NanoLuc luciferase, catalyzing the luminescence of a luciferin substrate detectable by a microplate reader. We have achieved stable overexpression of LgBiT protein in cells and utilized CRISPR-Cas9 gene editing technology to insert HiBiT tags at the N- or C-terminal of endogenous target genes. These cell models are suitable for both endpoint and pharmacokinetic studies. Using a luciferase substrate appropriate for live-cell detection, the luminescence value reflects the current level of HiBiT protein, which correlates with the level of the target protein. Additionally, employing a long-term luciferase substrate allows real-time monitoring of protein levels at each time point. Currently, these cell lines target proteins such as IRAK3, GSPT1, GSPT2, ELAVL1, and GRK2. They support high-throughput screening of PROTACs, molecular glues, lysosome-targeting chimeras (LYTACs), and antibody-based PROTACs (AbTACs), offering a robust platform for advancing drug discovery and development. Citation Format: Siyu Li, Shi Qin, Yao Peng, Yunpeng Zhai, Jinying Ning, Feng Hao. LgBiT-HiBiT engineered cell lines for protein degradation detection: Comprehensive PROTACs evaluation platform with endpoint and real-time kinetic analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6842.