Chapter 1 Introduction What is biochemical pharmacology? What is it? ◮ pharmacology, but with a focus on how drugs work, not on whether we should take them before or after dinner ◮ fascinating—you will love it, or double your money back What is it not? ◮ just molecular pharmacology—physiological context is important, too ◮ a claim that we completely understand the biochemical action modes of all practically useful drugs—we don’t On drugs and poisons: Paracelsus’ maxim “Alle Ding’ sind Gift und nichts ohn’ Gift; allein die Dosis macht, dass ein Ding kein Gift ist.” “All things are poison and nothing is without poison; only the dosage makes it so that something is not a poison.” “Dosis sola facit velenum.” Picture from wikimedia Image credit: Wikimedia A very small drug particle and a very large one Some drug molecules of more typical size OH H O O− HO N H N S O O N O OH O O− Acetylsalicylic acid Terbutaline Penicillin G O Functional classes of protein drug targets 1. Enzymes 2. Hormone and neurotransmitter receptors 3. Ion channels 4. Membrane transporters 5. Cytoskeletal proteins Non-protein drug targets 1. DNA: alkylating anti-tumor drugs 2. RNA: anti-ribosomal antibiotics, antisense oligonucleotides 3. Lipid membranes: antibiotics (amphotericin B, polymyxin); gaseous narcotics, alcohol? 4. Free space, or rather no target at all: osmolytes Histamine receptor antagonists Histamine NH2 N N Allergic H1-Receptor H2-Receptor Gastric acid, reaction ulcer H H N N S N N N N N N Cyclicine Cimetidine The development of H2-receptor blockers NH2 Histamine—physiological agonist N N H N NH2 Guanylhistamine—weak antagonist N N NH H H N N Methiamide—stronger antagonist S N N S H H N N Cimetidine—first clinical antagonist S N N N N NH2 S Famotidine—stronger clinical antagonist S N O H2N N N S O NH2 NH2 Angiotensin: Proteolytic release from angiotensinogen, and mode of action Angiotensinogen DRVYIHPF–HL–VIHN. Renin Angiotensin 1 DRVYIHPF–HL Angiotensin converting enzyme Angiotensin 2 DRVYIHPF Angiotensin receptor 2+ Gαq-GDP Gαq-GTP Ca contraction Vascular smooth muscle cell Two inhibitors of proteolytic angiotensin release O S O OH O HO O NH HOO N O H H H N N N N O OH Remikiren Enalaprilate Sequence of saralasin, a peptide inhibitor of the angiotensin 2 receptor Angiotensin Asp-Arg-Val-Tyr-Ile-His-Pro-Phe Saralasin Sar-Arg-Val-Tyr-Val-His-Pro-Ala Non-peptide ligands of peptide receptors N Cl N HO N NH O N N N N HO O OH N Losartan Morphine Fentanyl Arsphenamine, the first modern antibacterial drug EM photo credit: CDC image library H2N HO As As OH NH2 Treponema pallidum Arsphenamine Paul Ehrlich, the discoverer of both arsphenamine and the receptor concept Image credit: wikimedia Drug discovery by brute force: sulfamidochrysoidine H2N Sulfamidochrysoidine O N NH2 H2N S N O reductive metabolism H2N NH2 O H2N Sulfanilamide H2N S NH2 O Natural compounds and semisynthetic derivatives N ⊕N OH OH O N⊕ O O O O O Atropine Ipratropium Acetylcholine Protein structure-based drug discovery: HIV protease bound to its inhibitor saquinavir Structure of saquinavir, and its conformation in the active site of HIV protease NH2 O O OH H O N N N N H O NH Drug discovery by accident (1): From a letter by Reverend Edmund Stone to the Royal Society, 1763 Among the many useful discoveries, which this age hath made, there are very few which, better deserve the attention of the public than what I am going to lay before your Lordship. There is a bark of an English tree, which I have found by experience to be a powerful adstringent, and very efficacious in curing anguish and intermitting disorders. About six years ago, I accidentally tasted it, and was surprised at its extraordinary bitterness . As this tree delights in a moist or wet soil, where agues chiefly abound, the general maxim, that many natural maladies carry their cures along with them, or that their remedies lie not far from their causes, was so apposite to this particular case, that I could not help applying it; and that this might be the intention of Providence here, I must own had some little weight with me . The active ingredient of willow bark, and its more widely known derivative O OH O OH HO O O Salicylic acid Acetylsalicylic acid Drug discovery by accident (2): The discovery of penicillin Penicillium notatum Staphylococcus aureus Not all bacteria are susceptible to penicillin Escherichia coli Penicillium notatum Staphylococcus aureus Streptococcus pyogenes Neisseria gonorrhoeae Corynebacterium diphtheriae Haemophilus influenzae Drug development and approval ◮ preclinical, in-house: synthesis, in vitro and preliminary animal testing ◮ investigational drug application to Food and Drug Administration (FDA)—must be approved before clinical testing ◮ clinical trials in three phases: (1) Healthy volunteers; focus on pharmacokinetics, toxicity (2) Small number of patients with targeted disease (3) Larger patient collective (several hundred to several thousand), comparison to established reference therapies ◮ new drug application—review by FDA ◮ post-introduction market surveillance Chapter 2 Pharmacodynamics Pharmacodynamics: General principles of drug action ◮ Theory of drug-receptor interaction ◮ The two-state model of receptor activation ◮ Dose-effect relationships and their modulation by signaling cascades ◮ Potency, efficacy, and therapeutic index The invention of the receptor concept . I therefore assumed that the tetanus toxin must unite with certain chemical groupings in the protoplasm of cells . As these receptors, which may be regarded as lateral chains of the protoplasm . become occupied by the toxin, the relevant normal function of this group is eliminated . Paul Ehrlich, from his Nobel Lecture, 1908 How do drugs affect their receptors? ◮ Mode of binding: reversible vs. irreversible ◮ Binding site: orthosteric vs. allosteric ◮ Functional effect: activation vs. inhibition Labetalol as an example of stereoselective drug action NH2 OH NH2 OH H H N N O O HO HO R,R (β-antagonist) S,R (α-antagonist) NH2 OH NH2 OH H H N N O O HO HO R,S (inert) S,S (inert) Two natural stereoisomers with separate therapeutic uses O O N OH N OH N N Quinine Quinidine Mass action kinetics and receptor occupancy [L][Rfree] K = [LR] Receptor occupancy = Y = [LR] = [L] [Rtotal] [L]+K Linear and semi-logarithmic plots of receptor occupancy Linear plot Semilogarithmic plot 1 1 KI=0.1 µM 10 µM 0.5 0.5 0.1 µM 10 µM 1 mM 1 mM 0 0 Receptor saturation (Y) 0 20 40 60 80 100 10-4 10-2 100 102 104 Ligand (µM) Ligand (µM) The Scatchard plot One receptor, varying K Two receptors, varying K and n 100 K=1µM 120 R1, R2 80 R1 60 80 R2 40 2.5µM 40 20 10µM Ligand bound / free 0 0 0 20 40 60 80 100 0 50 100 150 200 250 Ligand bound (µM) Ligand bound (µM) Reversible and covalent receptor inhibition 1 no inhibitor reversible 0.5 Agonist Y covalent 0 10-4 10-2 100 Agonist (µM) Theory of competitive inhibition L I KL KI RL R RI Rtotal [RL] [L] [L] Y = = = R [I] L + ′ [ total] [L] + KL 1 + [ ] K KI Theory of irreversible or covalent inhibition L I KL RL R RI Runmodified [RL] [L] Yu = = [R]unmodified [L] + K L [RL] [L] [R]unmodified Yt = = [R]total [L] + K L [R]total Two inhibitors of α-adrenergic receptors Cl + NH3 HO N NH N O HO OH Norepinephrine Tolazoline Phenoxybenzamine Inhibition of spleen strip contraction by tolazoline and phenoxybenzamine 100 100 0 µM Tolazoline Phenoxy- benzamine 80 80 0 µM 60 60 0.4 µM 40 40 10 µM 20 20 µM 20 0.8 µM Contractile force (%) 0 0 1 8 64 512 0.25 1 4 16 64 Norepinephrine (µM) Norepinephrine (µM) Which of these inhibitors act(s) covalently? A. Tolazoline B. Phenoxybenzamine run Mechanism of covalent receptor blockade by phenoxybenzamine R R Aziridinium intermediate N N+ Cl R′ R′ – Cl Receptor—S− R N Covalent receptor adduct Receptor—S R′ The two-state model of receptor activation inactive receptor active receptor Kintr effect adapter protein Agonist behavior in the two-state model Agonist Kintr KA Active receptor Antagonists and partial agonists Antagonist Kintr KI Inactive receptor Partial agonist Kintr KI KA Inactive receptor Active receptor Dose-effect curves in the two-state model 1 Y receptor saturation (Y) full agonist 0.5 partial agonist neutral antagonist inverse agonist Active fraction / 0 10-5 10-4 10-3 10-2 10-1 100 Ligand concentration (µM) Application of the two-state model: Effects of aripiprazole on serotonin and dopamine receptors 5-HT1A 5-HT2B Dopamine D2L 100 160 ) 100 % 80 75 120 60 50 40 S binding ( 80 release (%) 35 25 3 - 20 γ IP 0 40 0 GTP- Reduction of cAMP (%) 10-3 100 103 10-3 100 103 101 103 105 Ligand (nM) Ligand (nM) Ligand (nM) dopamine or 5-HT aripiprazole Receptor behaviour not explained by the two-state model ◮ cooperativity of oligomeric receptors ◮ agonist-specific coupling ◮ β-arrestin-biased ligands ◮ refractory receptor states Cooperative behavior of oligomeric receptors Kintr L L KI KA Kintr L L L L L L KI KA KI KA L L L L L L L L KI KA L L L L L L Effect of cooperativity on receptor activity 1 1 n=1 n=1 n=3 n=3 0.5 0.5 Active fraction 0 0 10-4 10-3 10-2 10-1 10-4 10-3 10-2 10-1 Receptor occupancy Ligand concentration (µM) Ligand concentration (µM) Agonist-specific coupling Adapter 1 Adapter 2 Effect 1 Effect 2 Experimental example: 5-HT2 receptors 100 100 PLC PLC PLA2 PLA2 75 75 50 50 25 25 Response (% of 5-HT) 0 0 10-2 10-1 100 101 10-2 10-1 100 101 TFMPP (µM) DOI (µM) NH 2 NH NH2 N HO O N H O F F I F Serotonin (5-HT) DOI TFMPP vayssiere Some receptors have refractory states Reactivation Activation Dissociation Inactivation Dose-effect relationships in biochemical cascades Receptor = effector