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The determination and correlation of molecular and cellular equilibrium Kd and kinetic k(off) values for small molecule allosteric antagonists of LFA-1.
- Source :
-
Biochemical pharmacology [Biochem Pharmacol] 2007 Jun 30; Vol. 74 (1), pp. 98-106. Date of Electronic Publication: 2007 Mar 12. - Publication Year :
- 2007
-
Abstract
- Molecular K(d) and k(off) parameters are often used to define the molecular potency of drugs. These constants, however, are determined on purified target proteins, and their relationship to in vivo binding phenomena is poorly understood. Herein, we report two novel assays to determine the off-rates of allosteric antagonists from lymphocyte function-associated antigen 1 (LFA-1). The SPR assay involves using the non-blocking mAb TS2/4 to immobilize full-length LFA-1 on a hydrophilic chip surface, and the soluble, native ligand sICAM-1 to probe the fraction of free LFA-1. To determine the fraction of free LFA-1 on cell surfaces, a flow cytometry assay was developed utilizing the fluorophore-labeled Fab R3.1. The R3.1 antibody has been previously demonstrated to block the ability of both ICAM-1 and antagonists to bind to purified and cell-surface LFA-1. The molecular and ex vivo cellular parameters were determined for a set of nine structurally-related LFA-1 allosteric antagonists. The relationships between the parameters determined in the ELISA (K(d)), SPR (k(off)), and flow cytometry (k(off)) assays were shown to be linear with slopes approximately equal to 1, and a correlation analysis showed that the three assay datasets were equivalent at the alpha=0.05 level. These results were unexpected, as the ELISA and SPR assays involve high affinity LFA-1, and the flow cytometry assays involve cell surface LFA-1 in whole-blood, in which a distribution of affinity states would be expected. Nevertheless, the results presented herein show that the K(d) and k(off)'s determined in molecular assays can be used as predictors of LFA-1 receptor occupancy in ex vivo assays.
- Subjects :
- Allosteric Regulation drug effects
Allosteric Regulation physiology
Allosteric Site drug effects
Allosteric Site physiology
Antibodies, Monoclonal metabolism
Cell Adhesion Molecules chemistry
Flow Cytometry
Hydantoins chemistry
Hydantoins metabolism
Hydantoins pharmacology
Imidazolidines chemistry
Imidazolidines pharmacology
Integrins immunology
Intercellular Adhesion Molecule-1 pharmacology
Kinetics
Lymphocyte Function-Associated Antigen-1 chemistry
Reproducibility of Results
Cell Adhesion Molecules metabolism
Enzyme-Linked Immunosorbent Assay methods
Imidazolidines metabolism
Integrins antagonists & inhibitors
Lymphocyte Function-Associated Antigen-1 metabolism
Surface Plasmon Resonance methods
Subjects
Details
- Language :
- English
- ISSN :
- 0006-2952
- Volume :
- 74
- Issue :
- 1
- Database :
- MEDLINE
- Journal :
- Biochemical pharmacology
- Publication Type :
- Academic Journal
- Accession number :
- 17482579
- Full Text :
- https://doi.org/10.1016/j.bcp.2007.03.007