Pharmacodynamics II Drug Receptor Binding: Pharmacology and Therapeutics Prof. John Kelly and Dr. Miriam Moriarty 1 Drug Receptor Binding Competitive antagonists that bind at the agonist binding site (the orthosteric site) can be reversible or irreversible. For reversible competitive antagonists, adding increasing concentrations of antagonist shifts the receptor binding curve progressively to the right. The antagonist competes for binding sites on the receptor; the higher the antagonist concentration the more likely that a receptor binding site is taken up by an antagonist. You need to add more agonist to displace the antagonist, and get the same response as you did without any antagonist. For irreversible competitive antagonists, adding increasing concentrations of antagonists progressively lowers the maximum binding of agonist (because the antagonist takes up the binding site on the receptors and won’t let go). Learning outcomes: By the end of this practical you should be able to • Define what an antagonist is and the different types of antagonism • Prepare log concentration drug receptor binding curves for agonists with different concentrations of antagonists. • Differentiate between reversible and irreversible antagonist binding curves 2 Worked example Reversible competitive antagonism You have been provided with example data for reversible competitive antagonism. Table 1: Drug receptor binding data for the non-selective beta-adrenoceptor agonist isoprenaline, in the presence of increasing concentrations of the non-selective beta blocker, propranolol (Prop). Fractional Occupancy -8 -7 Agonist Concentration Isoprenaline (M) -6 1.0 x10 No antagonist -6 -5 -5 -4 -4 0.86 0.35 0.05 -3 0.92 0.55 0.11 -3 0.98 0.86 0.35 -2 0.99 0.92 0.55 -2 0.99 0.98 0.86 -1 0.99 0.99 0.92 0.99 0.98 5.0 x10 1.0 x10 5.0 x10 1.0 x10 5.0 x10 1.0 x10 5.0 x10 1.0 x10 5.0 x10 1.0 x10 0.01 10 M antag Prop 0 10 M antag Prop l 0 0.05 0.001 0.11 0.01 0 0 0.35 0.05 0.001 0.55 0.11 0.01 --1 5.0 x10 1 0.99 1. Plot the data on semi-log graph paper, and derive Kd and maximum occupancy. 2. Determine Kd for isoprenaline for each curve. Kd (0 propranolol) 8 x 10-5 M Kd (10-8 M propranolol) 9 x 10-4 M Kd (10-7 M propranolol 9 x 10-3 M 3. What is the maximum fractional occupancy for each concentration of antagonist? Max fractional occupancy (0 propranolol) 0.99 -8 0.99 -7 0.99 Max fractional occupancy (10 M propranolol) Max fractional occupancy (10 M propranolol 4. What is the effect of reversible (competitive) antagonism on binding and affinity? A reversible antagonist increases the Kd therefore decreases the affinity of the agonist for the receptor, but has no effect on maximum binding (occupancy) because adding more agonist will out-compete the antagonist 3 Figure 1: Reversible competitive antagonism - agonist concentration (M) on the X axis versus fractional receptor occupancy in the presence of increasing antagonist concentrations. 4 Irreversible competitive antagonism 1. You have been provided with example data for irreversible competitive antagonism. Table 2: Drug receptor binding data for the non-selective beta-adrenoceptor agonist isoprenaline, in the presence of increasing concentrations of the irreversible beta antagonist, MeB358764 (an experimental drug). -7 Agonist Concentration (M) Fractional Occupancy 1 x 10 M isoprenaline No antagonist MeB35876 -6 0.01 0.01 -6 0.05 0.05 -5 0.11 0.11 -5 0.35 0.25 0.55 0.4 0.86 0.58 0.92 0.63 0.98 0.7 0.99 0.7 0.99 0.7 0.99 0.7 1.0 x10 5.0 x10 1.0 x10 5.0 x10 -4 1.0 x10 -4 5.0 x10 -3 1.0 x10 -3 5.0 x10 -2 1.0 x10 -2 5.0 x10 -1 1.0 x10 1. Draw two curves on the same graph using the data in Table 2 (solution on next page). Answer the following questions: 2. Determine Kd for isoprenaline for each curve. Kd (0 MeB358764) 8 x 10-5 M Kd (10-7 M MeB358764) 8 x 10-5 M 3. What is the maximum fractional occupancy for each concentration of antagonist? Max fractional occupancy (0 MeB358764) 0.99 Max fractional occupancy (10-9 M MeB358764) 0.7 4. What is the effect of irreversible antagonists on binding and affinity? Irreversible antagonists decrease maximum binding but do not affect affinity. 5 Figure 2: Irreversible competitive antagonism - agonist concentration (M) on the X axis versus fractional receptor occupancy in the presence of increasing antagonist concentrations. 6 Sample Questions for Student Practise You have been provided with example data for receptor binding in the presence and absence of an antagonist. Table 3: Drug receptor binding data for the alpha adrenoceptor agonist phenylephrine, in the presence of increasing concentrations of the alpha-1 adrenoceptor antagonist, prazosin (Prz). -8 -7 Agonist Concentration (M) Fractional Occupancy 4 x 10 M 4 x 10 M Phenylephrine No antagonist Prz Prz 0.01 0 0 0.04 0 0 0.1 0.01 0 0.3 0.04 0 0.52 0.1 0.01 0.76 0.3 0.04 0.85 0.52 0.1 0.96 0.76 0.3 0.98 0.85 0.52 0.99 0.96 0.76 0.99 0.98 0.85 0.99 0.99 0.99 0.98 -9 1.0 x10 -9 5.0 x10 -8 1.0 x10 -8 5.0 x10 -7 1.0 x10 -7 5.0 x10 -6 1.0 x10 -6 5.0 x10 -5 1.0 x10 -5 5.0 x10 -4 1.0 x10 -4 5.0 x10 -3 1.0 x10 Answer the following questions: 1. Plot the data on semi-log graph paper, and derive Kd and maximum occupancy. 2. Determine Kd for phenylephrine for each curve. Kd (0 prazosin) Kd (4 x10-8 M prazosin) Kd (4 X10-7 M prazosin) 3. What is the maximum fractional occupancy for each concentration of antagonist? Max fractional occupancy (0 prazosin) Max fractional occupancy (10-8 M prazosin) Max fractional occupancy (10-7 M prazosin) 4. From your data state what kind of antagonist prazosin is. 5. Give reasons for your answer. You have been provided with example data for receptor binding in the presence and absence of an antagonist. 7 Table 4: Drug receptor binding data for the alpha adrenoceptor agonist phenylephrine, in the presence of increasing concentrations of the alpha adrenoceptor antagonist, phenoxybenzamine. -7 Agonist Concentration (M) Fractional Occupancy 5 x 10 M Phenylephrine No antagonist phenoxybenzamine -8 0.01 0.01 -8 0.03 0.03 -7 0.08 0.07 -7 0.3 0.15 -6 0.4 0.35 -6 0.65 0.4 -5 0.78 0.5 -5 0.88 0.58 -4 0.96 0.6 -4 0.99 0.6 -3 0.99 0.6 1.0 x10 5.0 x10 1.0 x10 5.0 x10 1.0 x10 5.0 x10 1.0 x10 5.0 x10 1.0 x10 5.0 x10 1.0 x10 Answer the following questions: 1. Plot the data on semi-log graph paper, and derive Kd and maximum occupancy. 2. Determine Kd for phenylephrine for each curve. Kd (0 prazosin) Kd (5 x 10-7 M phenoxybenzamine) 3. What is the maximum fractional occupancy for each concentration of antagonist? Max fractional occupancy (0 prazosin) Max fractional occupancy (5 x 10-7 M phenoxybenzamine) 4. From your data state what kind of antagonist prazosin is. 5. Give reasons for your answer. 8 9 10
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