Skp2, as a short-lived protein, is regulated by UPS during cell life and degraded by the ubiquitin-proteasome pathway. Because most of its substrates are associated with tumor inhibition, Skp2 is considered to be a cancer-related protein, and its high expression in most cancers also reveals that30. Skp2 plays an important role in different cancers due to its significant regulation of cell cycle progression and its direct and indirect influence on the initiation and development of tumors through the activation of multiple signaling pathways and transcription factors, such as the PI3K/AKT pathway, IL-6/JAK2/STAT3 pathway, SP1, FOXM1, etc31. The p27 is a cell cycle regulator and can lower the kinase activity of Cdk2 (cyclin-dependent kinase 2) by binging with the Cdk2/cyclin A complex, and causing cell block from G1 phase to S phase 32; 33. Previous studies revealed that Skp2 was upregulated in the G2 phase and involved in the degradation of p2734. Cyclin-dependent kinase subunit 1(Cks1) was a small cohesive protein and can greatly enhance the binding affinity of p27 to Skp218. When phosphorylated at threonine on p27 protein 188 sites, Skp2 interacts with Cks1 and leads to p27 degradation through the ubiquitin-proteasome system which promotes tumor proliferation and migration35. It was reported that Skp2 is highly expressed at protein and mRNA levels in a variety of cancer tissues and mediates tumorigenesis. And this was consistent with the results that downregulation of p27 predicted poor prognosis of tumors36; 37. The development of Skp2-Cks1 inhibitors might provide a novel and potential target for Skp2-overexpressed tumor patients38.
Skp2 has attracted increasing attention as an ideal antitumor drug target, and some small molecule inhibitors of Skp2 have been reported in the literature,such as SMIP004(Skp2)39, 6-O-angeloylplenolin (Skp1-Skp2)40, C1, C20(Skp2-p27)41, M1(Skp2-p300)42, NSC681152 (Skp2-Cks1), Compound 22d(Skp2-Cks1)25, etc. Most of these small molecule inhibitors act on the Skp1 / Skp2-Cks1-p27 axis, thereby triggering intracellular signaling and inhibiting the growth and proliferation of tumor cells. The high-throughput screening platform for most Skp2 inhibitors was found to screen compounds by detecting changes in the fluorescence signal of a single protein after drug action. Although this method can directly observe the influence of intracellular protein expression of the drug, it’s susceptible to other factors and lead to false-positive results.
HTRF technology-based Skp2-Cks1 high-throughput drug screening platform has been previously reported, but the detection reagents used are different from ours. At the same time, we also carried out some optimization and improvement based on the previous researches. In the previous literature, the reagents used were Eu with FLAG label and APC with GST label. APC (phycocyanin, XL665) is a large hetero hexameric edifice of 105 kDa, cross-linked after isolation for better stability and preservation of its photophysical properties in HTRF assays. But in this study, the recombinant proteins we used were 71kDa and 10kDa, respectively. The larger molecular weight of XL665 may cause a certain steric influence on the binding of the target protein, thus affecting the protein binding and HTRF value. The reagents we used were GST-Eu and His6-d2. The d2 is the second-generation receptor optimized based on XL665. Based on inheriting a series of photophysical properties of XL665, the molecular weight of d2 is only about 1kDa, which can also solve the possible influence of steric hindrance. After the optimization of reagents and reaction conditions, when the concentrations of GST-Skp2 and His6-Cks1 were 20nmol and 30nmol respectively, the HTRF value could reach the optimal level. However, in the previous literature, the concentrations of GST-Skp2 and FLAG-Cks1 were 140nmol and 160nmol respectively, which may be due to reduce the effect of steric hindrance and made the protein binding more fully. On the other hand, we optimized the purification method of the target protein, which greatly increased the purity and activity of the target recombinant protein.
When screening of Skp2-Cks1 target inhibitors, there were mainly two methods: AlphaScreen43 assay and HTRF17 assay, both were made up of donor beads, receptor beads, and the molecular complex of interaction. They can stimulate the donor beads by excitation light of specific wavelengths, thereby causing the energy cascade transfer reaction and the receptor beads emitting energy. Compared with the HTRF assay, the AlphaScreen assay had a relatively high window range because a donor bead can release more than 6,000 singlet oxygen molecules, and the AlphaScreen assay had a dynamic range of over 200 In the previous literature about the AlphaScreen system of Skp2-Cks1. AlphaScreen technology has its advantages, but there are limitations. The capture of monomer oxygen molecules by some compounds reduces the optical signal, and the photobleaching effect of donor beads makes it impossible to detect multiple times. The HTRF technology could greatly solve the deficiency of AlphaScreen Assay. Due to the long half-life of lanthanides, HTRF technology has a low background and can effectively avoid the interference of non-specific fluorescence and false-positive results. However, the disadvantage of HTRF is the small window range, which makes the sensitivity too low in the drug screening process. Combining the two screening methods to screen Skp2-Cks1 target inhibitors may be a good solution and promote the development of Skp2 small-molecule inhibitors.
We selected an inhibitor of Skp2 that has been reported to verify the accuracy of this screening platform, and the protein level and mRNA level results showed that the compound of Skp2-2 is the targeted inhibitor for Skp2. There are not currently Skp2-Cks1 inhibitors for the treatment of malignant tumors in clinic. The established High-throughput screening HTRF assay of Skp2-Cks1 was used for screening of compound libraries. After screening, we obtained the lead compound from 2000 compounds, and its IC50 was about 2.14 µM at the enzyme activity level, but it did not show a good effect in cells. Therefore, in the follow-up work, we will continue to optimize its structure to get better small molecule inhibitors.
In summary, we have built a small molecule inhibitor screening method based on HTRF technology, which can screen the target inhibitors of Skp2-Cks1. We will continue to optimize based on the screened lead compounds to find promising small molecule inhibitors of Skp2-Cks1 for clinical application. At the same time, based on analyzing different screening technologies, we try to combine HTRF with AlphaScreen, to make the high-throughput screening system for small molecule inhibitors of Skp2-Cks1 more efficient and convenient.