Structure-Based Discovery of a Novel Class of Small-Molecule Pure Antagonists of Integrin alpha V beta 3

Inhibitors of integrin alpha V beta 3 have therapeutic promise for a variety of diseases. Most alpha V beta 3-targeting small molecules patterned after the RGD motif are partial agonists because they induce a high-affinity, ligand-binding conformation and prime the receptor to bind the ligand without an activating stimulus, in part via a charge-charge interaction between their aspartic acid carboxyl group and the metal ion in the metal-ion-dependent adhesion site (MIDAS). Building upon our previous studies on the related integrin alpha IIb beta 3, we searched for pure alpha V beta 3 antagonists that lack this typical aspartic acid carboxyl group and instead engage through direct binding to one of the coordinating residues of the MIDAS metal ion, specifically beta 3 E220. By in silico screening of two large chemical libraries for compounds interacting with beta 3 E220, we indeed discovered a novel molecule that does not contain an acidic carboxyl group and does not induce the high-affinity, ligand-binding state of the receptor. Functional and structural characterization of a chemically optimized version of this compound led to the discovery of a novel small-molecule pure alpha V beta 3 antagonist that (i) does not prime the receptor to bind the ligand and does not induce hybrid domain swing-out or receptor extension as judged by antibody binding and negative-stain electron microscopy, (ii) binds at the RGD-binding site as predicted by metadynamics rescoring of induced-fit docking poses and confirmed by a cryo-electron microscopy structure of the compound-bound integrin, and (iii) coordinates the MIDAS metal ion via a quinoline moiety instead of an acidic carboxyl group.