High-throughput fragment screening identifies a new small molecule scaffold that modulates TREM2 Signaling.

Fragment-based drug discovery (FBDD) remains a powerful tool in drug development for targeting a wide range of proteins and identifying new small molecule-based scaffolds. Here, we explored a fragment library of 3200 compounds using a temperature-related intensity change (TRIC)-based high-throughput screening (HTS) approach, and successfully identified new scaffolds that bind to triggering receptor expressed on myeloid cells 2 (TREM2), a relevant target in neurodegenerative diseases and cancer immunotherapy. We first validated the hits with dose-dependent assays, then chose the three most promising compounds (2 M06, 6B10, 7G19) with binding affinities in the low to medium micromolar range for a "SAR by catalog" study. We screened 29 selected derivatives and subsequently evaluated them with dose-dependent experiments, a thermal shift assay (TSA), selectivity studies with off-targets (LAG-3, TREM1), and finally, in vitro TREM2 activation assays. In this SAR study, derivative 6B10-9 emerged as the lead compound with moderate TREM2 binding affinity (KD = 68.3 μM) and significant effects on TREM2-dependent phosphorylation of SYK and DAP12 in HEK cells as wells as on microglial phagocytosis in HMC3 cells. Additionally, an in silico analysis revealed that 6B10-9 forms a stable complex with TREM2 via hydrogen bonding, which maintains its structural integrity during extended molecular dynamics (MD) simulations. These results suggest that 6B10-9 could serve as a promising lead for future optimizations in the development of small molecule-based TREM2 modulators.