Abstract 20: HiBiT tagging system for high throughput chemical screening for chemotherapy

Abstract

Abstract Chemical screening is an essential procedure for finding novel drugs of cancer treatments. Especially, cell-based screenings are mostly used for discovery of novel compounds or drug repositioning of already-used drugs. Cell viability or cell proliferation are usually used for evaluation of effects of candidate novel compounds for cancer treatments. However, high throughput system for rapid evaluation of endogenous target protein expression changes has not been established so far. Therefore, an easy and quick method for detection of endogenous target protein expression changes has been strongly needed for drug discovery. Herein, we established a novel chemical compound screening system using the HiBiT-tagging system for this aim using PD-L1 as an example. HiBiT tag is a small peptide tag composed of only 11 amino acids forming a dimer with LgBiT, and functions as a Nanoluciferase. The shortness of the peptide tag is useful for insertion of tag using genome editing technology. Thereby, we established a rapid chemical screening method using HiBiT-tagged cell lines created by CRISPR-Cas9 system. With this method, we made it possible to detect endogenous target protein expression changes within 10 minutes by just adding lytic buffer and LgBiT. For validation of this method, we performed chemical screening of PD-L1 using a HiBiT-tagged lung adenocarcinoma cell line with 1,280 compounds. As a result, we identified microtubule inhibitors as compounds upregulating PD-L1, and validated its upregulation of PD-L1 with qPCR and western blotting. In conclusion, we succeeded in establishing the method for chemical compounds detecting endogenous target protein expression changes with HiBiT-tagging system. Furthermore, we validated this method by performing chemical screening with PD-L1, and succeeded in identifying microtubules inhibitors as up-regulators of PD-L1. Citation Format: Yutaro Uchida, Takahide Matsushima, Ryota Kurimoto, Tomoki Chiba, Yuki Inutani, Hiroshi Asahara. HiBiT tagging system for high throughput chemical screening for chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 20.