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

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.