Affinity Selection-Mass Spectrometry Coupled with Biophysical Validation Enables Proof-of-Concept Discovery of CHI3L1 Binders

Abstract

Chitinase-3-like protein 1 (CHI3L1) is a multifunctional extracellular glycoprotein implicated in tumor progression, immune suppression, and fibrosis, making it an attractive but challenging therapeutic target. To explore its chemical tractability, we applied an affinity selection-mass spectrometry (AS-MS) workflow to screen 10,000 small molecules for CHI3L1 binding. The screen yielded 124 initial hits with a hit rate of 1.24%, which were prioritized based on chemical suitability, and six candidates were advanced for validation using microscale thermophoresis (MST). Among these, compound A9 exhibited a clear, dose-dependent binding response in MST with a Kd of 182 {+/-} 18 {micro}M. Molecular docking supported these findings, revealing that A9 forms hydrophobic and hydrogen-bonding interactions within a defined pocket of the CHI3L1 structure. Although modest in affinity, A9 represents the first small molecule binder of CHI3L1 identified through AS-MS. This study provides a proof-of-concept demonstration that CHI3L1 can be chemically engaged using AS-MS, establishing a foundation for future medicinal chemistry optimization and the development of chemical probes targeting this previously undruggable extracellular protein. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=121 SRC="FIGDIR/small/681114v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@12959c2org.highwire.dtl.DTLVardef@1c3e3a4org.highwire.dtl.DTLVardef@1962a04org.highwire.dtl.DTLVardef@1a0a2c4_HPS_FORMAT_FIGEXP M_FIG C_FIG