Basic Principles of Neuropharmacology I 2024-25 Past Paper (PDF)

Basic principles of neuropharmacology I @Arturas Volianskis Basic principles of neuropharmacology Learning outcomes: 1. Define ligands and the concept of binding, introduce and explain different types of agonists and antagonists, and their int...

Basic principles of neuropharmacology I @Arturas Volianskis Basic principles of neuropharmacology Learning outcomes: 1. Define ligands and the concept of binding, introduce and explain different types of agonists and antagonists, and their interaction. 2. Understand and explain concentration response relationships; define affinity and efficacy, and potency. 3. Discuss spare receptors and their role for ligand efficacy, discuss selectivity. BI2432: Fundamental neuropharmacology Binding of ligands to receptors Ligand is any chemical that binds to (or combines with) a receptor. ++ -- Receptor is a cellular macromolecule - or assembly of macromolecules - concerned directly and specifically in ++ -- chemical signalling between and within cells. Binding of ligands to receptors is an active process, ++ ++ which happens due to the alignment of 3D shape and biophysical properties (forces) between the ligand and the binding site on the receptor: -- ++ –multiple points of interaction may be needed within a binding site for binding to occur –hydrophilic and hydrophobic, charge sites –van der waal, electrostatic, covalent -- ++ Binding happens with a force. Binding of one part of the molecule can facilitate or prevent the binding of another part (e.g. through shape changes). BI2432: Fundamental neuropharmacology Binding of ligands to receptors Ligand is any chemical that binds to (or combines with) a receptor. ++ -- Receptor is a cellular macromolecule - or assembly of macromolecules - concerned directly and specifically in chemical signalling between and within cells. Binding of ligands to receptors is an active process, ++ ++ which happens due to the alignment of 3D shape and biophysical properties (forces) between the ligand and the binding site on the receptor: -- ++ –multiple points of interaction may be needed within a binding site for binding to occur –hydrophilic and hydrophobic, charge sites –van der waal, electrostatic, covalent ++ -- -- ++ Binding happens with a force. Binding of one part of the molecule can facilitate or prevent the binding of another part (e.g. through shape changes). BI2432: Fundamental neuropharmacology Ligands and their binding to receptors Ligands that are produced naturally by the body (e.g. neurotransmitters and other molecules that can bind to receptors) are referred to as endogenous ligands. Endogenous ligands can also be synthesised in the lab or modified to change their properties. Endogenous ligands that are modified or designed by the chemists are termed exogenous ligands. The concept of “binding” of ligands to their receptors in tissues was developed in 1960s using radioligand binding assays to quantify the amount of ligand bound to receptors. A ligand (e.g neurotransmitter) radiolabelled and incubated with a tissue preparation, which is washed extensively to remove loosely bound drug. A radioactive atom, typically 3H, 14C, or 125I, must be added to the ligand without altering its binding properties. BI2432: Fundamental neuropharmacology The radioligand binding assay Radiolabelled Sample Tissue Ligand with Receptors Incubate Tissue & Ligand Together Bound Ligand Specific Binding WASH Nonspecific Binding The total binding of a ligand to the tissue preparation comprises two components: (1) specific binding, which is saturable, and (2) nonspecific binding, which is not saturable. BI2432: Fundamental neuropharmacology Specific and nonspecific binding Total binding is the total amount of binding observed. Nonspecific binding represents the nonsaturable portion of binding that is presumably not associated with the specific binding site under investigation. Ligand concentration Specific binding is calculated as the difference between total The total binding of a ligand to the tissue and nonspecific binding and preparation comprises two components: reflects the amount of (1) specific binding, which is saturable, radioligand bound to the and (2) nonspecific binding, which is not saturable. specific binding site. BI2432: Fundamental neuropharmacology Quantification of radioligand binding Radioligand concentration —> Log (radioligand concentration —>) BI2432: Fundamental neuropharmacology Quantification of radioligand binding The specific binding of a ligand to a tissue preparation, when the ligand is at equilibrium with the receptors, is quantified according to two properties: the affinity of the binding, which is expressed as a dissociation constant (Kd), and the total amount of binding (Bmax). Radioligand concentration —> Log (radioligand concentration —>) The Kd is defined as the concentration of ligand at which half of the maximal binding sites (Bmax) are occupied. BI2432: Fundamental neuropharmacology Quantification of radioligand binding The specific binding of a ligand to a tissue preparation, when the ligand is at equilibrium with the receptors, is quantified according to two properties: the affinity of the binding, which is expressed as a dissociation constant (Kd), and the total amount of binding (Bmax). In a perfect world inhibition of 50% of the binding sites corresponds to inhibition of 50% of receptors - i.e. 50% of receptor occupancy. Radioligand concentration —> Log (radioligand concentration —>) The Kd is defined as the concentration of ligand at which half of the maximal binding sites (Bmax) are occupied. BI2432: Fundamental neuropharmacology Autoradiography & protein distribution Autoradiography Tissue is incubated with a radiolabelled ligand to allow it to bind to its targets. The tissue is then exposed to photo film. The radiation emitted by the radioactive ligand exposes the film and creates a picture highlighting the specific pattern of binding in the brain. BI2432: Fundamental neuropharmacology All of this is great, but…. Binding studies describe the physical relationship between a drug and its target but do not directly assess the physiological or functional consequences of this association. BI2432: Fundamental neuropharmacology What is a biological response or effect? BI2432: Fundamental neuropharmacology Functional classification of ligands: agonists and antagonists Ligands, in simple terms, can be Max effect defined as agonists or antagonists, described in terms of effects and their ability to bind to their targets. Agonists evoke (produce) effects in biological tissue; they can be full, No effect partial or inverse. Antagonists do not have effects of their own on biological tissue but can block effects evoked by the agonists. Thus their effect is to antagonise the action of an agonist. Antagonists can Opposite be competitive and non-competitive. effect BI2432: Fundamental neuropharmacology Quantification of an agonist effect It is usually a biological response (in vitro or in vivo), which is elicited by an agonist, that we measure, and this is often plotted as a concentration- response curve (in vitro) or dose-response curve (in vivo). Emax Response (% max) Bigger Effect EC50 = 10mM More agonist Concentration (mM) Concentration-response curves allow us to estimate the maximal response that the drug can produce (Emax), and the effective concentration (EC50) or dose (ED50) needed to produce a 50% maximal response. BI2432: Fundamental neuropharmacology Quantification of an agonist effect It is usually a biological response (in vitro or in vivo), which is elicited by an agonist, that we measure, and this is often plotted as a concentration- response curve (in vitro) or dose-response curve (in vivo). Linear Plot Logarithmic Plot a b Response (% max) Response (% max) EC50 = 10mM EC50 = 10mM Concentration (mM) Log Concentration (mM) The logarithmic plot is the preferred method for visualising concentration- response relationships because it becomes easier to accurately determine the potency of he ligand: the EC50 value (the concentration which produces 50% of the maximum response) by placing it on the linear portion of the curve. BI2432: Fundamental neuropharmacology Comparing potency of different agonists Emax is the same for all Response (% max) A B C 3 agonists “Efficacy” All 3 agonists have different EC50 values Log Concentration (mM) “Potency” Concentration-response relationships for 3 agonists that vary in potency. Agonists A, B and C have the same efficacy, but differ in terms of their potency. The most potent agonist - A, has the lowest EC50 value (~5mM), and is approximately four times more potent than C (~20mM). BI2432: Fundamental neuropharmacology Comparing efficacy of different agents All 3 drugs have the same A Response (% max) EC50 agonists “Efficacy” Emax is different for all 3 agonists B C Log Concentration (mM) “Potency” Concentration-response relationships for 3 agonists that vary in efficacy. Each agonist has essentially the same EC50 value (equipotent), but they differ in terms of the maximum response they produce. For example, agonist A has a relative efficacy that is two times greater than agonist B. BI2432: Fundamental neuropharmacology Partial versus full agonist When a partial agonist binds to a receptor it elicits only a small response. This is because it lacks a portion of the molecule required for the full physiological effect or it binds to a slightly different site on the receptor. Nonetheless a partial agonist, whilst being less efficacious can be more potent than a full agonist! Full Agonist Response (% max) Partial Agonist Log Concentration (mM) BI2432: Fundamental neuropharmacology Partial versus full agonist When a partial agonist binds to a receptor it elicits only a small response. This is because it lacks a portion of the molecule required for the full physiological effect or it binds to a slightly different site on the receptor. Nonetheless a partial agonist, whilst being less efficacious can be more potent than a full agonist! Full Agonist Response (% max) In the presence of a full agonist, a partial agonist will act as a functional antagonist, competing with the full agonist for the same receptor. Partial Agonist This reduces the ability of the full agonist to produce its maximal effect. Log Concentration (mM) BI2432: Fundamental neuropharmacology Maximal Drug Responses and Spare Receptors Repeated application or use of agonists can lead to receptor ‘desensitisation’ or tolerance that may be overcome by increasing the dose. Why? Linear relationship Maximal response when all receptors occupied Hyperbolic relationship In most mammalian systems the relationship between receptor occupancy & drug response is hyperbolic Maximal responses at less than maximal receptor occupancy Some receptors are “spare” BI2432: Fundamental neuropharmacology Amplification of efficacy BI2432: Fundamental neuropharmacology Other types of concentration response curves An inverted U-shaped curve indicates that the biological response elicited by an agonist progressively increases as the agonist concentration increases and subsequently peaks at a moderate concentration; higher concentrations elicit progressively smaller responses. Response (% max) Increase in Decrease in “Efficacy” Efficacy Efficacy Log Concentration (mM) “Potency” BI2432: Fundamental neuropharmacology Functional classification of ligands: agonists and antagonists Ligands, in simple terms, can be Max effect defined as agonists or antagonists, described in terms of effects and their ability to bind to their targets. Agonists evoke (produce) effects in biological tissue; they can be full, No effect partial or inverse. Antagonists do not have effects of their own on biological tissue but can block effects evoked by the agonists. Thus their effect is to antagonise the action of an agonist. Antagonists can Opposite be competitive and non-competitive. effect BI2432: Fundamental neuropharmacology Competitive antagonists A competitive antagonist competes with an agonist (or endogenous ligand) for the same binding site on the receptor. The antagonist does not alter the efficacy of the agonist because the same number of receptors are available to both drugs. Rightward Shift (Less Potent) Response (% max) Agonist Alone Agonist + Antagonist Log Concentration (mM) That is primarily why, if you increase the concentration of the agonist, it will overcome the effects of a competitive antagonist. BI2432: Fundamental neuropharmacology Competitive antagonists A competitive antagonist competes with an agonist (or endogenous ligand) for the same binding site on the receptor. The antagonist does not alter the efficacy of the agonist because the same number of receptors are available to both drugs. Mirajkar. K. et al. J. Venom. Anim. Toxins incl. Trop. Dis. 2016 That is primarily why, if you increase the concentration of the agonist, it will overcome the effects of a competitive antagonist. BI2432: Fundamental neuropharmacology Noncompetitive antagonists A noncompetitive antagonist works at a completely different binding site and alters the configuration of the receptor for the agonist. It reduces the number of receptors available for the agonist to bind to. Response (% max) Reduction in Efficacy Agonist Alone Agonist + Antagonist Log Concentration (mM) The potency remains the same, but the efficacy is greatly reduced. BI2432: Fundamental neuropharmacology How do inert antagonists produce behavioural responses? By preventing the agonist action and therefore preventing the biological effect. Competitive antagonists compete with agonists (or endogenous ligands) for the same binding site on the receptor. Noncompetitive antagonists bind to an allosteric (non-agonist) site on the receptor to prevent activation of the receptor. BI2432: Fundamental neuropharmacology How do inert antagonists produce behavioural responses? By preventing the agonist action and therefore preventing the biological effect. Competitive antagonists compete with agonists (or endogenous ligands) for the same binding site on the receptor. Noncompetitive antagonists bind to an allosteric (non-agonist) site on the receptor to prevent activation of the receptor. In the presence of a constant concentration of an agonist (endogenous or exogenous), and by systematically changing the antagonist concentration we can quantify the inhibitory effect of the antagonist action on the agonist evoked response. The IC50 value (half maximal inhibitory concentration) indicates how much antagonist is needed to inhibit a biological process by half. IC50 values can be used to compare potencies of antagonists in different IC50 ︎= 0.3 μM IC50 = 1.8 μM tissues or to compare potencies of different antagonists. BI2432: Fundamental neuropharmacology How do inert antagonists produce behavioural responses? By preventing the agonist action and therefore preventing the biological effect. Competitive antagonists compete with agonists (or endogenous ligands) for the same binding site on the receptor. Noncompetitive antagonists bind to an allosteric (non-agonist) site on the receptor to prevent activation of the receptor. In the presence of a constant concentration of an agonist (endogenous or exogenous), and by systematically changing the antagonist concentration we can quantify the inhibitory effect of the antagonist action on the agonist evoked response. The IC50 value (half maximal inhibitory concentration) indicates how much antagonist is needed to inhibit a biological process by half. IC 50 values can be used to compare potencies of antagonists IC50 ︎ ~ 6 nM IC50 ︎= 0.3 μM IC50 = 1.8 μM in different tissues or to compare potencies of different antagonists. BI2432: Fundamental neuropharmacology How do inert antagonists produce behavioural responses? By preventing the agonist action and therefore preventing the biological effect. Competitive antagonists compete with agonists (or endogenous ligands) for the same binding site on the receptor. Noncompetitive antagonists bind to an allosteric (non-agonist) site on the receptor to prevent activation of the receptor. In the presence of a constant concentration of an agonist (endogenous or exogenous), and by systematically changing the antagonist concentration we can quantify the inhibitory effect of the antagonist action on the agonist evoked response. The IC50 value (half maximal inhibitory concentration) indicates how much antagonist is needed to inhibit a biological process by half. IC 50 values can are also used to compare potencies of different antagonists. IC 50 values can be used to compare potencies of antagonists IC50 ︎ ~ 6 nM IC50 ︎= 0.3 μM IC50 = 1.8 μM in different tissues or to compare potencies of different antagonists. BI2432: Fundamental neuropharmacology Summary: agonists, antagonists and modulators Receptor A Agonist B B alone Competitive Antagonist C Allosteric Ligands that alter the agonist (A) response may Potentiator activate the agonist binding site, compete with the agonist (competitive inhibitors, B), or act at D separate (allosteric) sites, increasing (C) or decreasing (D) the response to the agonist. Allosteric Inhibitor BI2432: Fundamental neuropharmacology Summary: affinity, potency and efficacy Affinity gets the ligand (agonist or antagonist) bound to the receptor, and efficacy refers to what happens with the agonist effect once agonist is bound. The term potency is used as a comparative term for distinguishing which agonist / antagonist has a higher affinity for a given receptor. Affinity (Kd) describes the strength of the binding between a ligand and its target substrate. Potency refers to the amount of Note: It is ligand required to achieve or possible for prevent a defined biological effect. a ligand to be potent Efficacy is a measure of the maximum but also to have low biological effect that a drug can produce efficacy. as a result of receptor binding. BI2432: Fundamental neuropharmacology Some real-world examples…. Isoprenaline + Propanolol 5-HT + Methysergide The potency of the agonist decreases Some antagonists bind covalently to the with a competitive antagonist because receptor and cannot be displaced by the agonist and antagonist compete for either competing ligands or washing the same binding site on the same (effects are irreversible), changing the receptors. efficacy of the agonist. Isoprenaline (analogue of adrenaline) is a β-adrenergic 5-HT is an agonist at serotonin receptors. receptor agonist. Methysergide is a 5-HT antagonist with actions at Propanalol is a “β-blocker”. adrenergic and dopaminergic receptors. BI2432: Fundamental neuropharmacology Most ligands have affinity to many receptors: the concept of selectivity Binding affinities for human (h), mouse (m), rat (r) receptors (filled circles = receptor named). pKi’s for 5-HT4 using an antagonist = red, and using an agonist, green Conlon et al 2018. Nonclinical cardiovascular studies of prucalopride, a highly selective 5-hydroxytryptamine 4 receptor agonist. J Pharmacol Exp Ther 364:156–69 BI2432: Fundamental neuropharmacology Study materials: BI2432: Fundamental neuropharmacology Example question L2: Which ligand can act as a functional antagonist? (A) Competitive antagonist (B) Partial agonist (C) Full agonist (D) Noncompetitive antagonist (E) Uncompetitive antagonist BI2432: Fundamental neuropharmacology Weekly schedule of the fundamental neuropharmacology Friday 29.11.2024 (13:10-14:00 & 14:10-15:00); C/-1.04 Meyer & Quenzer Psychopharmacology, Nestler, Hyman & Malenka’s Molecular Neuropharmacology L1. Introduction to fundamental neuropharmacology Rang & Dale’s Pharmacology, L2. Basic principles of neuropharmacology I & lecture materials Friday 06.12.2024 (13:10-14:00 & 14:10-15:00); C/-1.04 Meyer & Quenzer Psychopharmacology, Nestler, Hyman & Malenka’s Molecular Neuropharmacology L3. Basic principles of neuropharmacology II Rang & Dale’s Pharmacology L4. Techniques in neuropharmacology & lecture materials Friday 10.12.2024 (13:10-14:00 & 14:10-15:00); C/-1.04 Meyer & Quenzer Psychopharmacology, Nestler, Hyman & Malenka’s Molecular Neuropharmacology L5. Acetylcholine and Glutamate (and a bit of Glycine) Rang & Dale’s Pharmacology L6. GABA and Glycine & lecture materials Tuesday 07.01.2025 (13:10-14:00);C/-1.04 The Hippocampus Book pages 27-30 & lecture materials L7. Pharmacological dissection of field responses Friday 10.01.2025 (13:10-14:00 & 14:10-15:00); C/-1.04 Meyer & Quenzer Psychopharmacology, Nestler, Hyman & Malenka’s Molecular Neuropharmacology L8. Catecholamines Rang & Dale’s Pharmacology L9. Serotonin & lecture materials Friday 27.01.2025 (13:00-13:45 & 14:00-14:45); C/-1.04 Meyer & Quenzer Psychopharmacology, Nestler, Hyman & Malenka’s Molecular Neuropharmacology L10. Neuropharmacology of drug dependence and addiction I Rang & Dale’s Pharmacology L11. Neuropharmacology of drug dependence and addiction II & lecture materials Tuesday 21.01.2025 (13:10-14:00); C/-1.04 Tuesday 23.01.2025 Neuroanatomy L12. Exam preparation 2 and Neuropharmacology ICA BI2432: Fundamental neuropharmacology