Development of rapid screening system for targeted protein degradation using Flp-In and HiBiT system

다국어 초록 (Multilingual Abstract)

Target protein degradation is new therapeutic intervention for “undruggable” targets. There are two types of targeted protein degradation strategies: PROTAC and Molecular glue. PROteolysis TArgeting Chimeras (PROTACs) are bifunctional small molecules that simultaneously bind a target protein and an E3-ubiquitin ligase, thus causing ubiquitination and degradation of the target protein by the proteasome. Molecular glue induces direct interaction between two protein, thereby binding to ubiquitin E3 ligases and recruit proteins for degradation by the UPS(ubiquitin-proteasome system).
In this study, to find a rapid screening method of UPS-based compound, I used unique feature of Flp-In and HiBiT system. In combination with the Flp-in system, depends on Flp-mediated recombination between Flippase Recognition Target sites in host cell and the transfected plasmid, I established stable cells containing HiBiT-tagged IKZF2. As a result, I have established a method to measure the degradation of target proteins in cells with luminescence. This method provides the development of screening and application of technology in future therapies and drug discovery.

국문 초록 (Abstract)

표적 단백질 분해는 “약물 개발 불가” 표적에 대한 새로운 치료 기법이다. 표적 단백질 분해 전략에는 프로텍(PROTAC)과 분자 접착제 (Molecular glue)라는 두 종류가 있다. 프로텍은 표적 단백질과 E3 유비퀴틴 라이게이즈(E3 ubiquitin ligase)를 동시에 결합하는 소분자로 프로테아좀에 의한 목적 단백질의 분해를 유발한다. 분자 접착제는 두 단백질 사이의 직접적인 상호작용을 유도하여 E3 유비퀴틴 라이게이즈에 결합하고 유비퀴틴-프로테아좀 시스템에 의해 분해될 단백질을 매개한다.
본 연구에서는 유비퀴틴-프로테아좀 시스템을 기반으로한 화합물의 빠른 스크리닝 방법을 찾기 위해 Flp-In 과 HiBiT 시스템의 독특한 특징을 활용했다. 플립페이즈 인식 표적(FRT: Flippase Recognition Target) 부위를 가지고 있는 세포에 Flp-In 시스템을 사용해 지속적으로 HiBiT 태그가 붙어 IKZF2가 발현되는 세포를 만들었다. 그 결과 발광으로 세포 내 표적 단백질의 분해 정도를 측정하는 방법을 확립했다. 이 방법은 향후 치료 및 약물 발견에 있어 스크리닝 개발과 기술의 응용을 제공한다.

목차 (Table of Contents)

• 1. Introduction ···························································
• 1.1 The ubiquitin proteasome system ······························
• 1.2 Molecular glues and targeted protein degradation ··········
• 1.3 Flp-In system ·····················································
• 1.4 HiBiT System ····················································
• 1.5 Helios(IKZF2) as therapeutic targets··························
• 2. Materials & Methods ···············································
• 2.1 Cell Culture······················································
• 2.2 Expression Plasmids ···········································
• 2.3 Generation of Flp-In 293 HiBiT Stable cell line ···········
• 2.4 HiBiT Blotting and Antibodies ·······························
• 2.5 Lytic HiBiT Detection ·········································
• 3. Results···································································
• 3.1 Identification of a new IKZF2 degrader, TD-1120, using immunoblot-based chemical screening························
• 3.2 Establishment of IZKF2 tagging HiBiT Cell using Flp-In system ····························································
• 3.3 HiBiT assay is correlated with western blotting ············
• 3.4 Degradation of IKZF2 by TD-1120 through ubiquitin proteasome system ··············································
• 4. Discussion ······························································
• 5. Reference ······························································