Activity difference of three labdane diterpenoids on human constitutive androstane receptor.

The constitutive androstane receptor (CAR) regulates enzyme transcription related to drug metabolism; therefore, natural compound clarification in food that interacts with CAR is significant for drug development. We revealed that 13-epimanool, which is a compound found in the common sage, is bound to hCAR based on differential scanning fluorometry (DSF) measurements using recombinant hCAR protein. Similar labdane diterpenoids were examined, which revealed that manool and sclareol, which were both natural compounds contained in herbs, are bound to hCAR. They exhibited different effects for CAR activity in the luciferase assay despite the structural similarity. Manool was a partial agonist, 13-epimanool was a weak partial agonist, and sclareol was an antagonist. The activity of hCAR may be regulated by slight differences in the bound compound.

Expression, Purification and Characterization of CAR/NCOA-1 Tethered Protein in E. coli Using Maltose-Binding Protein Fusion Tag and Gelatinized Corn Starch.

The constitutive active/androstane receptor (CAR) is a nuclear receptor that functions as a xenobiotic sensor, which regulates the expression of enzymes involved in drug metabolism and of efflux transporters. Evaluation of the binding properties between CAR and a drug was assumed to facilitate the prediction of drug-drug interaction, thereby contributing to drug discovery. The purpose of this study is to construct a system for the rapid evaluation of interactions between CAR and drugs. We prepared recombinant CAR protein using the Escherichia coli expression system. Since isolated CAR protein is known to be unstable, we designed a fusion protein with the CAR binding sequence of the nuclear receptor coactivator 1 (NCOA1), which was expressed as a fusion protein with maltose binding protein (MBP), and purified it by several chromatography steps. The thus-obtained CAR/NCOA1 tethered protein (CAR-NCOA1) was used to evaluate the interactions of CAR with agonists and inverse agonists by a thermal denaturation experiment using differential scanning fluorometry (DSF) in the presence and absence of drugs. An increase in the melting temperature was observed with the addition of the drugs, confirming the direct interaction between them and CAR. DSF is easy to set up and compatible with multiwell plate devices (such as 96-well plates). The use of DSF and the CAR-NCOA1 fusion protein together allows for the rapid evaluation of the interaction between a drug and CAR, and is thereby considered to be useful in drug discovery.