Intracellular drug target validation and target engagement quantification have proven to be challenging, and all drugs have some degree of non-specific accumulation due to variable drug affinity, biodistribution, pharmacokinetics, and metabolism. A dynamic ratiometric imaging approach for estimating the concentration of cell surface receptors, termed Paired Agent Imaging, could have a revitalizing effect on quantitative intracellular protein receptor imaging, offering a potential means for quantifying intracellular drug target availability. We have developed a dynamic, fluorescence-based, three-compartment model termed intracellular paired-agent imaging that utilizes fluorophore labeled small molecule therapeutics as imaging agents to measure drug target availability in live cells and tissues.
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