* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Topic 4 Proteins as Drug Targets
Survey
Document related concepts
Polyclonal B cell response wikipedia , lookup
Evolution of metal ions in biological systems wikipedia , lookup
Western blot wikipedia , lookup
Mitogen-activated protein kinase wikipedia , lookup
Drug design wikipedia , lookup
Two-hybrid screening wikipedia , lookup
Ultrasensitivity wikipedia , lookup
Metalloprotein wikipedia , lookup
NMDA receptor wikipedia , lookup
Lipid signaling wikipedia , lookup
Biochemical cascade wikipedia , lookup
Ligand binding assay wikipedia , lookup
Endocannabinoid system wikipedia , lookup
Paracrine signalling wikipedia , lookup
G protein–coupled receptor wikipedia , lookup
Transcript
Topic 4 Proteins as Drug Targets Receptors-Chapters 5 and 6 Patrick and Corey 78-80 Contents 1. Structure and function of receptors 1.1. Chemical Messengers 1.2. Mechanism 2. 3. The binding site Messenger binding 3.1. Introduction 3.2. Bonding forces Overall process of receptor/messenger interaction Signal transduction 5.1. Control of ion channels 4. 5. 6. 7. 8. 9. 10. 5.2. Activation of signal proteins 5.3. Activation of enzyme active site Competitive (reversible) antagonists Non competitive (irreversible) antagonists Non competitive (reversible) allosteric antagonists Antagonists by umbrella effect Agonists 1. Structure and function of receptors • Globular proteins acting as a cell’s ‘letter boxes’ • Located mostly in the cell membrane • Receive messages from chemical messengers coming from other cells • Transmit a message into the cell leading to a cellular effect • Different receptors specific for different chemical messengers • Each cell has a range of receptors in the cell membrane making it responsive to different chemical messengers 1. Structure and function of receptors Nerve Nerve Signal Messenger Receptor Response Nucleus Cell Cell 1. Structure and function of receptors Chemical Messengers Neurotransmitters: Chemicals released from nerve endings which travel across a nerve synapse to bind with receptors on target cells, such as muscle cells or another nerve. Usually short lived and responsible for messages between individual cells Hormones: Chemicals released from cells or glands and which travel some distance to bind with receptors on target cells throughout the body • Chemical messengers ‘switch on’ receptors without undergoing a reaction 1. Structure and function of receptors Nerve 1 Blood supply Nerve 2 Hormone Neurotransmitters 1. Structure and function of receptors Mechanism • Receptors contain a binding site (hollow or cleft in the receptor surface) that is recognised by the chemical messenger • Binding of the messenger involves intermolecular bonds • Binding results in an induced fit of the receptor protein • Change in receptor shape results in a ‘domino’ effect • Domino effect is known as Signal Transduction, leading to a chemical signal being received inside the cell • Chemical messenger does not enter the cell. It departs the receptor unchanged and is not permanently bound 1. Structure and function of receptors Mechanism Induced fit Messenger Messenger Messenger Cell Membrane Receptor Receptor Cell Cell Receptor Cell message Message 2. The binding site • A hydrophobic hollow or cleft on the receptor surface - equivalent to the active site of an enzyme • Accepts and binds a chemical messenger • Contains amino acids which bind the messenger • No reaction or catalysis takes place Binding site Binding site ENZYME 3. Messenger binding 3.1 Introduction Messenger M Induced fit • Binding site is nearly the correct shape for the messenger • Binding alters the shape of the receptor (induced fit) • Altered receptor shape leads to further effects - signal transduction 3. Messenger binding 3.2 Bonding forces • • • Ionic H-bonding van der Waals Example: vdw interaction H-bond Binding site O Ser H ionic bond CO2 Asp Receptor Phe 3. Substrate binding 3.2 Bonding forces • Induced fit - Binding site alters shape to maximise intermolecular bonding Phe Phe O O H Ser Ser CO2 Asp Intermolecular bonds not optimum length for maximum binding strength Induced Fit H CO2 Asp Intermolecular bond lengths optimised 4. Overall process of receptor/messenger interaction M M M RE R RE Signal transduction • • • Binding interactions must be: - strong enough to hold the messenger sufficiently long for signal transduction to take place - weak enough to allow the messenger to depart Implies a fine balance Drug design - designing molecules with stronger binding interactions results in drugs that block the binding site - antagonists 5. Signal transduction 5.1 Control of ion channels • Receptor protein is part of an ion channel protein complex • Receptor binds a messenger leading to an induced fit • Ion channel is opened or closed • Ion channels are specific for specific ions (Na+, Ca2+, Cl-, K+) • Ions flow across cell membrane down concentration gradient • Polarises or depolarises nerve membranes • Activates or deactivates enzyme catalysed reactions within cell 5. Signal transduction 5.1 Control of ion channels Hydrophilic tunnel Cell membrane 5. Signal transduction 5.1 Control of ion channels Receptor Binding site Cell membrane Five glycoprotein subunits traversing cell membrane Messenger Induced fit Cell membrane ‘Gating’ (ion channel opens) Cationic ion channels for K+, Na+, Ca2+ (e.g. nicotinic) = excitatory Anionic ion channels for Cl- (e.g. GABAA) = inhibitory 5. Signal transduction 5.1 Control of ion channels: MESSENGER ION CHANNEL (closed) Cell membrane Cell Ion channel RECEPTOR BINDING SITE Lock Gate Ion channel ION CHANNEL (open) MESSENGER Induced fit and opening of ion channel Cell membrane Cell membrane Cell Ion channel Ion channel Cell membrane 5. Signal transduction 5.2 Activation of signal proteins • Receptor binds a messenger leading to an induced fit • Opens a binding site for a signal protein (G-protein) • G-Protein binds, is destabilised then split messenger induced fit closed open G-protein split 5. Signal transduction 5.2 Activation of signal proteins • G-Protein subunit activates membrane bound enzyme Binds to allosteric binding site Induced fit results in opening of active site • Intracellular reaction catalysed Enzyme Enzyme active site (closed) active site (open) Intracellular reaction 5. Signal transduction 5.3 Activation of enzyme active site • Protein serves dual role - receptor plus enzyme • Receptor binds messenger leading to an induced fit • Protein changes shape and opens active site • Reaction catalysed within cell messenger messenger induced fit closed active site open intracellular reaction closed 6. Competitive (reversible) antagonists M An An RE • • • • • • • R Antagonist binds reversibly to the binding site Intermolecular bonds involved in binding Different induced fit means receptor is not activated No reaction takes place on antagonist Level of antagonism depends on strength of antagonist binding and concentration Messenger is blocked from the binding site Increasing the messenger concentration reverses antagonism 7. Non competitive (irreversible) antagonists X Covalent Bond X OH OH O Irreversible antagonism • • • • • Antagonist binds irreversibly to the binding site Different induced fit means that the receptor is not activated Covalent bond is formed between the drug and the receptor Messenger is blocked from the binding site Increasing messenger concentration does not reverse antagonism 8. Non competitive (reversible) allosteric antagonists Binding site unrecognisable Binding site ACTIVE SITE (open) Receptor ENZYME Allosteric site Induced fit (open) Receptor ENZYME Antagonist • • • • • Antagonist binds reversibly to an allosteric site Intermolecular bonds formed between antagonist and binding site Induced fit alters the shape of the receptor Binding site is distorted and is not recognised by the messenger Increasing messenger concentration does not reverse antagonism 9. Antagonists by umbrella effect • • • • Antagonist binds reversibly to a neighbouring binding site Intermolecular bonds formed between antagonist and binding site Antagonist overlaps with the messenger binding site Messenger is blocked from the binding site messenger Binding site for antagonist Binding site for messenger Receptor Antagonist Receptor 10. Agonists • • • • • Agonist binds reversibly to the binding site Similar intermolecular bonds formed as to natural messenger Induced fit alters the shape of the receptor in the same way as the normal messenger Receptor is activated Agonists are often similar in structure to the natural messenger Agonist Agonist Agonist Induced fit RE R RE Signal transduction Contents Part 1: Sections 6.1 - 6.2 1. Receptor superfamilies 2. Ion channel receptors (Ligand gated ion channels) 2.1. General structure 2.2. Structure of protein subunits (4-TM receptor subunits) 2.3. Detailed structure of ion channel 2.4. Gating 1. Receptor superfamilies RESPONSE TIME • ION CHANNEL RECEPTORS • G-PROTEIN COUPLED RECEPTORS • KINASE LINKED RECEPTORS • INTRACELLULAR RECEPTORS msecs MEMBRANE BOUND seconds minutes 2. Ion channel receptors (Ligand gated ion channels) 2.1 General structure Receptor Binding site Messenger Cell membrane INDUCED FIT ‘GATING’ (ion channel opens) Cell membrane Five glycoprotein subunits traversing cell membrane Cationic ion channels for K+, Na+, Ca2+ (e.g. nicotinic) = excitatory Anionic ion channels for Cl− (e.g. GABAA) = inhibitory 2. Ion channel receptors (Ligand gated ion channels) Transverse view (nicotinic receptor) Binding sites Ion channel α β Cell membrane δ α α γ γ α β δ 2xα, β, γ, δ subunits Two ligand binding sites mainly on α-subunits 2. Ion channel receptors (Ligand gated ion channels) Transverse view (glycine receptor) Binding sites Ion channel α α β α Cell membrane β β α α α β 3xα, 2x 2x β subunits Three ligand binding sites on α-subunits 2. Ion channel receptors (Ligand gated ion channels) 2.2 Structure of protein subunits (4-TM receptor subunits) Neurotransmitter binding region Extracellular loop H2N CO2H Cell membrane TM1 Intracellular loop TM2 TM3 TM4 Variable loop 4 Transmembrane (TM) regions (hydrophobic) 2. Ion channel receptors (Ligand gated ion channels) 2.3 Detailed structure of ion channel Protein subunits TM4 TM1 TM2 TM3 TM1 TM2 TM2 TM4 TM3 TM3 TM1 TM3 TM4 TM4 TM2 TM2 TM1 TM1 TM3 TM4 Transmembrane regions Note: TM2 of each protein subunit ‘lines’ the central pore 2. Ion channel receptors (Ligand gated ion channels) 2.4 Gating Neurotransmitter binds Induced fit at binding site ‘Domino effect’ Rotation of 2TM regions of each protein subunit Ion flow TM2 Cell membrane TM2 TM2 TM2 TM2 TM2 TM2 TM2 Transverse view of TM2 subunits TM2 TM2 TM2 TM2 Closed Open Transverse view of TM2 subunits 2. Ion channel receptors (Ligand gated ion channels) 2.4 Gating • Fast response measured in msec • Ideal for transmission between nerves • Binding of messenger leads directly to ion flows across cell membrane • Ion flow = secondary effect (signal transduction) • Ion concentration within cell alters • Leads to variation in cell chemistry Contents Part 2: Sections 6.3 - 6.6 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 3.5. Receptor types and subtypes 3.6. Signal transduction pathway a) Interaction of receptor with Gs-protein b) Interaction of αs with adenylate cyclase c) Interaction of cyclic AMP with protein kinase A (PKA) 3.7. Glycogen metabolism - triggered by adrenaline in liver cells 3.8. GI proteins 3.9. Phosphorylation 3.10. Drugs interacting with cyclic AMP signal transduction 3.11. Signal transduction involving phospholipase C (PLC) 3.12. Action of diacylglycerol 3.13. Action of inositol triphosphate 3.14. Resynthesis of PIP2 3. G-protein-coupled receptors (7-TM receptors) 3.1 Structure - Single protein with 7 transmembrane regions Extracellular loops NH2 N -Terminal chain Membrane VII VI V IV III II I G-Protein binding region HO2 C C -Terminal chain Variable intracellular loop Intracellular loops Transmembrane helix 3. G-protein-coupled receptors (7-TM receptors) 3.2 Ligands • Monoamines e.g. dopamine, histamine, noradrenaline, acetylcholine (muscarinic) • Nucleotides • Lipids • Hormones • Glutamate • Ca++ 3. G-protein-coupled receptors (7-TM receptors) 3.3 Ligand binding site - varies depending on receptor type Ligand A B C D 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 3. G-protein-coupled receptors (7-TM receptors) 3.4 Bacteriorhodopsin & rhodopsin family • Rhodopsin = visual receptor • Many common receptors belong to this same family • Implications for drug selectivity depending on similarity (evolution) • Membrane bound receptors difficult to crystallise • X-Ray structure of bacteriorhodopsin solved - bacterial protein similar to rhodopsin • Bacteriorhodopsin structure used as ‘template’ for other receptors • Construct model receptors based on template and amino acid sequence • Leads to model binding sites for drug design • Crystal structure for rhodopsin now solved - better template 3. G-protein-coupled receptors (7-TM receptors) 3.4 Bacteriorhodopsin & rhodopsin family Common ance stor Monoamines muscarinic beta alpha Opsins, Rhodopsins Bradykinin, Endothelins Angiotensin.Tachyki nins Interleukin-8 2 4 5 Muscarinic Receptor types 3 1 H1 H2 Histamine 1 2A 2B 2C D4 D3 D2 D1A D1B D5 !-Adrenergic Dopaminergic 3 2 1 "-Adrenergic Receptor sub-types 3. G-protein-coupled receptors (7-TM receptors) 3.5 Receptor types and subtypes Reflects differences in receptors which recognise the same ligand Receptor Types Adrenergic Alpha (α) Beta (β) Acetylcholine Nicotinic Muscarinic Subtypes α1, α2A, α2B, α2C β1, β2, β3 Μ1−Μ5 3. G-protein-coupled receptors (7-TM receptors) 3.5 Receptor types and subtypes • Receptor types and subtypes not equally distributed amongst tissues. • Target selectivity leads to tissue selectivity Heart muscle - β1 adrenergic receptors Fat cells - β3 adrenergic receptors Bronchial muscle - α1& β2 adrenergic receptors GI-tract - α1 α2 & β2 adrenergic receptors 3. G-protein-coupled receptors (7-TM receptors) 3.6 Signal transduction pathway a) Interaction of receptor with Gs-protein GS-Protein - membrane bound protein of 3 subunits (α, β, γ) - αS subunit has binding site for GDP -GDP bound non covalently β γ α GDP 3. G-protein-coupled receptors (7-TM receptors) 3.6 Signal transduction pathway a) Interaction of receptor with Gs-protein Ligand Cell membrane Receptor ß γ α Ligand binding Induced fit G-protein binds ß γ α G Protein ß γ α GDP Binding site for G-protein opens = GDP Induced fit for G-protein GTP G-Protein alters shape GDP binding site distorted GDP binding weakened GDP departs 3. G-protein-coupled receptors (7-TM receptors) 3.6 Signal transduction pathway a) Interaction of receptor with Gs-protein ß γ α GTP binds Binding site recognises GTP γ ß γ α Fragmentation and release ß α Induced fit G-protein alters shape Complex destabilised • Process repeated for as long as ligand bound to receptor • Signal amplification - several G-proteins activated by one ligand • αs Subunit carries message to next stage 3. G-protein-coupled receptors (7-TM receptors) 3.6 Signal transduction pathway GTP GDP b) Interaction of αs with adenylate cyclase Binding site for αs subunit αs-subunit Adenylate cyclase GTP hydrolysed to GDP catalysed by αs subunit Binding Induced fit Active site (closed) P ATP cyclic AMP Active site (open) Signal transduction (con) αs Subunit recombines with β,γ dimer to reform Gs protein ATP cyclic AMP Active site (closed) αs Subunit changes shape Weaker binding to enzyme Departure of subunit Enzyme reverts to inactive state 3. G-protein-coupled receptors (7-TM receptors) 3.6 Signal transduction pathway b) Interaction of αs with adenylate cyclase • • • • Several-100 ATP molecules converted before αs-GTP deactivated Represents another signal amplification Cyclic AMP becomes next messenger (secondary messenger) Cyclic AMP enters cell cytoplasm with message NH2 NH2 N N O O O N N HO P O P O P O OH OH OH Adenylate cyclase O H H O H OH N N O H ATP N N P OH O H H Cyclic AMP O OH OH 3. G-protein-coupled receptors (7-TM receptors) 3.6 Signal transduction pathway c) Interaction of cyclic AMP with protein kinase A (PKA) • • • Protein kinase A = serine-threonine kinase Activated by cyclic AMP Catalyses phosphorylation of serine and threonine residues on protein substrates • Phosphate unit provided by ATP O O H N C Protein kinase A H N H N C H H OH O C HC Serine O OH H OH CH3 P HO O O Threonine Protein kinase A H N C HC H O CH3 HO P O OH 3. G-protein-coupled receptors (7-TM receptors) 3.6 Signal transduction pathway c) Interaction of cyclic AMP with protein kinase A (PKA) Adenylate cyclase ATP cyclic AMP Activation Protein kinase P Enzyme (inactive) Enzyme (active) Chemical reaction 3. G-protein-coupled receptors (7-TM receptors) 3.6 Signal transduction pathway c) Interaction of cyclic AMP with protein kinase A (PKA) Protein kinase A - 4 protein subunits - 2 regulatory subunits (R) and 2 catalytic subunits (C) cAMP C catalytic subunit C R cAMP binding sites R R R C C catalytic subunit Note Cyclic AMP binds to PKA Induced fit destabilises complex Catalytic units released and activated 3. G-protein-coupled receptors (7-TM receptors) 3.6 Signal transduction pathway c) Interaction of cyclic AMP with protein kinase A (PKA) C P Protein + ATP Protein + ADP Phosphorylation of other proteins and enzymes Signal continued by phosphorylated proteins Further signal amplification 3. G-protein-coupled receptors (7-TM receptors) 3.7 Glycogen metabolism - triggered by adrenaline in liver cells Adrenaline !s !s !-Adrenoreceptor adenylate cyclase cAMP Glycogen synthase (active) Protein kinase A Inhibitor (inactive) Catalytic C subunit of PKA Glycogen synthase-P (inactive) Phosphorylase kinase (inactive) Inhibitor-P (active) Phosphatase (inhibited) Phosphorylase kinase-P (active) Phosphorylase b (inactive) Phosphorylase a (active) Glycogen Glucose-1-phosphate 3. G-protein-coupled receptors (7-TM receptors) 3.7 Glycogen metabolism - triggered by adrenaline in liver cells 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) 3.8 GI proteins • Binds to different receptors from those used by Gs protein • Mechanism of activation by splitting is identical • αI subunit binds adenylate cyclase to inhibit it • Adenylate cyclase under dual control (brake/accelerator) • Background activity due to constant levels of αs and αi • Overall effect depends on dominant G-Protein • Dominant G-protein depends on receptors activated 3. G-protein-coupled receptors (7-TM receptors) 3.9 Phosphorylation • • • Prevalent in activation and deactivation of enzymes Phosphorylation radically alters intramolecular binding Results in altered conformations NH3 NH3 NH3 O O O H O Active site closed P O O O O O P O O O Active site open 3. G-protein-coupled receptors (7-TM receptors) 3.10 Drugs interacting with cyclic AMP signal transduction Cholera toxin - constant activation of cAMP - diarrhea Theophylline and caffeine - inhibit phosphodiesterases - phosphodiesterases responsible for metabolising cyclic AMP - cyclic AMP activity prolonged O O H3C H N H 3C N CH3 N N N N O N CH3 Theophylline O N CH3 Caffeine 3. G-protein-coupled receptors (7-TM receptors) 3.11 Signal transduction involving phospholipase C (PLC) • • • • • Gq proteins - interact with different receptors from GS and GI Split by same mechanism to give αq subunit αq Subunit activates or deactivates PLC (membrane bound enzyme) Reaction catalysed for as long as αq bound - signal amplification Brake and accelerator Active site (open) Active site (closed) α α PLC DG α PLC PLC PIP2 IP3 GTP hydrolysis DG α Phosphate PLC PIP2 IP3 Binding weakened Active site (closed) αq departs α PLC enzyme deactivated 3. G-protein-coupled receptors (7-TM receptors) 3.11 Signal transduction involving phospholipase C (PLC) R O C R C O O O CH2 CH O P H CH2 HO PLC O O P O H O O P H HO OH H H HO H + O P R R O C O C O O CH2 CH OH O P HO OH IP3 CH2 DG O P PIP2 Phosphatidylinositol diphosphate (integral part of cell membrane) R= long chain hydrocarbons Inositol triphosphate (polar and moves into cell cytoplasm) P = PO3 2- Diacylglycerol (remains in membrane) 3. G-protein-coupled receptors (7-TM receptors) 3.12 Action of diacylglycerol • • • • • Activates protein kinase C (PKC) PKC moves from cytoplasm to membrane Phosphorylates enzymes at Ser & Thr residues Activates enzymes to catalyse intracellular reactions Linked to inflammation, tumour propagation, smooth muscle activity etc Cell membrane DG Binding site for DG DG DG PKC Active site closed PKC Cytoplasm PKC moves to membrane Cytoplasm DG binds to DG binding site PKC Enzyme (inactive) Cytoplasm Enzyme (active) Chemical reaction Induced fit opens active site 3. G-protein-coupled receptors (7-TM receptors) 3.12 Action of diacylglycerol Drugs inhibiting PKC - potential anti cancer agents CHCO2Me CH3CH2CH2 CH CH CH Me CH C Me O O H O MeO2C CH Me H OH C Me O O H OH O O HO O H Me H Me C O OH H Bryostatin (from sea moss) 3. G-protein-coupled receptors (7-TM receptors) 3.13 Action of inositol triphosphate • IP3 - hydrophilic and enters cell cytoplasm • Mobilises Ca2+ release in cells by opening Ca2+ ion channels • Ca2+ activates protein kinases • Protein kinases activate intracellular enzymes • Cell chemistry altered leading to biological effect 3. G-protein-coupled receptors (7-TM receptors) 3.13 Action of inositol triphosphate Cell membrane IP3 Cytoplasm Calmodulin Calcium stores Calmodulin Ca++ Activation Protein kinase Enzyme (inactive) Ca++ Activation P Enzyme (active) Chemical reaction Protein kinase Enzyme (inactive) P Enzyme (active) Chemical reaction 3. G-protein-coupled receptors (7-TM receptors) 3.14 Resynthesis of PIP2 several steps IP3 + DG PIP2 Inhibition Li+ salts Lithium salts used vs manic depression Contents Part 3: Section 6.7 4. Tyrosine kinase linked receptors 4.1. Structure 4.2. Reaction catalysed by tyrosine kinase 4.3. Epidermal growth factor receptor (EGF- R) 4.4. 4.5. 4.6. Insulin receptor (tetrameric complex) Growth hormone receptor Signalling pathways 4. Tyrosine kinase linked receptors • Bi-functional receptor / enzyme • Activated by hormones • Over-expression can result in cancer 4. Tyrosine kinase linked receptors 4.1 Structure Extracellular N-terminal chain Ligand binding region NH2 Hydrophilic transmembrane region (α-helix) Cell membrane Catalytic binding region (closed in resting state) Intracellular C-terminal chain C O2 H 4. Tyrosine kinase linked receptors 4.2 Reaction catalysed by tyrosine kinase Tyrosine kinase Mg++ O Protein N C Protein OH Tyrosine residue ATP ADP O Protein N C Protein O Phosphorylated tyrosine residue P 4. Tyrosine kinase linked receptors 4.3 Epidermal growth factor receptor (EGF- R) EGF Ligand binding and dimerisation Cell membrane Phosphorylation OH HO OH Inactive EGF-R monomers OH OP PO ATP Induced fit opens tyrosine kinase active sites Binding site for EGF EGF - protein hormone - bivalent ligand Active site of tyrosine kinase ADP OP OP 4. Tyrosine kinase linked receptors 4.3 Epidermal growth factor receptor (EGF- R) • Active site on one half of dimer catalyses phosphorylation of Tyr residues on other half • Dimerisation of receptor is crucial • Phosphorylated regions act as binding sites for further proteins and enzymes • Results in activation of signalling proteins and enzymes • Message carried into cell 4. Tyrosine kinase linked receptors 4.4 Insulin receptor (tetrameric complex) Insulin Phosphorylation Cell membrane HO OH OH OH ATP Kinase active site opened by induced fit Insulin binding site Kinase active site ADP PO OP OP OP 4. Tyrosine kinase linked receptors 4.5 Growth hormone receptor Tetrameric complex constructed in presence of growth hormone GH GH binding & dimerisation Binding of kinases GH receptors (no kinase activity) Activation and phosphorylation ATP HO kinases OH HO OH OH OH OH OH ADP PO OP Kinase active site opened by induced fit http://en.wikipedia.org/wiki/Cytokine_receptor http://www.ebi.ac.uk/interpro/potm/2004_4/Page2.htm Growth hormone binding site Kinase active site(Janus, JAK kinase) OP OP Tales from the drug development trenchesTucson-John Kozarich,Ligand Pharmaceuticals Thrombocyte,i.e. platelet Eltrombopag,PROMACTA Binds to DIFFERENT site than thrombopoetin with Zn 2+ . http://www.ligand.com/collaborations.php#Leading Tales from the drug development trenches-Tucson http://en.wikipedia.org/wiki/Cytokine_receptor TPO and EPO receptors (cytokine type, also growth hormone) connected to Janus kinase (JAK) family of tyrosine kinases 4. Tyrosine kinase linked receptors 4.6 Signalling pathways Ligand P P P P P P Ligand P P P P signalling protein 4. Tyrosine kinase linked receptors 4.6 Signalling pathways 1-TM Receptors Tyrosine kinase inherent or associated Guanylate cyclase Signalling proteins PLCγ IP3 DG Ca++ PKC IP3 kinase PIP3 GAP cGMP Grb2 Others 4. Tyrosine kinase linked receptors 4.6 Signalling pathways Receptor binding site GROWTH FACTOR RECEPTOR Tyrosine kinase active site (inactive) HO OH HO OH 4. Tyrosine kinase linked receptors 4.6 Signalling pathways Growth factor 1) Binding of growth factor Dimerisation Phosphorylation 2) Conformational change HO HO HO OH OH HO OH HO HO OH OHHO PO OH OH PO PO OP OPPO Binding Ras and Grb2 Binding and phosphorylation of Grb2 HO OH OH OP PO PO PO OP OPPO Grb2 OP OP GTP/GDP exchange OP PO PO PO OP OPPO OP OP Ras GDP GTP OP OP 4. Tyrosine kinase linked receptors 4.6 Signalling pathways Gene transcription OP Ras PO PO PO OP OPPO OP OP Raf (inactive) Raf (active) Mek (inactive) Mek (active) Map kinase (inactive) Map kinase (active) Transcription factor (inactive) Transcription factor (active) Contents Part 4: Section 6.8 5. Intracellular receptors 5.1. Structure 5.2. Mechanism 5.3. Oestrogen receptor 5. Intracellular receptors • Chemical messengers must cross cell membrane • Chemical messengers must be hydrophobic • Example - steroids and steroid receptors 5. Intracellular receptors 5.1 Structure CO2H Steroid binding region Zinc DNA binding region (‘zinc fingers’) H2 N Zinc fingers contain Cys residues (SH) Allow S-Zn interactions 5. Intracellular receptors 5.2 Mechanism Receptor Co-activator protein DNA Messenger Receptor-ligand complex Dimerisation Cell membrane 1. Messenger crosses membrane 5. Complex binds to DNA 2. Binds to receptor 3. Receptor dimerisation 4. Binds co-activator protein 6. Transcription switched on or off 7. Protein synthesis activated or inhibited 5. Intracellular receptors 5.3 Oestrogen receptor Binding site H12 AF-2 regions Coactivator Coactivator Oestradiol DNA Oestrogen receptor Dimerisation & exposure of AF-2 regions Nuclear transcription factor Transcription 5. Intracellular receptors 5.3 Oestrogen receptor His 524 Me OH H H Glu353 H H O Hydrophic skeleton H2O Arg394 Oestradiol • • • • • H Phenol and alcohol of oestradiol are important binding groups Binding site is spacious and hydrophobic Phenol group of oestradiol positioned in narrow slot Orientates rest of molecule Acts as agonist 5. Intracellular receptors 5.3 Oestrogen receptor Asp351 N H Side chain His 524 O Glu353 O OH H O S Arg394 Raloxifene • • • • • • Raloxifene is an antagonist (anticancer agent) Phenol groups mimic phenol and alcohol of oestradiol Interaction with Asp351 is important for antagonist activity Side chain prevents receptor helix H12 folding over as lid AF-2 binding region not revealed Co-activator cannot bind 5. Intracellular receptors 5.3 Oestrogen receptor O Me2N CH2CH3 Tamoxifen (Nolvadex) - anticancer agent which targets oestrogen receptor Contents Case Study-LATER 6. Case Study - Inhibitors of EGF Receptor Kinase 6.1. The target 6.2. Testing procedures - In vitro tests - In vivo tests - Selectivity tests 6.3. Lead compound – Staurosporine 6.4. Simplification of lead compound 6.5. X-Ray crystallographic studies 6.6. Synthesis of analogues 6.7. Structure Activity Relationships (SAR) 6.8. Drug metabolism 6.9. Further modifications 6.10.Modelling studies on ATP binding 6.11.Model binding studies on Dianilinophthalimides 6.12.Selectivity of action 6.13.Pharmacophore for EGF-receptor kinase inhibitors 6.14.Phenylaminopyrrolopyrimidines 6.15.Pyrazolopyrimidines 6. Case Study - Inhibitors of EGF Receptor Kinase 6.1 The target - Epidermal growth factor receptor - Dual receptor / kinase enzyme role Extracellular space Receptor Binding site cell membrane Cell Kinase active site (closed) 6.1 The target Overexpression of erbB1 gene Excess receptor - KINASE INHIBITOR Excess sensitivity to EGF Excess signal from receptor Excess cell growth and division Tumours Potential anticancer agent 6.1 The target H N H Protein N N O O N N H O O O H P O P O O O N N HO O Tyrosine residue H OH OH Mg Tyrosine kinase H N N O H ATP H P Protein HN O O O N O O H H P H H OH OH ADP Protein O O P Protein HN O O O O O P O O Phosphorylated tyrosine residue 6.1 The target Inhibitor Design Possible versus binding site for tyrosine region Possible versus binding site for ATP Inhibitors of the ATP binding site Aims: To design a potent but selective inhibitor versus EGF receptor kinase and not other protein kinases. 6.2 Testing procedures In vitro tests Enzyme assay using kinase portion of the EGF receptor produced by recombinant DNAtechnology. Allows enzyme studies in solution. EGF-R cell membrane Cell Recombinant DNA Water soluble kinase 6.2 Testing procedures In vitro tests Enzyme assay Test inhibitors by ability to inhibit standard enzyme catalysed reaction ATP ADP OH OP Angiotensin II Angiotensin II kinase Assay product to test inhibition Inhibitors • • Tests inhibitory activity only and not ability to cross cell membrane Most potent inhibitor may be inactive in vivo 6.2 Testing procedures In vitro tests Cell assays • • • • Use cancerous human epithelial cells which are sensitive to EGF for growth Measure inhibition by measuring effect on cell growth - blocking kinase activity blocks cell growth. Tests inhibitors for their ability to inhibit kinase and to cross cell membrane Assumes that enzyme inhibition is responsible for inhibition of cell growth Checks • • • Assay for tyrosine phosphorylation in cells - should fall with inhibition Assay for m-RNA produced by signal transduction - should fall with inhibition Assay fast growing mice cells which divide rapidly in presence of EGF 6.2 Testing procedures In vivo tests • Use cancerous human epithelial cells grafted onto mice • Inject inhibitor into mice • Inhibition should inhibit tumour growth • Tests for inhibitory activity + favourable pharmacokinetics 6.2 Testing procedures Selectivity tests Similar in vitro and in vivo tests carried out on serinethreonine kinases and other tyrosine kinases 6.3 Lead compound - Staurosporine H N O N N O H3C H3C O NH H3C • • • • • Microbial metabolite Highly potent kinase inhibitor but no selectivity Competes with ATP for ATP binding site Complex molecule with several rings and asymmetric centres Difficult to synthesise 6.4 Simplification of lead compound H N O N N H 3C H 3C O Simplification Remove asymmetric ring H N O * * * O * Simplification Symmetry NH H 3C Staurosporine N H N H H N O N H Arcyriaflavin A • Symmetrical molecule • Active and selective vs PKC but not EGF-R O N H 6.4 Simplification of lead compound maleimide ring H N O O Bisindolylmaleimides PKC selective N H N H H N O O indole ring Phthalimide indole ring Simplification N H Aniline N H Simplification Aniline Dianilinophthalimide (CGP 52411) • Selective inhibitor for EGF receptor and not other kinases • Reversal of selectivity H N O N H O N H 6.5 X-Ray crystallographic studies Different shapes implicated in different selectivity Arcyriaflavin Planar O H N Bisindolyl-maleimides Bowl shaped O O H N Dianilino-phthalimides Propellor shaped asymmetric O N H N H O O NH N H H N N H HN 6.5 X-Ray crystallographic studies Propeller conformation relieves steric clashes Steric clash O H N O H N O HH HH Steric clash H Twist H H H NH NH O HN Planar HN Propeller shape 6.6 Synthesis of analogues O H 2C TMSCl, NEt3 DMF, O 100 oC H 2C O O H 3C Diels Alder Toluene Si (CH3) 3 O CH3 Anilines O O O (H3C) 3SiO OSi(CH3) 3 Acetic acid, 120 oC MeO 2C Si (CH3) 3 C C CO2Me H 3C O CH3 O O a) LiOH, MeOH O O O O NH3 or formamides R1 NR2 R 2N O 140-150 oC b) (Ac)2O, toluene R1 O R N R1 R1 NR2 R 2N R1 R1 NR2 R 2N 6.7 Structure Activity Relationships (SAR) O R N O R1 R1 NR2 R 2N • • • • • • R=H Activity lost if N is substituted Aniline aromatic rings essential (activity lost if cyclohexane) R1=H or F (small groups). Activity drops for Me and lost for Et R2=H Activity drops if N substituted Aniline N’s essential. Activity lost if replaced with S Both carbonyl groups important. Activity drops for lactam H N NH O HN 6.7 Structure Activity Relationships (SAR) Parent Structure: R=R1=R2=H chosen for preclinical trials IC50 = 0.7 µM H N O NH O HN CGP 52411 6.8 Drug metabolism Excretion H N O O Glucuronylation HO H N O NH Glucose O Glucose O Drug HN O Metabolism (man,mouse, rat, dog) NH HN CGP 52411 H N O Metabolism (monkey) HO NH O HN Glucuronylation OH Drug O Glucose Excretion 6.8 Drug metabolism Introduce F at para position as metabolic blocker H N O F Metabolic blocker NH O HN CGP 53353 F Metabolic blocker 6.9 Further modifications a) Chain extension H N O Chain extension NH O HN CGP58109 Activity drops Chain extension 6.9 Further modifications b) Ring extension / expansion extension ring expansion H N O NH HN O HN CGP 52411 (IC50 0.7µM) NH O O NH remove polar groups HN CGP54690 (IC50 0.12µM) Inactive in cellular assays due to polarity (unable to cross cell membrane) HN N O NH HN CGP57198 (IC50 0.18µM) Active in vitro and in vivo 6.9 Further modifications c) Simplification H N O NH O HN CGP52411 Simplification H N O NH O OH CGP58522 Similar activity in enzyme assay Inactive in cellular assay 6.10 Modelling studies on ATP binding • No crystal structure for EGF- receptor available • Make a model active site based on structure of an analogous protein which has been crystallised • Cyclic AMP dependant protein kinase used as template 6.10 Modelling studies on ATP binding Cyclic AMP dependant protein kinase + Mg + ATP + Inhibitor (bound at substrate site) Crystallise Crystals X-Ray Crystallography Structure of protein / inhibitor / ATP complex Molecular modelling Identify active site and binding interactions for ATP 6.10 Modelling studies on ATP binding • ATP bound into a cleft in the enzyme with adenine portion buried deep close to hydrophobic region. • Ribose and phosphate extend outwards towards opening of cleft • Identify binding interactions (measure distances between atoms of ATP and complementary atoms in binding site to see if they are correct distance for binding) • Construct model ATP binding site for EGF-receptor kinase by replacing amino acid’s of cyclic AMP dependent protein kinase for those present in EGF receptor kinase 6.10 Modelling studies on ATP binding Gln767 HN H-bond interactions empty pocket Thr766 H2NOC H N O O Leu768 H3C Met769 S H H3C H O N H H N O N H3C H N 1 6 N N O O N O O O P O O O O H 1N P O H H OH H OH is a H bond acceptor 6-NH2 is a H-bond donor Ribose forms H-bonds to Glu in ribose pocket 'ribose' pocket P O O 6.11 Model binding studies on Dianilinophthalimides Gln767 HN empty pocket Thr766 H-bond interaction H2NOC H N O Leu768 H3C Met769 S H H3C O H O N O H N O N H NH O H3C O HN 'ribose' pocket 6.11 Model binding studies on Dianilinophthalimides • Both imide carbonyls act as H-bond acceptors (disrupted if carbonyl reduced) • Imide NH acts as H bond donor (disrupted if N is substituted) • Aniline aromatic ring fits small tight ribose pocket • Substitution on aromatic ring or chain extension prevents aromatic ring fitting pocket • Bisindolylmaleimides form H-bond interactions but cannot fit aromatic ring into ribose pocket. • Implies ribose pocket interaction is crucial for selectivity 6.11 Model binding studies on Dianilinophthalimides Gln767 HN empty pocket Thr766 H-bond interaction H2NOC H N O O Leu768 H3C Met769 S H H3C H O N O HN H O N H3C O H N NH O HN 'ribose' pocket 6.11 Model binding studies on Dianilinophthalimides Gln767 HN empty pocket Thr766 H-bond interaction H2NOC H N O O Leu768 H3C Met769 S H H3C H O O N H N O N H NH O H3C NH O 'ribose' pocket 6.12 Selectivity of action POSERS ? • Ribose pocket normally accepts a polar ribose so why can it accept an aromatic ring? • Why can’t other kinases bind dianilinophthalimides in the same manner? 6.12 Selectivity of action Amino Acids present in the ribose pocket Hydrophobic Protein Kinase A EGF Receptor Kinase Hydrophilic Leu,Gly,Val,Leu Glu,Glu,Asn,Thr Leu,Gly,Val,Leu,Cys Arg,Asn,Thr 6.12 Selectivity of action • Ribose pocket is more hydrophobic in EGF-receptor kinase • Cys can stabilise and bind to aromatic rings (S-Ar interaction) Gln767 HN empty pocket Thr766 H2NOC H N O H H3C Leu768 H3C Met769 S O H O N O H N O N H NH O H3C O HN H S 'ribose' pocket • Stabilisation by S-Ar interaction not present in other kinases • Leads to selectivity of action 6.13 Pharmacophore for EGF-receptor kinase inhibitors O HBD HBD H N HBA NH O HBA HN Ar • • • Pharmacophore Ar Pharmacophore allows identification of other potential inhibitors Search databases for structures containing same pharmacophore Can rationalise activity of different structural classes of inhibitor 6.14 Phenylaminopyrrolopyrimidines CGP 59326 - Two possible binding modes for H-bonding Cl HBD H HBD HBA N H N H N N N HBA N Ar N N H Cl Mode I Mode II Only mode II tallies with pharmacophore and explains activity and selectivity 6.14 Phenylaminopyrrolopyrimidines empty pocket Cl O O H H N N H empty pocket N N N CGP59326 H N N N H N H N H CGP59326 H S S Cl 'ribose' pocket Binding Mode I like ATP (not favoured) 'ribose' pocket Binding mode II (favoured) Illustrates dangers in comparing structures and assuming similar interactions (e.g. comparing CGP59326 with ATP) 6.14 Phenylaminopyrrolopyrimidines HBD H HBD N HBA H N HBA N N Ar Ar Cl 6.15 Pyrazolopyrimidines i) Lead compounds Cl NH2 N NH2 N N N N N H 2N (I) EC50 0.80µM • • • N N H (II) EC50 0.22µM Both structures are selective EGF-receptor kinase inhibitors Both structures belong to same class of compounds Docking experiments reveal different binding modes to obey pharmacophore 6.15 Pyrazolopyrimidines ii) Structure I empty pocket O H Extra binding interactions HBD H HBA N H N H N N H Structure I N N N N N N N H S 'ribose' pocket Ar 6.15 Pyrazolopyrimidines ii) Structure I NH2 NH2 N N N N N N (I) EC50 0.80µM N N (III) EC50 2.7µM 6.15 Pyrazolopyrimidines iii) Structure II • Cannot bind in same mode since no fit to ribose pocket • Binds in similar mode to phenylaminopyrrolopyrimidines empty pocket Cl O H N N N NH Structure II N H N H 2N H S unoccupied 'ribose' pocket 6.15 Pyrazolopyrimidines Extra H-bonding interaction iv) Drug design on structure II Cl Cl Cl OH HBD HBD H N N H N NH H N N NH HBA N H N NH N NH HBA N Simplification N H 2N (II) EC50 0.22µM (remove extra functional group) Extension N N (add aromatic ring for ribose pocket) N NH N N NH N Ar (IV) EC50 0.16µM Activity increases Extension Cl Cl (V) EC50 0.033µM Activity increases Ar fits ribose pocket (VI) EC50 0.001µM Activity increases • Upper binding pocket is larger than ribose pocket allowing greater variation of substituents on the ‘upper’ aromatic ring