Establishment of high-throughput screening HTRF assay for identification small molecule inhibitors of Skp2-Cks1

Kaizhao Hu,Moges Dessale Asmamaw,Xiao-Jing Shi,Hong-Min Liu,Xiao-Jing Li

doi:10.1038/s41598-021-00646-3

Abstract

S-phase kinase associated protein 2 (Skp2), a member of the F-box family that constitute the largest known class of ubiquitin E3 specificity components, is responsible for recognizing and recruiting cyclin-dependent kinase inhibitor p27 for its ubiquitination in the presence of the small accessory protein cyclin-dependent kinase regulatory subunit 1(Cks1). Skp2 is overexpressed in esophageal carcinoma tissues and closely related with tumor poor prognosis, and perturbation of the Skp2-Cks1 interaction by inhibitors or RNAi could inhibit the proliferation and metastasis of tumor cells. Therefore, inhibition of Skp2 function by small-molecule compounds targeting Skp2-Cks1 interaction is emerging as a promising and novel anti-cancer strategy. In this study, we establish an improved high-throughput screening platform to screen Skp2 inhibitors targeting Skp2-Cks1interaction, which may provide a new therapeutic approach for the clinic.

Highlights

S-phase kinase associated protein 2 (Skp2), a member of the F-box family that constitute the largest known class of ubiquitin E3 specificity components, is responsible for recognizing and recruiting cyclindependent kinase inhibitor p27 for its ubiquitination in the presence of the small accessory protein cyclin-dependent kinase regulatory subunit 1(Cks[1])
An appropriate method to detect the binding of two proteins is very important for the high throughput screening platform based on the principle of protein–protein interaction (PPI)
Skp2/Skp[1] is expressed with glutathione S-transferase (GST)-tag which can firmly combine with anti-GST donor beads

Summary

Introduction

S-phase kinase associated protein 2 (Skp2), a member of the F-box family that constitute the largest known class of ubiquitin E3 specificity components, is responsible for recognizing and recruiting cyclindependent kinase inhibitor p27 for its ubiquitination in the presence of the small accessory protein cyclin-dependent kinase regulatory subunit 1(Cks[1]). Skp[2] is overexpressed in esophageal carcinoma tissues and closely related with tumor poor prognosis, and perturbation of the Skp2-Cks[1] interaction by inhibitors or RNAi could inhibit the proliferation and metastasis of tumor cells. Inhibition of Skp[2] function by small-molecule compounds targeting Skp2-Cks[1] interaction is emerging as a promising and novel anti-cancer strategy. Skp[2] mainly degrades the Cyclin-dependent kinase inhibitor (CKIs) which can inhibit the G1 to S phase transition of the cell cycle and subsequently regulates tumor proliferation and metastasis[7]. Inhibition of Skp[2] binding to Cks[1] can reduce ubiquitination degradation of p27, resulting in cycle arrest of tumor c ells[15]. Screening system decreased due to the low window value, high optimal protein concentration and steric hindrance, which may affect the screening of inhibitors

Methods

Results

Conclusion