Abstract Cyclin dependent kinase 2 (CDK2) is aberrantly regulated in a wide range of cancers and small molecule inhibitors should be beneficial in the treatment of cancer. Preclinical and clinical CDK2 inhibitors are ATP competitive and bind at the ATP site in a canonical mode. A novel binding mode for a new class of quinoline-based inhibitors of CDK2 has been discovered using the affinity-based Automated Ligand Identification System (ALIS) technology. While the inhibitors do bind to the ATP site the key mechanism of action for inhibition is the induction of a novel protein conformational change that enables the compound to bind into the core of the kinase domain. Global changes to CDK2 upon compound binding result in a conformation that can no longer bind to its activating protein cyclin A. Therefore, these inhibitors are both ATP competitive and are protein-protein interaction disrupters. The key strategy for discovering this class of protein-protein disrupter compounds was to screen the unphosphorylated monomer CDK2 in ALIS and to perform secondary binding and enzyme mechanism of action assays that identified quinoline-based compounds that only bound to the inactive CDK2 monomer and not the active CDK2/cyclin A heterodimer. Through a series of chemical modifications the affinity (Ki) of the original hit improved from 0.9 μM to 0.005 μM. The screening strategy utilized and the discovery of this new kinase inhibitor binding mode may be applicable to other kinase programs. Citation Format: {Authors}. {Abstract title} abstract. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2785. doi:10.1158/1538-7445.AM2011-2785