G protein-coupled receptors (GPCRs): advances in structures, mechanisms and drug discovery

G protein-coupled receptors (GPCRs), the largest family of human membrane proteins and an important class of drug targets, play a role in maintaining numerous physiological processes. Agonist or antagonist, orthosteric effects or allosteric effects, and biased signaling or balanced signaling, characterize the complexity of GPCR dynamic features. In this study, we first review the structural advancements, activation mechanisms, and functional diversity of GPCRs. We then focus on GPCR drug discovery by revealing the detailed drug-target interactions and the underlying mechanisms of orthosteric drugs approved by the US Food and Drug Administration in the past five years. Particularly, an up-to-date analysis is performed on available GPCR structures complexed with synthetic small-molecule allosteric modulators to elucidate key receptor-ligand interactions and allosteric mechanisms. Finally, we highlight how the widespread GPCR-druggable allosteric sites can guide structure- or mechanism-based drug design and propose prospects of designing bitopic ligands for the future therapeutic potential of targeting this receptor family.


INTRODUCTION
0][11][12][13] Advances in protein engineering, X-ray crystallography, and cryo-electron microscopy (cryo-EM), coupled with innovative technologies such as X-ray free electron lasers (XFELs) and nuclear magnetic resonance (NMR) spectroscopy, have revolutionized our understanding of GPCR structures and dynamics.These studies provide insights into ligand-receptor interactions, conformational changes, and signaling complexes, offering unprecedented opportunities for in-depth investigations into receptor activation, orthosteric/allosteric modulation, biased signaling, and dimerization.
Once activated by exogenous stimuli, GPCRs primarily employ heterotrimeric G-proteins and arrestins as transducers to produce second messengers and further initiate the downstream signaling, resulting in promiscuous signaling profiles within cells. 11Such spectrum of signaling is the prerequisite for function diversity of GPCRs and is fundamental in regulating physiological processes, including sensory perception, neurotransmission, and endocrine processes. 14,15The mutations and truncation of GPCRs; however, can dysregulate GPCR functionality by altering constitutive activity, influencing membrane expression and affecting posttranslational behaviors. 16Unraveling the mechanisms of stimuli-GPCR-effector coupling, as well as the concise regulation of GPCR dysfunction will bring about valuable therapeutic potentials and inspire the design of modulators with high potency, selectivity, or biased signaling.
Till date, approximately 34% of the US Food and Drug Administration (FDA)-approved drugs are targeted to GPCRs, with modulators in clinical trials or preclinical stages experiencing exponential growth. 17,18Among them, orthosteric ligands impose an effective alteration on GPCR activity and signaling process by competitively preventing the binding of endogenous ligands. 19owever, due to the sequence conservation of orthosteric sites, in most cases, subtype selectivity remains an intractable issue, which implies the inevitable side effects of orthosteric drugs. 202][23][24][25] Allosteric modulators are highlighted for their high subtype selectivity and low side effects.A progressive structural understanding of the detailed receptorligand interactions is paving the way for fragment-to-lead optimization in structure-based drug design (SBDD) (Fig. 1).Moreover, the knowledge of allosteric sites is useful for the design of bitopic ligands by creating a molecule attached to both an allosteric and orthosteric site.Bitopic ligands have several advantages of improved affinity and enhanced selectivity over a single allosteric or orthosteric ligand.In addition, elucidating allosteric mechanisms of GPCRs provides a viable strategy to develop biased ligands such as G protein-or β-arrestin-based allosteric modulators. 26Bitopic modulators have higher selectivity to reduce side effects since they exert pathway-specific effects on GPCR signaling. 26,27n this review, we first summarize the structural progression, activation mechanisms, and functional diversity of GPCRs.To delve into the advancement of GPCR drug discovery, we investigate the detailed drug-target interactions at the orthosteric sites, focusing on GPCR structures in complex with recent FDA-approved orthosteric drugs.Subsequently, allosteric modulators are extensively discussed, with a focus on recent breakthroughs in GPCR structures that bind to synthetic small molecules.Notably, peptides and antibodies are excluded from our analysis.Such investigation systematically clusters the location of allosteric sites in the extracellular vestibule, transmembrane domain, and intracellular surface, highlighting the key binding modes with their target receptors and allosteric mechanisms.This review aims to provide a deeper understanding of GPCR structures, mechanisms, and drug discovery, which has important implications for structure-or mechanism-based drug design and the design of bitopic ligands for the future therapeutic potential of targeting this receptor family.

STRUCTURE ADVANCES IN GPCRS
The low expression of membrane protein GPCRs, combined with their conformational flexibility, initially posed great challenges for high-resolution diffraction. 28The initial crystal structures of rhodopsin and the ligand-activated β2 adrenergic receptor (β2AR) were resolved in 2000 and 2007, respectively. 29,30Over the past two decades, considerable progress has been made in Fig. 1 Phylogenetic tree of GPCRs indicating GPCR structures that have been solved in complex with modulators.Nodes represent GPCRs named according to their UniProt gene name and are organized according to the GPCR database.GPCR structures bound to modulators are highlighted by color the engineering of proteins and the technique of X-ray crystallography. 31Notably, the use of GPCR engineering with fusion proteins, 32,33 antibody fragment crystallization 34,35 , and thermostabilizing mutations 36 , has produced numerous antagonist-or agonist-bound GPCR structures.However, only agonist-bound GPCRs frequently exist in an intermediate conformation because the fully active conformation requires stabilizing chaperones, including G proteins, G protein mimetics, conformationally specific nanobodies, and mini-G proteins. 37he first GPCR-G protein complex was determined in 2011 using X-ray diffraction; 38 however, the demanding nature of X-ray crystallography has rendered GPCR-G protein complex crystallization a difficult undertaking.Cryo-EM has developed to be an alternative technique, driving a novel trend in GPCR structural biology.Unlike X-ray crystallography, cryo-EM does not rely on crystals and has considerably superior potential to directly visualize detergent-or nanodisc-solubilized GPCRs.This capability enables the determination of previously intractable fully active states and larger protein complexes, including GPCR-G protein complexes. 39Since then, the number of cryo-EM structures depicting GPCRs in complex with intracellular partners has experienced exponential growth (Fig. 2).As of November 2023, the Protein Data Bank has accumulated 554 complex structures, of which 523 are resolved using cryo-EM. 40However, both crystallography and cryo-EM are limited to capturing the most stable and lowest energy conformations under crystallization conditions. 4oreover, the comprehensive characterization of intermediate states and transition kinetics remains elusive.Crystallographic, spectroscopic, and simulation techniques have offered complementary information on the conformational dynamics of GPCRs.
The advanced XFELs possess the potential to solve the missing information.The exceptional properties of XFELs, characterized by extreme brilliance and femtosecond short pulses, allow them to overcome radiation damage, facilitating the determination of GPCR structures with atomic-level information at femtosecond timescales. 41NMR spectroscopy offers a valuable technique to detect dynamic features of GPCRs in liquid environments. 42,43The number, position, and shape of signals in the NMR spectra are sensitive to changes in the micro-environment of stable-isotope "probes" incorporated into receptors.Double electron-electron resonance (DEER) spectroscopy enables the assessment of a distance distribution between two different probes.Fluorescence resonance energy transfer (FRET), a technique based on fluorescence, functions as an "atomic ruler" to detect the proximity between two labels, providing valuable data about the number of states and their relative populations. 44,45Among these, DEER and FRET provide only localized details regarding the chemical probes that have been inserted.7][48] Advances in the structural biology of GPCRs have revealed key information on ligand-receptor interactions, conformational changes, and signaling complexes, opening the opportunity for exploration of receptor activation, orthosteric/allosteric modulation, biased signaling, and dimerization.

MECHANISM OF GPCR ACTIVATION AND SIGNALING
Although the nature of GPCRs and activating stimuli may vary significantly, GPCRs primarily coordinate distinct downstream signaling responses through two types of transducers: heterotrimeric G proteins and arrestins.Human G proteins comprise four major families (G s , G i/o , G q/11, and G 12/13 ) and more than half of GPCRs activate two or more G proteins, each of which exhibits distinct efficacies and kinetics. 49,50The promiscuous coupling leads to fingerprint-like signaling profiles inside the cell, which contributes to the complexity of GPCR signaling.Fig. 2 Timeline of major advancements in GPCR structure study using X-ray crystallography and cryo-EM When bound to GDP, the Gαβγ heterotrimer is inactive.Agonist binding leads to the formation of an active conformation of GPCRs, which initiates signaling cascades involving the recruitment and activation of G-proteins.The activated GPCR catalyzes the GDP/GTP exchange on the Gα subunit, causing the dissociation of Gα from the Gβγ dimer.Due to high cellular concentrations of GTP, Gα rapidly binds a molecule of GTP at the nucleotidebinding site.Both Gα-GTP and Gβγ can modulate subsequent effector proteins.Gα-GTP can activate or inhibit enzymes such as adenylyl cyclase (AC), phospholipase C (PLC), or ion channels, depending on the specific G protein type.Gβγ can also modulate various signaling pathways and interact with target proteins.Activation of effector proteins by Gα-GTP or Gβγ generates second messengers, such as cyclic AMP (cAMP).The cellular response concludes with the Gα subunit hydrolyzing GTP to GDP, leading to its reassociation with Gβγ and G protein inactivation.Subsequently, the Gα subunit completes the G-protein activation cycle by reassociating with Gβγ.
To prevent sustained signaling, activated GPCRs may also undergo C-terminal phosphorylation facilitated by G-proteincoupled receptor kinases (GRKs).This multi-site GPCR phosphorylation determines β-arrestin binding affinity and induces receptor desensitization via steric hindrance, followed by clathrin-mediated endocytosis and ubiquitination of the receptor (Fig. 3a). 11,51,52The receptor-arrestin complex also serves as a scaffold for over 20 different kinases, including mitogen-activated protein (MAP) kinases, ERK1/2, p38 kinases, and c-Jun N-terminal kinases, activating G-protein independent signaling pathway.Four isoforms of arrestin (arrestins 1-4) and multiple GRK isoforms were discovered, with arrestins 1 and 4 being only found in the visual system.β-arrestins 1 and 2, also referred to as arrestins 2 and 3, interact with and regulate numerous non-visual GPCRs. 34riginally classified as monomers, GPCRs were subsequently recognized to engage in homo-or hetero-dimerization, displaying distinct properties in receptor activation, pharmacological cascades, and biological functions. 53,54Recent research indicates that GPCRs can bind to various single transmembrane accessory proteins to regulate their biological functions such as ligand binding, transducer coupling, and intracellular signaling. 55,56rominent examples include the family of receptor activitymodifying proteins (RAMPs) that majorly regulate the glucagon receptor (GCGR) and the melanocortin receptor accessory proteins (MRAPs) that regulate the melanocortin receptors (MC1R-MC5R). 57,58Currently, the interactions between the negative allosteric modulator RAMP2 and GCGR as well as the positive allosteric modulator MRAP1 and MC2R have been elucidated by cryo-EM. 59,60tructural changes within GPCRs facilitate their function as molecular conduits that transmit extracellular signals across membranes to elicit cellular responses.A distinctive feature of GPCR activation involves notable outward movement of the cytoplasmic end of TM6, creating an intracellular pocket to accommodate the downstream transducers.][63] Additionally, Na + acts as an endogenous negative allosteric modulator (NAM) of class A GPCR activation, stabilizing the inactive state through direct interactions. 64,65High resolution structures reveal that Na + interacts mainly with residues from Fig. 3 a Schematic representation of GPCR activation process.Upon agonist (red circle) binding, the receptor proceeds into a pre-activation state coupling with the G protein heterotrimer, where the exchange of GDP and GTP in G protein α subunit leads to G protein dissociation and mediate G protein signaling pathway.The phosphorylation of the receptor C-terminal tail by GRK binding promotes arrestin recruitment and signaling.When the antagonists (blue circle) bind, the receptor stabilizes in an inactive state.b Crosstalk of downstream pathway of Gs, Gq, Gi and arrestin TM1, TM2, TM3, and TM7 and these interactions vary across GPCRs. 66,67igands can regulate receptor activity by stabilizing distinct conformations.Since the diverse signaling pathways elicit distinct physiological effects, ligands that selectively induce beneficial pathways hold promising therapeutic value.These drugs are commonly referred to as "biased ligands."For instance, G proteinbiased μ-opioid receptor (μOR) agonists are of remarkable clinical relevance as they enhance analgesia effects and reduce adverse reactions associated with the activation of β-arrestin pathways, in contrast to morphine.9][70] Hence, unraveling the coupling mechanisms governing G proteins, GRKs, and arrestins will establish a robust foundation for designing biased ligands tailored to selectively activate or inhibit specific pathways.
In the absence of agonists, GPCRs may display different levels of constitutive activities.The efficacy of diverse ligands acting on a single GPCR in terms of activation or inactivation also varies widely.Considering both receptor constitutive activity and drug efficacy, GPCR ligands are categorized as (full) agonists, partial agonists, antagonists, and inverse agonists.These variations in efficacy significantly influence their therapeutic properties.

FUNCTIONAL DIVERSITY OF GPCRS Overview of GPCR subfamilies and their physiological functions
GPCRs can be categorized into class A, class B, class C, class F, and class T according to their structural and functional characteristics.Class A GPCRs, namely the rhodopsin-like family, is the superfamily with the largest proportion and the most extensive research. 71lass A GPCRs can further be divided by function into aminergic, peptide, protein, lipid, melatonin, nucleotide, steroid, dicarboxylic acid, sensory, and orphan subgroups, 72 with their corresponding indications ranging from hypertension, cardiovascular diseases, and pulmonary diseases, to depression and psychiatric disorders. 17Class B GPCRs are divided into secretin (B1) and adhesion (B2) subfamilies, with the former characteristic of large extracellular domains (ECD) and the latter possessing a unique long N-terminal motif and autoproteolysis-inducing domain. 73While glucagon-like peptide-1 receptor (GLP-1R) and glucagon receptor (GCGR) are emerging as the famous B1 GPCR targets in regulating blood glucose homeostasis and lipid metabolism; 74,75 the B2 subfamily is critical in modulating sensory, endocrine, and gastrointestinal systems. 76Class C GPCRs, the glutamate receptors, are unique in their large ECDs, conserved venus fly traps (VFTs), cysteine-rich domains (CRDs) on the ligand binding sites, and constitutive dimers for receptor activation. 77With mGluRs (metabotropic glutamate receptors) taking the lead in clinical transformation, the physiological functions of class C GPCRs are implicated in cancer, migraine, schizophrenia, and movement disorders. 77Class F GPCRs, comprising 10 frizzled receptors (FZDs) and one smoothened receptor (SMO), are distinctive in their conserved CRD regions and involvement in Hedgehog and Wnt signaling pathways.Therefore, they are mainly associated with cancer, fibrosis, and embryonic development. 78The current drug discovery is only focused on SMO, 79 leaving broad exploration space for the therapeutic potential of FZDs.Particularly, although taste 2 receptors (TAS2Rs), the receptors modulating taste perception of humans, show structure similarity with class A GPCRs, their low sequence homology (<20%) with the existing types of GPCRs isolates them to a novel category of class T GPCRs, 76 deepening our understanding of the entire GPCR family.
Involvement of GPCRs in sensory perception, neurotransmission, and endocrine regulation Rhodopsin, TAARs, and TASRs in sensory perception.One of the most significant physiological functions GPCRs exercise is mediating sensory information such as light perception, taste, olfaction, and pheromone sensation.Rhodopsin, which contributes to the first stage of visual activation in vertebrates, exhibits the typical and representative features of class A GPCRs.Upon absorbing photons, the orthosteric ligand of rhodopsin, retinal, experiences conformational flipping within picoseconds, thus rapidly triggering signal propagation from the receptor to G proteins, cGMP phosphodiesterase, or cGMP-gated ion channel. 80he covalent linkage of retinal with the receptor, and the instantaneous overturning and signaling serve as a paradigm for elucidating the efficiency of GPCRs in sensory perception.
Olfactory sensory receptors, which can be categorized into odorant receptors (ORs) and trace amine-associated receptors (TAARs), are a valuable medium for researchers to understand olfactory information encoding.Guo et al. 81 has recently revealed the universal mechanism of TAARs recognition of amine odor molecules and the structural basis of "combinatorial coding" of the olfactory receptor in ligand recognition.Notably, the selective coupling of mTAAR9 with Gs and Golf is also delineated, which serves as a pioneer in the field of mammalian olfactory recognition.Apart from selective G-proteins, the downstream transduction mechanism of olfactory receptors is also associated with adenylyl cyclase and cAMP-gated ion channel, 82 leaving favorable exploration opportunities.
To regulate the sensory function of taste, which is one of the most important sensations in human life, taste receptors (TASRs) are extensively studied from physiological and pharmacological perspectives.Among them, type I taste GPCRs function by forming heterodimeric complexes to stimulate sweet (TAS1R2/TAS1R3) and umami (TAS1R1/TAS1R3) sensation, whereas Type II are monomeric TAS2Rs that regulate bitter flavor. 83Tastant binding to the receptor activates downstream secondary messengers, resulting in depolarization and sensitizing the transient receptor potential (TRP) channel, which in turn innervates the gustatory cortex in the brain. 84Given the inapplicability of the previous GPCR expression techniques in TAS2Rs, 85 overcoming difficulties in the structural determination of taste receptors will further facilitate their physiological research.μOR and CBR in neurotransmission.Currently, neurological therapeutic demands mainly revolve around neuropathic pain alleviation, treatment of depression, psychiatric disorders, and Parkinson's diseases.μ-Opioid receptors (μORs), possessing a research history of over 50 years, have been extensively researched about their mechanism of analgesic action in the peripheral nervous system (PNS) and the central nervous system (CNS).For instance, μORs reduce the release of nociceptive substances and decrease Ca 2+ production following nerve injury by interacting with TRPV1, H1R, and NK1R in nociceptive receptors, 86 whereas in spinal dorsal horn neurons, μORs modulate 5-HT receptors, glycine receptors, and norepinephrine receptors to activate pain inhibitory pathway. 87rthosteric biased modulators, allosteric modulators, and bitopic modulators have been successively developed to exert analgesic effects while alleviating side effects like respiratory depression and addiction. 88Cannabinoid receptors (CBRs) are also representative targets involved in neurotransmission and neuropathic pain pathophysiology.The subtype CB1R is primarily found in presynaptic terminals of neurons in CNS, the activation of which inhibits neurotransmitter release and algesthesia transmission, 89 while CB2R is highly expressed in immune cells, the activation of which can inhibit inflammatory factors that promote pain sensitization. 90No-selective orthosteric CB1R and CB2R activators can produce an antinociceptive effect and improve sleep in several animal models, while selective positive allosteric modulators (PAMs) like ZCZ011 (40)  are rising as more promising ligands without inducing cannabis-like side effects. 91LP-1R and GPR120 in endocrine regulation.Endocrine syndrome has been rising as one of the most critical health issues in the 21 st century.Numerous metabolism-related GPCRs, which are usually activated by energy metabolites or substrates, are pivotal sensors of endocrine dysregulation.GLP-1R and GPR120 (also known as free fatty acid receptor 4), for example, are both promising therapeutic targets for the treatment of type 2 diabetes and obesity. 74,92Mechanistically, the endogenous ligand of GLP-1R, GLP-1, can reduce the secretion of glucagon in pancreatic α cells and promote insulin secretion in pancreatic β cells.For GPR120, however, the binding of omega-3 polyunsaturated fatty acids (ω3-FAs) and receptor activation can reduce inflammation of adipose tissue and protect against insulin resistance. 93The receptor's coupling with G q/11 subsequently stimulates the PI3K/Akt pathway, resulting in the uptake of glucose in adipocytes. 94As GLP-1R agonist liraglutide takes the lead in FDA-approved drugs treating type 2 diabetes and obesity, 95 drug development of more endocrine-related targets such as GPR35, GPR40, GPR41, GPR43, GPR81, and GPR119 are supposed to come into our view.
Receptor promiscuity and cross-talk between different signaling pathways GPCR receptors convert the extracellular stimuli to intracellular signals to control cellular function and phenotype.GPCR receptors convert the extracellular stimuli to intracellular signals to control cellular phenotype and function.These intracellular signaling pathways intersect with each other to enhance or downgrade relevant responses in a phenomenon known as "cross-talk."The promiscuity of the GPCR signaling network is consequently outlined, resulting in more extensive regulation, low selectivity, and possible adverse effects.
Promiscuity and cross-talk can occur at three levels, including the GPCR receptors, G-proteins/β-arrestins, and the downstream effectors.The receptor promiscuity lies in the formation of heterodimers, which can either be constituted of subtypes of the same receptor family or those of different families.A compelling case is the heterodimerization of GABA b(1) and GABA b(2) which leads to the functionality of modulating GIRK (Gprotein gate inward rectifying channel) potassium channels, whilst neither of them is functional when expressed as a monomer. 96nother well-established example is the plentiful interrelationship of adenosine receptors and dopamine receptors, where the activation of A 1A and A 2A adenosine receptors decreases dopamine binding to D1 and D2 dopamine receptors. 97The bivalent ligands that bind adenosine receptors and dopamine receptors at each end further demonstrate the occurrence and functionality of heterodimerization. 98The participants of heterodimerization are assumed to share a common G-protein pool, thus contributing to the redistribution of their interaction of G-proteins and reshaping the signaling landscape. 99Given this, by direct cross-talk between two GPCR receptors, ligands can be designed towards one receptor to modulate the affinity and efficacy of the other target, although certain pharmacological profiles remains unclear.
At the second stage of the hierarchical signaling of GPCRs, namely the recruitment of G s , G i , G q , G 12 , β-arrestin 1, and β-arrestin 2, a spectrum of coupling strengths ranging from highly selective coupling to promiscuous coupling is exhibited.MD simulations performed by Sandhu et al. revealed that engineered mutant GPCRs can alter the coupling of non-cognate G-proteins by reshaping the intracellular interface, 99 demonstrating that "dynamic structural plasticity" of the GPCR cytosolic pockets is the foundation of G-protein promiscuity.Mutants, orthosteric and allosteric modulators that exert long-range and delicate effects towards the cytosolic binding interface are therefore principal strategies to achieve selectivity of G-protein signaling.
The promiscuity of distinct downstream effectors, known as the third stage of signaling, is highly correlated with the cross-talk of G-proteins.Normally, stimulation of G s , G i, and G q results in the activation of AC, the inhibition of AC, and the stimulation of PLC, respectively. 100However, once distinct G-proteins are recruited near the membrane at a similar time, βγ subunits released from respective G-protein activation are "exchangeable" between diverse signaling pathways and can potentiate responses mediated by other G-proteins. 101The second messengers then phosphorylate, activate, or deactivate each other to construct a fine-tuning network (Fig. 3b).Albeit conducting a great deal of research, the precise control of GPCR promiscuity remains obscure.
Impact of GPCR mutations on human diseases and therapeutic implications Besides being involved in numerous physiological processes, mutations in GPCRs can be linked to manifold human diseases, underlying the necessity of GPCR genomics, and imposing therapeutic implications.Till date, over 2350 mutations in GPCR genes have been identified as the major causes of more than 60 inherited monogenic diseases in humans (Fig. 4a), with missense mutations harboring the maximum proportion (>60%) and small inserts/deletions ranking the second (>15%). 16assification of the effects of mutations on GPCR dysfunctions.The effects of mutations in GPCRs can be categorized into gain-offunction (GoF) and loss-of-function (LoF), corresponding to physiological hyperfunction and hypofunction, respectively.Recent studies have provided a more detailed explanation of the diverse underlying mechanisms of GoF and LoF mutations.Compared with the wild-type (WT) GPCR activation, the common pharmacological mechanisms of activating and inactivating mutations lie in three aspects: (1) Mutations transform microswitch cascades within the receptors and induce active/inactive conformations, thus altering the constitutive activity of GPCRs and affecting the recruitment of downstream effectors.(2) Mutations influence receptor expression directly or indirectly increasing/ decreasing receptors' intracellular transport, degradation, and recycling.(3) Some mutations affect ligand potency, specificity, or promiscuous recognition, thereby exerting regulatory functions by shifting the conformational population, redistributing the downstream couplings, or altering receptor dimerization.Furthermore, all variants are not pathogenic.This provides robust evidence for another classification of "driver" and "passenger" mutations (Fig. 4b). 102Computational approaches are recently emerging to predict the driver ability of mutations in GPCR-related diseases, based on abundant clinical data of mutations and relevant GPCR dysfunctions.
Correlation of GPCR mutations and human diseases.The genomic alterations induced by GPCR mutations serve as the major driver of various monogenic diseases.Some well-established examples include missense mutations in SMO receptor causing basal cell carcinoma, 103 missense and nonsense mutations in MC4R causing obesity 104 , and missense mutations in FSHR inducing ovarian hyperstimulation syndrome.The majority of mutations are highly conserved and thus in an advantageous position during evolution. 105Therefore, the pathological relevance between GPCR mutations and human diseases may be more effectively predicted taking the evolutionary conservation of a certain residue into consideration.
Therapeutic implications and approaches of GPCR pathologies.Terapeutic approaches of GPCR pathologies mainly include symptomatic and etiological treatment.As many GPCR dysfunctions ultimately result in endocrine diseases with endorgan resistance or cancer, the administration of hormones or chemotherapeutics may be considered to reduce pathologic phenotypes. 106,107ore state-of-the-art therapeutic implications, however, are oriented towards etiological treatment.Missense mutations in GPCRs can mislead protein folding and post-translational modifications to cause trafficking alterations, in which pharmacological chaperones are applicative therapeutic regimens. 108For receptor truncation resulting from nonsense mutations or frame-shifting mutations, RNA interference, gene replacement approaches, and the genome editing approach CRISPR/Cas9 may rescue the receptor integrality, provided that at least the first three transmembrane helices remain in the mutant receptor. 109F NMR. 111 Once the hits were obtained, structureactivity relationship (SAR) optimization in synergistic application of computational methodologies such as fragment-growing, property prediction, and MD simulations, was conducted to initiate the hit-to-lead and lead-to-drug campaign. 99erein, we specially emphasize on the interaction and signaling mechanism of synthetic small-molecule modulators bound to GPCRs, with the aim of enlightening the discovery of more ingenious molecules with high potency, selectivity, and potential biased effects.

Structure-based drug design targeting the orthosteric sites of GPCRs
Orthosteric small molecule modulators are the most universal non-peptide regulators of GPCRs.By competing with endogenous ligands, they interact with the orthosteric binding pocket (OBP) and exert a full agonistic 112,113 /partial agonistic 114 /antagonistic function 115,116 by triggering the conformational displacement of GPCR internal structures. 117,118Despite their relatively mature development, side effects derived from low subtype selectivity and promiscuous signaling remain the major hurdle. 49,119ver the past 30 years, the widespread use of X-ray and Cryo-EM has facilitated the characterization of GPCR-orthosteric ligand complexes, with 657 class A, 16 class B1, 6 class B2, 19 class C, 18 class F, and 1 class T structures solved (supplementary Table 1-5). 120ere, we meticulously selected five representative complexes in which ligands have been launched recently to elucidate the mechanisms of ligand recognition, specificity, and elaborate signaling transduction.Furthermore, we exemplified two cases to demonstrate the beneficial engagement of structural information in exploiting not only SAR but also the structure-functional selectivity relationship (SFSR).Considering these seven cases as a paradigm, we aimed to condense valuable hints based on a detailed analysis of approved drugs or selective compounds and provide a constructive outlook for the high-quality discovery of GPCR orthosteric modulators that may overcome the current dilemma.
μOR in complex with oliceridine.7][128] Oliceridine (2), a partial agonist binding at the orthosteric site of μOR, was approved by the FDA in 2020 for its ability to biased signaling via the G protein pathway and thus alleviating side effects (Fig. 5a, b). 129Therefore, casting light on the oliceridine-μOR complex structure and the underlying mechanism of biased signaling will provide insight in developing a novel generation of analgesic drugs.
By aligning the complex structures of μOR-oliceridine and μOR-fentanyl, a well superimposed binding mode in OBP above Trp295 6.48 was found.The only exception was that the pyridine ring of oliceridine tilts 35°toward TM2 relative to the n-aniline group of fentanyl, resulting in weaker hydrophobic interactions with TM6/7 than that with fentanyl.Based on the results of MD performed by Zhang et al., 130 extended interactions with TM6/7 can be inferred to have elicited inward movement of TM6 and TM7-H8 toward the TM core, shaping adaptive intracellular pocket conformation for both G-protein and β-arrestin coupling and thus leading to neutral signaling, whereas reduced interactions may have kept the intracellular end of TM6/7 relatively away from the TM core and therefore stabilize an intracellular pocket preferential for G protein binding and signaling.Two fentanyl-derived μOR agonists (3-4), which substituted the aniline group on fentanyl with n-propyl or isopropyl to reduce hydrophobicity with TM6/7, were thereupon designed as "proof-of-concept" to successfully achieve biased signaling via the G protein pathway (Fig. 5c).Different from the "trial-and-error" mode when developing biased ligand oliceridine, 131 the comprehensive study by Zhang et al. serves as a paradigm for dissecting co-crystallized complexes to understand the molecular basis of preferential signaling mechanisms initiated from the orthosteric pocket and broadens the avenue for designing biased modulators of ORs through SBDD strategies.
S1PR in complex with siponimod.3][134][135][136] Although Fingolimod received regulatory approval from the FDA in 2010 as a first-inclass S1PR agonist, 137 its low subtype selectivity has led to several "off-target" effects, including bradycardia and atrioventricular blockade. 138Therefore, a second-generation, highly subtypeselective S1PR modulator is crucially needed.Siponimod ( 5) was globally approved in 2019 for the treatment of adults with relapsing MS by selectively targeting S1PR1 and S1PR5. 139Insights into the mechanisms of drug recognition and receptor activation will provide a framework for understanding ligand selectivity and signal transduction in GPCRs. 140,141uan et al. presented the cryo-EM structures of siponimod-S1PR1-G i and siponimod-S1PR5 complexes, in which the ligands exhibited an identical linear conformation across a polar module and the deep hydrophobic cavity of the orthosteric pocket (Fig. 6a). 142Given that members of the S1PR family display different extracellular vestibules, distinct extracellular leaflets have been reported to have contributed to diverse access channels for ligand entry and thus relate to specificity among subtypes (Fig. 6b). 143Moreover, further careful comparison of the siponimod-S1PR1-G i complex with antagonist ML056-bound S1PR1 structure underlines the "twin toggle mechanism" during receptor activation. 144Upon ligand binding, Leu128 3.36 rotates 130°away from TM5 to form a direct interaction with the hydrophobic portion of siponimod, disrupting its previous interaction with Trp269 6.48 and triggering a synergistic downward movement of Trp269 6.48 .The dramatic displacement of the two residues can therefore loosen the interaction between TM3 and TM6, inducing a consequent outward movement of TM6 that can accommodate G protein binding (Fig. 6c, d).Similar activation mechanism involving corresponding mechanical switches can also be found in CB1 and MC4R, 145,146 which provides valuable hints that designing ligands forming elaborate hydrophobic interaction with residue 3.36 or directly inducing reconfiguration of 3.36-6.48may contribute to enhanced activation efficacy.
OX2R in complex with lemborexant.8][149] The two subtypes, OX1R and OX2R, dominate the respective regulatory behaviors, with OX1R involved in gating rapid eye movement (REM) sleep and OX2R involved in gating non-REM and REM sleep. 150Lemborexant (6), an orthosteric competitive antagonist approved by the FDA in 2019, exhibits outstanding inhibitory activity against OXRs. 151,152However, the most important features of lemborexants lie in two aspects: (1) Why lemborexants show moderate selectivity toward OX2R over OX1R, 152 which will facilitate the design of OX1R/OX2R-selective modulators that can be applied to REM and non-REM functionality studies?2) What is the basis of the dynamic parameters of lemborexant that may explain the relationship between druginduced improvement of sleep onset and a decrease in wake time after sleep?
To elucidate the mechanism of lemborexant subtype selectivity and provide guidance for anti-insomnia drug development, Asada et al. presented the crystal structure of the OX2R-lemborexant complex and compared its ligand-binding mode with that of the previously solved OX1R-lemborexant complex structure. 153espite the ligand's shared hydrogen bonds with Gln126 3.32 of OX1R and Gln134 3.32 of OX2R, lemborexant binds OX1R as a mixture of two orientations owing to the small side chain of Ala127 3.33 , whereas lemborexant binds OX2R in only one configuration because of the steric hindrance of Thr135 3.33 , which is inferred to be the primary cause of the difference in its affinity for OX1R and OX2R (Fig. 7a, b).In contrast, by simulating lemborexant in solution, the intramolecular stacking of two Fig. 5 a A bridged general view of fentanyl and oliceridine inducing distinct pharmacological profiles.b 2D structure of fentanyl and oliceridine shown for clarity.c Superimposed views of μOR-fentanyl (gray cartoon, gray sticks; PDB: 8EF5) and μOR-oliceridine (light green cartoon, light green sticks; PDB: 8EFB) complex structure, together with the comparison of ligand binding modes and arrestin coupling interfaces, are presented.2D structures of two designed biased modulators are also presented aromatic rings was observed to play a vital role in shaping the conformation of lemborexant close to the bound state before receptor binding, which explains the high k on value of the ligand.In addition, the higher binding free energy of lemborexant compared to other OXR modulators may contribute to a higher k off value.Collectively, these observations highlight the possibility of obtaining a high k on by optimizing the conformation of free molecules via intramolecular interactions (Fig. 7c, d).By extension, separately modulating the enthalpy of molecular binding to the receptor and entropy derived from the intramolecular structure may be important strategies for designing drugs with enhanced kinetics and dynamics.5-HT 1F in complex with lasmiditan.5][156] Although traditional targeted agonists have been clinically used as anti-migraine drugs for decades, side effects such as therapeutic vasoconstrictive actions owing to the non-selective activation of 5-HT 1B and 5-HT 1D remain a major hindrance. 157asmiditan (7), a potent and highly selective drug toward 5-HT 1F was approved by the FDA in 2019 because of its vasoconstrictive side effects and high-penetration properties. 158Elucidation of the scaffold features of lasmiditan and the mechanism of 5-HT 1Fselective activation will provide a template for the rational design of safer anti-migraine drugs.
Through the 5-HT 1F -lasmiditan-G i1 complex solved by Huang et al., an overview of the lasmiditan-binding mode was presented. 159In the orthosteric binding pocket, the primary amine on the methylpiperidine group largely contributes to the stability of lasmiditan by forming a canonical charge interaction with Asp103 3.32 of the receptor while simultaneously forming a Fig. 6 a Detailed binding modes of S1PR5 in complex with siponimod.Labels of the residues engaged in polar contacts with siponimod are colored in blue, with hydrogen bonds presented by orange dashes.The residues of the hydrophobic pocket that stabilizes ligand binding are marked with green labels, while residues that are critical for signal transduction are labeled in red.b Superimposed views of S1PR1 (orange cartoon, PDB: 7T6B), S1PR2 (light green cartoon, PDB: 7C4S), S1PR3 (light purple cartoon, PDB: 7YXA), and S1PR5 (yellow cartoon, PDB: 7TD4) GPCR structures, where TM1 and TM7 of S1PR5 are highlighted for clarity.c Superimposed views of active S1PR1-siponimod complex (cyan cartoon, cyan stick, PDB: 7TD4) and inactive S1PR1 structure (gray cartoon, gray stick, PDB: 3V2Y) to illustrate the "toggle switch" activation mechanism.d 2D structure of siponimod is shown for clarity hydrogen bond with Tyr337 7.42 .Notably, in the extended binding pocket (EBP), the trifluorobenzene group of lasmiditan forms additional hydrophobic interactions with Ile174 ECL2 and Pro158 4.60 and forms hydrogen bonds with residue Glu313 6.55 , Asn317 6.59 , Thr182 5.40 , and His176 ECL2 .Structural alignment of 5-HT 1F with other 5-HT1 receptor subtypes revealed that the TM4-TM5-ECL2 region, which is highly conserved in the other four subtypes, underwent a notable conformational change, thereby disrupting the interaction between lasmiditan and 5-HT 1A , 5-HT 1B , 5-HT 1D , and 5-HT 1E .Thus, designing ligands that accommodate EBP and form specific interactions with the TM4-TM5-ECL2 region may enable high 5-HT 1F selectivity (Fig. 8a, b).Activation mechanical analysis by Huang et al. revealed that lasmiditan triggers the downward movement of the toggle switch residue Trp 6.48 and then induces conformational rearrangement of the PIF, DRY, and NPxxY motifs.Particularly, structural comparison of 5-HT 1F -G i complex and other 5-HT 1 -G i/o showed that the αN of 5-HT 1F -bound G i shifts away from other 5-HT 1 receptor-bound G i/o , suggesting unique G i coupling and corresponding specific downstream effects (Fig. 8c).Therefore, designing modulators that interact with the toggle switch residue and optimize their blood-brain-barrier (BBB) penetration properties may yield effective and safer 5-HT 1F agonists.
GnRH1 in complex with elagolix.1][162] With the first availability of GnRH1R non-peptidic antagonist elagolix (8) on the market in 2018, 163 structural insights into the GnRH1R-elagolix complex have gained pharmaceutical interest. 164Additionally, unlike other class A GPCRs, GnRH1R lacks a C-terminal helix (helix 8) in the cytoplasmic region and harbors Asn 2.50 instead of the highly conserved Asp 2.50 present in other receptors, 165 leaving a wide space for different microswitches along the signaling cascade within 7TMD.
The crystal structure of the GnRH1R-elagolix complex studied by Yan et al. revealed that polar network residues composed of Lys121 3.32 and Asp98 2.61 play critical roles in forming polar interactions with the ligand, whereas Tyr283 6.51 and Tyr290 6.58 are engaged in ligand recognition by contributing to hydrophobic interactions (Fig. 9a).Notably, Elagolix is located closer to TM7, resulting in an enlarged orthosteric pocket that allows N-terminal entry and co-occupation of the site.Structural alignment and IP accumulation assays showed that, unlike some GPCRs in which ligands can contact residue Trp 6.48 directly and trigger the toggle switch, the special motif Tyr283 6.51 -Tyr284 6.52 -Trp280 6.48 -Phe276 6.44 in TM6 was suggested to be a critical structural motif involved in mediating the propagation of signal transmission (Fig. 9b).Moreover, only 4% of class A GPCRs, including GnRH1R, have asparagine at the 5.58 position, which is implicated in a polar interaction with Ser136 3.47 GnRH1R, thus leading to TM6 packing tightly with TM3 and TM5 in GnRH1R and exercising an antagonistic function.Collectively, these analyses highlight the distinctive features of GnRH1R in the binding of a representative antagonist and provide insights for structural biologists.
SFSR study utilizing structural information.7][168] This is partially due to the "activity-cliff" phenomenon which to some extent, undermines the profits from structural information.Nevertheless, the promising prospect still deserves expecting.Two examples are analyzed here to arouse future interest in SFSR studies utilizing structural information.
The first example is the efficient discovery and optimization of A 2 AR selective antagonist 1,2,4-triazine derivative 4d (9) via SBDD strategy.With Biophysical Mapping (BPM) approach and crystal structure analysis, compound 4d was revealed to be primarily stabilized by two hydrogen bonds between the triazine core and N253 6.55 , with ring A oriented towards TM2 and TM7 (Fig. 10a).Fig. 7 a Detailed binding mode of lemborexant in complex with OX2R (receptor: light orange, ligand: cyan, PDB: 7XRR), where steric hindrance of T135 3.33 only allows one orientation of the ligand.b Detailed binding mode of lemborexant in complex with OX1R (receptor: light pink, ligand: yellow, PDB: 6TOT), where small side chain of A127 3.33 accounts for two orientations of the ligand.c Abridged general view of employing MD simulation to predict the conformation of the ligand before receptor binding, to improve K on values.d 2D structure of lemborexant is shown for clarity Hence, the presence of a hydrogen bond acceptor at the para position of ring A to interact with His278 7.43 , as well as the introduction of one or more flanking lipophilic substituents on the same ring to interact with Ile66 2.64 was suggested as the focus of the SAR program.Introducing either a phenolic hydroxyl or 4-pyridyl nitrogen at the para position of ring A, and fine-tuning affinity by various combinations of small lipophilic substituents efficiently yielded compound 4k, which proves the best balance of potency and efficacy (Fig. 10b). 169Further research compared the binding pockets of A 2 AR in complex with adenosine (agonist), ZM241385 (antagonist), and compound 4e (antagonist).The hydrophobic sub-pocket in the lower chamber was observed to be occupied by the ribose ring system of adenosine analogs in agonist complexes, though was typically unoccupied when antagonists bound.The same region also allowed optimization of selectivity for A 2 AR over A 1 AR (Fig. 10c).Therefore, expanding chemotypes into this region may harvest a more efficient chemical series when designing selective and diverse functional modulators. 170he second paradigm entails the structure-based drug design of novel β-arrestin-biased D2R agonists commencing with aripiprazole, so as to alleviate the movement disorders associated with the adverse effects of antipsychotics. 171The dichlorophenylpiperazine portion of aripiprazole was first replaced with an indolepiperazine, leading to 12 that displayed comparable activity in both G i/o -mediated cAMP inhibition and β-arrestin2 recruitment assays.Molecular docking with a D 2 R homology model revealed that the indole NH of 12 formed a hydrogen bond with Ser5.42, which has been shown to mediate G-protein-dependent signaling in highly homologous β2 adrenergic receptors.A methyl group was thus attached to the NH of indole to fine-tune the binding conformation of 12 and thereby preclude TM5 engagement (Fig. 11a).Inspired by structural information from homologous 5-HT 2B receptor, where ligand interactions with hydrophobic residues on ECL2 appear to promote β-arrestin recruitment (Fig. 11a), a second methyl was introduced to position 2 of the indole ring, yielding 13 with a β-arrestin bias factor of 20 and potentially reduced side effects (Fig. 11b). 172To our knowledge, this is the first successful attempt at using structural information for the rational design of GPCR-biased ligands, underlining the necessity of interactive structural comparison in SFSR study.
Delineation of GPCR structures complexed with small-molecule allosteric modulators and allosteric signaling Over the past 10 years, allosteric drug discovery targeting GPCRs has witnessed significant progress in structural understanding, with the advance in knowledge of GPCR allostery. 173,174Till February 2024, the crystal structures of 59 allosteric smallmolecule modulators bound to GPCRs have been solved, including 33 class A, 7 class B, 18 class C, and 1 class F modulators.These structures reveal that despite the intrinsic dynamic nature of GPCRs and the structural diversity among different GPCRs, only limited locations function as allosteric pockets, and the same pockets are present in GPCRs with different homologies. 175Even within a single receptor, more than one allosteric site has been identified.In addition, druggable allosteric hotspots spread throughout the receptor and can be divided into the following sections: extracellular vestibule, transmembrane domain, intracellular surface, outside 7TMD, and inside 7TMD domains. 174,175As will be discussed, allosteric binding sites in all GPCRs are currently known to be located at 11 distinct locations with some consensus, 176 depicted in Fig. 12.In this figure, the locations of all pockets identified in different GPCRs are mapped onto the structure of an example GPCR to facilitate the comparison of these sites.
Based on the compounds' ability to affect the stimulatory activity of orthosteric ligands, allosteric ligands can be classified into several categories, including positive allosteric modulators (PAMs), negative allosteric modulators (NAMs), allosteric modulators, and allosteric inverse agonists. 22,177A PAM, such as cinacalcet, targets the calcium-sensing receptor (CaSR) and potentiates the response of the receptor to its orthosteric agonist.Conversely, NAM attenuates the response of the receptor to its orthosteric agonist, mavoglurant, which targets the metabotropic Fig. 10 a Detailed binding mode of A 2 AR in complex with compound 4d (receptor: light gray, ligand: orange, PDB: 3UZA).b SAR study of A 2 AR antagonist.c Comparison of the orthosteric binding site of A 2 AR-Adenosine complex (light blue, PDB: 2YDO), A 2 AR-ZM241385 complex (light yellow, PDB: 4EIY), A 2 AR-Compound 4e complex (light green, PDB: 3UZC), the difference in cavity occupation is highlighted by red circles and arrows glutamate receptor 5 (mGluR5). 178Ago allosteric modulators can activate or inhibit a receptor without an orthosteric agonist such as compound 2, which targets the glucagon-like peptide-1 receptor (GLP-1R).
Targeting of GPCR extracellular vestibule (outside and inside 7TMD).After the first FDA approval of cinacalcet (a PAM of CaSR) in 2004 as a treatment for hyperparathyroidism, 179 smallmolecule allosteric modulators bound to the extracellular vestibule have developed rapidly.Till date, four of these modulators have been approved by the FDA, and one has entered clinical trials, as summarized in Table 1.Resolved crystal structures have revealed three extracellular binding sites: the pocket outside helices I and II, the pocket outside helices II and III, and the pocket inside 7TMD (Fig. 13). 180,181Due to their proximity to the traditional active sites of class A and B GPCRs, such allosteric modulators may exert their effects by directly altering the binding of orthosteric ligands to the receptor.As GPCRs evolved from a common ancestor, this allosteric site, found on receptors, may represent the ancestral orthosteric site. 176,182g. 11 a Detailed binding mode of D 2 R in complex with compound 1 (12) (receptor: light gray, ligand: salmon, the receptor is modeled from PDB: 3PBL).TM5 of the receptor is colored in pink and ECL2 is colored in blue for clarity.4][185] For such peptide receptors, allosteric pockets on GPCRs may be easier to target for small-molecule drugs than orthosteric drugs. 186[189][190][191] LSN3160440 (14) (Fig. 14) is a small-molecule PAM targeted GLP-1R with an EC 50 of 1 μM to enhance the potency and efficacy of GLP-1(9-36) becoming a full agonist. 180The cryo-EM structure of GLP-1R in complex with LSN3160440, the orthosteric ligand GLP-1, and the Gs protein revealed a clear depiction of the U-shaped binding mode of LSN3160440.The allosteric site is formed by residues on helices I and II in the extracellular vestibule (Fig. 15a). 180Within the binding pocket, the benzimidazole moiety of LSN3160440 (Fig. 15c) formed hydrophobic contacts with Fig. 12 11 allosteric binding sites reported across GPCRs mapped onto representative class A GPCR CB1R.Gray pockets represent binding pockets within 7TMD, and white pockets represent binding pockets outside 7TMD.For each pocket, the number of unique ligands is indicated using boldface type, and the number of GPCRs containing the pocket is provided in parentheses.The boundary of the lipid bilayer is indicated by gray dashes Fig. 13 Three extracellular allosteric binding sites in GPCRs and the corresponding small-molecule allosteric modulators.Stick models of small-molecule ligands are mapped to representative members of outside 7TMD (I and II) (GLP-1R, PDB: 6VCB), outside 7TMD (II and III) (GPR101, PDB: 8W8S), and within 7TMD (M4R, PDB: 7V68) GPCRs.The position of an orthosteric ligand of M4R (shown in gray and sphere-andstick representation) is mapped onto the overview of allosteric modulators for comparison.For each pocket, the number of unique modulators is indicated in boldface type, and the number of GPCRs containing the pocket is indicated in parentheses Leu142 1.37 (the superscript represents the generic residue numbers of GPCRs) and engaged in aromatic interactions with Tyr145 1.40 (Fig. 15b).Mutation and molecular dynamics (MD) simulation results also suggest that water-mediated hydrogen bonds may form between N3 of benzimidazole and Lys202 2.72 . 192,193Notably, LSN3160440 interacts with GLP-1 and acts as a molecular glue. 194The 2,6-dichloro-3-methoxyl phenyl moiety of LSN3160440 forms van der Waals interactions with Phe12 GLP-1 , Val16 GLP-1 and Leu20 GLP-1 simultaneously.
6][197][198] Structural comparisons of these ligands with LSN3160440 revealed a shared region situated at the extracellular termini of the TM1-TM2 cleft, further suggesting that this is a promising area for lead optimization for both orthosteric and allosteric agonists.Within the binding site, the aromatic interactions with Tyr145 1.40 are conserved.
2) Outside 7TMD (TM I-II) GPR101-AA-14 structure GPR101 is an orphan class A GPCR that is highly expressed in the nucleus accumbens and the hypothalamus and has constitutive Gs and Gq activity. 199GPR101 gene duplication or mutation modulates its constitutive activity, rendering GPR101 a promising target for metabolic diseases. 200Recent studies have identified AA-14 (15) (Fig. 14) as an allosteric agonist of GPR101, demonstrating robust Gs activation activity and high subtype selectivity. 201In vivo studies have shown that AA-14 exerts rejuvenating effects by activating GPR101 in the pituitary.
LY2119620 (20) acts as a PAM that has activity at both the M2 and M4 receptors (Fig. 14) but is inappropriate for treatment, probably because of crossreactivity and cardiovascular liability. 215
Contrarily, in the M4 receptor, only Gln427 ECL3 formed a hydrogen bond with the modulator.
In addition, the structures of M2 receptor-iperoxo-LY2119620 (PDB: 4MQT) and M2 receptor-LY2119620 (PDB: 6U1N) are highly similar, with Trp422 7.35 perpendicular to the horizontal plane and forming a π-π stacking with LY2119620 (Fig. 17d).In contrast, Trp422 7.35 of the M2 receptor-iperoxo (PDB:4MQS), exhibits a parallel conformation, suggesting that the allosteric binding site is formed predominantly in the presence of an allosteric modulator.MD simulations have revealed that LY2119620 modulates the conformation of Trp422 7.35 , causing reorientation of Tyr426 7.39 within the orthosteric site. 219This reorientation may explain the observed increase in affinity for  220 For GPCRs that use other sites to bind endogenous ligands, the traditional orthosteric pocket is potentially druggable for allosteric modulators. 221Calcium-sensing receptors (CaSR), members of the family C GPCR, are found primarily in the parathyroid glands and kidneys to ensure strict control of calcium homeostasis. 222,223levated Ca 2+ levels trigger the activation of CaSR, leading to the inhibition of parathyroid hormone (PTH) secretion.5][226] Cinacalcet (27) (Fig. 14), an orally active allosteric agonist of CaSR, has been used for the treatment of secondary hyperparathyroidism 227,228 whereas calcilytic NPS-2143 (29) (Fig. 14) is a potent NAM-targeting CaSR that exhibits favorable in vitro and in vivo activity. 229hen bound to CaSR, PAM cinacalcet adopted extended and bent poses between CaSR homodimers (Fig. 18a).The naphthylethylamine moiety was bound to highly similar poses in both the extended and bent conformations (Fig. 18b, c).The naphthyl group engaged in hydrophobic interactions with Ile777 5.44 on one side and formed edge-to-face π-π interactions with Phe684 3.36 and Trp818 6.50 on the other, thereby effectively securing the side chain of Trp818 6.50 inside 7TM helical bundle.The NH group formed a hydrogen bond with Gln681 3.33 .In the extended conformation (Fig. 18b), the linker and phenyl group were parallel to TM VI, extending upward and driving Tyr825 6.57 to orient downward.In the bent conformation (Fig. 18c), the phenyl group folded between TM V and TM VI to form a parallel-displaced π-π stacking with the naphthyl group, whereas Tyr825 6.57 assumed a conformation perpendicular to TM VI and stabilized the ligand through a σ-π interaction.
In the CaSR-NPS-2143 complex, NPS-2143 exhibited the same crescent conformation as the homodimers (Fig. 19a).The naphthyl group at one end of NPS-2143 (Fig. 19c) was lined by residues Phe684 3.36 , Leu776 5.43 , Ile777 5.44 , Trp818 6.50 , and Ile841 7.37 in the interior of the pocket (Fig. 19b).Conversely, the 3-chloro-2-cyanophenyl ring of NPS-2143 protrudes out toward the lateral opening and forms hydrophobic contacts with Leu773 5.40 and π-π stacking interactions with Tyr825 6.57 .A single hydrogen bond was established between the hydroxyl group and Tyr825 6.57 .Moreover, the conformation of the NAM-bound CaSR agrees well under both active (in the presence of Ca 2+ and L-Trp) and inactive (no Ca 2+ ) conditions.
Despite having highly similar binding sites, NPS-2143 and cinacalcet exhibit quite different pharmacological properties, which may be explained by the conformation of the receptor residues.The conformations of NPS-2143-bound and Ca 2+ -bound CaSR were similar. 230Nevertheless, cinacalcet binding induced Fig. 17 a Superposition of PAM LY2119620 bound to M2 receptor (pink cartoon, pink sticks; PDB: 4MQT) and M4 receptor (yellow cartoon, yellow sticks; PDB: 7V68).b Detailed binding modes of M2 receptor bound to LY2119620.c Detailed binding modes of M4 receptor bound to LY2119620.Hydrogen bonds are presented as orange dashes and π-π stacking is presented as gray dashes.d Superimposed views of highlighted residue Trp422 7.36 on M2 receptor-iperoxo-LY2119620 (pink cartoon, pink sticks; PDB: 6U1N), M2 receptor-LY2119620 (purple cartoon, purple sticks; PDB: 4MQT), and M2 receptor-iperoxo (blue cartoon, blue sticks; PDB: 4MQS) structures.The orthosteric agonist iperoxo is presented in orange significant conformational changes in Trp818 6.50 , Phe821 6.53 , and Tyr825 6.57 within the allosteric pocket (Fig. 19d).Trp818 6.50 rotates inwardly from a vertical to a horizontal conformation, forming extensive π-π interactions with cinacalcet.Phe821 6.53 underwent an outward shift and was inserted into a crevice between TM6 and TM7 facing the dimer interfacial area.Simultaneously, Tyr825 6.57 flips down, driven by structural conflicts in the extended conformation of cinacalcet.In summary, cinacalcet induces a bent conformation of TM6 and stabilizes the homodimer interface, thereby contributing to receptor activation. 231Contrarily, NPS-2143 decreased agonist efficacy by enhancing TM VI helicity, which spatially hindered receptor activation.
Based on the special binding conformations of cinacalcet, Liu et al. conducted a virtual screening of 1.2 billion compounds to discover novel PAMs with potentially novel pharmacology.To respectively mimic the "extended" and "bent" conformation, extensive orientations and conformations of library molecules were sampled, which gave 682 trillion configurations overall and finally achieved a 3.8% and 13.6% hit rate.The hits were then optimized to a pharmacologically potent lead (36) via synergistic application of structural information, fragment hybridization, and stereochemistry separation (Fig. 19e). 232Such practice serves as a paradigm for its elaborate utility of solved GPCR structures and conformation sampling strategy and is generalizable in the discovery of CaSR NAMs and other allosteric modulators.
Targeting of GPCR transmembrane domain (outside 7TMD).As shown by their structures, GPCRs utilize the domain outside 7TMD at the lipid interface to bind allosteric modulators.Till date, five different binding sites outside 7TMD in the transmembrane domain have been defined by their crystal structures (Table 2): the pocket outside helices I-III, the pocket outside helices II-IV, the pocket outside helices III-V, the pocket outside helices V-VI, and the pocket outside helices I, VI, and VII (Fig. 20a, c).Allosteric modulator binding to these regions targets class A GPCRs.These sites are typically shallow and not as well surrounded by the 7TM helical bundle as the traditional orthosteric sites.Polar functional groups are commonly found in allosteric modulators at these sites where they anchor themselves to the pocket.Thus, hydrogen atom donor or acceptor groups exposed between the receptor and lipid bilayer are more likely to mediate the binding of such allosteric ligands.These modulators are also required to preserve their overall hydrophobic character to enter the transmembrane domain.][235] 1) TM I-III: P2Y 1 -BPTU Structure To the best of our knowledge, one small molecule allosterically targets this relatively shallow pocket.Because of the flat TM helical bundle surface and relatively narrow cavity of the binding pocket, rational drug design in this area may be challenging.
In this instance, the protein target was the P2Y 1 purinergic receptor.Agonists induce the activation of the P2Y 1 receptor, leading to the potentiation of platelet aggregation that triggers platelet secretion; 236,237 thus, antagonists targeting the P2Y 1 receptor offer a prospective approach to treat thrombosis. 238,239PTU (37) (Fig. 20b), a P2Y 1 antagonist, has been gaining attention as an antithrombotic treatment and is the first allosteric GPCR modulator located outside the helical bundle. 240BPTU blocks the P2Y 1 -induced platelet aggregation with nanomolar potency and presents good selectivity for P2Y 1 receptor and highly homologous P2Y 12 receptor (P2Y 1 K i = 75 nM, P2Y 12 K i > 70 μM). 241he binary complex structure of P2Y 1 − BPTU was determined and showed that the BPTU binding pocket consists mainly of residues in helices I − III (Fig. 21a). 242Notably, two crucial hydrogen bonds were formed between the two NH moieties of the urea group of BPTU and the main-chain carboxyl group of Leu102 2.55 (Fig. 21b).In terms of hydrophobic interactions, the BPTU pyridyl group makes contact with the residues Ala106 2.59 and Phe119 ECL1 .Hydrophobic interactions of the tert-butyl phenyl group occur within a distinct subpocket shaped by helices II and III, including residues Leu102 2.55 , Thr103 2.56 , Met123 3.24 , Leu126 3.27 , and Gln127 3.28 .On the opposite side of the ligand, the trifluoromethoxyphenyl group participated in hydrophobic interactions with Phe62 1.43 and Phe66 1.47 .
This case suggests an effective shape-complementary mechanism for allosteric ligands: bulges on 7TM helices can be utilized as anchors for fixation.In this instance, Pro105 2.58 serves as the corresponding anchor, which is conserved in 74% of nonolfactory class A GPCRs. 243 A comparison between the P2Y 1 receptor bound to the agonist 2MeSADP and the allosteric antagonist BPTU revealed that the BPTU induces a 1.4 Å shift in Tyr100 2.53 toward TM3 (Fig. 21c), preventing the conformational change of Phe131 3.32 . 244Thus, Phe131 3.32 interacts with Phe276 6.51 and limits the TM6 transition, which is required for the activation of class A GPCRs. 245 In addition, MD simulations have demonstrated that the binding of BPTU stabilizes the helical bundle, leading to an increase in lipid order. 246This, in turn, stabilizes the ionic lock formed between Lys46 1.46 and Arg195 ECL2 in the inactive receptor.
2) TM II−IV: CB1R−ORG27569, CB1R − ZCZ011, and PAR2 − AZ3451 Structures Cannabinoid receptors, which consist of two subtypes, CB1R and CB2R, are activated by neurotransmitter endocannabinoids and play key modulatory roles in synaptic transmission. 247,248mong them is the most abundant GPCR in the human brain is CB1R. 249][252][253] ORG27569 (39) (Fig. 20b) is the first and most comprehensively studied NAM of CB1R. 254,255However, when tested in vivo, Org27569 was not sufficiently effective in modulating the effects of orthosteric cannabinoids. 256The co-crystal structure of CB1R and NAM ORG27569, together with its orthosteric CP55940 agonist, has been solved. 257ORG27569 occupied an allosteric site outside helices II and IV in the inner lobe of the phospholipid bilayer (Fig. 22a). 258Specifically, the chloro-indole ring of ORG27569 establishes a key aromatic interaction with the indole group of Trp241 4.50 and buries in a hydrophobic pocket surrounded by His154 2.41 and Val161 2.48 .Cys238 4.47 , Trp241 4.50 , Thr242 4.51 , and Ile245 4.54 supply hydrophobic interactions for the amide-linked piperidinylphenyl chain (Fig. 22b).
The recently developed ZCZ011 (Fig. 20b) is also an indole derivative that exerts PAM and partial agonist effects in vivo assays. 259,260ZCZ011 showed good shape complementarity with the pocket outside 7TMD comprising helices II−IV (Fig. 22a).The indole group in ZCZ011 is anchored by Phe191 3.27 and forms a hydrogen bond with the main chain of Phe191 3.27 while forming π−π stacking interactions with the side chain.(Fig. 22c). 261In addition, the indole group interacted with Leu165 2.52 , Ile169 2.56 , Ile245 4.54 , and Val249 4.58 from TM II and TM IV, respectively.NAM ORG27569 and AZ3451 (38), as well as PAM ZCZ011, bind to the same TM II−IV surface, however, exert opposite allosteric effects. 262Unlike traditional NAMs, ORG27569 enhances the affinity of the agonist though reduces the activity of G i turnover. 263Structurally, the inactivating efficacy of ORG27569 acts by stabilizing the "activation switch" formed by Phe155 2.42 and Phe237 4.46 in CB1R (Fig. 22d). 264,265Furthermore, a hydrogen bond was formed between ORG27569 and Trp241 4.50 , which, together with the hydrogen bond formed between Trp241 4.50 , Ser158 2.45 , and Ser206 3.42 (Fig. 22b), created a polar network that contributed to maintaining the inactive conformation. 266However, the precise mechanism through which ORG27569 augments agonist affinity remains to be elucidated.Upon comparing the CB1 structures bound to the PAM ZCZ011 and the antagonist AM6538, a notable shift of Ile169 2.56 in TM2 towards TM3 was observed.This shift results in the contraction of the receptor"s active site (Fig. 22e), and is believed to be associated with activation. 267,268Additionally, Ser173 2.60 undergoes notable inward movement and forms a hydrogen bond with CP55940, thereby stabilizing the agonist binding.
3) TM III-V: GPR40-compound 1, GPR40-AP8, β2AR-AS408, β2AR-Cmpd-6FA, C5aR1-NDT9513727, C5aR1-avacopan, and DRD1-LY3154207 structures A deep pocket is present outside the transmembrane helices III-V among GPCRs, which allows for the presence of a population of allosteric regulators bound to this pocket.In this case, allosteric agonists and PAMs (i.e., compound 1 (41), AP8 (42), Cmpd-6FA (43), and LY3154207 (47)) localize to regions near ICL2 and stabilize the ICL2 α helix through direct interactions, facilitating the inward movement of Pro ICL2 .This movement leads to a ~3°i nward displacement of TM III, which in turn determines the outward shift of TM V together with TM VI, which is a hallmark of GPCR activation. 269,270Thus, the binding of allosteric modulators increases the proportion of receptors that adopt active conformations, thereby increasing their affinity for agonists.Contrarily, allosteric antagonists and NAMs (i.e., AS408 (44), NDT9513727 (45), and avacopan ( 46)) bind to regions far from ICL2.As these ligands are bound close to the proline kink of TM V, the receptorligand interactions collectively inhibit the interhelical movements and rotations within TM III, TM IV, and TM V, which are required for receptor activation. 271he dopamine D1 receptor is a prototypical example.Dopamine functions as an essential catecholamine neurotransmitter that signals via the dopamine D1 to D5 receptors. 272,273The dopamine D1 receptor (DRD1) regulates neuronal growth, memory, and learning in the central nervous system. 274,275LY3154207 (Fig. 20b) is a selective PAM of the dopamine D1 receptor that improved motor symptoms associated with Lewy body dementia in a 2022 phase 2 clinical trial. 2768][279][280] In both binding modes, LY3154207 was localized at the receptor-lipid bilayer interface surrounded by TM III, TM IV, and ICL2.
In the upright conformation, the tertiary alcohol group of LY3154207 extends toward TM III, establishing a single hydrogen bond with Cys115 3.44 (Fig. 23b).The central tetrahydroisoquinoline ring of LY3154207 engages in extensive hydrophobic interactions   45 .Additionally, the dichlorophenyl group forms a π-cation interaction with the side chain of Arg130 ICL2 .In the boat conformation, the dichlorophenyl group participates in π-cation interactions with the side chains of Arg130 ICL2 , and interacts sandwich-like π-π stacking with Trp123 3.52 (Fig. 23c).The tetrahydroisoquinoline ring of LY3154207 established hydrophobic interactions with neighboring residues, including Met135 ICL2 , Ala139 4.41 , Ile142 4.44 , Leu143 4.45 , and the alkyl chain of Lys134 ICL2 .In addition, two hydrogen bonds were observed between LY3154207 and the polar residues Arg130 ICL2 and Lys138 4.40 .Superposition of the D1R-LY3154207-dopamine and D1R-dopamine structures showed near-identical binding positions for dopamine and the surrounding residues.Instead, LY3154207 interacted with Arg130 ICL2 and Lys134 ICL2 and stabilized ICL2, which interacts directly with G-proteins, thereby increasing the population of D1R adopting active conformations (Fig. 23b, c). 279nother example of an allosteric modulator that binds outside 7TMD formed by helices III-V is the allosteric antagonist avacopan of C5a receptor 1. Human C5a receptor 1 (C5aR1), which binds to the pro-inflammatory mediator C5a, is primarily expressed on the surfaces of various immune cells, such as neutrophils, eosinophils, and dendritic cells. 281,282Overactivation of the C5aR1-C5a axis will cause uncontrolled inflammation; 283 thus, C5aR1 antagonists are ideal candidates for treating various inflammatory conditions, including sepsis COVID-19, etc. [284][285][286] Avacopan (Fig. 20b) is an orally administered allosteric antagonist of C5aR1 approved by the FDA in 2021 to treat severe autoantibody (ANCA)-ANCA-associated vasculitis (granulomatosis with polyangiitis and microscopic polyangiitis). 287,2880][291] The co-crystal structure of C5aR1 was reported with avacopan, highlighting the binding site outside 7TMD between helices III and V (Fig. 24a).The cyclopentane group of avacopan extended into the crevice between helices III and IV and occupied the hydrophobic pocket consisting of Leu125 3.41 , Val159 4.48 , Leu163 4.52 , and Leu167 4.56 (Fig. 24b).The o-methyltrifluoromethylbenzene group exhibited hydrophobic and aromatic interactions mediated by residues Ile124 3.40 , Leu125 3.41 , Leu209 5.45 , Trp213 5.49 , Pro214 5.50 , and Leu218 5.45 in the binding cleft between helices III and V.The m-methylfluorobenzene group lies deeper and forms non-polar interactions with residues Phe135 3.52 , Ile220 5.56 , Cys221 5.57 , and Phe224 5.60 in C5aR1.Only one hydrogen bond was observed between the carbonyl substituent of the amide bond of avacopan and Trp213 5.49 .Additionally, there is a water-mediated polar interaction between avacopan and Thr217 5.53 .
In the inactive C5aR1 structure, Trp213 5.49 undergoes a conformational transition to accommodate avacopan binding, in contrast to the active C5aR1 structure (Fig. 24c).Avacopan may stabilize the conformation of the residues Ile124 3.40 , Pro214 5.50 , and Phe251 6.44 in their inactive states through direct hydrophobic interactions (Fig. 24b).This stabilization, in turn, hinders conformational changes in transmembrane helices TM5 and TM6, which are necessary for receptor activation. 292,293) TM V-VI: CXCR3-SCH546738 structure C-X-C chemokine receptor type 3 (CXCR3), a class A GPCR, is highly expressed on effector T cells and is activated by chemokines CXCL9, CXCL10 and CXCL11.294 Due to the critical role of CXCR3 in type 1 immunity, agonists and antagonists targeting CXCR3 have been synthesized to treat infection, autoimmune diseases, allograft rejection and cancers.[295][296][297] Among these, SCH546738 (48) (Fig. 25c) has shown remarkable efficacy in several preclinical trials by effectively inhibiting the activation of T cell chemotaxis with an affinity of 0.4 nM.298 In the CXCR3-SCH546738 structure, SCH546738 is trapped in a narrow hydrophobic pocket surrounded by TM3, TM5 and TM6 (Fig. 25a).299 The head of SCH546738 is surrounded by a hydrophobic pocket formed by residues Phe135 3.36 , Ala139 3.40 , Phe224 5.47 , Leu228 5.51 , Met231 5.54 , Ile261 6.41 , Ala273 6.53 (Fig. 25b).The tail of SCH546738 stretches out to the lipid bilayer and interacts with Tyr235 5.58 , Leu239 5.62 and Leu258 6.38 .Given the unique allosteric site of SCH546738, the interposition of SCH546738 may weaken the repacking between TM5-TM6, maintaining the receptor in an inactive state.
Comparing the ADO-A1R-G i2 and MIPS521-ADO-A1R-G i2 structures showed that MIPS521 has minimal impact on the binding position of ADO and receptor conformations.Mechanistically, the binding of MIPS521 may stabilize the active conformation by interacting with the allosteric site, which, in turn, promotes the collapse of the Na + pocket (a nearby conserved class A activation motif). 245,306rgeting of GPCR transmembrane domain (inside 7TMD).An empty pocket is present in the middle of the GPCR transmembrane domain that serves as an allosteric site inside 7TMD, as shown in Fig. 27.Allosteric modulators bound to this pocket primarily interact with helices other than TM I and TM IV.To date, a PAM of FFAR3, an allosteric antagonist of CRF1R, an allosteric agonist of PTH1R, and five NAM and one PAM of mGluR5 have been reported to bind to this region (Table 3).In contrast, the binding of NAMs and an allosteric antagonist blocks the outward movement of TM VI, thereby acting as an antagonist.Notably, the binding of only the allosteric agonist stabilizes the G protein through direct interactions.
1) Inside 7TMD: FFAR3-AR420626, CRF1R-CP-376395, mGluR5 -mavoglurant, mGluR5-compound 14, mGluR5-HTL14242, mGluR5-Fenobam, mGluR5-M-MPEP, and PTH1R-PCO371 structures The metabotropic glutamate (mGlu) receptor type 5 is among the eight most widely expressed mGlu receptors in the brain. 3077][318] NAMs of the mGlu5 receptors may serve to normalize excessive glutamate activity without blocking the physiological roles of the brain; thus, they are regarded as prospective agents for the treatment of multiple neurological disorders. 319,320he key binding determinants of these NAMs were identified in TM III, VI, and VII (Fig. 29a).Among these, HTL14242 (57) (Fig. 28) is an advanced and orally active NAM that progresses in early clinical testing for the potential treatment of amyotrophic lateral sclerosis (ALS) (Fig. 29).Crystal structures of mGluR5 with the lead compound of HTL14242, compound 14 (56), demonstrate the underlying basis for SAR optimization.With the pyrimidine linker traversing the narrow channel in the allosteric pocket formed by Tyr659 3.44 , Ser809 7.39 , Val806 7.36 , and Pro655 3.40 , the benzene ring and pyrazole ring of compound 56 respectively sit in two sub-pockets.The presence of the nitrile moiety on the benzene ring is crucial due to its delicate orientation that mediates the formation of hydrogen bonds with Val740 5.40 via the bridging of a water molecule (Fig. 29b).Therefore, removal or replacement of the nitrile moiety caused a substantial drop in affinity, underlying the significance of maintaining this moiety during SAR studies (proved by  29c).Notably, the conformation of the pyrazole end allows slightly bulkier rings and small substitutes, as long as the hydrogen bond between the nitrogen atom and Ser809 7.39 , and the surrounding polar network is maintained (Fig. 29b).Such SBDD analysis thereupon yielded HTL14242, which harbors highly similar binding modes with 56 while simultaneously benefiting from its favorable physicochemical and pharmacokinetic properties (Fig. 29c). 321Trp785 6.50 within the FxxCWxP 6.50 motif serves as a "toggle switch" in class A receptors, undergoing a conformation change during activation. 321,326It is plausible that this residue may also play a role in class C GPCR activation, though further investigation is required to elucidate its involvement. 178Other allosteric inhibitors bound to the transmembrane domain include allosteric PTH1R agonists.9][330] Recently, a highly selective PTH1R agonist, the orally active non-peptidic small molecule PCO371 (54) (Fig. 28), was identified and is currently undergoing phase 1 clinical trials to treat hypoparathyroidism. 331,332PCO371 consists of four chemical modules, from left to right: trifluoromethoxyphenyl, spiro-imidazolone, dimethylphenyl, and dimethylhydantoin. 333he solved crystal structure complex of PTH1R-PCO371 reveals important information regarding the PCO371 binding cavity, 334 which was observed to reside in a pocket surrounded by residues on TM II, TM III, TM VI, and TM VII, analogous to mGluR5 (Fig. 30a). 335,336In the PCO371 binding site, the NH moiety of the spiroimidazolone in PCO371 is engaged in a hydrogen binding interacts with Tyr459 7.57 (Fig. 30b).The carbonyl group of dimethylhydantoin in PCO371 forms a salt bridge interaction with the protonated nitrogen of Arg219 2.46 .In addition to polar contacts, the trifluoromethoxyphenyl group formed nonpolar interactions with Met414 6.46 , Leu416 6.48 , and Phe454 7.52 ; the spiroimidazolone group formed hydrophobic interactions with Leu226 2.53 , Ile299 3.47 , Pro415 6.47 , and Phe417 6.49 ; the dimethylphenyl group formed hydrophobic interactions with His223 2.50 , Leu306 3.54 , Val412 6.44 , Leu413 6.45 and Tyr459 7.57 ; and the dimethylhydantoin group formed hydrophobic interactions with Asn463 8.47 .Notably, Glu394 G.H5.24 in the Gs protein was hydrogenbonded to the carbonyl group of dimethylhydantoin, and Tyr393 G.H5.23 formed a hydrophobic interaction with dimethylhydantoin, thus stabilizing the ternary PCO371-PTH1R-Gs complex.
As the binding of PCO371 precludes the endogenous ligand from occupying the core of the 7TM helical bundle, only structures in which PCO371 alone binds to PTH1R exist.The presence of PCO371 induced inward displacement of the extracellular and cytoplasmic termini of TM6 cells (Fig. 30c).Comparison of the structures of PCO371-PTH1R and CP-376395-CRF1R revealed that PCO371 has a deeper binding site and therefore does not impede the formation of the conserved Pro 6.47 -X-X-Gly 6.50 kink, as CP-376395 does.In addition, the binding site of PCO371 is shallower than that of allosteric modulators that bind to the intracellular surface inside 7TMD and therefore does not impede G-protein or  Targeting of GPCR Intracellular (outside and inside 7TMD).In these instances, allosteric modulators bind to interfaces located between the cytoplasmic terminus of the 7TM helical bundle and downstream transducers, 337 including the outside and inside of 7TMD. 46Till date, only two different binding sites in the intracellular surface have been identified using crystal structures (Table 4), namely the pocket outside helices V − VII and the pocket inside 7TMD (Fig. 31).The reported allosteric sites on the intracellular surface outside 7TMD are all attached to TM VI, located between helices V and VII, suggesting that TM VI plays a functional role in transmitting ligand-binding information to the orthosteric pocket. 338G-protein coupling requires outward motion of the intracellular end of TM VI, a key signature for activating GPCR, 339 which inspired the design of small-molecule allosteric modulators that target this site.
The GABA B receptor, classified as a heterodimeric class C GPCR, comprises two distinct subunits, GB1 and GB2, which can be activated by γ-aminobutyric acid (GABA), a primary inhibitory neurotransmitter in the central nervous system. 340,3413][344] Dimerization of GABA B receptors results in the emergence of this new dimer-specific allosteric binding site (Fig. 33a), [345][346][347][348][349] which prevents GS39783 (69) and BHFF (70) from acting on the monomer, but instead specifically controls GPCR dimer activity.BHFF (Fig. 32), a potent PAM of the GABA B receptor that enhances receptor potency only when activated by orthosteric agonists, lacks efficacy in mouse experiments. 350Determination of the BHFF −GABA B receptor co-crystal structure clearly shows that BHFF is bound between TM V−TM VI of GB1 and TM VI of GB2 within the intracellular tips (Fig. 33a). 351The 3-hydroxy group and ketone of BHFF formed two hydrogen bonds with the Lys792 ICL3 side chain of GB1 (Fig. 33b).For hydrophobic interactions, BHFF is buried in a cavity defined by the residues Ala788 5.58 , Tyr789 5.59 , Met807 6.41 , Tyr810 6.44 of GB1, and Tyr691 6.38 and Met694 6.41 GB2.
Compared with GS39783, another PAM molecule for the GABA B receptor, both PAM molecules bind to highly similar locations within the dimer interface, and the overall complex structure is almost identical.In contrast to the binding of the orthosteric agonist baclofen alone, the PAM induces a straightening and inward shift of TM3 and TM5 in GB2 on the intracellular side and stabilizes the TM6-mediated dimerization.[354]   Compound 2 (68) (Fig. 32) is a ago-PAM of GLP-1R that covalently binds to Cys347 6.36 on helices VI and displays partial and almost full agonism. 355,356clinical trials of compound 2 failed because of its poor pharmacokinetic properties. 357n the co-crystal structure of GLP-1R complexed with compound 2, it was demonstrated that compound 2 was located on the TM VI membrane-facing surface and formed interactions only with residues on TM VI (Fig. 33d), among which a covalent disulfide bond was formed between the sulfonic group of compound 2 and Cys347 6.36 . 358In addition, the tert-butyl group of compound 2 extended toward TM VII and formed nonpolar interactions with Ala350 6.39 and Lys351 6.40 (Fig. 33e).The dichloroquinoxaline group was directed toward ICL3, forming van der Waals contacts with Lys346 6.35 and Cys347 6.36 .
The binding of compound 2 alone, GLP-1 alone, and compound 2 − GLP-1 together all led to a similar extent of outward movement in the intracellular terminus of TM6 (Fig. 33c).Nonetheless, in the compound 2-GLP-1-GLP-1R-G s structure, two additional salt bridge interactions have arisen (R176 2.46 and E408 8.49 , E423 8.64 and R46 in Gβ) (Fig. 33f), offering the potential to strengthen G-protein binding through long-range allosteric communications. 358Furthermore, the N-terminal α-helix of the GLP-1 R extends downward into the orthosteric pocket upon binding to compound 2, which may serve to stabilize the active conformation (Fig. 33c). 3591][362] This suggests that the protruding free cysteine residues in the transmembrane helix provide an opportunity to develop irreversible GPCR allosteric modulators.
4][365] In this case, the protruding TM VI can serve as a base for the attachment of modulators in a clamp shape.The binding of such allosteric modulators requires certain conditions, such as a cleft between helices for the insertion of the pincers and adequate hydrophobic contacts with the surrounding residues for stable binding.The outward displacement within the TM VI required for transducer binding is restricted, preventing receptor activation. 366he glucagon receptor belongs to the class B GPCR and plays a critical role in the maintenance of glucose homeostasis. 367,3680][371] In support of this concept, a novel allosteric antagonist targeting GCGR, MK-0893 (Fig. 32), has advanced into phase 2 clinical studies to treat type 2 diabetes mellitus; 372 however, side effects, including increased LDL-C, have hindered its clinical use. 373he X-ray crystal structure of MK-0893 with GCGR was solved, revealing the atomic details of allosteric modulator binding. 363In this structure, MK-0893 is situated within an intracellular pocket outside helices V-VII (Fig. 34a).In terms of polar interactions, the terminal anionic carboxylic acid moiety of MK-0893 (Fig. 34c) established a salt-bridge interaction with Arg346 6.37 (Fig. 34b). 374t contributes to the establishment of a hydrogen-bonding network involving the side chain of Asn404 7.61 and the mainchain amine of Lys405 7.62 .It also forms a water-mediated hydrogen bond with the side chain of Ser350 6.44 .Additionally, the amide group of the ligand formed hydrogen bonds with the backbone carbonyl groups of Ser350 6.41 , Leu399 7.56 , and the side chain of Lys349 6.40 .Regarding hydrophobic interactions, the Fig. 31 Two intracellular allosteric binding sites in GPCRs and the corresponding small-molecule allosteric modulators.Stick models of smallmolecule ligands are mapped to representative members of outside 7TMD (V−VII) (GCGR, PDB: 5EE7) and inside 7TMD (CCR2, PDB: 5T1A) GPCRs.For each pocket, the number of unique modulators is indicated in boldface type, and the number of GPCRs containing the pocket is provided in parentheses methoxynaphthalene moiety wedges into a cavity formed by helices V and VI and with residues Leu329 5.61 , Phe345 6.36 , Leu352 6.43 and Thr353 6.44 , and the alkyl chain of Lys349 6.40 .The phenylethylpyrazole core scaffold engages in hydrophobic interactions with residues Thr353 6.44 , and Leu399 7.56 , and forms a π −cation interaction with Lys349 6.40 .This structure closely resembles other inactive structures (PDB:5XEZ, 8JRV, and 8JRU) 375 and does not exhibit specific conformations induced by MK-0893.
2) Inside 7TMD: β2AR-Cmp-15PA, CCR2-CCR2-RA-[R], CCR9-vercirnon, CCR7-Cmp2105, CXCR2-00767013, GPR61-Compound 1, and NTSR1-SBI-553 structures In contrast to orthosteric ligands, GPCRs utilize their intracellular surface inside 7TMD to directly mediate downstream signaling via G proteins or arrestins.This site has also been found to act as a binding site for allosteric regulators and is quite similar in all solved complexes (a cavity composed of the cytoplasmic region of helices I, II, VI, and VII) (Fig. 31). 24,376,377Allosteric antagonists bound to this region inhibit GPCR-mediated signaling through a novel dual mechanism, which appears to be a powerful way to antagonize the receptor.These allosteric modulators not only compete with G-proteins or arrestins by occupying their binding sites but also display π-π interactions (parallel or T-shaped) with Tyr 7.53 from the conserved NPxxY motif in TM VII, acting as a molecular glue to hold together the cytoplasmic ends of the helical bundle, further preventing conformational transitions associated with activation.Therefore, targeting this druggable allosteric pocket in receptors using small-molecule allosteric antagonists may provide new opportunities for the discovery of GPCR drugs.
][383] Vercirnon (73) (Fig. 32), an allosteric antagonist of CCR9 used in Crohn's treatment, has progressed to Phase 3 clinical studies.However, its efficacy is limited due to the requirement for significantly high doses to effectively block receptor activation. 384his uncertainty may stem from the fact that drug molecules might not necessarily bind to the intracellular G protein region upon entering the cytoplasm. 271This challenge could be common among this class of modulators, but only vercirnon has entered clinical trials at present.
The binding site of vercirnon has been shown via X-ray crystallography to be the center of the 7TM helical bundle on the intracellular side (Fig. 35a).The sulfone group (Fig. 35c) of vercirnon engages in a trivalent hydrogen bond with the mainchain amino groups of three neighboring residues: Glu322 8.48 , Arg323 8.49 , and Phe324 8.50 . 385Additionally, the pyridine-N-oxide group is hydrogen-bonded to the side chain of Thr81 ICL1 , and the ketone group forms a hydrogen bond with the side chain of Thr256 6.37 (Fig. 35b).Regarding hydrophobic interactions, the tertbutylphenyl group is deeply buried in the hydrophobic pocket surrounded by Val69 1.53 , Val72 1.56 , Tyr73 1.57 , Leu87 2.42 , Tyr317 7.53 , and Phe324 8.50 .The chlorophenyl group was situated within a narrow crevice formed by the hydrophobic parts of residues Leu87 2.43 , Ile140 3.46 , Val259 6.40 , and Tyr317 7.53 .Furthermore, the pyridine-N-oxide group is enclosed within a polar cavity formed by residues Thr81 ICL1 , Thr83 2.39 , Asp84 2.40 , Arg144 3.50 , and Arg323 8. 49 .

CONCLUSIONS AND PERSPECTIVES
7][388][389] Till date, 388 orthosteric modulators, as well as 717 complex structures GPCRs bound to the modulators, have been reported.In addition, 53 different allosteric small-molecule modulators bound to GPCR complex structures and nine different allosteric sites were also solved.In this study, we first review the structure advances, signaling mechanisms, and functional diversity of GPCRs to outline the current profile and upto-date development in this field.Importantly, by means of the aforementioned crystallographic advancements, orthosteric and allosteric modulators are discussed separately in terms of their complex structures, signaling mechanisms, and consequential implications for drug discovery.Such in-depth investigation underlines the significance of understanding GPCR structures and mechanisms for developing effective therapeutics.
Because of the relatively large amount of orthosteric modulators and their shared functional mechanism of competing with the endogenous ligands, representative cases of orthosteic drugs launched within the past five years have been carefully selected and dissected, including μ-OR-oliceridine complex (stand-out for G-protein biased signaling), S1PR-siponimod complex (stand-out for subtype selectivity and "toggle switch" activation mechanism), OX2R-lemborexant complex (stand-out for kinetics and dynamics parameters), 5-HT 1F -lasmiditan complex (distinctive for subtype selectivity), and GnRH1-elagolix complex (distinctive for signal transmission based on atypical receptor structure).Structural analysis revealed that modulators are stabilized within the orthosteric pockets by key polar interactions with residues on the TM bundles, in which residues that are unconserved among the family subtypes are often determinants of selectivity.Furthermore, the "toggle switch", PIF, DRY, and NPxxY motifs are critical mechanical switches that transmit extracellular stimuli to the intracellular regions, and some specific polar interactions between TM3 and TM5/6 may also serve as a boost for the displacement of TM5/6, which is a hallmark of receptor activation/ deactivation.
Since the binding patterns and action mechanisms of allosteric modulators tend to be unique, we placed special emphasis on the depiction of allosteric regulators. 390From the analysis of these structural complexes, the extracellular vestibule inside 7TMD was identified as the most prevalent binding site for allosteric modulators, and induced-fit shape matching and charge matching are the determinants of allosteric ligand accommodation.The binding of allosteric modulators alters the free-energy landscape and stabilizes the different dominant conformations of the receptor. 391Specifically, we established a novel classification of  allosteric modulators into two categories.First, the allosteric effects of the modulators can be realized by directly modulating the binding of orthosteric ligands or intracellular transducer proteins to receptors.In this instance, allosteric modulators may occupy a pocket above the orthosteric binding pocket and interact directly with orthosteric ligands and/or surrounding residues to alter association or dissociation.Allosteric modulators may also occupy the binding pockets of G-proteins and arrestins to impede their binding or occupy the pockets above their binding sites to regulate binding.Second, allosteric modulators regulate the ability of receptor complexes to interact with intracellular transducer proteins by indirectly altering their activation pathways.In this instance, allosteric modulators appear to act as steric wedges, stabilizing or destabilizing certain interactions to restrict or facilitate conformational rearrangements of the receptor.
In addition to the commonalities summarized above, it is of broad pharmaceutical interest to understand how the drug recognition basis and delicate mechanistic regulation of orthosteric/allosteric modulators can expand upon drug design and why we need to pursue this line of research. 117,392,393From a structural and chemical biology perspective, understanding the mechanical switch cascade assists in acquiring insights into fine-tuning regulation and enables human control of different downstream functions of GPCRs. 62Therefore, GPCRs after mutagenesis may be employed as biosensors to initiate distinct intracellular signaling events. 394edicinal chemists can be inspired by five perspectives based on the analysis presented in this review: ][397][398] Concerning with enhancement of affinity and efficacy, designing ligands that constitute "anchors" and "drivers" has been suggested. 399,400The "anchors" represent the primary parts of the ligands that contribute to affinity by binding with utmost residues and remain nearly unchanged during the transition between different states of the receptor.The "drivers" should be designed to form certain interactions with "toggle switches" and thus trigger activation/deactivation signaling.The "anchors" provide a foundation that allows the "drivers" to exert a "pull" and/or "push" action that shifts the receptor population, thereby enhancing efficacy.This "mechanism-based drug design" concept, expanding upon the traditional "structure-based drug design" strategy, may shed light on effective and efficient GPCR drug discovery. 401,402Regarding the improvement of selectivity, although the orthosteric sites are highly conserved in one family, capturing slight differences in loop/TM bundle/key residue conformation from other receptor subtypes and specifically interacting with these hotspots may result in a multifold increase in selectivity, thus alleviating side effects.7][408][409][410][411][412][413][414] Based on the identified allosteric sites, allosteric modulators can be designed using a threedimensional (3D) molecular generation algorithm that uses the topological surface and geometric structure. 415lthough the low conservation of allosteric sites may result in a lack of a general formula concerning the scaffold of allosteric modulators, 416 it can be concluded that while the hydrophobic nature of allosteric ligands will contribute to receptor binding and regulation in most instances, polar groups can also interact, especially when the modulators bind outside 7TMD and require immobilization to the receptor.Moreover, an "allosteric-like" rule condensed by our group (molecular weight ≤600; 2≤ number of rotatable bonds ≤6; number of rings ≤5; number of rings in the largest ring system= 1 or 2, 3≤ SlogP ≤7) can serve as a filter criteria when designing molecules. 4173) With the GPCR-ligand complex structural information in hand, the topic of whether the kinetic and dynamic properties of ligands can also be modified by medicinal chemists is considered. 177,418,419Based on the desirable k on and k off values of lemborexant analyzed above, it is inferred that designing a ligand that can assemble to its receptorbound state in advance may help achieve a high k on value, in which case a simple MD simulation of designed ligands in a solvent can acquire the preferential conformation of the ligand and guide our selection.In contrast, estimating the binding free energy of the designed ligands may help predict and rank their k off values, thus providing valuable guidance for ligand optimization from a kinetic perspective. 420,4213][424][425][426][427][428] With the design of G-proteinbiased modulators of μ-OR as a paradigm, it is suggested that comparison of the biased and unbiased ligands binding modes may help locate the key residues or regions that contribute to biased signaling.A "mechanism-based drug design" that designs ligand forming/avoiding interactions with hotspots may lead to the successful distinguishing of promiscuous signals.3][434][435][436][437] With GPCR bitopic ligands occupying a relatively blank zone, linker optimization while immobilizing fragments at both ends, as well as fixing the orthosteric fragment while attaching a warhead to the allosteric fragment to anchor it in an allosteric pocket containing nucleophilic amino acids, may prove to be promising strategies for exploiting this field.
Despite significant technological advances and studies on GPCR structure and drug discovery, [438][439][440][441][442] obstacles still exist.4][445] For example, modeling from AlphaFold is not precise in residue orientation and can thus mislead the analysis of signaling mechanisms. 446Moreover, MD simulations do not guarantee the identification of novel cryptic allosteric sites in allosteric drug design. 447Therefore, other novel perspectives such as sequence, coarse-grained topology, 448 and evolution are urgently required and may alleviate structural dependencies. 449One possibility is the use of available large data of sequences and end-to-end concepts in deep learning to develop "sequence-to-mechanism" or "sequence-to-drug discovery" methodologies, 450 which can help avoid error accumulation from various models if the experimental strategies fail to provide the crystal structures.
In summary, we are at a stage of in-depth research on the orthosteric and allosteric modulation of GPCRs.Although our understanding of drug-target interactions, binding hotspots, and mechanisms for small-molecule modulators has become increasingly clear, some aspects still require further study.The present study contributes to a better understanding of the ligand recognition and regulatory mechanisms of GPCRs.Furthermore, by proposing a novel classification of the mechanism of allosteric modulators and an innovative concept of "mechanism-based drug discovery," we aim to outline the latest landscape of GPCRs and interest researchers to facilitate this field.More effective, selective, and safer small-molecule therapeutics for GPCRs should be the focus of future studies in this field.

Fig. 4 a
Fig. 4 a Categories of Representative human diseases caused by GPCR dysfunctions.b Classification of the effects of mutations on GPCR dysfunctions

Fig. 18 a
Fig. 18 a Schematic representation of PAM cinacalcet bound to CaSR (PDB: 7M3F).b Detailed binding modes of CaSR bound to cinacalcet in extended conformations.c Detailed binding modes of CaSR bound to cinacalcet in bent conformations.Hydrogen bonds are presented as orange dashes and π-π stackings are presented as gray dashes

Fig. 20 a
Fig. 20 a Five allosteric binding sites in the transmembrane domain outside 7TMD of GPCRs and the corresponding small-molecule allosteric modulators.Stick models of small-molecule ligands are mapped to representative members of outside 7TMD (I-III) (P2Y 1 , PDB: 4XNV), outside 7TMD (II-IV) (CB1R, PDB: 6KQI), outside 7TMD (III and V) (C5aR1, PDB: 6C1R), outside 7TMD (V and VI) (CXCR3, PDB: 8HNN), and outside 7TMD (I, VI, and VII) (A1R, PDB: 7LD3) GPCRs.For each pocket, the number of unique modulators is indicated in boldface type, and the number of GPCRs containing the pocket is provided in parentheses.b 2D chemical structures of synthetic small-molecule allosteric ligands targeting the transmembrane domain outside 7TMD of GPCRs.c Extracellular view of the five allosteric sites

Fig. 23 a
Fig. 23 a Superposition of the cocrystal structures of PAM LY3154207 bound to dopamine D1 receptor in upright (pink, PDB: 7CKZ) and boat (yellow, PDB: 7LJC and 7X2F) conformations.b Detailed binding modes of dopamine D1 receptor binding to LY3154207 in upright conformations.c Detailed binding modes of dopamine D1 receptor binding to LY3154207 in boat conformations.Hydrogen bonds are presented as orange dashes; π-π stacking and π-cation stacking interactions are presented as gray dashes

Fig. 26 a
Fig. 26 a Schematic representation of PAM MIPS521 bound to A1R (PDB code 7LD3).b Detailed binding modes of A1R binding to MIPS521.Hydrogen bonds are presented as orange dashes.c 2D structure of small-molecule allosteric ligand MIPS521 provided for clarity

Fig. 27
Fig. 27 Allosteric binding sites in the transmembrane domain within 7TMD of GPCRs and corresponding small-molecule allosteric modulators.Stick models of small-molecule ligands are mapped to representative member CRF1R, PDB: 4K5Y.The number of unique modulators is indicated in boldface type, and the number of GPCRs containing the pocket is provided in parentheses

Fig. 28
Fig. 28 2D chemical structures of synthetic small-molecule allosteric ligands targeting the transmembrane domain within 7TMD of GPCRs

Fig. 30 a
Fig. 30 a Schematic representation of the allosteric agonist PCO371 bound to PTH1R (PDB: 8GW8).b Detailed binding modes of PTH1R binding to PCO371.Hydrogen bonds are presented as orange dashes.c Superposition of the PTH1R bound to PCO371 (displayed in pink) and PTH (displayed in blue) reveals conformational changes upon PCO371 binding

Fig. 34 aFig. 35 a
Fig. 34 a Schematic representation of the allosteric antagonist MK-0893 bound to GCGR (PDB: 5EE7).b Detailed modes of GCGR binding to MK-0893.Hydrogen bonds are presented orange dashes.c 2D structure of small-molecule allosteric ligand MK-0893 shown for clarity

b
Classification of the effects of mutations on GPCR dysfunctions

Table 1 .
Solved GPCR structures complexed with synthetic allosteric modulators bound to the extracellular vestibule 2 R

Table 2 .
Solved GPCR structures complexed with synthetic allosteric modulators bound to the transmembrane domain outside 7TMD

Table 3 .
Solved GPCR structures complexed with synthetic allosteric modulators bound to the transmembrane domain inside 7TMD Till date, PCO371 is probably the only receptor allosteric agonist reported to stabilize the active state through direct interactions with downstream transducers.

Table 4 .
Solved GPCR structures complexed with synthetic allosteric modulators bound to the intracellular surface