High-Throughput CETSA Identifies Small Molecule Modulators of ILT3 (LILRB4) with Functional Activity in Human iPSC-Derived Microglia for Alzheimer’s Disease

Immune inhibitory signaling in microglia contributes to impaired amyloid-β (Aβ) clearance and neuroinflammation in Alzheimer’s disease (AD), yet small molecule modulators targeting these pathways remain largely unexplored. Here, we report the development of a high-throughput cellular thermal shift assay (HT-CETSA) platform for identification of small molecule binders targeting the inhibitory immune receptor ILT3 (LILRB4). Screening of ∼40 000 compounds yielded multiple validated hits, including IB15C, a submicromolar ILT3 binder identified through preliminary structure–activity relationship optimization. Orthogonal validation by microscale thermophoresis, surface plasmon resonance, docking, and site-directed mutagenesis confirmed direct and target-specific ILT3 engagement. Functionally, IB15C disrupted the ILT3-ApoE interaction and restored microglial activity in human iPSC-derived microglia, reducing SHP1/2 and NF-κB signaling, suppressing IL-1β secretion, and enhancing Aβ uptake. IB15C also demonstrated favorable in vitro pharmacokinetic and safety properties, supporting further development of ILT3-targeted neuroimmune therapeutics.