Development of BODIPY FL Vindoline as a Novel and High-Affinity Pregnane X Receptor Fluorescent Probe

Abstract

The pregnane X receptor (PXR) regulates the metabolism and excretion of xenobiotics and endobiotics by regulating the expression of drug-metabolizing enzymes and transporters. The unique structure of PXR allows it to bind many drugs and drug leads, possibly causing undesired drug–drug interactions. Therefore, it is crucial to evaluate whether chemicals or drugs bind to PXR. Fluorescence-based assays are preferred because of their sensitivity and nonradioactive nature. On the basis of our previously characterized 4 (BODIPY FL vinblastine), a high-affinity PXR probe, we developed 20 (BODIPY FL vindoline) and showed that it is a novel and potent PXR fluorescent probe with Kd of 256 nM in a time-resolved fluorescence resonance energy transfer (TR-FRET) binding assay with PXR. By using 20 (BODIPY FL vindoline) in the PXR TR-FRET assay, we obtained a more than 7-fold signal-to-background ratio and high signal stability (signal was stable for at least 120 min, and Z′-factor > 0.85 from 30 to 240 min). The assay can tolerate DMSO up to 2%. This assay has been used to evaluate a panel of PXR ligands for their PXR-binding affinities. The performance of 20 (BODIPY FL vindoline) in the PXR TR-FRET assay makes it an ideal PXR fluorescent probe, and the newly developed PXR TR-FRET assay with 20 (BODIPY FL vindoline) as a fluorescent probe is suitable for high-throughput screening to identify PXR-binding ligands.

Glucose-dependent regulation of pregnane X receptor is modulated by AMP-activated protein kinase

“…The normalized results for each chemical were fitted into a one-site competitive binding equation with GraphPad Prism to derive the IC 50 values. In the test, the positive control T0901317 had an IC 50 value of 95.9 ± 7.0 nM, which is consistent with the value of 101.6 nM previously recorded under similar assay conditions49.…”

SPA70 is a potent antagonist of human pregnane X receptor

“…We performed statistical calculations by using one-way analysis of variance with Tukey’s multiple-comparison test or a paired t -test when comparing two groups. Sample sizes were chosen according to previous studies that showed statistical significance 27 , 35 , 47 , which also contained information on assay validation and determination of Z’ factor (a measure of statistical effect size) 47 . No data were excluded from the analyses.…”

Marine Invertebrate Xenobiotic-Activated Nuclear Receptors: Their Application as Sensor Elements in High-Throughput Bioassays for Marine Bioactive Compounds

“…Numerous NR LBD-based biosensors have been developed, principally in the context of drug development [ 166 , 190 ]. Among the established xenobiotic receptors, the human PXR LBD has been successfully used as the sensor element in a number of differing biosensor formats [ 111 , 191 , 192 ]. Such biosensors have confirmed previously known human PXR ligands such as hyperforin, clotrimazole, ginkgolide A, SR12813, and 5b-pregnane-3,20-dione [ 111 , 112 , 191 ].…”

“…Among the established xenobiotic receptors, the human PXR LBD has been successfully used as the sensor element in a number of differing biosensor formats [ 111 , 191 , 192 ]. Such biosensors have confirmed previously known human PXR ligands such as hyperforin, clotrimazole, ginkgolide A, SR12813, and 5b-pregnane-3,20-dione [ 111 , 112 , 191 ]. The successful development of human PXR LBD-based biosensors supports the theoretical feasibility of using marine invertebrate XANR LBDs in biosensor formats to screen for marine bioactive compounds.…”