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Competition between intercellular adhesion molecule-1 and a small-molecule antagonist for a common binding site on the l subunit of lymphocyte function-associated antigen-1

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 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 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 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.

⁵ Present addresses: DNA Gateway International, San Francisco, CA 94108, USA;

⁶ DNAX-Schering-Plough, Palo Alto, CA 94304, USA;

⁷ SARcode, Oakland, CA 94611, USA.

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