Key features and details
- Assay type: Quantitative
- Detection method: Fluorescent
- Platform: Microplate reader
- Sample type: Inhibitor compounds
Product nameCYP3A4 Inhibitor Assay Kit (Fluorometric)
See all CYP3A43 kits
Sample typeInhibitor compounds
CYP3A4 Inhibitor Screening Kit (Fluorometric) (ab211077) allows rapid screening of drugs and other new chemical entities (NCEs) for CYP3A4-compound interaction in a reliable, high-throughput fluorescence-based assay. The kit provides a yeast microsomal preparation of human CYP3A4 and cytochrome P450 reductase (CPR) enzymes. The assay utilizes a non-fluorescent CYP3A4 substrate that is converted into a highly fluorescent metabolite detected in the visible range (Ex/Em = 535/587 nm), ensuring a high signal-to-background ratio with little interference by autofluorescence. The concentration of fluorogenic substrate used for screening is roughly equivalent to its Km for CYP3A4, facilitating detection of weak competitive inhibitors.
The kit contains a complete set of reagents sufficient for performing 200 reactions in a 96 well plate format.
Cytochrome P450 3A4 (CYP3A4, EC 22.214.171.124) is a member of the cytochrome P450 monooxidase (CYP) family of microsomal xenobiotic metabolism enzymes. CYPs are membrane-bound hemeproteins responsible for Phase I biotransformation reactions, in which lipophilic drugs and other xenobiotic compounds are transformed to more hydrophilic products to facilitate excretion from the body. CYP3A4 is expressed in high levels in the liver and intestines, where it catalyzes oxidation of an extraordinarily wide variety of structurally distinct ligands. More than half of all small molecule drugs commonly used by humans are metabolized by CYP3A4 and inhibition of CYP3A4-mediated metabolism is a common cause of adverse drug/drug and drug/food interactions and toxicity. In addition, for drugs whose pharmacological activity requires metabolism from a pro-drug form, CYP3A4 inhibition can lead to decreased drug efficacy.
Storage instructionsStore at -20°C. Please refer to protocols.
Components 200 tests β-NADP+ Stock 100X (5 mg) 1 vial CYP3A4 Assay Buffer 1 x 100ml CYP3A4 Inhibitor (Ketoconazole) (2 mg) 1 vial CYP3A4 Substrate (2 mg) 1 vial NADPH Generating System 100X (2 mg) 1 vial Recombinant Human CYP3A4 (2 x 5 mg) 2 vials Resorufin Standard (5 mM in DMSO) 1 x 50µl
RelevanceCYP3A43 is a member of the cytochrome P450 superfamily of enzymes which are monooxygenases which catalyze many reactions involved in drug metabolism and synthesis of cholesterol, steroids and other lipids. CYP3A43 exhibits low testosterone 6 beta hydroxylase activity.
Cellular localizationEndoplasmic reticulum membrane; Peripheral membrane protein. Microsome membrane; Peripheral membrane protein.
- Cytochrome P450 family 3 subfamily A polypeptide 43
- Cytochrome P450 polypeptide 43
- Cytochrome P450 subfamily IIIA polypeptide 43
Typical Resorufin standard calibration curve. One mol of Resorufin corresponds to the metabolism of one mol of CYP3A4 substrate.
Reaction kinetics of recombinant human CYP3A4 enzyme at 25°C in the presence and absence of the indicated inhibitors (solvent control reaction includes final concentration of 0.7% acetonitrile).
Dose-response curves for various CYP3A4 inhibitors of differing structural and mechanistic classes: the canonical competitive CYP3A4 inhibitor Ketoconazole, the selective mechanism-based CYP3A4 inhibitors azamulin and cyp3cide, and bromocriptine, a CYP3A4 substrate that acts as a weak enzyme inhibitor. For dose-response curves, percent activity was calculated for each concentration of inhibitor by comparison to activity of reactions containing no inhibitor. For each CYP3A4 inhibitor, IC50 values were derived by 4-parameter logistic curve fitting and represent the mean ± SEM of at least three replicate experiments. Assays were performed according to the kit protocol.
ab211077 has not yet been referenced specifically in any publications.