G- Protein Coupled Receptors

GPCRs are the most dynamic and most abundant all the receptors. The G protein-coupled receptor (GPCR) superfamily comprises the largest and most diverse group of proteins in mammals. 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. GPCRs 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.
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.

1. G-Protein-Coupled Receptors Dr. Prashant Shukla Junior Resident Dept of Pharmacology

2. 2 Contents Introduction Historical Background GPCR - Basics GPCR as Targets for Drug Designing GPCR associated Diseases Concept of Orphan GPCR Future Prospects & Conclusions

3. Introduction 3 Receptors 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.

4. 4 Types of receptors

5. 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, and G protein–linked receptors (GPLR). The human genome encodes >800 GPCRs. 5 GPCRs

6. GPCRs  It 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

7. Receptors associated with GPCRs 1. GABA B Receptors GABA B R 1 and GABA B R 2 2. Taste Receptors T 1 R 3 and T 1 R 2 3. Adrenergic Receptors Three subfamilies ( α 1 , α 2 and β ) Family A ( rhodopsin -like) GPCRs 4. Opioid Receptors Three cloned subtypes: δ , қ and μ 7

8. Receptors associated with GPCRs 5. Somatostatin Receptors Five subtypes (SSTR 1-5 ) 6. Purinergic Receptors Neurotransmitters in the CNS, CVS, immune system, and other tissues i.e. adenosine and ATP 7. Olfactory Receptors Represent the largest family of GPCRs, with >300 members 8. Vasopressin, Oxytocin and Other Receptors 8

9. The importance of GPCRs Number ( 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) 9

10. Historical background 10

11. Robert Lefkowitz   and Brian Kobilka : the 2012 Nobel Prize in Chemistry for groundbreaking discoveries that reveal the inner workings of an important family of receptors: G-protein–coupled receptors . 11 Historical background

12. 12 Common Experimental Tools used to Study GPCRs

13. GPCR- basics 13 Structure Classification Signal molecules/ Ligands Physiological role G proteins Mechanism of action

14. GPCR- Basic structure CYTOSOL EXTRACELLULAR 14 Extracellular loops Intracellular loops Plasma membrane

15. 15 GPCR- Basic structure

16. 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

17. Based on phylogenetic origin: The  GRAFS classification system has been proposed  Glutamate Rhodopsin Adhesion Frizzled/Taste Secretin 17 GPCR: Classification

18. 18 GPCR Tree

19. Signal molecules/ Ligands of GPCRs GPCRs 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

20. Signal molecules 20 Biogenic amines : Adrenaline, noradrenaline , dopamine, 5-HT, histamine, acetylcholine Amino acids and ions : Glutamate, Ca2+, GABA Lipids : PAF, prostaglandins, leukotrienes, anandamine

21. 21 Peptides / 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, opiates Signal molecules…

22. Physiological roles 22 Visual sense : Rhodopsin Sense of smell : Olfactory receptor Behavioral and mood regulation : Serotonin, dopamine, GABA and glutamate Immune system activity and inflammation : Chemokine receptors, histamine receptors ANS transmission : β adrenergic receptors Apoptosis

23. Structure of G Protein G proteins , also known as  guanine 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 to GDP. When they bind GTP, they are 'on', and, when they bind GDP, they are 'off ' . 23

24. G protein complexes are m ade up of  20 alpha (α) 6 beta (β) 12 gamma (γ)  subunits . Beta and gamma subunits can form a stable dimeric complex referred to as the  beta-gamma complex. α subunit β subunit γ subunit 24

25. Types of G Proteins 25

26. G protein cycle 26 Basal state Activated state

27. GPC Receptors G Protein Receptors Signaling Pathway G S Beta adrenergic receptors, glucagon, histamine, serotonin Increase CAMP Excitatory effects G i Alpha 2 adrenergic receptors, mAchR , opioid , serotonin Decrease CAMP Cardiac K + channel open- decrease heart rate G q mAchR , H1, α 1, Vasopressin type 1, 5HT 1C PLC- IP 3 , DAG Increase Cytoplasmic Ca G t Rhodopsin and colour opsins in retinal rod and cone cells Increase cGMP phosphodiesterase . Decrease cGMP 27

28. G Protein Mediated Pathways Secondary messenger Systems Involved In Signal Transduction: Adenylate cyclase cAMP mediated pathway P hospholipase mediated pathway GPCR s can also directly activate the ion channels 28

29. cAMP Mediated P athway T he   cAMP -dependent pathway , also known as the  adenylyl cyclase  pathway , is a G protein-coupled receptor triggered  signaling cascade used in cell communication . G s cAMP Dependent Pathway G i cAMP Dependent Pathway 29

30. GTP GDP  GDP GTP  ATP cAMP Cell response AT Protein kinase ADP P Inactive protein Active protein hormone Adenylate cyclase Signaling System AC R S Inhibitor R i   CYTOSOL EXTRACELLULAR 30 G s cAMP Dependent Pathway

31. G i cAMP Dependent Pathway 31

32. 32 CYTOSOL EXTRACELLULAR Gq Protein Coupled Receptor

33. 33 Gt PCR: involved in photo transduction. Gt Protein Coupled Receptor

34. Signal Amplification through G proteins 34

35. 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. 35

36. 36 Desensitization mechanisms include “down-regulation” or reduction of receptor number “sequestration” or apparent shielding of the receptors from interacting ligands “uncoupling” from G-proteins. Regulation of GPCRs

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

38. 38

39. Homologous desensitization The activated state of GPCRs serves not only as an activator of G proteins, but also as the substrate for GPCR kinases (GRKs). 39

40. 40

41. 41 Homologous desensitization

42. Based on feedback regulation of receptors by the second-messenger-regulated kinases . Eg . Upon stimulation , β- receptors leads to ↑ cAMP , which activates PKA. PKA can then phosphorylate the β- 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. 42 Heterologous desensitization

43. Receptor Drugs and some key indications AT 1 angiotensin II receptor Antagonists e.g . losartan in treatment of HT or CHF α 1 A -c receptor Antagonists e.g . tamsulosin to treat disorders asso . with enlarged prostate β 1 - receptor Antagonists e.g . propranolol, atenolol , metoprolol , carvedilol to treat essential HT or CHF β 2 - receptor Agonists e.g . terbutaline , salbutamol , formoterol for treatment of COPD or Bronchial asthma D 2 receptor Antagonists e.g. Haloperidol & clozapine to treat schizophrenia Agonists e.g. levodopa for Parkinsonism 43 GPCR as drug targets

44. 44 Receptor Drugs and some key indications D 3 receptor Antagonists e.g . haloperidol in schizophrenia 5-HT 2A receptor Antagonists e.g . clozapine for schizophrenia. Indirect agonists e.g . fluvoxamine for depression 5-HT 2C receptor Antagonists e.g . clozapine for schizophrenia CCR 5 Associated with progression of AIDS e.g. Aplaviroc and maraviroc M 3 Antagonists e.g. Atropine to dilate pupil; Scopolamine for motion sickness Neuropeptide S receptor Asthma susceptibility e.g. Neuromedin and neurotensin P2Y 12 Associated with bleeding diathesis e.g. Clopidogrel GPCR as drug targets…

45. 45 GPCR as drug targets…

46. Diseases associated with G-proteins Abnormal G protein signalling can result by Bacterial toxins (Cholera and pertussis ) Gene mutations Loss of function mutations Gain of function mutations Altered GPCR folding 46

47. Mutations in GPCR Mutations in genes encoding are an important cause of human disease Help 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 signaling Mimic states of hormone excess 47

48. Receptor Disease Inheritance Cone opsins Colour blindness X-linked, AR Rhodopsin Retinitis pigmentosa AD; AR V 2 vasopressin Diabetes insipidus X-Linked ACTH Familial ACTH resistance AR LH ♂ pseudohermaphrodite AR TSH Cong. hypothyroidism AR TRH Central hypothyroidism AR Diseases caused by Loss of function Mutation 48

49. 49 Receptor Disease Inheritance FSH Hypergonadotropic Ovarian failure AR Ca2 + sensing Hypocalciuric hypercalcaemia AD Ca 2 + sensing Neonatal hyperthyroidism AR GHRH G H deficiency AR GnRH Central hypogonadism AR Endothelin -B Hirschsprung disease Complex Melanocortin 4 Extreme obesity Co-dominant PTH / PTHrP Chondrodysplasia AR Diseases caused by Loss of function Mutation

50. 50 Receptor Disease Inheritance Rhodopsin Congenital night blindness AD LH Familial ♂ precocious puberty AD LH Sporadic Leydig cells tumours Somatic TSH Familial non-autoimmune hyperthyroidism AD TSH Sporadic hyperfunctional thyroid adenomas Somatic Ca2 + sensing Familial hypocalcaemia AD PTH / PTHrP Jansen metaphyseal chondrodysplasia AD Diseases caused by Gain of function Mutation

51. 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

52. 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 Ca 2+ sensing NPS R-568 Diseases due to GPCR misfolding 52

53. POLYMORPHISMS OF GPCR Variations 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 found 53

54. POLYMORPHISMS.... Challenges Ahead Whether such differences are important in individual variation in drug response ( pharmacogenomics ) Whether they could confer susceptibility to disease. 54

55. Allosteric Modulators of G-protein Bind 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. 55

56. ORPHAN GPCRs Lack their pharmacological identities Pre-genome era : Most GPCRs were found by sequence similarity using nucleic acid-based homology screening approaches After genome sequencing : 150 Orphan GPCRs using bio- informatic analysis First Orphan GPCR was G21, later found to be 5HT 1A receptor in 1988 Focus of intense research effort, both in academia and in industry 56

57. 57 GPCR types No. of members Orphan receptors Glutamate-class GPCRs 22 Two third (15) Rhodopsin -class GPCRs 701 63 Adhesion-class GPCRs 33 Majority Frizzled/ taste GPCRs 36 (11 frizzled and 25 taste) None among frizzled ; Most taste Secretin -class GPCRs 15 None

58. The de- Orphanization of GPCRs Evolutionarily conserved and thus are expected to be active Reverse Pharmacological Approaches based on receptor reactivity & receptor binding are applied Isolating natural ligand provides a first hint of function, structural cues for lead design Once de- orphanised , GPCRs can be used for designing new drugs. 58

59. 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 59

60. Search available orphans that have an effect on a specific therapeutic area Analyze Orphans using: 1 . Laser capture micro-dissection to determine the localization 2. 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 60

61. Issues of Orphan GPCR research Deorphanization is a risky, lengthy and demanding endeavour GPCRs exist not only as monomers but as dimers or higher oligomers Concentration of transmitters in their natural environment. 61

62. GPCR Screening Cell-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

63. Recent developments Ligand -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

64. 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 Tyr 8  of 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. 64

65. 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. 65

66. 66

67. GPCR signaling independent of G proteins There 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

68. An example is angiotensin II at its AT 1  receptor activating both β- 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 β- arrestin and activates ERK. 68 GPCR signaling independent of G proteins…

69. Constitutively active receptors G-protein-coupled receptors may also be constitutively (i.e. spontaneously) active in the absence of any agonist. This was first shown for β- adrenoceptor . Histamine H3 receptor also shows constitutive activity. It means that inverse agonists can play a role here. 69

70. GPCRs and drug discovery Regarded 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

71. Novel pancreatic β-cell GPCRs About 20 GPCRs have been found in pancreatic β-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 β-cell. Activation of GPCRs such as GLP1 can enhance the amount of intracellular calcium, for example through activation of Gα q /11  and subsequent generation of IP3 and release of Ca++ from intracellular stores, thereby potentiating glucose stimulation of insulin secretion. Among the other GPCRs identified in β-cells are the newly discovered free fatty acid receptors, GPR40, 43, and 41 . GPR40 couples to Gα q /11 , so free fatty acid would enhance the calcium response of the β-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

72. GPCRs as new therapeutic targets for type 2 diabetes GPCRs that have received recent attention in the field of diabetes therapeutics include Incretin receptors: GLP1R, GIPR (GPR119) Free fatty acid receptor:FFAR1 (GPR40), FFAR4 (GPR120) Bile acid receptor: GPBAR1 (TGR5) 72

73. Novel neuroendocrine GPCRs regulating reproduction There 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 (NK B ) and its receptor (TACR3), which give rise to hypogonadotropic hypogonadism and pubertal failure. The discovery of NK B , 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

74. Role of H4 receptor in asthma H4 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

75. 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

76. Permeate plasma membrane Enter cells Correct folding Allowing routing to plasma membrane How Chaperones Work ? 76 Bind selectively to misfolded proteins

77. Deorphanisation of GPR55 GPR55 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

78. 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 . 78 Future Prospects & Conclusions

79. Future Prospects & Conclusions Ever 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 β- arrestin recruitment assays for known and orphan GPCRs instead of second messenger signaling assays 79

80. Further studies are needed to explore Advances in novel forms Methods to rescue function of misfolded or truncated GPCRs Complexity demands collaborative approaches between persons of medicinal chemistry, analytical pharmacologists & bioinformatic experts 80 Future Prospects & Conclusions

81. GPCRs were once considered highly tractable targets. Current targets much lower success rates Low hanging fruit largely picked Lack of Hits Hits have high molecular weight Poor PK/in vivo activity Difficult to optimize 81 Future Prospects & Conclusions

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83. 83 THANK YOU FOR YOUR PATIENCE