Biased signaling

Hans Bräuner-Osborne, University of Copenhagen, Denmark

Detailed signaling map of the orphan GPR139 receptor

GPR139 is an orphan G protein-coupled receptor, which is activated in vitro by physiological levels of the aromatic amino acids L-Trp and L-Phe and supraphysiological levels of ACTH/a-MSH-related peptides, but still needs to be linked to an endogenous ligand in vivo. We and several pharmaceutical companies have developed potent and selective surrogate agonists targeting GPR139, and several lower potency antagonists have also been developed. The receptor shows the highest expression in the striatum, thalamus, hypothalamus, pituitary and habenula of the human, rat and mouse CNS, indicating that the receptor could by a drug target in e.g. Parkinsons disease, eating disorders and schizophrenia. Indeed, the agonist TAK-041 (Takeda) is currently in clinical trials for treatment of negative symptoms associated with schizophrenia. GPR139 mainly signals through the G q/11 pathway to elevate intracellular levels of inositol phosphates and calcium. However, some studies have also reported activation of G i/o , G s and G 12/13 signaling pathways using a range of assays, cell types and ligands. It has thus been unclear how the receptor signals and to which extend ligands might be biased towards different pathways. Recently, the group of Dr. Kirill Martemyanov used a proximal G protein activation assay to demonstrate that the agonist JNJ-63533054 activates G q/11 and G i/o/z subtypes, but not G s and G 12/13 subtypes. As expected, this led to increased calcium levels, but paradoxically also increased cAMP levels, presumably mediated by calcium-activated adenylate cyclases. To elaborate on these findings, we have tested a broad range of agonists to generate a detailed signaling map using this G protein activation assay and downstream 2 nd messenger assays. Moreover, in collaboration with the groups of Dr. Zhi-Jie Liu and Dr. David Gloriam we have directly demonstrated the ability of GPR139 to couple to both Gq and Gi proteins and delineated the binding-site of JNJ-63533054 by cryo-EM structures and receptor mutagenesis.

Overall, we confirm that GPR139 couples to G q/11 and G i/o/z subtypes and show that a diverse range of ligands show a similar signaling map.

Alena Randáková,

Czech Academy of Sciences, Prague, Czech Republic

Biased agonists of muscarinic receptors

The disruption of muscarinic signalling is frequently involved in various pathophysiological conditions, including neuropathic pain, neurological and psychiatric disorders, e.g., Alzheimer's disease or schizophrenia. To target these particular conditions, selective modulation of individual muscarinic subtypes to avoid undesired side effects is necessary. High homology of the orthosteric binding site among all muscarinic subtypes makes a finding of orthosteric agonists that bind selectively to individual muscarinic subtypes virtually unattainable. Selective targeting at a particular G-protein mediated signalling pathway by biased agonists, via agonist-specific conformation, can be the way to achieve functional selectivity among individual subtypes of muscarinic receptors. Specifically, the binding of an agonist to one or a subset of functional hot spots within the binding site results in activation of a subset of signalling pathways and thus in ligand-mediated signalling bias. An agonist relatively small in size has a greater chance to bind to a smaller number of functional hot spots than a larger agonist. We demonstrate that newly developed tetrahydropyridin based muscarinic agonists activate an only subset of signalling pathways. They display agonist-specific activation profiles of individual G-protein isoforms, that differ among subtypes. Our results prove that selective activation of individual subtypes of muscarinic receptors by biased agonists can be achieved.

Sabina Podlewska,

Maj Institute of Pharmacology Polish Academy of Sciences, Kraków, Poland

Signaling bias of the selected mu-opioid receptor agonists – behavioral and molecular modeling studies

Numerous research campaigns are currently carried out with the aim to find new analgesics. Classical opioid drugs are commonly used in the pain management; however, the opioid-based treatment (especially when being long-term) is related to the wide range of side effects [1].

Recent studies suggest that analgesic and side effects are mediated by activation of distinct signaling pathways (G protein and beta-arrestin, respectively). In this study, we confronted the outcome of behavioral studies on selected G protein-biased opioid receptor agonists (SR-14968, SR-17018, PZM21) and morphine with results of molecular modeling experiments (docking and molecular dynamics simulations carried out for both activated and inactivated receptor forms). The molecular modeling study did reveal some amino acids, interaction with which may be related to the particular behavior of SR-agonists in comparison to PZM21 and morphine.There was also a clear evidence that the stability of compound pose in the binding site of the receptor may play a role in the observed compound activity [3].


The study was funded by the grant 2018/31/B/NZ7/03954 financed by the National Science Centre, Poland (


[1] Al-Hasani, R.; Bruchas, M.R. Molecular Mechanisms of Opioid Receptor-Dependent Signaling and Behavior. Anesthesiology 2011, 115, 1363–1381.

[2] Faouzi, A.; Varga, B.R.; Majumdar, S. Biased Opioid Ligands. Molecules 2020, 25, 4257.

[3] Kudla, L.; Bugno, R.; Podlewska, S.; Szumiec, L.; Wiktorowska, L.; Bojarski, A.J.; Przewlocki, R. Comparison of an addictive potential of μ-opioid receptor agonists with G protein bias: behavioral and molecular modeling studies, Pharmaceutics, 2022, 14, 55

Abdul-Akim Guseinov, Queen's University Belfast, Northern Ireland

Neurotensin receptor 1 allosteric sites explored by probe molecular dynamics simulations and BRET-based assays

Affiliation: 1School of Pharmacy, Queen’s University Belfast (UK), 2NQuiX Ltd (UK), 3Wellcome-Wolfson Institute for Experimental Medicine, Queen’s University Belfast (UK)

Probe molecular dynamics (MD) simulations is a promising method to identify allosteric sites. It consists in simulating the target protein in presence of probe molecules (drug fragments or ligands). In probe-confined MD, the probes are confined to a specific area of interest to increase the efficiency of binding pocket detection [1].

Neurotensin is a peptide and its receptor (Neurotensin receptor 1; NTSR1) activates both Gq and β-arrestin pathways. Activation of the β-arrestin pathway by NTSR1 has been reported to be beneficial for treatment of drug abuse. However, early agonists failed to be viable treatments because NTSR1-mediated Gq activation plays an important role in regulating blood pressure, body temperature and locomotion [2]. For this reason, allosteric biased ligands specifically increasing β-arrestin recruitment by NTSR1 without increasing its Gq pathway is an interesting therapeutic avenue. To find such allosteric sites on NTSR1 we applied probe-confined MD simulations using several NTSR1 structures available.

To evaluate the ability of the predicted allosteric sites to affect NTSR1 signalling upon ligand binding, we designed NTSR1 mutants bearing several point mutations in the vicinity of each of the studied sites. β-arrestin2 recruitment to the plasma membrane and Gq activation upon neurotensin stimulation were monitored using bioluminescence resonance energy transfer (BRET)-based assays. The characterisation of these mutant receptors allows to suggest the possibility to design NTSR1 ligands targeting these corresponding allosteric sites to promote biased signalling.


A. Ciancetta et al. ACS Cent. Sci. 2021 7(11):2374-7943

L. Ferraro et al. J. Pharmacol. 2016 30(2):112-127