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G-Protein-Coupled ReceptorsDr. Prashant ShuklaJunior ResidentDept of Pharmacology

2ContentsIntroductionHistorical Background GPCR - Basics GPCR as Targets for Drug DesigningGPCR associated DiseasesConcept of Orphan GPCRFuture Prospects & Conclusions

Introduction 3Receptors are the sensing elements in the system of chemical communications that coordinates the function of all the different cells in the body.

The chemical messengers can be hormones, transmitters and other mediators.

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Types of receptors

The G protein-coupled receptor (GPCR) superfamily comprises the largest and most diverse group of proteins in mammals.

Synonym: seven-transmembrane (7-TM), serpentine receptors, heptahelical receptors,serpentine receptor, andG proteinlinked receptors(GPLR).

The human genome encodes >800 GPCRs.5GPCRs

GPCRsIt is involved in information transfer (signal transduction) from outside the cell to the cellular interior.

GPCRs are responsible for every aspect of human biology from vision, taste, sense of smell, sympathetic and parasympathetic nervous functions, metabolism, and immune regulation to reproduction.

~45% of all pharmaceutical drugs are known to target GPCRs.6

Receptors associated with GPCRs1. GABAB ReceptorsGABABR1 and GABABR2 2. Taste ReceptorsT1R3 and T1R23. Adrenergic ReceptorsThree subfamilies ( 1, 2 and ) Family A (rhodopsin-like) GPCRs4. Opioid ReceptorsThree cloned subtypes: , and

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Receptors associated with GPCRs5. Somatostatin ReceptorsFive subtypes (SSTR1-5) 6. Purinergic Receptors Neurotransmitters in the CNS, CVS, immune system, and other tissues i.e. adenosine and ATP 7. Olfactory ReceptorsRepresent the largest family of GPCRs, with >300 members8. Vasopressin, Oxytocin and Other Receptors

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The importance of GPCRsNumber (C.elegans 1100; H. sapiens, ~1000; D. melanogaster, 160; reflects number of olfactory receptor genes in worm [~1000] and mammal [several hundreds]), a few % of genome; 300-400 non-olfactory GPCRs)

Diversity (mostly small molecule ligands)

Evolutionarily conserved yeast to man (yeast Ga 45% identical to mammalian Gia)

Pharmaceutical importance: ~500 known molecular targets of drugs, 60% of these are cell surface receptors, 75% of these are GPCRs (GPCRs = ~45% of all known drug targets)

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Historical background10

Robert Lefkowitzand Brian Kobilka: the 2012 Nobel Prize in Chemistry for groundbreaking discoveries that reveal the inner workings of an important family of receptors: G-proteincoupled receptors.

11Historical background

12Common Experimental Tools used to Study GPCRs

GPCR- basics13Structure ClassificationSignal molecules/ LigandsPhysiological roleG proteinsMechanism of action

GPCR- Basic structure

CYTOSOLEXTRACELLULAR

14Extracellular loopsIntracellular loopsPlasmamembrane

15GPCR- Basic structure

GPCR: Classification Based on Sequence homology and functional similarity Class A (or 1) (Rhodopsin-like)Class B (or 2) (Secretin receptor family)Class C(or 3) (Metabotropic glutamate/ pheromone)Class D (or 4) (Fungal mating pheromone receptors)Class E (or 5) (Cyclic AMP receptors)Class F (or 6) (Frizzled/Smoothened)16

Based on phylogenetic origin: TheGRAFSclassification system has been proposedGlutamateRhodopsin Adhesion Frizzled/TasteSecretin17GPCR: Classification

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GPCR Tree

Signal molecules/ Ligands of GPCRsGPCRs interact with a number of ligands ranging from photons, ions, amino acids, odorants, pheromones, eicosanoids, neurotransmitters, peptides, proteins, and hormones.

Nevertheless, for the majority of GPCRs, the identity of their natural ligands is still unknown, hence remain orphan receptors.19

Signal molecules20Biogenic amines: Adrenaline, noradrenaline, dopamine, 5-HT, histamine, acetylcholine

Amino acids and ions: Glutamate, Ca2+, GABA

Lipids : PAF, prostaglandins, leukotrienes, anandamine

21Peptides / proteins : GnRH, angiotensin, bradykinin, thrombin, bombesin, glucagon, calcitonin, vasoactive intestinal peptides, PTH, FSH, LH, TSH

Nucleotides : Adenosine nucleotides, adenine nucleotides, uridine nucleotides

Others : Light, odorants, pheromones, opiatesSignal molecules

Physiological roles22Visual sense: RhodopsinSense of smell: Olfactory receptorBehavioral and mood regulation: Serotonin, dopamine, GABA and glutamateImmune system activity and inflammation: Chemokine receptors, histamine receptors ANS transmission: adrenergic receptorsApoptosis

Structure of G ProteinG proteins, also known asguanine nucleotide-binding proteins,involved in transmitting signals and function as molecular switches.

Their activity is regulated by factors that control their ability to bind to and hydrolyze GTP toGDP. When they bind GTP, they are 'on', and, when they bind GDP, they are 'off '.

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G protein complexes are made up of20 alpha () 6 beta () 12 gamma ()subunits.

Beta and gamma subunits can form a stable dimeric complex referred to as thebeta-gamma complex.

subunit subunit subunit24

Types of G Proteins25

G protein cycle26

Basal stateActivated state

GPC ReceptorsG ProteinReceptorsSignaling Pathway GSBeta adrenergic receptors, glucagon, histamine, serotonin Increase CAMPExcitatory effectsGiAlpha2 adrenergic receptors, mAchR, opioid, serotonin Decrease CAMP Cardiac K+ channel open- decrease heart rateGqmAchR, H1, 1, Vasopressin type 1, 5HT1CPLC- IP3 , DAGIncrease Cytoplasmic CaGtRhodopsin and colour opsins in retinal rod and cone cellsIncrease cGMP phosphodiesterase.Decrease cGMP

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G Protein Mediated PathwaysSecondary messenger Systems Involved In Signal Transduction:Adenylate cyclase cAMP mediated pathway Phospholipase mediated pathway

GPCR s can also directly activate the ion channels

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cAMP Mediated Pathway

ThecAMP-dependent pathway, also known as theadenylyl cyclasepathway, is aG protein-coupled receptor triggeredsignaling cascade used incell communication.

Gs cAMP Dependent Pathway

Gi cAMP Dependent Pathway 29

GTPGDP GDPGTP

ATP

cAMP

Cell responseATProtein kinase

ADP

PInactive proteinActive protein

hormoneAdenylate cyclase Signaling SystemACRSInhibitorRi

CYTOSOLEXTRACELLULAR30Gs cAMP Dependent Pathway

Gi cAMP Dependent Pathway 31

32CYTOSOLEXTRACELLULARGq Protein Coupled Receptor

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Gt PCR: involved in photo transduction.

Gt Protein Coupled Receptor

Signal Amplification through G proteins 34

Regulation of GPCRs Turning GPCRs Off A cell must also be able to stop responding to protect overstimulation

High activation of a receptor leads to a reduced ability to be stimulated in the future (desensitization)

Can also significantly limit therapeutic usefulness of many receptor agonists.

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36Desensitization mechanisms includedown-regulation or reduction of receptor number

sequestration or apparent shielding of the receptors from interacting ligands

uncoupling from G-proteins.Regulation of GPCRs

Homologous desensitization: The activation dependent regulation of receptors.

Heterologous desensitization: Receptor activation-independent regulation of receptors.37Regulation of GPCRs

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Homologous desensitizationThe activated state of GPCRs serves not only as an activator of G proteins, but also as the substrate for GPCR kinases (GRKs).

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41Homologous desensitization

Based on feedback regulation of receptors by the second-messenger-regulated kinases.

Eg. Upon stimulation, - receptors leads to cAMP, which activates PKA. PKA can then phosphorylate the - receptors themselves, even those particular receptor proteins that were not activated by the current stimulation. These PKA-phosphorylated receptors are less able to mount a response. 42Heterologous desensitization

ReceptorDrugs and some key indicationsAT1 angiotensin II receptor Antagonists e.g. losartan in treatment of HT or CHF1A-c receptor Antagonists e.g. tamsulosin to treat disorders asso. with enlarged prostate1- receptorAntagonists e.g. propranolol, atenolol, metoprolol, carvedilol to treat essential HT or CHF2- receptorAgonists e.g. terbutaline, salbutamol, formoterol for treatment of COPD or Bronchial asthma D2 receptorAntagonists e.g. Haloperidol & clozapine to treat schizophreniaAgonists e.g. levodopa for Parkinsonism

43GPCR as drug targets

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44ReceptorDrugs and some key indicationsD3 receptor Antagonists e.g. haloperidol in schizophrenia5-HT2A receptor Antagonists e.g. clozapine for schizophrenia.Indirect agonists e.g. fluvoxamine for depression5-HT2C receptor Antagonists e.g. clozapine for schizophreniaCCR5 Associated with progression of AIDS e.g. Aplaviroc and maraviroc M3Antagonists e.g. Atropine to dilate pupil; Scopolamine for motion sicknessNeuropeptide S receptor Asthma susceptibility e.g. Neuromedin and neurotensinP2Y12 Associated with bleeding diathesis e.g. Clopidogrel

GPCR as drug targets

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Diseases associated with G-proteinsAbnormal G protein signalling can result by

Bacterial toxins (Cholera and pertussis)

Gene mutationsLoss of function mutationsGain of function mutations

Altered GPCR folding

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Mutations in GPCRMutations in genes encoding are an important cause of human diseaseHelp to define critical structure-function relationships

Two types Loss-of-function : Block signalling in response to the corresponding agonist(s)Hormone resistance, mimicking hormone deficiency

Gain-of-function : Lead to constitutive, agonist-independent activation of signalingMimic states of hormone excess47

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Receptor Disease Inheritance Cone opsinsColour blindnessX-linked, AR RhodopsinRetinitis pigmentosaAD; AR V2 vasopressinDiabetes insipidusX-Linked ACTHFamilial ACTH resistanceAR LH pseudohermaphroditeAR TSHCong. hypothyroidismAR TRHCentral hypothyroidismAR

Diseases caused by Loss of function Mutation48

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49Receptor Disease Inheritance FSHHypergonadotropic Ovarian failureAR Ca2+ sensingHypocalciuric hypercalcaemiaAD Ca2+ sensingNeonatal hyperthyroidismAR GHRHG H deficiencyAR GnRHCentral hypogonadismAR Endothelin-BHirschsprung disease Complex Melanocortin 4Extreme obesityCo-dominant PTH/PTHrPChondrodysplasiaAR

Diseases caused by Loss of function Mutation

50Receptor Disease Inheritance RhodopsinCongenital night blindnessAD LHFamilial precocious pubertyAD LHSporadic Leydig cells tumoursSomatic TSHFamilial non-autoimmune hyperthyroidismAD TSHSporadic hyperfunctionalthyroid adenomasSomatic Ca2+ sensingFamilial hypocalcaemiaAD PTH/PTHrPJansen metaphyseal chondrodysplasiaAD

Diseases caused by Gain of function Mutation

Mis-folded GPCRs Point mutations resulting in protein sequence variations may result in production of mis-folded and disease-causing proteins

Retain proper function but end up in parts of cell where function is inappropriate, or even deleterious, to cell function. 51

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Disease/Abnormality GPCR Pharmacoperones

Retinitis pigmentosa Rhodopsin 9-cis-retinal, 11-cis-retinal, 11-cis-7-ring retinal Nephrogenic diabetes insipidus V2R SR121463 (satavaptan), SR49059 (relcovaptan), VPA-985, YM087, OPC41061 (tolvaptan), OPC31260 Hypogonadotropic hypogonadism GnRHR Indoles, quinolones, erythromycin-derived macrolides Familial hypocalciuric hypercalcemia Ca2+ sensingNPS R-568

Diseases due to GPCR misfolding52

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POLYMORPHISMS OF GPCRVariations in GPCR gene sequence can have important consequences beyond causing Mendelian diseases

As more polymorphisms are discovered more examples of variations in GPCR gene sequence will be found53

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POLYMORPHISMS.... Challenges Ahead Whether such differences are important in individual variation in drug response (pharmacogenomics)

Whether they could confer susceptibility to disease.54

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Allosteric Modulators of G-proteinBind receptor domains topographically distinct from orthosteric site, altering biological activity of orthosteric ligand by changing its binding affinity, functional efficacy or both. Potential for engendering greater GPCR subtype-selectivity Challenge for detecting /validating allosteric behaviors Contribute to physical or pathophysiological processes.

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ORPHAN GPCRs Lack their pharmacological identitiesPre-genome era: Most GPCRs were found by sequence similarity using nucleic acid-based homology screening approachesAfter genome sequencing: 150 Orphan GPCRs using bio-informatic analysisFirst Orphan GPCR was G21, later found to be 5HT1A receptor in 1988

Focus of intense research effort, both in academia and in industry56

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57GPCR typesNo. of membersOrphan receptorsGlutamate-class GPCRs 22Two third (15)Rhodopsin-class GPCRs 70163Adhesion-class GPCRs 33MajorityFrizzled/ taste GPCRs36 (11 frizzled and 25 taste)None among frizzled ; Most tasteSecretin-class GPCRs15None

The de-Orphanization of GPCRsEvolutionarily conserved and thus are expected to be active Reverse Pharmacological Approaches based on receptor reactivity & receptor binding are appliedIsolating natural ligand provides a first hint of function, structural cues for lead design Once de-orphanised, GPCRs can be used for designing new drugs.

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Tools for de-orphanization High -throughput screening GPCR over expressing cells

Ligand libraries: chemicals, serum, peptides

Finding a robust marker: Measure receptor binding or Receptor reactivity

Finding an endogenous ligand

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Search available orphans that have an effect on a specific therapeutic area

Analyze Orphans using: 1. Laser capture micro-dissection to determine the localization2. Microarray to compare the level of transcript expression

Screening against Compound Libraries

Identify compound hits and optimize for pre-clinical and, if successful, clinical trials

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Issues of Orphan GPCR research Deorphanization is a risky, lengthy and demanding endeavourGPCRs exist not only as monomers but as dimers or higher oligomersConcentration of transmitters in their natural environment.

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GPCR ScreeningCell-based screens performed with calcium-sensitive or membrane-potential-sensitive dyes

Gs- and Gi-coupled GPCRs are assayed via cAMP determinations using either a cell-based real time cAMP assay or other validated cAMP assay platform

All screens include positive controls and a comprehensive report. 62

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Recent developmentsLigand-induced selective signaling (LiSS): It states that different ligands selectively recruit different intracellular signaling proteins to produce different phenotypic effects in cells .

Terry Kenakin proposed this concept and is rapidly becoming a generic theme for GPCRs.

This phenomenon is referred to by different groups using a variety of terms such as: functional selectivity, biased agonism, ligand-selective agonism, agonist-directed trafficking of signaling, or agonist-receptor trafficking. 63

It has important implications in specific drug development and in minimizing side effects. E.g. the effects of the two naturally occurring GnRHs, GnRH I and GnRH II, operating through the single GnRH type I receptor. GnRH I is much more potent in generating inositol phosphate than in its antiproliferative effects on certain cells, whereas GnRH II does not show much difference between these two effects. An extreme example is a GnRH antagonist, which has no intrinsic stimulation of inositol phosphate generation but has potent antiproliferative activity. It has been shown that the Tyr8of GnRH II is the main determinant of selective antiproliferative effects and identified residues in the TM domains and ECLs of the GnRH receptor that determine selectivity for ligand binding.

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The LiSS concept has now been demonstrated for many GPCRs and is creating a new level of sophistication, which challenges the dogma that ligand engagement of a GPCR consistently elicits a specific intracellular signal. Instead, it has become increasingly clear that the nature of the ligand and the dynamically changing intracellular environment alter the flavor of the signaling. Indeed, it appears that there is a new era of drug discovery on our doorstep, in which screening for novel ligands will not simply involve receptor binding and/or the most convenient high-throughput functional signal output but instead will screen for the appropriate intracellular signal, which reflects the desired phenotypic response of a cell for a disease state or pathophysiology. Equally, appropriate cells will have to be used to ensure an appropriate intracellular context. Although these challenges are substantial, we believe they will bear fruit in the longer term efforts of GPCR drug discovery in the spin-off benefits of reduced failure in the clinic through lack of specificity and off-target effects.

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GPCR signaling independent of G proteinsThere are many ways in which GPCRs can signal independently of G proteins. So, a case has been made for abandoning the term G protein-coupled receptors and referring to them as seven-transmembrane receptors.

The first convincing evidence for the existence of GPCR-independent signaling came from the works of Lefkowitz.67

An example is angiotensin II at its AT1receptor activating both -arrestin and G proteins. When antagonists such as angiotensin II-receptor blockers (losartan and valsartan) engage the binding site, neither signal is propagated. However, another type of antagonist (SII) does not activate the G protein pathway but exclusively recruits -arrestin and activates ERK. 68GPCR signaling independent of G proteins

Constitutively active receptorsG-protein-coupled receptors may also be constitutively (i.e. spontaneously) active in the absence of any agonist.

This was first shown for -adrenoceptor.Histamine H3 receptor also shows constitutive activity.

It means that inverse agonists can play a role here.69

GPCRs and drug discoveryRegarded as Drug Discovery Engines of 21st Century

G protein-coupled receptors (GPCRs) represent 50-60% of the current drug targets.

The pace of GPCR-targeted new molecular entities (NMEs) approved by the USFDA in the recent years still remains to a level near its historical average, with five in 2010, five in 2011, seven in 2012, six in 2013, and eight in 2014. 70

Novel pancreatic -cell GPCRsAbout 20 GPCRs have been found in pancreatic -cells, all of which can potentially stimulate or inhibit insulin secretion. The glucagon-like peptide 1 (GLP1) receptor is one of these. Insulin secretion is stimulated by glucose transport through the glucose transporter 2 into the -cell.

Activation of GPCRs such as GLP1 can enhance the amount of intracellular calcium, for example through activation of Gq/11and subsequent generation of IP3 and release of Ca++ from intracellular stores, thereby potentiating glucose stimulation of insulin secretion.

Among the other GPCRs identified in -cells are the newly discovered free fatty acid receptors, GPR40, 43, and 41. GPR40 couples to Gq/11, so free fatty acid would enhance the calcium response of the -cell to glucose and increase insulin secretion. Insulin responses to glucose are improved in mutant mice overexpressing the GPR40 receptor and in normal rats treated with GRP40 agonists . 71

GPCRs as new therapeutic targets for type 2 diabetesGPCRs that have received recent attention in the field of diabetes therapeutics includeIncretin receptors: GLP1R, GIPR (GPR119)

Free fatty acid receptor:FFAR1 (GPR40), FFAR4 (GPR120)

Bile acid receptor: GPBAR1 (TGR5)72

Novel neuroendocrine GPCRs regulating reproductionThere have been a number of breakthroughs in neuroendocrinology in the last year.

After the seminal discovery that kisspeptin/GPR54 acts as a major whole-body sensor mediating diverse effects on the GnRH neuron described mutations in neurokinin B (NKB) and its receptor (TACR3), which give rise to hypogonadotropic hypogonadism and pubertal failure.

The discovery of NKB, dynorphin A, and GnIH as neuroendocrine regulators has provided new opportunities for research on novel GPCRs in fine tuning the hypothalamic-pituitary-gonadal axis and provides new pathways in which to interrogate feedback mechanisms and metabolic, photoperiod, and behavioral influences on the reproductive system.73

Role of H4 receptor in asthmaH4 receptor was discovered with an orphan GPCR gene sequence followed by pharmacology characterization.

Animal models suggested a role for the H4 receptor in mediating asthma and chronic pruritus associated with conditions such as atopic dermatitis.

TheH4 antagonist JNJ 39758979 has recently been found to have efficacy in preclinical models of pruritus, dermatitis, asthma, and arthritis. Several other H4 antagonists have also been entered into clinical trials for these indications. 74

Concept of pharmacopherones Pharmacoperones or Chaperone

Small nonpeptide molecules do scaffolding in order to promote correct folding.

Regulation of routing of cellular proteins will provide opportunity for novel drug development. 75

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Permeate plasma membrane Enter cells

Correct folding Allowing routing to plasma membrane How Chaperones Work ?

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Bind selectively to misfolded proteins

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Deorphanisation of GPR55GPR55 has been recently deorphanized to be a receptor for lysophophatidylinositol. Other GPR55 ligands identified so far are neither cannabinoids nor bind to the cannabinoid CB1 and CB2 receptors.

GPR55 has been implicated in three therapeutic areas, including the regulation of energy intake and expenditure, resorption of bone, and agonist pro- carcinogensis.77

The simple dogma that underpins much of our current understanding of GPCRs, namely,

one GPCR gene one GPCR protein one functional GPCR one G protein one response

is showing distinct signs of wear.78Future Prospects & Conclusions

Future Prospects & ConclusionsEver expanding field of research

Concept is diverting from a linear signaling to increasingly complex signaling networks

Next generation platforms for studying internalisation and heterodimerisation is to adopt novel, universal -arrestin recruitment assays for known and orphan GPCRs instead of second messenger signaling assays 79

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Further studies are needed to explore Advances in novel formsMethods to rescue function of misfolded or truncated GPCRs

Complexity demands collaborative approaches between persons of medicinal chemistry, analytical pharmacologists & bioinformatic experts

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GPCRs were once considered highly tractable targets.

Current targets much lower success ratesLow hanging fruit largely pickedLack of HitsHits have high molecular weightPoor PK/in vivo activityDifficult to optimize81Future Prospects & Conclusions

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