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INFLAMMATION AND IMMUNOPHARMACOLOGY

A Small Molecule ₄₁/₄₇ Antagonist Differentiates between the Low-Affinity States of ₄₁ and ₄₇: Characterization of Divalent Cation Dependence

Pharmacology (L.A.E., J.C., U.K., P.A.D.), High Throughput Screening (C.M.), Immunology and Rheumatology (G.V.R., E.M., R.A.M.), Medicinal Chemistry (L.S.L., S.E.d.L., D.N.Y., G.Y., W.K.H.), Drug Metabolism (D.C.D., C.E.R., M.A.W., A.N.J.), Merck & Co., Rahway, New Jersey; Aventis (J.A.S.), Bridgewater, New Jersey; and Biogen, Inc. (R.B.P., D.M.S., W.-C.L., M.A.C.), Cambridge, Massachusetts

An ₄₁/₄₇ 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 ₄ integrins. In the presence of 1 mM each Ca²⁺/Mg²⁺, ³⁵S-compound 1 bound to several cell lines expressing both ₄₁ and ₄₇, but 2S-[(1-benzenesulfonyl-pyrrolidine-2S-carbonyl)-amino]-4-[4-methyl-2S-(methyl-{2-[4-(3-o-tolyl-ureido)-phenyl]-acetyl}-amino) pentanoylamino]-butyric acid (BIO7662), a specific ₄₁ antagonist, completely inhibited ³⁵S-compound 1 binding, suggesting that ₄₁ was responsible for the observed binding. ³⁵S-Compound 1 bound RPMI-8866 cells expressing predominantly ₄₇ 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 ₄₇. With Ca²⁺/Mg²⁺, ³⁵S-compound 1 bound Jurkat cells expressing primarily ₄₁ 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 ₄₁/₄₇ antagonists to block binding of activated ₄₁ or ₄₇ 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 ₄₁ in Ca²⁺/Mg²⁺ was used to identify nonselective antagonists among these four. These studies demonstrate that ₄₁ and ₄₇ 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.

Address correspondence to: Dr. Linda A. Egger, Merck & Co., Inc., Pharmacology, P.O. Box 2000, RY80N-A26, Rahway, NJ 07065. E-mail: linda_egger{at}merck.com