title: A small molecule alpha4beta1/alpha4beta7 antagonist differentiates between the low-affinity states of alpha4beta1 and alpha4beta7: characterization of divalent cation dependence.
abstract: An alpha4beta1/alpha4beta7 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 alpha4 integrins. In the presence of 1 mM each Ca2+/Mg2+, 35S-compound 1 bound to several cell lines expressing both alpha4beta1 and alpha4beta7, 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 alpha4beta1 antagonist, completely inhibited 35S-compound 1 binding, suggesting that alpha4beta1 was responsible for the observed binding. 35S-Compound 1 bound RPMI-8866 cells expressing predominantly alpha4beta7 with a KD 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 alpha4beta7. With Ca2+/Mg2+, 35S-compound 1 bound Jurkat cells expressing primarily alpha4beta1 with a KD 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 alpha4beta1/alpha4beta7 antagonists to block binding of activated alpha4beta1 or alpha4beta7 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 alpha4beta1 in Ca2+/Mg2+ was used to identify nonselective antagonists among these four. These studies demonstrate that alpha4beta1 and alpha4beta7 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.
authors.affiliation: Pharmacology, Merck & Co, Inc, Rahway, NJ 07065, USA. linda_egger@merck.com
authors.forename: Linda A
authors.initials: LA
authors.lastname: Egger
authors.forename: Jin
authors.initials: J
authors.lastname: Cao
authors.forename: Christine
authors.initials: C
authors.lastname: McCallum
authors.forename: Usha
authors.initials: U
authors.lastname: Kidambi
authors.forename: Gail
authors.initials: G
authors.lastname: Van Riper
authors.forename: Ermengilda
authors.initials: E
authors.lastname: McCauley
authors.forename: Richard A
authors.initials: RA
authors.lastname: Mumford
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Lanza
authors.forename: Linus S
authors.initials: LS
authors.lastname: Lin
authors.forename: Stephen E
authors.initials: SE
authors.lastname: de Laszlo
authors.forename: David N
authors.initials: DN
authors.lastname: Young
authors.forename: Ginger
authors.initials: G
authors.lastname: Yang
authors.forename: Dennis C
authors.initials: DC
authors.lastname: Dean
authors.forename: Conrad E
authors.initials: CE
authors.lastname: Raab
authors.forename: Mike A
authors.initials: MA
authors.lastname: Wallace
authors.forename: Allen N
authors.initials: AN
authors.lastname: Jones
authors.forename: William K
authors.initials: WK
authors.lastname: Hagmann
authors.forename: John A
authors.initials: JA
authors.lastname: Schmidt
authors.forename: R Blake
authors.initials: RB
authors.lastname: Pepinsky
authors.forename: Daniel M
authors.initials: DM
authors.lastname: Scott
authors.forename: Wen-Cherng
authors.initials: WC
authors.lastname: Lee
authors.forename: Mark A
authors.initials: MA
authors.lastname: Cornebise
authors.forename: Patricia A
authors.initials: PA
authors.lastname: Detmers
mesh_terms: D001665:Binding Sites; D002413:Cations, Divalent / Q000378:metabolism*; D002460:Cell Line; D004151:Dipeptides / Q000737:chemistry / Q000494:pharmacology*; D006801:Humans; D039041:Integrin alpha4beta1 / Q000037:antagonists & inhibitors* / Q000378:metabolism; D016023:Integrins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D019169:Jurkat Cells; D020014:K562 Cells; D007700:Kinetics; D008024:Ligands; D010649:Phenylalanine / Q000031:analogs & derivatives / Q000737:chemistry / Q000494:pharmacology*; D010671:Phenylurea Compounds / Q000737:chemistry / Q000494:pharmacology*; D011485:Protein Binding; D011869:Radioligand Assay; D013462:Sulfur Radioisotopes; D014407:Tumor Cells, Cultured; D019010:Vascular Cell Adhesion Molecule-1 / Q000378:metabolism
chemical_list: C438014:2-((1-benzenesulfonylpyrrolidine-2-carbonyl)amino)-4-(4-methyl-2-(methyl-(2-(4-(3-o-tolylureido)phenyl)acetyl)amino)pentanoylamino)butyric acid; D002413:Cations, Divalent; D004151:Dipeptides; D039041:Integrin alpha4beta1; D016023:Integrins; D008024:Ligands; C459380:N-(2,6-dichlorobenzoyl)-4-(2',6'-bismethoxyphenyl)phenylalanine; D010671:Phenylurea Compounds; D013462:Sulfur Radioisotopes; D019010:Vascular Cell Adhesion Molecule-1; C071777:integrin alpha4beta7; D010649:Phenylalanine