Molecular mechanism of distinct chemokine engagement and functional divergence of the human Duffy antigen receptor.

The Duffy antigen receptor is a seven-transmembrane (7TM) protein expressed primarily at the surface of red blood cells and displays strikingly promiscuous binding to multiple inflammatory and homeostatic chemokines. It serves as the basis of the Duffy blood group system in humans and also acts as the primary attachment site for malarial parasite Plasmodium vivax and pore-forming toxins secreted by Staphylococcus aureus. Here, we comprehensively profile transducer coupling of this receptor, discover potential non-canonical signaling pathways, and determine the cryoelectron microscopy (cryo-EM) structure in complex with the chemokine CCL7. The structure reveals a distinct binding mode of chemokines, as reflected by relatively superficial binding and a partially formed orthosteric binding pocket. We also observe a dramatic shortening of TM5 and 6 on the intracellular side, which precludes the formation of the docking site for canonical signal transducers, thereby providing a possible explanation for the distinct pharmacological and functional phenotype of this receptor.

The impact of nanobodies on GPCR structural biology and their potential as therapeutic agents.

The family of human G protein-coupled receptors (GPCRs) is comprised of about 800 different members, with about 35% of current pharmaceutical drugs targeting GPCRs. However, GPCR structural biology, necessary for structure-guided drug design, has lagged behind that of other membrane proteins, and it was not until the year 2000 when the first crystal structure of a GPCR (rhodopsin) was solved. Starting in 2007, the determination of additional GPCR structures was facilitated by protein engineering, new crystallization techniques, complexation with antibody fragments, and other strategies. More recently, the use of camelid heavy-chain-only antibody fragments (nanobodies) as crystallographic chaperones has revolutionized the field of GPCR structural biology, aiding in the determination of more than 340 GPCR structures to date. In most cases, the GPCR structures solved as complexes with nanobodies (Nbs) have revealed the binding mode of cognate or non-natural ligands; in a few cases, the same Nb has acted as an orthosteric or an allosteric modulator of GPCR signaling. In this review we summarize the multiple ingenious strategies that have been conceived and implemented in the last decade to capitalize on the discovery of nanobodies to study GPCRs from a structural perspective. Significance Statement G protein-coupled receptors (GPCRs) are major pharmacological targets, and the determination of their structures at high resolution has been essential for structure-guided drug design and for insights about their functions. Single domain antibodies (nanobodies) have greatly facilitated the structural determination of GPCRs, by forming complexes directly with the receptors or indirectly through protein partners.

Nanodomain cAMP signalling in cardiac pathophysiology: potential for developing targeted therapeutic interventions.

3', 5'-cyclic adenosine monophosphate (cAMP) mediates the effects of sympathetic stimulation on the rate and strength of cardiac contraction. Beyond this pivotal role, in cardiac myocytes cAMP also orchestrates a diverse array of reactions to various stimuli. To ensure specificity of response, the cAMP signaling pathway is intricately organized into multiple, spatially confined, subcellular domains, each governing a distinct cellular function. In this review, we describe the molecular components of the cAMP signalling pathway, how they organized are inside the intracellular space and how they achieve exquisite regulation of signalling within nanometer-size domains. We delineate the key experimental findings that lead to the current model of compartmentalised cAMP signaling and we offer an overview of our present understanding of how cAMP nanodomains are structured and regulated within cardiac myocytes. Furthermore, we discuss how compartmentalized cAMP signaling is affected in cardiac disease and consider the potential therapeutic opportunities arising from understanding such organization. By exploiting the nuances of compartmentalized cAMP signaling, novel and more effective therapeutic strategies for managing cardiac conditions may emerge. Finally, we highlight the unresolved questions and hurdles that must be addressed to translate these insights into interventions that may benefit patients.

Structural basis of psychedelic LSD recognition at dopamine D1 receptor.

Understanding the kinetics of LSD in receptors and subsequent induced signaling is crucial for comprehending both the psychoactive and therapeutic effects of LSD. Despite extensive research on LSD's interactions with serotonin 2A and 2B receptors, its behavior on other targets, including dopamine receptors, has remained elusive. Here, we present cryo-EM structures of LSD/PF6142-bound dopamine D1 receptor (DRD1)-legobody complexes, accompanied by a ß-arrestin-mimicking nanobody, NBA3, shedding light on the determinants of G protein coupling versus ß-arrestin coupling. Structural analysis unveils a distinctive binding mode of LSD in DRD1, particularly with the ergoline moiety oriented toward TM4. Kinetic investigations uncover an exceptionally rapid dissociation rate of LSD in DRD1, attributed to the flexibility of extracellular loop 2 (ECL2). Moreover, G protein can stabilize ECL2 conformation, leading to a significant slowdown in ligand's dissociation rate. These findings establish a solid foundation for further exploration of G protein-coupled receptor (GPCR) dynamics and their relevance to signal transduction.

Regulation of cannabinoid and opioid receptor levels by endogenous and pharmacological chaperones.

Cannabinoid and opioid receptor activities can be modulated by a variety of posttranslational mechanisms including the formation of interacting complexes. This study examines the involvement of endogenous and exogenous chaperones in modulating the abundance and activity of cannabinoid CB1 receptor (CB1R), delta opioid receptor (DOR), and CB1R-DOR interacting complexes. Focussing on endogenous protein chaperones namely receptor transporter proteins (RTPs), we examined relative mRNA expression in the mouse spinal cord and found RTP4 to be expressed at higher levels compared to other RTPs. Next, we assessed the effect of RTP4 on receptor abundance by manipulating RTP4 expression in cell lines. Overexpression of RTP4 causes an increase and knock-down causes a decrease in the levels of CB1R, DOR, and CB1R-DOR interacting complexes; this is accompanied by parallel changes in signaling. The ability of small molecule lipophilic ligands to function as exogenous chaperones was examined using receptor-selective antagonists. Long term treatment leads to increases in receptor abundance and activity with no changes in mRNA supporting a role as pharmacological chaperones. Finally, the effect of cannabidiol (CBD), a small molecule ligand and a major active component of Cannabis, on receptor abundance and activity in mice was examined. We find that CBD administration leads to increases in receptor abundance and activity in mouse spinal cord. Together, these results highlight a role for chaperones (proteins and small molecules) in modulating levels and activity of CB1R, DOR, and their interacting complexes potentially through mechanisms including receptor maturation and trafficking. Significance Statement This study highlights a role for chaperones (endogenous and small membrane-permeable molecules) in modulating levels of CB1R, DOR, and their interacting complexes. These chaperones could be developed as therapeutics for pathologies involving these receptors.

New Target(s) for RNF43 Regulation: Implications for Therapeutic Strategies.

Cancer cells depend on specific oncogenic pathways or present a genetic alteration that leads to a particular disturbance. Still, personalized and targeted biological therapy remains challenging, with current efforts generally yielding disappointing results. Carefully assessing onco-target molecular pathways can, however, potently assist with such efforts for the selection of patient populations that would best respond to a given drug treatment. RNF43, an E3 ubiquitin ligase that negatively regulates Wnt/frizzled (FZD) receptors by their ubiquitination, internalization, and degradation, controls a key pathway in cancer. Recently, additional target proteins of RNF43 were described, including p85 of the PI3K/AKT/mTOR signaling pathway and protease-activated receptor 2 (PAR2), a G-protein-coupled receptor that potently induces ß-catenin stabilization, independent of Wnts. RNF43 mutations with impaired E3 ligase activity were found in several types of cancers (e.g., gastrointestinal system tumors and endometrial and ovarian cancer), pointing to a high dependency on FZD receptors and possibly PAR2 and the PI3K/AKT/mTOR signaling pathway. The development of drugs toward these targets is essential for improved treatment of cancer patients.

KLS-13019, a novel structural analogue of cannabidiol (CBD) and GPR55 receptor antagonist, Prevents and Reverses Chemotherapy-Induced Peripheral Neuropathy (CIPN) in Rats.

Neuropathic pain is a form of chronic pain that develops because of damage to the nervous system. Treatment of neuropathic pain is often incompletely effective, and most available therapeutics have only moderate efficacy and present side effects that limit their use. Opioids are commonly prescribed for the management of neuropathic pain despite equivocal results in clinical studies and significant abuse potential. Thus, neuropathic pain represents an area of critical unmet medical and novel classes of therapeutics with improved efficacy and safety profiles are urgently needed. The cannabidiol (CBD) structural analogue and novel antagonist of GPR55, KLS-13019, was screened in rat models of neuropathic pain. Tactile sensitivity associated with chemotherapy exposure was induced in rats with once daily 1mg/kg paclitaxel injections for 4 days or 5 mg/kg oxaliplatin every third day for one week. Rats were then administered KLS-13019 or comparator drugs on day 7 in an acute dosing paradigm or days 7-10 in a chronic dosing paradigm and mechanical or cold allodynia was assessed. Allodynia was reversed in a dose-dependent manner in the rats treated with KLS-13019, with the highest dose reverting the response to pre-paclitaxel injection baseline levels with both I.P. and P.O. administration after acute dosing. In the chronic dosing paradigm, 4 consecutive doses of KLS-13019 completely reversed allodynia for the duration of the phenotype in control animals. Additionally, co-administration of KLS -13019 with paclitaxel prevented the allodynic phenotype from developing. Together, these data suggest that KLS-13019 represents a potential new drug for the treatment of neuropathic pain. Significance Statement Chemotherapy-induced neuropathic pain (CIPN) is a common, debilitating side effect of cancer treatment with no known cure. GPR55 antagonist KLS-13019 represents a novel class of drug for this condition that is a potent, durable inhibitor of allodynia associated with CIPN in rats in both prevention and reversal dosing paradigms. This novel therapeutic approach addresses a critical area of unmet medical need.

Unlocking the potential of higher-molecular-weight 5-HT7R ligands: Synthesis, affinity, and ADMET examination.

An increasing number of drugs introduced to the market and numerous repositories of compounds with confirmed activity have posed the need to revalidate the state-of-the-art rules that determine the ranges of properties the compounds should possess to become future drugs. In this study, we designed a series of two chemotypes of aryl-piperazine hydantoin ligands of 5-HT7R, an attractive target in search for innovative CNS drugs, with higher molecular weight (close to or over 500). Consequently, 14 new compounds were synthesised and screened for their receptor activity accompanied by extensive docking studies to evaluate the observed structure-activity/properties relationships. The ADMET characterisation in terms of the biological membrane permeability, metabolic stability, hepatotoxicity, cardiotoxicity, and protein plasma binding of the obtained compounds was carried out in vitro. The outcome of these studies constituted the basis for the comprehensive challenge of computational tools for ADMET properties prediction. All the compounds possessed high affinity to the 5-HT7R (Ki below 250 nM for all analysed structures) with good selectivity over 5-HT6R and varying affinity towards 5-HT2AR, 5-HT1AR and D2R. For the best compounds of this study, the expression profile of genes associated with neurodegeneration, anti-oxidant response and anti-inflammatory function was determined, and the survival of the cells (SH-SY5Y as an in vitro model of Alzheimer's disease) was evaluated. One 5-HT7R agent (32) was characterised by a very promising ADMET profile, i.e. good membrane permeability, low hepatotoxicity and cardiotoxicity, and high metabolic stability with the simultaneous high rate of plasma protein binding and high selectivity over other GPCRs considered, together with satisfying gene expression profile modulations and neural cell survival. Such encouraging properties make it a good candidate for further testing and optimisation as a potential agent in the treatment of CNS-related disorders.

An atlas of GPCRs in dopamine neurons: Identification of the free fatty acid receptor 4 as a regulator of food and water intake.

Midbrain dopaminergic neurons (DANs) are subject to extensive metabotropic regulation, but the repertoire of G protein-coupled receptors (GPCRs) present in these neurons has not been mapped. Here, we isolate DANs from Dat-eGFP mice to generate a GPCR atlas by unbiased qPCR array expression analysis of 377 GPCRs. Combined with data mining of scRNA-seq databases, we identify multiple receptors in DAN subpopulations with 38 of these receptors representing the majority of transcripts. We identify 41 receptors expressed in midbrain DANs but not in non-DAN midbrain cells, including the free fatty acid receptor 4 (FFAR4). Functional expression of FFAR4 is validated by ex vivo Ca2+ imaging, and in vivo experiments support that FFAR4 negatively regulates food and water intake and bodyweight. In addition to providing a critical framework for understanding metabotropic DAN regulation, our data suggest fatty acid sensing by FFAR4 as a mechanism linking high-energy intake to the dopamine-reward pathway.

Insight into the regulatory mechanism of ß-arrestin2 and its emerging role in diseases.

ß-arrestin2, a member of the arrestin family, mediates the desensitization and internalization of most G protein-coupled receptors (GPCRs) and functions as a scaffold protein in signalling pathways. Previous studies have demonstrated that ß-arrestin2 expression is dysregulated in malignant tumours, fibrotic diseases, cardiovascular diseases and metabolic diseases, suggesting its pathological roles. Transcription and post-transcriptional modifications can affect the expression of ß-arrestin2. Furthermore, post-translational modifications, such as phosphorylation, ubiquitination, SUMOylation and S-nitrosylation affect the cellular localization of ß-arrestin2 and its interaction with downstream signalling molecules, which further regulate the activity of ß-arrestin2. This review summarizes the structure and function of ß-arrestin2 and reveals the mechanisms involved in the regulation of ß-arrestin2 at multiple levels. Additionally, recent studies on the role of ß-arrestin2 in some major diseases and its therapeutic prospects have been discussed to provide a reference for the development of drugs targeting ß-arrestin2.

Molecular Mechanism of the ß3AR Agonist Activity of a ß-Blocker.

Development of subtype-selective drugs for G protein-coupled receptors poses a significant challenge due to high similarity between subtypes, as exemplified by the three ß-adrenergic receptors (ßARs). The ß3AR agonists show promise for treating the overactive bladder or preterm birth, but their potential is hindered by off-target activation of ß1AR and ß2AR. Interestingly, several ß-blockers, which are antagonists of the ß1ARs and ß2ARs, have been reported to exhibit agonist activity at the ß3AR. However, the molecular mechanism remains elusive. Understanding the underlying mechanism should facilitate the development of ß3AR agonist drugs with improved selectivity and reduced off-target effects. In this work, we determined the structures of human ß3AR in complex with the endogenous agonist epinephrine or with a synthetic ß3AR agonist carazolol, which is also a high-affinity ß-blocker. Structure comparison, mutagenesis studies and molecular dynamics simulations revealed that the differences on the flexibility of D3.32 directly contribute to carazolol's distinct activities as an antagonist for the ß2AR and an agonist for the ß3AR. The process is also indirectly influenced by the extracellular loops (ECL), especially ECL1. Taken together, these results provide key guidance for development of selective ß3AR agonists, paving the way for new therapeutic opportunities.

Molecular regulation of calcium-sensing receptor (CaSR)-mediated signaling.

Calcium-sensing receptor (CaSR), a family C G-protein-coupled receptor, plays a crucial role in regulating calcium homeostasis by sensing small concentration changes of extracellular Ca2+, Mg2+, amino acids (e.g., L-Trp and L-Phe), small peptides, anions (e.g., HCO3 - and PO4 3-), and pH. CaSR-mediated intracellular Ca2+ signaling regulates a diverse set of cellular processes including gene transcription, cell proliferation, differentiation, apoptosis, muscle contraction, and neuronal transmission. Dysfunction of CaSR with mutations results in diseases such as autosomal dominant hypocalcemia, familial hypocalciuric hypercalcemia, and neonatal severe hyperparathyroidism. CaSR also influences calciotropic disorders, such as osteoporosis, and noncalciotropic disorders, such as cancer, Alzheimer's disease, and pulmonary arterial hypertension. This study first reviews recent advances in biochemical and structural determination of the framework of CaSR and its interaction sites with natural ligands, as well as exogenous positive allosteric modulators and negative allosteric modulators. The establishment of the first CaSR protein-protein interactome network revealed 94 novel players involved in protein processing in endoplasmic reticulum, trafficking, cell surface expression, endocytosis, degradation, and signaling pathways. The roles of these proteins in Ca2+-dependent cellular physiological processes and in CaSR-dependent cellular signaling provide new insights into the molecular basis of diseases caused by CaSR mutations and dysregulated CaSR activity caused by its protein interactors and facilitate the design of therapeutic agents that target CaSR and other family C G-protein-coupled receptors.

Application of FRET- and BRET-based live-cell biosensors in deorphanization and ligand discovery studies on orphan G protein-coupled receptors.

Bioluminescence- and fluorescence-based resonance energy transfer assays have gained considerable attention in pharmacological research as high-throughput scalable tools applicable to drug discovery. To this end, G protein-coupled receptors represent the biggest target class for marketed drugs, and among them, orphan G protein-coupled receptors have the biggest untapped therapeutic potential. In this review, the cases where biophysical methods, BRET and FRET, were employed for deorphanization and ligand discovery studies on orphan G protein-coupled receptors are listed and discussed.

Free Fatty Acids and Free Fatty Acid Receptors: Role in Regulating Arterial Function.

The metabolic network's primary sources of free fatty acids (FFAs) are long- and medium-chain fatty acids of triglyceride origin and short-chain fatty acids produced by intestinal microorganisms through dietary fibre fermentation. Recent studies have demonstrated that FFAs not only serve as an energy source for the body's metabolism but also participate in regulating arterial function. Excess FFAs have been shown to lead to endothelial dysfunction, vascular hypertrophy, and vessel wall stiffness, which are important triggers of arterial hypertension and atherosclerosis. Nevertheless, free fatty acid receptors (FFARs) are involved in the regulation of arterial functions, including the proliferation, differentiation, migration, apoptosis, inflammation, and angiogenesis of vascular endothelial cells (VECs) and vascular smooth muscle cells (VSMCs). They actively regulate hypertension, endothelial dysfunction, and atherosclerosis. The objective of this review is to examine the roles and heterogeneity of FFAs and FFARs in the regulation of arterial function, with a view to identifying the points of intersection between their actions and providing new insights into the prevention and treatment of diseases associated with arterial dysfunction, as well as the development of targeted drugs.

Protein-Protein Docking Approach to GPCR Oligomerization.

G-protein-coupled receptors (GPCRs), the largest family of human membrane proteins, play a crucial role in cellular control and are the target of approximately one-third of all drugs on the market. Targeting these complexes with selectivity or formulating small molecules capable of modulating receptor-receptor interactions could potentially offer novel avenues for drug discovery, fostering the development of more refined and safer pharmacotherapies. Due to the lack of experimentally derived X-ray crystallography spectra of GPCR oligomers, there is growing evidence supporting the development of new in silico approaches for predicting GPCR self-assembling structures. The significance of GPCR oligomerization, the challenges in modeling these structures, and the potential of protein-protein docking algorithms to address these challenges are discussed. The study also underscores the use of various software solutions for modeling GPCR oligomeric structures and presents practical cases where these techniques have been successfully applied.

Class B1 GPCRs: Insights into Multi-Receptor Pharmacology for the Treatment of Metabolic Disease.

The secretin-like, class B1 sub-family of seven transmembrane-spanning G protein coupled receptors (GPCRs) consists of 15 members that coordinate important physiological processes. These receptors bind peptide ligands and utilize a distinct mechanism of activation that is driven by evolutionarily conserved structural features. For the class B1 receptors, the C-terminus of the cognate ligand is initially recognized by the receptor via a large N-terminal extracellular domain that forms a hydrophobic ligand binding groove. This binding enables the N-terminus of the ligand to engage deep into a large volume, open transmembrane pocket of the receptor. Importantly, the phylogenetic basis of this ligand-receptor activation mechanism has provided opportunities to engineer analogues of several class B1 ligands for therapeutic use. Among the most successful of these are drugs targeting the glucagon-like peptide-1 (GLP-1) receptor for the treatment of type 2 diabetes and obesity. Recently, multi-functional agonists possessing activity at the GLP-1 receptor and the glucose-dependent insulinotropic polypeptide (GIP) receptor, such as tirzepatide, and others that also contain glucagon receptor activity, have been developed. In this article, we review members of the class B1 GPCR family with focus on receptors for GLP-1, GIP, and glucagon, including their signal transduction and receptor trafficking characteristics. The metabolic importance of these receptors is also highlighted, along with the benefit of poly-pharmacologic ligands. Further, key structural features and comparative analyses of high-resolution cryogenic electron microscopy structures for these receptors in active-state complex with either native ligands or multi-functional agonists are provided, supporting the pharmacological basis of such therapeutic agents.

G protein-coupled receptors and traditional Chinese medicine: new thinks for the development of traditional Chinese medicine.

G protein-coupled receptors (GPCRs) widely exist in vivo and participate in many physiological processes, thus emerging as important targets for drug development. Approximately 30% of the Food and Drug Administration (FDA)-approved drugs target GPCRs. To date, the 'one disease, one target, one molecule' strategy no longer meets the demands of drug development. Meanwhile, small-molecule drugs account for 60% of FDA-approved drugs. Traditional Chinese medicine (TCM) has garnered widespread attention for its unique theoretical system and treatment methods. TCM involves multiple components, targets and pathways. Centered on GPCRs and TCM, this paper discusses the similarities and differences between TCM and GPCRs from the perspectives of syndrome of TCM, the consistency of TCM's multi-component and multi-target approaches and the potential of GPCRs and TCM in the development of novel drugs. A novel strategy, 'simultaneous screening of drugs and targets', was proposed and applied to the study of GPCRs. We combine GPCRs with TCM to facilitate the modernisation of TCM, provide valuable insights into the rational application of TCM and facilitate the research and development of novel drugs. This study offers theoretical support for the modernisation of TCM and introduces novel ideas for development of safe and effective drugs.

Quantitative Monitoring of GPCR-Mediated Spatiotemporal IP3 Dynamics Using Confocal Fluorescence Microscopy.

Activation of G protein-coupled receptors upon chemoattractant stimulation induces activation of multiple signaling pathways. To fully understand how these signaling pathway coordinates to achieve directional migration of neutrophils, it is essential to determine the dynamics of the spatiotemporal activation profile of signaling components at the level of single living cells. Here, we describe a detailed methodology for monitoring and quantitatively analyzing the spatiotemporal dynamics of 1,4,5-inositol trisphosphate (IP3) in neutrophil-like HL60 cells in response to various chemoattractant fields by applying Förster resonance energy transfer (FRET) fluorescence microscopy.

Neuroprotective compounds alter the expression of genes coding for proteins related to mitochondrial function in activated microglia.

A hallmark of neuroinflammatory disorders is mitochondrial dysfunction. Nevertheless, the transcriptional changes underlying this alteration are not well-defined. Microglia activation, a decrease in mitochondrion biogenesis and a subsequent alteration of the redox are common factors in diseases coursing with neuroinflammation. In the last two decades, components of the adenosinergic system have been proposed as potential therapeutic targets to combat neuroinflammation. In this research, we analyzed by RNAseq the gene expression in activated microglia treated with an adenosine A2A receptor antagonist, SCH 582561, and/or an A3 receptor agonist, 2-Cl-IB-MECA, since these receptors are deeply related to neurodegeneration and inflammation. The analysis was focused on genes related to inflammation and REDOX homeostasis. It was detected that in the three conditions (microglia treated with 2-Cl-IB-MECA, SCH 582561, and their combination) more than 40 % of the detected genes codified by the mitochondrial genome were differentially expressed (FDR < 0.05) (14/34, 16/34, and 13/34) respectively, being almost all of them (>85 %) upregulated in the microglia treated with adenosinergic compounds. Also, we analyzed the differential expression of genes related to mitochondrial function and oxidative stress codified by the nuclear genome. Additionally, we evaluated the oxygen consumption rate (OCR) of mitochondria in microglia treated with LPS and IFN-γ, both alone and in combination with adenosinergic compounds. The data showed an improvement in mitochondrial function with the antagonist of the adenosine A2A receptor, compared to the effects of pro-inflammatory stimulus, confirming a functional effect consistent with the RNAseq data.

Voltage dependence of M2 muscarinic receptor antagonists and allosteric modulators.

Muscarinic receptors are G protein-coupled receptors (GPCRs) that play a role in various physiological functions. Previous studies have shown that these receptors, along with other GPCRs, are voltage-sensitive; both their affinity toward agonists and their activation are regulated by membrane potential. To our knowledge, whether the effect of antagonists on these receptors is voltage-dependent has not yet been studied. In this study, we used Xenopus oocytes expressing the M2 muscarinic receptor (M2R) to investigate this question. Our results indicate that the potencies of two M2R antagonists, atropine and scopolamine, are voltage-dependent; they are more effective at resting potential than under depolarization. In contrast, the M2R antagonist AF-DX 386 did not exhibit voltage-dependent potency.Furthermore, we discovered that the voltage dependence of M2R activation by acetylcholine remains unchanged in the presence of two allosteric modulators, the negative modulator gallamine and the positive modulator LY2119620. These findings enhance our understanding of GPCRs' voltage dependence and may have pharmacological implications.