Using receptor kinetics to quantitatively measure agonist bias at Montana Molecular G-protein coupled receptors Scott Martinka 1, Sam Hoare 2, Kevin Harlen 1, Anne Marie Quinn 1, Paul Tewson 1, Thom Hughes 1 1 Montana Molecular, 2 Pharmechanics
Agonist Agonist Agonist Adenylyl yclase (A )
P G ensor Gs Angiotensin P P2 Receptor Vasopressin 2+ β Receptor Ca -Arrestin β rrestin ATP cAMP P3 ensor c is ensor C Ca ensor
We created a green fluorescent β-arrestin biosensor. Different biased compounds at the Angiotensin II receptor Vasopressin and Oxytocin produce different response (AT1R) produce responses with different kinetics. kinetics at the Vasopressin receptor.
GPCR By testing hundreds of prototypes, GPCR ß-arrestin cAMP we found a sensor that was bright DAG ß-Arrestin 1.1 enough to be used on a plate 2.2 reader with an excellent Z’ value β-Arrestin 1.00 1.0 for detection of AT1R activation 1.0 2.0 0.95 AngII with angiotensin II. 0.9 1.8 S II 0.9 Oxytocin 0.90 TRV120026 AngII 1.6 PBS TRV120045 Vasopressin 0.85 0.8 0.8 S II TRV120055 PBS (no drug) 1.4 0.80 TRV120026 TRV120045 0.7 0.7 1.2 0.75 TRV120055 Angiotensin II S II 1.0 0.6 0.6 0 200 400 600 800 0 200 400 600 800 0 100 200 300 400 0 100 200 300 400 1.4 Norm. DAG sensor Red Fluorescence seconds seconds Norm. Arrestin Sensor Green Fluorescence seconds seconds 1.3 The human vasopressin receptor (human AVPR2) signals through cAMP and β-arrestin. Oxytocin and Vasopressin both activate the receptor and elevate 1.2 Red DAG sensor Red DAG sensor cAMP. The β-arrestin responses are consistently different. 1.1 Ca2+ ß-Arrestin 5 1.0 5 Fluorescence 1.0 Kinetics Can be Used to Measure Bias 0.9 4 ß-Arrestin sensor ß-Arrestin sensor 4 0.9 3 Quantifying bias involves comparing the responses 0.8
2 0.8 of the G-protein based second messengers with 40 3 the β-arrestin sensor. The kinetic data that all of 0 100 200 300 400 500 0 100 200 300 400 500 1 Initial rate (kTau) Ang II 0.7 seconds seconds TRV120026 80 120 160 200 the sensors produce can be used to accurately TRV120045 seconds 30 0.6 determine the initial rate of the reaction at saturating Cells expressing the AT1R receptor are activated by either angiotensin II or S II. 2 TRV120055 Angiotensin II produces a robust increase in DAG and fast β-arrestin response, while the β-arrestin S II 20 concentrations of the agonist. These initial rates can 0.5 biased ligand S II produces no detectable DAG response and a slow β-arrestin response. then be compared to arrive at the bias ratio. A full 1 0.4 10 description of this approach can be found at: G-protein signaling Plateau × 0.693 0 200 400 600 800 1000 0 200 400 600 800 1000 Initial rate = Half time seconds seconds Hoare, S.R.J., Tewson, P.H., Quinn, A.M., and Hughes, T.E. (2019). 0 A new kinetic method for measuring agonist efficacy and ligand bias using high resolution biosensors and a kinetic data Angiotensin II S II 0 5 10 15 20 analysis framework. β-arrestin sensor can follow receptor desensitization Time (min) bioRxiv doi: https://doi.org/10.1101/772293 1.0
First Drug First Drug Second Drug Arrestin Diacylglycerol Calcium 0.9 Wash Wash 317 nM 1.0 2+ 10 nM kτ kτ kτ kτ Arrestin/DAG kτ kτ Arrestin/Ca -1 1 -1 1 -1 1 S II, then S II Ligand (NFU.min ) (% AngII) (NFU.min ) (% AngII) kτ ratio (NFU.min ) (% AngII) kτ ratio 0.8 0.9 (% AngII ratio) (% AngII ratio) S II 0.8 S II, then Ang II AngII 0.40 0.03 100 1.7 0.2 100 1.00 2.2 0.2 100 1.0 0.7 31 nM 1 M Ang II, 0.7 Angiotensin II then Ang II TRV120055 0.37 0.02 92 2.1 0.1 120 0.74 2.5 0.2 110 0.81 100 nM 0.6 0.6 TRV120045 0.38 0.06 93 0.66 0.02 30 3.1 Normalized Fluorescence 0 20 40 60 80 100 0 20 40 60 80 100 TRV120026 0.25 0.03 62 0.46 0.08 21 3.0 200 400 600 800 1000 1200 1400 1600 200 400 600 800 1000 1200 1400 1600 minutes minutes seconds seconds To explore receptor desensitization, we first washed out the drug. Cells expressing the angiotensin SII 0.20 0.04 49 0.25 0.06 12 4.2 receptor (AT1R) and activated with angiotensin II did not return to baseline. However if they were The arrestin sensor reliably reports differences in agonist concentration treated with S II, they did recover. A second application then reactivated the β-arrestin sensor. through a change in kinetics as well as the amplitude.
This work is supported by the NIH (R44 GM125390-02) #646.29