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Allosteric modulation of G-protein Coupled Receptors conformational landscape

Periodic Reporting for period 2 - AlloGPCR (Allosteric modulation of G-protein Coupled Receptors conformational landscape)

Reporting period: 2020-09-01 to 2021-08-31

G protein–coupled receptors (GPCRs) mediate the majority of cellular responses to external stimuli. Ligand binding triggers a series of receptor conformational rearrangements, enabling the coupling to intracellular partners and the activation of signaling cascades. However, the molecular details of such mechanism remain elusive and additional data are required to shed light on the complex pharmacology of these receptors. The scope of the project is to address some fundamental open questions in the field: i) GPCR conformational dynamics and the impact of ligands, lipids and intracellular effectors ii) the role of the membrane environment on GPCR modulation. These aspects of GPCR signaling are still poorly understood but contribute to the complexity of the receptor signaling pathway, being crucial for the regulation of the spatial and temporal GPCR biological response in different tissues and cell types. Two receptor systems were used to address these questions: the beta2-adrenergic receptor (b2AR), a prototypical class A GPCR, and the ghrelin receptor or ghrelin hormone secretagogue receptor (GHSR). b2AR mediates its function in the heart and lungs and is the target of many drug compounds such as beta-blockers and anti-asthma medications. The ghrelin hormone secretagogue receptor (GHSR) is involved in important physiological processes and major diseases such as obesity and diabetes. No drugs are available at the moment to target the GHSR. Our efforts are directed to a better and more comprehensive understanding of the GPCR system. Our study contribute to the identification of drug candidates at the GHSR for the treatment of obesity and the development of the new generation drugs for the b2AR.
Ligand agonists usually bind GPCRs to activate both G protein and arrestin mediated pathways, with sometimes very different signaling outputs. Bias agonist are ligands that are able to only select one pathway over the other and represent a very interesting pharmacology target for both academia and industry. We studied the efficacy profile of b2AR ligands for the coupling to the G protein subtypes Gs and Gi. In the heart, b2AR couples primarily to the stimulatory G protein Gs, mediating heart contraction. However, b2AR can also bind to Gi, the inhibitory G protein, counteracting the effects of Gs. This dual mechanism of the b2AR has important pharmacological implications as binding to Gi can attenuate the overstimulation of Gs, a condition that leads to heart failure. We identified salmeterol and its derivatives as bias ligands for the Gi stimulation pathway, meaning they preferentially activate Gi signaling at the b2AR. We obtained a Cryo-EM structure of the b2AR-Gi complex. We also investigated the role of lipids in the binding of Gi to the b2AR and found that this interaction is favored in the presence of neutral lipids and cholesterol.
By reconstituting the GHSR in lipid discs with various lipid composition we confirmed the role of lipids as allosteric GPCR modulators. We then used the GHSR to study the conformational rearrangements of the receptor using fluorescence spectroscopy. We investigated the importance of water molecules and the hydration state of the receptor during the activation process. Our findings, backed by molecular dynamic (MD) simulations, suggest a role for the hydration network of the receptor during activation. In particular, we reported changes in the hydration patterns at specific receptor regions that are known to undergo conformational changes during receptor activation. Interestingly, the changes observed were different in the case of bias agonists, suggesting distinct conformations of the receptor when bound to different classes of ligands. This study represents one of the first attempts, beside structure determination and MD simulations, to link the water network of interactions with the activation process of GPCRs.
In summary, our work has highlighted the importance of lipids for GPCR function. We have gained additional insights into bias agonism at the b2AR and GHSR, with important implications in the context of drug design. Our studies also unveiled the allosteric role of hydration during GPCR activation, only hypothesized so far. These results have been presented at international conferences and meetings both by the fellow and the supervisors. One publication, describing the importance of the hydration network, is already available as open access while a second manuscript is in preparation.
Despite the incredible advancement in GPCR structure characterization prompted by X-ray-crystallography and more recently cryo-electron microscopy (Cryo-EM), there are still plenty of challenges in the GPCR field. Indeed, these structures only represent static snapshots that provide little insight into GPCRs dynamics and conformational heterogeneity, which plays a fundamental role on GPCR function and signaling. Additional biophysical studies are therefore absolutely required to fully understand the complex mechanism of GPCR signaling. Moreover, very little is known about GPCR function in their native environment, and how the interaction with ligands and allosteric partners modulate GPCR dynamics and function. Throughout the project, our efforts have been directed towards a better understanding of GPCR pharmacology and conformational dynamics. We have also addressed the role of the membrane lipids and other allosteric modulators such as the hydration state of the receptor.
Our results are of great interest in the context of drug design. For what concerns the b2AR, there is the need for better tolerated and safer drugs to prevent heart failure, the most common cause of death in the western countries. To achieve this goal we need more selective drugs, that only target one receptor subtype, and at the same time bias drugs, that only activate one signaling pathway. The compounds we have identified in our study are very selective for the b2AR and show bias for the hinibitory G protein Gi. They can be used as scaffolds to design the next generation of b2AR drugs.
The GHSR is a very interesting pharmacology target, implicated in various biological processes such as energy homeostasis, food intake and hormones secretion. GHSR is a main target for the treatment of obesity, but despite intense research no compounds have been identified yet to target this receptor. Issues in identifying such compounds include scarce drugs bioavailability and unexpected side effects. Additionally, the pharmacology of the GHSR is only partially understood. Our investigations aim at a better understanding of the GHSR as a system, to delineate the important aspects of its modulation in terms of ligands, lipids and allosteric modulation. Our findings deepen our knowledge of the conformational changes experience by GHSR during activation. In particular, bias ligands seem to drive distinct receptor conformational changes. These different receptor conformations are likely the key to GPCR modulation and their characterization is of fundamental interest for the design of future drugs.
signaling pathways at the b2AR, a model GPCR