P450-Glo CYP3A4 Screening System
The P450-Glo CYP3A4 Assay System provides a homogeneous, luminescent method for measuring CYP3A4 activity. The assays are designed to measure the activities of P450s from recombinant and native sources and for testing the effects of analytes such as drugs and new chemical entities on P450 activities. These luminescent assays exhibit exquisite sensitivity, low background signals and broad dynamic range. P450-Glo Assays employ luminogenic P450 substrates that are derivatives of beetle luciferin, a substrate for luciferase enzymes. The derivatives are not substrates for luciferase but are converted by P450s to luciferin, which in turn reacts with luciferase to produce light that is directly proportional to the activity of the P450. The P450-Glo Assays generate a glow-type luminescent signal, produced using derivatized luciferins as P450 substrates and a recombinant stabilized luciferase (Ultra-Glo Luciferase) coupled with a proprietary buffer system. The half-life of the luminescent output is greater than two hours, eliminating the need for luminometers with injectors and allowing batch plate processing. The formulation also minimizes the incidence of false positives due to inhibition of luciferase by analytes when screening for cytochrome P450 inhibitors. Drug-induced changes in expression of CYP450 genes are a key cause of drug-drug interactions. The ability to measure enzymatic activity of the specific human isoforms that are induced is critical for developing safer drugs. Currently, the most important inducible human isoforms are CYP1A2, CYP2B6 and CYP3A4. With the introduction of the new P450-Glo CYP2B6 Assay we now have assays to measure the activities of all three of these important inducible isoforms. The luciferin-based substrates are readily taken up by cells and rapidly converted into luciferin inside the cell, which reduces the incubation time required (typically 30-60 minutes). The low background and high signal-to-noise ratios produced mean less starting material is required. Dimethyl sulfoxide (DMSO), a common solvent used to solubilize chemical compounds, can significantly inhibit the activity of the 3A4 isoform of cytochrome P450, even at low concentrations (<0.1%). The P450-Glo CYP3A4 System (Luciferin-PPXE) DMSO-Tolerant Assay is specifically designed to tolerate DMSO in the 3A4 reaction. The assay exhibits little to no change in the signal-to-background ratio in the presence of 0.2% DMSO compared to a no-DMSO control.