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.

Melatonin receptor structure and signaling.

Melatonin (5-methoxy-N-acetyltryptamine) binds with high affinity and specificity to membrane receptors. Several receptor subtypes exist in different species, of which the mammalian MT1 and MT2 receptors are the best-characterized. They are members of the G protein-coupled receptor superfamily, preferentially coupling to Gi/o proteins but also to other G proteins in a cell-context-depending manner. In this review, experts on melatonin receptors will summarize the current state of the field. We briefly report on the discovery and classification of melatonin receptors, then focus on the molecular structure of human MT1 and MT2 receptors and highlight the importance of molecular simulations to identify new ligands and to understand the structural dynamics of these receptors. We then describe the state-of-the-art of the intracellular signaling pathways activated by melatonin receptors and their complexes. Brief statements on the molecular toolbox available for melatonin receptor studies and future perspectives will round-up this review.

Melatonin drugs inhibit SARS-CoV-2 entry into the brain and virus-induced damage of cerebral small vessels.

COVID-19 is a complex disease with short- and long-term respiratory, inflammatory and neurological symptoms that are triggered by the infection with SARS-CoV-2. Invasion of the brain by SARS-CoV-2 has been observed in humans and is postulated to be involved in post-COVID state. Brain infection is particularly pronounced in the K18-hACE2 mouse model of COVID-19. Prevention of brain infection in the acute phase of the disease might thus be of therapeutic relevance to prevent long-lasting symptoms of COVID-19. We previously showed that melatonin or two prescribed structural analogs, agomelatine and ramelteon delay the onset of severe clinical symptoms and improve survival of SARS-CoV-2-infected K18-hACE2 mice. Here, we show that treatment of K18-hACE2 mice with melatonin and two melatonin-derived marketed drugs, agomelatine and ramelteon, prevents SARS-CoV-2 entry in the brain, thereby reducing virus-induced damage of small cerebral vessels, immune cell infiltration and brain inflammation. Molecular modeling analyses complemented by experimental studies in cells showed that SARS-CoV-2 entry in endothelial cells is prevented by melatonin binding to an allosteric-binding site on human angiotensin-converting enzyme 2 (ACE2), thus interfering with ACE2 function as an entry receptor for SARS-CoV-2. Our findings open new perspectives for the repurposing of melatonergic drugs and its clinically used analogs in the prevention of brain infection by SARS-CoV-2 and COVID-19-related long-term neurological symptoms.

Melatonin-vorinostat hybrid ligands show higher histone deacetylase and cancer cell growth inhibition than vorinostat.

Anticancer drug conjugates are an emerging approach for future cancer treatment. Here, we report a series of hybrid ligands merging the neurohormone melatonin with the approved histone deacetylase (HDAC) inhibitor vorinostat, using melatonin's amide side chain (3a-e), its indolic nitrogen (5a-d), and its ether oxygen (7a-d) as attachment points. Several hybrid ligands showed higher potency thanvorinostat in both HDAC inhibition and cellular assays on different cultured cancer cell lines. In the most potent HDAC1 and HDAC6 inhibitors, 3e, 5c, and 7c, the hydroxamic acid moiety of vorinostat is linked to melatonin through a hexamethylene spacer. Hybrid ligands 5c and 7c were also found to be potent growth inhibitors of MCF-7, PC-3M-Luc, and HL-60 cancer cell lines. As these compounds showed only weak agonist activity at melatonin MT1 receptors, the findings indicate that their anticancer actions are driven by HDAC inhibition.

Simufilam Reverses Aberrant Receptor Interactions of Filamin A in Alzheimer's Disease.

Simufilam is a novel oral drug candidate in Phase 3 clinical trials for Alzheimer's disease (AD) dementia. This small molecule binds an altered form of filamin A (FLNA) that occurs in AD. This drug action disrupts FLNA's aberrant linkage to the α7 nicotinic acetylcholine receptor (α7nAChR), thereby blocking soluble amyloid beta1-42 (Aß42)'s signaling via α7nAChR that hyperphosphorylates tau. Here, we aimed to clarify simufilam's mechanism. We now show that simufilam reduced Aß42 binding to α7nAChR with a 10-picomolar IC50 using time-resolved fluorescence resonance energy transfer (TR-FRET), a robust technology to detect highly sensitive molecular interactions. We also show that FLNA links to multiple inflammatory receptors in addition to Toll-like receptor 4 (TLR4) in postmortem human AD brains and in AD transgenic mice: TLR2, C-X-C chemokine receptor type 4 (CXCR4), C-C chemokine receptor type 5 (CCR5), and T-cell co-receptor cluster of differentiation 4 (CD4). These aberrant FLNA linkages, which can be induced in a healthy control brain by Aß42 incubation, were disrupted by simufilam. Simufilam reduced inflammatory cytokine release from Aß42-stimulated human astrocytes. In the AD transgenic mice, CCR5-G protein coupling was elevated, indicating persistent activation. Oral simufilam reduced both the FLNA-CCR5 linkage and the CCR5-G protein coupling in these mice, while restoring CCR5's responsivity to C-C chemokine ligand 3 (CCL3). By disrupting aberrant FLNA-receptor interactions critical to AD pathogenic pathways, simufilam may promote brain health.

Genetic and biased agonist-mediated reductions in ß-arrestin recruitment prolong cAMP signaling at glucagon family receptors.

Receptors for the peptide hormones glucagon-like peptide-1 (GLP-1R), glucose-dependent insulinotropic polypeptide (GIPR), and glucagon (GCGR) are important regulators of insulin secretion and energy metabolism. GLP-1R agonists have been successfully deployed for the treatment of type 2 diabetes, but it has been suggested that their efficacy is limited by target receptor desensitization and downregulation due to recruitment of ß-arrestins. Indeed, recently described GLP-1R agonists with reduced ß-arrestin-2 recruitment have delivered promising results in preclinical and clinical studies. We therefore aimed to determine if the same phenomenon could apply to the closely related GIPR and GCGR. In HEK293 cells depleted of both ß-arrestin isoforms the duration of G protein-dependent cAMP/PKA signaling was increased in response to the endogenous ligand for each receptor. Moreover, in wildtype cells, "biased" GLP-1, GCG, and GIP analogs with selective reductions in ß-arrestin-2 recruitment led to reduced receptor endocytosis and increased insulin secretion over a prolonged stimulation period, although the latter effect was only seen at high agonist concentrations. Biased GCG analogs increased the duration of cAMP signaling, but this did not lead to increased glucose output from hepatocytes. Our study provides a rationale for the development of GLP-1R, GIPR, and GCGR agonists with reduced ß-arrestin recruitment, but further work is needed to maximally exploit this strategy for therapeutic purposes.

Pharmacological evidence for transactivation within melatonin MT2 and serotonin 5-HT2C receptor heteromers in mouse brain.

Association of G protein-coupled receptors into heterodimeric complexes has been reported for over 50 receptor pairs in vitro but functional in vivo validation remains a challenge. Our recent in vitro studies defined the functional fingerprint of heteromers composed of Gi -coupled melatonin MT2 receptors and Gq -coupled serotonin 5-HT2C receptors, in which melatonin transactivates phospholipase C (PLC) through 5-HT2C . Here, we identified this functional fingerprint in the mouse brain. Gq protein activation was probed by [35 S]GTPγS incorporation followed by Gq immunoprecipitation, and PLC activation by determining the inositol phosphate levels in brain lysates of animals previously treated with melatonin. Melatonin concentration-dependently activated Gq proteins and PLC in the hypothalamus and cerebellum but not in cortex. These effects were inhibited by the 5-HT2C receptor-specific inverse agonist SB-243213, and were absent in MT2 and 5-HT2C knockout mice, fully recapitulating previous in vitro data and indicating the involvement of MT2 /5-HT2C heteromers. The antidepressant agomelatine had a similar effect than melatonin when applied alone but blocked the melatonin-promoted Gq activation due to its 5-HT2C antagonistic component. Collectively, we provide strong functional evidence for the existence of MT2 /5-HT2C heteromeric complexes in mouse brain. These heteromers might participate in the in vivo effects of agomelatine.

Therapeutic potential of melatonin and melatonergic drugs on K18-hACE2 mice infected with SARS-CoV-2.

As the COVID-19 pandemic grows, several therapeutic candidates are being tested or undergoing clinical trials. Although prophylactic vaccination against SARS-CoV-2 infection has been shown to be effective, no definitive treatment exists to date in the event of infection. The rapid spread of infection by SARS-CoV-2 and its variants fully warrants the continued evaluation of drug treatments for COVID-19, especially in the context of repurposing of already available and safe drugs. Here, we explored the therapeutic potential of melatonin and melatonergic compounds in attenuating COVID-19 pathogenesis in mice expressing human ACE2 receptor (K18-hACE2), strongly susceptible to SARS-CoV-2 infection. Daily administration of melatonin, agomelatine, or ramelteon delays the occurrence of severe clinical outcome with improvement of survival, especially with high melatonin dose. Although no changes in most lung inflammatory cytokines are observed, treatment with melatonergic compounds limits the exacerbated local lung production of type I and type III interferons, which is likely associated with the observed improved symptoms in treated mice. The promising results from this preclinical study should encourage studies examining the benefits of repurposing melatonergic drugs to treat COVID-19 and related diseases in humans.

Upregulated Apelin Signaling in Pancreatic Cancer Activates Oncogenic Signaling Pathways to Promote Tumor Development.

Despite decades of effort in understanding pancreatic ductal adenocarcinoma (PDAC), there is still a lack of innovative targeted therapies for this devastating disease. Herein, we report the expression of apelin and its receptor, APJ, in human pancreatic adenocarcinoma and its protumoral function. Apelin and APJ protein expression in tumor tissues from patients with PDAC and their spatiotemporal pattern of expression in engineered mouse models of PDAC were investigated by immunohistochemistry. Apelin signaling function in tumor cells was characterized in pancreatic tumor cell lines by Western blot as well as proliferation, migration assays and in murine orthotopic xenograft experiments. In premalignant lesions, apelin was expressed in epithelial lesions whereas APJ was found in isolated cells tightly attached to premalignant lesions. However, in the invasive stage, apelin and APJ were co-expressed by tumor cells. In human tumor cells, apelin induced a long-lasting activation of PI3K/Akt, upregulated ß-catenin and the oncogenes c-myc and cyclin D1 and promoted proliferation, migration and glucose uptake. Apelin receptor blockades reduced cancer cell proliferation along with a reduction in pancreatic tumor burden. These findings identify the apelin signaling pathway as a new actor for PDAC development and a novel therapeutic target for this incurable disease.

Acylation of the Incretin Peptide Exendin-4 Directly Impacts Glucagon-Like Peptide-1 Receptor Signaling and Trafficking.

The glucagon-like peptide-1 receptor (GLP-1R) is a class B G protein-coupled receptor and mainstay therapeutic target for the treatment of type 2 diabetes and obesity. Recent reports have highlighted how biased agonism at the GLP-1R affects sustained glucose-stimulated insulin secretion through avoidance of desensitization and downregulation. A number of GLP-1R agonists (GLP-1RAs) feature a fatty acid moiety to prolong their pharmacokinetics via increased albumin binding, but the potential for these chemical changes to influence GLP-1R function has rarely been investigated beyond potency assessments for cAMP. Here, we directly compare the prototypical GLP-1RA exendin-4 with its C-terminally acylated analog, exendin-4-C16. We examine relative propensities of each ligand to recruit and activate G proteins and ß-arrestins, endocytic and postendocytic trafficking profiles, and interactions with model and cellular membranes in HEK293 and HEK293T cells. Both ligands had similar cAMP potency, but exendin-4-C16 showed ∼2.5-fold bias toward G protein recruitment and a ∼60% reduction in ß-arrestin-2 recruitment efficacy compared with exendin-4, as well as reduced GLP-1R endocytosis and preferential targeting toward recycling pathways. These effects were associated with reduced movement of the GLP-1R extracellular domain measured using a conformational biosensor approach and a ∼70% increase in insulin secretion in INS-1 832/3 cells. Interactions with plasma membrane lipids were enhanced by the acyl chain. Exendin-4-C16 showed extensive albumin binding and was highly effective for lowering of blood glucose in mice over at least 72 hours. Our study highlights the importance of a broad approach to the evaluation of GLP-1RA pharmacology. SIGNIFICANCE STATEMENT: Acylation is a common strategy to enhance the pharmacokinetics of peptide-based drugs. This work shows how acylation can also affect various other pharmacological parameters, including biased agonism, receptor trafficking, and interactions with the plasma membrane, which may be therapeutically important.

Amyloid Beta Peptide Is an Endogenous Negative Allosteric Modulator of Leptin Receptor.

INTRODUCTION: Metabolic dysfunction is now recognized as a pivotal component of Alzheimer's disease (AD), the most common dementia worldwide. However, the precise molecular mechanisms linking metabolic dysfunction to AD remain elusive. OBJECTIVE: Here, we investigated the direct impact of soluble oligomeric amyloid beta (Aß) peptides, the main molecular hallmark of AD, on the leptin system, a major component of central energy metabolism regulation. METHODS: We developed a new time-resolved fluorescence resonance energy transfer-based Aß binding assay for the leptin receptor (LepR) and studied the effect of Aß on LepR function in several in vitro assays. The in vivo effect of Aß on LepR function was studied in an Aß-specific AD mouse model and in pro-opiomelanocortin (POMC) neurons of the hypothalamic arcuate nucleus. RESULTS: We revealed specific and high-affinity (Ki = 0.1 nM) binding of Aß to LepR. Pharmacological characterization of this interaction showed that Aß binds allosterically to the extracellular domain of LepR and negatively affects receptor function. Negative allosteric modulation of LepR by Aß was detected at the level of signaling pathways (STAT-3, AKT, and ERK) in vitroand in vivo. Importantly, the leptin-induced response of POMC neurons, key players in the regulation of metabolic function, was completely abolished in the presence of Aß. CONCLUSION: Our data indicate that Aß is a negative allosteric modulator of LepR, resulting in impaired leptin action, and qualify LepR as a new and direct target of Aß oligomers. Preventing the interaction of Aß with LepR might improve both the metabolic and cognitive dysfunctions in AD condition.

Melatonin controversies, an update.

Melatonin was discovered more than 60 years ago. Since then, several seminal discoveries have allowed us to define its function as a neuroendocrine hormone and its molecular targets in mammals and many other species. However, many fundamental issues have not yet been solved such as the subcellular localization of melatonin synthesis and the full spectrum of its molecular targets. In addition, a considerable number of controversies persist in the field, mainly concerning how many functions melatonin has. Altogether, this illustrates how "immature" the field still is. The intention of this opinion article is to note the controversies and limitations in the field, to initiate a discussion and to make proposals/guidelines to overcome them and move the field forward.

Journal of pineal research guideline for authors: Defining and characterizing melatonin targets.

A multitude of effects has been attributed to melatonin at pmol/L to mmol/L concentrations. More than fifteen targets have been proposed for melatonin but only few of them are well characterized. The current guidelines intend to provide a framework to improve and rationalize the characterization of melatonin targets and effects. They should be considered as mandatory guidelines and minimum requirements for manuscripts submitted to the Journal of Pineal Research.

Melatonin promotes regeneration of injured motor axons via MT1 receptors.

Melatonin is an ancient multi-tasking molecule produced by the pineal gland and by several extrapineal tissues. A variety of activities has been ascribed to this hormone in different physiological and pathological contexts, but little is known about its role in peripheral neuroregeneration. Here, we have exploited two different types of injury to test the capability of melatonin to stimulate regeneration of motor axons: (a) the acute and reversible presynaptic degeneration induced by the spider neurotoxin α-Latrotoxin and (b) the compression/transection of the sciatic nerve. We found that in both cases melatonin administration accelerates the process of nerve repair. This pro-regenerative action is MT1 -mediated, and at least in part due to a sustained activation of the ERK1/2 pathway. These findings reveal a receptor-mediated, pro-regenerative action of melatonin in vivo that holds important clinical implications, as it posits melatonin as a safe candidate molecule for the treatment of a number of peripheral neurodegenerative conditions.

GPR50-Ctail cleavage and nuclear translocation: a new signal transduction mode for G protein-coupled receptors.

Transmission of extracellular signals by G protein-coupled receptors typically relies on a cascade of intracellular events initiated by the activation of heterotrimeric G proteins or ß-arrestins followed by effector activation/inhibition. Here, we report an alternative signal transduction mode used by the orphan GPR50 that relies on the nuclear translocation of its carboxyl-terminal domain (CTD). Activation of the calcium-dependent calpain protease cleaves off the CTD from the transmembrane-bound GPR50 core domain between Phe-408 and Ser-409 as determined by MALDI-TOF-mass spectrometry. The cytosolic CTD then translocates into the nucleus assisted by its 'DPD' motif, where it interacts with the general transcription factor TFII-I to regulate c-fos gene transcription. RNA-Seq analysis indicates a broad role of the CTD in modulating gene transcription with ~ 8000 differentially expressed genes. Our study describes a non-canonical, direct signaling mode of GPCRs to the nucleus with similarities to other receptor families such as the NOTCH receptor.

Melatonin MT1 and MT2 receptor ERK signaling is differentially dependent on Gi/o and Gq/11 proteins.

G protein-coupled receptors (GPCRs) transmit extracellular signals into cells by activating G protein- and ß-arrestin-dependent pathways. Extracellular signal-regulated kinases (ERKs) play a central role in integrating these different linear inputs coming from a variety of GPCRs to regulate cellular functions. Here, we investigated human melatonin MT1 and MT2 receptors signaling through the ERK1/2 cascade by employing different biochemical techniques together with pharmacological inhibitors and siRNA molecules. We show that ERK1/2 activation by both receptors is exclusively G protein-dependent, without any participation of ß-arrestin1/2 in HEK293 cells. ERK1/2 activation by MT1 is only mediated though Gi/o proteins, while MT2 is dependent on the cooperative activation of Gi/o and Gq/11 proteins. In the absence of Gq/11 proteins, however, MT2 -induced ERK1/2 activation switches to a ß-arrestin1/2-dependent mode. The signaling cascade downstream of G proteins is the same for both receptors and involves activation of the PI3K/PKCζ/c-Raf/MEK/ERK cascade. The differential G protein dependency of MT1 - and MT2 -mediated ERK activation was confirmed at the level of EGR1 and FOS gene expression, two ERK1/2 target genes. Gi/o /Gq/11 cooperativity was also observed in Neuroscreen-1 cells expressing endogenous MT2 , whereas in the mouse retina, where MT2 is engaged into MT1 /MT2 heterodimers, ERK1/2 signaling is exclusively Gi/o -dependent. Collectively, our data reveal differential signaling modes of MT1 and MT2 in terms of ERK1/2 activation, with an unexpected Gi/o /Gq/11 cooperativity exclusively for MT2 . The plasticity of ERK activation by MT2 is highlighted by the switch to a ß-arrestin1/2-dependent mode in the absence of Gq/11 proteins and by the switch to a Gi/o mode when engaged into MT1 /MT2 heterodimers, revealing a new mechanism underlying tissue-specific responses to melatonin.

A novel leptin receptor antagonist uncouples leptin's metabolic and immune functions.

Leptin links body energy stores to high energy demanding processes like reproduction and immunity. Based on leptin's role in autoimmune diseases and cancer, several leptin and leptin receptor (LR) antagonists have been developed, but these intrinsically lead to unwanted weight gain. Here, we report on the uncoupling of leptin's metabolic and immune functions based on the cross talk with the epidermal growth factor receptor (EGFR). We show that both receptors spontaneously interact and, remarkably, that this complex can partially overrule the lack of LR activation by a leptin antagonistic mutein. Moreover, this leptin mutant induces EGFR phosphorylation comparable to wild-type leptin. Exploiting this non-canonical leptin signalling pathway, we identified a camelid single-domain antibody that selectively inhibits this LR-EGFR cross talk without interfering with homotypic LR signalling. Administration in vivo showed that this single-domain antibody did not interfere with leptin's metabolic functions, but could reverse the leptin-driven protection against starvation-induced thymic and splenic atrophy. These findings offer new opportunities for the design and clinical application of selective leptin and LR antagonists that avoid unwanted metabolic side effects.

Dual role of mitochondria in producing melatonin and driving GPCR signaling to block cytochrome c release.

G protein-coupled receptors (GPCRs) are classically characterized as cell-surface receptors transmitting extracellular signals into cells. Here we show that central components of a GPCR signaling system comprised of the melatonin type 1 receptor (MT1), its associated G protein, and ß-arrestins are on and within neuronal mitochondria. We discovered that the ligand melatonin is exclusively synthesized in the mitochondrial matrix and released by the organelle activating the mitochondrial MT1 signal-transduction pathway inhibiting stress-mediated cytochrome c release and caspase activation. These findings coupled with our observation that mitochondrial MT1 overexpression reduces ischemic brain injury in mice delineate a mitochondrial GPCR mechanism contributing to the neuroprotective action of melatonin. We propose a new term, "automitocrine," analogous to "autocrine" when a similar phenomenon occurs at the cellular level, to describe this unexpected intracellular organelle ligand-receptor pathway that opens a new research avenue investigating mitochondrial GPCR biology.

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.