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  • G0- ↓ ca entry into cells
  • Guanine exchange factors
  • To form heterotrimer
  • GEF- Guanine exchange factors
  • Conformational change
  • substrates
  • The protein is an inhibitor of cardiac muscle sarcoplasmic reticulum Ca++-ATPase (SERCA) in the unphosphorylated state, but inhibition is relieved upon phosphorylation of the protein. The subsequent activation of the Ca++ pump leads to shorter intervals between contractions, thereby contributing to the inotropic response
  • stimulate lipase[
  • Brain-derived neurotrophic factorbinds to certain DNA sequences called cAMP response elements (CRE) thereby increasing or decreasing the transcription of the downstream genes
  • GEFs: proteins involved in the activation of small GTPases
  • More efficacious & mimic action of several neurotransmitters; in neurodegenrative disorders
  • desensitization
  • gram positive and negative bacteria,tumor cells and heterologous antigens; involved in transplantation
  • Inhibition of IP3 receptors by phosphorylation of IP3 receptor associated cGMPkinase substrate… Phosphorylation of phospholamban- stimulate SERCA (sarcoendoplasmic reticulum pump Ca ATPase
  • Stable, prinzmetals; esophageal spasm, biliary colic, cyanide poisoning; aortic dissection;
  • administered intravenously usually by bolus followed by IV infusion
  • which is caused by this 2nd messengers
  • Resistance-atrerioles, small arteries and capillaries; Capacitance- veins ie they hold blood
  • When activated hydrolyse minor membrane phospholipidPhosphotidyl-inositol 4,5 biphosphate to generate 2 intracellular signals: IP3( Inositoltriphosphate) & Diacylglycerol(DAG)
  • Gq
  • Protein kinase G (cGMP)
  • Increasein the sensitivity of RyRs to Ca2+
  • the gitsecretes digestive enzymes & gastric acid, the lungsecretes surfactants & sebaceous glands secrete sebum
  • Via activation of kinases mek1/2 & erk1/2; activation of trascription factors of AP1(activator protein) family if fos/jun- growth factors
  • OncogenicRas
  • Viz present in the plasma membrane; which causes apoptosis by forming holes in mitochondria,
  • Mammilian target of rapamycin
  • mechanisms that defend the host from infection by other organisms in a non-specific manner.does not confer long-lasting or protective immunity to the host; TIR no enzymatic activity
  • Myeloid differention protein 88
  • TRADD recruits TRAF2 and RIP. TRAF2in turn recruits the multicomponent protein kinase IKK, enabling the serine-threoninekinase RIP to activate it.TRADD binds FADD, which then recruits the cysteine protease caspase-8

Second Messenger Systems Second Messenger Systems Presentation Transcript

  • Second MessengerSystemsDr. Kunal A. Chitnis2nd Yr ResidentT.N.M.C.16th July 11
  •  Molecules that relay signals from receptors on the cell surfaceto target molecules inside the cell i.e. cytoplasm or nucleus Relay the signals of hormones like epinephrine, growth factors& others; causing some kind of change in the activity of the cell The term was coined upon the discovery of these substancesin order to distinguish them from hormones & other moleculesthat function outside the cell as “first messengers” in thetransmission of biological information
  •  Earl Wilbur Sutherland Jr.discovered second messengerswon the 1971 Nobel Prize in Medicine He saw that epinephrine wouldstimulate the liver to convert glycogento glucose in liver cells, butepinephrine alone would not convertglycogen to glucose He found that epinephrine had to trigger asecond messenger, cyclic AMP for the liver to convertglycogen to glucose View slide
  • cAMPSystemcGMPSystemPhospho-inositolSystemTyrosineKinaseSystemLigands EpinephrineAchANP, NO Oxytocin PDGFPrimaryEffectorAdenylcyclaseGuanylatecyclasePhospho-lipase CReceptorTyrosinekinaseSecondarymessengercAMP cGMP IP3 & DAG;Ca2+- View slide
  • G- Proteins Guanine nucleotide binding proteins which act as a Transducerbetween a receptor & an effector Discovered by Alfred Gilman & Martin Rodbell in 1990 Significance:• Of the top 100 drugs,26 directed at GPCRs• 60 % act through GPCRs• 40 % of prescriptions• 3rd largest family of genes (865)• Present in almost every organ system
  • Endogenous Ligands:Sensory signal mediators: Light & Olfactory stimulatory moleculesBiogenic amines: Dopamine, Adr, NA, Ach, HistaminePeptide hormones: FSH, GnRH, calcitonin, TRH, OxytocinMediators of inflammation: Prostaglandins, PAF, leukotrienes,chemokines
  • Structure: Embedded in the plasma membrane 7 transmembrane -helices Rhodopsin was first of theseto have its structure confirmed byX-ray crystallography-AdrenergicReceptorPDB 2RH1Lysozymeinsertligand
  • Binding Domains Small molecular ligandsbind to sites within thehydrophobic core formed bytransmembrane α helices Protein and peptide agonistsbind to N terminus &extracellular hydrophilicloops joining thetransmembrane domain G-proteins bind either tosecond and thirdcytoplasmic loop which islargest or to carboxy terminus
  •  Molecular Switch: On/Off Heterotrimericα-subunitβ-subunitγ-subunit α subunit: specific recognition ofreceptors & effectors;GTP binding site βƔ subunit: Membrane localisation by prenylation of Ɣ subunit Coded by of genes:α – 23, β-7, Ɣ-12
  • Functions of α subunit & βƔ dimer
  • Receptors CouplersMuscarinic Gi, Go, GqDopamine D2 Gi, Goβ-adrenergic Gs, Giα2-adrenergic Gi, Gs, GoGABA-B Gi, Go5-HT Gi, Gq, Gs
  • G Protein Activation Conformational changein receptor Transmitted from ligandbinding pocket to 2nd & 3rdintracellular loop α subunit exchange GDPwith GTP Presence of GEF‟s(Guanine exchange factors) Release of GTP boundα subunit & βƔ dimer
  • Inactivation Activated α subunit isinactivated by hydrolysisof GTP to GDP by GAPs(GTPase ActivatingProteins) Rebinds to βƔ complex Modulated byRegulators of Gproteins Signaling (RGSs) Acclerate hydrolysis ofGTP & potential drugtargets
  • Toxins Cholera toxin catalyzes covalent modification of Gs• ADP-ribose is transferred from NAD+ to an arginine residueat the GTPase active site of Gs• ADP-ribosylation prevents GTP hydrolysis by Gs• The stimulatory G-protein is permanently activated. Pertussis toxin catalyzes ADP-ribosylation at a cysteine residueof the inhibitory Gi, making it incapable of exchanging GDP forGTP• The inhibitory pathway is blocked. ADP-ribosylation is a general mechanism by which activity ofmany proteins is regulated, in eukaryotes & prokaryotes.
  • Receptor Desensitization Activated receptor isphosphorylated via aG-protein Receptor Kinase Binds to a protein arrestinpromotes removal of thereceptor from the membrane byclathrin-mediated endocytosis May also bind a cytosolicPhosphodiesterase, bringingthis enzyme close to wherecAMP is being produced,contributing to signal turnoff
  • Adenyl Cyclase- cAMP Pathway
  • Adenyl Cyclase 9 membrane bound, 1 soluble isoform (AC1-10) 120kDa Basal enzymatic activity modulated byGTP liganded α subunit Gs & Gi Other regulatory interactions: βƔ subunits, Ca2+, proteinkinase & diterpene forskolin Dephophorylation of ATP by removal of 2 phosphatemolecules→ cAMP
  • cAMP has following major targets:1. cAMP dependent Protein Kinase A (PKA)2. cAMP regulated Guanine nucleotide exchange factorstermed EPACs (Exchange Proteins Activated by cAMP)3. CREB (cAMP responsive element binding protein)
  • Protein Phosphorylation→ most common form of post-translational modification in natureProtein function altered by addition of a negatively chargedphosphate group to a Ser, Thr or Tyr residue by Protein Kinase:• Binding properties• Enzymatic activity if a catalytic proteinProtein phosphatase catalyzes removal of the Pi by hydrolysisProtein OH + ATP Protein O POOO+ ADPPi H2OProtein KinaseProtein Phosphatase
  • 1. Protein Kinase A Holoenzyme: 2 Regulatory(R) & 2 Catalytic(C) subunits Heterotetramer complex R2C2 ↓ cAMP, R inhibits C→ inactive ↑ cAMP→ 4 cAMP mols bind to R2C2, 2 to each R→ lowersaffinity to C → Activation
  • Active C subunits→ Phosphorylate serine/threonine residues on proteinsAs protein expression varies from cell type to cell type,proteins that are available for phosphorylation will depend uponthe cell type Phosphorylation of ion channels→ Regulation(Ca2+ activated K+ channel activation & Na+/K+ ATPase) Inhibition of Myosin Light Chain Kinase
  • Cell Type Stimulators Inhibitors EffectsHepatocyte Epinephrine(β),GlucagonProduce glucose:stimulate glycogenolysis,inhibit glycogenesis,stimulate gluconeogenesis,inhibit glycolysis.Skeletal -MyocyteEpinephrine(β) Produce glucose:stimulate glycogenolysis,inhibit glycogenesis,stimulate glycolysisCardio-myocyteNorepinephrine(β)Sequester Ca2+ insarcoplasmic reticulum,PhosphorylatesphospholambanActions of Protein kinase A
  • Smooth musclemyocyteβ2 agonist (β2)Histamine (H2)ProstacyclinProstaglandinD2/E2Muscarinic(M2)VasodilationAdipocyte Epinephrine (β),GlucagonEnhancelipolysisNeurons inNucleusaccumensDopamine (D3) ActivaterewardsystemPrincipal Cells Vasopressin(V2)Synthesis &Exocytosis ofAquaporin 2JuxtaglomerularCellsAdrenergic (β1)Dopamine(D1)GlucagonReninsecretion
  • 2. cAMP ResponseElement-Binding(CREB) Cellular transcription factor Genes: c-fos, the neurotrophinBDNF, tyrosine hydroxylase &many neuropeptides Neuronal plasticity & long-termmemory formation Development & progression ofHuntington‟s Chorea
  • 3. Exchange Proteins Activatedby cAMP (EPAC) cAMP Regulated Guanine NucleotideExchange Factors Bind to GDP liganded GTPase,exchange of GDP for GTP Activation of PKC Cell differentiation/proliferation, cytoskeletalorganization, vesicular trafficking & nuclear transport Additional effector system Potential target for cancer therapy
  • Therapeutic ApplicationsSelected Drugs that target cAMP signallingDrug MOA cAMP TherapeuticApplicationSalmeterol β2 Agonist ↑ Asthma, COPDRimonabant CB-1 Antagonist ↑ ObesityHaloperidol D2,D3,D4 Antag ↑ SchizophreniaMetoclopramide D2, 5HT4 Antag ↑ Nausea,VomitingDesmopressin V2 ReceptorAgonist↑ Diabetesinsipidus
  • Metoprolol β1 Antag ↓ Angina,Hypertension,CHFMorphine μ Agonist ↓ PainSumatriptan 5-HT1D/5-HT1B ↓ MigraineIbuprofen Non selectiveInhibitor of COX↓ Inflammation,PainRanitidine H2 Antag ↓ Peptic ulcer,GERDMisoprostol PG ReceptorAgonist↓ Prevention ofNSAID ulcersCabergoline D2 Agonist ↓ Parkinson‟sdisease
  • Novel Drug Targets: Analgesia• ↑ cAMP→ ↑nociception• AC1 & AC5 involved• Selective AC1 & AC5inhibitors→ Analgesics• Preclinical stages Drug dependence• Repeated opiod exposure→ upregulation of AC activity• 3 specific isoforms- AC1, AC5 & AC8• Selective inhibitors for opiod dependence Neurodegenerative disorders• Target second messengers used my multiple neurotransmitters• AC1 is most attractive target• AC1 activators→ used for cognitive decline
  •  Chronic Heart Failure:• Alterations in β adrenoreceptor- AC- cAMP pathway• Downregulation of β1 receptors• Upregulation of inhibitory G proteins &G-Protein coupled receptor kinases• Catecholamine refractoriness, Exercise intolerance• Protective phenomenon→ shields myocytes fromarrhythmogenic, hypertrophic & apoptotic effects ofcatecholamines• Genetic variant of G-Protein Coupled Receptor Kinase 5(GRK5)→Accelerated desensitization→ Better prognosis Sperm function:• sAC regulates sperm motility & capacitation• ↑cAMP→ mediates capacitation• Inhibitors for male contraception in early stages
  • Cyclic GMP Pathway
  •  Unlike cAMP, cGMP has established signaling roles in onlyfew cell types Signaling pathways that regulate synthesis include:1. Nitric Oxide2. Hormonal Regulation (ANP/BNP/CNP)
  • 1. Nitric Oxide Nitric oxide: diatomic free radical gas Lipid soluble Very small→ easy passage between cell membranes Short lived, degraded or reacted within a few seconds Synthesis:Synthesized from L-arginine catalyzed byreaction of NO-synthaseto form NO and L-citrulline
  •  Nitric Oxide Synthase (NOS) exists as 4 isoforms neuronal type I isoform (nNOS) inducible type II isoform (iNOS) endothelial type III isoform (eNOS) mitochondrial isoform (mtNOS)
  • NOS constitutively expressed in: eNOS :• Endothelium, cardiac myocytes, renal mesangial cells,osteoblasts, platelets nNOS :• CNS and NANC nerves
  • Control exerted in following ways: Endothelium-dependent agonists (Ach, bradykinin, substance P)↑ cytoplasmic concentration of Ca2+ ↑calcium-calmodulin eNOS or nNOS activation Shear stress in resistance vessels→ Sensed bymechanoreceptors→ Transduced via a serine threonine protein kinasecalled Protein kinase B/ Akt → Phosphorylation of specific residues oneNOS
  •  iNOS:• Macrophages, Kuffer cells, neutrophils, fibroblasts, vascularsmooth muscle cells & endothelium• Activity independent of Ca2+• Positive inducers : IFNγ, TNFα, IL-1, IL-2, LPS, antigens• Inhibitory cytokines: TGF-β, IL-4, IL-10, glucocorticoids
  • 2. Natriuretic Peptide Receptors3 small peptide ligands: Atrial Natriuretic Peptide (ANP):• Released from atrial storage granules• ↑ Intravascular volume/ Stimulation with pressor hormones Brain Natriuretic Peptide (BNP):• Synthesized and released from ventricular tissue• Volume overload
  •  C-type Natriuretic Peptide (CNP):• Synthesized in brain & endothelial cells• Growth factors/ stress on vascular endothelial cells Major physiological effects: ↓ BP (ANP/BNP) ↓ Cardiac hypertrophy & fibrosis (BNP)
  • Receptors with intrinsic enzymatic activity3 types of receptors:1) ANP Receptor (NPR-A) Binds to ANP & BNP Maintaining normal state of CVS2) BNP Receptor (NPR-B) Binds to BNP Role in bone function3) CNP Receptor (NPR-C) No enzymatic activity
  •  Ligand brings juxtamembrane regions together Phosphorylation of serine residues→Stimulation of guanyl cyclase
  • Guanylate Cyclase: Membrane-bound (type 1) & soluble (type 2) forms Membrane bound→ activated by ligands Soluble→ activated by NO Catalyzes reaction ofGTP to cGMP
  • Downstream reactions of cGMP: Activation of Protein Kinase G cGMP gated ion channels cGMP-modulated Phosphodiesterase
  • cGMP-dependent protein kinase or Protein Kinase G(PKG) Serine/threonine kinases PKG I → cytoplasm, PKG II → membrane associated PKG I→ smooth muscles, platelets, brainPKG II→ intestines, bones, kidney Regulatory and catalytic domains contained on a singlepolypeptide chain Upon activation dimerizes to form PKG holoenzyme
  •  Actions of PKG I: Inhibits of Gq/G11→ Inhibition of Phospholipase C→ ↓ Ca2+ Inhibits G12/G13→ Inhibits Ca2+ sensitizing mechanisms Phosphorylates and inhibits the Myosin light chainkinase which normally phosphorylates the myosin lightchains
  •  Effects: Relaxes all smooth muscle types Inhibits platelet aggregation & granule secretion Functions of PKG II: Stimulates in chloride & water secretion in small intestine Normal endochondral bone development Inhibits renin secretion
  • Therapeutic Applications1. NO Donors Organic nitrates• Short acting: Nitroglycerin (NTG)• Long acting: Isosorbide mononitrate, Isosorbide dinitrate,Pentaerythritol tetranitrate• Use: Treatment of Angina• A/E: Tolerance (Nitrate free interval)
  •  Sodium Nitroprusside• Hypertensive emergencies Nicorandil• K+ channel opner and NO donor• Antianginal CCB’s: Nitrendipine, Nifendipine, Lacidipine• Dihydropyridine• Release NO• Retard atherosclerosis• Use : Hypertension, angina
  •  β Blockers: Nebivolol, Celiprolol• Additional vasodilatory effects2. Natriuretic Peptides Nesiritide:• Synthetic Brain Natriuretic Peptide• Promotes vasodilation, natriuresis & diuresis• Use: Acutely decompensated congestive heart failure Ecadotril:• Neural Endopeptidase (NEP) catalyses BNP degradation• It inhibits NEP• Use: CHF (Phase 2)
  • BAY series of compounds8-bromo-cGMP8-bromoPET-cGMP8-pCPT-cGMP Specific activators & inhibitors of cGKI & cGKII Improve the specificity & availability of treatment Evaluated for:• Asthma• Graft survival after liver & lung transplantation
  • Phosphodiesterases (PDE) PDE comprise family of enzymes expressed in almost all cellsof the body & of prime importance in cellular functioning Hydrolysis of phosphodiester bond in the second messengersc AMP & c GMP → inactive forms 5 AMP and 5 GMPrespectively
  •  Reversal of activation of cellular protein kinases Inactivation leads to termination of intracellular signals Regulators of cyclic nucleotide signaling & responsible fordiverse physiological functions PDEs comprise a super family composed of 25 genesCategorized into 11 sub families i.e. PDE 1 – PDE 11 Substrate specificity:c AMPSpecificc GMPSpecificcAMP & cGMPSpecific• PDE 4• PDE 7• PDE 8• PDE 5• PDE 6• PDE 9• PDE 1• PDE 2• PDE 3• PDE 10• PDE 11
  •  Act by modulating the levels of 2nd messengersTherapeutic Applications: Congestive heart failure: PDE-3 Inhibitors→ Amrinone, Milrinone ↑ In force of contraction Direct vasodilation of both the resistance & capacitance vessels Inodilators
  •  Erectile dysfunction: PDE-5 Inhibitors→ Sildenafil, Tadalafil, Vardenafil Never combined with nitrates:• Nitrates produce vasodilatation by NO dependent elevationof cGMP in vascular smooth muscle• Thus PDE 5 inhibitor if given along with of a NO donors cancause profound & extreme hypotension• Pts should be asked for a history of nitrate consumption withinprevious 24 hrs
  •  Bronchial Asthma: Theophylline, Aminophylline Non selective PDE inhibitor Narrow therapeutic range (5 – 20 mcg/ml)Other uses: COPD, Premature apnoea in infants Peripheral Vascular Disease: Pentoxyphylline PDE 3 inhibitor Rheologic modifier improves the flexibility of RBCs& ↓ blood viscosity Improves microcirculation Cilastazol PDE 3 inhibitor Promotes vasodilatation & inhibition of platelet aggregation
  •  Antiplatelets Dipyridamole Inhibits PDE 5 Prevents uptake & degranulation of adenosine Was introduced for angina pectoris but was a failure due to„coronary steal phenomenon‟ Anagrelide PDE 3 inhibitor Other uses: Essential thrombocytosis, Thrombocytopenia in Polycythmia Vera Zaprinast PDE 6 Inhibitor Antiproliferative & proapoptic property Vasoproliferative disorders
  •  Antispasmodics Drotaverine Inhibits PDE 4 Selective for smooth muscles Intestinal, biliary, renal colic; Irritable bowel syndrome;Dysmenorrhea; Acceleration of labor
  • Phospholipase C: IP3-DAG Pathway
  • Three Types of Inositol phospholipids:PI, PI(4)P, PI(4,5)P2
  • Phospholipase C exists as 2 isoforms:1. Phospholipase Cβ:• Activated by GPCRs couple to Gi/Gq→ release GTP boundα subunit & βƔ dimer→ both activate2. Phospholipase CƔ:• Activated by Receptor/Non Receptor Tyrosine Kinases Cytosolic enzymes Translocate to plasma membrane on receptor activation Ligands:AGT, GnRH, GHRH, Oxytocin, TRH
  • Phospholipase C forms Diacylglycerol & Inositol 1,3,5- triphosphatefrom Phosphotidyl Inositol 4,5-bisphosphate
  • Protein kinase C: Regulatory domain & catalyticdomain tethered together by ahinge region C1 domain, present in all of theisoforms of PKC has a binding sitefor DAG C2 domain acts as a Ca2+ sensor Catalytic Region brings aboutphosphorylation Ser/Thr a.a. ofproteins Upon activation, translocated to theplasma membrane
  • Cell type Activators EffectsSmooth muscle(vascular)5HT(5HT2A)Adrenergic(α1)VasoconstrictionSmooth muscle(GIT)5HT(5HT2A/5HT2B)Adrenergic(α1)ContractionSmooth muscle(bronchi)5HT(5HT2A)Adrenergic(α1)Ach(M1/M3)BronchoconstrictionSmooth muscle(ureter/ urinary bladder/urethral sphincter)Adrenergic(α1) ContractionSmooth muscleIris dilator Adrenergic(α1) ContractionIris constrictor/ Ciliary Ach(M3) ConstrictionPlatelets 5HT(5HT2A) Aggregation
  • Cell type Activators EffectsCardiomyocytes Adrenergic(α1) Positive ionotropiceffectHepatocyte Adrenergic(α1) Glycogenolysis,GluconeogenesisAdipocyte Adrenergic(α1) Glycogenolysis,GluconeogenesisProximalConvoluted tubuleAngiotensin II (AT1)Adrenergic (α1)Stimulate NHE3→H+ secretion & Na+reabsorptionStimulatebasolateral Na+-K+ATPase →Na+ reabsorption
  •  IP3 Receptor Ligand gated Ca2+ channel High conc. in membrane of ER Ligands which regulate:1. PKA:↑ Ca2+ release by phosphorylation2. PKG:• Inhibits Ca2+ release3. IP3:• ↑ Ca2+ release4. Ca2+:• Conc of 100-300nM→ ↑ Ca2+ release• Conc of 1000nM→inhibits Ca2+ release• Oscillatory pattern of Ca2+ release
  •  Ryanodine receptor Present in skeltetal & cardiac muscles& neurons Major cellular mediator ofcalcium-induced calcium release Ca2+ enters through L-type Ca Channels Conformational change in RyR receptors Release of Ca2+ from SR into cytosol Agonist: Xanthines (Caffeine, Pentoxyfylline) Antagonist: Dantrolene
  •  Calcium Reuptake Na+/Ca2+ exchanger on plasma membrane Ca2+ pump on ER membrane Ca2+ binding molecules Ca2+ pump on Mitochondia
  • Cell Type EffectSecretory Cells (mostly) ↑secretion (vesicle fusion)Juxtaglomerular cells ↓secretionParathyroid chief cells ↓secretionNeuronstransmission (vesiclefusion)T-cellsactivation in response toantigen presentationMyocytes Contraction (TroponinC)Effects of Ca2+ :
  • Calmodulin binds Ca2+ & activates 5 differentCalmodulin-dependent kinases1. Myosin light-chain kinase→ Phosphorylates myosin→Contraction in smooth muscle2. CaMK I → synaptic function3. CaMK II → neurotransmitter secretion, transcription factorregulation & glycogen metabolism4. CaMK III → protein synthesis5. Calcineurin, a phosphatase that inactivates Ca2+ channelsby dephosphorylating prominent role in activating T cells →inhibited by some immunosuppressants
  • Therapeutic Applications:Drug Mechanism ofActionIP3/DAGLevelsTherapeuticApplicationPrazosin α1 blocker ↓ Hypertension/ProstateHyperplasiaChlorpheni-ramineH1 blocker ↓ Allergies/Common coldIpratropiumbromideM3 blocker ↓ Asthma/ COPDLosartan AT1 Receptorblocker↓ Hypertension/MI/ DiabeticNephropathyMontelukast LT C4/D4 blocker ↓ AsthmaOxytocin Direct action onGq↑ Labour induction/Uterine inertia
  • Cell surface receptors recruit activity of protein kinases in two general ways:Receptor Tyrosine Kinases:Possess an intrinsic tyrosine kinase activity that is part of the receptor proteinExamples include receptors for growth factors (PDGF, EGF, insulin, etc.)Non-receptor tyrosine kinases:Receptors lacking self-contained kinase functionrecruit activities of intracellular protein kinases to the plasma membrane
  • Receptor Tyrosine Kinases Implicated in diverse cellular responses:Cell division, Differentiation & Motility At least 50 RTKs identified:Subdivided into 10 subclasses based ondifferences within extracellular, ligand-binding domain of receptor
  • Structure:Four common structural features sharedamong RTKs Extracellular ligand-binding domain Single transmembrane domain Cytoplasmic tyrosine kinase domain(s) Regulatory domains
  • Receptor DimerisationThree ways in which signaling proteins can cross-link receptorchains1. Dimer ligand2. Monomer but brought together by proteoglycan3. Cluster on membrane
  •  Receptor dimerization leads to activation ofcatalytic domains causingautotransphosphorylation Receptor autotransphosphorylation:• Further stimulates kinase activity• Leads to phosphorylation of additionalproteins involved in receptorsignalling pathway
  • Provides “docking sites” for downstreamsignalling proteins(Grb2, PI3-kinase, phospholipase C , etc.)Src homology (SH) 2 & SH3 domains:SH2 domains: bind P-Tyr-containing sequencesSH3 domains: bind to pro-rich (PxxP) sequencesActivates Phospholipase CƔThe binding of SH2-containing intracellular signalingproteins to an activated PDGF receptor
  • RTK mediated pathways:1. Ras-Raf-MAP kinase pathwayThe activation of Ras by RTK signaling
  • The MAP-kinase regulated by Ras
  • Ras-Raf-MAP kinase pathway in Cancer Ras gene mutation→defective Ras protein GAP binds to GTP bound Ras, butnot able to provide domain for GTPase GTP is not lysed & the GTP boundRas remains continuously active Thus permanently activated MAP kinase pathwayresults in growth factors transcription causing continuous cellproliferation Development of Cancer
  • 2. PI3 Kinase Pathway Activated PI3 docks at thephosphorylated RTK Brings about phosphorylation ofPIP2→PIP3 Downstream effects:• Inhibits proapoptotic protein BAX• Translation of tumour proteins byactivation of mTOR• Phosphorylation of FOXFO, antitumour protein,→ ubiquitinisation→ degradation
  • Therapeutic ApplicationsI. Receptor Tyrosine Kinase Inhibitors:A. Epidermal Growth Factor Receptor Inhibitor/ HER 1 InhibitorGeftinib:• Inhibits EGFR tyrosine kinase activity• Blocks ATP binding site• NSCLC pts. who have failed with std. chemotherapy Erlotinib• Similar mechanism of action• Locally advanced or metastatic NSCL & Pancreatic Ca
  •  Cetuximab:• Monoclonal antibody to extracellular domain of EGFR• Combined with Radiation for Locally advancedSquamous Cell Ca of head & neck• EGFR positive metastatic Colorectal Ca Panitumumab:• Recombinant fully humanized IgG to extracellular domainof EGFR• EGFR positive metastatic Colorectal Ca
  • B. HER2/neu Inhibitor Lapatinib• Inhibits EGFR & HER2/neu Kinase activity• ATP binding pocket• Approved for Trastuzumab Refractory breast Ca withCapecitabine Trastuzumab:• Humanized monoclonal Antibody to external domainof HER2/neu receptor• Her2/neu overexpressing metastatic breast Cawith Paclitaxel
  • II. mTOR inhibitors: IL-2 stimulates immune system by activation of T Cells viaactivation of mTORSirolimus (Rapamycin) Binds to mTOR & inhibits action of IL-2 Immunosuppressive agent in organ transplant & GVHD Cardiac stents to ↓ chances of re-occlusion A/E: thrombocytopenia, hyperlipidemia, HUSEverolimus Short t1/2 Cardiac transplants
  •  JAK-STAT Receptor Pathway Ligands: Interferon Ɣ, Growth hormone, Prolactin Receptors have no intrinsic activity Intracellular domain binds intracellular tyrosine kinase→Janus Kinase (JAK) Receptor mediated dimerization → phosphorylation ofSignal transducers & activators of transcription (STAT) STATs translocate to nucleus & regulate transcription 4 JAKs & 6 STATs combine differently depending onCell type & signal Eg: Prolactin→ JAK1, JAK2 & STAT5
  • Therapeutic Application: Lestaurtinib:• Janus Kinase 2 Inhibitor• JAK/STAT signaling exaggerated in MPNs• Polycythemia vera, essential thrombocythemia& primary myelofibrosis• Mutant JAK2 activity• Inhibits wild type JAK2 kinase activity• Inhibits proliferation MPD cells• Phase II AML & Myeloproliferative disorders
  •  Receptor Serine-Threonine Kinases: Anologous to RTK except they haveSerine/Threonine kinase domain in cytoplasmic region Ligand: TGFβ Dimerizes in presence of ligand→Phosphorylation of kinase domain→ activation Phosphorylation of gene regulatory protein termedSmad on serine residue Dissociates from receptor→ associates with transcription factors→ Morphogenesis & transformation Inhibitory Smads: Smad6/7
  •  Toll like Receptors Signaling related to innate immunity Family of 10 receptors Structure:• Single polypeptide chain• Large extracellular ligand binding domain• Short membrane spanning domain• Cytoplasmic TIR domain Ligands: Pathogens (lipids, peptidoglycans, lipopeptides,viruses) Inflammatory response to pathogen
  • Signaling: Receptor induced dimerisation Recruitment of Mal & MyD88 to TIR Recruits Interleukin-associated kinases(IRAKs) Auotphosphorylates & complex with MyD88 Also recruits TRAF6 Interact with protein kinase TAK1 & Adaptor TAB1 Activates NF-КB Trasncription of inflammatory genes
  • TNFα Receptors Structure:• Single membrane spanning receptor• Extracelluler ligand binding domain• Transmembrane domain• Cytoplasmic domain (death domain) 2 types: TNF1 (most cells) & TNF 2 (immune cells) Activated by trimerisation
  • Has following effects: Activation of NF-κB:• Transcription of proteins involved in cell survival andproliferation, inflammatory response & anti-apoptotic factors Activation of the MAPK pathways:• The JNK pathway is involved in cell differentiation,proliferation & is pro-apoptotic Induction of death signalling:• Cell apoptosis
  • Therapeutic Application Promotes inflammatory response & associated withautoimmune disorders:(Rheumatoid arthritis, ankylosing spondylitis, inflammatorybowel disease, psoriasis, hidradenitis suppurativa andrefractory asthma) Treated by using a TNF inhibitor:• Monoclonal antibodies such asInfliximab, Adalimumab & certolizumab pegol• Circulating receptor fusion protein such as etanercept
  • Pharmacodynamic Interactions in a MulticellularContext Consider the vascular wall of an arteriole Several cells interact at this site:Smooth muscle cells(SMC), endotheial cells, platelets & post-ganglionic parasympathetic neurons Effects produced• SMC contraction→ Ang II, NE• SMC relaxation→ NO, BNP, epinephrine• Alter gene expression→ PDGF, Ang II, NE, Eicosanoids
  •  Patient with hypertension• ↑ levels of AngII• ↑ activity of sympathetic nervous system• ↓ NO production Pharmacotherapy directed towards:• ↓ BP• Prevent long term changes in vessel wall Drugs used to treat hypertension• ↓ Ang II (Atenolol/Aliskiren/Enalapril/Losartan)• α1 blockers→ ↓ NE binding on SMCs• ↑ NO production (Na Nitroprusside)
  • Conclusion: Second messengers have shown to play physiological &pathophysiological settings Evolved as targets for drug develoment for numerous diseasesHowever, availability of compounds acting on specific targets isthe biggest challenge Also is their difficult expression & purification Such specific drugs are still in their early stages of development„Today‟s drug targets, tomorrow‟s blockbusters‟