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Merck: Multiplexed Imaging, Transfluor Beats FLIPR at Predicting GPCR Activity

Researchers at Merck Research Laboratories have found that a multiplexed Transfluor assay sold by Molecular Devices provides more detailed information than the firm’s fluorometric imaging plate reader, and is more predictive of a compound’s antagonistic activity at the level of receptor-ligand interaction, versus the FLIPR assay.
The researchers multiplexed the Transfluor assay with an immunofluorescence-based quantification of G protein-coupled receptor internalization to screen approximately 2,000 compounds.
Using this technique, they were able to identify and classify 377 compounds interfering with agonist-induced activation of the Transfluor assay, receptor internalization, or both. The investigators also used the Transfluor imaging assay, biochemical receptor binding assays, and the FLIPR technology to analyze a subset of compounds for their effect on GPCR activity.
The findings suggest that a multiplexed imaging assay for this GPCR is a good predictor of direct biochemical inhibition and may be used in compound structure-activity relationship studies. The investigators also provided a method for rescuing false negatives and eliminating false positives.
“When working with GPCR modulators, it is important to know if the compound has the potential to act as an agonist, or a modifier, or induces the changes in signaling that you would expect, because it may change the trafficking of the receptor itself,” Francesca Santini, a research fellow in automated biotechnology at Merck Research Laboratories, told CBA News this week.
The research, which was published online recently in the Journal of Biomolecular Screening, also showed that by combining the β-arrestin as a measure of receptor activation and monitoring localization of the GFP:β-arrestin construct, the researchers could generally determine the type of pathway that the GPCR takes and the type of GPCR signaling that is initiated.

“Using high-content cellular imaging, we could, in one assay, address cellular function and phenotypic response.”

In addition, by monitoring the receptor itself and localizing the receptor with an antibody, “we could see if the action of the compound is restricted to the plasma membrane and if the compound was having an effect on the known receptor pathway, or targeting the receptor toward a different subcellular compartment,” Santini said.
In addition to the compounds that the investigators were looking at, their approach highlighted many compounds with “interesting phenotypes that we could use as tools for further characterization and for other campaigns.”
She added that with imaging, the investigators could correlate a functional assay such as FLIPR with another assay such as the Transfluor, and that an imaging assay is more informative, because “we could gain an understanding of a compound’s mechanism of action.”
The researchers could see if the compounds were bound to the receptor, if they had the ability to modify the path of the receptor, and if they had the ability to modify the signaling that originated from the receptor, Santini said.
“Using high-content cellular imaging, we could, in one assay, address cellular function and phenotypic response,” she said.
The finding that a direct biochemical binding assay is better than FLIPR at correlating an imaging assay, and that the imaging assay provides a context to interpret otherwise non-target-specific FLIPR data, “did not surprise us” because the FLIPR assay only reports on one parameter, said Santini.
“The beauty of cell imaging is that you can report on many parameters,” she said, “so the information is all integrated.”
By integrating all of the information that the imaging assay provides, “you can better predict the findings of the biochemical assay,” said Santini. “That is why we use that assay for funneling.” In drug discovery, one starts with the assays that are simple, but “you need to identify good leads and funnel down to the compounds that will have the best success,” she explained.
Imaging assays provide that funneling while also providing good-quality, informative data regarding the ability of the compound to do what it has been predicted to do, Santini said. Imaging assays also provide information on how the cell responds, because “you are doing this in a cellular context.”
With FLIPR, one is also running the assay and looking at the effects of a compound in a cellular context, but “you are looking at only one parameter, so there are many false negatives,” she added. In addition, the biochemical assay is done in vitro, “so you get yes or no, it binds or does not bind, but it does not provide comprehensive information about the compound’s effects.”   
The investigators said that additional testing is necessary to determine if the correlation between the data from the multiplexed imaging assay for GPCRs and the direct biochemical binding assay is an isolated case, and whether it is a general feature of all GPCRs or is only applicable to certain classes of GPCRs.

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