Pilot Screening of TREM1 Inhibitors in Cell-Based Reporter Assays Reflecting TREM1/DAP12 Receptor Assembly and Functionality

Proinflammatory TREM1 receptors expressed on myeloid-derived cells have recently been recognized as a new oncogenic target in cancer, including gliomas. They are established chemotherapeutic targets in neurodegenerative Parkinson's and Alzheimer's diseases, and they also contribute to stroke and sepsis severities. TREM1 activation requires the TREM1/DAP12 interaction for receptor clustering and signal transduction coordinated by TREM1 ligands. Here, we established the quantitative cell-based high-throughput split luciferase assays of DAP12 dimerization, TREM1 dimerization, and TREM1/DAP12 interaction that allow screening of the inhibitory compounds with quantitative dose-responses, IC₅₀ values, and specificity evaluation. The assays are based on the reconstitution of firefly luciferase activity during DAP12 dimerization, TREM1 dimerization, and TREM1/DAP12 interaction, leading to robust luminescence signals in the presence of luciferin. The ligand-dependent and -independent SCHOOL TREM1 inhibitory peptides were utilized for assay validation. Our pilot screen identified several compound scaffolds disrupting DAP12 dimerization, TREM1 dimerization, and the TREM1/DAP12 interaction. The compound potential mechanisms of action and binding sites in the TREM1 and DAP12 complexes were revealed using CB-Dock2 docking software. To our knowledge, this is the first report providing the first generation of pharmacological modulators for TREM1 receptors.