Authors:

Andrew M Strawhacker

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About Paper:

A hyperactive variant of protein phospholipase C gamma 2 (PLCG2), known as S707Y, has been genetically linked to autoinflammatory immune dysregulation (APLAID), characterized by severe tissue lesioning and immunodeficiency, as well as to resistance to the leading leukemia treatment ibrutinib. Currently, there are no PLCG2-targeted therapies for these conditions. This project aims to address this gap by identifying and optimizing inhibitors of PLCG2 S707Y through an integrated approach of kinetic characterization, structural analysis, and computational modeling. Using a fluorescent substrate analog, the S707Y variant was confirmed to exhibit approximately 7-fold higher catalytic activity compared to the wild-type enzyme, consistent with its proposed pathogenic role. To search for inhibitors, high-throughput screening by affinity selection mass spectrometry (ASMS) identified several PLCG2-specific binding compounds, which were subsequently evaluated using the fluorescence platform. One of these compounds inhibited PLCG2 S707Y in vitro, with its IC50 and maximum inhibition determined at a 5nM enzyme concentration. Derivative molecules were then explored for chemical moieties that increase inhibitor potency. To guide inhibitor optimization, the crystal structures of PLCG2 S707Y with and without the initial lead compound were determined by X-ray diffraction at Argonne National Laboratory's Advanced Photon Source. These structures were analyzed alongside binding predictions generated by MIT's novel Boltz-2 machine learning platform, providing an integrated foundation for structure-based inhibitor refinement. These findings provide the first structural characterization of PLCG2 S707Y and identify an early-stage inhibitor, representing a first step in PLCG2-targeted therapies for APLAID and ibrutinib-resistant leukemia. Keywords: PLCG2; APLAID; Ibrutinib Resistance; X-Ray Crystallography; Drug Design † Presenting Undergrad Author; ‡ Contributing Undergrad Author; * Undergrad Acknowledgment

Source:

Purdue University / 2025

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Co-authors:

Andrew M Strawhacker