ABSTRACT The two PLC-gamma isozymes (PLC-gamma1, -gamma2) are central to the relay and amplification of signals originating from numerous receptor tyrosine kinases and immune receptors, including the B and T cell receptors. When this control is dysregulated, the PLC-gamma isozymes contribute to inflammatory and immunological diseases as exemplified by various leukemias and lymphomas. For example, PLC-gamma1 is the most frequently (~40%) mutated protein in patients with adult T cell leukemia/lymphoma; the mutated proteins are invariably constitutively active. Similarly, mutated, constitutively active forms of PLC-gamma2 arise in response to treatment of B cell leukemias and lead to refractory disease. Despite the obvious causality between the PLC-gamma isozymes and hematologic cancers, there are no chemical probes or breakthrough therapeutics that specifically target the PLC-gamma isozymes. This deficiency arises mainly from our incomplete understanding of how the PLC-gamma isozymes are regulated as well as inadequate tools to monitor this regulation in a manner amenable to high-throughput screens. We recently determined the first structure of a full-length PLC-gamma isozyme at atomic resolution and used it to develop an allosteric model of its regulation. In addition, we created XY-69, the first fluorescent, membrane-bound analog of PIP2 that reliably captures the allosteric activation of PLC-gamma isozymes in response to either extracellular stimuli or mutation. In preliminary studies, we show that XY-69 can be used to identify both orthosteric and allosteric inhibitors in high-throughput screens. Consequently, the overall objective of this proposal is to identify selective inhibitors of the PLC-gamma isozymes to be used as chemical probes and leads for drug development. Two Specific Aims will be pursued. In Aim 1, we will use a novel high-throughput screen to identify drug-like compounds that selectively inhibit the PLC-gamma isozymes. In Aim 2, these compounds will be profiled and prioritized for further optimization using a battery of biochemical, biophysical and cellular assays that emphasize T cell biology and chemotaxis. Ultimately, these inhibitors will be essential, high-demand probes used to dissect cellular processes controlled by the PLC-gamma isozymes. Moreover, these inhibitors will be promising leads for the development of drugs to treat various hematologic cancers driven by the PLC-gamma isozymes.