Introduction to Receptors and Pharmacology (PDF)

RCSI Royal College of Surgeons in Ireland Coláiste Ríoga na Máinleá in Éirinn FFP1-62 Introduction to Receptors: G protein-coupled receptors Prof Will Ford 337 [email protected]...

RCSI Royal College of Surgeons in Ireland Coláiste Ríoga na Máinleá in Éirinn FFP1-62 Introduction to Receptors: G protein-coupled receptors Prof Will Ford 337 [email protected] Dr. Roger Preston  Learning Outcomes Outline the concept and nature of receptor signalling Explain the structure of G-protein-coupled receptors Explain the nature of the signalling cascades G- protein coupled proteins can generate Describe the mechanisms by which G- proteins regulate the effector enzymes Receptor response theory Four types of receptor – others are available  G-protein coupled receptor (GPCR) structure Monomeric proteins MW 35K- 70K Pass through the membrane 7 times At least 500 different receptors Include light, taste and smell Overview of how GPCRs work Signal amplification  Key points about G-proteins 1. An enzyme composed of 3 subunits: , ,  2. Bind to and hydrolyse GTP to GDP 3. Inactive when GDP bound 4. Active when GTP bound 5. Acquires high What does each part of the G- protein do? The activity status of G proteins is determined by the  subunit 4 families of G proteins based on structural similarities – Gs, Gi, Gq and G12 Main purpose is to regulate amplifier or effector protein activity – βγ exist as dimers – 6 different β and 11 different γ – Can also exert signalling activity FYI  GPCR signalling The  and  of the G-protein anchor it to the membrane in its “inactive” or “unbound” state G-protein is not linked to the receptor G-protein has GDP-bound Lodish et al. Molecular Cell Biology  Ligand binding and activation Agonist-induced activation induces conformational change Conformational change reveals binding site for - subunit Lodish et al. Molecular Cell Biology  G-protein binding to receptor G-protein binds to receptor Lodish et al. Molecular Cell Biology  Signal transduction begins GDP is replaced with GTP GTP-Gα dissociates from Gβγ Lodish et al. Molecular Cell Biology  Second messengers are produced GTP-Gα and / or Gβγ activate the effector Meanwhile the agonist has dissociated Lodish et al. Molecular Cell Biology  The system resets GTPase activity returns the system to resting The cycle can start again Lodish et al. Molecular Cell Biology G-protein-linked effectors Liu et al, 2024, Circ Res 135(1). https://doi.org/10.1 161/CIRCRESAHA.1 24.323067 Regulatory control of GPCRs Guanine-nucleotide exchange factors (GEF) GDI Ligand-bound receptor acts GEF (Accelerates signalling) Guanine nucleotide dissociation inhibitor (GDI) βγ acts as GDI preventing GDP release (Inhibits signalling) GTPase-accelerating proteins (GAPs) Stimulate GTPase activity (Turn off signalling) GPCR desensitisation FYI 1) Uncoupling Phosphorylation uncouples receptor stopping recruitment of G-protein. P-receptor has lower affinity for agonist (dissociation) 2) Internalisation Sustained stimulation allows binding of β- arrestin leading to receptor internalization and activation other transduction pathways. 3) Downregulation Continued stimulation of receptor traffics receptor to lysosomes where it is Albert et al. (2005). The Neuroscientist 10. 575-93. Activation of adenylyl cyclase AC generates cAMP, an important ‘second messenger’ in cells Activated by Gs Inhibited by Gi Cell Signalling Biology - Michael J. Berridge - www.cellsignallingbiology.org - 2009 Ga subunits control AC activation Gαs activates adenylyl cyclase Activated by cholera toxin Inhibited by pertussis toxin Gai inhibits adenylyl cyclase Receptors regulating adenylyl cyclase Lodish et al. Molecular Cell Biology Chapter 13  Ga subunits control AC activation 160,00 deaths / year  Role of cyclic adenosine monophosphate cAMP activates protein kinases A (PKAs) Protein kinases phosphorylate proteins* cAMP-dependent kinases have many substrates ‐ ion channels and ‐ metabolic pathways cAMP is metabolised When AC activity is disrupted E. coli toxin – E. coli ‘traveller’s diarrhoea’ toxin Covalent modification of Gαs - can’t hydrolyse GTP (locked ‘ON’) Elevated cAMP levels in colonic epithelium cause efflux of water and ions Severe diarrhea and dehydration Treatment for this disruption Loperamide/Imodium E. coli Loperamide* acts as a μ-opioid toxin receptor agonist in large intestine Treatment – opiate receptor coupled to Gi Another example of functional antagonism  Activation of phospholipase C Classically activated by Gαq/11 Hydrolyses phosphoinositide (PIP2) from the membrane 1. Diacylglycerol (DAG) 2.Produces second Inositol 1,4,5-trisphosphate (IP3) messengers PLC-generated signal transduction Ca2+ calmodulin Activation of kinases Phosphorylation cellular proteins Change in cellular function  FYI Diversity of GPCRs Gs Gi Gq/11 5-HT4, 5-HT7 5-HT1, 5-HT5 5-HT2 ACh M2, M4 ACh M1, M3, Adenosine A2 Adenosine A1, A3 M5 Adrenergic β1, Adrenergic α2 2, 3 Cannabinoid CB Adrenergic α1 Dopamine D2, 3, 4 Dopamine D1, Glutamate D5 mGlu2, 3, 4, 6, 7, 8 Glutamate Histamine H3, 4 mGlu1, 5 Opioid δ, κ, μ Histamine H1 Histamine H2 Prostanoid EP3 Vasopressin Vasopressin V V1  FYI Diversity of GPCR signalling  FYI Even more signaling pathways!!!!! Pharmacology, Rang & Dale p 67-8  Further reading and viewing https://www.nature.com/scitable/topicpage/gpcr-1404747 1 Katzung Rang Medical Pharmacology at a glance https://portlandpress.com/pages/cell_signalling_biology  What we have learned The concept and nature of receptor signalling The structure of G-protein-coupled receptors The nature of the signalling cascades G- protein coupled proteins can generate The mechanisms by which G-proteins regulate the effector enzymes – adenylate cyclase and phospholipase C  RCSI Royal College of Surgeons in Ireland Coláiste Ríoga na Máinleá in Éirinn FFP1-54 Introduction to Pharmacology Prof Will Ford 337 [email protected] Dr. Roger Preston  Learning outcomes Explain the differences between chemistry-led and target-led drug discovery Describe the advantages and disadvantages of protein-based therapeutics Explain the generation, activity and applications of monoclonal antibodies Discuss new areas of therapeutics that overlap with pharmacology, such as cell therapies Discuss new areas of therapeutics that overlap with pharmacology, such gene-based therapies Recommended Reading  What is pharmacology? What happens to Pharmacokinetics PHARMACOLOG drugs in the body Pharmacodynamics What drugs do in the body PharmacotherapeuticsHow drugs are used Y Study of drugs from Pharmacognosy naturally occurring sources Toxicology Study of adverse effects  The evolution of pharmacology 3400BC Egyptian inhalation of herbs to relieve asthma symptoms 1000BC Ma huang ingested to alleviate asthma symptoms Chemical structures of morphine, cocaine determined ynthesis of aspirin 1897  What is a drug? “A drug is a chemical that when applied to a physiological system affects it in a specific way.” Rang and Dale’s Pharmacology 2018, Chapter 2, p29. Where do drugs come from? Source Drug Plant Foxglove Digitalis Animal Viper Eptifibati de Penicillium notatum Microbe (fungus) Penicillin  Drugs vs herbal vs ‘alternative’ medicine Drugs Herbal Homeopathy Biologically active Biologically active No biological activity 1 active component >1 active component No active component (?) Dose is known Dose is an estimate Dose = 0 (?) Manufacture/sale Manufacture/sale Manufacture/sale regulated generally unregulated unregulated Biological > placebo Biological > placebo effect Placebo effect only effect Link to information about homeopathy: https://www.nccih.nih.gov/health/homeopathy  What can drugs target? Receptors Enzymes Ion channels Transporters  Drug discovery approaches Chemistry-led discovery Target-led discovery  Identify chemical with biological  Identify drug target of interest activity  Screen chemicals for binding to  Modify chemical structure to target make library of analogues  Design chemical entities based  Screen analogues for biological on computer modelling of ligand activity docking to 3D protein structures  Generate structure-activity  Mechanism known and effect relationships to inform further predicted development  Mechanism often unknown and Example treatment basedof receptor structure modelling. Rang & Dale onthbiological Pharmacology, 9 edition 2018 p59 effect  Mepyramine binding to H1 receptors Wang et al. Molecular mechanism of antihistamines recognition and regulation of the histamine H1 receptor. Nat Commun 15, 84 (2024)  An example of chemistry-based drug development: Ranitidine [Sir James Black] 1948 Histamine antagonists already clinically used (H1 receptor selective) Histamine caused gastric acid secretion – insensitive to available antagonists Proposal of another histamine receptor (H2) Clinical utility for treatment of peptic ulcers (none available at that time) 1964 Program established at SKF with Sir James Black to develop antagonists of H2 receptors Synthesis of histamine analogues testing for H2 selectivity  Compound development 1. Dissociation constants CH2CH2NH2 H1 H2 H2/H1 Histamine 0.5 μM 0.5 μM 1 N N H CH2CH2CH2CH2NH-C-NHCH3 S Burimamide 320 μM 70 nM 5,000 N N H Poor oral absorption H3C CH2SCH2CH2NH-C-NHCH3 S N N Metiamide >500 μM 2.5 μM >200 H Granulocytopenia  Compound development 2. Dissociation constants H3C CH2SCH2CH2NH-C-NHCH3 H1 H2 H2/H1 NCN Cimetidine 450 μM 33 μM 14 N N H Cyp450 drug interactions CH2SCH2CH2NH-C-NHCH3 CHNO2 O Ranitidine >500 μM 1 μM >500 CH2NCH2CH3 General features Aromatic Polar H- Flexible chain ring bonding group Functional evidence of selectivity  Modern drug development Conventional pharmacology uses small synthetic molecules Origins in early 19th century with the purification of morphine, quinine, strychnine and cocaine One of the first drugs to be synthesized was  Small molecule drugs - problems While most drugs are ‘small molecules’ it has not always been possible to develop small molecule agents: – Many interactions are between proteins, and small molecule inhibitors are not possible – Chemistry is too difficult  Current drug approaches Recent advances in biotechnology has completely changed the options for therapy New therapies include: – Recombinant engineered proteins – Nucleic acid-based therapeutics – Gene therapy approaches – Cell-based therapies  Protein therapies Proteins have long ADVANTAGES: been used as plentiful, effective drugs and easy to isolate? Purification of insulin and its use DISADVANTAGES: to treat diabetes in require blood 1922 donation, infection risk, difficult to Since then, other further modify hormones and coagulation factors Recombinant have been purified proteins solve this and used problem  Recombinant protein therapies Protein not isolated, but protein- encoding gene cloned into cell line Cells generate ‘recombinant’ protein is expressed and purified Limited potential for viral contamination – Insulin – Erythropoietin – Interferon – Factor VIII and IX – Growth factors – Hirudin (Brassica napus)  Monoclonal antibody therapies Köhler and Milstein discovered a method to immortalize antibody-producing cells (1975) Antibody-producing cells from the spleen fused with myeloma cells (cancer cells) Using monoclonal antibodies to fight cancer  Value of monoclonal antibody therapies ADVANTAGES: DISADVANTAGES: Single epitope on a Expensive to make single antigen Have to be given IV It is not necessary to use animals in the production* Large amounts of antibody can be produced Predictable batch properties  Monoclonal antibody nomenclature Mouse Moabs -omab – Tositumomab Chimeric antibodies -ximab – Infliximab Humanised antibodies -zumab – Natalizumab, Trastuzumab Human antibodies -umab – Adalimumab  Antivenin / antivenom Antivenin is used to treat snake and spider bites Usually, serum from a horse that has been inoculated with snake venom is used* Horse serum is foreign and can trigger immune reactions Anti-horse antibodies  Nucleic acids as drugs Nucleic acids can be used as drugs: 1. ‘Antisense’ molecules mRNA exists as a PATISARAN single “sense” FDA-approved RNAi molecule therapy for hereditary amyloidosis with Complementary polyneuropathy (or mRNA chain can malfunction of bind and inhibit nerves) transcription 2. Aptamers are  Gene therapy There are a number of diseases in which a protein is not expressed due to a genetic mutation In other diseases the expression of a gene is  Cell-based therapies ‘Cell-based’ therapy can involve: The introduction of genetically-modified cells to treat disease …or the application of stem cells to renew existing cell defects or deficits – Embryonic, pluripotent – Self, multipotent  Stem cell therapy Stem cells have the potential to differentiate into an unlimited number of mature tissue-specific cell types Lots of potential uses, but most commonly used for treatment of leukaemias Stem cell grafts can be  Adoptive cell transfer therapy ‘Living drugs’ Chimeric antigen receptor (CAR)-T cell therapy: use patients own T cells being isolated and genetically modified to express synthetic receptors on their surface that recognise tumour antigens The CAR-T cells are then better able to destroy the malignant cells  Are medical devices drugs? Devices are regulated by different legislation to drugs Recently the divide between the two areas has blurred Use of stents is very important in cardiology where vessel re- occlusion is problematic – Drug-coated stents are  What we have learned… Explain the differences between chemistry-led and target-led drug discovery Describe the advantages and disadvantages of protein-based therapeutics Explain the generation, activity and applications of monoclonal antibodies Discuss new areas of therapeutics that overlap with pharmacology, such as cell therapies Discuss new areas of therapeutics that overlap with pharmacology, such gene-based therapies  RCSI Royal College of Surgeons in Ireland Coláiste Ríoga na Máinleá in Éirinn FFP1-68 Enzyme-linked (, ion-gating) and intracellular receptors Prof Will Ford 337 [email protected]  Learning Outcomes 1. Explain the structure and function of ligand- gated ion channels 2. Explain the structure and function of tyrosine kinase-linked receptors 3. Explain the structure and function of intracellular receptors 4. Outline and compare the signalling cascade for each of these receptor types Four types of receptor – others are available Ligand-gated ion channels Ligand-gated ion channels Structure of ligand-gated ion channels Ligand-gated ion channels contain receptor subunits around a ‘central pore’ Receptor is also the ion channel Allows flow down a concentration gradient Timescale - ms  Ligand-gated ion channel examples Ligand LGICs GPCRs 5-HT 5-HT3 5-HT1, 2, 4, 5, 6, 7 ACh nicotinic muscarinic GABA GABAA GABAB Glutamate* AMPA, Kainate, NMDA mGluR Purinergic P2X P2Y  Ligand-gated ion channel regulation desensitised state tends to have higher affinity for the agon  Ligand-gated ion channel biophysics amidas & Lynch. Cell Mol Life Sci (2013) 70:1241-1253 Receptor tyrosine kinases (RTK) How do receptor tyrosine kinases ‘receive’ and ‘transmit’ signals? Extracellular domain: responsible for capturing or binding signal (‘ligand’) Single transmembrane helix Intracellular domain: tyrosine phosphorylation pattern drives signal  RTK signal transduction Adapter proteins link signalling molecules together, but don’t signal Growth receptor binding protein 2 (Grb2) is an adapter protein One ‘arm’ (SH2 domain) of Grb2 recognises a pY on active receptor = tyrosine, pY = phosphorylated tyrosine Other arm (SH3 domain) recognises proline on signalling Ras cell signalling Active Ras triggers the activation of numerous cell signalling pathways changes in protein activity and gene expression Cell proliferation Tyrosine kinase-linked receptors: Variation on a theme Nuclear exclusion motif hidden, and nuclear localisation motif exposed Nuclear exclusion motif exposed Nuclear receptors  Nuclear receptor types Homodimeric Heterodimeric  Nuclear receptors Homodimers exist in the cytoplasm Examples include oestrogen, progesterone, androgen, glucocorticoid, mineralocorticoid receptors Heterodimers exist in the nucleus already – join with retinoid X receptor. Examples include thyroid hormone, retinoic acid (vitamin A) All bind DNA as dimers Receptors require phosphorylation for activation  Homodimeric nuclear receptors agonist Nucle us R R HS HS P P HS HS R P P -ve ↓ gene R R R RE transcription Change R R in protein R expressio +ve ↑ gene n R RE transcription Cellular Cytoplasm respons e  Heterodimeric nuclear receptors Plasma membrane Retinoid X receptor Ligand nucleus Cellular response Protein expression  Nuclear receptors Homodimers Heterodimers Each subunit of dimer binds RXR – common nuclear one repeat receptor monomer Half-sites are inverted Bind direct repeat half-sites repeats (palindromes) Common example: Common example: AGGACA(Nx)TGTCCT TGACCT(Nx)TGACCT TCCTGT ACAGGA ACTGGA ACTGGA Recognition determined by Recognition determined by spacing (Nx or number of spacing base pairs) Receptors for vitamin D, Receptors for progesterone, retinoic acid, 9-Cis retinoic acid glucocorticoids, androgens (binds to RXR), triiodothyronine  Summ ary Ligand-gated G-protein Tyrosine-kinase Intracellular ion channels coupled linked receptor receptor receptor  What we have learned… 1. The concept and nature of receptor signalling 2. The structure and function of tyrosine kinase- linked receptors 3. The structure and function of ligand-gated ion channels 4. The structure and function of intracellular receptors 5. The signalling cascade for each of these receptors  Further reading and viewing https://www.khanacademy.org/test-prep/ mcat/organ-systems/biosignaling/v/enzy me-linked-receptors https://www.khanacademy.org/test-prep/m cat/organ-systems/biosignaling/v/membra ne-receptors  Further reading and viewing Background Science Clinical  90 Learning outcomes 1. Differentiate between pharmacokinetics and pharmacodynamics 2. List the four key processes involved in pharmacokinetics: Absorption, distribution metabolism and elimination (ADME) 3. Compare the common routes of administration 4. Describe how absorption can influence plasma drug levels and define the term 'Bioavailability' 5. Describe first pass metabolism and its importance  91 Factors determining the response of a patient to a drug Pharmacodynamics (what happens to the receptor after attachment) Actions of the drug on the body physiological & biochemical effects of drug interactions with macromolecular ‘targets’ Specific to drug or drug class Pharmacokinetics (getting the drug to the receptor site) Effects of the body on the drug Drug movement movement of drugs into, around and out of the body Non-specific, general processes PK-PD underpins relationship between Dose and Effect * Clinically one looks at how the conditions change with time after therapeutic intervention  93 Four basic processes of pharmacokinetics - ADME Absorption – the transfer of drug from the site of administration to the general circulation – Bioavailability (F) Distribution (how do you get it to the target?) – the transfer of drug from the general circulation into different organs of the body – Apparent volume of distribution (Vd) Metabolism + Excretion = Elimination – the removal of drug from the body – this may involve metabolism or excretion or both – Clearance (Cl) – Plasma Half -Life (t1/2) ADME overview  95 Drug passage across membranes (absorption processes) Of importance to absorption, distribution and excretion: Lipid Bilayer Through pores or Passive Carrier- Pinocytosis ion channels diffusion mediated By diffusing through aqueous Diffusing directly process Transport by pores formed by aquaporins though the lipid vesicles. Rare 1. Facilitated that traverse lipid bilayer. Passive movement for drugs diffusion, e.g., Passive movement along along concentration e.g., IgG levodopa & blood- concentration gradient. gradient. Drug must brain barrier Water soluble small be somewhat lipid 2. Active transport, molecules, e.g., lithium soluble e.g., 5-fluorouracil  96 Passive diffusion  97 Drug passage across membranes (absorption processes) Carrier-mediated processes: Solute carrier transporters (SLCs) Facilitate transport DOWN a concentration gradient Organic anion transporters (OATs) Organic cation transporters (OCTs) ATP binding cassette transporters (ABCs) Require ATP to pump material (resistance in bacteria, c e.g., P-glycoproteins Common cause of multidrug resistance  98 Drug absorption Routes of administration can broadly be divided into: 1. Topical: local effect, substance is applied directly where its action is desired 2. Enteral: desired effect is systemic (non-local), substance is given via the digestive tract 3. Parenteral: desired effect is systemic; substance is given by routes other than the digestive tract The U.S. Food & Drug Administration (FDA) recognises >100 distinct routes of administration  99 Examples of common routes of administration Topical – Application to epithelial surfaces (e.g., skin, cornea, vagina, nasal mucosa, lung) Enteral – Oral - (per os / PO), by mouth – Sublingual - placed under the tongue – Rectal - (per rectum), suppositories or enemas Parenteral – Inhalation – Injection:  IM injections given in a large muscle mass (deltoids or gluteals) – best for larger volumes and when faster absorption desired, e.g. antibiotic Subcutaneously (SC/SQ) - below the dermis and epidermis – when a slower, more prolonged effect is desired, e.g. insulin, many immunizations, heparin (an anticoagulant) Intravenous (IV) - directly into a vein Intradermal (ID) - into dermis, just below the epidermis – longest absorption time of all the parenteral routes, used for allergy tests and local anaesthesia  101 Oral administration: Most common & convenient but drugs also face most barriers to entry into the systemic circulation Factors influencing drug absorption from the gut: Drug structure: – Highly polar/ionized (insoluble in water) compounds poorly absorbed – Weak acids and weak bases undergo pH partitioning – Peptides broken down by digestive enzymes (insulin) Formulation: – Capsule/tablet must disintegrate – Modified release formulations Gastric emptying can affect rate but not quantity – Food, generally slows absorption rate due to delayed gastric emptying and stimulation of gastric acid secretion – Fasting, malnutrition  102 Oral administration: Most common & convenient but drugs also face most barriers to entry into the systemic circulation Aspirin uncharged in stomach – good absorption Pethidine charged in stomach – variable absorption  103 First pass metabolism (or presystemic metabolism) Drug is absorbed from GI tract, passes via portal vein into liver where many are metabolised − propranolol, lignocaine, glyceryl trinitrate, aspirin Only a proportion of drug reaches the circulation Alternative routes of administration (e.g., intravenous, intramuscular, sublingual) avoid first-pass effect  104 First pass metabolism (or presystemic metabolism) First-pass metabolism: Intestinal lumen (digestive enzymes) Intestinal wall (MAO) Liver (multiple enzymes) – CYP450 Lung (MAO, peptidases) (note: an example of first pass metabolism but not relevant to oral absorption)  105 Drugs not given orally Route Example Intravenous Lignocaine / lidocaine (analgesic) – 100% bioavailable Sublingual Glyceryl trinitrate (angina attacks) (also buccal) – straight into systemic circulation without entering portal system so escapes first-pass metabolism. Also avoids intestinal enzymes & low pH Per rectum Diazepam (epilepsy) – either to produce local effect or to produce systemic effects. Absorption unreliable but useful in patients vomiting, fitting or postoperatively. Avoids intestinal enzymes & low pH Transdermal Nicotine patches, oestrogen hormone replacement – usually used for local effect but absorption can lead to systemic effects. Only suitable for lipid-soluble drugs and expensive Intramuscular Adrenaline Inhalation Bronchodilators, gaseous anaesthetics  106 Advantages of various routes of administration Avoids first Controlled Rapid onset Localized pass release effect metabolism Oral - + -/++ rarely Intravenous +++ - +++ - Sublingual ++ - +++ - Intramuscular +++ +++ + -/+ Transdermal ++ +++ - -/+ Per rectum ++ ++ + -/+ Inhalation +++ - +++ +++  107 Route of administration is determined by: The physical characteristics of the drug The speed which the drug is absorbed and / or released The need to bypass hepatic metabolism and achieve high concentrations at particular sites  Drug plasma concentration versus time plots Typical plasma level curve after Typical plasma level curve after administration of an IV bolus a single oral dose of a drug dose Maximum plasma concentration Note maximum plasma (Cmax) is reached at a time tmax concentration occurs at time = 0, after administration immediately after dosing  Drug [plasma] - time curve after oral administration maximum safe concentration (MSC) minimum effective concentration (MEC) Onset of action occurs when plasma level reaches MEC Plasma levels should remain below the MSC to minimise adverse reactions Duration of action is the time period for which the plasma level is at or above MEC  Area under the curve (AUC) AUC is a measure of the total amount of drug that enters the body after administration It is the actual area calculated from a plasma concentration–time curve The units are concentration × time (e.g., mg.hr L−1). AUC after IV and oral administration of the same dose of a drug; shaded regions show the AUC for each Note - the AUC and maximum plasma concentration are much higher with IV dosing  111 Extent of absorption Bioavailability (F) is defined as “the fraction of the administered dose that reaches the systemic circulation as intact drug” Bioavailability determines dose required by different routes of administration Determination of bioavailability is by comparison of plasma levels of a drug obtained after administration (e.g., oral / IM) with plasma levels following IV administration Bioavailability (F): amount of drug reaching the systemic circulation as parent drug % Bioavailability = AUCoral / AUCIV x 100 Fractional availability = F Has no units Quote as percentage 25% or as decimal 0.25 For i.v.: 100% and non i.v.: ranges from 0 to 100%  Bioavailability (F) Why do we care about bioavailability? The true dose is not the amount swallowed, but is the drug available to exert its effects Propranolol 80 mg / day oral (F 25%) – Sublingual – 40 mg (F 63%) – IV – 20 mg (F 100%)  RCSI Royal College of Surgeons in Ireland Coláiste Ríoga na Máinleá in Éirinn FFP1-58 Drug Receptor Interactions: Agonists Prof Will Ford 337 [email protected] Dr. Roger Preston  Learning outcomes Define the terms ‘ligands’ and ‘receptors’ Describe the way in which receptors are defined Explain the concept of a dose-response relationships Explain quantitative concepts [Kd; Bmax; IC50 / EC50]  Receptor-ligand interactions Analogous to enzyme substrate interactions: “Drugs do not act unless bound” (Paul Ehrlich, 1913) Many drugs bind to receptors  Receptors and enzymes Drug + target Effect Ligand + receptor Modified receptor activated Substrate + enzyme Modified substrate Different types of receptor  What is a receptor? Receptors possess affinity for ligands Receptors are saturable and finite (limited number of binding sites) Receptors are discontinuously distributed Agonist binding to a receptor causes a response The same receptor may generate a different physiological response in different tissues  How do ligands bind selectively to receptors?  Selectivity defined as relative affinity.  Ligand-receptor binding is not specific a ligand will bind to other receptors as the dose/concentration is increased – see therapeutic index xample: β1- and β2-adrenoceptor subtypes in the heart Stimulation of cardiac β1 adrenoceptors increases heart rate (HR) Stimulation of cardiac β2 adrenoceptors increases coronary flow (CF)  The dose-response relationship Tissue responses are generally directly 120 proportional to the 110 fraction of the Heart rate receptors occupied 100 with agonist (bpm) 90 The more occupied receptors, the bigger 80 the signaling response 70 0 20 40 60 80 100 Dose-response ED50 curve – ED50 Adrenaline (ng/Kg/min) Linear versus log dose-response curves 100 % Maximum } Tissue Response Linear Threshold ED50 ED50 Log Dose [A] Linear scale Log scale Sub-threshold concentration Effective concentration 50% (EC50) Maximum response  How do you assess how drugs interact with receptors? Functional studies Measure response of tissue to drug Binding studies Directly measure the binding of radiolabeled drug to tissue. Ligand only?  Necessary assumptions… Ligands bind specifically and reversibly to a receptor Binding is SATURABLE (at a certain concentration no more binding is possible) and REVERSIBLE All receptors are equally accessible to ligands Receptors are EITHER (i) free or (ii) bound to drug SATURATION BINDING - PRINCIPLES Radioactive ligand R Receptor P Membrane protein Unbound label WASH P R R TOTAL BINDING SPECIFIC BINDING NON-SPECIFIC BINDING SPECIFIC BINDING Only interested in binding to the receptor (specific) Specific binding is SATURABLE – i.e. there is an upper limit – the number of receptors present Non-specific binding is non-saturable – i.e. will always increase with increasing addition of ligand Total binding = specific binding + non-specific binding How can specific binding be worked out from this? NON-SPECIFIC BINDING Radioactive ligand R Receptor P Membrane protein ‘cold’ ligand WASH P R R Non-specific binding remains SATURATION BINDING ANALYSIS Radioactivity Total Specific = total – non-specific Max specific binding Specific Non-specific 50% specific binding KDConcentration of radioligand COMPETITION BINDING Add ONE concentration of radioligand Displace radioligand from receptors with unlabelled ligand Radioligand and ‘cold’ ligand can be the same Affinity is calculated based on the RELATIVE affinities and concentration of radioligand used  ANALYSIS OF COMPETITION BINDING 1  D * K A* KA  IC 50 Radioactivity You do not need Specific IC50 to learn this formula Non-specific Concentration of ‘cold’ ligand  How do we calculate drug potency? Potency is a Emax measure of drug 100 Response (%) activity 80 60 Expressed in A B C 40 terms of the 20 amount required 0 to produce an 1 10 100 1000 10000 effect of given [Agonist] µM intensity Isoprenaline (A) 20 μM > E.g., 50% of its Adrenaline (B) 80 μM > Noradrenaline (C) 300 μM maximal response  What is a ‘partial agonist’? Efficacy or intrinsic activity (α) is the ability to produce a response Partial agonists exhibit efficacy of greater than 0, but α=1: Full agonist less than 1 1>α>0: Partial agonist Partial agonists will α=0: antagonist still occupy all the  Full versus partial agonists Full Partial agonis agonist t recept recept or or conformational REDUCED conformational change change FULL REDUC respon ED se respons e Agonist - a ligand which Partial agonist - a chemical binds to a receptor and that binds to a receptor, but causes a biological induces reduced signalling response response  Example of partial agonism: ‘Painkillers’ - Opioid receptors Opioid receptors –GPCRs important for pain transmission and neurotransmission Opioids are agonists for mu opioid receptors Full agonists include fentanyl, morphine, methadone Partial agonists include buprenorphine, codeine*, butorphanol**, and  Example of partial agonism: Nicotine Replacement Therapy Blocking or desensitizing nicotinic receptors prompts greater receptor expression on neurons Nicotinic partial agonists (e.g., varenicline)  Difference between varenicline & nicotine 100 100 No tobacco No tobacco 80 Nicopatch 80 Var alone % Max reward % Max reward NP with cigarette Together 60 60 40 40 20 20 0 0 0 50 100 150 200 250 0 50 100 150 200 250 Time (hours) Time (hours)  What are ‘spare receptors’? Maximum signalling response often observed without full occupancy of available receptors Receptors are ‘spare’ whenever maximum response at less than full agonist binding Has the effect of increasing tissue sensitivity to agonists Presence of spare receptors mean it is possible to get a What is the difference between ‘efficacy / intrinsic activity’ and ‘potency’? Drug A has GREATER POTENCY than Drug B, but SAME POTENCY as Drug C Drug A and Drug B have the SAME EFFICACY as each other, BOTH have GREATER EFFICACY than Drug C Although Drug C has lower efficacy than B, It  1. Summary – New terminology Ligand – Binds its target Receptor – Next week Agonist – Full – Partial Antagonist – Next lecture  2. Summary – New terminology Affinity – the ability to bind a target Kd , K i K no Dose – amount given w ED50 yo ur Concentration – amount per unit volumeu EC , IC ni 50 50 ts Potency Efficacy / intrinsic activity Partial agonists Spare receptors  What we have learned… Define the terms ‘ligands’ and ‘receptors’ Describe the way in which receptors are defined Explain the concept of a dose-response relationships Explain quantitative concepts [Kd; Bmax; IC50 / EC50]  RCSI Royal College of Surgeons in Ireland Coláiste Ríoga na Máinleá in Éirinn FFP1-67 Drug Receptor Interactions: Antagonists Prof Will Ford 337 [email protected] Dr. Roger Preston  Learning outcomes Define 'Antagonists’ Explain the difference between reversible, irreversible, competitive, non–competitive antagonists Describe how each type of antagonist affects the dose-response curve Explain population studies for 'all-or-none’ responses  What is an ‘antagonist’? antagoni agonis st t recept recept or or respon No se response Agonist - a Antagonist - binds ligand which to a receptor but… binds to a receptor and (i) has no effect causes a (ii) prevents the biological effects of an  Intrinsic activity/efficacy Intrinsic activity (α) is the ability to produce a response Also called Efficacy α =1: Full agonist 1> α >0: Partial agonist α =0: antagonist α < 0: inverse agonist  What is a ‘competitive’ antagonist? Binds to the same site as the agonist and the two compete with each other – Orthosteric If binding is reversible, the effect is surmountable by excess agonist If binding is  How does a competitive antagonist affect a log concentration response curve? EC50 values With increasing antagonist concentration:  Parallel right-ward shifts of the concentration-response curve  EC50 increases with increasing antagonist concentration  Degree of shift is directly proportional to the concentration of antagonist  Maximal response stays the same  What is a ‘non-competitive’ antagonist? Antagonist binds to a different site from agonist Called an ‘allosteric’ antagonist or inhibitor Consequences appear the same as irreversible competitive Differences between competitive and non-competitive antagonism on log dose response curve EC50 EC50 values values EC50 increases with EC50 decreases with competitive antagonist competitive antagonist concentration concentration Log concentration curve Emax reduces with shifts to the right but Emax noncompetitive antagonist unaffected concentration  How does a non-competitive antagonist affect a log dose response curve? No spare receptors Spare receptors present EC50 values EC50 values Max response falls with increasing Max response only falls with increasing antagonist concentration antagonist concentration when receptor reserve is used up EC50 increases with increasing antagonist concentration EC50 increases with increasing antagonist concentration  Antagonism that does not affect agonist binding 1) Affinity for the receptor is unchanged – EC50 unchanged 2) Size of response ↓ as 2nd messenger ↓ Example of functional antagonism Target enzyme – Adenylyl cyclase, converts ATP into cyclic AMP Ri – muscarinic Rs – β- adrenergic m2, m4 β 1, β 2, β 3 Antagonists - Classifications Physiologic Pharmacokineti al c Chemical Competitive Non-competitive  Antagonists - overview Classified by how and where they bind Binding strength ‐ Reversible ‐ Irreversible (truly or effectively) Binding site ‐ Competitive ‐ Non-competitive  Antagonists - examples Competitive – Reversible e.g., beta blockers, antihistamines – Irreversible e.g., clopidogrel, phenoxybenzamine Non-competitive – Allosteric site e.g., channel blockers – diltiazem, verapamil  Antagonists - examples Chemical – Binding endogenous ligand e.g., etanercept, TNF-α and TNF-β Pharmacokinetic – Increasing metabolism e.g., St John’s wort, oestrogen Physiological – Actions oppose each other e.g., salbutamol in asthma  What is an inverse agonist? An inverse agonist Results with induces an opposite antagonists are signalling outcome to predictable The two-state model  The two-state model Inv Ag Agonist R R* IR AR* Agonists have higher affinity for R* Inverse agonists have higher affinity for R  The two-state model Antagonist R R* AnR AnR* Antagonists have equal affinity for R and R* No change in the equilibrium  Range of agonist and antagonist FYI drugs Benzodiazepine and GABA receptor  Loss of drug response or desensitisation Desensitisation / tachyphylaxis: the effect of a drug diminishes when it is given repeatedly or continuously Tolerance: similar, but develops more slowly – Refractoriness can due to: change or loss of receptors (most agonists) Exhaustion of mediators (amphetamine) Increased metabolic degradation (alcohol) Physiological adaption (diuretics-> RAS) What might happen with long-term antagonist treatment?  Just one more concept and then something different A receptor can switch on more than one signalling pathway – Conventional agonism A biased agonist will selectively trigger one of these signalling cascades  What if drug response is not linear or easily measurable? A graded dose-response curve can be constructed for responses that are measured on a continuous scale, e.g., heart rate Sometimes effects are ‘all-or-nothing’ i.e., quantal rather than graded – e.g., sleep, death, infection, pregnancy, presence or absence of epileptic seizures So how can we measure potency, and safety in these drugs? Frequency distribution curve A frequency distribution or quantal dose- response curve can be constructed for drugs that elicit an ‘all-or-none’ response Thus the response (y) axis is % people who respond to a  What is the median effective dose 50% (ED50)? Helps identify drug dose required to elicit therapeutic benefit The median ED50 is the dose required to produce a therapeutic effect in 50% of the  What is the median effective dose 50% (ED50)? Dose required to produce a specified response in 50% of a population But which response?  The difference between therapeutic index and therapeutic windowNOAEL no observed adverse effect = level NOAEL  Summary (1) Five types of antagonism (others exist) ‐ Competitive ‐ Non-competitive ‐ Chemical ‐ Pharmacokinetic ‐ Physiological They bind the orthosteric or an allosteric site Their effects on dose-response curves of agonists  Summary (2) ugs can be agonists, antagonists or inverse ago Selective affinity for R or R* ased agonism occurs sponses can be graded or quantal Both can be handled the same mathematically Quantal – ED50 states 50% of the population… erapeutic index erapeutic window  What we have learned… Define 'Antagonists’ Explain the difference between reversible, irreversible, competitive, non–competitive antagonists Describe how each type of antagonist affects the dose-response curve Explain population studies for 'all-or-none’ responses