Primary screening and hit follow up to identify the first selective inhibitors of PLC?1 and PLC?2 - PROJECT SUMMARY Dysfunction of either of the two phospholipase C gamma family members, PLCγ1 and PLCγ2 have been implicated as driving oncogenesis through activating mutants or oncofusion protein-induced upregulation. Additionally, PLCγ1 has been identified as an interaction partner with PD-L1, suggesting PLCγ1 may be a synergistic target for PD-L1 immunotherapies. While modulation of these two enzymes is a tantalizing target for a variety of cancers, unfortunately, no specific, potent inhibitors of PLCγ1 or PLCγ2 are known. One of the reasons for this is that HTS-amenable substrates have not been readily available to screen PLCγ1 or PLCγ2. Our lab has developed a novel fluorescent substrate, called C8CF3-coumarin, and currently has rapid synthetic access to up to multiple grams of the substrate. Additionally, our lab has worked with a chemical supplier to make an additional substrate, XY-69, commercially available. This substrate is incorporated into a liposome and for this reason it better mimics the enzymes' natural membrane-bound PIP2 substrate. To identify small molecule inhibitors of PLCγ1 and PLCγ2, we will employ the already developed and optimized HTS-amenable solution assay using our new C8CF3-coumarin to screen 200,000 compounds against both PLCγ enzymes. Primary hit molecules will be retested in the same assay conditions to confirm their activity. After eliminating any known promiscuous binders and compounds that interfere with the assay, these hits will be confirmed using the now commercially available XY-69 substrate in a liposomal assay. The confirmed hits then will undergo further testing to determine their dose-response, mode of action, and activity in other orthogonal assays including biophysical interaction and cellular assays. Compounds that advance through all of the orthogonal testing workflow will be designated as validated hits and an apparent path for future development will be determined by performing a preliminary structure-activity-relationship (SAR) study. Chemical scaffolds with a promising forward path will be designated as validated hit scaffolds. These validated hit scaffolds are the primary outcome of this project and will result in chemical matter that can be further developed into useful chemical probes or potential therapeutics upon further investment. To successfully identify validated hit scaffolds, the following specific aims will be employed: Specific Aim 1 – Identification of small molecule inhibitors of PLCγ1 and PLCγ2 via already developed and optimized biochemical high-throughput screening assays Specific Aim 2 – Validation of identified confirmed hit molecules via orthogonal assays Specific Aim 3 – Generation of preliminary structure-activity-relationship studies to determine the presence of a promising forward path towards the generation of chemical probes and/or therapeutic leads
