Evaluating Cellular Uptake of Drugs with Fluorescent Sensor Proteins

Abstract

Here we introduce a qualitative approach to evaluate cellular uptake of inhibitors with spatiotemporal resolution in living cells. The approach is based on con-verting the protein target of a given class of inhibitors into a fluorescent biosensor. By measuring the affinity and kinetics of binding of different inhibitors to their cognate biosensor in live cells and comparing these values to those measured in vitro, the cellular uptake and concentrations of the inhibitors can be ranked. The approach is label-free and does not require the measurement of a biological read-out of the inhibition. We demonstrate the feasibility of the approach by evaluating cellular uptake of two different classes of inhibitors into the cytosol of living cells: inhibitors of the enzyme human carbonic anhydrase II and inhibitors of the protein-protein interaction between p53 and HDM2