Rapid and high-throughput screening of proteolysis targeting chimeras using a dual-reporter system expressing fluorescence protein and luciferase.

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.