Background: Many studies link G protein-coupled receptors (GPCRs) to cancer. Some endocrine tumors are unresponsive to standard treatment and/or require long-term and poorly tolerated treatment. This study explored, by bioinformatics analysis, the tumoral profiling of the GPCR transcriptome to identify potential targets in these tumors aiming at drug repurposing. Methods: We explored the GPCR differentially expressed genes (DEGs) from public datasets (Gene Expression Omnibus (GEO) database and The Cancer Genome Atlas (TCGA)). The GEO datasets were available for two medullary thyroid cancers (MTCs), eighty-seven pheochromocytomas (PHEOs), sixty-one paragangliomas (PGLs), forty-seven pituitary adenomas and one-hundred-fifty adrenocortical cancers (ACCs). The TCGA dataset covered 92 ACCs. We identified GPCRs targeted by approved drugs from pharmacological databases (ChEMBL and DrugBank). Results: The profiling of dysregulated GPCRs was tumor specific. In MTC, we found 14 GPCR DEGs, including an upregulation of the dopamine receptor (DRD2) and adenosine receptor (ADORA2B), which were the target of many drugs. In PGL, seven GPCR genes were downregulated, including vasopressin receptor (AVPR1A) and PTH receptor (PTH1R), which were targeted by approved drugs. In ACC, PTH1R was also downregulated in both the GEO and TCGA datasets and was the target of osteoporosis drugs. Conclusions: We highlight specific GPCR signatures across the major endocrine tumors. These data could help to identify new opportunities for drug repurposing.

In vertebrates, the octopeptide angiotensin II (AngII) is an important in vivo regulator of the cardiovascular system. It acts mainly through two G protein-coupled receptors, AT1 and AT2. To better understand distinctive features of these receptors, we carried out a phylogenetic analysis that revealed a mirror evolution of AT1 and AT2, each one split into two clades, separating fish from terrestrial receptors. It also revealed that hallmark mutations occurred at, or near, the sodium binding site in both AT1 and AT2. Electrostatics computations and molecular dynamics simulations support maintained sodium binding to human AT1 with slow ingress from the extracellular side and an electrostatic component of the binding free energy around -3kT, to be compared to around -2kT for human AT2 and the δ opioid receptor. Comparison of the sodium binding modes in wild type and mutated AT1 and AT2 from humans and eels indicates that the allosteric control by sodium in both AT1 and AT2 evolved during the transition from fish to amniota. The unusual S7.46N mutation in AT1 is mirrored by a L3.36M mutation in AT2. In the presence of sodium, the N7.46 pattern in amniota AT1 stabilizes the inward orientation of N3.35 in the apo receptor, which should contribute to efficient N3.35 driven biased signaling. The M3.36 pattern in amniota AT2 favours the outward orientation of N3.35 and the receptor promiscuity. Both mutations have physiological consequences for the regulation of the renin-angiotensin system.

Environmental factors, including mechanical stress and surrounding lipids, can influence the response of GPCRs, such as the mechanosensitive angiotensin II type 1 receptor (AT1). To investigate the impact of these factors on AT1 activation, we developed a steered molecular dynamics simulations protocol based on quaternion formalism. In this protocol, a pulling force was applied to the N-terminus of transmembrane helix 6 (TM6) to induce the TM6 opening characteristic of activation. Subsequently, the simulations were continued without constraints to allow the receptor to relax around the novel TM6 conformation under different conditions. We analyzed the responses of AT1 to membrane stretching, modeled by applying surface tension, in different bilayers. In phosphocholine bilayers without surface tension, we could observe a transient atypical structure of AT1, with an outward TM7 conformation, at the beginning of the activation process. This atypical structure then evolved toward a pre-active structure with outward TM6 and inward TM7. Strikingly, the presence of anionic phosphoglycerol lipids and application of surface tension synergistically favored the atypical structure, which led to an increase in the cross-section area of the receptor intracellular domain. Lipid internalization and H-bonds between lipid heads and the receptor C-terminus increased in phosphoglycerol vs phosphocholine bilayers, but did not depend on surface tension. The difference in the cross-section area of the atypical and pre-active conformations makes the conformational transition sensitive to lateral pressure, and favors the atypical conformation upon surface tension. Anionic lipids act as allosteric modulators of the conformational transition, by stabilizing the atypical conformation. These findings contribute to decipher the mechanisms underlying AT1 activation, highlighting the influence of environmental factors on GPCR responses. Moreover, our results reveal the existence of intermediary conformations that depend on receptor environment and could be targeted in drug design efforts.

Les récepteurs de l’angiotensine II de type 1 (AT1) et de type 2 (AT2) sont deux récepteurs qui appartiennent à la superfamille des RCPG. Ces récepteurs sont impliqués fortement dans l’homéostasie cardiovasculaire et la pression artérielle. En absence de leur ligand naturel, certains RCPG sont activables par des forces mécaniques (forces de cisaillement ou étirement membranaire) ou par des lipides anioniques tels que les DOPG mais nous ignorons encore comment ces facteurs externes modifient les mécanismes moléculaires d’activation des récepteurs de l’angiotensine. Nous avons donc réalisé des simulations de dynamique moléculaire classique et dirigée des récepteurs AT1 et AT2 en modélisant le stress mécanique sous forme de tension de surface, mimant ainsi l’effet d’étirement. Nous avons examiné les mécanismes d’activation de ces deux récepteurs en absence et en présence de l’ion sodium, un régulateur allostérique négatif, ainsi que dans des environnements lipidiques différents (DOPG/POPC). Nos simulations dynamiques classiques montrent que l’activation du récepteur AT1 par l’effet des lipides anioniques DOPG couplé à une tension de 20 dyn/cm conduit à l’adoption d’une conformation active particulière « non-canonique ». En utilisant les simulations dynamiques dirigées, nous avons montré les étapes mécanistiques d’activation des récepteurs AT1 et AT2 et nous avons pu ainsi identifier la structure non canonique d’AT1 comme une structure intermédiaire d’activation. Cette structure est modulée par les interactions électrostatiques entre les hélices 6 et 7, en particulier les interactions entre D6.32 et K7.58. Par conséquence, toute perturbation de ces interactions par des facteurs externes peut initier l’activation du récepteur d’AT1 en absence de son ligand. Ainsi, l’activation du récepteur AT1 par les lipides anioniques peut être expliquée par l’interaction des DOPG avec les résidus chargés positivement de l’hélice 7 et 8 tel que les lysines K7.58 et K7.61. Ces résultats mettent en évidence un site allostérique qui pourrait être ciblé en drug design.

Oxytocin (OT) and its receptor (OT-R) are implicated in the aetiology of autism spectrum disorders (ASDs) and OT-R is a potential target for therapeutic intervention. We report here the design and pharmacological activity of a second-generation OT-R agonist that is more potent and selective than OT itself both in vitro and in vivo, in two animal models of autism after peripheral and oral administration. The preclinical potential of this new lead compound, LIT-002, is also evaluated and discussed.