Discovery of Small Molecule CHI3L1 Inhibitors by SPR-Based High-Throughput Screening

Chitinase-3-like 1 (CHI3L1) is a secreted glycoprotein implicated in carcinogenesis and tumor immune evasion. Elevated CHI3L1 expression is frequently detected in cancer patients, highlighting it as a promising therapeutic target. To overcome the limited availability of small molecule CHI3L1 inhibitors, we established a surface plasmon resonance (SPR)–based high-throughput screening platform and applied it to a focused chemical library of small molecules. Primary screening identified seven hits, with compounds 1-4 and 1-7 validated as CHI3L1 binders ( Kd = 10.4 ± 1.0 μM and 7.40 ± 0.78 μM, respectively). Both compounds disrupted the CHI3L1–galectin-3 interaction in AlphaLISA assays and engaged the CHI3L1 binding pocket in docking and molecular dynamics (MD) simulations. Importantly, functional evaluation in a multicellular 3D glioblastoma (GBM) spheroid model demonstrated that compound 1-7 potently reduced spheroid viability and inhibited STAT3 phosphorylation, outperforming both compound 1-4 and the known CHI3L1–STAT3 disruptor hygromycin B (HB). These findings validate SPR as a robust primary screening platform for CHI3L1 and demonstrate that the identified small molecule binders exert functional activity in a physiologically relevant multicellular GBM spheroid model.