Abstract 6919: Cell line panel with HIBIT-tagged endogenous proteins to accelerate PROTAC drug discovery

Abstract

Abstract Small molecular drugs have been an important part of disease treatment, but there are also some problems along with time and intrinsic disadvantages of small molecular drugs. In recent years, the emergence of proteolysis targeting chimeras (PROTACs) is expected to overcome some defeats of traditional small molecules. PROTACs, also known as hetero-bifunctional compounds, are a new kind of therapeutic, that consists of a specific ligand to bind the target protein, a suitable linker, and an E3 ubiquitin ligase substrate. After binding to target proteins, PROTACs recruit E3 ligase and induce degradation of target proteins through the ubiquitin-proteasome system. Therefore, PROTACs are a good choice for some protein targets that have small molecular binding ligands but poor small molecular efficacy, which expands druggable targets. Meanwhile, the protein degradation induced by PROTACs destroys not only the enzymatic but also the non-enzymatic functions of targets, rather than only inhibiting the protein activity, which reduces drug resistance. In this study, in order to detect the degradation of intracellular target proteins by PROTACs, Kyinno has designed and constructed several HiBiT-tagged cell line models for different target proteins and their mutations, including KRA, CDK2, LDHA, and HPK. HiBiT is a small tag with 11 amino acids that don’t influence the expression, folding, and localization of target proteins, and luminesces with a detection buffer. All these cell lines with endogenous protein mutations and HiBiT knock-ins are constructed by CRISPR-Cas9 technology and validated by siRNA and PROTACs. Compared with overexpressed exogenous HiBiT-tagged target proteins, endogenous proteins can more accurately and stably reflect the efficacy of PROTACs. Our cell models can be used for high-throughput screening not only for PROTAC drugs but also for molecular glues, lysosome-targeting chimeras (LYTACs), and antibody-based PROTACs (AbTACs). Citation Format: Yunpeng Zhai, Jiayi Ma, Yu Wang, Meng Liang, Yao Peng, Jinying Ning, Feng Hao. Cell line panel with HIBIT-tagged endogenous proteins to accelerate PROTAC drug discovery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6919.