Discovering New Metallo-Deubiquitinase CSN5 Inhibitors by a Non-Catalytic Activity Assay Platform
- J Med Chem. 2024 Aug 22;67(16):14649-14667. doi: 10.1021/acs.jmedchem.4c01514.
- 1. Key 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.
- 2. College of Food and Bioengineering, Xihua University, Chengdu 610039, China.
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.