Real-Time Kinetic Analysis of GPCR Signaling in Stable PathHunter® Cell Lines Paul Tewson (Montana Molecular), Sam Hoare (Pharmechanics), Thom Hughes (Montana Molecular), Anne Marie Quinn (Montana Molecular), Paul Shapiro (Eurofins DiscoverX), Nurulain T. Zaveri (Astraea Therapeutics) Overview Investigate GPCR biology with a simple protocol that Kinetic measurements make it possible to extract combines DiscoverX cells and Montana Molecular the initial rate parameter (kTau), which can be used • Eurofins DiscoverX stable cell lines are widely used biosensors to quantify agonist activity at GPCRs such as the tools in GPCR drug discovery, providing endpoint DAY 1 TRANSDUCE AND PLATE CELLS Nociceptin Opioid (OPRL1) assays for G- signaling, GPCR internalization, STEP 1 The green cADDis Sensor 2 and β-arrestin recruitment. Prepare cells can be used in DiscoverX cells expressing the Nociceptin to detect • Montana Molecular offers genetically-fluorescent 2+ + + Gi signaling. Dose response biosensors to detect cAMP, DAG, PIP2 , Ca , c GMP, STEP 2 BacMam Media HDAC 6 2 measurements to the agonist β-arrestin, and cell stress. Prepare viral transduction reaction Inhibitor Transduction Mix nociceptin/orphanin FQ were made, and the kinetic data was 2 used use determine the EC50 • The fluorescent sensors can be used effectively in both by calculating the initial rate STEP 3 2 PathHunter® and cAMP Hunter® CHO-K1 cell lines to 2 of activity (kTau) at each dose. Mix cells and transduction reaction + The initial rate of signaling monitor signaling kinetics of G-protein and β-arrestin (kTau) is a direct measure of pathways in live cells, in real time. 2 6 ligand efficacy. DA MEASRE RESCECE • A new method of kinetic analysis provides a robust 1.00 SEP Initial rate dose response The kinetic method described kinetic parameter (kTau), which simplifies the quantifi- 0.75 above was used to evaluate the activity of three additional cation of agonist activity and bias at particular GPCRs, 0.50 agonists at the OPRL1 and could provide a new method for studying G-protein 0.25 receptor: AT-090, AT-127, and AT-403, developed by and/or β-arrestin recruitment in opioid, angiotensin II, 0.00 400 450 500 550 600 650 NOFQ Astraea Therapeutics and receptor cell lines. 2 AT-090

Emax/kTau Ligand EC50 (pM) AT-127 %NOFQ Genetically-encoded, fluorescent sensors for High-resolution kinetic measurements of AT-403 NOFQ 100 870 AT-090 94 220 detecting G-protein and β-arrestin signaling cAMP signaling in Eurofins DiscoverX cell lines (β-2 , ADRB2) 2 6 AT-127 100 300 AT-403 100 120

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The signaling kinetics of the G-protein and β-arrestin pathways can be compared to measure agonist bias M) 6 in a simple, easy to implement approach. 2 β2 2 6

2 ∆ 6 2 2 M) cADDis Sensor ® Vasopressin (30 PathHunter CHO-K1 ADRB2 cells express- The cADDis Sensor can be used to 2 2 ing the green cADDis Sensor, responding generate reliable concentration response (30 to the addition of 1 µM isoproterenol. curves in PathHunter® cells. The change in fluorescence indicates GPCR an increase in cAMP production upon GPCR β activation of the -2 adrenergic receptor.

2 6 2 2 2 β-Arrestin High-resolution kinetic measurements of Discover X cells expressing the The cADDis Sensor was used diacylglycerol signaling in Eurofins DiscoverX cell 2 (cAMP Hunter® to measure the cAMP signal CHO-K1 AVPR2) were activated by produced when the V2R receptor β Arrestin Sensor converts lines (Angiotensin II, type 1 receptor, AGTR1) vasopressin or oxytocin. β-arrestin was activated with vasopressin or GPCR binding to changes in 2 2 recruitment was monitored with the green oxytocin. fluorescence β-Arrestin Sensor, which revealed a β-Arrestin Sensor 2 2 difference in signaling kinetics between the M) two ligands. 226 2 2

Vasopressin Oxytocin Arrestin response 100 66 kTau (% vaso.) cAMP response 100 108 kTau (% vaso.) cAMP : Arrestin 2 2 1 1.64 Bias Factor Cells expressing the Angiotensin II The β-arrestin and cAMP responses 2 The DAG Sensor was used to measure DAG Sensor receptor (PathHunter® CHO-K1 AGTR1) produced by each agonist, reported as the activity of 5 ligands at the AT1 recep- are activated by angiotensin II, producing kTau (% vaso.), were used to generate a tor, three of which are known to be biased an increase in diacylglycerol that can be bias factor, which indicated that oxytocin toward the β-arrestin pathway. detected with the green DAG Sensor. is biased toward the Gs pathway.