Quantifying intracellular drug target validation and target engagement has presented significant challenges, as drugs tend to accumulate non-specifically due to variations in drug affinity, biodistribution, pharmacokinetics, and metabolism. We have developed a ratiometric imaging approach, termed intracellular Paired Agent Imaging (iPAI), for estimating the concentration of protein receptors. This innovative technique holds great potential for revitalizing quantitative intracellular protein receptor imaging, offering a potential solution to quantify the availability of intracellular drug targets. Our approach involves the utilization of fluorophore-labeled small molecule therapeutics as imaging agents. We show it is now possible to use small molecule fluorophore labeled therapeutic inhibitors to visualize their targeted proteins intracellularly, providing a chemical tool kit to generate maps of bound and unbound inhibitors at the single-cell level both in vitro and ex vivo. iPAI platform provides a powerful new opportunity to study numerous other drugs in cells and tissues, resulting in a comprehensive spatial view of drug distribution and binding for improved personalized therapy.
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