A High-Throughput Screening Platform For The Identification Of Small Molecule Inhibitors Of Map2k4

Prostate cancer (PCa) is the second highest cause of cancer death in United States males. If the metastatic movement of PCa cells could be inhibited, then mortality from PCa could be greatly reduced. Mitogen-activated protein kinase kinase 4 (MAP2K4) has previously been shown to activate pro-invasion signaling pathways in human PCa. Recognizing that MAP2K4 represents a novel and validated therapeutic target, we sought to develop and characterize an efficient process for the identification of small molecules that target MAP2K4. Using a fluorescence-based thermal shift assay (FTS) assay, we first evaluated an 80 compound library of known kinase inhibitors, thereby identifying 8 hits that thermally stabilized MAP2K4 in a concentration dependent manner. We then developed two in vitro MAP2K4 kinase assays to evaluate kinase inhibitory function, one a western-blot based assay employing the biologically relevant downstream substrates, JNK1 and p38 MAPK, and the other an Alphascreen based activity assay. In this manner, we validated the performance of our initial FTS screen. Finally, by coupling our structure-activity relationship data to MAP2K49s crystal structure, we constructed a model for inhibitor binding. It predicted binding of our identified inhibitors to MAP2K49s ATP pocket. We next applied this approach in a high-throughput fashion to over 2200 chemically diverse compounds and identified new inhibitors of MAP2K4. Herein we report the creation of a robust inhibitor-screening platform with the ability to inform the discovery and design of new and potent MAP2K4 inhibitors. Citation Format: Sankar Narayan Krishna, Chi-Hao Luan, Matthew R. Clutter, Rama K. Mishra, Karl A. Scheidt, Wayne F. Anderson, Raymond C. Bergan. A high-throughput screening platform for the identification of small molecule inhibitors of MAP2K4. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5394. doi:10.1158/1538-7445.AM2014-5394