PCL3000H Pharmacodynamics Lecture 3 PDF

PCL3000H Session 3 Oct 31 2024 Pharmacodynamics Pharmacodynamics what the drug does to the body (i.e., the action of the drug) the relationship between drug concentrations and responses Most drugs act by initially binding to target proteins called “receptors” present on the cell surface or...

PCL3000H Session 3 Oct 31 2024 Pharmacodynamics Pharmacodynamics what the drug does to the body (i.e., the action of the drug) the relationship between drug concentrations and responses Most drugs act by initially binding to target proteins called “receptors” present on the cell surface or within the cell Some targets of drugs are non-protein such as ions (chelators), lipids, DNA, water (laxatives) Drugs that bind to receptors are manipulating or ‘hijacking’ the ability of these proteins to bind to endogenous ligands that are responsible for directing a particular biological process or signal. https://media.pharmacologycorner.com/wp- content/uploads/2009/01/types-of-drug-receptors.jpg  The Druggable Genome Subsets of genes in the human genome expressing proteins with the ability to bind drug-like molecules have been termed the “Druggable Genome”. Hopkins, AL and Groom, CR. Nature Reviews Drug Dis. 1:727, 2002 entire portfolio of pharmaceutical industry 3051 of predicted 30,000 genes in human 483 drug targets genome encode a ‘druggable’ protein 130 protein families represent all drug targets Drug targets druggable protein -not necessarily a drug target How do receptors function and how do we manipulate their signaling with drugs to achieve therapeutic benefit? Receptor Classes 1. Ion channels 2. G protein-coupled receptors 3. Receptor Tyrosine Kinase 4. Steroid receptor superfamily 5. Others  Ion Channels (Ionotropic Receptors) Drugs can mimic, block or modulate the actions of endogenous ligands that control the flow of ions through plasma membrane channels Fastest mechanism--cellular response can occur within milliseconds Often associated with signaling at neuronal synapses https://doi.org/10.1016/bs.apha.2014.06.019 Ion Channels – GABA (γ-Aminobutyric acid) and GABAA receptor 1. GABA can bind to extracellular sites of the GABAA receptor (subunit diversity) 2. triggers opening of chloride ion-selective pore 3. Increased chloride conductance causes hyperpolarization of plasma 4. blocks firing of new action potentials. 5. This mechanism is responsible for the sedative effects of GABAA allosteric agonists.  G Protein-Coupled Receptors 7 transmembrane domains AND receptors can oligomerize Ligand binding to extracellular region of the receptor may cause conformational change, activates a G-protein and initiates a signaling cascade Several G-proteins and effectors Second fastest response--occurs within seconds and lasts seconds to minutes Some Ligands (GABA, acetylcholine) can act on ionotropic receptors or G protein-coupled receptors In unstimulated cells, the state of G alpha (orange circles) is defined by its interaction with GDP, G beta-gamma (purple circles), and a G-protein-coupled receptor (GPCR; light green loops). Upon receptor stimulation by a ligand called an agonist, 1) G alpha dissociates from the receptor and G beta-gamma 2) GTP is exchanged for the bound GDP, which leads to G alpha activation. The Molecule Pages database. Nature 420, 716-717 3) G alpha then goes on to activate other (2002 molecules in the cell.  G protein signaling 1. Different GPCRs can be coupled to different G protein types 2. G proteins – different α versions can stimulate or inhibit a given signaling effect 3. Adenylyl cyclase – increased cAMP is an amplification of signal 4. The dissociated Gβ and Gγ subunits act together and can signal independently of the Gα subunits https://doi.org/10.1007/978-981-13-1571-8_1  G-protein coupled receptors that bind dopamine 1. Different dopamine binding GPCRs can be coupled to different G protein types 2. Drugs that interact with dopamine receptors affinity will be receptor-specific 3. Dopamine receptors expression 4. Oligomerization of receptors can result in novel downstream sequalae http://www.frontiersin.org/Cellular_Neuroscience/editorialboard Receptor Tyrosine Kinase (RTKs) Cytosolic enzyme activity Binding of a ligand to an extracellular domain leads to an increase or decrease in enzyme activity Most are associated with tyrosine kinase activity-- activated when ligand binds to two receptors and leads to phosphorylation of tyrosine residues of specific proteins Duration of responses to receptor stimulation is on the order of minutes to hours 1. Ligand binding to both receptors, receptors form dimer 2. Tyrosine kinases activated, each phosphorylates tyrosines on the tail of the other 3. Receptor proteins now recognized by ‘relay proteins’ inside the cell 4. Relay proteins bind to phosphorylated tyrosines, activate transduction pathways Intracellular Receptors Drug must enter the cell to interact with the receptor, so ligand is usually hydrophobic Binding of ligand usually results in dissociation of a repressor protein ligand-receptor complex moves to the nucleus and bind to specific DNA sequences that result in the regulation of gene expression Longest onset (> 30 minutes) and duration of response is (hours to days)  Summary of Drug Targets Many drugs with many distinct mechanisms of action Most drugs act by one of four common receptor-based mechanisms -- binding of the drug to an ion channel, GPCR, enzyme-linked receptor or intracellular receptor Understanding how a drug exerts its effects can assist in the prediction of how long it will take to cause a therapeutic response and how long that response will last  Drug – Receptor Binding Irreversible Covalent Binding formation of a shared electron pair bond (and possibly effect) can last long after you stop taking the drug e.g., aspirin and ibuprofen reduce pain by binding to cyclooxygenase (acetylation at Ser5290 which is involved in the production of prostaglandins, cytokines, etc. https://doi.org/10.4236/ojfd.2021.114010 Reversible Binding typical of most drug-receptor interactions e.g., G protein-coupled receptors DOI:10.1039/c2mb25429h Drug – Receptor Binding Drug-Receptor Binding …How do/did we study this? Use radioactive or fluorescent ligand… RADIOLIGANDS – untagged receptors in tissue/cells..binding assays rely on use of scintillation proximity (separation of bound ligand from free ligand, followed by detection of bound ligand using scintillation counter) FLUORESCENT LIGANDS - binding to unlabelled GPCR can be through microscopy, using plate readers to detect fluorescence, or through fluorescent polarization and flow cytometry, use of fluorescent ligands and tagged GPCRs for FRET, BRET assays https://www.celtarys.com/science-highlights/the-advantages-to-using-fluorescence-vs-radioactivity-in- receptors-kinetic-and-binding-assays.html  Radioligand Binding Assay So… if we want to study a drug and receptor using a Radioligand Binding Assay, what criteria should we be concerned with? Specific Activity – we need to be able to be able to detect ligand – receptor interactions in tissue that can be detected using scintillation assays… how does this work: a scintillator bead gets excited and emits light when it binds or is in close proximity to a radioactive isotope. Any radioactive material goes through decay, during this radioactive decay it emits energy in the form of beta-particles that can be electrons or positrons; in this technique the energy of the radioactive decay is converted into light photons. The beta- particles transfer their energy to the scintillators while traveling through an organic scintillator medium. These scintillators get excited and emit light which can be detected by scintillator counters or CCD imagers. Affinity – we need the radioligand to avidly bind to the receptor to a high degree, ideally we want less than 10% of the radioligand to dissociate from the receptor when we add the ligand to the receptor in the tissue bound to the filter (process takes less than 1 minute) Selectivity – the ligand should want to bind to the receptor being studied to a high degree and not want to bind to other receptors or other proteins/interaction sites + = Radioligand Binding Assay Binding (moles per mg of tissue) Label drug of interest with radioactive isotope, add to tissue to measure amount of drug that is ‘bound’ and compare to amount that is ‘free’ … use to get info on # of specific binding sites 1. Homogenize brain and divide into 2 sets of tubes 2. First set of tubes - Incubate samples with various concentrations of radiolabelled drug 3. Filter and wash sample to only leave behind drug bound to Radioactive drug concentration brain tissue (see graph top right of slide) 4. Second set of tubes – Incubate samples with same Binding (moles per mg of tissue) concentrations of radiolabelled drug AND large excess of cold non-radioactive drug 5. Filter and wash sample to only leave behind radioactive drug 6. Measure amount of radioactivity per sample For the second set of tubes – the large excess of ‘cold drug’ binds to all receptor sites present, essentially leaves no specific sites for radioactive drug, only nonspecific sites bound by drug (bottom right of slide) Radioactive drug concentration  Radioligand Binding Assay – “Saturation Binding” Binding (moles per mg of tissue) B max D+R DR Receptor-mediated binding – should be saturated at higher concentrations – means all receptors are occupied Total Binding – Nonspecific Binding = Specific Binding Radioactive drug concentration Kd Bmax – maximum number of specific binding sites in tissue Kd – the molar concentration of drug required to occupy 50% of the receptors, known as the Dissociation Constant  Kd Dissociation Constant Thus, when [D] = Kd the reaction is at equilibrium and the concentration of drug D is just enough to bring the reaction to half-maximal completion, i.e. half of all receptors are occupied. The mass action equation for drug/receptor interaction is therefore: [DR] = [D][RT] / ([D] + Kd) where RT is the total number of receptors and [DR] is the concentration of the complex of drug and receptor The fractional occupancy is the proportion of the receptors occupied by the drug (which would be [DR]/[Rt]) if we rearrange the equation like so: [DR] / [Rt] = [D] / ([D] + Kd) Scatchard plots (Rosenthal plots) With the hyperbolic saturation curve, we can determine Kd but we can only ‘estimate’ Bmax We usually get this information from a nonlinear regression analysis known as a Scatchard analysis (‘Bound is plotted on the X axis, Bound/Free is plotted on Y axis) Then we get a straight line 1. The slope of the line is -1/Kd and the X-intercept is the Bmax  Comparing the ability of different drugs to bind to a receptor and elicit a response Potency It’s easier to compare effects of different drugs if we plot on Semi-Log Dose-Response curves The y-axis might measure ‘binding’ or ‘response’ Potency – reflects dose/concentration needed to give a particular response/fraction of binding Can use ED50 values to compare relative potency, the higher the potency the less drug needed to elicit a given effect The y-axis might measure ‘binding’ or ‘response’ Comparing the ability of different drugs to bind to a receptor and elicit a response - Efficacy Efficacy – measuring the biological response of a drug that is binding to Receptor # is regulated – it is possible to a receptor achieve a maximal response without engaging The higher the maximal response, the more efficacious the drug is every receptor (some are spare receptors)  Comparing the ability of different drugs to bind to a receptor and elicit a response Agonist vs. Antagonist Agonist – a ligand or drug that has affinity for a receptor that produces/alters a signal and leads to altered cell function – an agonist drug usually mimics the action of an endogenous ligand Antagonist – a ligand or drug that binds to a receptor but doesn’t alter activity -its binding can block action of endogenous agonist ligand or another drug from binding When conducting “Saturation binding experiments”, the ligand to be used will likely be an antagonist instead of an agonist In general, antagonists bind receptors with higher affinity than agoinsts -agonist induce conformational changes in receptor-effector complexes, can cause ligand-receptor complexes to exist in multiple states with different binding characteristics https://images.app.goo.gl/MMVwudQx3JBGRUdP8  Competitive vs. Noncompetitve Antagonism Competitive – reversible competitive inhibitors Produce a parallel shift to Noncompetitive – irreversible right of agonist inhibitors produce a shift but NOT -extent of the shift parallel to binding curve of agonist rightward is related to the -cannot be overcome by increasing Affinity of the antagonist to the agonist concentration – the insurmountable could be allosteric antagonist: binds to site on receptor DISTINCT from agonist but changes binding affinity for agonist  Competition Binding Plots Measure equilibrium binding of a single concentration of radioligand at various concentrations of unlabeled contributor(s) … learn about the affinity of the competitor Most ligands for receptors are not available in a radioactive form As the concentration of unlabeled ligand increases, the amount of radioligand bound to the receptor decreases  Types of Agonists Full Agonist – prefers active state of the receptor and produces full biological response at saturating concentrations Partial Agonist – has less efficacy at full receptor occupancy than a full agonist -can function like an antagonist in the presence of a full agonist Inverse Agonist – has preferential affinity for the inactive state of a receptor (less efficacy than the basal state)  Biased Agonism Biased signaling. Biased agonism is the ligand-dependent selective activation of a subset of all GPCR-controlled signal pathways, compared to a reference agonist. Binding of different ligands (red, orange) to the same GPCR (green) activates the primary GPCR effectors (G proteins, arrestin) to different degrees (shaded arrows), leading to ligand-specific outputs. DOI:10.1016/j.bbamcr.2018.11.015