Ligandability Assessment of the LAG-3 D1 Domain Enables Discovery of a Small-Molecule Inhibitor

LAG-3 is an emerging immune checkpoint whose extracellular D1 domain engages MHC class II through a broad protein-protein interface traditionally considered difficult to modulate with small molecules. To evaluate the ligandability of this region, we combined 100-ns molecular dynamics (MD) simulations, structure-based virtual screening, and biophysical and biochemical assays. MD sampling of the isolated D1 domain revealed a recurrent, transient cavity adjacent to the MHCII-binding surface. A representative pocket-open conformation was used to screen a [~]10,240-compound diversity library, yielding a single validated hit, N05. N05 bound the D1 domain with micromolar affinity measured by microscale thermophoresis (Kd = 59.2 {micro}M, TRIC/MST channel) and by spectral-shift detection (Kd = 56.1 {micro}M), and it partially inhibited the LAG-3/MHCII interaction (EC50 = 42.9 {micro}M; maximal inhibition [~]76%). A 30-ns MD simulation of the LAG-3-N05 complex showed stable ligand engagement within the MD-identified cavity and consistent stabilizing interactions with residues forming the pocket. These results demonstrate that the LAG-3 D1 domain possesses an accessible, dynamically formed binding site capable of accommodating small molecules, providing a structural and biophysical foundation for future exploration of LAG-3 ligandability. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=151 SRC="FIGDIR/small/692800v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@177b446org.highwire.dtl.DTLVardef@1dc9efdorg.highwire.dtl.DTLVardef@14a2d6aorg.highwire.dtl.DTLVardef@ebca69_HPS_FORMAT_FIGEXP M_FIG C_FIG Overview of the computational and experimental workflow used to identify and validate a small-molecule binder of the LAG-3 D1 domain.