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Updated: Jun 29, 2025
Assays for the Degradation of Misfolded Proteins in Cells
Published on: August 28, 2016
A rapid and accurate method for evaluating the degradation of pan-Akt in cells by PROTACs using NanoLuc luciferase
Xiaojun Ji1,2,3, Lei Miao4,2, Yebin Wu1
1Innovation Department of the Research Institute, Nanjing Chia-Tai Tianqing Pharmaceutical Co. Ltd., Nanjing 210046, PR China.
Abstract:
Proteolysis targeting chimera (PROTAC) is a protein degradation technique that has been increasingly used in the development of new drugs in recent years. Akt is a classical serine/threonine kinase, and its role outside of the kinase has gradually gained attention in recent years, making it one of the proteins targeted by PROTACs. Currently, there are many methods used for the evaluation of intracellular protein degradation, but each has its own advantages or disadvantages. This study aimed to investigate the feasibility of evaluating the degradation of pan-Akt proteins in cells by PROTACs (MS21 and MS170) using the NanoLuc luciferase method. After conducting a thorough comparison between this method and the classical western blot assay in various cells, as well as testing the stability of the experiments between multiple batches, we found that NanoLuc luciferase is a highly accurate, stable, low-cost and easy-to-operate method for the evaluation of intracellular pan-Akt degradation by PROTACs with a short cycle time and high cellular expandability. Given the numerous advantages of this method, it is hypothesized that it could be extended to evaluate the degradation of more target proteins of PROTACs. In summary, the NanoLuc luciferase is a suitable method for early protein degradation screening of PROTAC compounds.
Insights
The NanoLuc luciferase assay offers a highly accurate and efficient method for evaluating protein degradation by Proteolysis Targeting Chimeras (PROTACs). This technique is ideal for screening PROTAC compounds targeting proteins like pan-Akt, demonstrating superior performance over traditional western blot assays.
Area of Science:
- Biochemistry
- Molecular Biology
- Drug Discovery
Background:
- Proteolysis Targeting Chimeras (PROTACs) are an emerging drug development technology.
- Akt, a serine/threonine kinase, is increasingly targeted by PROTACs due to its non-kinase roles gaining attention.
- Existing methods for assessing intracellular protein degradation have limitations.
Purpose of the Study:
- To evaluate the feasibility of using the NanoLuc luciferase assay for assessing intracellular pan-Akt protein degradation induced by PROTACs (MS21 and MS170).
- To compare the NanoLuc luciferase method with the classical western blot assay for protein degradation evaluation.
- To assess the stability and efficiency of the NanoLuc luciferase assay across multiple experimental batches.
Main Methods:
- Utilized PROTACs MS21 and MS170 to induce pan-Akt degradation in various cell types.
- Employed the NanoLuc luciferase assay for quantitative measurement of protein degradation.
- Validated results against the traditional western blot assay.
Main Results:
- The NanoLuc luciferase assay demonstrated high accuracy, stability, and cost-effectiveness in evaluating intracellular pan-Akt degradation by PROTACs.
- This method offers a short cycle time and high cellular expandability compared to western blotting.
- The assay proved to be easy to operate and highly reliable across multiple batches.
Conclusions:
- The NanoLuc luciferase assay is a suitable and advantageous method for evaluating intracellular protein degradation induced by PROTACs, specifically for pan-Akt.
- Its efficiency and accuracy suggest potential application for screening other PROTAC target proteins.
- This method represents a valuable tool for early-stage screening of PROTAC compounds in drug discovery.
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