Fuchs, N.; Kuncewicz, K.; El gaamouch, F.; Gabr, M.

Triggering receptor expressed on myeloid cells 2 (TREM2) is an immunomodulatory receptor implicated in both neurodegenerative diseases and cancer. Depending on the context, TREM2 agonists or inhibitors hold therapeutic potential. To date, the majority of TREM2-targeted strategies have centered on monoclonal antibodies (mAbs), which face limitations such as poor tissue penetration and potential immunogenic side effects. To overcome these challenges and expand the chemical space for TREM2-targeting agents, we developed a high-throughput screening (HTS) platform to identify novel small molecule TREM2 binders. Using temperature-related intensity change (TRIC) technology in a 384-well plate format (NanoTemper Dianthus), we screened two focused compound libraries comprising over 1,200 molecules. From this screen, 18 preliminary hits (1.44% hit rate) were identified and subsequently validated by dose-response binding studies using microscale thermophoresis (MST), yielding four validated hits (0.32% hit rate) with binding affinities in the high to medium micromolar range (e.g., T2337, KD = 22.4 M). The binding of the top hit, T2337, was further validated using surface plasmon resonance (SPR). Additionally, we assessed the functional activity of all four validated hits in a cellular assay measuring TREM2-mediated Syk phosphorylation in HEK293 cells co-expressing human TREM2 and its adaptor protein DAP12. These findings establish a robust and scalable platform for the discovery of small molecule TREM2 modulators and serve as a proof-of-concept for broader HTS campaigns targeting TREM2.