A Small Molecule α4β1/α4β7 Antagonist Differentiates between the Low-Affinity States of α4β1 and α4β7: Characterization of Divalent Cation Dependence

An α4β1/α4β7 dual antagonist, 35S-compound 1, was used as a model ligand to study the effect of divalent cations on the activation state and ligand binding properties of α4 integrins. In the presence of 1 mM each Ca2+/Mg2+, 35S-compound 1 bound to several cell lines expressing both α4β1 and α4β7, but 2 S -[(1-benzenesulfonyl-pyrrolidine-2 S -carbonyl)-amino]-4-[4-methyl-2 S -(methyl-{2-[4-(3- o -tolyl-ureido)-phenyl]-acetyl}-amino) pentanoylamino]-butyric acid (BIO7662), a specific α4β1 antagonist, completely inhibited 35S-compound 1 binding, suggesting that α4β1 was responsible for the observed binding. 35S-Compound 1 bound RPMI-8866 cells expressing predominantly α4β7 with a K D of 1.9 nM in the presence of 1 mM Mn2+, and binding was inhibited only 29% by BIO7662, suggesting that the probe is a potent antagonist of activated α4β7. With Ca2+/Mg2+, 35S-compound 1 bound Jurkat cells expressing primarily α4β1 with a K D of 18 nM. In contrast, the binding of 35S-compound 1 to Mn2+-activated Jurkat cells occurred slowly, reaching equilibrium by 60 min, and failed to dissociate within another 60 min. The ability of four α4β1/α4β7 antagonists to block binding of activated α4β1 or α4β7 to vascular cell adhesion molecule-1 or mucosal addressin cell adhesion molecule-1, respectively, or to 35S-compound 1 was measured, and a similar rank order of potency was observed for native ligand and probe. Inhibition of 35S-compound 1 binding to α4β1 in Ca2+/Mg2+ was used to identify nonselective antagonists among these four. These studies demonstrate that α4β1 and α4β7 have distinct binding properties for the same ligand, and binding parameters are dependent on the state of integrin activation in response to different divalent cations.