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

An α 4 β 1 /α 4 β 7 dual antagonist, 35 S-compound 1, was used as a model ligand to study the effect of divalent cations on the activation state and ligand binding properties of α 4 integrins. In the presence of 1 mM each Ca 2+ /Mg 2+ , 35 S-compound 1 bound to several cell lines expressing both α 4 β 1 and α 4 β 7 , 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 α 4 β 1 antagonist, completely inhibited 35 S-compound 1 binding, suggesting that α 4 β 1 was responsible for the observed binding. 35 S-Compound 1 bound RPMI-8866 cells expressing predominantly α 4 β 7 with a K D of 1.9 nM in the presence of 1 mM Mn 2+ , and binding was inhibited only 29% by BIO7662, suggesting that the probe is a potent antagonist of activated α 4 β 7 . With Ca 2+ /Mg 2+ , 35 S-compound 1 bound Jurkat cells expressing primarily α 4 β 1 with a K D of 18 nM. In contrast, the binding of 35 S-compound 1 to Mn 2+ -activated Jurkat cells occurred slowly, reaching equilibrium by 60 min, and failed to dissociate within another 60 min. The ability of four α 4 β 1 /α 4 β 7 antagonists to block binding of activated α 4 β 1 or α 4 β 7 to vascular cell adhesion molecule-1 or mucosal addressin cell adhesion molecule-1, respectively, or to 35 S-compound 1 was measured, and a similar rank order of potency was observed for native ligand and probe. Inhibition of 35 S-compound 1 binding to α 4 β 1 in Ca 2+ /Mg 2+ was used to identify nonselective antagonists among these four. These studies demonstrate that α 4 β 1 and α 4 β 7 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.