DOI: 10.1186/s13550-016-0249-9Pages: 1-13

New approaches for the reliable in vitro assessment of binding affinity based on high-resolution real-time data acquisition of radioligand-receptor binding kinetics

1. Radiopharmacy and Experimental Nuclear Medicine, Department of Biomedical Imaging and Image-guided Therapy, Division of Nuclear Medicine

2. University of Applied Sciences Wiener Neustadt, Faculty of Engineering

3. University of Vienna, Department of Inorganic Chemistry

4. University of Vienna, Department of Pharmaceutical Chemistry

5. University of Vienna, Department of Pharmaceutical Technology and Biopharmaceutics

6. Ludwig Boltzmann Institute for Applied Diagnostics

Correspondence to:
Markus Mitterhauser
Tel: (0043) 1 40 400 55340
Email: markus.mitterhauser@meduniwien.ac.at

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Abstract

Background

Resolving the kinetic mechanisms of biomolecular interactions have become increasingly important in early-phase drug development. Since traditional in vitro methods belong to dose-dependent assessments, binding kinetics is usually overlooked. The present study aimed at the establishment of two novel experimental approaches for the assessment of binding affinity of both, radiolabelled and non-labelled compounds targeting the A3R, based on high-resolution real-time data acquisition of radioligand-receptor binding kinetics. A novel time-resolved competition assay was developed and applied to determine the Ki of eight different A3R antagonists, using CHO-K1 cells stably expressing the hA3R. In addition, a new kinetic real-time cell-binding approach was established to quantify the rate constants kon and koff, as well as the dedicated Kd of the A3R agonist [125I]-AB-MECA. Furthermore, lipophilicity measurements were conducted to control influences due to physicochemical properties of the used compounds.

Results

Two novel real-time cell-binding approaches were successfully developed and established. Both experimental procedures were found to visualize the kinetic binding characteristics with high spatial and temporal resolution, resulting in reliable affinity values, which are in good agreement with values previously reported with traditional methods. Taking into account the lipophilicity of the A3R antagonists, no influences on the experimental performance and the resulting affinity were investigated.

Conclusions

Both kinetic binding approaches comprise tracer administration and subsequent binding to living cells, expressing the dedicated target protein. Therefore, the experiments resemble better the true in vivo physiological conditions and provide important markers of cellular feedback and biological response.

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  • Accepted: Dec 15, 2016
  • Online: Mar 7, 2017

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