1. 1 © Patrick An Introduction to Medicinal Chemistry 3/e Chapter 6 PROTEINS AS DRUG TARGETS: RECEPTOR STRUCTURE & SIGNAL TRANSDUCTION Part 2: Sections 6.3 - 6.6

2. 1 © Contents 3.G-protein-coupled receptors (7-TM receptors) 3.1.Structure - Single protein with 7 transmembrane regions 3.2.Ligands 3.3.Ligand binding site - varies depending on receptor type 3.4.Bacteriorhodopsin & rhodopsin family (2 slides) 3.5.Receptor types and subtypes (2 slides) 3.6.Signal transduction pathway a)Interaction of receptor with Gs-protein (3 slides) b)Interaction of  s with adenylate cyclase (2 slides) c)Interaction of cyclic AMP with protein kinase A (PKA) (4 slides) 3.7.Glycogen metabolism - triggered by adrenaline in liver cells (2 slides) 3.8.GI proteins 3.9.Phosphorylation 3.10.Drugs interacting with cyclic AMP signal transduction 3.11.Signal transduction involving phospholipase C (PLC) (2 slides) 3.12.Action of diacylglycerol (2 slides) 3.13.Action of inositol triphosphate (2 slides) 3.14.Resynthesis of PIP2 [29 slides]

3. 1 © 3. G-protein-coupled receptors (7-TM receptors) 3.1 Structure - Single protein with 7 transmembrane regions Transmembrane helix C-Terminal chain G-Protein binding region Variable intracellular loop Extracellular loops Intracellular loops N-Terminal chain HO 2 C NH 2 VIIVI VIVIIIIII Membrane

4. 1 © 3.2 Ligands Monoamines e.g. dopamine, histamine, noradrenaline, acetylcholine (muscarinic)Monoamines e.g. dopamine, histamine, noradrenaline, acetylcholine (muscarinic) NucleotidesNucleotides LipidsLipids HormonesHormones GlutamateGlutamate Ca ++Ca ++ 3. G-protein-coupled receptors (7-TM receptors)

5. 1 © 3.3 Ligand binding site - varies depending on receptor type A) Monoamines - pocket in TM helices B) Peptide hormones - top of TM helices + extracellular loops + N-terminal chain C) Hormones - extracellular loops + N-terminal chain D) Glutamate - N-terminal chain Ligand BDCA 3. G-protein-coupled receptors (7-TM receptors)

6. 1 © 3.4 Bacteriorhodopsin & rhodopsin family Rhodopsin = visual receptorRhodopsin = visual receptor Many common receptors belong to this same familyMany common receptors belong to this same family Implications for drug selectivity depending on similarity (evolution)Implications for drug selectivity depending on similarity (evolution) Membrane bound receptors difficult to crystalliseMembrane bound receptors difficult to crystallise X-Ray structure of bacteriorhodopsin solved - bacterial protein similar to rhodopsinX-Ray structure of bacteriorhodopsin solved - bacterial protein similar to rhodopsin Bacteriorhodopsin structure used as ‘template’ for other receptorsBacteriorhodopsin structure used as ‘template’ for other receptors Construct model receptors based on template and amino acid sequenceConstruct model receptors based on template and amino acid sequence Leads to model binding sites for drug designLeads to model binding sites for drug design Crystal structure for rhodopsin now solved - better templateCrystal structure for rhodopsin now solved - better template 3. G-protein-coupled receptors (7-TM receptors)

7. 1 © 3.4 Bacteriorhodopsin & rhodopsin family

8. 1 © 3.5 Receptor types and subtypes Reflects differences in receptors which recognise the same ligand ReceptorTypesSubtypes Alpha (  ) Beta (  )  1,  2A,  2B,  2C  1,  2,  3 AdrenergicMuscarinic NicotinicMuscarinic     3. G-protein-coupled receptors (7-TM receptors)

9. 1 © Receptor types and subtypes not equally distributed amongst tissues.Receptor types and subtypes not equally distributed amongst tissues. Target selectivity leads to tissue selectivityTarget selectivity leads to tissue selectivity Heart muscle -  1 adrenergic receptors Fat cells -  3 adrenergic receptors Bronchial muscle -  1 &  2 adrenergic receptors GI-tract -  1  2 &  2 adrenergic receptors 3. G-protein-coupled receptors (7-TM receptors) 3.5 Receptor types and subtypes

10. 1 © 3.6 Signal transduction pathway a) Interaction of receptor with G s -protein G S -Protein - membrane bound protein of 3 subunits (  ) -  S subunit has binding site for GDP -GDP bound non covalently   GDP 3. G-protein-coupled receptors (7-TM receptors)

11. 1 © ß   GDPGTP LigandbindingInducedfit G-proteinbindsInduced fit for G-protein G-Protein alters shape GDP binding site distorted GDP binding weakened GDP departs ß   Ligand Receptor G Protein Cell membrane ß   Binding site for G-protein opens = GDP a) Interaction of receptor with G s -protein 3. G-protein-coupled receptors (7-TM receptors) 3.6 Signal transduction pathway

12. 1 © ß   Binding site recognises GTP GTP binds Induced fit G-protein alters shape Complex destabilised Fragmentation and release ß   Process repeated for as long as ligand bound to receptor Process repeated for as long as ligand bound to receptor Signal amplification - several G-proteins activated by one ligand Signal amplification - several G-proteins activated by one ligand  s Subunit carries message to next stage  s Subunit carries message to next stage ß   a) Interaction of receptor with G s -protein 3. G-protein-coupled receptors (7-TM receptors) 3.6 Signal transduction pathway

13. 1 © b) Interaction of  s with adenylate cyclase  s Subunit recombines with  dimer to reform G s protein Active site (closed) Binding site for  s subunit cyclic AMP ATP Binding Induced fit Active site (open) P cyclic AMP ATP GTP hydrolysed to GDP catalysed by  s subunit  s -subunit Adenylate cyclase GTP GDP  s Subunit changes shape Weaker binding to enzyme Departure of subunit Enzyme reverts to inactive state Active site (closed) Signaltransduction(con) 3. G-protein-coupled receptors (7-TM receptors) 3.6 Signal transduction pathway

14. 1 © Several 100 ATP molecules converted before  s -GTP deactivatedSeveral 100 ATP molecules converted before  s -GTP deactivated Represents another signal amplificationRepresents another signal amplification Cyclic AMP becomes next messenger (secondary messenger)Cyclic AMP becomes next messenger (secondary messenger) Cyclic AMP enters cell cytoplasm with messageCyclic AMP enters cell cytoplasm with message b) Interaction of  s with adenylate cyclase 3. G-protein-coupled receptors (7-TM receptors) 3.6 Signal transduction pathway

15. 1 © c) Interaction of cyclic AMP with protein kinase A (PKA) Protein kinase A = serine-threonine kinaseProtein kinase A = serine-threonine kinase Activated by cyclic AMPActivated by cyclic AMP Catalyses phosphorylation of serine and threonine residues on protein substratesCatalyses phosphorylation of serine and threonine residues on protein substrates Phosphate unit provided by ATPPhosphate unit provided by ATP 3. G-protein-coupled receptors (7-TM receptors) 3.6 Signal transduction pathway

16. 1 © cyclic AMP ATP Adenylatecyclase Enzyme(active)P Enzyme(inactive) Chemical reaction Proteinkinase Activation c) Interaction of cyclic AMP with protein kinase A (PKA) 3. G-protein-coupled receptors (7-TM receptors) 3.6 Signal transduction pathway

17. 1 © c) Interaction of cyclic AMP with protein kinase A (PKA) Protein kinase A Protein kinase A - 4 protein subunits - 2 regulatory subunits (R) and 2 catalytic subunits (C) Cyclic AMP binds to PKA Induced fit destabilises complex Catalytic units released and activated NoteCC R R cAMP cAMPbindingsites catalytic subunit R R C C 3. G-protein-coupled receptors (7-TM receptors) 3.6 Signal transduction pathway

18. 1 © Phosphorylation of other proteins and enzymes Signal continued by phosphorylated proteins Further signal amplification C Protein + ATP Protein + ADP P c) Interaction of cyclic AMP with protein kinase A (PKA) 3. G-protein-coupled receptors (7-TM receptors) 3.6 Signal transduction pathway

19. 1 © 3.7 Glycogen metabolism - triggered by adrenaline in liver cells 3. G-protein-coupled receptors (7-TM receptors)

20. 1 © Coordinated effect - activation of glycogen metabolism - inhibition of glycogen synthesis Adrenaline has different effects on different cells - activates fat metabolism in fat cells 3. G-protein-coupled receptors (7-TM receptors) 3.7 Glycogen metabolism - triggered by adrenaline in liver cells

21. 1 © 3.8 G I proteins Binds to different receptors from those used by G s proteinBinds to different receptors from those used by G s protein Mechanism of activation by splitting is identicalMechanism of activation by splitting is identical  I subunit binds adenylate cyclase to inhibit it  I subunit binds adenylate cyclase to inhibit it Adenylate cyclase under dual control (brake/accelerator)Adenylate cyclase under dual control (brake/accelerator) Background activity due to constant levels of  s and  iBackground activity due to constant levels of  s and  i Overall effect depends on dominant G-ProteinOverall effect depends on dominant G-Protein Dominant G-protein depends on receptors activatedDominant G-protein depends on receptors activated 3. G-protein-coupled receptors (7-TM receptors)

22. 1 © 3.9 Phosphorylation Prevalent in activation and deactivation of enzymesPrevalent in activation and deactivation of enzymes Phosphorylation radically alters intramolecular bindingPhosphorylation radically alters intramolecular binding Results in altered conformationsResults in altered conformations O NH 3 O P O O O O NH 3 H O Active site closed open NH 3 O O P O O O 3. G-protein-coupled receptors (7-TM receptors)

23. 1 © 3.10 Drugs interacting with cyclic AMP signal transduction Cholera toxin - constant activation of c.AMP - diahorrea Theophylline and caffeine - inhibit phosphodiesterases - phosphodiesterases responsible for metabolising cyclic AMP - cyclic AMP activity prolonged 3. G-protein-coupled receptors (7-TM receptors)

24. 1 © 3.11 Signal transduction involving phospholipase C (PLC) G q proteins - interact with different receptors from G S and G I G q proteins - interact with different receptors from G S and G I Split by same mechanism to give  q subunit Split by same mechanism to give  q subunit  q Subunit activates or deactivates PLC (membrane bound enzyme)  q Subunit activates or deactivates PLC (membrane bound enzyme) Reaction catalysed for as long as  q bound - signal amplification Reaction catalysed for as long as  q bound - signal amplification Brake and accelerator Brake and accelerator  Active site (closed)PLC (open) PLC  PLC PIP 2 Binding weakened GTP hydrolysis  q departs Active site (closed)enzymedeactivated  PLC DG IP 3  PLC PIP 2 DG IP 3 Phosphate 3. G-protein-coupled receptors (7-TM receptors)

25. 1 © 3.11 Signal transduction involving phospholipase C (PLC) Phosphatidylinositol diphosphate (integral part of cell membrane) Inositol triphosphate (polar and moves into cell cytoplasm) Diacylglycerol (remains in membrane) R= long chain hydrocarbons 3. G-protein-coupled receptors (7-TM receptors)

26. 1 © 3.12 Action of diacylglycerol Activates protein kinase C (PKC)Activates protein kinase C (PKC) PKC moves from cytoplasm to membranePKC moves from cytoplasm to membrane Phosphorylates enzymes at Ser & Thr residuesPhosphorylates enzymes at Ser & Thr residues Activates enzymes to catalyse intracellular reactionsActivates enzymes to catalyse intracellular reactions Linked to inflammation, tumour propagation, smooth muscle activity etcLinked to inflammation, tumour propagation, smooth muscle activity etc PKC DG Binding site for DG Cell membrane Cytoplasm PKC moves to membrane PKC DG Cytoplasm DG binds to DG binding site Active site closed PKC DG Cytoplasm Induced fit opens active site Enzyme (inactive) Enzyme (active) Chemical reaction 3. G-protein-coupled receptors (7-TM receptors)

27. 1 © Drugs inhibiting PKC - potential anti cancer agents 3.12 Action of diacylglycerol Bryostatin (from sea moss) 3. G-protein-coupled receptors (7-TM receptors)

28. 1 © 3.13 Action of inositol triphosphate IP 3 - hydrophilic and enters cell cytoplasm IP 3 - hydrophilic and enters cell cytoplasm Mobilises Ca 2+ release in cells by opening Ca 2+ ion channels Mobilises Ca 2+ release in cells by opening Ca 2+ ion channels Ca 2+ activates protein kinases Ca 2+ activates protein kinases Protein kinases activate intracellular enzymes Protein kinases activate intracellular enzymes Cell chemistry altered leading to biological effect Cell chemistry altered leading to biological effect 3. G-protein-coupled receptors (7-TM receptors)

29. 1 © IP 3 Calciumstores Ca ++ Calmodulin Calmodulin Activation Proteinkinase Activation Proteinkinase Enzyme(inactive)Enzyme(active)P Cytoplasm Cell membrane Enzyme(active)Enzyme(inactive)P Chemicalreaction Chemicalreaction 3.13 Action of inositol triphosphate 3. G-protein-coupled receptors (7-TM receptors)

30. 1 © 3.14 Resynthesis of PIP 2 IP 3 + DG PIP 2 severalsteps Li + salts Inhibition Lithium salts used vs manic depression 3. G-protein-coupled receptors (7-TM receptors)