g protein coupled receptor and pharmacotherapeutics

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G-protein coupled receptors and drugs modulating them DR. NISHIKANT SHARMA DR. PRIYANKA KUMAWAT

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Page 1: G protein coupled receptor and pharmacotherapeutics

G-protein coupled receptors and drugs

modulating them

DR. NISHIKANT SHARMA

DR. PRIYANKA KUMAWAT

Page 2: G protein coupled receptor and pharmacotherapeutics

• General description of Receptors and signaling

• G- Protein coupled receptor and its mechanism

• Classes of GPCR

• Second messenger and its applied

pharmacology

• Recent development

• Tools for drug discovery

• Conclusion

Brief outline

Page 3: G protein coupled receptor and pharmacotherapeutics

History

1967. Ragnar Granit, Haldan Keffer Hartline and George Wald-Physiological and chemical processes underlying photoreception.

1971. Earl W. Sutherland, Jr.-cyclic AMP (cAMP).

1988. Sir James W. Black-Discovery of propranolol, which blocks the β-adrenergic receptor, and the H2 histamine receptor blocker cimetidine.

1994. Martin Rodbell and Alfred G. Gilman-Heterotrimeric G-proteins.

2004. Linda B. Buck and Richard Axel- Odourant receptors.

2012. Brain kobilka and Robert Lefkowitz-Studies of G-protein coupled receptors.

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RECEPTORS

INTRACELLULAR RECEPTORS- Cytoplasmic

Nuclear receptors

CELL SURFACE RECEPTORS

ION CHANNEL RECEPTOR

• Ligand gated ion channels

• Controlled by neurotransmitters

• Present in neurons• Eg: Ach cation

channel

G-PROTEIN LINKED RECEP

• Act via second messengers-cAMP, IP3/DAG,cGMP

ENZYME LINKED RECEP

• Eg: Protein kinaseTyrosine kinaseTyrosine phosphotaseSerine/threonine kinaseGuanylyl cyclaseHistidine kinase

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Concept of Cell Signaling

Process in which cells sense the extracellular stimuli through membranous or intracellular receptors, transduce the signals via intracellular molecules -Regulate the biological function of the cells.

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Features of signal transduction

Specificity- Signal molecules fits binding site on its complementary receptor, other signals do not.

Affinity- High affinity of receptors for signal molecules

Amplification-Signal receptor activate many molecules of second enzyme, which activates many molecules of the third enzyme and so on

Desensitization –Feedback circuit that shuts off the receptor or remove it from the cell

Integration – Two signals with opposite action on second messenger, the regulatory outcome results from integrated output from both the receptors

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Signals to which cell respond

Antigen

Cell surface glycoproteins/ oligosaccharides

Extracellular matrix component

Growth factors

Hormones

Neurotransmitters

Light

Mechanical touch

Nutrients

Odorants

Pheromones

Tastants

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Primary

Messengers

Secondary

Tertiary

Transmit the signal from receptor to the enzyme and activate it to produce secondary messenger

Eg:Gα,Gβγ

Transmit signals in form of either direct cellular response eg:cAMP, cGMPOr activate further enzymes to produce response eg:IP3,DAG

Release after action of second messenger on an organelle (ER) and act directly or in conjunction to give cellular responses

Eg:Ca+2

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G-protein coupled receptor structure

Seven transmembrane (7TM) α helices coupled to effecter system (enzyme/ channel) through GTP/GDP binding protein called G-proteins

An extracellular domain which binds to the ligand (drug/ neurotransmitter)

An intracellular domain which couples to G-protein

Page 12: G protein coupled receptor and pharmacotherapeutics

G- proteinA family of membrane proteins anchored to the

membrane.

Recognize activated GPCR’s and pass the message to the effector system.

Named as G-protein because of their interaction with guanine nucleotides (GTP/GDP)

Consist of three subunits: α, β and γ. Guanine nucleotides bind to the α subunit, has GTPase enzymic activity

Functions as a molecular switches. when bind with GTP they are “on” & when with GDP they are “off”.

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Types of G-protein

1. “Large" G proteins (Heterotrimeric)Activated by GPCRsMade up of alpha (α), beta (β), and gamma

(γ) subunits.

2.  ”Small" G proteins- Belong to the Ras superfamily of small GTPases. Homologous to the alpha (α) subunitAlso bind GTP and GDP and are involved in signal

transduction.

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G-protein subunits with second messenger

β γ α

Gs Gi Gq

cAMP stimulationβ receptorHistamineSerotoninDopamine

cAMP inhibitionα2 receptorM2 receptorOpioid receptorD2 receptor5HT1 receptor

PLC(IP3 & DAG)α1

M1

AT1

5HT2

Vasopressin

•Activate potassium channels• Inhibit voltage-gated calcium channels• Activate mitogen-activated protein kinase cascade.

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Golf-Odorant receptor,Adenylyl cyclase

Gt- cGMP phosphodiesterase , cGMP

Gα12/13 -Rho family GTPase signaling and control

cell cytoskeleton remodeling and regulating cell migration.

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Mechanism of GPCR The ligand comes to the extracellular binding site of receptor

make some conformational changes in receptor which attract G-protein

Coupling of the α subunit to an agonist-occupied receptor causes the bound GDP to exchange with intracellular GTP

α-GTP complex then dissociates from the receptor and from the βγ complex

This α-GTP complex interacts with a target protein (target- adenylyl cyclase/ ion channel/PLC)

The βγ complex may also activate a target protein (target)

These effectors then form the second messengers to initiate the cell responses e.g.; cAMP 2nd messenger for Adenylyl cyclase, IP3/DAG for PLC and cGMP for guanylyl cyclase

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GTP

GDP

GDPGTP

4 ATP

4 cAMP

Cell response

AT

Protein kinase

ADP

PInactive protein

Active protein

hormone

Adenylate cyclase Signaling System

AC

RS

Inhibitor

Ri

Page 18: G protein coupled receptor and pharmacotherapeutics

Phospholipase-c signaling systemPIP2

IP3 DAG

Release of Ca+2

from ER

intracellular Ca+2

Along with Ca+2 Activate Protein Kinase-C

Cellular functions- Proliferation, differentiation, apoptosis, cytoskeletal Remodeling, vesicular trafficking, ion channels conductance,neurotransmission

PLC

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GPCR classes Class A- Rhodopsin like-receptors e.g.: Retinal, odorants,

catecholamine(β2),adenosine(A2), opiates, enkephalins,

anandamide, thrombin.

Class B- Secretin like- Secretin, Glucagon, PTH, Calcitonin, VIP

Class C- Metabotropic glutamate- Glutamate

Class D- Pheromone- Used for chemical communication

Class E- cAMP receptor(Dietyostelium)

Class F- Frizzled/smoothened family-Wnt binding, a key regulator of animal development (embryonic life)

Ocular albinism proteins

Putative families- Vomeronasal receptors (V1R & V2R),Taste

receptors(T2R)

Orphan GPCR- putative unclassified

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Second messengers

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Targets of G proteinsAdenylyl cyclase

IP3/DAG Phospolipase C system

Ion channels esp. potassium and calcium

Rho a/ Rho kinase system

Page 24: G protein coupled receptor and pharmacotherapeutics

The Adenylyl cyclase/cAMP system

cAMP is a nucleotide

Synthesized within the cell from ATP by membrane-bound, adenylyl cyclase

Produced continuously

Inactivated by hydrolysis to 5´-AMP, by the Phosphodiesterases

Common mechanism, namely the activation of protein kinases

Involved in Energy metabolism Cell division and cell differentiation Ion transport, ion channels Contractile proteins in smooth muscle

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Cyclic AMP dependent protein kinase

Best understood target of cyclic AMP

Can phosphorylate a diverse array of physiological targets Metabolic enzymes Transport proteins Numerous regulatory proteins including other protein

kinases Ion channels Transcription factors

For example cAMP response element–binding protein(CREB) leads to Tyrosine hydroxylase, iNOS, AchR, Angiotensinogen,

Insulin, the glucocorticoid receptor, and CFTR

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Cyclic Amp–Regulated Guanine Nucleotide Exchange Factors

(Gefs)

Monomeric GTPases and key regulators of cell function

Integrate extracellular signals from membrane receptors with cytoskeletal changes

EPAC pathway provides an additional effector system for cAMP signaling and drug action that can act independently or cooperatively with PKA

Activation of diverse signaling pathways, regulate Phagocytosis Progression through the cell cycle Cell adhesion Gene expression Apoptosis

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PhosphodiesterasesHydrolyze the cyclic 3',5'-phosphodiester bond

in cAMP and cGMP

>50 different PDE proteins divided into 11 subfamilies

Drug targets for Asthma Cardiovascular diseases such as heart failure Atherosclerotic coronary and peripheral arterial disease Neurological disorders

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Energy metabolism

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cAMP and Immunomodulation

Am J Respir Cell Mol Biol Vol 39. pp 127–132, 2008

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The Phospholipase C/ inositol phosphate system

1950s by Hokin and Hokin

PIP2 is the substrate for a membrane-bound enzyme, phospholipase Cβ (PLCβ),

Which splits it into DAG and inositol (1,4,5) trisphosphate (IP3)

Both function as second messengers

After cleavage of PIP2, the status quo is restored

Lithium blocks this recycling pathway

IP3 receptor- a ligand-gated calcium channel present on the membrane of the endoplasmic reticulum

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Diacylglycerol and protein kinase C

DAG, unlike the inositol phosphates, is highly lipophilic and remains within the membrane

Binds to a specific site on the PKC molecule, which migrates from the cytosol to the cell membrane in the presence of DAG, thereby becoming activated

10 different mammalian PKC subtypes

Kinases in general play a central role in signal transduction, and control many different aspects of cell function

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Ca2+

IP3 receptor – a ligand-gated Ca2+ channel found in high concentrations in the membrane of the ER

10-9 m range enhance Ca2+ release, but concentrations near 10-9 m inhibit release

Phosphorylation of the IP3 receptor by PKA enhances Ca2+ release,

Phosphorylation of an accessory protein, IRAG, by PKG inhibits Ca2+ release

In smooth muscle, this effect of PKG represents part of the mechanism by which cyclic GMP relaxes vessel tone

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Ca2+

In skeletal and cardiac muscle - Ca2+ release from intracellular stores occurs through a process -Ca2+-induced Ca2+ release

Primarily mediated by the ryanodine receptor (RyR)

Ca2+ entry into a skeletal or cardiac myocyte through L-type Ca2+ channels causes conformational changes in the ryanodine receptor

Induce release of large quantities of Ca2+ into the sarcoplasm.

Drugs that activate the RyR include caffeine; drugs that inhibit the RyR include Dantrolene

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Ion channels as targets for G-proteins

Directly by mechanisms that do not involve second messengers

In cardiac muscle, for example, mAChRs are known to enhance K+ permeability

Opiate analgesics reduce excitability by opening potassium channels

Actions are produced by direct interaction between the βγ subunit of G0 and the channel, without the involvement of second messengers

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The Rho/Rho kinase system

Activated by certain GPCRs (and also by non-GPCR mechanisms), which couple to G-proteins of the G12/13 type

Rho-GDP, the resting form, is inactive

When GDP-GTP exchange occurs, Rho is activated

In turn activates Rho kinase

Smooth muscle contraction and proliferation, angiogenesis and synaptic remodeling

Important in the pathogenesis of pulmonary hypertension

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DesensitizationReceptor phosphorylation

Phosphorylation by PKA and PKC Not very selective, receptors other than that for the

desensitizing agonist will also be affected Heterologous desensitization

Phosphorylation by GRKs Receptor-specific to a greater or lesser degree Affects mainly receptors in their activated (i.e. agonist-

bound) state Homologous desensitization

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RECENT DEVELOPMENTS

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GPCR dimerisation The conventional view first overturned by work on the GABAB receptor

Most, if not all, GPCRs exist as oligomers

Within the opioid receptor family, stable and functional dimers of κ and δ receptors have been found whose pharmacological properties differ from those of either parent

Functional dimeric complexes between angiotensin (AT1) and

bradykinin (B2) receptors occur in human platelets

Show greater sensitivity to angiotensin than 'pure' AT1 receptors

Pre-eclampsia number of these dimers increases due to increased

expression of B2 receptors

Resulting-paradoxically- in increased sensitivity to the vasoconstrictor action of angiotensin

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Constitutively active receptors

Spontaneously active in the absence of any agonist

β-adrenoceptor, histamine H3

Inverse agonists, which suppress this basal activity, may exert effects distinct from those of neutral antagonists, which block agonist effects without affecting basal activity.

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Agonist specificityCellular effects are qualitatively different with

different ligands

Existence of probably many-R* states

Agonist trafficking or protean agonism

If substantiated, it will add a new dimension to the way in which we think about drug efficacy and specificity

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GPCR and arrestinsFollowing continued agonist binding to GPCR

Cytosolic GRKs are induced to translocate to GPCR

This phosphorylation attracts -arrestins to the receptors

Compete with G proteins for binding to the cytoplasmic site of the receptor

Arrestins uncouple GPCRs from G proteins

Causing desensitization, internalization of GPCR

Universal response to agonist activation and is critical for the inactivation of GPCRs and the termination of neurotransmitter and hormone action

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GPCR and arrestinsShown to have in vivo physiological roles in

mediating the functions of GPCRs

Implicated in development of tolerance to and dependence on drugs

Safety mechanisms to prevent the over stimulation of GPCRs

Could be important targets for the development of drugs to prevent tolerance development to established drugs and prolong the therapeutic activity

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Orphan GPCRs200 or so known GPCRs whose endogenous

ligands and functions are not known

Attempts have been made to deorphanise these receptors

Evidence that some recently deorphanised GPCRs, such as orexin receptor, may dimerise or associate with more classical GPCRs

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British Journal of Pharmacology (2008) 153 S339–S346

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GPCR mutations, disease and novel drug discovery

Loss of function mutations in GPCRs involved in the control of endocrine systems

Homozygous loss of function mutations in the type 5 chemokine receptor provides resistance to HIV infection

Critical for the infectivity of this virus

Gain of function mutations in GPCRs also cause disease

 Mutations in GPCRs could be responsible for variations in drug sensitivities among different populations

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mAbs 2:6, 594-606; November/December 2010; © 2010 Landes Bioscience

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Tools for GPCR drug discovery

Receptor binding assay

G-protein dependent functional assaysGTPγS binding assaycAMP assayIP3/IP1 and Ca2+ assaysReporter assay

Generic G-protein independent functional assaysReceptor internalization assayβ-arrestin recruitment assayLabel-free whole cell assaysReceptor dimerization assay

Acta Pharmacol Sin. 2012 March; 33(3): 372–384

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Conclusion  Nearly 40% of the drugs approved for marketing by

the FDA target GPCRs

 800-1,000 different GPCRs and the drugs that are marketed target less than 50 GPCRs

GPCR will continue to be highly important in clinical medicine because of their large number, wide expression and role in physiologically important responses

Future discoveries will reveal new GPCR drugs, in part because it is relatively easy to screen for pharmacologic agents that access these receptors and stimulate or block receptor-mediated biochemical or physiological responses

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REFERENCES Goodman and Gilman’s Pharmacological basis of therapeutics,

12thed

Rang and Dale’s pharmacology, 7th edition

Alexander SPH, Mathie A, Peters JA (2011). Guide to Receptors and Channels (GRAC), 5th edn. Br J Pharmacol 164 (Suppl. 1): S1–S324.

Gurevich, E.V., et al., G protein-coupled receptor kinases: More than just kinases and not only for GPCRs,JPT Elsevier doi:10.1016j.pharmthera.2011.08.001JPT-06382;

GLIDA-GPCR ligand database version 2.04 10/10/2010

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Let the future begin THANK YOU

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