From Virtual\nScreens to Cellular Target Engagement:\nNew Small Molecule Ligands for the Immune Checkpoint LAG‑3

Herein, we performed a virtual screening study to discover\nnew\nscaffolds for small molecule-based ligands of the immune checkpoint\nlymphocyte-activation gene 3 (LAG-3). Molecular dynamics (MD) simulations\nusing the LAG-3 structure revealed two putative binding sites for\nsmall molecules: the antibody interface and the lipophilic canyon.\nA 3D pharmacophore screening resulted in the identification of potential\nligands for these binding sites and afforded a library of 25 compounds.\nWe then evaluated the screening hits for LAG-3 binding via microscale\nthermophoresis (MST) and surface plasmon resonance (SPR). Our biophysical\nscreening identified two binders with <i>K</i><sub>D</sub> values in the low micromolar range, compounds <b>3</b> (antibody\ninterface) and <b>25</b> (lipophilic canyon). Furthermore, we\ninvestigated the ability of LAG-3 hits to engage LAG-3 on a cellular\nlevel using a cellular thermal shift assay (CETSA). In summary, compound <b>3</b> shows potential as a lead but is not yet a development candidate.