A novel high-content imaging-based technique for measuring binding of Dickkopf-1 to low-density lipoprotein receptor-related protein 6.

Autor: Priestley RS; Alzheimer's Research UK Oxford Drug Discovery Institute, University of Oxford, NDM Research Building, Old Road Campus, Roosevelt Drive, Oxford OX3 7FZ, UK. Electronic address: richard.priestley@ndm.ox.ac.uk., Cheung J; Alzheimer's Research UK Oxford Drug Discovery Institute, University of Oxford, NDM Research Building, Old Road Campus, Roosevelt Drive, Oxford OX3 7FZ, UK. Electronic address: jonathan.cheung@ndm.ox.ac.uk., Murphy EJ; Alzheimer's Research UK Oxford Drug Discovery Institute, University of Oxford, NDM Research Building, Old Road Campus, Roosevelt Drive, Oxford OX3 7FZ, UK. Electronic address: emma.murphy@ndm.ox.ac.uk., Ehebauer MT; Alzheimer's Research UK Oxford Drug Discovery Institute, University of Oxford, NDM Research Building, Old Road Campus, Roosevelt Drive, Oxford OX3 7FZ, UK. Electronic address: matthias.ehebauer@ndm.ox.ac.uk., Davis JB; Alzheimer's Research UK Oxford Drug Discovery Institute, University of Oxford, NDM Research Building, Old Road Campus, Roosevelt Drive, Oxford OX3 7FZ, UK. Electronic address: john.davis@ndm.ox.ac.uk., Di Daniel E; Alzheimer's Research UK Oxford Drug Discovery Institute, University of Oxford, NDM Research Building, Old Road Campus, Roosevelt Drive, Oxford OX3 7FZ, UK. Electronic address: elena.didaniel@ndm.ox.ac.uk.
Jazyk: angličtina
Zdroj: Journal of pharmacological and toxicological methods [J Pharmacol Toxicol Methods] 2019 Jan - Feb; Vol. 95, pp. 47-55. Date of Electronic Publication: 2018 Nov 23.
DOI: 10.1016/j.vascn.2018.11.003
Abstrakt: Introduction: Dickkopf-related protein 1 (Dkk1) is a secreted protein ligand of low-density lipoprotein receptor-related protein 6 (LRP6), which antagonises canonical Wnt signalling. Elevated Dkk1 levels have been linked to Alzheimer's disease (AD), with protein blockade protective in pre-clinical AD models, suggesting inhibitors of Dkk1-LRP6 binding may have therapeutic utility against AD. Cell-based Dkk1-LRP6 assays reported in the literature use either modified Dkk1 protein and/or do not possess suitable throughput for drug screening. Here we report a novel immunocytochemical-based assay utilising high-content imaging (HCI) and automated data analysis suitable for the screening of protein and small-molecule inhibitors of Dkk1-LRP6 binding.
Methods: We developed an immunocytochemical (ICC) protocol to detect specific binding of exogenous human Dkk1 protein to human LRP6 transiently expressed in HEK293 cells. Images were generated using the PerkinElmer Operetta HCI System, after which quantitative data was generated using the PerkinElmer Columbus™ System.
Results: Our ICC technique and analysis pipeline allowed measurement of cell membrane-localised, LRP6-specific Dkk1 binding, normalised at individual cellular events. Saturation binding demonstrated concentration-dependent Dkk1 binding to LRP6, with a K D in keeping with reported values. Association kinetic experiments demonstrated the utility of the technique to investigate Dkk1 binding kinetics. Human Dkk members Dkk2 and Dkk4 fully displaced Dkk1 binding in a competition assay, while Dkk3 and Soggy-1/DkkL1 exhibited non-complete displacement of Dkk1. Finally gallocyanine, a previously reported inhibitor of Dkk1-LRP6 binding, fully displaced Dkk1 near the expected IC 50 .
Discussion: In conclusion, we provide a validated cell-based assay, suitable for the screening of inhibitors of Dkk1-LRP6 binding, and provide the basis for additional assay development, investigating Dkk1-LRP6 pharmacology.
(Copyright © 2018 Elsevier Inc. All rights reserved.)
Databáze: MEDLINE
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