Pharmacology 4001 Lecture 8 PDF

Optional accompanying reading Pharmacology 4001: Mechanisms of Drug Action Lecture 8: How Drugs Act Feb 8, 2024 Chapter 3 Discussion of Research Paper #1 Next Tuesday 2/13 Discussion of Research Paper #1 If you would like additional slides to be part of your presentation (optional), please send them...

Optional accompanying reading Pharmacology 4001: Mechanisms of Drug Action Lecture 8: How Drugs Act Feb 8, 2024 Chapter 3 Discussion of Research Paper #1 Next Tuesday 2/13 Discussion of Research Paper #1 If you would like additional slides to be part of your presentation (optional), please send them to me by Monday 2/12 Discussion of Research Paper #1 s Check out the rubric available on Canva Note: You are welcome to bring up a notecard, or paper, or laptop with notes Information on Exam #1 Information on Exam #1 Finishing Our Discussion of Clinical Trials t s r s t s s Why do a Phase I trial? Things to conside – Controlled Environmen – Limitation – Ethic Characteristics of well-executive Phase II and Phase III Trial – Patients randomized to treatment group – Controlled using a placebo group and/or current standard-of-care treatmen – Evaluates not just a drug, but also the specific formulation, route, dose, and dosing regimen Learning Objectives 1. Define types of ligands: agonists, inverse agonists, antagonists 2. List intermolecular interactions that may contribute to drug binding. 3. Explain how radioligand binding can be used to determine a drug’s affinity for a target and be able to look at a specific binding curve and ball-park binding affinity (KD).. 4. Describe the what structure-activity relationship studies are and why they are performed. Defining a ‘Receptor’ In pharmacology, the term receptor is used broadly to refer to the macromolecule with which a drug interact – Can be a transmembrane or nuclear receptor or a transporter or an enzyme s Figure 1.2 of JF Clark, SF Queener and VB Karb “Pharmacological Basis of Nursing Practice” 2000, Mosby. Affinity vs. Efficacy Receptors are in a dynamic equilibrium between inactive and active conformations R Inactive conformation ensemble R* Active conformation ensemble Receptors that spend enough time in the active conformation in the absence of agonist to active transducers are said to have constitutive activity R Inactive conformation ensemble R* Active conformation ensemble Constitutive (basal) activity is defined as ligand-independent activity Agonists preferentially bind and stabilize the active conformation R Inactive conformation ensemble R* Active conformation ensemble Antagonists block the ability of agonists to bind, but do not change the equilibrium R Inactive conformation ensemble R* Active conformation ensemble Antagonists do not stabilize either the active or the inactive conformation Antagonists block the ability of agonists to bind, but do not change the equilibrium R Inactive conformation ensemble R* Active conformation ensemble Antagonists do not stabilize either the active or the inactive conformation ◆ ◆ ◆ Bind equally to both conformations Equilibrium is not affected Pharmacological effect results from preventing natural signaling molecules or an added agonist from binding and shifting equilibrium to the active state. Inverse agonists preferentially bind and stabilize the inactive conformation R Inactive conformation ensemble R* Active conformation ensemble Many compounds originally characterized as antagonists have been re-categorized as inverse agonists based on structural data suggesting that they preferentially bind the inactive receptor conformation Types of Interactions by Which a Drug Can Interaction with a Receptor Ioni Covalent (some irreversible, like aspirin Hydrogen bond Hydrophobic interactions ) s c Multiple types in the same drugreceptor interactions! Key Points No compound is 100% specific for a receptor. That is, all drugs are capable of binding multiple targets at some concentration. The potential for drug interactions with receptors that cause toxicity increases with the drug concentration. So, how do you determine which receptor is mediating the effects of a drug? 1. Compare drug affinity and potency at a target to the concentrations at which the drug produces the pharmacological benefit. 2. Conduct structure-activity relationship studies.. 3. Use genetically modified organisms and assess the effect of the drug on organisms lacking the hypothesized target KD is a measure of drug affinity for a target. It is the concentration of drug needed to occupy 50% of receptors. Drug-Receptor Drug (D-R) (D) KD = + [D] [R] [D-R] This is a dissociation constant. The lower the KD, the higher the affinity of a drug. Receptor (R) Drug-Receptor Drug (D-R) (D) KD = [D] [R] + Receptor (R) To show that this is true, let’s set the concentration of drug [D] equal to the KD [D-R] Brackets [], indicate ‘the concentration of’ whatever is inside. For example, [D] is the concentration of drug (unbound drug) and [D-R] is the concentration of drug-receptor complex (bound drug) Drug-Receptor Drug (D-R) (D) KD = + Receptor (R) [KD] [R] [D-R] Brackets [], indicate ‘the concentration of’ whatever is inside. For example, [D] is the concentration of drug (unbound drug) and [D-R] is the concentration of drug-receptor complex (bound drug) Drug-Receptor Drug (D-R) (D) KD = + Receptor (R) [KD] [R] [D-R] Brackets [], indicate ‘the concentration of’ whatever is inside. For example, [D] is the concentration of drug (unbound drug) and [D-R] is the concentration of drug-receptor complex (bound drug) Drug-Receptor Drug (D-R) (D) 1= + Receptor (R) [R] [D-R] Brackets [], indicate ‘the concentration of’ whatever is inside. For example, [D] is the concentration of drug (unbound drug) and [D-R] is the concentration of drug-receptor complex (bound drug) Drug-Receptor Drug (D-R) (D) [R] Unoccupied receptor = + Receptor (R) [D-R] Occupied receptor Since all the receptors are either free or bound to ligand, this means that half the receptors are free and half are bound to ligand. In other words, when the concentration of ligand equals the Kd, half the receptors will be occupied at equilibrium. If the receptors have a high affinity for the ligand, the Kd will be low, as it will take a low concentration of ligand to bind half the receptors. Is the drug’s binding affinity consistent with the concentration resulting in the pharmacological benefit? How can you measure a drug’s affinity for a target (KD)? Saturation radioligand binding! The first approach and often still most definitive approach to demonstrate receptor binding Let’s talk about how this works… Take tissue with the hypothesized target and make a homogenate/solubilize it Your homogenate will contain impurities…that’s ok Add your drug tagged with a radioisotope Incubate Some drug will bind specifically Some drug will bind non-specifically to debris or other targets Some drug will remain unbound Now, pass your homogenate with radioligand through a filter Now, pass your homogenate with radioligand through a filter Now, pass your homogenate with radioligand through a filter Unbound Bound Put this into a tube and count how much radioactivity there is This is the ‘Total Binding’ It will have a shape like this Note that this curve has an initial bend, and then become a straight line. Counts per Minute (CPM) 3000 [3H]Drug (Total) 2000 1000 0 0 5 10 3 [ H]Drug (nM) 15 This includes both specific and nonspecific binding…we need to determine specific binding. Counts per Minute (CPM) 3000 [3H]Drug (Total) 2000 1000 0 0 5 10 3 [ H]Drug (nM) 15 Specific binding is rarely measured experimentally. Instead, it is calculated from total and nonspecific Binding. Counts per Minute (CPM) 3000 [3H]Drug (Total) 2000 1000 0 0 5 10 3 [ H]Drug (nM) 15 To measure nonspecific binding, add an abundance (10 – 100 times that of your radiolabeled compound) of an unlabeled alternative ligand for your target (best) or a saturating concentration of your unlabeled experimental drug (less ideal alternative). To measure nonspecific binding, add an abundance (i.e., 100 – 1000s times its KD) of an unlabeled alternative ligand for your target (best) or a saturating concentration of your unlabeled experimental drug (less ideal alternative). This unlabeled compound will bind all your specific binding sites, leaving your radiolabeled ligand with only nonspecific sites available. Filter to separate bound from unbound ligands Filter to separate bound from unbound ligands But this into a tube and count how much radioactivity is there This is the ‘Nonspecific Binding’ It will have a shape like this Note that this a straight line. Nonspecific binding increases linearly with the amount of radiolabeled drug. Counts per Minute (CPM) 3000 [3H]Drug (Nonspecific Binding) 2000 1000 0 0 5 10 3 [ H]Drug (nM) 15 Now we have both Total Binding and Nonspecific Binding 3000 [3H]Drug (Total) Counts per Minute (CPM) [3H]Drug (Nonspecific Binding) 2000 1000 0 0 5 10 [ 3H]Drug (nM) 15 Specific binding is calculated by subtracting nonspecific binding from total binding 3000 [3H]Drug (Total) [3 H]Drug (Nonspecific Binding) Note that the specific binding curve is a rectangular hyperbola. Specific binding is saturable, while total and nonspecific binding is not. Counts per Minute (CPM) [3 H]Drug (Specific Binding) 2000 1000 0 0 5 10 3 [ H]Drug (nM) 15 Specific binding is calculated by subtracting nonspecific binding from total binding Based on the specific activity of the isotope and the amount of material in the assay, CPM can be converted into fmol of bound receptor / mg of tissue Counts per Minute (CPM) 3000 2000 [3H]Drug (Specific Binding) KD = Measure of affinity, concentration of drug needed to occupy 50% of the specific binding sites Bmax = Total number of specific binding sites 1000 0 0 Specific Binding Bmax = 1273 ± 52 CPM Kd = 0.52 ± 0.1 nM R2 = 0.9120 5 10 3 [ H]Drug (nM) 15 What if your drug of interest isn’t or can’t be radiolabeled? You can conduct competition radioligand binding experiments and calculate an (inhibitory concentration) IC50 value. IC50 values can provide important information on ligand affinity when KD cannot be calculated. Note that to calculate an IC50 value you must have two ligands competing to occupy the same binding site on the receptor. Structure-Activity Relationship (SAR) Studies Is there a correlation between derivatives of a drug that bind to the presumptive receptor and the pharmacological effect? (COX-1, COX-2 ) Example: Determining Aspirin’s Site of Action Physiological Effects of Prostaglandins h : Pain: PGI2 and PGE2 sensitize nerve endings to bradykinin, histamine and substance P Inflammation: PGI2, PGD2 and PGE2 are vasodilators (edema, erythema) Protection of the gastric mucosa: PGE2, PGI2 Maintenance of renal blood flow: PGE2 , PGI2 Fever: PGE2 Platelets: TXA2 stimulates platelet aggregation Uterus: PGF2a contracts uterus Other PGE2 keeps ductus arteriosus open following birt Aspirin SAR Indomethacin Aspirin Salicylic Acid Aspirin = acetylsalicylic acid Learning Objectives 1. Define types of ligands: agonists, inverse agonists, antagonists 2. List intermolecular interactions that may contribute to drug binding. 3. Explain how radioligand binding can be used to determine a drug’s affinity for a target and be able to look at a specific binding curve and ball-park binding affinity (KD).. 4. Describe the what structure-activity relationship studies are and why they are performed. Homework Assignments for Jan 16 – Feb. 13 Complete Quiz #7 by Tuesday at 9:05 am. On Canvas. Two attempts NEXT CLASS. : RESEARCH PAPER #1: Druker at al. (1996). Effects of a specific inhibitor of the Abl tyrosine kinase on growth of Bcr-Abl positive cells. Nature Medicine 2, 561-564. – Start reading and prepare to discuss during lecture on Feb. 13 (First student presentations will be on this day. Figure/Table assignments are available on Canvas.)