Society for Neuroscience 2019 – Poster Presentation

Biased agonism is a crucial concept in the search for drugs that produce analgesia without addiction. G-protein coupled receptors can signal through two different pathways: the G-proteins for which they were named, and β-arrestin. These two different pathways can mediate very different cellular responses, and some agonists at a particular receptor can be biased towards the activation of one of the pathways, a phenomenon known as biased agonism. This bias is typically measured by comparing two different end-point assays for G-protein and β-arrestin signaling. Such a comparison is difficult, and different results can be produced by measuring at different time points 1. We recently created a green fluorescent β-arrestin sensor that can be used to follow the signaling over time in living cells. This kinetic measurement can be compared to G-protein signaling measured with sensors to Ca2+, cAMP, or DAG. To determine whether the kinetics of the responses of these pathways could be used to determine bias, we measured the responses of the Angiotensin II receptor over time (~ 15 min.) to a panel of known biased agonists (at saturating concentrations). Treating the receptor as an enzyme, and fitting the responses enabled us to extract the initial rate of the reaction (kTau) for the activation of the β-arrestin or G-protein signaling 2. The ratio of kTau produced a precise, reproducible measure of bias that was consistent with the literature. We are currently testing whether this approach will be useful in the search for biased agonists at receptors that are involved in pain and addiction. 1. Klein Herenbrink, C. et al. The role of kinetic context in apparent biased agonism at GPCRs. Nat. Commun. 7, 10842 (2016). 2. Hoare, S. R. J., Pierre, N., Moya, A. G. & Larson, B. Kinetic operational models of agonism for G-protein-coupled receptors. J. Theor. Biol. 446, 168-204 (2018).

S. MARTINKA¹, S. HOARE², K. HARLEN¹, A. QUINN¹, P. TEWSON¹, *T. E. HUGHES¹;¹Montana Mol., Bozeman, MT; ²Pharmechanics, Owego, NY