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

Immune inhibitory signaling in microglia contributes to impaired amyloid clearance and neuroinflammation in Alzheimers 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, suppressing cytokine secretion, and enhancing amyloid uptake. IB15C also demonstrated favorable in vitro pharmacokinetic and safety properties, supporting further development of ILT3-targeted neuroimmune therapeutics.