title: Binding site elucidation of hydantoin-based antagonists of LFA-1 using multidisciplinary technologies: evidence for the allosteric inhibition of a protein--protein interaction.
abstract: The binding site on the lymphocyte function-associated antigen-1 (LFA-1) of a class of hydantoin-based antagonists of leukocyte cell adhesion has been identified. This site resides in the inserted-domain (I-domain) of the CD11a chain at a location that is distal to residues known to be required for interactions with the intercellular adhesion molecules. This finding supports the hypothesis that the molecules are antagonizing cell adhesion via an allosteric modification of LFA-1. The binding site was identified using an integrated immunochemical, chemical, and molecular modeling approach. Antibodies that map to epitopes on the I-domain were blocked from binding to the purified protein by the hydantoins, indicating that the hydantoin-binding site resides on the I-domain. Photoaffinity labeling of the I-domain followed by LC/MS and LC/MS/MS analysis of the enzymatic digest identified proline 281 as the primary amino acid residue covalently attached to the photoprobe. Distance constraints derived from this study coupled with known SAR considerations allowed for the construction of a molecular model of the I-domain/inhibitor complex. The atomic details of the protein/antagonist interaction were accurately predicted by this model, as subsequently confirmed by the X-ray crystal structure of the complex.
authors.affiliation: Research and Development Center, Boehringer Ingelheim Pharmaceuticals, 900 Ridgebury Road, P.O. Box 368, Ridgefield, Connecticut 06877, USA.
authors.forename: K
authors.initials: K
authors.lastname: Last-Barney
authors.forename: W
authors.initials: W
authors.lastname: Davidson
authors.forename: M
authors.initials: M
authors.lastname: Cardozo
authors.forename: L L
authors.initials: LL
authors.lastname: Frye
authors.forename: C A
authors.initials: CA
authors.lastname: Grygon
authors.forename: J L
authors.initials: JL
authors.lastname: Hopkins
authors.forename: D D
authors.initials: DD
authors.lastname: Jeanfavre
authors.forename: S
authors.initials: S
authors.lastname: Pav
authors.forename: C
authors.initials: C
authors.lastname: Qian
authors.forename: J M
authors.initials: JM
authors.lastname: Stevenson
authors.forename: L
authors.initials: L
authors.lastname: Tong
authors.forename: R
authors.initials: R
authors.lastname: Zindell
authors.forename: T A
authors.initials: TA
authors.lastname: Kelly
mesh_terms: D000495:Allosteric Site; D000911:Antibodies, Monoclonal; D001667:Binding, Competitive; D020650:Combinatorial Chemistry Techniques; D018604:Epitope Mapping; D006801:Humans; D006827:Hydantoins / Q000138:chemical synthesis / Q000378:metabolism / Q000494:pharmacology*; D018799:Intercellular Adhesion Molecule-1 / Q000737:chemistry / Q000378:metabolism; D016169:Lymphocyte Function-Associated Antigen-1 / Q000187:drug effects* / Q000276:immunology / Q000378:metabolism; D013058:Mass Spectrometry; D008958:Models, Molecular; D019920:Photoaffinity Labels; D011485:Protein Binding; D013237:Stereoisomerism; D013329:Structure-Activity Relationship
chemical_list: D000911:Antibodies, Monoclonal; D006827:Hydantoins; D016169:Lymphocyte Function-Associated Antigen-1; D019920:Photoaffinity Labels; D018799:Intercellular Adhesion Molecule-1