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Allosteric modulation of G-protein Coupled Receptors conformational landscape

Periodic Reporting for period 1 - AlloGPCR (Allosteric modulation of G-protein Coupled Receptors conformational landscape)

Reporting period: 2018-09-01 to 2020-08-31

G protein coupled receptors (GPCRs) are membrane proteins that convert external stimuli into cellular responses. GPCRs bind a wide range of ligands (hormones, neurotransmitters, peptides, lipids, etc..); after ligand binding, the receptor undergoes structural changes which trigger the binding to intracellular signaling partners (e.g. G proteins, arrestins and GRKs). Despite being the largest receptor family with over 800 members, GPCRs bind to only four main G protein families named Gs, Gi/o, Gq/11, G12/13, which activate distinct downstream signaling cascades. While the coupling of GPCRs to specific G proteins is important to for different signaling responses, the molecular determinants of G protein specificity and selectivity remain largely elusive.
In recent years, we have gained an incredible amount of information from the GPCR-G protein complex structures that have been obtained, especially with the advances in single-particle cryo-EM microscopy. However, these structures do not explain the basis of G protein specificity and further investigations are required to understand the molecular basis of this mechanism. To this aim, we focused on the beta-2 adrenergic receptor (b2AR), a widely studied model GPCR whose pharmacology is well-known. The dual G protein selectivity of the b2AR is best characterized in the cardiac muscle, where b2AR couples primarily to the G protein subtype Gs resulting in cardiac muscle contraction. b2AR is also able to trigger an alternative signaling pathway through binding to Gi, an inhibitor G protein subtype, which in turn decreases the contraction rate.
From a therapeutic point of view, it is of great interest to understand the interplay between Gs and Gi activation, as Gi stimulation can counteract some effects of chronic Gs stimulation during heart failure. However, increased Gi signaling has also been linked to some models of heart failure, underlying the importance of deciphering the molecular basis of the switch between Gs and Gi signaling.
We adopted an integrative structural and functional approach towards the achievement of the project objectives.
Our data highlighted a surprising efficacy profile for some of the b2AR ligands, and we have implemented a comprehensive functional, structural and signaling characterization of G protein specificity at the b2AR. We are focusing on conformational studies and kinetic measurements of receptor activation and association/dissociation to different G proteins in order to get a clearer picture of how the conformational rearrangements at the receptor triggered by ligands can help selecting one G protein subtype over the other.
These complementary investigations have highlighted important aspects of ligands pharmacology that reflect the complex behavior of receptor activation. More broadly, work is ongoing towards a deeper understanding of the role played by receptor conformational states in G protein specificity and selectivity
We are directing our efforts towards the understanding of the mechanism of action of some b2AR ligands and how it affects G protein specificity.
Our focus is in particular on b2AR partial agonists, which are intensively studied in the GPCR field because of their pharmacological properties and efficacy profile, which make them usually better tolerated than full agonists.
One example application involving the b2AR is the need to find better tolerated and safer drugs for the treatment of asthma. For the past 15 years, it has been challenging to find new-generation compounds, that ideally have to be selective for the b2AR in the lungs and potentially less effective at triggering a response in the hearth, where the b2AR is also expressed and can cause harmful side effects.
Understanding the mode of action of these ligands and their signaling implications will be important for the design of better ligands as new therapeutic agents with a more desirable pharmacological profile and less adverse effect.