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.

A Small Molecule Agonist of an Integrin, αLβ2

“…A humanized antibody to ␣ L ␤ 2 that blocks its binding to the ligand ICAM-1 has been approved by the FDA for treatment of psoriasis, a T cellmediated autoimmune disease of the skin (9, 10). Furthermore, small molecule antagonists of ␣ L ␤ 2 have been discovered and are in development (11)(12)(13)(14)(15)(16)(17).␣ L ␤ 2 contains two von Willebrand factor-type A domains, the inserted (I) domains in the ␣ L and the ␤ 2 subunits (18 -20). Both ␣ L I and ␤ 2 I domains have a Rossman fold (i.e.…”

“…One group of antagonists binds the hydrophobic pocket underneath the ␣7 helix of the ␣ L I domain (e.g. LFA703 or BIRT377), blocks the downward axial movement of the ␣7 helix, and inhibits ligand binding of ␣ L ␤ 2 allosterically by stabilizing the ␣ L I domain in the low affinity conformation (11)(12)(13)(14)34). These antagonists are called ␣ I allosteric inhibitors.…”

“…A humanized antibody to ␣ L ␤ 2 that blocks its binding to the ligand ICAM-1 has been approved by the FDA for treatment of psoriasis, a T cellmediated autoimmune disease of the skin (9,10). Furthermore, small molecule antagonists of ␣ L ␤ 2 have been discovered and are in development (11)(12)(13)(14)(15)(16)(17).…”

Photoaffinity labeling combined with mass spectrometric approaches as a tool for structural proteomics

“…Subsequent proteolytic digestion of the isolated protein-ligand complex and MS-based sequencing can, in turn, reveal the sequence of the amino acids in the ligand-binding region and provide valuable information regarding the potential binding chemistry. These techniques have been used to define a wide variety of binding sites on protein receptors including those for allosteric regulation of enzyme substrate specificity [29], bile acid transport protein [30], alkyl glucoside inhibitors of glucose cotransport protein [31], allosteric inhibitors of protein-protein interactions [32], inhibitors of enzyme activity [33], visual cycle enzyme substrates [34], substrate-binding sites of cytochrome P450 [35], and substrate-binding domains of the drug-efflux mediator P-glycoprotein [36][37][38]. In addition to identifying and characterizing binding sites in known protein receptors, several groups have demonstrated the promise of combining proteomics with PAL to identify protein targets for candidate drugs [39,40].…”

“…Site-directed mutagenesis of the full-length integrase followed by in vitro inhibition assays implicate two specific residues, C130 and W132, as being involved in inhibitor binding. The approach we used here is particularly useful for characterizing the stoichiometry of protein-ligand complexes that are difficult to analyze by X-ray crystallography and NMR techniques.With regard to drug development and design, once information regarding the location and stoichiometry of protein-ligand interactions is obtained, the next step is to develop 3D models of the complex [32,38,46]. Structural models of protein-binding sites can then be used to conduct molecular modeling experiments to identify novel compounds that may have high affinity for the binding site, or screen the affinity of existing compounds computationally.…”