A large group of enzymes known as cytochromes P450 (CYP or P450) are responsible for the oxidative metabolism of many xenobiotic, hydrophobic chemicals, which include most therapeutic drugs. P450 metabolism can inﬂuence the clearance rate of drugs, their toxicity, and their interactions with other drugs. For drug discovery, researchers need to determine how new drug molecules are metabolized by P450 enzymes and how they may alter P450 activity. Some of these determinations can be achieved by using known P450 substrates as probes that change in a measurable way when they react with a P450. If the reactivity of the probe is altered in the presence of a drug, it can be concluded that the drug has an impact on P450 activity. As described herein, novel P450 inhibitors can be found if they reduce the reactivity of a P450 enzyme with a probe substrate.
BMG LABTECH microplate readers are ﬂexible multi-functional microplate readers that can be equipped with a dedicated luminescence detection system. This system allows for the measurement of a strong luminescent signal, while keeping the background noise low.
P450-Glo™ Assays employ luminogenic P450 probe substrates that are derivatives of beetle luciferin, a substrate for luciferase enzymes. The derivatives are not substrates for luciferase but are converted by different P450 isoenzymes to luciferin (Figure 1), which in turn reacts with luciferase to produce a measurable amount of light that is directly proportional to the P450 activity.
A luminogenic substrate is incubated with an active P450 preparation. The P450 activity is stopped and luciferin detected by adding Ultra-Glo™ Luciferase, a recombinant, stable luciferase in a proprietary buffer system that generates a “glow-type” luminescent signal (Figure 2). The half-life of the luminescent output is greater than two hours, allowing batch plate processing.
Materials & Methods
- BMG LABTECH microplate reader
- Opaque, ﬂat-bottom 96-well Costar® plates
- Promega’s P450 GloTM Screening System (contains baculovirus membrane preparation with human cytochrome P450 enzyme, luminogenic substrate (Fig. 1), NADPH regeneration solution, luciferin detection reagent, reaction buffer, and luciferin-free water) for: CYP1A2 (catalog # V9770), CYP2C9 (#V9790), CYP3A4 (#V9800), CYP2C19 (# V9880), and CYP2D6 (# V9890) D
P450 assays were done with membrane preparations from insect cells co-expressed with P450 reductase or P450 reductase plus cytochrome b5. For background measurements membrane fractions with no P450 enzymes were used. P450 reactions were performed in opaque 96-well Costar® plates using a 50μl volume in KPO4 buffer (pH 7.4). P450 enzymes were incubated with their substrates for 10-30 minutes at 37°C. Reactions were initiated by the addition of an NADPH regenerating solution. P450 activity was stopped and luminescence was initiated by adding Luciferin Detection Reagent. Luminescence was read directly on the BMG LABTECH microplate reader in luminescence mode. The instrument’s incubation chamber and onboard injectors can be easily used to automate the process. Each data point represents the average of three wells.
Results & Discussion
Using the BMG LABTECH reader, P450-Glo™ Assays can detect dose-dependent inhibition of recombinant P450s by known P450 inhibitors. Figure 3 shows dose related inhibition activity curves of the top ﬁve drug metabolizers (CYP1A2, 2C9, 2C19, 2D6, and 3A4) and their respective inhibitors. The determined IC50 values are similar to values obtained with conventional probe substrates. This data demonstrates that a range of concentrations of a single compound can be screened to measure IC50 values, but it also implies that single concentrations of multiple test compounds can be screened against a chemical library.
P450-Glo™ Assays provide a rapid, sensitive and highly reproducible approach to P450 screening. The systems are compatible with conventional P450 assay protocols in automated or manual multiwell plate formats. P450-Glo™ Luminescent Assays have several unique properties:
- Speed - The luminescent reaction is immediate and eliminates the need for time-consuming analyses
- Great Sensitivity - Low background levels and a large dynamic range result in sensitive assays that require less P450 enzyme
- Soluble Substrates - P450-Glo™ substrates are highly soluble in aqueous solutions
- Single readout - Multiple P450 isoforms with multiple substrates can be assayed at a single instrument setting
- Low false-positive rate - Proprietary stabilized ﬁreﬂy luciferase minimizes the number of false positives due to inhibition of luciferase by analytes when screening for P450 inhibitors
BMG LABTECH microplate readers provide the perfect platform to perform Promega’s P450-GloTM Assay. With a dedicated luminescence detection system the BMG LABTECH reader minimizes the background signal, thereby increasing the conﬁdence in the data. Furthermore, with two optional onboard injectors and a standard 45°C incubation chamber, the BMG LABTECH reader can easily become fully automated to perform all of your P450 screening needs.