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High-Throughput CETSA Identifies Small Molecule Modulators of ILT3 (LILRB4) with Functional Activity in Human
Somaya A Abdel-Rahman1, Moustafa Gabr1
1Department of Radiology, Molecular Imaging Innovations Institute (MI3), Weill Cornell Medicine, New York, New York 10065, United States.
Abstract:
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.
Insights
Researchers developed a new assay to find drugs targeting immune cells in Alzheimer's disease. They identified IB15C, a compound that restores microglial function and enhances amyloid-beta clearance.
Area of Science:
- Neuroimmunology
- Drug Discovery
- Alzheimer's Disease Pathogenesis
Background:
- Microglial immune inhibitory signaling impairs amyloid-beta (Aβ) clearance and promotes neuroinflammation in Alzheimer's disease (AD).
- Targeting these inhibitory pathways with small molecules is an underexplored therapeutic strategy for AD.
Purpose of the Study:
- To develop a high-throughput screening platform for identifying small molecule modulators of the inhibitory immune receptor ILT3 (LILRB4).
- To validate and characterize novel ILT3 binders for potential therapeutic applications in AD.
Main Methods:
- Development and application of a high-throughput cellular thermal shift assay (HT-CETSA) for screening compound libraries.
- Orthogonal validation using microscale thermophoresis, surface plasmon resonance, molecular docking, and site-directed mutagenesis.
- Functional assessment in human induced pluripotent stem cell (iPSC)-derived microglia to evaluate effects on signaling pathways, cytokine secretion, and Aβ uptake.
Main Results:
- Screening of approximately 40,000 compounds identified multiple ILT3 binders, including IB15C, a potent submicromolar inhibitor.
- IB15C demonstrated direct, target-specific engagement with ILT3, disrupting the ILT3-ApoE interaction.
- IB15C restored microglial function by reducing SHP1/2 and NF-κB signaling, suppressing IL-1β secretion, and enhancing Aβ uptake in iPSC-derived microglia.
Conclusions:
- The developed HT-CETSA platform is effective for identifying small molecule modulators of ILT3.
- IB15C represents a promising lead compound for developing novel neuroimmune therapeutics targeting ILT3 in Alzheimer's disease.
- Further preclinical development of IB15C is warranted due to its demonstrated efficacy and favorable properties.
