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G-Protein-Coupled Receptors

Dr. Prashant Shukla


Junior Resident
Dept of Pharmacology
Contents
 Introduction

 Historical Background

 GPCR - Basics

 GPCR as Targets for Drug Designing

 GPCR associated Diseases

 Concept of Orphan GPCR

 Future Prospects & Conclusions

2
Introduction

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.
3
Types of receptors

4
GPCRs
 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).
5
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
6
Receptors associated with
GPCRs
1. GABAB Receptors
 GABABR1 and GABABR2
2. Taste Receptors
 T1R3 and T1R2
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
Receptors associated with
GPCRs
5. Somatostatin Receptors
 Five subtypes (SSTR1-5)
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
The importance of GPCRs
1. 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)

2. Diversity (mostly small molecule ligands)

3. Evolutionarily conserved yeast to man (yeast


Ga 45% identical to mammalian Gia)

4. 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
Historical background

10
Historical background

 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
Common Experimental Tools used to
Study GPCRs

12
GPCR- basics
 Structure
 Classification
 Signal molecules/ Ligands
 Physiological role
 G proteins
 Mechanism of action

13
GPCR- Basic structure
Extracellular loops

EXTRACELLULA
R
Plasma
membrane

CYTOSOL

Intracellular loops
14
GPCR- Basic structure

15
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
GPCR: Classification

Based on phylogenetic origin:


The GRAFS classification system has
been proposed
1. Glutamate
2. Rhodopsin
3. Adhesion
4. Frizzled/Taste
5. Secretin

17
GPCR
Tree

18
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
Signal molecules
Biogenic amines: Adrenaline,
noradrenaline, dopamine, 5-HT,
histamine, acetylcholine

Amino acids and ions: Glutamate,


Ca2+, GABA

Lipids : PAF, prostaglandins,


leukotrienes, anandamine
20
Signal molecules…
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
21
Physiological roles
1. Visual sense: Rhodopsin
2. Sense of smell: Olfactory receptor
3. Behavioral and mood regulation:
Serotonin, dopamine, GABA and glutamate
4. Immune system activity and
inflammation: Chemokine receptors,
histamine receptors
5. ANS transmission: β adrenergic receptors
6. Apoptosis

22
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
G protein complexes are made up of
 20 alpha (α) γ subunit

 6 beta (β)
α subunit
 12 gamma (γ) subunits.

 Betaand gamma subunits


can form a stable dimeric
complex referred to as the
beta-gamma complex.
β subunit

24
Types of G Proteins

25
G protein cycle
Basal state

Activated state

26
GPC Receptors
G Receptors Signaling Pathway
Protein
GS Beta adrenergic receptors, Increase CAMP
glucagon, histamine, serotonin Excitatory effects
Gi Alpha2 adrenergic receptors, Decrease CAMP
mAchR, opioid, serotonin Cardiac K+ channel
open- decrease heart rate
Gq mAchR, H1, α1, Vasopressin PLC- IP3 , DAG
type 1, 5HT1C Increase Cytoplasmic Ca

Gt Rhodopsin and colour opsins Increase cGMP


in retinal rod and cone cells phosphodiesterase.
Decrease cGMP

27
G Protein Mediated
Pathways
Secondary messenger Systems
Involved In Signal Transduction:
Adenylate cyclase cAMP mediated
pathway
 Phospholipase mediated pathway

GPCR s can also directly activate the


ion channels

28
cAMP Mediated Pathway

The cAMP-dependent pathway, also known


as the adenylyl cyclase pathway, is a G
protein-coupled receptor triggered signaling
cascade used in cell communication.

 Gs cAMP Dependent Pathway

 Gi cAMP Dependent Pathway

29
Gs cAMP Dependent Pathway
hormone Inhibitor
EXTRACELLULA
R

RS Ri

AC
GDP 
GTP

 GDP
CYTOSOL

GTP ATP

AT P
Inactive
Protein protein
cAMP kinase

Adenylate cyclase ADP


Active
Signaling protein
System Cell response30
Gi cAMP Dependent Pathway

31
Gq Protein Coupled Receptor
EXTRACELLULA
R

CYTOSOL

32
Gt Protein Coupled Receptor
Gt PCR: involved in photo transduction.

33
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.
35
Regulation of GPCRs

Desensitization mechanisms include


1. “down-regulation” or reduction of
receptor number

2. “sequestration” or apparent shielding


of the receptors from interacting
ligands

3. “uncoupling” from G-proteins.


36
Regulation of GPCRs

 Homologous desensitization: The


activation dependent regulation of
receptors.

 Heterologous desensitization: Receptor


activation-independent regulation of
receptors.

37
38
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
Homologous desensitization

41
Heterologous desensitization
 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
GPCR as drug targets

Receptor Drugs and some key indications

AT1 angiotensin Antagonists e.g. losartan in treatment of HT or


II receptor CHF
Antagonists e.g. tamsulosin to treat disorders
α1A-c receptor
asso. with enlarged prostate
Antagonists e.g. propranolol, atenolol, metoprolol,
β1- receptor
carvedilol to treat essential HT or CHF
Agonists e.g. terbutaline, salbutamol, formoterol for
β2- receptor
treatment of COPD or Bronchial asthma
Antagonists e.g. Haloperidol & clozapine to treat
D2 receptor schizophrenia
Agonists e.g. levodopa for Parkinsonism

43
GPCR as drug targets…

Receptor Drugs and some key indications

D3 receptor Antagonists e.g. haloperidol in schizophrenia


Antagonists e.g. clozapine for schizophrenia.
5-HT2A receptor Indirect agonists e.g. fluvoxamine for
depression
5-HT2C
Antagonists e.g. clozapine for schizophrenia
receptor
Associated with progression of AIDS e.g.
CCR5
Aplaviroc and maraviroc
Antagonists e.g. Atropine to dilate pupil;
M3
Scopolamine for motion sickness
Neuropeptide S Asthma susceptibility e.g. Neuromedin and
receptor neurotensin
44
Associated with bleeding diathesis e.g.
GPCR as drug targets…

45
Diseases associated with G-
proteins
Abnormal G protein signalling can result
by

1. Bacterial toxins (Cholera and pertussis)

2. Gene mutations
 Loss of function mutations
 Gain of function mutations

3. Altered GPCR folding


46
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
Diseases caused by Loss of function
Mutation

Cone opsins Colour blindness X-linked, AR

Rhodopsin Retinitis pigmentosa AD; AR

V2 Diabetes insipidus X-Linked


vasopressin
ACTH Familial ACTH resistance AR
LH ♂ pseudohermaphrodite AR

TSH Cong. hypothyroidism AR


TRH Central hypothyroidism AR

48
Diseases caused by Loss of function
Mutation

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

Rhodopsin Congenital night blindness AD


LH Familial ♂ precocious AD
puberty
LH Sporadic Leydig cells Somatic
tumours
TSH Familial non-autoimmune AD
hyperthyroidism
TSH Sporadic hyperfunctional Somatic
thyroid adenomas
Ca2+ sensing Familial hypocalcaemia AD
PTH/PTHrP Jansen metaphyseal AD
chondrodysplasia 50
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
Diseases due to GPCR misfolding

Disease/ Pharmacoperones
GPCR
Abnormality

9-cis-retinal, 11-cis-retinal, 11-cis-


Retinitis pigmentosa Rhodopsi
7-ring retinal
n
SR121463 (satavaptan),
Nephrogenic SR49059 (relcovaptan), VPA-985,
V2R
diabetes insipidus YM087, OPC41061 (tolvaptan),
OPC31260
Hypogonadotropic Indoles, quinolones,
GnRHR
hypogonadism erythromycin-derived macrolides
Familial hypocalciuric Ca2+
NPS R-568
hypercalcemia sensing
52
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
POLYMORPHISMS.... Challenges Ahead

Whether such differences are


important in individual variation
in drug response
(pharmacogenomics)

Whether they could confer


susceptibility to disease.
54
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
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 5HT1A receptor in 1988

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

56
GPCR types No. of members Orphan receptors
Glutamate- 22 Two third (15)
class GPCRs
Rhodopsin- 701 63
class GPCRs
Adhesion- 33 Majority
class GPCRs
Frizzled/ taste 36 (11 frizzled and None among
GPCRs 25 taste) frizzled ; Most
taste
Secretin-class 15 None
GPCRs
57
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
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
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
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
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
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 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 Tyr8 of GnRH II is the main determinant of
selective antiproliferative effects and identified
residues in the TM domains and ECLs of the
64
 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
65
of specificity and off-target effects.
66
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 67
GPCR signaling independent of
G proteins…
 An example is angiotensin II at its
AT1 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
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
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
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 Gαq/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 Gαq/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
GPCRs as new therapeutic targets
for type 2 diabetes
GPCRs that have received recent
attention in the field of diabetes
therapeutics include
1. Incretin receptors: GLP1R, GIPR
(GPR119)

2. Free fatty acid receptor:FFAR1


(GPR40), FFAR4 (GPR120)

3. Bile acid receptor: GPBAR1 (TGR5) 72


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 (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, 73
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 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
How Chaperones Work ?
Permeate plasma membrane

Enter cells

Bind selectively to misfolded proteins

Correct folding

Allowing routing to plasma membrane

76
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
Future Prospects & Conclusions

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
 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
Future Prospects & Conclusions
 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

 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
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