Decreased levels of cell cycle inhibitor p27Kip1 due to excessive degradation occur in a variety of aggressive human tumors.Since reduced p27Kip1 expression has been associated with a poor prognosis in many human cancers and resistance to certain anti-tumor therapies, it has been postulated that elevation of p27Kipl expression could improve prognosis and perhaps even provide a cure for malignant cancers.However, this concept has not been proven or rigorously tested largely due to the absence of specific small molecule inhibitors that perturb abnormal reduction in p27Kip1 levels.The abundance of p27Kip1 is primarily controlled by the ATP-dependent ubiquitin-proteasome pathway.Ubiquitination of p27Kip1 is primarily catalyzed by SCFskp2, a multi-protein E3 ligase complex and requires specific interaction between Skp2 and Cks 1.Mutations in Skp2 or Cks 1 that disrupt the Cks 1-Skp2 interaction perturb p27Kip1 degradation.We have developed a non-radioactive, robust, high throughput screening assay for the Skp2-Cks1 interaction based on the Amplified Luminescent Proximity Homogeneous Assay Technology (AlphaScreenTM).Using this assay we screened small molecule compound libraries for inhibitors that disrupt Skp2-Cks 1 binding.Several compounds were identified and shown to be able to inhibit p27 ubiquitination both in vitro and in cancer cells.Identifying specific small molecule inhibitors of p27Kip1 degradation is a first step towards evaluating whether inhibition of p27Kip1 degradation would be an effective anti-cancer therapy approach and translating what we have learned about the basic mechanisms of p27Kip1 degradation into potential new drug leads in cancer biology.