Discovering New Metallo-Deubiquitinase CSN5 Inhibitors by a Non-Catalytic Activity Assay Platform
Yu-Hang Yan1, Liu-Liu Wei1, Jing-Wei Wu1
1Key Laboratory of Drug-Targeting and Drug Delivery System of the Education Ministry and Sichuan Province, Department of Medicinal Chemistry, West China School of Pharmacy, Sichuan University, Chengdu 610041, China.
Abstract:
COP9 signalosome catalytic subunit CSN5 plays a key role in tumorigenesis and tumor immunity, showing potential as an anticancer target. Currently, only a few CSN5 inhibitors have been reported, at least partially, due to the challenges in establishing assays for CSN5 deubiquitinase activity. Here, we present the establishment and validation of a simple and reliable non-catalytic activity assay platform for identifying CSN5 inhibitors utilizing a new fluorescent probe, CFP-1, that exhibits enhanced fluorescence and fluorescence polarization features upon binding to CSN5. By using this platform, we identified 2-aminothiazole-4-carboxylic acids as new CSN5 inhibitors, which inhibited CSN5 but slightly downregulated PD-L1 in cancer cells. Furthermore, through the integration of deep learning-enabled virtual screening, we discovered that shikonins are nanomolar CSN5 inhibitors, which can upregulate PD-L1 in HCT116 cells. The binding modes of these structurally distinct inhibitors with CSN5 were explored by using microsecond-scale molecular dynamics simulations and tryptophan quenching assays.
Insights
Researchers developed a new assay to find COP9 signalosome subunit 5 (CSN5) inhibitors. This led to the discovery of novel 2-aminothiazole-4-carboxylic acids and shikonins as potential anticancer agents targeting CSN5.
Area of Science:
- Biochemistry
- Molecular Biology
- Cancer Research
Background:
- COP9 signalosome catalytic subunit 5 (CSN5) is crucial in tumorigenesis and tumor immunity, making it a promising anticancer target.
- Developing effective CSN5 inhibitors is challenging due to difficulties in assaying its deubiquitinase activity.
Purpose of the Study:
- To establish and validate a novel, reliable non-catalytic activity assay platform for identifying CSN5 inhibitors.
- To discover new CSN5 inhibitors using the developed assay and computational methods.
Main Methods:
- Development of a fluorescent probe (CFP-1) for a non-catalytic activity assay platform.
- Utilizing the platform to screen for CSN5 inhibitors, identifying 2-aminothiazole-4-carboxylic acids.
- Employing deep learning-enabled virtual screening to identify shikonins as CSN5 inhibitors.
- Conducting molecular dynamics simulations and tryptophan quenching assays to explore inhibitor binding modes.
Main Results:
- A simple and reliable non-catalytic activity assay platform for CSN5 inhibitors was successfully established and validated.
- 2-aminothiazole-4-carboxylic acids were identified as novel CSN5 inhibitors, showing slight PD-L1 downregulation in cancer cells.
- Shikonins were discovered as potent nanomolar CSN5 inhibitors via virtual screening, capable of upregulating PD-L1 in HCT116 cells.
Conclusions:
- The developed assay platform is effective for identifying CSN5 inhibitors.
- Both 2-aminothiazole-4-carboxylic acids and shikonins represent promising chemical scaffolds for developing novel CSN5-targeted anticancer therapies.
- Further investigation into the distinct effects of these inhibitors on PD-L1 expression is warranted.
More Related Videos
07:05Measuring Enzymatic Activity of Neurodevelopmental Disorder-Associated Deubiquitylating Enzymes via an In Vitro Ubiquitin Chain Cleavage Assay
Published on: September 27, 2024
09:45Method for Measuring the Activity of Deubiquitinating Enzymes in Cell Lines and Tissue Samples
Published on: May 10, 2015