Receptor Pharmacology Lecture Notes PDF

Receptor Pharmacology 1. Introduction to Receptor Pharmacology 2. Ligand-receptor interactions and Dose Response Relationships 3. Agonist, antagonist and Inverse Agonist 4. Cooperativity, allosteric interactions Dr. Ly P. Vu Assistant Professor, Faculty Pharmaceu...

Receptor Pharmacology 1. Introduction to Receptor Pharmacology 2. Ligand-receptor interactions and Dose Response Relationships 3. Agonist, antagonist and Inverse Agonist 4. Cooperativity, allosteric interactions Dr. Ly P. Vu Assistant Professor, Faculty Pharmaceutical Sciences Scientist, Terry Fox Laboratory, BC Cancer Who is Dr. Ly Vu [email protected] @Vuphuongly Full name: Ly Phuong Vu or Vu Phuong Ly Originally from Hanoi, Vietnam U Laboratory BSc in Molecular Biology, Vietnam National University, Hanoi PhD in Cancer Biology, Memorial Sloan Kettering Cancer Center, NY, NY, USA – thesis in transcriptional control and histone modifying enzymes in blood stem cells and leukemia. Post-doc in Molecular Pharmacology and Cancer Biology, Memorial Sloan Kettering Cancer Center, NY, NY, USA Joined Terry Fox Laboratory, BC Cancer July 2019 Joined Faculty of Pharmaceutical Sciences, UBC January 2022 Teach PHRM-100 (this lecture) and PHRM 333- Pharmacotherapy in Oncology Introduction to Receptor Pharmacology  What is receptor pharmacology – why do we need to study this subject? Ø Receptor pharmacology is the study of the interactions of receptors with drugs/pharmaceuticals and other xenobiotics (substances that are foreign to the body). Ø Majority of drugs target receptors act through their interaction with specific receptors. Ø Understanding receptor pharmacology is fundamental for study of effects of drugs, drug mechanisms of action and pharmacological features of drugs. Learning Objectives Define and describe different types of “receptors” and their “ligands” Understand and describe the history of “Receptor Theory” Name and describe examples of ligands/drugs that act on different types of receptors Definition of Receptors ! Receptor is the component of a cell that interacts with a specific ligand (e.g. drug) and initiates the chain of events leading to the observed effect (adapted from Katzung, 2011) ! Receptors are usually intracellular or membrane-bound proteins which produce a pharmacological effect after binding with a specific ligand (e.g. drug) ! Macromolecules as receptors: protein; DNA and RNA Why cells need Receptors ? External Environment DNA/gene Genotype A Cell mRNA Phenotype Protein Why cells need Receptors ? External Environment DNA/gene Interact with neural system Genotype Interact with mRNA other cells A Cell within tissues Phenotype Interact with Protein Immune system Why cells need Receptors ? Signals External Environment DNA/gene Interact with Genotype neural system Interact with mRNA other cells A Cell Phenotype within tissues Receptors “receive” signals for cell to ”know”: Interact with ü what they are Protein Immune system ü where they are ü what they do Mode of delivering signals to receptors 5 types: Ø Paracrine signaling molecules: target neighboring cells close to the release site. Ø Endocrine (hormones): released by endocrine cells and reach target cells in one or more distant body locations via blood circulation. Ø Autocrine signaling molecules: target the same cell that secreted them. Ø Neurotransmitters: are released by neurons and affect other neurons or effector cells near the release site. Ø Cell- cell interaction: between cells without chemical messengers Why cells need Receptors ? Signals External Environment DNA/gene Interact with Ligands Genotype neural system Interact with mRNA other cells A Cell Phenotype within tissues Receptors “receive” signals for cell to ”know”: Interact with ü what they are Protein Immune system ü where they are ü what they do Definition of Ligands ! Ligand is a molecule that binds to a receptor to send signals leading to a change in cell signaling/activities, and ultimately, cell behavior or structure. ! Ligands bind specific receptors ! Ligands can be classified by different criteria ! There are two main types of ligands based on their location: Ø Intracellular ligands: ligands that bind to receptors inside the cell Ø Extracellular ligands: ligands that bind to receptors outside the cell Types of Ligands Based on chemical properties of the ligands: Ø water-soluble (hydrophilic) – e.g. peptide hormones; neurotransmitters; cytokines; nucleotides and nucleosides Ø lipid-soluble (hydrophobic) – e.g. steroid hormones (cortisol, testosterone, and estrogen etc); thyroid hormones; eicosanoids; nitric oxide Based on nature of the ligands: Ø Gas Ø Cholesterol Ø Amino acids and Peptides Ø Proteins Ø Nucleotides and nucleosides Types of Receptors Based on their location in cell Ø Cell surface receptors – typically trigger downstream cell signaling pathways Ø Intracellular receptors – cytoplasmic/nuclear receptors – typically directly trigger downstream transcriptional activities of gene activation/inhibition  Cell surface receptors 3 types of cell surface receptors Ø Are transmembrane proteins embedded in the plasma membrane of target cells. Ø Consist of : (i) an extracellular domain containing the ligand-binding site, (ii) a transmembrane domain and (iii) an intracellular domain that transmits the signal inside G protein the cell. Ø Receive a water-soluble ligand and initiate a signal transduction pathway that alters cellular function. Ø 3 main types based on structural and functional G protein–coupled receptors Enzyme-linked receptors Ion channel receptors similarities https://theory.labster.com/signaling-receptor/ G protein coupled receptors Ligand Inactive Active Ø GPCRs consist of a single polypeptide embedded in a cell's plasma membrane- seven-transmembrane receptors and the intervening portions loop both inside and outside the cell. Ø GPCRs interact with G proteins in the plasma membrane. Ø An external signaling molecule binding to GPCR causes a conformational change in the GPCR. This change then triggers the interaction between the GPCR and a nearby G protein. Ø G proteins are specialized proteins with the ability to bind the nucleotides guanosine triphosphate (GTP) and guanosine diphosphate (GDP). Ø Responses are fast – within seconds Adapted from https://www.nature.com/scitable/ G protein coupled receptors Second messenger Muscle relaxation Examples of GPCRs include adrenergic receptors, muscarinic acetylcholine receptors, dopamine receptors, and opioid receptors. Enzyme linked receptors Ø Enzyme-linked receptors usually has only one transmembrane domain. Ø Enzyme-linked receptors have cytosolic domain with either an intrinsic enzyme activity or associated directly with an enzyme. Ø Responses to signals are slow (minutes to hours) and require many intracellular signaling steps that eventually lead to changes in gene expression. Ø Enzyme-linked receptors response to extracellular signal proteins that promote the growth, proliferation, differentiation, or survival of cells in animal tissues. Adapted from https://www.nature.com/scitable/ Enzyme linked receptors Ø 6 classes of of enzyme-linked receptors Seven subfamilies of receptor tyrosine kinases 1. Receptor tyrosine kinases - Largest group 2. Tyrosine-kinase-associated receptors 3. Receptor like tyrosine phosphatases 4. Receptor serine/threonine kinases 5. Receptor guanylyl cyclases 6. Histidine-kinase-associated receptors Adapted from https://www.nature.com/scitable/  Enzyme linked receptors Mechanisms – Oncology f Major Classes of Cell Surface Receptors Neighbor cell Dimeric Cytokine ligand Ligands Delta Hedgehog WNT Notch r n TGFβ LRP P P P P P P PTCH SMO Frizzled P P P P Dishevelled P P P STAT Kinase Smads Signaling TF TF cascades P P P P P P TF P P P P P TF P Cytokine Receptor TGFβ Hedgehog Wnt Notch s receptors tyrosine kinases receptors (Hh) receptors receptors receptors Activate cytosolic Activate cytosolic Activate Smad Hh binding causes release Release an activated Cytosolic domain of Notch STAT transcription kinases that translocate transcription factors in the of transcription factor transcription factor from a released by proteolysis factors by to the nucleus and cytosol by phosphorylation (TF) from cytosolic multiprotein complex in acts in association with phosphorylation activate transcription complex the cytosol nuclear transcription factors by factors phosphorylation Adapted from https://www.nature.com/scitable/ Ion channel receptors Ø Ion channel receptors are multimeric proteins located in the plasma membrane. Ø Ion channel receptors forms a passageway or pore extending from one side of the membrane to the other. Ø Ion channels have the ability to open and close in response to chemical or mechanical signals. Ø Responses are very fast - within few milliseconds Ø Individual ion channels are specific to particular ions, meaning that they usually allow only a single type of ion to pass through them. Ø Most well characterized –Nicotinic receptor for neurotransmitters Adapted from https://www.nature.com/scitable/ Ion channel receptors Nicotinic receptor Ø Consists of a pentamer of protein subunits, with two binding sites for acetylcholine – when bound alter the receptors’ configuration, resulting in the open of the internal pore Ø The pore allows Na+ ions to flow down their electrochemical gradients Intracellular receptors Ø Intracellular receptors are typically globular proteins that located in the cytoplasm or nucleus of target cells Ø Intracellular receptors bind lipid-soluble chemical messengers e.g. steroids (corticosteroids, vitamin D etc); Retinoic acid (vitamin A); hormones Ø Intracellular receptors once activated induce gene expression changes by binding to “response elements” on DNA. Ø The effects are slow (minutes to hours) and can be long- lasting. Ø The persistence of effect from enzymes/proteins can remain active in cells for hours or days after they are synthesized. Intracellular receptors Adapted from https://www.open.edu/ Drugs as ligands for receptors to elicit responses Receptors G Protein Enzyme Ion channel Intracellular coupled linked receptors receptor receptors receptors Drug groups Adrenergic receptor agonists or antagonists Insulin and estrogen/progesterone agonists Neuronal receptor modulators Steroid drugs The importance of Receptor Theory/Concept “The receptor concept is to pharmacology as homeostasis is to physiology, or metabolism to biochemistry. They provide the basic framework, and are the ‘Big Ideas' without which it is impossible to understand what the subjects are about.” The receptor concept: pharmacology's big idea by H P Rang1,2006 The concept of Receptor helps explain many aspects of drug actions. Receptors Ø Largely determine the quantitative relations between dose or concentration of drug and pharmacologic effects Ø Are responsible for selectivity of drug action Ø Mediate the actions of pharmacologic agents i.e. agonists and antagonists Introduction of Receptor Concept In mid-1870s, Langley proposed physiological action of the drug alkaloid pilocarpine and atropine can “form compounds” with physiological substances similar to how two inorganic chemical substances combine with each other. 1905- in his work on the effect of nicotine on muscles, Langley described ” …nicotine (activates muscles) John N. Langley- Professor of Physiology, and curare (induces muscle paralysis) act on the University of Cambridge “receptive substance” of muscle cells” Introduction of Receptor Concept Ehrlich’s groundbreaking article “Aus Theorie und Praxis der Chemotherapie”, was the basis for histological staining techniques– using dyes to visualize cellular structures. 1878- Ehrlich’s famous article on 'side-chain theory of immunity” describing the binding of toxins to cells- certain side-chains of the cells could bind certain toxins with selectivity and specificity. Paul Ehrlich_Nobel prize 1908 ” …specific chemical characteristics of a cell responsible for specific binding ….” These receptors are either associated with cells or distributed more abundantly in the blood stream in response to antigen interaction. Introduction of Receptor Concept v In 1900, Ehrlich replaced the term 'side chain' with 'receptor’ v In 1907, Ehrlich proposed “chemoreceptors for drugs” v Ehrlich won the Nobel prize in 1908 for his landmark immunological insights v The receptor concept was extensively used in Ehrlich’s chemotherapy work on “magic bullet concept”: drugs that go straight to their intended cell- structural targets. History of Receptor Theory/Concept The emergence of the drug receptor theory _Nature reviews 2002 Ligand/drug-receptor interactions and Dose Response Relationships Learning Objectives Understand and describe drug-receptor interactions Name and describe techniques to measure ligand-receptor interaction Define dose–response relationship Describe the use and implications of dose-response relationships Define drug specificity versus selectivity Describe the occupancy theory and its modifications Understand the basis and use of dose-response curve Define Efficacy versus Potency List the definitions of ED50, TD50, therapeutic index Describe and explain the different factors (Vmax/Emax, Km/Kd etc.) influencing the dose–response curves  Recognition of the ligand at receptor site Lock and Key model Lock and Key model for Enzymes recognize their substrates Ligand-Receptor interactions as a lock receives a key. Emil Fisher Ligand/Drug Molecule Ravel et al,. 2022 Recognition of the ligand at receptor site Induced Fit theory Substrate (for enzyme) or ligand (for receptor) binding causes a subsequent conformational change in the complex Daniel Koschland Specificity and Flexibility Boggula et al,. 2023 Drug-Receptor binding forces Interaction between a drug and receptor is formed via multiple bonds. Four basic types of binding: i. hydrophobic bond; ii. Hydrogen bond iii. Ionic bond iv. Covalent bond Drug-binding forces vary in strength. Hydrophobic binding is weak, whereas covalent binding can be strong. Most drugs reversibly bind to their receptors. Duration of engagements vary from minutes to hours. Chapter1_2017_Pharmacology-and-Therapeutics-for-Dentistry Drug specificity vs. selectivity The strength and duration of the interaction between a drug and its target receptor and the intended actions are characterized by the selectivity and specificity of the drug Selectivity refers to a drug's strong preference for its intended target over other targets. i.e. a drug which is highly selective binds one/few receptors while a non-selective drug binds many receptors Specificity describes the extent to which a drug produces only the desired therapeutic effect without causing any other physiological changes. i.e. a drug with high specificity exhibits a strong drug–receptor interaction, ensuring targeted action and minimal side effects while drugs with low specificity may produce unintended consequences due to their weak drug-receptor interaction. Drug specificity vs. selectivity Factors influence selectivity and specificity: Affinity of drug-receptor interaction Uniqueness of interaction sites Presence/absence of receptors in other tissues/cells Ability of ligands/drugs to interact with other molecules/structures *** No drug is entirely specific – NO 1 drug-1receptor Measuring ligand-receptor interaction Ligand binding assays directly capture the engagement and binding of ligand-receptor – the first step of ligand-receptor interaction –, measuring the physicochemical properties and kinetics of ligand- receptor complex formation. Functional assays measure the actual biological response (electrical or biochemical or physical) evoked by the ligand via its receptor – indirect readout of ligand-receptor engagements and its effectiveness Dose-Response Relationship A fundamental aspect of drug action is the relationship between the dose administered and the effect/response obtained. Ø Dose: amount of drug administrated in the patient e.g. 500 mg of tylenol Ø Response: effect shown by the body to a particular drug e.g. reduced pain Ø The relationship used to analyze a kind of response obtained after administering specific dose of drug Ø Dose vs. plasma concentration relationship Ø Plasma concentration vs. response relationship Dose-Response Relationship A fundamental aspect of drug action is the relationship between the dose administered and the effect obtained. The dose–effect relationship of a drug is NOT a linear function throughout the entire dose range. Ø Below a minimum threshold, there will be no observable effect. Ø Above a certain ceiling, even a large dose would exert no additional effect - the maximal effect has already been reached.  Occupancy Theory/ Occupation Concept Ø 1926, A.J. Clark published his classical papers on the actions of acetylcholine and atropine on the frog heart ….concluded: ‘‘The hypothesis that the concentration-action curve of acetylcholine expresses an adsorption process of the type described by Langmuir appears to involve fewer improbable assumptions than any alternative hypothesis’’ – ie, neurotransmitters, hormones and drugs interact with receptors. Ø Clark provided the first quantitative study of the antagonism of acetylcholine by atropine and described the ‘parallel shift’ of the log concentration-response curve produced by a competitive antagonist. Alfred J. Clark, Chair of Pharmacology University of Edinburgh (1926-1942) Ø It was from this point forward that the receptor concept began to take hold quantitatively – generation of dose-response curve (Clark_ log concentration-effect curve) What is dose response curve? Relationship of dose (or concentration – in consideration of actual plasma concentration vs. dose administrated) to response can be illustrated by a graph which is called as dose response curve. 2 required components to build a dose response curve: 1. Doses of drug 2. Corresponding percentage of response groups to specific dosages Occupancy Theory/ Occupation Concept After binding (DR) has occurred, an effect (E) follows. Drug effects (E) can be quantified: Emax : Maximal Effect (response) EC50 : Concentration yielding 50% of maximal response The effect of the drug is predictably and Threshold: lowest concentration to elicit a measurable response quantitatively dependent on the drug Kd : The dissociation constant concentration. A geometric relationship Inverse of Kd The equilibrium association constant (Ka) (rectangular hyperbola) exists when graphing E versus [D]. Chapter1_2017_Pharmacology-and-Therapeutics-for-Dentistry Fractional occupancy Assumption of the occupancy theory ~ law of mass action All receptors are equally accessible to ligands. Receptors are either free or bound to ligand. No more than one affinity state, or states of partial binding. Binding does not alter the ligand or receptor. Binding is reversible. Fractional occupancy: is the fraction of all receptors that are bound to ligand. Practical equation Fractional occupancy Practical equation When [Ligand]=Kd, fractional occupancy is 50%. Utility of the [log] dose response curve Saturation Emax Slope Threshold Utility of the [log] dose response curve Saturation Emax Slope Threshold The range of concentration needed to fully depict the dose-response relationship (~3log units) is too wide to visualize in linear format. Utility of the [log] dose response curve à Wide range of drug doses can be displayed on graph à Comparison between different drugs is easier/clearer Graded dose response curve The dose-response relationship in which the magnitude of biological response is proportional to the dose of drug i.e., as the concentration of a drug increases, its biologic effect gradually increases until all the receptors are occupied (maximal response). The pharmacological effect of drug is quantitatively measured and expressed in numeric units A graded dose–response curve has the general shape of a rectangular hyperbola (or transformed to sigmoid-log curve). Understanding Dose response curve Graded dose response curve provides 4 important values: 1. Potency 2. Efficacy 3. Slope 4. Variability Drug Potency and Efficacy Potency is the concentration (EC50) or dose (ED50) of a drug required to produce 50% of that drug’s maximal effect. Efficacy (Emax) is the maximum effect which can be expected from this drug (i.e. when this magnitude of effect is reached, increasing the dose will not produce a greater magnitude of effect). Drug Potency and Efficacy https://derangedphysiology.com/ Drug Potency and Efficacy Drug A and Drug B achieve the same Drug A achieves a higher maximum effect maximum effect, i.e. they have equal than Drug B. Drug A is therefore said to be efficacy. However, Drug A achieves this more efficacious (higher efficacy). effect at a lower dose (i.e Drug A has higher potency than Drug B). https://derangedphysiology.com/ Slope and Variability Slope reflects the effect of incremental Variability reflects reproducibility/consistency increase in doses vs. response of data – which can be variable between different testing subjects Slope and Variability (A) Steep – small window of response (Left) Variable: highly dependent on subjects – (B) Wide - large window of response e.g. individual responses to drugs differ (Right) Consistent Reading the curves https://derangedphysiology.com/ Reading the curves Both Drug A and Drug B achieve the same maximum effect, i.e. they have equal efficacy. At EC50, Drug B is more potent than drug A. Both Drug A and Drug B achieve the maximum effect (Emax) at the same dose (i.e. they have equal maximum potency). Drug B achieves EC50 effect with a lower dose. Drug A has a steeper dose-response curve than Drug B - a rise from EC50 to Emax is accomplished with a relatively small increase in dose. https://derangedphysiology.com/ Modifications of the Occupancy Theory 1954, Ariens’ modification: the maximal drug response is not equal to the maximal occupancy à modifier α: intrinsic activity where response = α x [R] 1956, Stephensen's modification: the concepts of stimulus and efficacy. non-linear relationship between receptor activation vs. response à modifier ε: intrinsic efficacy where efficacy = ε × [R] Quantal dose response curve Birkett (1995) wrote: "An alternative way of constructing a concentration effect curve is to determine the percentage of a population of patients showing a defined response at various drug concentrations... These are called quantal (population) concentration response curves and have the same shape and parameters as the graded concentration response curves" The quantal dose response is the dose-response relationship in which a defined drug effect which is either present or absent. In a population, there is usually some variation of doses required to achieve the defined drug effect The distribution of these dose tends to be a normal Gaussian distribution (i.e a bell curve) Quantal dose response curve The cumulative percentage of the population responses to increasing doses can be plotted as a curve (which assumes a sigmoid shape) Median effective dose (ED50) ED50 and LD50 The median effective dose (ED50) is the dose at which 50% of individuals exhibit the specified quantal effect The median lethal dose (LD50) is the dose required to kill 50% of subjects TD50 and Therapeutic index The median toxic dose (TD50) is the dose Toxic effect required to produce a defined toxic effect in 50% of subjects The therapeutic index (TI) is a numeric measure of the selectivity of the drug for its desired effect * TD50 can be LD50 TD50 Implications and Caveats of Therapeutic index Implications: TI guides the need for therapeutic level monitoring. Drugs with a narrow therapeutic index should be monitored more closely. IT guides the dosing interval. The drug with a narrow index would need to be given more frequently and in smaller doses. Caveats: Difficulties to determine LD50 or toxic effects for TD50 Misinterpretation of risk Therapeutic range/window Therapeutic range/window The therapeutic window is the range between the minimum toxic dose and the minimum therapeutic dose, or the range of doses over which the drug is effective for most of the population and the toxicity is acceptable. Q: For drugs, do we aim for small or large therapeutic window?