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bioRxiv · 10.64898/2026.05.19.726368

Development of a High-Throughput TR-FRET Assay for Identification of Small Molecule Inhibitors of the LILRB4 (ILT3)-SCG2 Immune Checkpoint Interaction

Abdel-Rahman, S.·Gabr, M.
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Abstract

Leukocyte immunoglobulin-like receptor B4 (LILRB4, ILT3) is an inhibitory immune checkpoint expressed on myeloid cells, where it contributes to immunosuppression within the tumor microenvironment. Secretogranin 2 (SCG2) has recently been identified as a functional ligand of LILRB4, yet small molecule modulators of this interaction remain unexplored. Here, we report the development of a high-throughput time-resolved fluorescence resonance energy transfer (TR-FRET) assay to interrogate the LILRB4 (ILT3)-SCG2 interaction. The assay demonstrated robust performance and was validated using a blocking anti-LILRB4 antibody, consistent with orthogonal ELISA measurements. Pilot screening of chemical libraries identified 23 primary hits, of which two compounds, BMS-813160 and PSB-603, showed reproducible, dose-dependent inhibition with TR-FRET IC50 values of 26.7 {+/-} 1.03 {micro}M and 37.2 {+/-} 2.14 {micro}M, respectively. Activity was confirmed by ELISA, supporting the robustness of the assay. This platform enables high-throughput discovery of first-in-class small molecule modulators of the LILRB4-SCG2 immune checkpoint and provides a foundation for targeting myeloid-driven immunosuppression.

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Abdel-Rahman, S., Gabr, M.. 2026-05-21. Development of a High-Throughput TR-FRET Assay for Identification of Small Molecule Inhibitors of the LILRB4 (ILT3)-SCG2 Immune Checkpoint Interaction. https://doi.org/10.64898/2026.05.19.726368

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