2023 Julius Axelrod Symposium: Plant-Derived Molecules Acting on G Protein-Coupled Receptors.

Plant extracts have played a significant role in traditional medicine for centuries, contributing to improved health and the treatment of various human illnesses. G protein-coupled receptors (GPCRs) are crucial in numerous physiologic functions, and there is growing evidence suggesting their involvement in the therapeutic effects of many plant extracts. In recent years, scientists have identified an expanding number of isolated molecules responsible for the biologic activity of these extracts, with many believed to act on GPCRs. This article critically reviews the evidence supporting the modulation of GPCR function by these plant-derived molecules through direct binding. Structural information is now available for some of these molecules, allowing for a comparison of their binding mode with that of endogenous GPCR ligands. The final section explores future trends and challenges, focusing on the identification of new plant-derived molecules with both orthosteric and allosteric binding modes, as well as innovative strategies for designing GPCR ligands inspired by these plant-derived compounds. In conclusion, plant-derived molecules are anticipated to play an increasingly vital role as therapeutic drugs and serve as templates for drug design. SIGNIFICANCE STATEMENT: This minireview summarizes the most pertinent publications on isolated plant-derived molecules interacting with G protein-coupled receptors (GPCRs) and comments on available structural information on GPCR/plant-derived ligand pairs. Future challenges and trends for the isolation and characterization of plant-derived molecules and drug design are discussed.

Pistacia vera Extract Potentiates the Effect of Melatonin on Human Melatonin MT1 and MT2 Receptors with Functional Selectivity.

Melatonin is a tryptophan derivative synthesized in plants and animals. In humans, melatonin acts on melatonin MT1 and MT2 receptors belonging to the G protein-coupled receptor (GPCR) family. Synthetic melatonin receptor agonists are prescribed for insomnia and depressive and circadian-related disorders. Here, we tested 25 commercial plant extracts, reported to have beneficial properties in sleep disorders and anxiety, using cellular assays (2─[125I]iodomelatonin binding, cAMP inhibition, ERK1/2 activation and ß-arrestin2 recruitment) in mock-transfected and HEK293 cells expressing MT1 or MT2. Various melatonin receptor-dependent and -independent effects were observed. Extract 18 (Ex18) from Pistacia vera dried fruits stood out with very potent effects in melatonin receptor expressing cells. The high content of endogenous melatonin in Ex18 (5.28 ± 0.46 mg/g extract) is consistent with this observation. Ex18 contains an additional active principle that potentiates the effect of melatonin on Gi protein-dependent pathways but not on ß-arrestin2 recruitment. Further active principles potentiating exogenous melatonin were detected in several extracts. In conclusion, we identified plant extracts with various effects in GPCR-based binding and signalling assays and identified high melatonin levels and a melatonin-potentiating activity in Pistacia vera dried fruit extracts that might be of therapeutic potential.

Melatonin in type 2 diabetes mellitus and obesity.

Despite considerable advances in the past few years, obesity and type 2 diabetes mellitus (T2DM) remain two major challenges for public health systems globally. In the past 9 years, genome-wide association studies (GWAS) have established a major role for genetic variation within the MTNR1B locus in regulating fasting plasma levels of glucose and in affecting the risk of T2DM. This discovery generated a major interest in the melatonergic system, in particular the melatonin MT2 receptor (which is encoded by MTNR1B). In this Review, we discuss the effect of melatonin and its receptors on glucose homeostasis, obesity and T2DM. Preclinical and clinical post-GWAS evidence of frequent and rare variants of the MTNR1B locus confirmed its importance in regulating glucose homeostasis and T2DM risk with minor effects on obesity. However, these studies did not solve the question of whether melatonin is beneficial or detrimental, an issue that will be discussed in the context of the peculiarities of the melatonergic system. Melatonin receptors might have therapeutic potential as they belong to the highly druggable G protein-coupled receptor superfamily. Clarifying the precise role of melatonin and its receptors on glucose homeostasis is urgent, as melatonin is widely used for other indications, either as a prescribed medication or as a supplement without medical prescription, in many countries in Europe and in the USA.

Endospanin 1 Determines the Balance of Leptin-Regulated Hypothalamic Functions.

Endospanin 1 (Endo1), a protein encoded in humans by the same gene than the leptin receptor (ObR), and increased by diet-induced obesity, is an important regulator of ObR trafficking and cell surface exposure, determining leptin signaling strength. Defective intracellular trafficking of the leptin receptor to the neuronal plasma membrane has been proposed as a mechanism underlying the development of leptin resistance observed in human obesity. More recently, Endo1 has emerged as a mediator of "selective leptin resistance." The underlying mechanisms of the latter are not completely understood, but the possibility of differential activation of leptin signaling pathways was suggested among others. In this respect, the expression level of Endo1 is crucial for the appropriate balance between different leptin signaling pathways and leptin functions in the hypothalamus and is likely participating in selective leptin resistance for the control of energy and glucose homeostasis.

Enabling STD-NMR fragment screening using stabilized native GPCR: A case study of adenosine receptor.

Structural studies of integral membrane proteins have been limited by the intrinsic conformational flexibility and the need to stabilize the proteins in solution. Stabilization by mutagenesis was very successful for structural biology of G protein-coupled receptors (GPCRs). However, it requires heavy protein engineering and may introduce structural deviations. Here we describe the use of specific calixarenes-based detergents for native GPCR stabilization. Wild type, full length human adenosine A2A receptor was used to exemplify the approach. We could stabilize native, glycosylated, non-aggregated and homogenous A2AR that maintained its ligand binding capacity. The benefit of the preparation for fragment screening, using the Saturation-Transfer Difference nuclear magnetic resonance (STD-NMR) experiment is reported. The binding of the agonist adenosine and the antagonist caffeine were observed and competition experiments with CGS-21680 and ZM241385 were performed, demonstrating the feasibility of the STD-based fragment screening on the native A2A receptor. Interestingly, adenosine was shown to bind a second binding site in the presence of the agonist CGS-21680 which corroborates published results obtained with molecular dynamics simulation. Fragment-like compounds identified using STD-NMR showed antagonistic effects on A2AR in the cAMP cellular assay. Taken together, our study shows that stabilization of native GPCRs represents an attractive approach for STD-based fragment screening and drug design.

Endospanin1 affects oppositely body weight regulation and glucose homeostasis by differentially regulating central leptin signaling.

The hypothalamic arcuate nucleus (ARC) is a major integration center for energy and glucose homeostasis that responds to leptin. Resistance to leptin in the ARC is an important component of the development of obesity and type 2 diabetes. Recently, we showed that Endospanin1 (Endo1) is a negative regulator of the leptin receptor (OBR) that interacts with OBR and retains the receptor inside the cell, leading to a decreased activation of the anorectic STAT3 pathway. Endo1 is up-regulated in the ARC of high fat diet (HFD)-fed mice, and its silencing in the ARC of lean and obese mice prevents and reverses the development of obesity. OBJECTIVE: Herein we investigated whether decreased Endo1 expression in the hypothalamic ARC, associated with reduced obesity, could also ameliorate glucose homeostasis accordingly. METHODS: We studied glucose homeostasis in lean or obese mice silenced for Endo1 in the ARC via stereotactic injection of shRNA-expressing lentiviral vectors. RESULTS: We observed that despite being leaner, Endo1-silenced mice showed impaired glucose homeostasis on HFD. Mechanistically, we show that Endo1 interacts with p85, the regulatory subunit of PI3K, and mediates leptin-induced PI3K activation. CONCLUSIONS: Our results thus define Endo1 as an important hypothalamic integrator of leptin signaling, and its silencing differentially regulates the OBR-dependent functions.

Hypothalamic tanycytes are an ERK-gated conduit for leptin into the brain.

Leptin secreted by adipocytes acts on the brain to reduce food intake by regulating neuronal activity in the mediobasal hypothalamus (MBH). Obesity is associated with resistance to high circulating leptin levels. Here, we demonstrate that peripherally administered leptin activates its receptor (LepR) in median eminence tanycytes followed by MBH neurons, a process requiring tanycytic ERK signaling and the passage of leptin through the cerebrospinal fluid. In mice lacking the signal-transducing LepRb isoform or with diet-induced obesity, leptin taken up by tanycytes accumulates in the median eminence and fails to reach the MBH. Triggering ERK signaling in tanycytes with EGF reestablishes leptin transport, elicits MBH neuron activation and energy expenditure in obese animals, and accelerates the restoration of leptin sensitivity upon the return to a normal-fat diet. ERK-dependent leptin transport by tanycytes could thus play a critical role in the pathophysiology of leptin resistance, and holds therapeutic potential for treating obesity.

Anti-obesity phenotypic screening looking to increase OBR cell surface expression.

The leptin receptor, OBR, is involved in the regulation of whole-body energy homeostasis. Most obese people are resistant to leptin and do not respond to the hormone. The prevention and reversal of leptin resistance is one of the major current goals of obesity research. We showed previously that increased OBR cell surface expression concomitantly increases cellular leptin signaling and prevents obesity development in mice. Improvement of OBR cell surface expression can thus be considered as an interesting anti-obesity therapeutic strategy. To identify compounds that increase the surface expression of OBR, we developed a cell-based, phenotypic assay to perform a high-content screen (HCS) against a library of 50,000 chemical compounds. We identified 67 compounds that increased OBR cell surface expression with AC50 values in the low micromolar range and no effect on total OBR expression and cellular toxicity. Compounds were classified into 16 chemical clusters, of which 4 potentiated leptin-promoted signaling through the JAK2/STAT3 pathway. In conclusion, development of a robust phenotypic screening approach resulted in the discovery of four new scaffolds that demonstrate the desired biological activity and could constitute an original therapeutic solution against obesity and associated disorders.

Suprachiasmatic nucleus neuropeptide expression in patients with Huntington's Disease.

STUDY OBJECTIVE: To study whether sleep and circadian rhythm disturbances in patients with Huntington's disease (HD) arise from dysfunction of the body's master clock, the hypothalamic suprachiasmatic nucleus. DESIGN: Postmortem cohort study. PATIENTS: Eight patients with HD and eight control subjects matched for sex, age, clock time and month of death, postmortem delay, and fixation time of paraffin-embedded hypothalamic tissue. MEASUREMENTS AND RESULTS: Using postmortem paraffin-embedded tissue, we assessed the functional integrity of the suprachiasmatic nucleus in patients with HD and control subjects by determining the expression of two major regulatory neuropeptides, vasoactive intestinal polypeptide and arginine vasopressin. Additionally, we studied melatonin 1 and 2 receptor expression. Compared with control subjects, the suprachiasmatic nucleus contained 85% fewer neurons immunoreactive for vasoactive intestinal polypeptide and 33% fewer neurons for arginine vasopressin in patients with HD (P = 0.002 and P = 0.027). The total amount of vasoactive intestinal polypeptide and arginine vasopressin messenger RNA was unchanged. No change was observed in the number of melatonin 1 or 2 receptor immunoreactive neurons. CONCLUSIONS: These findings indicate posttranscriptional neuropeptide changes in the suprachiasmatic nucleus of patients with HD, and suggest that sleep and circadian rhythm disorders in these patients may at least partly arise from suprachiasmatic nucleus dysfunction.

A role for the melatonin-related receptor GPR50 in leptin signaling, adaptive thermogenesis, and torpor.

The ability of mammals to maintain a constant body temperature has proven to be a profound evolutionary advantage, allowing members of this class to thrive in most environments on earth. Intriguingly, some mammals employ bouts of deep hypothermia (torpor) to cope with reduced food supply and harsh climates [1, 2]. During torpor, physiological processes such as respiration, cardiac function, and metabolic rate are severely depressed, yet the neural mechanisms that regulate torpor remain unclear [3]. Hypothalamic responses to energy signals, such as leptin, influence the expression of torpor [4-7]. We show that the orphan receptor GPR50 plays an important role in adaptive thermogenesis and torpor. Unlike wild-type mice, Gpr50(-/-) mice readily enter torpor in response to fasting and 2-deoxyglucose administration. Decreased thermogenesis in Gpr50(-/-) mice is not due to a deficit in brown adipose tissue, the principal site of nonshivering thermogenesis in mice [8]. GPR50 is highly expressed in the hypothalamus of several species, including man [9, 10]. In line with this, altered thermoregulation in Gpr50(-/-) mice is associated with attenuated responses to leptin and a suppression of thyrotropin-releasing hormone. Thus, our findings identify hypothalamic circuits involved in torpor and reveal GPR50 to be a novel component of adaptive thermogenesis in mammals.

Expression of the orphan GPR50 protein in rodent and human dorsomedial hypothalamus, tanycytes and median eminence.

The melatonin receptor family is composed of three members, MT(1) and MT(2) receptors that bind melatonin with high affinity and the orphan GPR50 that does not bind melatonin but shares significant sequence homology with the two other subtypes. In the absence of any known ligand for this orphan receptor, little is still known about its function. We recently reported the development of the first anti-GPR50 antibodies that reliably recognized the recombinant human GPR50. We here used these antibodies to study the expression of GPR50 in mouse, rat and human hypothalamus, a region reported to express GPR50 mRNA. GPR50 immunoreactivity (ir) was observed in dorsomedial hypothalamic (DMH) cells co-stained with the neuronal marker HuC/D. GPR50-ir was also observed in cells of the ependymal layer of the third ventricle that co-stained with vimentin. More specifically, its localization in the lower region of the third ventricle and along the long basal processes contacting portal blood vessels in the median eminence (ME) suggested expression of GPR50 in tanycytes. Consistent staining patterns were observed in all three species with two different antibodies. Taken together, our study validates two GPR50-specific antibodies for the use in rodent and human tissue. Evolutionary conserved expression of GPR50 in DMH neurons and tanycytes, together with previously reported expression of the receptor in the pituitary, support the potentially important role of GPR50 in key hypothalamic functions, including regulation of the hypothalamo-pituitary axes.

Silencing of OB-RGRP in mouse hypothalamic arcuate nucleus increases leptin receptor signaling and prevents diet-induced obesity.

Obesity is a major public health problem and is often associated with type 2 diabetes mellitus, cardiovascular disease, and metabolic syndrome. Leptin is the crucial adipostatic hormone that controls food intake and body weight through the activation of specific leptin receptors (OB-R) in the hypothalamic arcuate nucleus (ARC). However, in most obese patients, high circulating levels of leptin fail to bring about weight loss. The prevention of this "leptin resistance" is a major goal for obesity research. We report here a successful prevention of diet-induced obesity (DIO) by silencing a negative regulator of OB-R function, the OB-R gene-related protein (OB-RGRP), whose transcript is genetically linked to the OB-R transcript. We provide in vitro evidence that OB-RGRP controls OB-R function by negatively regulating its cell surface expression. In the DIO mouse model, obesity was prevented by silencing OB-RGRP through stereotactic injection of a lentiviral vector encoding a shRNA directed against OB-RGRP in the ARC. This work demonstrates that OB-RGRP is a potential target for obesity treatment. Indeed, regulators of the receptor could be more appropriate targets than the receptor itself. This finding could serve as the basis for an approach to identifying potential new therapeutic targets for a variety of diseases, including obesity.

Decreased MT1 melatonin receptor expression in the suprachiasmatic nucleus in aging and Alzheimer's disease.

The pineal hormone melatonin is involved in the regulation of circadian rhythms and feeds back to the central biological clock, the hypothalamic suprachiasmatic nucleus (SCN) via melatonin receptors. Supplementary melatonin is considered to be a potential treatment for aging and Alzheimer's disease (AD)-related circadian disorders. Here we investigated by immunocytochemistry the alterations of the MT1 melatonin receptor, the neuropeptides vasopressin (AVP) and vasoactive intestinal peptide (VIP) in the SCN during aging and AD. We found that the number and density of AVP/VIP-expressing neurons in the SCN did not change, but the number and density of MT1-expressing neurons in the SCN were decreased in aged controls compared to young controls. Furthermore, both MT1-expressing neurons and AVP/VIP-expressing neurons were strongly diminished in the last neuropathological stages of AD (Braak stages V-VI), but not in the earliest stages (Braak stages I-II), compared to aged controls (Braak stage 0). Our study suggests that the MT1-mediated effects of melatonin on the SCN are disturbed during aging and even more so in late stage AD, which may contribute to the clinical circadian disorders and to the efficacy of therapeutic melatonin administration under these conditions.

Distribution of MT1 melatonin receptor immunoreactivity in the human hypothalamus and pituitary gland: colocalization of MT1 with vasopressin, oxytocin, and corticotropin-releasing hormone.

Melatonin is implicated in numerous physiological processes, including circadian rhythms, stress, and reproduction, many of which are mediated by the hypothalamus and pituitary. The physiological actions of melatonin are mainly mediated by melatonin receptors. We here describe the distribution of the melatonin receptor MT1 in the human hypothalamus and pituitary by immunocytochemistry. MT1 immunoreactivity showed a widespread pattern in the hypothalamus. In addition to the area of the suprachiasmatic nucleus (SCN), a number of novel sites, including the paraventricular nucleus (PVN), periventricular nucleus, supraoptic nucleus (SON), sexually dimorphic nucleus, the diagonal band of Broca, the nucleus basalis of Meynert, infundibular nucleus, ventromedial and dorsomedial nucleus, tuberomamillary nucleus, mamillary body, and paraventricular thalamic nucleus were observed to have neuronal MT1 receptor expression. No staining was observed in the nucleus tuberalis lateralis and bed nucleus of the stria terminalis. The MT1 receptor was colocalized with some vasopressin (AVP) neurons in the SCN, colocalized with some parvocellular and magnocellular AVP and oxytocine (OXT) neurons in the PVN and SON, and colocalized with some parvocellular corticotropin-releasing hormone (CRH) neurons in the PVN. In the pituitary, strong MT1 expression was observed in the pars tuberalis, while a weak staining was found in the posterior and anterior pituitary. These findings provide a neurobiological basis for the participation of melatonin in the regulation of various hypothalamic and pituitary functions. The colocalization of MT1 and CRH suggests that melatonin might directly modulate the hypothalamus-pituitary-adrenal axis in the PVN, which may have implications for stress conditions such as depression.

Techniques: new pharmacological perspectives for the leptin receptor.

The function of leptin, initially confined to its role in energy homeostasis and obesity, has now expanded to the regulation of reproduction, glucose homeostasis, bone formation, wound healing and the immune system. Both stimulation and inhibition of the molecular target of leptin, the leptin receptor (LR), might find applications in disease treatment. Recent advances in the understanding of LR activation mechanisms have led to the design of LR antagonists. Several assays have been developed for the screening and evaluation of LR ligands. Both the extracellular and the intracellular domains of the LR are potential drug targets. The bioluminescence resonance energy transfer technique can be used to screen for compounds that target the extracellular part of the LR, and we propose that the novel reverse mammalian protein-protein interaction trap technique can be used to screen compounds that affect intracellular aspects of LR signalling. These assays can be easily adapted to other pharmacologically relevant receptors.