Design, Synthesis and Testing of Novel Small Molecule Inhibitors of S-phase Kinase-Associated Protein 2

Abstract

S-Phase Kinase-Associated Protein 2 (Skp2), an oncoprotein, is overexpressed in numerous human cancers and plays a critical role in cell cycle progression, senescence, metabolism, cancer progression and metastasis, through promoting the ubiquitination of regulatory protein substrates, such as p27KIP1, and targeting them for degradation by the 26S proteasome. Increased expression of Skp2, accompanied by decreased levels of p27KIP1, is often associated with an aggressive phenotype and poor prognosis in many cancers. This project aimed to develop small molecules which disrupt the ubiquitination ligase activity of Skp2 on p27, a process which is facilitated by the accessory protein Cks1. In silico screening of commercial compound databases, the Specs normal (approximately 539,783 compounds) and natural compounds library (approximately 721 compounds) (http://www.specs.net/), were carried out in Maestro (Schrödinger). Lead compounds (10 compounds) were selected based upon their docking scores and their physico-chemical properties. Several of the lead compounds were then synthesised and others were purchased from SPECs. Biological evaluations were carried out by cell cytotoxic assay and cell cycle assays in breast cancer and melanoma cell lines; p27 expression was also assessed in breast cell lines through Western blot studies. Compound (36), a novel small molecule synthesised in house exhibited potent GI50 values in breast cancer and melanoma cell lines, with some of the GI50 values were in the nanomolar range; e.g. in the NM179, a NRAS mutant melanoma cell line the GI50 was 5 nM. Cell cycle studies in these cell lines were not consistent and the results were not conclusive enough to conclude that this compound acts on the Skp2/p27 pathway. Nevertheless, Western blot studies in a breast cancer cell line (MDA-MB-231) did show initial change in p27/tubulin ratio and further investigation is warranted.