Study on drug-mediated protein–protein interaction in single living cells by fluorescence cross-correlation spectroscopy

Abstract

Drug-mediated protein–protein interaction and drug–protein interaction form the basis of drug development and pharmacological research. How to obtain the information of drug–protein or protein–protein interaction in living cells is still a big challenge. In this work, we reported a new method for studying drug-mediated protein–protein interaction in living cells by using fluorescence cross-correlation spectroscopy (FCCS). We used the mammalian target of rapamycin (mTOR) as a model and studied drug-mediated FRB protein–FKBP12 protein interaction in living cells. The FRB protein covers amino acid residues of mTOR from 2015 to 2114 and FKBP12 is a receptor-binding protein. First, FRB was fused with the green fluorescent protein EGFP (FRB–EGFP), and FKBP12 was fused with the red fluorescent protein mCherry (FKBP12–mCherry) using genetic engineering technology. Then, FCCS was used to obtain information on drug-mediated FRB protein–FKBP12 protein interaction in living cells. According to the autocorrelation curves and cross-correlation curves, we can obtain cross-correlation (CC) values of the interaction between two proteins. The CC value was positively correlated with the interaction between two proteins. Furthermore, we developed a method for measuring IC₅₀ for evaluating drug efficacy in living cells based on CC values. Compared with the current methods, our method can be used to study drug-mediated protein–protein interaction and evaluate effects of drugs on protein–protein interaction in living cells, and may become a useful tool for drug development and pharmacological research.