Researchers at Amgen have used DiscoveRx’s PathHunter technology to monitor G protein-coupled receptor activation and validated the approach using a Gαi-coupled GPCR somatostatin receptor 2, to show that it could be used with high-throughput screens in both agonist and antagonist screening modes.
Traditionally, GPCRs were pathway-specific because the receptor depends on what G protein it couples to: Gαi, Gαs, or Gαq. It is this coupling that ultimately results in second messenger signaling such as chemiluminescence, Ca2+ flux, or cAMP level change. As a result, researchers traditionally use aequorin, FLIPR, and cAMP assays to look at how the GPCR is activated.
Some GPCRs can elicit a robust signal, said Sharon Zhao, senior scientist in the lead discovery department at Amgen. However, problems working with Gi-coupled receptors have occurred because, as it is downstream of the signal and inhibits cAMP accumulation, its response is small. This deficiency has caused investigators to look for assays that work better with Gi-coupled receptors.
The PathHunter assay “offers an assay format with which some Gi-coupled receptors could potentially work very well,” Zhao said. She and her colleagues published an evaluation of the assay online last month in the Journal of Biomolecular Screening.
The PathHunter assay uses DiscoveRx’s proprietary β-galactosidase enzyme fragment complementation technology to monitor β-arrestin translocation. The mutated amino-terminal fragment of β-galactosidase, called ProLink, is fused to the carboxyl-terminus of GPCR, and an N-terminal deletion mutant of β-galactosidase, or the enzyme acceptor, is fused to the C-terminus of the β-arrestin molecule.
In cells that stably express these fusion proteins, ligand stimulation triggers association between the β-arrestin and activated GPCR, driving complementation of the β-galactosidase fragments and enabling researchers to detect GPCR activation by using various β-galactosidase substrates, including luminogenic ones.
In their paper, the Amgen researchers evaluated the PathHunter for its ability to screen the SSTR2 receptor in 384- and 1,536-well formats. They used the stable β-arrestin cell line, SSTR2 PathHunter CHOK1. The luminescent signal was detected 60 minutes after the addition of 12 µL of PathHunter detection mix by reading 384-well plates on a PerkinElmer EnVision multimode plate reader. The 1,536-well plates were read using a PerkinElmer Viewlux.
“I think that DiscoveRx has been a pioneer in converting the high-content β-arrestin translocation assay for GPCRs into a high-throughput, and that is very interesting to us, so we were evaluating their technology, which is how this work started,” said Zhao. “This represents another tool at our disposal, in cases where other assay formats do not work well.”
She said the PathHunter assay has certain advantages over cAMP, FLIPR, and aequorin assays, including the fact that it is not G protein-specific and it’s a robust, user-friendly assay format. “Usually, it is hard to have one assay format apply to all members of a receptor family,” she told CBA News last week. “This one has potential, but it is also receptor specific.”
Although β-arrestin translocation technologies such as Molecular Devices’ Transfluor and Invitrogen’s Tango assays have been widely used for GPCRs, the monitoring of β-arrestin translocation has so far been limited to the microscope-based high-content format, which is sensitive but requires an image-based system and a large database.
“Usually, its use is limited to low-throughput or small compound libraries,” said Zhao, who said that by converting to a high-throughput assay such as PathHunter, she was able to perform high-throughput screening.
“It would definitely be a good assay format for primary screening, because versus the cAMP and FLIPR assay, it is pretty robust and simple to use,” said Zhao. In their paper, the Amgen investigators wrote that the PathHunter assay could potentially complement other β-arrestin translocation-based assays for GPCRs, such as Transfluor and Tango.
The PathHunter assays are now implemented globally in the “top 15” pharmas and biotechs, Keith Olson, vice president of product and market development for DiscoveRx, told CBA News this week.