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Updated: May 26, 2025
High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay
Published on: June 1, 2014
Rapid and high-throughput screening of proteolysis targeting chimeras using a dual-reporter system expressing
Shuai Meng1, Yuan Meng2, Xuena Yang2
1Department of Pharmacy, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer, Tianjin'S Clinical Research Center for Cancer, Key Laboratory of Cancer Prevention and Therapy, Tianjin, 300060, China.
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.
Insights
This study introduces a novel dual-reporter system for high-throughput screening of Proteolysis Targeting Chimeras (PROTACs). The method efficiently identifies PROTAC candidates, accelerating drug discovery for target protein degradation.
Area of Science:
- Biochemistry
- Molecular Biology
- Drug Discovery
Background:
- Proteolysis targeting chimeras (PROTACs) offer a novel strategy for targeted protein degradation.
- Current methods for PROTAC screening, such as Western blotting, face limitations in throughput.
- There is a need for efficient, high-throughput methodologies to accelerate PROTAC development.
Purpose of the Study:
- To develop and validate a high-throughput dual-reporter system for screening PROTACs.
- To assess the efficacy of the developed system in identifying PROTACs targeting androgen receptor (AR) and Sirtuin 2 (SIRT2).
Main Methods:
- A dual-reporter system was constructed using Renilla luciferase (RLUC)-fused target protein and enhanced green fluorescent protein (EGFP) as a reference.
- Fluorescence and luminescence signals were measured using a plate-based reader.
- The system's feasibility was validated using a known AR-targeting PROTAC (ARV110) and subsequently applied to screen for SIRT2 degraders.
Main Results:
- The dual-reporter system demonstrated dose-dependent degradation of AR by ARV110, correlating with Western blot results.
- The platform successfully identified two potent SIRT2 degraders (Compounds 128 and 129) from a small library.
- These compounds induced dose-dependent degradation of endogenous SIRT2 and cell growth arrest in MCF-7 cells.
Conclusions:
- A novel dual-reporter system combining fluorescence and chemiluminescence for PROTAC screening has been successfully established.
- This method effectively identified potent PROTAC candidates targeting SIRT2.
- The developed dual-reporter system has the potential to significantly accelerate the drug discovery process for PROTACs.
