Rapid and High-throughput Screening of Proteolysis Targeting Chimeras Using a Dual-reporter System Expressing Fluorescence Protein and Luciferase

Overview

Journal BMC Biol

Publisher Biomed Central

Specialty Biology

Date 2025 Feb 21

PMID 39985000

Authors

Shuai Meng

Yuan Meng

Xuena Yang

Wenwen Yu

Bole Li

Tianjun Liu

Jie Zhang

Xiubao Ren

Lin Zhang

Affiliations

Soon will be listed here.

Abstract

Background: Proteolysis targeting chimera (PROTAC), a novel drug discovery strategy, utilizes the ubiquitin-proteasome system to degrade target proteins in cells. While Western blotting, mass spectrometry, and Lumit Immunoassay have been instrumental in determining protein levels, the rapid screening of PROTACs continues to pose challenges, necessitating the development of alternative methodologies.

Results: We herein reported an alternative high-throughput method for screening PROTACs using a dual-reporter system expressing a Renilla luciferase (RLUC)-fused target protein and enhanced green fluorescent protein (EGFP). EGFP served as an internal reference and RLUC as an indicated target protein degradation. Rapid measurement of EGFP or RLUC light signals was achieved using a fluorescence/luminescence plate-based reader in the endpoint mode. The feasibility of the screening model was tested using ARV110, a clinical trial-stage PROTAC targeting the androgen receptor (AR). In EGFP/RLUC-tAR-expressing modal cells treated with varying concentrations of ARV110, normalized RLUC luminescence decreased dose-dependently, as confirmed via western blotting detection of AR expression. Then the platform was used to practically screen Sirtuin 2 (SIRT2) degraders from a small group of PROTACs that we built. Normalized RLUC luminescence changes in model cells expressing EGFP/RLUC-SIRT2 reflected the degradation efficiencies of PROTACs. Compounds 128 and 129 exhibited the highest degradation efficacies, leading to dose-dependent degradation of endogenous SIRT2 protein in the MCF-7 cell line and inducing cell growth arrest.

Conclusions: The dual-reporter system using both fluorescence and chemiluminescence was successfully constructed. Using this method, we identified effective candidate PROTACs against SIRT2. The dual-reporter system may accelerate drug discovery during PROTAC development.

References

Cao C, He M, Wang L, He Y, Rao Y . Chemistries of bifunctional PROTAC degraders. Chem Soc Rev. 2022; 51(16):7066-7114. DOI: 10.1039/d2cs00220e. View

Yen H, Elledge S . Identification of SCF ubiquitin ligase substrates by global protein stability profiling. Science. 2008; 322(5903):923-9. DOI: 10.1126/science.1160462. View

Alves J, Schwinn M, Machleidt T, Goueli S, Cali J, Zegzouti H . Monitoring phosphorylation and acetylation of CRISPR-mediated HiBiT-tagged endogenous proteins. Sci Rep. 2024; 14(1):2138. PMC: 10810970. DOI: 10.1038/s41598-024-51887-x. View

Peng X, Hu Z, Zeng L, Zhang M, Xu C, Lu B . Overview of epigenetic degraders based on PROTAC, molecular glue, and hydrophobic tagging technologies. Acta Pharm Sin B. 2024; 14(2):533-578. PMC: 10840439. DOI: 10.1016/j.apsb.2023.09.003. View

Neklesa T, Winkler J, Crews C . Targeted protein degradation by PROTACs. Pharmacol Ther. 2017; 174:138-144. DOI: 10.1016/j.pharmthera.2017.02.027. View

Alfayomy A, Ashry R, Kansy A, Sarnow A, Erdmann F, Schmidt M . Design, synthesis, and biological characterization of proteolysis targeting chimera (PROTACs) for the ataxia telangiectasia and RAD3-related (ATR) kinase. Eur J Med Chem. 2024; 267:116167. DOI: 10.1016/j.ejmech.2024.116167. View

Corson T, Aberle N, Crews C . Design and Applications of Bifunctional Small Molecules: Why Two Heads Are Better Than One. ACS Chem Biol. 2008; 3(11):677-692. PMC: 2925120. DOI: 10.1021/cb8001792. View

Iglesias-Ara A, Osinalde N, Zubiaga A . Detection of E2F-Induced Transcriptional Activity Using a Dual Luciferase Reporter Assay. Methods Mol Biol. 2018; 1726:153-166. DOI: 10.1007/978-1-4939-7565-5_14. View

Riching K, Mahan S, Corona C, McDougall M, Vasta J, Robers M . Quantitative Live-Cell Kinetic Degradation and Mechanistic Profiling of PROTAC Mode of Action. ACS Chem Biol. 2018; 13(9):2758-2770. DOI: 10.1021/acschembio.8b00692. View

10.

Feng L, Lu W, Ma Y, Guo W, Wang Y, Sun Q . A novel dual-luciferase assay for anti-HIV drug screening based on the CCR5/CXCR4 promoters. J Virol Methods. 2018; 256:17-23. DOI: 10.1016/j.jviromet.2018.02.016. View

11.

Sehta P, Wilhelm A, Lin S, Urman M, MacNeil H, Fuchs G . A Split NanoLuc Reporter Quantitatively Measures Circular RNA IRES Translation. Genes (Basel). 2022; 13(2). PMC: 8871761. DOI: 10.3390/genes13020357. View

12.

Zhou B, Hu J, Xu F, Chen Z, Bai L, Fernandez-Salas E . Discovery of a Small-Molecule Degrader of Bromodomain and Extra-Terminal (BET) Proteins with Picomolar Cellular Potencies and Capable of Achieving Tumor Regression. J Med Chem. 2017; 61(2):462-481. PMC: 5788414. DOI: 10.1021/acs.jmedchem.6b01816. View

13.

Wilking-Busch M, Ndiaye M, Liu X, Ahmad N . RNA interference-mediated knockdown of SIRT1 and/or SIRT2 in melanoma: Identification of downstream targets by large-scale proteomics analysis. J Proteomics. 2017; 170:99-109. PMC: 5673532. DOI: 10.1016/j.jprot.2017.09.002. View

14.

Jiang T, Du L, Li M . Lighting up bioluminescence with coelenterazine: strategies and applications. Photochem Photobiol Sci. 2016; 15(4):466-80. DOI: 10.1039/c5pp00456j. View

15.

Bekes M, Langley D, Crews C . PROTAC targeted protein degraders: the past is prologue. Nat Rev Drug Discov. 2022; 21(3):181-200. PMC: 8765495. DOI: 10.1038/s41573-021-00371-6. View

16.

Lin H, Riching K, Lai M, Lu D, Cheng R, Qi X . Lysineless HiBiT and NanoLuc Tagging Systems as Alternative Tools for Monitoring Targeted Protein Degradation. ACS Med Chem Lett. 2024; 15(8):1367-1375. PMC: 11318018. DOI: 10.1021/acsmedchemlett.4c00271. View

17.

Nieuwenhuijsen B, Huang Y, Wang Y, Ramirez F, Kalgaonkar G, Young K . A dual luciferase multiplexed high-throughput screening platform for protein-protein interactions. J Biomol Screen. 2004; 8(6):676-84. DOI: 10.1177/1087057103258287. View

18.

Smith D, Lunghi M, Olafsson E, Hatton O, Di Cristina M, Carruthers V . A High-Throughput Amenable Dual Luciferase System for Measuring Bradyzoite Viability after Drug Treatment. Anal Chem. 2022; 95(2):668-676. PMC: 9850410. DOI: 10.1021/acs.analchem.2c02174. View

19.

Jiang T, Song J, Zhang Y . Coelenterazine-Type Bioluminescence-Induced Optical Probes for Sensing and Controlling Biological Processes. Int J Mol Sci. 2023; 24(6). PMC: 10049153. DOI: 10.3390/ijms24065074. View

20.

Daniels D, Riching K, Urh M . Monitoring and deciphering protein degradation pathways inside cells. Drug Discov Today Technol. 2019; 31:61-68. DOI: 10.1016/j.ddtec.2018.12.001. View