IDM - Pharmacology - Drug Targets

1. How do drugs work ?: - induce physiological changes by interacting with the body at a chemical level 2. Drug: natural or synthetic substance that alters the physiological state of a living organism. 3. Target proteins: - Receptors - Enzymes - Carrier molecules - Ion channels 4. What are receptors ?: - specific protein molecules expressed by cells - mainly located on the surface of the cell membrane - they are characterised by the selective binding of specific substances (agonists) 5. what is the function of receptors ?: - Interacting with chemical messengers - initiating a cellular response - enable extracellular signals to be relayed to the inside 6. What are the four main types of receptors ?: Ligand-gated ion channels G-protein coupled receptors Kinase-linked receptors Nuclear/ intracellular receptors 7. ligand-gated ion channel (ionotropic receptors): - they are plasma membrane proteins - contain a ligand binding site - receptor site is directly coupled to the ion channel - quick acting = response measured in milliseconds - involved with neurotransmission Occur: NMJ Autonomic ganglia CNS 8. Where are ligand-gated ion channels located?: plasma membrane of cells 9. Examples of ligand-gated ion channels: - nicotinic acetylcholine receptors - gamma-aminobutyric acid type A (GABAa) receptor - GABAc receptor - glutamate receptors 10. ligand-gated ion channel structure: - Pentamer of 4 different subunits (2 ±, ²,and ³ ´) - When clustered together the subunits form a pore. - 0.7 nm in diameter - Each receptor has two ACh binding sites. - Both sites must bind ACh in order to activate the receptor 1 / 11 11. Function of ligand-gated ion channels: - ACh binds to nicotinic receptor = causes the ion channel to open very quickly = increases the plasma membrane permeability to Na+ and K+ - intracellular side of a plasma membrane is negatively charged compared to the extracellular surface - Na+ rush inwards = this results in cellular depolarization 12. What is the function of the NMJ ?: to transmit action potentials conducted along motor (somatic) nerves across the synaptic clefts that occur in motor endplates. 13. Explain the synthesis of ACh ?: - occurs in the motor neurone terminal - catalysed by the cytosolic enzyme = choline acetyltransferase (C.A.T) - Acetyl groups are donated by acetylcoenzyme A (acetyl CoA) - acetyl groups are then covalently bonded to choline = form ACh - ACh is then stored in vesicles - action potentials conducted along the motor nerves reach their nerve terminals - depolarisation causes influx of Ca2+ ions = triggers release of ACh release into synaptic cleft - vesicles now fuse with the terminal membrane 14. How does the released ACh cause muscle contraction ?: - released ACh diffuses across the synaptic cleft, binds to nicotinic receptors on the sarcolemma - causes a depolarisation = large enough can stimulate an action potential in the muscle fibre - causes muscle to contract this is referred to as the endplate potential 15. Agonist drugs - NMJ: - ACh - Nicotine - Suxamethonium - Decamethonium 16. Agonist drug - Autonomic ganglia: - ACh - Nicotine - Lobeline - Epibatidine 17. Antagonist - NMJ: - Tubocurarine - Pancuronium - Bungarotoxin - Atracurium - Vecuronium 2 / 11 18. Antagonist - Autonomic ganglia: - Trimetaphan - Hexamethonium 19. Agents reducing ACh release: - Hemicholinium - Botulinum toxin - Mg2+, Mn2+ and Co2+ ions - Aminoglycoside Antibiotics 20. Competitive Neuromuscular blocking Ambenonium agents: - Tubocurarine - Gallamine - Pancuronium - Alcuronium - Atracurium - Vecuronium 21. Depolarizing: Neuromuscular blocking agents: - Suxamethonium - Decamethonium 22. Transmission potentiating agents: - Anticholinesterases - Pyridostigmine - Neostigmine - Edrophonium - Distigmine - Ambenomiun - Physostigmine ( eserine) - Dyflos 23. Choline carrier inhibiting agent (synthesis inhibitor):: - Hemicholinium 24. ACh release enhancing agent: 4-aminopyridine 25. What is Tubocurarine ?: - a competitive Neuromuscular blocking agent - found as a mixture of alkaloid substances called 'curare' - extracted from Chondrodendron tomentosum plant, grows in the rain forests of South America - Native Indians use curare to treat their arrow tips before using them for hunting. 26. What is myasthenia gravis ?: It is an autoimmune disease that affects neuromuscular transmission. - it is characterised by abnormal fatigability and weakness of skeletal muscles. IgG antibodies cause the loss of nicotinic receptors in the NMJ. 27. What range of people does Myasthenia gravis mainly affect ?: - It chiefly affects adolescents and young adults under 40yrs 28. Name some symptoms of myasthenia gravis ?: - ptosis = drooping of the upper eyelid - double vision - dysarthria = a speech disorder in which pronunciation is unclear 3 / 11 29. What happens to muscles if fatigue experienced becomes extreme (Myasthenia gravis) ?: The fatigue experienced might become so extreme that muscles become temporarily paralysed. 30. How is Myasthenia gravis treated ?: - administering anticholinesterases - using prednisolone or azathioprine - performing plasmaphoresis - surgically removing the thymus 31. Describe G-protein coupled receptors ?: - can also be called metabotropic, 7-transmembrane spanning receptors - work on a 'seconds' time scale - they are indirectly connected to the processes that they govern - they use G-proteins to form the link between the receptor itself and the target process that is under control. 32. What is an advantage of GPCRs ?: They can amplify a signal: meaning 1 agonist/receptor complex can activate several alpha subunits that can then go on to stimulate several targets = producing amplification. 33. Name examples of G-protein coupled receptors ?: - Muscarinic - Adrenergic - Dopaminergic - 5-hydroxytryptamine (5-HT) - Purine (adenosine, ATP) - Opiate & many receptors for peptides and olfaction are also GPCRs. 34. Describe the structure of GPCRs ?: - they have a basic structure consisting of seven transmembrane spanning regions, connected by extracellular and intracellular loops - 3 dimensional structures - they arrange their extracellular loops + some transmembrane regions into pockets that can accept ligands - contain many disulphide bonds = maintains the three dimensional structure. 35. Describe G-proteins ?: - made up of alpha, beta and gamma subunits. - they form links between GPCRs and their targets - beta & gamma subunits form a complex together - the complex is very hydrophobic so it is found residing in the intracellular surface of plasma membrane = can freely move about. 36. Describe the properties of the alpha subunit in G-proteins ?: - binds to guanine nucleotides - forms a loose association with the beta/gamma complex - catalyse the hydrolysis of GTP to GDP 4 / 11 37. Describe the G-protein in the resting state ?: - GPCR binding site remains unoccupied - subunits of G-proteins are associated together - alpha subunit binds GDP - the target is inactive 38. What happens when an agonist binds to a G-protein receptor ?: - conformation change is induced within the receptor - this allows the alpha subunit of a G-protein to bind to the receptor - alpha subunit exchanges its GDP for intracellular GTP 39. Describe the G-protein activation of target ?: - activated alpha subunit dissociates from the receptor + beta & gamma complex - it then diffuses across the surface of the membrane - alpha subunit interacts with the target protein activating it - target response induces a cellular response 40. Describe the termination of G-protein activation ?: - alpha subunit increases GTPase activity by associating with the target - GTP bound to the alpha subunit is hydrolysed to GDP + phosphate - this terminated alpha subunit activation & the target's activation - alpha subunit can now re-associate with the beta & gamma complex and whole process is repeated 41. What are the three main types of G-proteins ?: Gs - stimulatory Gi - inhibitory Gq - Activate phospholipase C 42. What are the main targets of G-proteins ?: - Ion channels - adenylate cyclase/ cyclic AMP (cAMP) system - phospholipase C/inositol phosphate system 43. What are muscarinic receptors ?: G-protein coupled receptors that bind ACh and on cardiac muscle cells increase membrane permeability to K+ ions. 44. GPCRs: Where are Muscarinic receptors found ?: - on cells and tissues innervated by the parasympathetic nervous system 45. What happens when G-proteins from cardiac muscarinic receptors are activated ?: - they cause K+ channels to open - K+ ions diffuse out - this results in cardiac muscle cell hyperpolarization - the intracellular compartment becomes more negatively charged - hyperpolarisation makes it harder for cells to depolarize and contract - The heart rate and force of contraction then decreases 46. GPCRs: What happens when the muscarinic receptors on the cardiac muscle cells are stimulated ?: this reduces the heart rate and force of the contraction 5 / 11 47. Name agonist drugs acting on muscarinic receptors ?: - ACh - Carbachol - Pilocarpine 48. Name antagonist drugs acting on muscarinic receptors ?: - Atropine - Hyoscine - Ipratropium 49. Describe Adrenergic receptors ?: - involved with the sympathetic nervous system - can be divided into alpha (±1, ±2) and beta (²1and ²2)subtypes - the subtypes can be distinguished from one another by the different levels of responses obtained by different drugs selectively binding to them - use a system of secondary messengers 50. What are secondary messengers ?: Agents whose intracellular concentrations vary = depending on levels of receptor stimulation. Common examples: - Ca2+ ions - cyclic adenosine monophosphate (cAMP) - inositol 1,4,5-triphosphate (InsP3) - diacylglycerol (DAG) 51. Describe the Guanylate cyclase/cyclic guanosine monophosphate system ?: - cyclic guanosine monophosphate (cGMP) is a secondary messenger - guanylate cyclase = enzyme that catalyses the synthesis of cGMP - membrane bound form of guanylate cyclase has a similar structure to the tyrosine kinase family = it is now hypothesised that when peptide agonists e.g., atrial natriuretic peptide binds to its receptor, the production of cGMP occurs via a tyrosine phosphorylation process. 52. What is the most famous activator of guanylate cyclase ?: Nitric oxide 53. What happens when the adrenergic receptors on the heart are stimulated ?: The rate and force of heart contraction increases 54. ±1adrenoceptors: Stimulatory in the blood vessels = cause vasoconstriction. 55. ±2adrenoceptors: Generally found on presynaptic nerve terminals - negatively feedback on neurotransmitter release. 56. ²1-adrenoceptors: Mostly concentrated in cardiac tissue and stimulate the heart to beat faster with more force. 57. ²2-adrenoceptors: Generally induce tissues to relax e.g., bronchodilation in lung airways and vasodilation in blood vessels that supply muscles. 58. What happens when ²-adrenoceptors are stimulated ?: - cAMP is formed from ATP by the enzyme adenylate cyclase 6 / 11 - cAMP then activates the enzyme protein kinase A - this then phosphorylates proteins to produce a physiological effect examples of the proteins: - Enzymes, muscle fibres, ion channels 59. What do Gs proteins do to cAMP levels ?: Receptors linked to Gs proteins stimulate adenylate cyclase = increase intracellular cAMP levels 60. What do Gi proteins do to cAMP levels ?: Receptors linked to Gi proteins inhibit adenylate cyclase, so this reduces cAMP levels. 61. Activation of phospholipase C results in the production of ?: DAG and inositol -1, 4, 5-triphosphate(Ins (1,4,5)P3) - These are examples of secondary messengers 62. What are kinase linked receptors ?: - receptors often linked to tyrosine kinase - some ligands that binds include cytokines & growth factors - found bound to membranes 63. What are cytokines ?: - small proteins (or biological factors) that are released by cells - they have special effects on cell-to-cell interactions = important for co-ordinating cellular communication and behaviour - they lack the intracellular tyrosine kinase domain - they still use tyrosine phosphorylation mechanism - cytokine ligands bind to these receptors - receptor dimerization once again occurs - after which cytosolic tyrosine kinase units are then able to bind - SH2 containing proteins then bind and become activated, so producing the cellular response 64. What is the most well known ligand that binds to a kinase receptor ?: Insulin 65. Describe the structure of a kinase -linked receptor ?: - each domain has 400-700 amino acid residues - possess extracellular and intracellular domains - domains are connected together by a single alpha helix that spans the thickness of the membrane. - extracellular domain contains the receptor's ligand binding site 66. What do extracellular domains form ?: The receptor's ligand binding site 67. What do intracellular domains form ?: The tyrosine kinase domains 68. What is the extracellular domain of a insulin receptor made from ?: - A single polypeptide chain that is then attached, via disulphide bridges to a second polypeptide chain that forms the transmembrane spanning alpha helix and the intracellular domain. 7 / 11 69. Where is the binding site for ATP on the kinase linked receptor ?: the intracellular domain ATP molecules bound to the intracellular domains, provides a source of phosphate groups 70. What do kinase-linked receptors require to enable them to work ?: Phosphate groups 71. Explain the role of kinase enzymes ?: They are responsible for catalysing the phosphorylation (addition of phosphate groups) to various cellular components. 72. Why does ligand binding to the extracellular domain of kinase linked receptors not result in a conformation change through the rest of the receptor helix connects these two domains together molecule ?: - only a very simple ±Dimerization occurs instead: - where two receptors pair up to form a dimer 73. Ligand binding and receptor activation (Kinase) - Resting state:: - receptors are not couples together in a dimer Receptor activation = agonist have to bind to pairs of receptors 74. Ligand binding and receptor activation (kinase): What occurs when ligands bind to the receptors ?: - Ligand binding results in a conformation change - Receptors can then pair up forming dimers - intracellular domain contains tyrosine residues - Dimer forms = process of auto-phosphorylation occurs = which the tyrosine residues become phosphorylated - phosphate groups for this process are donated by the bound ATP - Phosphorylated tyrosine's become high affinity binding sites for various intracellular proteins 75. SH2 domains:: - the intracellular proteins that interact with kinase receptors all contain highly conserved SH2 domains SH2 = special domains that contain about 100 amino acid residues - the bound protein becomes activated, eliciting a response - it was first identified in products obtained from Src oncogenes 76. What is an oncogene ?: a mutated or over - expressed version of a normal gene in a cell which is free from normal growth restraints. It can therefore convert the cell into a tumour cell. 77. What are Src oncogenes ?: The transforming sarcoma inducing gene of the Rous sarcoma virus. This gene produces a cytoplasmic protein with specific tyrosine kinase activity - this then associates with the cytoplasmic face of the plasma membrane 78. What is Insulin ?: A polypeptide hormone (6000 MW) 8 / 11 79. Describe the Insulin receptor ?: - a complex of two ±-and two ²-subunits. - subunits contain cysteine residues = enable formation of disulphide bridges - they are held together by the disulphide bridges - alpha subunits = extracellular, form the insulin binding site - beta subunits = transmembrane spanning proteins (they have both extracellular and intracellular domains) - the intracellular portion of the beta units possess tyrosine kinase activity 80. Describe what happens when insulin binds to its receptors ?: - beta subunits autophosphorylate - this enhances the kinase activity of other target proteins 81. How and where is insulin produced ?: - By beta cells in the islets of Langerhans in the pancreas 82. What is the role of insulin ?: regulates blood glucose levels and high blood sugar levels stimulate its secretion 83. How do insulin receptors contribute to insulin resistance ?: - when insulin binds, clusters of ligand/receptor complexes form - these are then internalizes in vesicles - this results in a loss of insulin receptors from the cell surface contributing to insulin resistance. 84. Describe the short term action of cellular events that were triggered by activated insulin receptors ?: These are the immediate metabolic effects e.g.,: - recruiting glucose receptors - increasing glucose uptake - increasing glycogen synthesis - reducing both gluconeogenesis & glycogen breakdown these effects are governed by regulating kinase and phospholipase enzymes 85. Describe the Long term actions of cellular events that were triggered by activated insulin receptors ?: This involves regulating the synthesis of specific mRNA molecules: - therefore controlling the synthesis of structures such as enzymes = this regulates cellular processes. 86. What are Nuclear Receptors ?: Their receptors are not expressed on membranes, instead they are intracellular & involved with regulating DNA transcription (synthesis of RNA) 87. State ligands for nuclear receptors ?: - steroid hormones - thyroid hormones - agents e.g., Vitamin D & Retinoic acid 9 / 11 88. Describe how Hormones are produced ?: - produced by the endocrine glands - they are then passed into the blood and distributed around the body eventually binding to their receptors 89. Describe the nuclear receptor structure ?: - large monomeric proteins containing 400-1000 amino acid residues (just one subunit) - middle portion of ~60 residues is highly conserved - contains 2 finger shaped loops of peptide chain, each are 15 residues long = 'zinc fingers' - each contains a Zn2+ ion - zinc ions are held in place by four cysteine residues 90. What does the nuclear receptor carboxyl end contain ?: The steroid binding domain 91. Describe the zinc finger region ?: This forms the DNA binding region as the fingers are believed to be able to wrap around DNA helixes. 92. What does the nuclear receptor amino end contain ?: the receptors regulatory domain, that can activate the transcription of specific genes. 93. How many domains does a steroid receptor contain ?: contains 3 domains: - ligand binding domain - zinc finger domain - regulatory domain 94. Ligand binding and mechanism of action: How do steroid hormones and drugs operate ?: - they trigger the transcription of specific genes - this leads to the synthesis of proteins 95. Are nuclear receptors intracellular or extracellular?: Intracellular - their ligands have to be lipid soluble to pass through plasma membrane - steroid molecules are highly lipid soluble + easily gain access to the intracellular compartment where they can bind to their receptors in the nucleus. 96. In Nuclear receptors - What results from the activation of intracellular receptor ?: - Binding of activated ligand/receptor complex to DNA. 97. What happens after the ligand/receptor complex binding to DNA?: - within minutes of binding = increase in RNA polymerase - mRNA is then produced - mRNA leaves the nucleus to form a complex with ribosomes - this enables the synthesis of new protein Actual physiological responses maybe delayed by several hours or days = as the whole process is relying on protein synthesis. 98. Name some agents that bind to nuclear receptors ?: - glucocorticoids - mineralocorticoids - sex hormones - thyroid hormones 10 / 11 - vitamin D3 - retinoic acid 99. What do Glucocorticoids do ?: - stimulate the production of annexin-1 (lipocortin-1) - this inhibits the activity of phospholipase A2 - reducing the formation of arachidonic acid - this may account for its anti-inflammatory effects 100. What is arachidonic acid the precursor of ?: - prostanoids - leukotrienes - platelet activating factor 101. What do Mineralocorticoids do?: - stimulate the production of various transport proteins that are in involved in renal tubular function. 102. What happens when a steroid binds to its receptor ?: - conformation change induced - the receptor unfolds to expose the zinc finger domain - ligand/receptor complex can now bind to specific portions of DNA - Binding positions are well defined regions of the DNA that contain 'hormone response elements' - hormone response elements = ~200 DNA base pairs upstream from the genes that are regulated. - within minutes of binding = increase in RNA polymerase - mRNA is then produced 103. What is the effect of anticholinesterase, neostigmine ?: Prolongs the action of acetylcholine. 104. What are Zinc fingers are associated with ?: Intracellular receptors 11 / 11