Characterization of Divalent Cation Dependence

An 4 1/ 4 7 dual antagonist, 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 4 integrins. In the presence of 1 mM each Ca /Mg , S-compound 1 bound to several cell lines expressing both 4 1 and 4 7, but 2S-[(1-benzenesulfonyl-pyrrolidine-2S-carbonyl)-amino]-4-[4methyl-2S-(methyl-{2-[4-(3-o-tolyl-ureido)-phenyl]-acetyl}amino) pentanoylamino]-butyric acid (BIO7662), a specific 4 1 antagonist, completely inhibited S-compound 1 binding, suggesting that 4 1 was responsible for the observed binding. S-Compound 1 bound RPMI-8866 cells expressing predominantly 4 7 with a KD 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 4 7. With Ca /Mg , S-compound 1 bound Jurkat cells expressing primarily 4 1 with a KD of 18 nM. In contrast, the binding of S-compound 1 to Mn -activated Jurkat cells occurred slowly, reaching equilibrium by 60 min, and failed to dissociate within another 60 min. The ability of four 4 1/ 4 7 antagonists to block binding of activated 4 1 or 4 7 to vascular cell adhesion molecule-1 or mucosal addressin cell adhesion molecule-1, respectively, or to S-compound 1 was measured, and a similar rank order of potency was observed for native ligand and probe. Inhibition of S-compound 1 binding to 4 1 in Ca /Mg was used to identify nonselective antagonists among these four. These studies demonstrate that 4 1 and 4 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. Lymphocyte recruitment in the vasculature is regulated by the differential expression and activation of homing receptors (selectins and integrins) on lymphocytes that interact with counter-receptors of the Ig superfamily on endothelial cells. This interaction mediates a multistep process, involving rolling and tethering of leukocytes to endothelial ligands, rapid activation of integrins by locally released chemokines, stable adhesion of activated integrins to endothelial ligands, and transendothelial migration through the vessel wall (Bargatze et al., 1995). Although all integrins expressed on leukocytes can mediate firm adhesion during normal lymphocyte trafficking and in response to inflammatory stimuli, 4 1 and 4 7 are members of a small subset of integrins that can also mediate rolling (Bargatze et al., 1995; Berlin et al., 1995). In vivo studies with monoclonal antibodies or inhibitory peptides demonstrate the pathophysiological role of 4 1 and 4 7 in leukocyte-mediated inflammation in animal models (Foster, 1996; Butcher, 1999), and clinical trials with Antegren (anti4, Elan/Biogen) resulted in remission for Crohn’s disease patients (Gordon et al., 2001). 4 integrins are constitutively expressed on a variety of leukocytes and can bind to shared or distinct binding partners. 4 1 is expressed on lymphocytes, eosinophils, and monocytes and mediates adhesion to vascular cell adhesion molecule-1 (VCAM-1) expressed on the endothelium and to Dr. Linda A. Egger, Merck and Co., Inc., Pharmacology, P.O. Box 2000, RY80W-206, Rahway, NJ 07065. E-mail: [email protected] Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. DOI: 10.1124/jpet.102.047704. ABBREVIATIONS: VCAM-1, vascular cell adhesion molecule-1; MAdCAM-1, mucosal addressin cell adhesion molecule-1; HPLC, high-performance liquid chromatography; DMSO, dimethyl sulfoxide; NSB, nonspecific binding; FACS, fluorescence-activated cell sorting; CS-1, connecting segment-1; mAb, monoclonal antibody; cmpd, compound. 0022-3565/03/3063-903–913$7.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 306, No. 3 Copyright © 2003 by The American Society for Pharmacology and Experimental Therapeutics 47704/1085413 JPET 306:903–913, 2003 Printed in U.S.A. 903 at A PE T Jornals on A uust 5, 2017 jpet.asjournals.org D ow nladed from the connecting segment-1 (CS-1) subdomain of human fibronectin in the extracellular matrix. 4 1 and 4 7 are coexpressed on peripheral blood leukocytes, and 4 7 is highly expressed on a discrete subpopulation of gut-homing memory T and B lymphocytes, mediating lymphocyte adhesion within the vasculature of the gastrointestinal tract, where its major ligand, mucosal addressin cell adhesion molecule-1 (MAdCAM-1), is preferentially expressed on high endothelial venules (Butcher, 1999). Although both 4 7 and 4 1 can bind VCAM-1 and CS-1, 4 1 does not bind MAdCAM-1, and 4 7 binds to MAdCAM-1 with higher affinity than to VCAM-1 or to CS-1 (Berlin et al., 1993). Key motifs for the binding of 4 7 and 4 1 to native ligands have been defined as leucine-aspartic acid-threonine in MAdCAM-1 (Viney et al., 1996), isoleucine-aspartic acidserine in VCAM-1 (Wang et al., 1995), and leucin-aspartic acid-valine in CS-1 (Wayner and Kovach, 1992). Small molecule antagonists of 4 7 that mimic the LDT motif have been described that block the binding of 4 7-expressing cells to MAdCAM-Ig in the presence of Mn (Carson et al., 1997; Shroff et al., 1998; Martin et al., 1999; Harriman et al., 2000; Egger et al., 2002). Similarly, antagonists of 4 1 have been reported to block binding of Mn -activated (Jackson et al., 1997; Vanderslice et al., 1997; Lin et al., 1998; Hagmann et al., 2001; Muller et al., 2001) and unactivated 4 1 (Chen et al., 1999, 2001) to ligand in vitro. The essential role of cation-binding sites in regulating integrin function is known, but the coordination of each cationbinding site and the individual role of different metal cations is not well understood (Leitinger et al., 2000). All integrin -subunits have seven homologous 60-amino acid repeats at the N terminus that have been predicted to fold into a -propeller structure (Shimaoka et al., 2002), and three to four putative Ca binding sites are located within repeats 4 through 7. A metal ion-dependent activation site motif is a unique Mg /Mn binding site located in the I-domain of the -subunit, and divalent cation bound at this site has a structural role in coordinating the binding of ligand to the I-domain containing integrins. Although 4 does not contain an I-domain, an I-like domain that contains a metal iondependent activation site-like motif is present in the -chain of all integrins. Although the effect of divalent cations on 4 7-ligand interactions has not been extensively characterized, recent studies have shown that Ca is essential to support rolling under shear flow, whereas Mg can promote firm adhesion of cells expressing 4 7 to MAdCAM-1 (de Chateau et al., 2001). To assess the effect of divalent cations on the activation state of 4 integrins expressed on human lymphocytes, we used a novel dual 4 1/ 4 7 antagonist, S-compound 1 (Fig. 1), as a model ligand. A similar approach has been used to study multiple activation states of 4 1 through their different affinities for a small molecule ligand (Chen et al., 1999, 2001), but the binding of a small molecule ligand to different activation states of 4b7 has not been described. These studies provide new information that 4 1 and 4 7 have distinct binding affinities for the same small molecule ligand, and binding is dependent on the state of integrin activation in response to different divalent cations. Materials and Methods Compounds. Compound 1, N-[N-benzenesulfonyl-4(R)-cyclopropylamino-2(S)-prolyl]-(L)-4-(2 ,6 -bismethoxyphenyl)phenylalanine; compound 2, N-{[N-(3,5-dichlorobenzenesulfonyl)]-2(S)-methylprolyl}(L)-4-(2 ,6 -bismethoxyphenyl)phenylalanine; compound 3, N-{[N-(3,5dichlorobenzenesulfonyl)]-2(S)-methylprolyl}-(L)-4-(2 ,6 -bishydroxyphenyl)phenylalanine; and compound 4, N-{[N-(3,5-dichlorobenzenesulfonyl)]-2(R)-methylprolyl}-(D)-phenylalanine, were synthesized as described previously (Hagmann et al., 2001; Kopka et al., 2002; Lin et al., 2002) or by using similar methods. TR14035, N-(2,6-dichlorobenzoyl)-(L)-4-(2 ,6 -bis-methoxyphenyl) phenylalanine, was synthesized as described previously (Sircar et al., 1999a). BIO7662, 2S-[(1-benzenesulfonyl-pyrrolidine-2S-carbonyl)-amino]-4-[4-methyl-2S-(methyl-{2-[4-(3o-tolyl-ureido)-phenyl]-acetyl}-amino) pentanoylamino]-butyric acid, was synthesized as described previously (Chen et al., 2001). The structures of all compounds are shown in Fig. 1. S-Compound 1 was synthesized by reacting [S]PhSO2Cl with an appropriately diprotected amine precursor in dichloromethane in the presence of ditertbutylmethylpyridine. After sequential deprotection of the resulting [S]sulfonamide intermediate, crude S-compound 1 was purified by semipreparative HPLC and formulated as a solution in methanol with a concentration of 1.14 mCi/ml. The radiochemical purity of the compound was 95% as measured by reverse phase HPLC, and the specific activity was measured to be 722 Ci/mmol by liquid chromatography/ mass spectrometry. The radioligand in MeOH was aliquoted and stored at 20°C. The binding affinity for the radiolabeled compound was indistinguishable from that of the unlabeled compound as measured by the ability of compound to block the binding of native ligands to cells expressing 4 7 and 4 1. Antibodies and Cell Lines. The following purified monoclonal antibodies were obtained from BD PharMingen (San Diego, CA): 4B4 (mouse anti-human 1), FIB27 (rat anti-mouse 7 that cross-reacts with human 7), and isotype controls (mouse IgG1, rat IgG2b). HP2/1 (mouse anti-human 4) was obtained from Coulter/Immunotech (Hialeah, FL). The following cell lines were used: RPMI-8866 cells (human B cell line) obtained from John A. Wilkins (University of Fig. 1. Structure of antagonists of 4 1/ 4 7 integrins. The structures of compounds 1, 2, 3, and 4 (Hagmann et al., 2001; Kopka et al., 2002; Lin et al., 2002), TR14035 (Sircar et al., 1999a), and BIO7662 (Chen et al., 2001) are shown. Chemical names are given in “Compounds”. 904 Egger et al. at A PE T Jornals on A uust 5, 2017 jpet.asjournals.org D ow nladed from Manitoba, Winnipeg, Canada); Jurkat and HUT-78 (human T cell lines) from American Type Culture Collection (Manassas, VA); and K562/ 4 7 cells, a stably transfected human erythroleukemia cellline obtained from David J. Erle (University of California, San Francisco, San Francisco, CA). Binding of Native Ligands to Cells Expressing 4 7 and 4 1. A ligand binding assay for Mn 2 -activated 4 7 has been described previously (Egger et al., 2002) and was performed by incubating RPMI-8866 cells (7.5 10 cells/well) with 200 pM iodinated human MAdCAM-Ig. Similarly, a ligand binding assay for activated 4 1 was performed by incubating Jurkat cells (5 10 5 cells/well) expressing 4 1 with 100 pM iodinated VCAM-Ig in the presence of Mn , as described previously (Hagmann et al., 2001). Purified VCAM-Ig and MAdCAM-Ig were labeled with I using Bolton Hunter reagent and purified using HPLC gel filtration chromatography, and specific radioactivities were in excess of 1,100 Ci/mmol. Compounds were evaluated by incubating radioligand, compound (prepared in DMSO; 1% DMSO final concentration), cells, and binding buffer (25 mM HEPES, 150 mM NaCl, 3 mM KCl, 2 mM glucose, and 0.1% bovine serum albumin, pH 7.4) containing 1 mM MnCl2 at 25°C for 30 min ( 4 1 assays) or 45 min ( 4 7 assays) in a 96-well Millipore (Bedford, MA) multiscreen MHVBN filtration plate. After filtration and a single wash with binding buffer, the filtration plates were dried and transferred to adaptor plates. After adding 100 l of Microscint-20 (PerkinElmer Life Sciences, Boston, MA) to each well, the plates were sealed, placed on a shaker for 1 min, and counted on a PerkinElmer Top-Count. Wells containing cells radioligand 1 M compound or DMSO alone served as controls to calculate 100 and 0% inhibition, respectively. Binding of a Dual 4 1/ 4 7 Antagonist Probe, S-Compound 1, to Cells Expressing 4 7 or 4 1. Equilibrium binding studies were performed by incubating either RPMI-8866 cells (7.5 10 cells/tube) expressing 4 7 or Jurkat cells (5 10 5 cells/tube) expressing 4 1 in binding buffer containing either 1 mM MnCl2 or 1 mM each CaCl2 and MgCl2 with 0 to 30 nM (for 4 7 studies) or 0 to 60 nM (for 4 1 studies) S-compound 1 in siliconized Eppendorf microfuge tubes for 1 h at 4°C. All S-compound 1 binding studies with RPMI-8866 cells were conducted in the presence or absence of a specific 4 1 antagonist, 100 nM BIO7662 (Chen et al., 2001), to selectively block 4 1. The cells were pelleted by centrifugation at 20,000g for 3 min, washed twice with binding buffer at 4°C, transferred to a scintillation vial containing 5 ml of CytoScint (ICN Pharmaceuticals, Costa Mesa, CA), and cell-associated S-compound 1 was measured by scintillation counting. Tubes containing cells radioligand 1 M compound 1 or DMSO alone served as controls to calculate 100 and 0% inhibition, respectively. Data were analyzed by nonlinear regression to calculate Bmax and KD values. Kinetic analysis of S-compound 1 binding to cells expressing 4 was performed by incubating RPMI-8866 cells (7.5 10 cells/tube) expressing 4 7 or Jurkat cells (5 10 5 cells/tube) expressing 4 1 in binding buffer containing either 1 mM MnCl2 or 1 mM CaCl2 and 1 mM MgCl2 with 6.5 nM S-compound 1 and 5 nM unlabeled compound 1 in siliconized Eppendorf microfuge tubes for 2 to 120 min at 4°C. RPMI-8866 cells were pretreated with 100 nM BIO7662 (Chen et al., 2001) as described above. Binding was terminated by adding 5 M compound 1 at each time point. Cells were immediately transferred to an ice-bath for 10 min, pelleted by centrifugation at 20,000g for 3 min, and cell associated S-compound 1 was determined by scintillation counting as described above. When the rate of S-compound 1 dissociation was evaluated, reaction mixtures were incubated with 6.5 nM S-compound 1 and 5 nM unlabeled compound 1 for 1 h at 4°C, followed by the addition of 5 M compound 1 for another 2 to 120 min. At each time point, the cells were pelleted by centrifugation, washed twice with 4°C binding buffer containing either 1 mM MnCl2 or 1 mM each CaCl2 and MgCl2, and counted for cell-associated S-compound 1 as described above. On rates (kon), off rates (koff), and KD values for the binding of S-compound 1 were determined from kinetic measurements. Prism 3.0 software was used to calculate kobs (min ) and koff (min ) values from the on and off rate binding curves, respectively: kon (min 1 nM ) (kobs koff)/[ligand], and KD (M) koff/kon. A S-compound 1 binding assay for activated or unactivated 4 was performed by incubating either RPMI-8866 cells (7.5 10 cells/well), K562/ 4 7 cells (1 10 5 cells/well), HUT-78 cells (5 10 cells/well), or Jurkat cells (5 10 cells/well) in binding buffer containing either 1 mM MnCl2 or 1 mM CaCl2 and 1 mM MgCl2 with less than 150 pM S-compound 1 for 4 7 or 4 1 studies. RPMI8866 cells were pretreated with 100 nM BIO7662 (Chen et al., 2001) as described above. Test compounds were evaluated by incubating radioligand, compound, cells, and binding buffer at 25°C for 45 min in a 96-well multiscreen filtration plate on a shaking platform. After filtration and a single wash with binding buffer containing either 1 mM MnCl2 or 1 mM CaCl2 and 1 mM MgCl2, the plates were processed and counted as described above for the binding of native ligand to cells expressing 4. Nonspecific binding (NSB) was determined by the addition of 1 M compound 1. Quantitative FACS Analysis. A total of 10 RPMI-8866 or Jurkat cells were incubated for 30 min on ice in FACS buffer (phosphate-buffered saline containing 1 mM each CaCl2 and MgCl2, 5% fetal bovine serum, 100 g/ml goat IgG, and 0.05% sodium azide) containing saturating levels of the following phycoerythrin-conjugated antibodies: FIB504 rat anti-mouse 7 (2.4 g/ml; cross-reacts with human 7), MAR4 mouse anti-human 1 (80 g/ml), 9F10 mouse anti-human 4 (10 g/ml), mIgG1 isotype, and rIgG2a isotype controls. All phycoerythrin-conjugated antibodies were obtained from BD PharMingen. Cells were washed in FACS buffer and resuspended in FACS buffer containing 1 g/ml propidium iodide. Cells were analyzed by a FACScan flow cytometer (BD Biosciences, Franklin Lakes, NJ). Standardized quantum R-phycoerythrin microbeads (Flow Cytometry Standards Corp., Fishers, IN) were analyzed by flow cytometry and used to create a calibration curve that relates mean fluorescence intensities to molecules of equivalent soluble fluorescence for use in calculating receptor density values. Statistical Analysis. Curve fits and statistics were performed using KaleidaGraph (Synergy, Reading, PA) and GraphPad Prism (GraphPad Software Inc., San Diego, CA) with a one-way analysis of variance (nonparametric test) followed by a Tukey’s post test if overall P 0.05, and a paired t test was used when comparing only two sets of data. Data were analyzed by nonlinear regression with an equation for one-site binding to calculate Bmax and KD values. Nonlinear regression analysis was used with equations for one-phase exponential association with no weighting or one-phase exponential decay with no weighting to obtain curve fits for association and dissociation plots, respectively. R values were used as an indication of the goodness of fit, and both singleand double-phase exponential equations were compared to obtain the best fit. Double reciprocal plots were analyzed by linear regression analysis.