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Jason Y. Chang, Brian S. McKay, W Daniel Stamer; Plate assay for glaucoma drugs. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1980.
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© ARVO (1962-2015); The Authors (2016-present)
The goal of this study was to develop a medium throughput assay to screen small molecules, peptides and/or antibodies for their capacity to interfere with vascular endothelial (VE) cadherin-cadherin binding at the level of Schlemm's canal inner wall for development of novel conventional outflow drugs.
Chimeric human VE-cadherin-Fc proteins (Kd = ~8 mM in vivo) were used in the development of the assay; consisting of free VE-cadherin-Fc chimeric proteins (10nM-500nM) that were labeled with Alexa Fluor ® 488 hydrazide and VE-cadherin chimeras that were bound to immobilized protein A coated 96-well plates (1µg/well). As a control for specificity we also tested immobilized N-cadherin FC chimeras, which do not bind VE-cadherin. Anti-VE-cadherin IgGs that recognize either intracellular (#2158; Cell Signaling) or extracellular (9H7) VE-cadherin epitopes (used at 30-100 ng/ml final in 2mM calcium binding buffer) or calcium-free binding buffer were tested for ability to inhibit cadherin-cadherin binding. A fluorescence microtiter-plate reader (493ex/517em; FlexStation 3; Molecular Devices) was used to monitor binding interactions.
Control experiments validated the calcium dependence of VE-cadherin-cadherin binding (5nM - 25nM protein concentration) (n=4, p<0.05). Results also showed a linear relationship between VE-cadherin-cadherin binding activity and increased protein concentration (0.01µM - 5µM, r2=0.993). As expected, labeled VE-cadherin did not bind to the immobilized N-cadherin chimera. While both VE-cadherin antibodies (intracellular and extracellular) were able to compete against the VE-cadherin chimera for protein-protein binding at low concentrations of chimeric protein (50nM), only the extracellular antibody inhibited VE-Cadherin chimera binding at physiological proteins concentrations.
Our VE-cadherin assay possesses great potential for glaucoma drug discovery and development. Results show that the assay can detect interference of VE-cadherin-cadherin binding; although due to low (mM) affinity of VE-cadherin-cadherin binding, high concentrations of protein are required for specific VE-cadherin binding. Future studies to improve the sensitivity of the assay and better distinguish between specific and non-specific interactions are ongoing.
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