Complex Pharmacology of Free Fatty Acid Receptors

G protein-coupled receptors (GPCRs) are historically the most successful family of drug targets. In recent times it has become clear that the pharmacology of these receptors is far more complex than previously imagined. Understanding of the pharmacological regulation of GPCRs now extends beyond simple competitive agonism or antagonism by ligands interacting with the orthosteric binding site of the receptor to incorporate concepts of allosteric agonism, allosteric modulation, signaling bias, constitutive activity, and inverse agonism. Herein, we consider how evolving concepts of GPCR pharmacology have shaped understanding of the complex pharmacology of receptors that recognize and are activated by nonesterified or “free” fatty acids (FFAs). The FFA family of receptors is a recently deorphanized set of GPCRs, the members of which are now receiving substantial interest as novel targets for the treatment of metabolic and inflammatory diseases. Further understanding of the complex pharmacology of these receptors will be critical to unlocking their ultimate therapeutic potential

Discovery of Novel Receptors for Lipid Mediators - a study leading to the identification of receptors involved in metabolism and the immune system

Intercellular communication is of crucial importance in regulating physiology and G-protein coupled receptors (GPCRs) have evolved as an important mechanism in this process. Of the approximately 800 human GPCRs, about 160 are still considered to be “orphan” receptors for which an endogenous ligand remains to be identified. Since an estimated 50% of all clinical drugs act on 30 known GPCRs, the remaining orphan receptors provide excellent, potential new drug targets. Orphan receptors were selected using known receptor sequences as templates and subsequently cloned into expressing plasmids that were then stably transfected into luciferase-based reporter cells. An orphan receptor was found to be the second GPCR, BLT2, activated by the pro-inflammatory molecule leukotriene B4. Through use of a library of orphan receptors, potential ligands were screened for activity by applying reversed pharmacology. This approach led to the discovery of the novel receptor (FFA1R) for medium- to long-chain free fatty acids, previously known as the orphan receptor GPR40. Significantly, this receptor was found to be expressed on e.g. pancreatic beta-cells and to mediate the fatty acid augmentation of glucose stimulated insulin secretion. The clinically used anti-diabetic drugs, thiazolidinediones, also activate FFA1R expressed on reporter cells. It was discovered that FFA2R and FFA3R (GPR43 and GPR41) are activated by short-chain fatty acids (SCFAs). Being abundantly expressed on blood leukocytes, FFA2R may act as the mediator in SCFA-induced immune suppression in the intestinal tract. A recent proposal links FFA3R to leptin secretion by adipose tissue

Mechanism of PTH CA2+ Sensing on G-Protein Interactions with PTH1R

Parathyroid hormone 1 receptor (PTH1R) is a family-B GPCR that plays a crucial role in bone remodeling. Previous studies show extracellular Ca2+ is a positive allosteric modulator for one PTH1R ligand, parathyroid hormone (PTH), which is approved by US FDA to treat severe osteoporosis. Moreover, PTH residues E19 & E22 have shown to be involved in Ca2+ sensing. However, the effects of PTH Ca2+ sensing on intracellular G-protein binding are unknown. Here, we used FRET-based SPASM sensors to study the interaction between PTH1R and different Ga peptides. SPASM sensors, which are isolated in native HEK293T membranes through optimized protocol, contain PTH1R followed by the acceptor fluorophore, a flexible linker, the donor fluorophore, and a peptide from a Ga subunit that mimics the interaction of the full G-protein heterotrimer. In the current study, two SPASM sensor preparation methods, Giant Plasma Membrane Vessiculation and native membrane preparation, were employed. The quality and integrality of the SPASM sensors isolated through each method were evaluated and compared to one another. We performed FRET experiments to quantify the activation of different Ga isoforms by PTH and its Ca2+ sensing mutant, PTHE19AE22A. PTH binding to PTH1R SPASM sensors causes differential interactions between PTH1R and the Gs, Gq and Gi peptides. PTHE19AE22A activation of PTH1R-SPASM sensors leads to distinct interaction profiles between each G-peptide isoforms, which were further modulated by the presence of extracellular Ca2+. Quantifying the differential activation of the specific Ga isoforms by PTH and PTHE19AE22A in the presence and absence of Ca2+ will delineate mechanistic details of PTH1R activation and its role in bone-related diseases. Further, understanding the extracellular Ca2+ modulation of PTH signaling will provide insight for developing treatments for chronic hypocalcemia associated with hypoparathyroidism, while uncovering PTH1R novel regulation in bone remodeling

Electrophysiology-based investigations of G protein-coupled receptor pharmacology

G protein-coupled receptors (GPCRs) constitute targets for ~34% of approved drugs. The muscarinic acetylcholine M2 receptor (M2R) activates G protein-coupled receptor inward rectifying potassium (GIRK) channels in the central nervous system and heart. Membrane potential modulates agonist potency at several GPCRs. However, the mechanism underlying the voltage sensitivity remains debated. A highly conserved aspartate residue (D2.5069) has been proposed to mediate the voltage-sensitivity of the M2R, although the low expression of D69 mutants has complicated further functional investigations. Dopamine D2 and D3 receptors (D2R and D3R) are pre- and postsynaptic inhibitory receptors in the central nervous system, involved in locomotion, cognition and endocrine functions. D2R antagonists and weak partial agonists are used clinically as antipsychotics but are associated with several side effects. Various strategies have been suggested to reduce the side-effect profile of novel antipsychotic drugs. One such strategy includes the selective targeting of non-canonical signaling pathways, e.g., the β-arrestin pathway, while leaving the classical, G protein pathway, undisturbed. Additionally, binding affinity and kinetics at the D2R, as well as ligand lipophilicity, have been suggested to be of significance in determining the side-effect liability of antipsychotics. In the thesis, M2R, D2R and D3R were investigated using two-electrode voltage-clamp in Xenopus laevis oocytes co-expressing the respective receptor and GIRK channels. M2R carrying a charge-neutralizing D69N mutation demonstrated a voltage-dependent shift of agonist-potency, similar to the wild type M2R. This finding is in line with a recent alternative hypothesis, which implicates three tyrosine residues in the M2R voltage sensor. The proposed β-arrestin-selective partial D2R agonist, UNC9994, was found to be a weak partial- and almost full agonist at D2R and D3R mediated GIRK activation, respectively. These findings are incongruent with β-arrestin-selectivity and suggest that the promising effects of UNC9994 in animal models of psychosis may be related, at least in part, to involvement of the D3R. Finally, the partial D2R agonist positron emission tomography ligand, SV-III-130, demonstrated an insurmountable, yet competitive, binding mechanism at the D2R. Mutations of residues in a secondary binding pocket, engaging the secondary pharmacophore, abolished the insurmountable binding. Kinetic models incorporating an irreversible, SV-III-130-bound state captured the experimentally observed data. Molecular dynamics simulations suggested that D2R extracellular linkers participate in an induced-fit binding mechanism. In summary, the thesis addresses the mechanism of voltage-dependent agonist-potency at GPCRs and contradicts earlier reports of a β-arrestin-selective action of the experimental antipsychotic, UNC9994, at the D2R. Finally, a two-step induced-fit binding mechanism was demonstrated for the aripiprazole analogue, SV-III-130, at the D2R. The findings may guide further mechanistic investigations and provide insights for the development of novel diagnostic and therapeutic GPCR ligands

Quantitative Systems Pharmacology and Biased Agonism at Opioid Receptors: A Potential Avenue for Improved Analgesics

Chronic pain is debilitating and represents a significant burden in terms of personal and socio-economic costs. Although opioid analgesics are widely used in chronic pain treatment, many patients report inadequate pain relief or relevant adverse effects, highlighting the need to develop analgesics with improved efficacy/safety. Multiple evidence suggests that G protein-dependent signaling triggers opioid-induced antinociception, whereas arrestin-mediated pathways are credited with modulating different opioid adverse effects, thus spurring extensive research for G protein-biased opioid agonists as analgesic candidates with improved pharmacology. Despite the increasing expectations of functional selectivity, translating G protein-biased opioid agonists into improved therapeutics is far from being fully achieved, due to the complex, multidimensional pharmacology of opioid receptors. The multifaceted network of signaling events and molecular processes underlying therapeutic and adverse effects induced by opioids is more complex than the mere dichotomy between G protein and arrestin and requires more comprehensive, integrated, network-centric approaches to be fully dissected. Quantitative Systems Pharmacology (QSP) models employing multidimensional assays associated with computational tools able to analyze large datasets may provide an intriguing approach to go beyond the greater complexity of opioid receptor pharmacology and the current limitations entailing the development of biased opioid agonists as improved analgesics

Biosensing Techniques in Yeast: G-Protein Signaling and Protein-Protein Interaction Assays for Monitoring Ligand Stimulation and Oligomer Formation of Heterologous GPCRs

Guanine nucleotide-binding proteins (G-proteins) act as transducers of external stimuli for intracellular signaling, and control various cellular processes in cooperation with seven transmembrane G-protein-coupled receptors (GPCRs). Because GPCRs constitute the largest family of eukaryotic membrane proteins and enable the selective recognition of a diverse range of molecules (ligands), they are the major molecular targets in pharmaceutical and medicinal fields. In addition, GPCRs have been known to form heteromers as well as homomers, which may result in vast physiological diversity and provide opportunities for drug discovery. G-proteins and their signal transduction machinery are universally conserved in eukaryotes; thereby, the yeast Saccharomyces cerevisiae has been used to construct artificial in vivo GPCR biosensors. In this chapter, we focus on the yeast-based GPCR biosensors that can detect ligand stimulation and oligomer formation, and summarize their techniques using the G-protein signaling and protein-protein interaction assays

Direct regulation of HCN Ion channels by cannabinoids

Les cannabinoïdes sont une large classe de molécules qui agissent principalement sur les neurones, affectant la sensation de douleur, l'appétit, l'humeur, l'apprentissage et la mémoire. Des récepteurs cannabinoïdes spécifiques (CBR) ont été identifiés dans les neurones et d'autres types de cellules. Cependant, l'activation des CBR ne peut pas modifier directement l'excitabilité électrique des neurones, car les CBR ne génèrent pas de signaux électriques par eux-mêmes. Au lieu de cela, le potentiel membranaire et la signalisation électrique dans toutes les cellules excitables, y compris les neurones, sont générés par des canaux ioniques intégrés dans la membrane cellulaire. Récemment, il a été démontré que le cannabinoïde synthétique WIN55,212-2 affecte la mémoire en activant les récepteurs CB1, entraînant des changements de signalisation qui affectent le courant Ih généré par les canaux cycliques (HCN) activés par l'hyperpolarisation. Cependant, il a également été démontré que les cannabinoïdes régulent directement la fonction de plusieurs canaux ioniques, indépendamment de l'activation du CBR. Nous examinons ici si les cannabinoïdes, le 9-tétrahydrocannabidiol (THC) et le cannabidiol (CBD), que l'on trouve dans le cannabis sativa, peuvent réguler directement les canaux HCN1. En utilisant une pince de tension à deux électrodes (TEVC), sur des ovocytes de Xenopus, qui n'expriment pas de CBR, nous surveillons les changements dans la relation courant-tension, la cinétique de déclenchement et la dépendance à la tension des courants HCN1 dans des concentrations croissantes de cannabinoïdes. Nos données suggèrent que le CBD et le THC modulent directement le courant de HCN1. Étant donné que les cannabinoïdes sont des molécules thérapeutiques prometteuses pour le traitement de plusieurs troubles neurologiques, comprendre quelles cibles ils affectent, le mécanisme de leur régulation et comment ils se lient à des cibles potentielles sont des étapes essentielles de leur utilisation en tant que thérapies efficaces et du développement de cibles plus puissantes et plus efficaces médicaments spécifiques.Cannabinoids are a broad class of molecules that act primarily on neurons, affecting pain sensation, appetite, mood, learning and memory. Specific cannabinoid receptors (CBRs) have been identified in neurons, and other cell types. However, activating CBRs cannot directly alter electrical excitability in neurons, since CBRs do not generate electrical signals on their own. Instead, membrane potential and electrical signaling in all excitable cells, including neurons, are generated by ion channels embedded in the cell membrane. Recently, it has been shown that the synthetic cannabinoid WIN55,212-2 effects memory by activating CB1 receptors, leading to signaling changes that affect the Ih current generated by hyperpolarization-activated cyclic-nucleotide gated (HCN) channels. However, cannabinoids have also been shown to directly regulate the function of several ion channels, independently of CBR activation. Here we examine whether cannabinoids, 9-tetrahydrocannabidiol (THC) and cannabidiol (CBD), which are found in cannabis sativa, can directly regulate HCN1 channels. Using two-electrode voltage clamp (TEVC), on Xenopus oocytes, which do not express CBRs, we monitor changes in the current-voltage relationship, gating kinetics, and voltage-dependence of HCN1 currents in increasing concentrations of cannabinoids. Our data suggests CBD and THC directly modulate HCN1 current. Since cannabinoids are promising therapeutic molecules for the treatment of several neurological disorders, understanding what targets they affect, the mechanism of their regulation, and how they bind to potential targets are critical steps in their use as effective therapies and the development of more potent and target specific drugs

Subcellular location defines GPCR signal transduction

Intracellular G protein-coupled receptors (GPCRs) can be activated by permeant ligands, which contributes to agonist selectivity. Opioid receptors (ORs) provide a notable example, where opioid drugs rapidly activate ORs in the Golgi apparatus. Our knowledge on intracellular GPCR function remains incomplete, and it is unknown whether OR signaling in plasma membrane (PM) and Golgi apparatus differs. Here, we assess the recruitment of signal transducers to mu- and delta-ORs in both compartments. We find that Golgi ORs couple to Gαi/o probes and are phosphorylated but, unlike PM receptors, do not recruit β-arrestin or a specific Gα probe. Molecular dynamics simulations with OR–transducer complexes in bilayers mimicking PM or Golgi composition reveal that the lipid environment promotes the location-selective coupling. We then show that delta-ORs in PM and Golgi have distinct effects on transcription and protein phosphorylation. The study reveals that the subcellular location defines the signaling effects of opioid drugs

The Free Fatty Acid Receptor GPR40 - expression and role in islet hormone secretion

Type 2 diabetes (T2D) is a serious condition of growing proportions. Developing via an increasing imbalance between insulin sensitivity in the peripheral tissues and insulin release from pancreatic beta-cells, it ultimately renders the individual incapable of regulating the blood glucose concentration, e.g. after a meal. The increased prevalence of T2D is associated with an increase in the prevalence of obesity, with obesity being the single largest risk factor for the development of T2D. This work describes a molecule in the border zone between T2D and obesity. Our initial characterization of GPR40 identified it as a receptor for medium- to long-chain free fatty acids (FFAs). With a marked expression in pancreatic beta-cell lines, we expected GPR40 to be involved in FFA-mediated augmentation of insulin release. This was confirmed when we examined the dose-response relationship between FFA stimulation of GPR40 and both intracellular second messengers in a beta-cell line and insulin release from isolated pancreatic islets. A similarly increased glucagon secretion from alpha-cells was demonstrated after we established that these cells also express GPR40. Antisense knock-down of GPR40 abolished the effect of FFA stimulation on hormone secretion from both cell types. In the final part of this work, FFAs that activate GPR40 were shown to negatively regulate its mRNA expression, indicating a mechanism of protection from detrimental effects of sustained GPR40 stimulation. FFAs mediate effects on both alpha- and beta-cells that are potentially harmful in the development of T2D and it is possible that at least part of those occur via GPR40