From Virtual Screens to Cellular Target Engagement: New Small Molecule Ligands for the Immune Checkpoint LAG-3

Herein, we performed a virtual screening study to discover new scaffolds for small molecule-based ligands of the immune checkpoint lymphocyte-activation gene 3 (LAG-3). Molecular dynamics (MD) simulations using the LAG-3 structure revealed two putative binding sites for small molecules: the antibody interface and a lipophilic canyon. A 3D pharmacophore screening resulted in the identification of potential ligands for these binding sites and afforded a library of 25 compounds. We then evaluated the screening hits for LAG-3 binding via microscale thermophoresis (MST) and surface plasmon resonance (SPR). Our biophysical screening identified two binders with K _D values in the low micromolar range, compounds 3 (antibody interface) and 25 (lipophilic canyon). Furthermore, we investigated the ability of LAG-3 hits to en-gage LAG-3 on a cellular level using a cellular thermal shift assay (CETSA), where compound 3 emerged as a promising candidate for future development.