title: Competition between intercellular adhesion molecule-1 and a small-molecule antagonist for a common binding site on the alphal subunit of lymphocyte function-associated antigen-1.
abstract: 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.
authors.affiliation: Department of BioAnalytical Research and Development, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, USA. keating.susan@gene.com
authors.forename: Susan M
authors.initials: SM
authors.lastname: Keating
authors.forename: Kevin R
authors.initials: KR
authors.lastname: Clark
authors.forename: Lisa D
authors.initials: LD
authors.lastname: Stefanich
authors.forename: Fred
authors.initials: F
authors.lastname: Arellano
authors.forename: Caroline P
authors.initials: CP
authors.lastname: Edwards
authors.forename: Sarah C
authors.initials: SC
authors.lastname: Bodary
authors.forename: Steven A
authors.initials: SA
authors.lastname: Spencer
authors.forename: Thomas R
authors.initials: TR
authors.lastname: Gadek
authors.forename: James C
authors.initials: JC
authors.lastname: Marsters
authors.forename: Maureen H
authors.initials: MH
authors.lastname: Beresini
mesh_terms: D000494:Allosteric Regulation; D001665:Binding Sites; D001667:Binding, Competitive; D003432:Cross-Linking Reagents; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007074:Immunoglobulin G / Q000378:metabolism; D018799:Intercellular Adhesion Molecule-1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007668:Kidney / Q000378:metabolism; D008024:Ligands; D016169:Lymphocyte Function-Associated Antigen-1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism
chemical_list: D003432:Cross-Linking Reagents; D007074:Immunoglobulin G; D008024:Ligands; D016169:Lymphocyte Function-Associated Antigen-1; D010446:Peptide Fragments; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D018799:Intercellular Adhesion Molecule-1