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 triplicate 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 μM, TRIC/MST channel) and by spectral-shift detection (Kd = 56.1 μM), and it partially inhibited the LAG-3/MHCII interaction (EC₅₀ = 42.9 μM; maximal inhibition ∼76%). Triplicate 30-ns MD simulations 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.