Pharmacology and Physiology, Pharmacology Lectures BIOL243 / BMSC 213

1. Pharmacology and Physiology, Pharmacology Lectures BIOL243 / BMSC 213 Dr Paul Teesdale-Spittle School of Biological Sciences KK713 Phone 6094

2. Introduction What is pharmacology The study of the interaction between exogenous chemicals (or xenobiotics) and living organisms. 1. How an organism affects a xenobiotic. Transport, distribution and metabolism. 2. How a xenobiotic affects an organism. Molecular targets, mode of action and toxicology. Pharmacology is not just descriptive, but also quantitative. Mathematical quantification and physical chemistry. The purpose of the pharmacology lectures is to introduce the principles of the subject and show how it can be used to understand modulation of physiological function through the action of drugs.

3. Pharmacology is important for… • Chemists • designing new drugs. • Clinicians • administering drugs. • Toxicologists • explaining toxicity. • Biochemists, Physiologists, Psychologists • using drugs to modify the normal functioning of cells or organisms.

4. Definitions Xenobiotic: A chemical that is not endogenous to an organism. Endogenous: Made within. Drug: A chemical taken that is intended to modulate the current physiological status quo. Ligand: A compound that binds to another molecule, such as a receptor protein. Bioavailability: The amount or proportion of drug that becomes available to the body following its administration. Pharmacokinetics: What the body does to a drug. Pharmacodynamics: What a drug does to the body .

5. History Ancient knowledge of the materials that could relieve pain, alter moods and perceptions, aid against infection, poison etc. The first written treatises were generated by the Chinese e.g. Pen Tsao was written ~2700 B.C., describing uses and classifications of medicinal plants. The ancient Egyptians by 1550 B.C. had written prescriptions using a range of pharmaceutically active ingredients and vehicles for their delivery. At about the same time, similar medical advances were being made in Babylonia and India. Between about 400-300 B.C. the Greeks made enormous advances in the knowledge of anatomy and physiology.

6. Philippus Theophrastus Bombastus von Hohenheim (1493-1541), also known as Aureolus Paracelcus, took up the pharmacological baton. • He is often referred to as the ‘grandfather of pharmacology’ (and also the ‘grandfather of toxicology’) because of his impact on the understanding between dose and response – “All things are poisons, for there is nothing without poisonous qualities. It is only the dose which makes a thing a poison”. • No real further advances until the sciences of chemistry and physiology had developed: • To provide pure compounds. • Allow careful monitoring of their physiological effects. • This combination of circumstances arose in the early 19th Century.

7. Thus, the history of pharmacology has shown the importance of the subjects of: • Biochemistry • Chemistry and • Physiology. • The main aims of the subject are to: • Evaluate the mode and site of action of drugs • Their distribution, metabolism and elimination and • The molecular interactions by which they function.

8. Drug action • A drug is a compound that can modify the response of a tissue to its environment. • A drug will exert its activity through interactions at one or more molecular targets. • The macromolecular species that control the functions of cells. • May be surface-bound proteins like receptors and ion channels or • Species internal to cells, such as enzymes or nucleic acids.

9. Receptors • Receptors are the sites at which biomolecules such as hormones, neurotransmitters and the molecules responsible for taste and odour are recognised. • A drug that binds to a receptor can either: • Trigger the same events as the native ligand - an agonist. • Or • Stop the binding of the native agent without eliciting a response - an antagonist. • There are four ‘superfamilies’ of receptors.

10. Type 1. These have 4 or 5 membrane-spanning helical subunits. Their N- and C-terminii are found in the extracellular fluid. This family includes ion channels. Type 2. These have 7 helical transmembrane regions. Their N- terminal is extracellular and the C-terminal in intracellular. This family is coupled to the action of G-proteins: They are known as the G-protein coupled receptors. Type 3. These are tyrosine kinase-linked receptors with a single transmembrane helix. The insulin and growth factor receptors fall within this family. Type 4. These receptors are found in the cell nucleus and are transcription factors. They have looped regions held together by a group of four cysteine residues coordinating to a zinc ion. These motifs are called zinc fingers. The receptor ligands include steroids and thyroid hormones.

11. Read the sections on ion channels G-protein coupled receptors • Then answer the following… • Name two therapeutic uses for ion channel blockers. • Are channel blockers agonists or antagonists? • How many transmembrane helices are there in GPCRs? • Where does GTP bind? • Why GTP and not ATP? • How do receptors amplify the signal of a single ligand?

12. Enzymes • They are proteins that catalyse the reactions required for cellular function. • Generally specific for a particular substrate, or closely related family of substrates. • Molecules that restrict the action of the enzyme on its substrate are called inhibitors. • Inhibitors may be irreversible or reversible. • Reversible inhibitors may be: • Competitive. • Non-competitive. • Enzyme inhibitors might be seen to allow very ‘fine control’ of cellular processes.

13. Nucleic acids • Potentially the most exciting and valuable of the available drug targets. • BUT designing compounds that can distinguish target nucleic acid sequences is not yet achievable. • There are compounds with planar aromatic regions that bind in-between the base pairs of DNA or to the DNA grooves. • These generally inhibit the processes of DNA manipulation required for protein synthesis and cell division. • Suitable as drugs for applications where cell death is the goal of therapy - such as in the case of the treatment of cancer. • Name another use where cell death is desirable.

14. Mechanisms and Specificity of Drug Binding • The majority of binding and recognition occurs through non-covalent interactions. • These govern: • The folding of proteins and DNA. • The association of membranes. • Molecular recognition (e.g. interaction between an enzyme and its substrate or the binding of an antibody). • They are generally weak and operate only over short distances. • As a result large numbers of these interactions are necessary for stability, requiring a high degree of complementarity between binding groups and molecules.

15. Covalent bonds • The ‘sharing’ of a pair of electrons between two atoms. • These electrons largely occupy the space between the nuclei of the two atoms. • A very stable interaction • Requires hundreds of kilojoules to disrupt. • Compounds that inhibit enzymes through formation of covalent interactions are called ‘suicide inhibitors’. • Not all covalent bond formation is irreversible • Hydrolysis. • Action of repairing proteins.

16. Non-covalent interactions • The forces involved are: • Hydrogen bonds • van der Waals forces • Ionic / electrostatic interactions • Hydrophobic interactions. • Generally, such interactions are weak • vary from 4-30 kJ/mol.

17. van der Waals • Weak, but significant over many atoms. • Attractive over short distances • A strong repulsive force at very short distances. • From temporary dipole - induced dipoles • Every atom • No directionality • Less entropically unfavourable

18. Alignment of l.p. of A with H d+ d- D H :A “Acceptor” group An electronegative atom “Donor” group An electronegative atom e.g. O, N, S • Hydrogen bonds • Strong • Longer distance • Directional • Most common is between the C=O and NH groups on the peptide backbone.

19. Electrostatic • Two common classes of electrostatic interactions • Ionic and dipolar. • Ionic interactions arise between basic and acidic functionalities, typically amines and carboxylic acids. • Can be spread over more than one atom. • ‘Salt bridges’. • These are the strongest non-covalent interaction. • Dipolar interactions are also extremely important. • Interaction of partially charged regions of molecules or as a result of aromatic -systems. • Dipolar interactions are much weaker than ionic interactions.

20. Hydrophobic interaction • “Water hating” - oil / water principle • Why? • DG = DH – TDS • Either • Water H-bonding disrupted (DH +ve but DS -ve)Or • Water forms an ordered clathrate cage around solute (DH -ve but DS +ve) • In both cases DG +ve. • At low temps, formation of clathrate cages least unfavourable

21. Smaller Surface area • Combinbation of 2 clathrate cages gives a smaller overall surface area. • Leads to smaller amount of ordered H-bonding surface • and therefore less unfavourable DG. • Hydrophobic moieties tend to combine

22. Recognition • Forces of interactions are weak • Need many co-operative forces as binding entropically unfavourable. • i.e. lots of small -ve DH’s to make DG -ve. • So need good complementarity between binding groupsand molecules • lock & key • Only a small range of conditions under which most molecularassemblies will operate • e.g. Effected by temp, pH, metal concentration etc.

23. Conformation effects • Binding also locks a mobile, flexible molecule into a restricted conformation. • These losses of motion are entropically unfavourable (S negative). • Since G = H -TS, then the entropic energy loss must be compensated for by the enthalpic contribution. • Configuration effects • Differences in configuration (e.g. stereochemistry) can lead to startling differences in the biological effect. • e.g. The L enantiomer of penicillamine is highly toxic and only the (S) enantiomer of indomethacin acts as an anti-inflammatory agent. • The wrong configuration will lack required interactions or add undesired ones

24. Protein Surface X-Ray Diffraction Structure Of Hiv-1 Protease Complexed With SB203238 (Drawn from: Brookhaven database file: 1hbv.pdb. K.A.Newlander, J.F.Callahan, M.L.Moore, T.A.Tomaszek, W.F.Huffman A Novel Constrained Reduced-Amide Inhibitor Of HIV-1 Protease Derived From The Sequential Incorporation Of Gamma-Turn Mimetics Into A Model Substrate J.Med.Chem. 1993, 36, 2321.)

25. Selectivity, toxicity and therapeutic index Drugs may bind to both their desired target and to other molecules in an organism. If interactions with other targets are negligible then a drug is said to be specific. In most cases drugs will show a non-exclusive preference for their target - selective. The interaction with both their intended target and other molecules can lead to undesirable effects (side effects). Establish the concentrations at which the drug exerts its beneficial effect and where the level of side effects becomes unacceptable. Commonly used values are ED50 and LD50. For obvious reasons LD50 tests are not carried out on human volunteers!

26. One measure of the margin of safety is the therapeutic index. Therapeutic index = LD50 / ED50 • Drugs with low therapeutic indices are only used in ‘life or death’ type situations. • Exercise: it can be argued that the ratio LD1 / ED99 might be a more realistic estimate of safety. Why? • There are other side effects of drugs that are undesirable. • e.g. Drowsiness, nausea, impairment of immune functions and so on. • The protective index is defined as the ratio of ED50’s of the desired and undesired effects. • Should be >>1

27. Agonists & antagonists • Activity of a drug is the result of two independent factors: • Affinity is the ability of a drug to bind to its receptor. • Efficacy describes the ability of the bound drug to elicit a response. • The ‘two state model’. Receptors can at rest or activated. • An agonist stabilises the active state preferentially. • An antagonist shows no preference or it stabilises the resting state. • The efficacy of a compound in the two state model is the degree of selectivity for stabilising the active or resting state of the receptor.

28. The degree of selectivity can be expressed in terms of the ratio of the equilibrium binding constant, K for each receptor state. • Kactive / Kresting > 1, then the compound is an agonist. The higher the ratio, the higher will be the efficacy. • Kactive / Kresting 1, then the compound is an antagonist. The smaller the ratio, the higher will be the efficacy. • There is not a direct proportionality between receptor occupancy and response. • The maximum possible cellular response may occur at levels of lower than 100% receptor occupancy with a strong agonist. • Due to the amplification inherent in receptor response

29. There are 2 classes of agonist: • Full agonists – which elicit the maximum possible response at some concentration • Partial agonists – which never elicit the maximum possible response from the receptor. • There are also 2 classes of antagonist: • Competitive antagonists – which compete for the agonist binding site, and require higher agonist concentration to elicit a given response. • Non-competitive agonists – these bind at a site other than the agonist binding site, or even to a completely different molecular target. The result is the lowering of the maximum possible response in addition to the usual antagonist effect of ‘displacing’ agonist activity to higher concentration.

30. Concnvs response curves • The amount of drug could be expressed in terms of: • 1. Amount of drug administered • 2. Dose per unit bodyweight of the subject • 3. Concentration of drug in plasma or serum • Usually expressed in terms of 2. (clinically useful) or 3. (useful in research). • The monitored effect might be • Quantised (e.g. dead/alive, cured/not cured) • Continuous (e.g. Days of remission, percentage reduction in swelling)

31. Response Response log (dose) dose • In either case, data is often normalised • Responses given as a fraction (or percentage) of the group as a whole for quantised data or of the maximum response for an individual subject for continuous data. • It is common to use a logarithmic scale for response curves (i.e. plotting log(dose) or plotting dose on a logarithmic scale).

32. 1.0 0.5 0 Fraction of animals killed LD50 log (dose) (mg/kg) Quantised data Here there is a specific response being measured as an effect of concentration. Each subject will demonstrate that response at some concentration. The data can be represented by a graph of the cumulative fraction of animals displaying the response at a given concentration. Requires large numbers of test subjects and repeated experiments to be statistically valuable.

33. Continuous data A continuum of level of effect as concentration changes. Drug administered until the effect becomes saturated with a single individual. The data is plotted normalised to the maximum effect, to give a concentration vs fraction of maximum effect curve. Different test subjects usually give responses that are shifted along the concentration axis relative to each other. Use large test sets. ED50can be calculated as a mean from this data, along with a measure of distribution, such as standard deviation.

34. Fraction maximum effect 1.0 0.5 0 log (dose) (mg/kg) ED50’s

35. Exercise: Try constructing dose-response curves for the following systems: • 3 different full agonists of differing activity. • A full agonist and a partial agonist. • An agonist in the presence of no antagonist and 2 increasing doses of a competitive antagonist • An agonist in the presence of no antagonist and 2 competitive antagonists of differing activity • An agonist in the presence of no antagonist and 2 increasing doses of a non-competitive antagonist

36. k 1 Agonist + Receptor Agonist-Receptor complex A + R AR k -1 Some Physical Chemistry It is possible to explain these response curves based on the equilibrium associations involved. Response and occupancy are not always directly proportional, however this is assumed for the sake of simplicity. Agonist binding RateF = k1[A].[R] RateR = k-1[AR] The equilibrium constant K is given by the ratio K = k1/k-1

37. [AR] = F occ [Rtot] Consider [A] = 1/K. The equilibrium constant, K, is the inverse of the ligand concentration required to produce 50% occupancy of the receptor. Do not be surprised to find texts where the equilibrium constant is defined in terms of the reverse process, [AR] = K [A].[R] [AR] = F occ + [R] [AR] K.[A] [A] [AR]/[R] = = F = F occ + + 1 K.[A] [A] 1/K occ + 1 ([AR]/[R]) [AR]/[R] = K.[A] [Rtot] = [R] + [AR] Consider situation if A = 0, small, big,  As K increases, then the dependence of the response on K decreases. Focc becomes progressively a function of concentration. When K is very high, 1/K becomes effectively zero, so Focc becomes 1.

38. See Excell graphs

39. k 1 Agonist + Receptor Agonist-Receptor complex A + R AR k -1 k 1 Antagonist + Receptor Antagonist-Receptor complex N + R NR k -1 [AR] [Rtot] = [R] + [AR] + [NR] = F occ [R ] tot [AR] = F occ + + [R] [AR] [NR] [AR]/[R] = F /[R] occ + + 1 ([AR]/[R]) ([NR]/[R]) Competitive Antagonists

40. K.[A] = F [AR] occ + + 1 K.[A] K .[N] = K N [A].[R] If KN and [N] constant, this does not alter the shape of the curve. The effect is merely to shift the curve to apparent higher agonist concentration. This can be used to determine what increase in concentration of agonist, A, is required to restore the occupancy of the receptor found without the antagonist. [NR] = K N [N].[R] [AR]/[R] = F occ + + 1 ([AR]/[R]) ([NR]/[R]) [AR]/[R] = K.[A] [NR]/[R] = KN.[N]

41. [A]' [A]' = = r or [A] [A] r K. ([A]'/r) K.[A]' K.[A] K.[A]' = = + + 1 K. ( [A]' /r) 1 K.[A]' K .[N] + + + + 1 K.[A] 1 K.[A]' K .[N] N N /K[A]’ 1 1/r 1 1 1 = = + + + K. ( [A]' /r) 1 K.[A]' K .[N] + + + r K. ( [A]' ) 1 K.[A]' K .[N] N r + K.[A]’ = 1 + K.[A]’ + KN.[N] N Define the ratio, r, of new ([A]’) to old ([A]) concentrations as Setting Focc equal for situation with and without antagonist r = 1 + KN.[N]log(r-1) = log[N] + log KN

42. Example Problem: An agonist, A, provides 50% of its maximum response at a concentration of 30 M. Calculate the required concentration to reproduce this response in the presence of an antagonist, N, whose equilibrium binding constant, KN, is 6x104 M when the concentration of N is 20 M. Solution: The required increase in A is given by: r = 1 + KN.[N] In this case, KN = 6x104 M and [N] = 2x10-5 M (notice the conversion back to molarity, the equation would not work otherwise). So r = 1 + (6 x 104).(2 x 10-5) = 1 + 12 x10-1 = 2.2 So the required concentration of A is 2.2 times the concentration required to produce the same effect without the antagonist, i.e. 66 M.

43. See Excell graphs

44. Summary • A ligand for a receptor may be a full agonist, a partial agonist, a competitive antagonist or a non-competitive agonist. • An agonist stabilises the active state of the receptor. • An antagonist stabilises its resting state. • The degree of stabilisation reflects the efficacy of a ligand. • Ligand-response curves generally demonstrate a saturating response with increasing agonist concentration. • A competitive antagonist shifts the curve to higher agonist concentration. • A non-competitive agonist lowers the maximum possible response. • These effects can be quantified in simple models using physical chemistry.