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The constitutive androstane receptor (CAR, NR1I3) is responsible for the transcription of multiple drug metabolizing enzymes and transporters. There are two possible methods of activation for CAR, direct ligand binding and a ligand-independent method, which makes this a unique nuclear receptor. Both of these mechanisms require translocation of CAR from the cytoplasm into the nucleus. Interestingly, CAR is constitutively active in immortalized cell lines due to the basal nuclear location of this receptor. This creates an important challenge in most in vitro assay models because immortalized cells cannot be used without inhibiting the basal activity. In this book chapter, we go into detail of how to perform quantitative high-throughput screens to identify hCAR1 modulators through the employment of a double stable cell line. Using this line, we are able to identify activators, as well as deactivators, of the challenging nuclear receptor, CAR.
The constitutive androstane receptor (CAR, NR1I3), is a well-known transcription factor found mainly in the liver and intestine which modulates the expression of drug metabolism genes, such as oxidation and conjugation enzymes, while also regulating certain transporters [1,2]. Through this gene regulation, CAR has the potential to play a major role in drug-drug interactions. Drugs which activate CAR, for example, can induce cytochrome P450 (CYP) 2B6 and CYP3A4 which in turn will increase the clearance of any drug metabolized by these two enzymes. Alternately, when CAR is deactivated and CYP2B6 and CYP3A4 protein expression is inhibited, a previously prescribed drug can become toxic to the body due to the decreased metabolism. Recently, it has also been implicated that CAR plays a pivotal role in energy metabolism [3,4]. Therefore, it is not only important to identify compounds causing potential drug-drug interactions, but also drugs which can have a therapeutic effect through hCAR activation.
One of the unique features of CAR is the differing initial localization found when comparing human primary hepatocytes (HPH) and immortalized cell lines. This nuclear receptor has basal localization in the cytoplasm until activation occurs. Once stimulated, CAR will translocate into the nucleus and begin the activation process. However, in immortalized cell lines, CAR constantly resides inside the nucleus without stimulation. When performing in vitro assays, this nuclear localization results in high constitutive activity and difficulty to increase the signal further even after xenobiotic stimulation [5–7]. Because of the high cost and low availability of HPH, it is not an ideal option to use them in high-throughput screenings (HTS). Using immortalized cells is an excellent choice when working with HTS because of their easy culturing and relatively cheap characteristics.
Due to the basal activity of hCAR in immortalized cells as stated previously, a low concentration of a known deactivator can be co-treated with a test compound to decrease the constitutive activity and allow for the prediction of an activator. The most well-known deactivators of CAR are 1-(2-chlorophenylmethylpropyl)-3-isoquinoline-carboxamide (PK11195), meclizine, and clotrimazole [8–10]. However, along with deactivating CAR, meclizine has inconsistently been reported as a hCAR inverse agonist, a mCAR agonist, and also to have no effect on hCAR [8,11]. There is also conflicting clotrimazole data. This drug’s deactivation effects came into question when confirmation could not be completed along with varying results in different cell lines [12,13]. Deactivation of hCAR by PK11195 has not been called into question; however, it has also been shown to be an activator of PXR in HPH. This activation of PXR overrides its deactivation of CAR in HPH, proving PK11195 to be a viable option of hCAR deactivation only in immortalized cell lines .
The luciferase reporter gene assay is a common technique utilized to determine modulation of receptors [14,15]. A basic way to perform this assay is to transfect an expression plasmid containing the nuclear receptor along with a vector containing the promoter region of its target gene with a downstream luciferase reporter into a cell line of choice. However, when performing a HTS using 1536-well plates, the amount of cells per well is an important aspect of each experiment. Because transfection rates are not 100%, it is difficult to use transient transfection in a HTS. Therefore, stably transfecting the cells is an important first step in this protocol.
There are significant issues to overcome when identifying hCAR modulators using in vitro methods. This newly adopted quantitative HTS approach overcomes many of the difficulties residing throughout the luciferase assay . However, there are still limitations for this HTS. For instance, this assay is more likely to identify direct activators than indirect activators. Further studies should be completed to confirm actual hCAR modulation. Here, we outline step-by-step instructions to generate this HepG2-CYP2B6-hCAR stable cell line alongside the quantitative HTS luciferase method.
1Once coated, the plates can be kept in 4°C up to 2 months.
2Use plate 1 to perform the luciferase assay to determine if the cells have both hCAR1 and CYP2B6 transfected properly into them and use plate 2 to keep colony growing.
3The experiment can either proceed immediately to the next step or the entire plate with lysate can be stored in a −20°C freezer until the assay is ready to be completed.
4For example, in the agonist mode, add 1 μL of 4.5 μM PK11195, diluted in culture medium, to make a final concentration of 0.75 μM PK11195 inside each well using the Bioraptr. For the antagonist mode, add 1 μL of 300 nM CITCO, diluted in culture medium, to make a final concentration of 50 nM CITCO inside each well also using the Bioraptr.