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Hoffmann-La Roche Assay Uses FLIPR to Seek Positive Allosteric Modulators of GPCRs

SOUTH SAN FRANCISCO, Calif. — Scientists at Hoffmann-La Roche have designed and conducted a high-throughput screening campaign that used Molecular Devices’ FLIPR platform to identify positive allosteric modulators of a G protein-coupled receptor, a company official said this week.
Small-molecule positive allosteric modulators are attractive for targeting GPCRs, particularly in cases where success with agonists is unlikely, Anthony Aglione, an associate principal scientist for new lead discovery at Hoffmann-La Roche’s Nutley, NJ, facility, said during GTCbio’s third annual Assay Development and Screening Technologies conference, held here this week.
Examples of such cases include receptors where it is difficult to program acceptable potency, selectivity, and ADME properties into one molecule; and large peptide ligands, against which small-molecule agonists are unlikely to be identified.
Advantages of positive allosteric modulators include saturable effects, which pose less risk of overdose or adverse events and an increased duration of action by increasing the dose.
Other advantages of the modulators include the fact that they mimic the normal physiological state of tissue specificity and temporal specificity, and that desensitization is unlikely because withdrawal and tolerance have been observed after chronic treatment with GPCR agonists such as opiates.  
Aglione said that his research group at Hoffman-La Roche stably transfected CHOK1 cells with hemagglutinin-tagged GqGPCR and Neo DNAs, followed by selection in G418. Drug-resistant clones were tested for staining with anti-HA antibody. More than 100 clones tested without any positive HA staining.  
These clones were tested for their response to the agonist on MDS’ FlexStation, and the presence of a receptor was confirmed by real-time PCR and sequencing, Aglione said. Therefore, the receptor was present and functional, but the HA tag was masked.

“The selective compounds will be tested in agonist dose-response assays, where we will be looking for a greater than 3-fold leftward shift in agonist dose response in the presence of compound.”

The researchers incubated 384-well plates for an hour prior to the addition of the agonist, and evaluated calcium dyes for robustness of signal, percent variability, and EC (effect concentration) 50 consistency, Aglione said. MDS’ Calcium-4 dye was selected, as it performed the best over the series of assays.
Cells were seeded in 384-well plates at 6,000 cells per well and incubated overnight at 37 °C. The next day, the growth media was replaced with 25 µL of assay buffer and 25 µL of Calcium-4 dye. The plates were then incubated for one hour at 37 °C. Twenty µL of the putative compound at a concentration of 10 µM was transferred to the plates on FLIPR and read to detect agonist activity.
The plates were then incubated at room temperature for 30 minutes, and retuned to FLIPR and where the agonist at EC10 concentration was added to the cell plate. This was followed by a second read to detect a positive allosteric modulator effect.
Aglione said that the hit criterion was 33 percent or greater of the maximal response of the EC95 percent average for eight wells. For example, if the EC95 average was approximately 14,000 fluorescence changes, then a hit would have a value of 4,665 or greater.
Initial hits were then confirmed by being retested in triplicate on three independent cell plates. Confirmed hits then went to EC50 testing so that dose-response curves for positive allosteric modulators could be generated, Aglione said.
Positive allosteric modulator candidates were tested against members of the same family of GPCRs and some non-related GPCRs as well, he said. Aglione added that for their purposes, he and his team defined allosteric activation as a three-fold or greater leftward shift of the agonist dose-response curve in the presence of the positive allosteric modulator at up to a 30 µM concentration, and/or an increase in percent maximum efficacy.
Aglione said that overall, 2,292 plates, or 806,784 compounds, were screened and 9,139 hits were identified, yielding a 1.13 percent hit rate. He added that he used GeneData software to perform quality control of the data.
Primary hit lists were sent to Roche’s analytical chemists for preliminary analysis, and in all, 8,962 compounds were tested in triplicate for confirmation, of which 3,945 compounds were confirmed hits for a 44-percent hit rate.
A 10-point dose-response curve was generated in duplicate for the validated hits, Aglione said. He added that 361 compounds showed an EC50 of less than 5 µM. 
These compounds have been prioritized by the medicinal chemists and will be tested on four or five other GPCR-bearing cell lines to assess selectivity.
“The selective compounds will then be tested in agonist dose-response assays where we will be looking for a greater-than-3-fold leftward shift in agonist dose-response in the presence of compound,” Aglione said. 
“Further positive allosteric modulator high-throughput screening campaigns using FLIPR or cAMP detection formats are underway to identify compounds that selectively bind the allosteric site of other GPCRs that use large peptide cognate ligands,” he added.    

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