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Published online before print December 29, 2005, 10.1110/ps.051583406
Protein Science (2006), 15:290-303. Published by Cold Spring Harbor Laboratory Press. Copyright © 2006 The Protein Society
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Competition between intercellular adhesion molecule-1 and a small-molecule antagonist for a common binding site on the {alpha}l subunit of lymphocyte function-associated antigen-1

Susan M. Keating1, Kevin R. Clark1, Lisa D. Stefanich1, Fred Arellano2, Caroline P. Edwards3,5, Sarah C. Bodary3,6, Steven A. Spencer2, Thomas R. Gadek4,7, James C. Marsters, Jr.4 and Maureen H. Beresini1

1 Departments of BioAnalytical Research and Development, 2 Protein Chemistry, 3 Immunology, and 4 Medicinal Chemistry, Genentech, Inc., South San Francisco, California 94080, USA

(RECEIVED May 17, 2005; FINAL REVISION November 1, 2005; ACCEPTED November 1, 2005)

The lymphocyte function-associated antigen-1 (LFA-1) binding of a unique class of small-molecule antagonists as represented by compound 3 was analyzed in comparison to that of soluble intercellular adhesion molecule-1 (sICAM-1) and A-286982, which respectively define direct and allosteric competitive binding sites within LFA-1’s inserted (I) domain. All three molecules antagonized LFA-1 binding to ICAM-1-Immunoglobulin G fusion (ICAM-1-Ig) in a competition ELISA, but only compound 3 and sICAM-1 inhibited the binding of a fluorescein-labeled analog of compound 3 to LFA-1. Compound 3 and sICAM-1 displayed classical direct competitive binding behavior with ICAM-1. Their antagonism of LFA-1 was surmountable by both ICAM-1-Ig and a fluorescein-labeled compound 3 analog. The competition of both sICAM-1 and compound 3 with ICAM-1-Ig for LFA-1 resulted in equivalent and linear Schild plots with slopes of 1.24 and 1.26, respectively. Cross-linking studies with a photoactivated analog of compound 3 localized the high-affinity small-molecule binding site to the N-terminal 507 amino acid segment of the {alpha} chain of LFA-1, a region that includes the I domain. In addition, cells transfected with a variant of LFA-1 lacking this I domain showed no significant binding of a fluorescein-labeled analog of compound 3 or ICAM-1-Ig. These results demonstrate that compound 3 inhibits the LFA-1/ICAM-1 binding interaction in a directly competitive manner by binding to a high-affinity site on LFA-1. This binding site overlaps with the ICAM-1 binding site on the {alpha} subunit of LFA-1, which has previously been localized to the I domain.

Keywords: LFA-1; ICAM-1; antagonist; competitive binding; photoaffinity labeling; Schild analysis

Abbreviations: LFA-1, lymphocyte function-associated antigen-1 • ICAM-1, intercellular adhesion molecule-1 • I domain, inserted domain • MIDAS, metal ion-dependent adhesion site • IDAS, I domain allosteric site • ICAM-1-Ig, ICAM-1-Immunoglobulin G fusion • sICAM-1, soluble ICAM-1 • HRP, horseradish peroxidase • FITC, fluorescein isothiocyante • FP, fluorescence polarization • BGG, bovine {gamma}globulins • PBS, phosphate-buffered saline • BSA, bovine serum albumin • TMB, tetramethylbenzidine • GuHCl, guanidine hydrochloride • SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.051583406.

Reprint requests to: Susan M. Keating, Department of BioAnalytical Research and Development, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, USA; e-mail: keating.susan{at}gene.com; fax: (650) 225-5337.


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