Project Summary/Abstract Recent studies have shown that PIk1-interacting checkpoint helicase (PICH) is a potential cancer therapeutic target. PICH has critical functions in mitosis for the fidelity of chromosome segregation and without PICH there is an increased frequency of Ultra-Fine DNA Bridges (UFBs) in anaphase, which results in multi-nucleation and chromosome loss. Other proteins with key functions in mitosis, and that are required for accurate chromosome segregation, have proven to be effective targets of cancer drugs. For example, drugs that target microtubule dynamics. Importantly, PICH was found to be over-expressed in breast cancer cells and depletion of PICH was found to be extremely toxic to triple negative breast cancer cells, while not affecting matched normal tissue. Because PICH is over-expressed in a range of cancers, it is likely that many cancer types require PICH function for survival. Therefore, small molecule PICH inhibitors have potential for improved patient outcomes in cancer therapy. However, no inhibitors of PICH have been identified. Here we propose to identify selective PICH inhibitors and determine their cytotoxicity towards a range of cancer types in which PICH is over- expressed. Since PICH is a dsDNA-dependent ATPase, it has an activity suitable for high-throughput screening (HTS) for inhibitors using diverse small molecule libraries. We have established biochemical purification of active PICH and we have devised strategies and generated the tools that will allow primary hits from the HTS to be efficiently subjected to secondary screening, to validate bona fide PICH inhibitors and to eliminate non-specific inhibitors. The cellular consequences of PICH inhibition will be determined to fully characterize the effects on normal and cancer cells. Cytotoxicity studies will determine which cancer types can be selectively killed by the inhibitors and we will define the dosages required. The outcomes will be selective small molecule PICH inhibitors that will be valuable research tools and will be lead compounds for development of optimally potent molecules ahead of studies in animal cancer models.