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 Receptor second messenger effector

L L

E EL R RL M2-ase

functional effect Pre-M2 M2 inactivated M2

E EM2

functional effect The response of a biochemical cascade depends on receptor density

1

Active fraction

[Rtotal] = 100 pM 0.5 10 pM 1 pM 0.1 pM 0.01 pM Effect/active fraction 0 10-6 10-4 10-2 100 Ligand concentration (µM)

phenoxybenzamine Potency and efficacy

100 Red Forte 75

50 Blue Mite

25

0 Perfection of karma (%) 10-6 10-4 10-2 100 102 Drug concentration (µM) Therapeutic and toxic drug effects

benefit Drug receptor effector toxicity

effector1 benefit Drug receptor effector2 toxicity

receptor1 effector1 benefit Drug receptor2 effector2 toxicity Chapter 3

Pharmacokinetics The scope of pharmacokinetics

Pharmacokinetics deals with the following questions:

◮ Will the drug reach its intended site of action? If not, can we improve the drug’s uptake and distribution to help it reach its target? ◮ After uptake, how long will the drug stay in the system? How is it eliminated from the system? Stages of drug transport

◮ Absorption: Uptake of the drug from the compartment of application into the blood plasma ◮ Distribution: Equilibration of the drug between the blood plasma and the rest of the organism ◮ Elimination: Excretion or metabolic inactivation of the drug Absorption depends on the route of application

Route Advantage Disadvantage

Oral Convenience—route of Multiple barriers between choice where possible intestine and circulation Intra- No barriers to absorp- Involved; risk of infection; al- venous tion lergic reactions more severe Pulmonary Fast, quantitative up- Limited to gases (mostly nar- take cotics) The need for distribution varies with the location of the drug target

Location of target Example

Inside circulation, outside cells Proteases in blood coagulation and fibrinolysis Inside circulation, inside cells Chemotherapy of malaria parasites Outside circulation, on cell Histamine receptors surfaces Outside circulation, inside Cyclooxygenase cells Sections of the blood circulation

Arteries Arterioles Capillaries Venules Veins

intravascular pressure Capillaries as barriers to drug transport

Regular capillary Capillary in the brain

Endothelial cells

Glia cell Basal membrane Tight junction The intestinal epithelium as a barrier to drug absorption

Gut lumen

Apical cell membrane Tight junction

Basolateral cell membrane Basal membrane Mechanisms of solute transport across cell membranes

1. Active transport a) Primary: ATP-coupled b) Secondary: driven by ion gradients

2. Passive transport a) Facilitated diffusion: protein-mediated transport, not coupled to ATP or ion gradients b) Non-facilitated diffusion of lipophilic compounds; non-ionic diffusion Structure of a phosphatidylcholine bilayer

N⊕

O O P O− O

O O O O The polarity of drug molecules affects their rate of diffusion across lipid bilayers

Hydrophilic drug Hydrophobic Very hydrophobic The membrane permeability of drugs can be improved by removing polar functional groups

Epinephrine Ephedrine Metamphetamine

HN HN HN OH OH

HO OH Resorption esters can improve the diffusion of drugs across membranes

NH2 N H N S

O N O O O O O O O O O O O Bacampicillin Heroin History of heroin Ionizable drug molecules may cross bilayers by non-ionic diffusion

⊕ Acetylcholine ⊕ O O ⊕ N Hexamethonium N N

H⊕

Mecamylamine

⊕ HN H2N Gastric acid promotes accumulation of acetylsalicylic acid in the cells of the mucous membrane

Stomach lumen (pH 2) Cytosol (pH 7)

O O⊖ O O⊖

O O

O O

H⊕ H⊕

O OH O OH

O O

O O Inward- and outward-facing conformations of ABC transporters The functional cycle of ABC transporters

Extracellular space

Cytosol ABC transporters and the blood brain barrier (1)

O O N N N N N NH O

OH HN N O N O NH O H N N

N F F O

Imatinib Zosuquidar Elacridar ABC transporters and the blood brain barrier (2)

0.8

0.6

0.4

0.2 Imatinib, CSF/blood ratio

0

Control Elacridar BCRP –/– Zosuquidar MDR 1 –/– Cyclosporin A l-DOPA reaches the brain by specific transport

BBB HO O HO O

NH2 NH2

l-DOPA

HO HO OH OH

CO2

NH2 NH2

Dopamine HO HO OH OH The portal circulation Tissue structure of the liver

A B

C Portal vein branch

Bile duct tributary

Central vein Liver artery branch

Sinusoid

A–C reproduced with permission from pathorama.ch. Blood flow and bile flow in the liver lobule

Liver artery branch

Portal vein branch

Bile duct tributary

Liver vein tributary Propranolol and the first-pass effect

O HO HO HO NH NH OH

O O O

OH

Active metabolite Propranolol Inactive metabolite Outline of lung anatomy

Capillary

Bronchioli Trachea Alveoli O2 Bronchi CO2

Alveolus

Lung Major compartments of drug distribution

Intravascular volume (5%) Drug evenly distributed (uncommon) Intracellular volume (40%)

Interstitial volume (15%)

Fat (several %)

Drug confined to circulation Drug excluded by cell membranes Drug enriched in fat (very large drug molecules) (very polar drug molecules) (lipophilic drugs) Hydrophobic drugs tend to bind to proteins The volume of distribution: two alternate definitions

n Vd = (in absolute terms and in liters) [Drug]plasma

n Vd = (relative to body weight, l/kg) [Drug]plasma×body weight Example drugs and their uptake and distribution parameters

Drug Structure Vd (l/kg) Protein-bound Oral availability

Ibuprofen 0.15 99% 80% OH

O

S Olanzapine N 16.4 93% 60%

N H N

N

NH Amoxicillin 2 H 0.21 18% 93% N S O N HO O O HO Kinetics of thiopental distribution

100 fat O 75 H N muscle S 50 N liver, brain H 25 O eliminated 0

Tissue level (% of max.) 0 15 30 45 60 Thiopental Time (min) Overview of drug elimination

Hydrophilic drug molecule Hydrophobic drug molecule

Liver

Kidney Hydrophilic drug metabolite

Urine Bile Location and perfusion of the kidneys Overview of kidney function

Urine is “distilled” from blood plasma in several stages: 1. Ultrafiltration: 10-20% of the blood plasma volume flow is squeezed out; macromolecules are retained 2. Solute reuptake: glucose, salts, amino acids etc. are recovered from the primary filtrate by active transport 3. Water reuptake: driven by osmotic gradient 4. Solute secretion: some substrates are actively secreted into the nascent urine The nephron

Macula densa

Glomerulus

Distal tubule Proximal tubule Collecting duct

Loop of Henle Cross sections of glomeruli and tubules in kidney

From pathorama.ch with permission Plasma ultrafiltration in the glomerulus Filtration, reuptake, and active secretion A drug’s rate of urinary excretion depends on its membrane permeability

Vfiltrate Permeant drug Excluded drug flow

Vurine Vreabsorbed Curine = Cfiltrate nurine = nfiltrate Inulin, a model compound that is quantitatively filtrated and retained in the urine

... O Fructose O HO

OH OH O O HO

OH OH O HO O HO O OH Glucose OH OH O OH Definition of the renal clearance

dVurine [Drug] urine clearance Drug = × dt [Drug] plasma

Intuitive meaning: what volume of plasma is being “cleared” of the drug per unit of time? The volume flow of glomerular filtration can be measured from the clearance of inulin

V filtrate × [Inulin]filtrate = V urine × [Inulin]urine

[Inulin]urine V filtrate = V urine × [Inulin]filtrate

dV filtrate = dV urine × [Inulin]urine dt dt [Inulin]filtrate

= dV urine × [Inulin]urine dt [Inulin]plasma The GFR can be approximately determined using the creatinine clearance

glycine guanidinoacetate creatine

+ + NH2 NH2

arginine ornithine C NH2 SAM SAH C NH2

NH2 NH N CH3

CH2 CH2 CH2 COO− COO− COO−

ATP creatine kinase ADP

+ NH2 HN C NH P NH N CH3 N H C O CH2 3 P COO− creatinine creatine phosphate Tubular secretion of p-aminohippurate

p-Aminohippurate (pAH) O O− 2 K+ H ⊖ N ATP + 3 Na H N ⊖ 2 x Na+ O α-KG2– ⊖ H C O O α-KG2– Cl– 7 3 N S – – pAH pAH − H7C3 O O

Probenecid Tubular secretion of p-aminohippurate is almost quantitative The p-aminohippurate clearance measures the renal plasma flow

dn p-AH, urine ≈ dn p-AH, plasma dt dt

np-AH = [p-AH] × V

dV urine × ≈ dV plasma × dt [p-AH]urine dt [p-AH]plasma

dV plasma ≈ dV urine × [p-AH]urine dt dt [p-AH]plasma Non-equilibrium kinetics of drug elimination

n = [D]plasma × Vd × body weight

dn − × dt = k [D]plasma

− k [D]plasma,t × t = e Vd body weight [D]plasma,0

− k × t1 0.5 = e Vd body weight /2

× × Vd body weight t1/2 = ln 2 k

Vd definition Repeated drug application can result in accumulation

3 t1/2 = 24h

2

1

t1/2 = 6h Plasma concentration (A.U.) 0 0 24 48 72 96 120 Time (h) Chapter 4

Drug metabolism The place of metabolism in drug elimination

Hydrophilic drug molecule Hydrophobic drug molecule

Liver

Kidney Hydrophilic drug metabolite

Urine Bile Types of reactions in drug metabolism

1. Oxidation 2. Conjugation 3. Reduction 4. Hydrolysis Functional outcomes of drug metabolism

1. Inactivation and accelerated elimination of drugs 2. Activation of prodrugs 3. Formation of active metabolites with similar or novel activity 4. Detoxification of toxic xenobiotics 5. Toxification of non-toxic xenobiotics A hydrolytic metabolite of cocaine can be detected in wastewater

Cocaine N

O

O

O O

Milan OH⊖ Turin

Sampling MeOH site N

O

O

⊖O O

Benzoylecgonine Metabolism of phenobarbital

O NADPH+H+ NADP+ O OH H H N N O O N N H H O O2 H2O O

UDP-glucuronide PAPS

UDP 3′-P-AMP

O OH O− O O O O S H OH H O O N OH N O − O O O N N H H O O With many drugs, metabolic transformation facilitates excretion

Drug molecule

phase 1 phase 2 Reactive metabolite Conjugate

Urine

Deconjugation, reuptake Hydrophobicity does not strongly predict the extent of metabolism

100

75

50

25

Urinary excretion (%) 0 -6 -4 -2 0 2 4 6 8 10 logP (hydrophobicity) Major types of drug-metabolizing enzymes

Phase I

◮ Cytochrome P450 enzymes ◮ Diaphorase (NADH:quinone oxidoreductase) Phase II

◮ UDP-glucuronosyltransferases ◮ Sulfotransferases ◮ Glutathione-S-transferases ◮ N- and O-acetyltransferases Mode of action of cytochrome P450 enzymes

Reductase Cytochrome P450 NADPH+H+

FAD FMNH2 Heme R–OH O NADP+ 2

FADH2 FMN H2O Heme -Oá R–H

ER membrane Drug metabolism occurs to a large degree in the smooth endoplasmic reticulum

reproduced from medcell.med.yale.edu/histology, with permission Reactions catalyzed by cytochrome P450

[O] R−H R−OH Carbon oxidation

− [O] − + RCH2 OH RCH−O H2O [O] RCH−O RCOOH

− [O] − R2N H R2N OH Heteroatom oxidation [O] R3N R3N O [O] − R2S R2S−O

− [O] + − RO CH2R ROH O−CHR Dealkylation − [O] + − R2N CH2R R2NH O−CHR O [O] R−HC−CH−R R HC CH R Epoxide formation Formation of active metabolites by CYP450 enzymes

Carbamazepine Carbamazepine-10,11-epoxide Terfenadine

O

CYP450 Fexofenadine HO N N

H2N O H2N O N HO

HO N

Cl Cl HO

CYP450 N N N NH HO O O CYP450

HO O Diazepam Oxazepam Erythromycin bound to the active site of cytochrome P450

N

HO O O O HO O OH

O O O HO O

OH Ketoconazole bound to the active site of cytochrome P450

Cl

Cl O N N O

O

N O N Superposition of the erythromycin- and the ketoconazole-bound structures Transcriptional induction of drug metabolism

Cytochrome P450, PXR phase II enzymes, ABC transporters Transcriptional induction of cytochrome P450: Rifampicin bound to the pregnane X receptor

RXR Interaction of nuclear hormone receptors with DNA

5′ 3′ 5′ 3′ 5′ 3′

A C T G G A A G G T C A A G G T C A A G G T C A

3′ 5′ Conjugation reactions

Functional group Enzyme Cosubstrate

Glucuronic acid UDP-glucuronosyl- UDP-glucuronide transferases Sulfate Sulfotransferases 3′-Phosphoadenosine-5′- phosphosulfate (PAPS) Glutathione Glutathione-S- Free glutathione transferases / spon- taneous Acetate N-acetyltransferases Acetyl-CoA Methyl N-, S-, and O-methyl- S-adenosylmethionine transferases (SAM) Amino acids Amino acid trans- Free amino acids/ATP ferases Morphine skips phase I and is conjugated directly

N N UDP-glucuronide

O − O HO OH UDP O O O OH

O OH Morphine Morphine-3-glucuronide HO OH Epoxides of aromatic hydrocarbons can intercalate and covalently react with DNA

Benzopyrene

CYP1A1

DNA O

OH OH Benzopyrene–DNA adduct Enzymatic detoxification of benzopyrene epoxy-derivatives

O

OH G−SH H O OH OH 2 NH O 2

OH O NH OH S O OH NH OH OH OH O OH OH Hepatic metabolism of acetaminophen

HN O Acetaminophen NAPQI

G–SH SG HN O N O OH

CYP450

OH O HN O

Protein–SH

S Protein OH Activation of canfosfamide by glutathione-S-transferase

GST GST

OH

NH2 O O⊖ Cl OH Cl Alkylating drug

H Cl Cl O NH O N N O ⊖O O S P Cl P Cl HO H N N O O O NH O Cl Cl O R S Canfosfamide O Byproduct

alkylants Acetylation of INH by N-acetyltransferase 2 (NAT 2)

O O Acetylhydrazine NH NH2 NH H O O NH O NH 2 NH2 NAT2

O OH N N

Isoniazid Isonicotinic acid

N Bimodal distribution of INH acetylation speed

Fast, slow acetylators 25

20

15

10

5

Frequency (individuals) 0 0 1 2 3 4 5 6 7 8 9 INH in plasma (µg/mL) after 6h Metabolic activation of arylamine carcinogens

O H C CH NAT 3 N N H H

CYP CYP

O O H C CH C CH NAT 3 ST 3 N N N O OH OH O S O− O

NAT NAT 2– SO4

O

H H C CH3 N CH N N 3 ⊕ ⊕ O C – O CH3COO Glutamine conjugation of phenylacetate

Glutamine

H2N O Phenylacetic Conjugate acid H2N O O H2N O O OH O CoA–SH CoA O OH S N H OH

ATP AMP+PPi CoA–SH

urea cycle Reductive drug metabolism

◮ important functional groups in substrates: nitro, azo, sulfoxide, quinones ◮ diverse enzymology ◮ “incidental”—most enzymes that cause reductive drug metabolism primarily serve other roles in metabolism ◮ some reductive reactions can occur without enzyme catalysis Reduction of sulindac by thioredoxin

SH S O S S Thioredoxin Thioredoxin SH S

F O F O H2O OH OH Redox-active ingredients of Vicia faba

O O HO NH NH

H2N N O H2N N O H H

Divicine Isouramil Redox cycling of isouramil

O 2 H2O GSSG NADPH O NH 3 4

+ HN N O + NADP 2 GSH H H2O2 2 GSH NADP

4 1 2

O NADPH GSSG O HO 2 NH

H2N N O H Glucose-6-phosphate dehydrogenase deficiency leads to favism

◮ Most patients are healthy most of the time—hemolytic crises triggered by drugs or food ingredients that cause redox cycling ◮ Manifest in red blood cells because these cells lack protein synthesis—no replacement of deficient enzyme molecules during the lifetime of the cell ◮ Affords partial protection against malaria—similar to sickle cell anemia and other hemoglobinopathias Redox cycling of 5-hydroxyprimaquine

O2 H2O2

Primaquine OH O O O O CYP450

N N N NH NH N H2N H2N H2N

GSSG 2 GSH The anticancer prodrug CB1954 bound to quinone reductase 2 Two-step activation of the anticancer prodrug CB1954

+ + N 2 NADH 2 NAD N NADH NAD N H NO2 N NH2 OH O O O H2O H2O H2N NO2 H2N NO2 H2N NO2

acetyl-CoA CoA–SH N NH2

NH DNA N N ⊕ H N O O

O N N NH2 O acetate H N N H N NO 2 2 N NH O O

H2N NO2 Chapter 5

G protein-coupled receptors Drugs that act on G protein-coupled receptors: Some examples

Drug Major receptor Drug action Clinical use

salbutamol β2-adrenergic partial agonist bronchodilation

fexofenadine histamine H1 inhibitor antiallergic atropine muscarinic inhibitor pupil dilation haloperidol dopamine inhibitor antipsychotic morphine opioid agonist pain killer losartan angiotensin inhibitor antihypertensive clopidogrel adenosine inhibitor anticoagulation Rhodopsin as a model system of GPCR structure and function Membrane disks in the outer segments of retinal photoreceptors Light harvesting by stacked disks in photoreceptors Rhodopsin in the ground state and the activated state GPCR structure The G protein cycle The fluorophore in green-fluorescent protein forms autocatalytically

O O (C’) O H O (C’) N 2 2 2 N H O N O HO HN O HO (N’) N (N’) H N H O H 2 OH OH FRET detection of G protein binding to adenosine receptors

Adenosine 4

(% change) 2 CFP

FRET /I YFP I 0 0 0.25 0.5 0.75 Donor emission Time (seconds) FRET detection of G protein dissociation

Adenosine

4

(% change) 2 CFP /I YFP I 0 0 1 2 3 FRET Donor emission Time (seconds) G protein effector mechanisms: adenylate cyclase G protein effector mechanisms: the phospholipase C cascade

OH −2 O3PO

2− OPO3 2− OPO3 OH OH

Inositol triphosphate (IP3) Summary of G protein effector mechanisms

Class Effectors and Effects Some activating GPCRs

Gαs stimulation of adenylate cyclase β-adrenergic, 5-HT4, 5-HT6, (various types) 5-HT7,D1,D5; ACTH

Gαi/o inhibition of adenylate cyclase; α2-adrenergic, 5-HT1,D2, activation of extracellular signal- D3,D4 regulated kinase (ERK)

Gαq/11 stimulation of Phospholipase Cβ α-adrenergic, 5-HT2,H1, (various subtypes) GABAB

Gα12/13 indirect activation of RhoA GTPase 5-HT4, AT1, protease- acti- and of phospholipase A2 vated receptors Agonist-specific coupling with GPCRs

G protein 1 G protein 2

Effect 1 Effect 2 Agonist-specific coupling of 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 Substance P and its competitive antagonist CP-96345

NH2 H2N S N O NH H2N N 2 H O NH O N O O H N O 2 N NH H CP-96345 O O O NH O O H N N H2N N N H H H N O O

Substance P H2N O N Using receptor chimeras to locate the ligand binding sites of NK receptors

NK1 receptor NK1 receptor

100 100 80 80 60 60 40 40 20 20

0 Eledoisin bound (%) 0 -4 -2 0 -2 0 2 Substance P bound (%) 10 10 10 10 10 10 CP 96345 (µM) CP 96345 (µM) Engineered disulfide bonds pinpoint helix movements involved in GPCR function Protonation of residue E134 of rhodopsin in response to light stimulation

Light

E134Q pH ∆

wild type

-10 0 10 20 Time (ms) Protease-activated GPCRs Pharmacological inhibition of protease-activated receptors

CF3 )

Ω 20 Control (0 mg/kg) N N 10 1 mg/kg

Resistance ( 3 mg/kg H 0

O 0 1 2 3 4 5 6 Time after drug application (h) H H O GPCR oligomerization

◮ Oligomers can comprise identical or different subunits ◮ Potential for cooperativity ◮ Potential for novel ligand specificity ◮ Different mediators can reinforce or inhibit each other at the cell surface, reducing “noise” inside the cell Functional specialization in GABAB receptor heterodimers

100 100

50 50 released (%) 3 IP

CGP64213 bound (%) 0 0 Bivalent agonists of muscarinic acetylcholine receptors

15 S N N S basal)

N N x 10 S (CH2)n S

5

N N response ( 3 0 2 4 6 8 10 12

Max. IP Spacer length (methylene groups) Novel receptor specificity: selective activation of κδ opioid receptor hetero-oligomers by a monovalent ligand

900 δ 700 κ N κ/δ OH 500

release (A.U.) 300 ++ NH Ca HO O N N 100 H H -3 -2 -1 0 1 NH2 10 10 10 10 10 6-GNTI (µM) Could receptor heterodimers be targeted with heterodimeric drugs? GPCR deactivation by phosphorylation and endocytosis GPCR kinase 2 knockout attenuates tachyphylaxis of cardial β-receptors

2

GPCR kinase 2 k.o. 1.6

1.2 Control Pressure rise (MPa/s) 0.8 0 10 20 30 40 Time (min) Knock-out of arrestin may reduce GPCR-mediated signals

100 No inhibitor + H-89 (PKA inhibitor) + β-arrestin siRNA

50

0 0 10 20 30 ERK activation by isoproterenol (%) Time (min) Chapter 6

Pharmacology of cell excitation Clinical applications of drugs that influence excitable cell function

◮ blockade of nerve conduction for local anesthesia ◮ reduction of nerve cell excitability in the brain in epilepsy ◮ stabilization of mood in the treatment of bipolar disorder ◮ reduction of vascular smooth muscle tone to reduce blood pressure ◮ suppression of aberrant excitation in cardiac arrhythmia The nature of cell excitation

◮ all cells have an electrical potential across the cytoplasmic membrane, such that the cell interior is electrically negative relative to the outside (~−70 mV) ◮ in non-excitable cells, this membrane potential is stable; in excitable cells, it forms the resting potential ◮ cell excitation consists in transient reversals of the membrane potential, called action potentials, which spread rapidly across the entire cell membrane ◮ action potentials are spontaneously generated by some cells and transmitted between cells through chemical or electrical synapses Neurons and synapses

Chemical synapse Axon

Dendrite

Ca++

Presynaptic action potential Activation of postsynaptic receptors Some nerve cells have huge dendrites and axons

from Piersol, G.A., “Human Anatomy”, Lippincott, 1908 Other types of excitable cells

Heart excitation- conduction system Motoneuron Neuron Electrical synapse

Skeletal Gland cell muscle fibres Heart muscle cells The two driving forces that generate diffusion potentials across membranes

∆E = RT ln [cation]left zF [cation]right Equilibrium potentials for the major salt ions

◦ Ion Cytosolic Extracellular E0 at 37 C

K+ 150 mM 6 mM – 86 mV Na+ 15 mM 150 mM + 62 mV Ca++ 100 nM 1.2 mM + 126 mV Cl – 9 mM 150 mM – 70 mV Specific channels control ion permeabilities Diffusion potentials with multiple ions: the Goldman equation

Permeabilities change as ion channels open and close

RT P [K+] + P [Na+] + P [Cl−] ∆ = ln K outside Na outside Cl inside E + + − zF P K [K ] inside + P Na [Na ] inside + P Cl [Cl ] outside

Concentrations do not change significantly Structure of a voltage-gated K+ channel

Extracellular

Intracellular Structure of the K+ selectivity filter Voltage-gated sodium channels sustain and spread the action potential Voltage-gated potassium channels extinguish the action potential Voltage-gated channels and action potentials in the heart

Sinoatrial node

CaV T CaV LKV

KV (fast)

NaV CaV L KV (slow) Measuring ion fluxes across single channels using planar lipid bilayers The patch clamp technique Fast and slow channel block Diethylamine and phenol resemble parts of the lidocaine molecule

Diethylamine

N + H2N O N+ H O O O HN O OH

Cocaine Lidocaine Phenol Effects of diethylamine and of phenol on NaV channel conductance

Control, -40 mV Diethylamine, −40 mV Diethylamine, +40 mV

1 pA

0.5 s

Control, +40 mV Phenol, +40 mV

1 pA

5 s Drugs and poisons that act on NaV channels

OH OH N O O OH NH O O N O H HO HO N H N HO H OH O Tetrodotoxin Batrachotoxin OH

O NH 2 O HN NH O OH O N N

N H2N O

Mexiletine Phenytoin Carbamazepine Quinidine KATP channels regulate the tone of smooth muscle cells KATP channels in pancreatic β cells regulate insulin secretion

Glucose Insulin

Ca2+ Ca2+ Glucose

Membrane ADP + P Insulin i ATP potential K+ K+

ATP ATP Sulfonylurea receptor H2O, CO2 Drugs that act on K+ channels

Iptakalim O H N HN O

H2N HN HN N HO HN O N O N HN O S O S NH O + H2N N NH2 O O O NH S S Cl O− O O F Tolbutamide Diazoxide Minoxidil sulfate Sotalol Retigabine Two calcium channels control the contraction of striated muscle cells Entry of Ca++ through the DHPR is necessary in the heart, but not in skeletal muscle cells Inhibitors of voltage-gated calcium channels

Verapamil (L-type) Mibefradil (L,T-type)

O F O O N O O N N

O NH N O NO2 O O

O O O N O H N Nifedipine (L-type) H Ethosuximide (T-type)

heart Ca channels Structure of ω-conotoxin, an inhibitor of N-type CaV channels

synapse sketch Transient receptor potential channels

◮ activated by physical stimuli such as heat and mechanical tension ◮ conduct multiple cations (in hydrated form) ◮ function in various modes of sensory perception ◮ may be activated by ligands also Agonists of transient receptor potential (TRP) channels

O O N Capsaicin H HO

O HO H O O Cl N S N N S GSK1016790A N O H O Cl Na+/K+-ATPase maintains the ion gradients across the cytoplasmic membrane

2 K+

ATP

3 Na+

3 Na+ Na+ Ca++ Amino acid

K+ K+ Cl– Functional cycle of Na+/K+-ATPase

3 Na+ 2 K+

+ Na K+ Na+ + Na+ K ATP ATP P— P— ATP

ADP P ATP 3 Na+ i 2 K+ Na+/K+-ATPase activity as a function of KCl and NaCl concentrations

g) NaCl 40 mM µ 30

20 20 mM

10 3 mM 10 mM 0 mM 0 Phosphate released ( 0 30 60 90 120 KCl (mM) Effects of Rb+ and of Na+ on the phosphorylation state of Na+/K+-ATPase

3 Na+

Rb+ Na+

ATP P— 100 ADP 3 Na+ 75

50 2 K+ 25 P-enzyme (%)

32 0 K+ 0 15 30 45 60 + K Time (seconds) P—

Pi ATP is required to release Rb+ from tight binding to Na+/K+-ATPase

− 1.5 ATP

+ K 1 K+ ATP 0.5 + ATP ATP 0

+ Rb bound/mg protein 0 0.2 0.4 0.6 0.8 1

2 K 86 RbCl (mM) Structures of the Na+/K+-ATPase inhibitors ouabain and digitoxin

O O

Ouabain OH OH OH

HO O OH O HO O O OH OH Digitoxin

OH OH HO O O OH O O O O OH

NaKATPase function Function of a Presynaptic action potential opens CaV channels chemical synapse Purkinje cell Ca2+

Synthesis Presynaptic Vesicular feedback Reuptake transport

Intracellular breakdown

Release

Extracellular breakdown Retrograde feedback Postsynaptic action Summation of postsynaptic potentials

A B Ca2+

CD Neurotransmitter receptor families

1. Ligand-gated channels a) Cys-loop family b) Glutamate receptors c) Purine P2X receptors

2. G protein-coupled receptors Postsynaptic GPCRs can signal through cyclic nucleotide-gated (CNG) channels Structure of the nicotinic acetylcholine receptor

ACE

β δ

α α

γ

BDF

Trp α149 Trapping the nicotinic acetylcholine receptor in the open state

Forceps

NAR in closed state in membranes, on EM grid

Sample is dropped, nebulizer is triggered

Acetylcholine Sample is sprayed with acetylcholine nebulizer Acetylcholine opens channels

Liquid ethane (−160°C) Channels are flash-frozen while open Photoaffinity labeling of the acetylcholine binding site

Photactivatable N+ O N acetylcholine analog N (TDBzcholine) O CF3

hν Photoactivation N−N

N+ O Carbene radical C O CF3

Receptor−H Covalent reaction

N+ O H Receptor adduct Receptor O CF3 Isolation of affinity-labeled polypeptide chains and fragments

3H-TDBzcholine 3H-TDBzcholine + carbamoylcholine

40 20

30 15

20 10

10 5 H-label bound (cpm/100) 3 0 0 α β γ δ N 46 173 339 C Polypeptide chain V8 cleavage points (residues) Identification of affinity-labeled amino acid residues

1.5 Cys193 TDBzcholine only + carbamoylcholine

1 Pro194 Cys192

0.5 H-label bound (cpm/100)

3 0 0 5 10 15 20 25 30 35 Edman degradation cycle Desensitization of the nicotinic acetylcholine receptor Functional states of the nicotinic acetylcholine receptor

Acetylcholine Resting, bound

Resting, free Open, bound

Inactivated, free Inactivated, bound

Acetylcholine

Cholinesterase

Acetate + choline Ionotropic receptors in the cys-loop family

Receptor Ion selectivity Effect Comments

nicotinic acetyl- cations excitatory pharmacologically distinct choline subtypes, various applications

5-HT3 serotonin cations excitatory inhibitors are used to treat emesis

GABAA chloride inhibitory major drug target in narcosis, epilepsy, psychoses glycine chloride inhibitory regulates motor activity Drugs that interact with GABA receptors and transporters

OH

O NH2 HO O NH2 HN O HOO O N O O GABA NH N Muscimol 2 N Cl F S F N N O F N S F F N Cl Isoflurane Etomidate Pentylenetetrazole Baclofen Tiagabine

phenobarbital diazepam Drugs that interact with glycine receptors and transporters

O O OH N HO O O OH H O O H O O H O O N H O O O O OH N Strychnine O Ivermectin Org 25935 Chemical structures of subtype-selective glutamate receptor ligands

O O HO O O HO O O HO O OH OH N OH OH H NH

H2N H O H2N H NH

l-Glutamate AMPA Kainate NMDA

− O O O O OH P O NH2 HO O O HN N OH H O H2N H OH

OH O H2N H

ACPD Quisqualate l-AP4 Biosynthesis of the catecholamines and of serotonin

O OH O OH NH2 NH2 HN HO HO NH2 NH2 1 2 3 4

HO HO HO HO OH OH OH OH OH

l-tyrosine l-DOPA Dopamine Norepinephrine Epinephrine

O O OH OH NH2

NH NH 2 5 HO 2 2 HO

N N N H H H Tryptophan 5-Hydroxytryptophan Serotonin Drugs that interact with dopaminergic and serotoninergic synapses

Aripiprazole Haloperidol H Cl O N O N N N HO

Cl F O O Cl Ondansetron N N

O N O OH N N NH2 N O N Br H N OH HO H N O N NH Cl HO O N OH

Bromocriptine Clozapine 6-Hydroxy-l-DOPA Organization of the autonomic nervous system

Eyes, salivary glands

Bronchi Forebrain

Brain stem

Heart, blood vessels

Spinal cord Digestive tract

Adrenal glands Urinary tract, sexual organs

Blood– Sympathetic and brain parasympathetic barrier ganglia Transmitter receptors in the peripheral autonomic nervous system

Subsystem 1st Synapse 2nd Synapse

sympathetic nicotinic α- or β-adrenergic parasympathetic nicotinic muscarinic Receptor agonists or antagonists and false transmitters at adrenergic synapses

synapse

Isoproterenol (β ↑) Terbutaline (β2 ↑) Xylometazoline (α ↑) Clonidine (α2 ↑)

HN HN HN HN HO HO N HN N Cl Cl

HO HO OH OH

O HN O NH HO N OH 2 H2N N NH2 N O O N H2N N N

HO O OH O O NH2

Atenolol (β1 ↓) Prazosine (α1 ↓) α-Methyldopa Guanethidine Methyldopa is a false transmitter in noradrenergic synapses

OH CO2 H2N NH2 O

HO HO OH OH Drugs that act on the membrane transport of monoamine transmitters

F Cocaine FluoxetineF F Imipramine MDMA N N O H N N O O

O O N O O

N NH2 NH2 O N H O O O O O O O OH O Reserpine Amphetamine Tyramine Degradation of norepinephrine

NH2 O O O HO HO HO HO OH MAO COMT ADH

HO HO O O

OH OH H3C OH H3C OH

Norepinephrine Vanillylmandelate Reaction mechanism of monoamine oxidase (MAO)

H2O2 O2

− R−CH2−NH2 R−CH−O

MAO MAO−H2 NH3

•⊖ ⊖ MAO MAO−H H2O

•⊕ − ⊕ R−CH2−NH2 R−CH−NH2 Mechanism-based inhibition of MAO by tranylcypromine

MAO MAO•⊖

⊕ NH2 NH ⊕ NH2 NH2

• MAO MAO-induced toxicity of MPTP (N-methyl-4-phenyl-tetrahydropyridine)

N

MAO O

O ⊕ N N

Meperidine MPTP MPP+ Structures of cholinergic receptor agonists

O O N+ N+ O N N OH

Acetylcholine Muscarine Nicotine

N O O NH2 N N+ N + N O O

Carbamoylcholine Pilocarpine Dimethylphenylpiperazinium

O O +N +N O O

Succinylcholine Structures of cholinergic receptor antagonists

+ N OH N OH N

O O O O O Atropine Ipratropium Benztropine

O HO O O O N + N+ N O +

N HO O Pancuronium O d-Tubocurarine S+

+N +N N N

TrimethaphanO Hexamethonium The catalytic mechanism of cholinesterase

Ser Ser O H OH HO H ⊖O O N N O Acetate

His Glu

Ser ⊕ Ser O H O⊖ O N+ OH O O⊖ Acetylcholine

Ser H ⊕ O H O H H ⊖O O O N N N+ Ser O⊖ O Glu O His HO Choline N+ Covalent inactivation of cholinesterase by DFP, and its reactivation by pralidoxime

Ser OH O O⊖ O⊖ O N N N F P O

⊕ ⊕ O N N N H+

F– Ser O O⊖ O Ser O P O O P O O R N Ser O O O P O O⊖ R N O Structures of cholinesterase inhibitors and of a reactivator

O O O S P O O S P O OH S O N O O CYP450 O O ⊕ N O O

Malathion Malaoxon Pralidoxime

N N+

N N O O HO

HN O N O

Physostigmine Rivastigmine Edrophonium Purine receptor agonists and antagonists

O Adenosine Tecadenoson R = CH3: Caffeine R = H: Theophylline NH2 NH

N N N N O R N N N N N N O O O N N HO HO OH OH HO HO

Cl Cl O N CYP450 N OH S SH

Ticlopidine Opioid receptor agonists and antagonists

HO HO HO

O O O N N N N OH HO O

Morphine Naloxone Pentazocine Methadone Chapter 7

Hormones Major types of hormone receptors

◮ G protein-coupled receptors

◮ Many peptide hormones: glucagon, hypothalamic and hypophyseal hormones ◮ Epinephrine, norepinephrine

◮ Receptor tyrosine kinases

◮ Insulin ◮ Growth hormone and growth factors

◮ Nuclear hormone receptors

◮ Steroid hormones ◮ Thyroid hormones The hypothalamic-pituitary axis

Hypothalamus

CRH

Pituitary anterior posterior

ACTH

Adrenal cortex GH Oxytocin, vasopressin

Cortisol

peripheral tissues Peripheral glands and hormones controlled by anterior pituitary

Gland Stimulated by Hormones produced

thyroid gland thyroid-stimulating tri-, tetraiodothyronine

hormone (TSH) (T3,T4) adrenal glands adrenocorticotropic gluco-, mineralocorti- (cortex) hormone (ACTH) coids; androgens testicles / follicle-stimulating hor- androgens, estrogens, ovaries mone (FSH), luteinizing progestins hormone (LH) diffuse growth hormone growth factors mammary prolactin — gland Regulatory patterns in the hypothalamic-pituitary axis

CRH GHRH Somatostatin Hypothalamus

ACTH Growth hormone Pituitary

Cortisol IGF-1 Peripheral glands or tissues The posterior lobe of the hypophyseal gland produces oxytocin and vasopressin

l Oxytocin H2N-Cys--Tyr--Ile--Gln--Asn--Cys--Pro-- -Leu--Gly-CO-NH2

l Vasopressin H2N-Cys--Tyr--Phe--Gln--Asn--Cys--Pro-- -Arg--Gly-CO-NH2

d Desmopressin H-Cys--Tyr--Phe--Gln--Asn--Cys--Pro-- -Arg--Gly-CO-NH2 Thyroid hormones

O OH O OH

NH2 NH2

I I I I I O I O

HO HO I

Triiodothyronine (T3) Thyroxine (T4) Thyroid hormones activate cognate nuclear hormone receptors

5′ 3′ 5′ 3′ 5′ 3′

A C T G G A A G G T C A A G G T C A A G G T C A

3′ 5′ Tissue structure of the thyroid gland, and localization of hormone synthesis

Follicular epithelium Colloid I–

O2 I–

I–

TG TGTPO TPO

TGmod

TGmod

T3,T4

Capillary C cell T3,T4 Tyrosine side chain iodination by thyroid peroxidase

– H2O2 I

E Fe E Fe−O E Fe−OI–

H2O

OH– OH OH I Coupling of two iodinated tyrosine side chains

2e–

•

I I I I I I OH OH O• O

I CH2 I

O O I I O I I OH Thyroid hormones and pharmacotherapy

◮ Goiter: iodide supplementation ◮ Hyperthyroidism due to anti-TSH-receptor autoantibodies or hormone-producing tumors:

◮ thyroid peroxidase inhibitors 131 ◮ radioiodine ( I)

◮ Lowering blood lipid levels: TR-β-selective agonists (KB-141) ◮ Interference with thyroid hormone release or conversion: lithium and amiodarone Drugs that influence thyroid hormone function

Methimazole Sulfenic acid Sulfinic acid

N NH TPO N TPO OH OH N S N S N S O

I O HO Cl OH O I N O O Cl KB-141 O Amiodarone

T3,T4 Steroid hormones

Class Major members Glands

Glucocorticoids Cortisol, cortisone Adrenal glands Mineralocorticoids Aldosterone Adrenal glands Androgens Testosterone, dihy- Testicles drotestosterone Estrogens Estradiol, estriol Ovary Progestins Progesterone Ovary, placenta Effector mechanisms

Mechanism/target Receptor binds to Example

Transcriptional induc- DNA directly Induction of enzymes of tion (transactivation) gluconeogenesis by cortisol Transrepression Other proteins that Inhibition of transcription regulate transcription factors AP-1 and NF-κB by cortisol Adrenal steroid hormones: functional classes

Class Physiological function

Glucocorticoids Metabolic regulation, strong anti-inflammatory action Mineralocorticoids Control of sodium and potas- sium elimination in the kidneys Androgens Precursors for gonadal androgen and estrogen synthesis Synthesis of adrenal steroids

Cholesterol Pregnenolone Progesterone

O O

CYP11A1 3β-HSD

HO HO O

CYP17

OH OH O O O O OH HO HO

HO O O

DHEA Cortisol Aldosterone Glucocorticoids control inflammation via transrepression

Infections, autoimmune reactions

ACTH Cortisol AP-1, NF-κB

CRH Interleukins, TNF-α iNOS, Cox-2

Inflammation Glucocorticoid receptor agonists and antagonists

DexamethasoneOH Prednisolone OH O O 100 OH OH IL-1β ↓ HO HO ↑ 75 TAT

F 50 O O 25 N OH O 0 HO Effect (% dexamethasone)

RU 24858 Prednisolone Mifepristone F O MifepristoneO RU 24858 11-β-Hydroxysteroid dehydrogenase prevents non-specific mineralocorticoid receptor activation by cortisol

Cortisol Cortisone OH OH O O OH OH HO O 11-β-HSD

OH OH OH O O O HO O O O HO O 11-β-HSD

Aldosterone hemiacetal Aldosterone Mineralocorticoid receptor ligands

FludrocortisoneOH Spironolactone Eplerenone O O O OH O O HO O

F O O O S O O O Gonadal biosynthesis of androgens and estrogens

O O 3β-HSD

DHEA Androstenedione HO O

17α-HSD Aromatase

OH O

Testosterone Estrone O HO

5α-Reductase Aromatase 17α-HSD

OH OH OH 16α-Hydroxylase OH

Dihydro- Estradiol Estriol O testosterone HO HO Synthetic analogues of gonadal steroids

Ethinylestradiol Norgestrel Diethylstilbestrol Clomiphene OH OH OH Cl

N HO O HO O

H OH O N OH

H F3C N N O O N O N O2N MethyltestosteroneH StanozololH Finasteride Flutamide Formation and resorption of bone matrix

ABC

Alkaline phosphatase

DE

Pyrophosphate

Phosphate H+ Proteases Calcium

Matrix proteins Hormones that affect bone mineralization and bone matrix

◮ Parathyroid hormone (PTH)

◮ mobilizes calcium and phosphate from the bone ◮ promotes calcium retention and phosphate elimination in the kidneys ◮ promotes activation of vitamin D by hydroxylation

◮ Calcitonin: promotes deposition of calcium and phosphate in the bone ◮ Calcitriol (activated vitamin D)

◮ promotes intestinal calcium and phosphate uptake ◮ inhibits PTH secretion

◮ Estrogens: sustain bone matrix Endogenous biosynthesis of calcitriol

OH

hν 25-OH 1-OH HO

HO HO OH

7-Dehydrocholesterol Cholecalciferol 1,25-Dihydroxycholecalciferol Drugs that influence calcium balance and bone mineralization

OH

Pyrophosphate O HN O− O− HO P O P OH O O O− OH O− HO P P OH O O O− H O− F F HO P P OH

F O H O H2N HO OH Cinacalcet Medronate Alendronate 22-Oxacalcitriol Sites of action of statins and bisphosphonates

Statins

Acetyl-CoA HMG-CoA Mevalonate C5-,C10-prenoids

Bisphosphonates

Cholesterol 7-Hydroxycholesterol Farnesyl-PP

hν

Cholecalciferol Farnesylated proteins (Ras, Rho . . . )

bone matrix cartoon Chapter 8

Pharmacology of nitric oxide Physiological significance of nitric oxide (NO)

◮ Powerful vasodilator—NO-releasing drugs are used in the treatment of cardiovascular disease ◮ Neurotransmitter—signaling in the CNS and the autonomic nervous system ◮ Inflammatory mediator—inhibition of NO synthesis is of interest as a therapeutic strategy in infection and chronic inflammation Cholinergic and adrenergic nerve endings in a blood vessel wall The endothelium is required for vascular relaxation in response to acetylcholine The nitric oxide synthase reaction

O O O

NH2 NH2 NH2 HO HO HO

+ + + + NADPH + H NADP 1/2(NADPH+H ) 1/2NADP

N N NH

O2 H2O O2 H2O H2N NH2 H2N NH H2N O + OH N−O

arginine N-hydroxyarginine citrulline Activation of NOS by calcium and calmodulin

2+ Ca , CaM Reductase domains

Oxidase domains NO activates soluble guanylate cyclase (sGC) Signaling effects of cGMP NO-induced relaxation of smooth muscle cells is mediated by cGK

PLC cascade S-nitrosylation of cysteine residues in proteins by NO

• − N−O O2

Protein Protein OH Protein O SH N N S • S HO−O• •N−O Superoxide dismutase

1 1 /2H2O2 + /2O2 HO O N O Transfer of nitrosyl groups between proteins by glutathione

G−S–

S−N−O S–

G−S−N−O

S−N−O S–

G−S– Identification of cysteines affected by S-nitrosylation: The biotin switch method

O S S SH SH S S CH3 GS−NO O SH S NO S NO

S S S GS−H S S O S S H O ascorbate

HNO dehydroascorbate O S S biotin

S S CH3 O S S CH3

S S biotin SH

S O S S S H S O Identification of proteins subject to nNOS-dependent S-nitrosylation

Total nitrosylated total nitrosylated nNOS + k.o. + k.o. + k.o. + k.o. Neurofilament Na+/K+-ATPase heavy chain α2 subunit

NMDA receptor β-Tubulin subunit 2A NMDA receptor Creatine kinase subunit 1 Retinoblastoma gene product β/γ-Actin Glycogen phosphorylase GAPDH Heat shock protein 72 Role of NO and iNOS in the killing of microbes by phagocytes

iNOS Arginine NO

– O2

– ONO2 NO-releasing drugs

O 2– O O N O N O O N O O N O O N N C C Fe O C C N N O O O C N N O O O N O

Nitroglycerin Isosorbide dinitrate Nitroprusside Bioactivation of nitroglycerin by mitochondrial aldehyde dehydrogenase

SH SNO 2 100 E + R−ONO2 E + R−OH SH SH 80

60

40 DTT control 20 + cyanamide

S Aortic strip relaxation (%) 0 -1 0 1 2 3 4 E + HNO2 10 10 10 10 10 10 S Nitroglycerine (nM) Bioactivation of nitroglycerin and ISDN by human cytochrome P450 isoforms

300 ISDN Nitroglycerin 200

100 release (mol/mol)

2 0

NO 2J2 2E1 1A2 2A6 2C9 2D6 3A44A11 NO release by molsidomine

Molsidomine

O N + N 12 N N O N N O − O O N

min)] SOD

× 8 + – H2O NO,H ,O2 GSH

CO2, ethanol O2 M/(mg µ 4

No antioxidant

HN + N cGMP [ N N O N N O 0 O N− O N 10-5 10-3 10-1 Linsidomine Linsidomine (mM) Antiinflammatory effect of iNOS inhibition

NH O 1 wild type H2N N O iNOS k.o. L-NAME NH2 0.5

GW274150 O

Foot swelling (mL) 0 S H N N 2 OH 22 24 26 28 30 32 GW274150 NH2 Days after collagen injection Structure and mode of action of sildenafil (Viagra™)

O

N N O N N H

O S O PDE 5 N

N Chapter 9

Eicosanoids and related drugs Eicosanoids

◮ “Local hormones”—sphere of action often limited to same anatomical site or tissue ◮ Involved in control of inflammation, fever, blood coagulation, pain perception, labor ◮ Derived from arachidonic acid and other polyunsaturated fatty acids, which occur in membrane phospholipids ◮ Most effects mediated by G protein-coupled receptors ◮ Some effects mediated by ion channels and nuclear hormone receptors Pathways and key enzymes in eicosanoid synthesis

Cox-2 Endocannabinoids Prostamides Prostaglandin D2

FAAH

cPLA2 Cox-1, Cox-2 Phospholipids Arachidonic acid Prostaglandin H2 Prostaglandin E2

Lox P450 Lox Leukotrienes EET Prostaglandin F2 Thromboxane A2

Lipoxin A4 Prostaglandin I2 Calcium signals activate cPLA2 and initiate the synthesis of prostaglandins and leukotrienes

HO HO P P P OH

O O O O Biosynthetic pathways of prostaglandins and thromboxanes

arachidonic acid prostaglandin H2 O thromboxane A2 OH O OH O OH 1 2

O O O O OH OH 3 6 4 5

prostaglandin D2 prostaglandin E2 prostaglandin F2 prostaglandin I2 O O O O OH HO OH O OH HO OH HO O

O OH HO OH HO OH HO Eicosanoids synthesized by lipoxygenases

arachidonic acid 5-HPETE 5-HETE O O OH O OH O OH 7OH 8 OH

9,8 7

15-HETE leukotriene A4 leukotriene B4 O O OH OH OH O OH OH O

10 OH

7 11

lipoxin A leukotriene C leukotriene E 4 4 OH O 4 OH O HO OH O OH OH OH S S Glutathione OH H N OH 12,13 2 O The two steps of the cyclooxygenase reaction

O Arachidonic acid OH

2 O2 Cyclooxygenase site

O Prostaglandin G2 O OH O

O 2 GSH OH Peroxidase site GSSG O Prostaglandin H2 O OH O

OH Reactions in the cyclooxygenase site

Arachidonic acid Prostaglandin G2 O O O• OH O OH H H O

O OH •O

O O OH OH O• OH O • O • O•

O• O• H O Reduction of prostaglandin G2 to prostaglandin H2

O

O OH O Fe(III) GSSG

O OH H2O

+ • O Fe(IV)−O2– 2 GSH O OH O

OH Interaction between the cyclooxygenase and peroxidase sites

Heme Tyr385

Arachidonic acid His388 Priming of the active site tyrosine radical, and the action mode of acetaminophen

CH3

N O

R-O-OH Fe(III) NAPQI NAPQI

Acetaminophen O

+ • á Tyr-O CH3

R-OH Fe(IV)−O2– Tyr-OH HN O

Acetaminophen

OH Noncovalent cyclooxygenase inhibitors

Cl

O Cl O OH N N N N H H HO Cl N S HO O O O O O Indomethacin Diclofenac Piroxicam

F F O O S N F N O O O

SC 560Cl Rofecoxib Conformation of arachidonic acid and of diclofenac in the active site

Y385 Arachidonic acid

S530

Diclofenac

Y355 R120 Cox inhibitors and Cox mutants

Inhibitor Relative IC50 R120A Y355F S530A

Indomethacin > 240 > 240 1.1 Diclofenac 3.3 1.8 > 650 Piroxicam > 109 > 109 > 109 Acetylsalicylic acid is a covalent Cox inhibitor

Y385 O Y385 O

O S530 O O S530 H

+ O H

O O−

O O

O− O−

Acetylsalicylic acid Salicylic acid Rationale of low-dose acetylsalicylic acid treatment Cox inhibition can promote the synthesis of leukotrienes

Phospholipids

cPLA2

5-Lox Arachidonic acid Leukotrienes

Cox

Prostaglandin H2 Inhibitors that act downstream of Prostaglandin H2

HO O O

HO N N

O O N S F N S F H O O Ramatroban Cay10471 Endocannabinoids

◮ Arachidonate-containing, membrane-derived mediators ◮ Synthesis on demand, activated by calcium ◮ Mediate negative synaptic feedback

◮ CB1 receptors involved in pain perception both in the peripheral and the central nervous system

◮ CB2 receptors found on immune cells Feedback inhibition of synaptic transmission by endocannabinoids

OH OH OH Presynaptic action potential HN O O O Ca2+

2+ Ca βγ

CB1 AEA, 2-AG

FAAH, Anandamide 2-Arachidonyl- Ca2+ MAGL (AEA) glycerol (2-AG)

conotoxin Biosynthesis of endocannabinoids

Membrane stimulus PC PE

Ca2+ N-Acyltransferase

Lyso-PC N-Arachidonyl-PE

PLD GPCR activation Phosphatidic acid Anandamide

Gαq PLCβ

DAGL PIP2 Diacylglycerol 2-Arachidonylglycerol Degradation of endocannabinoids

Anandamide 2-Arachidonylglycerol

Cox-2 FAAH MAGL Cox-2

PGH2-ethanolamide Arachidonic acid PGH2-glyceryl ester Drugs that interact with the endocannabinoid system

OH HU-308 ∆9-Tetrahydrocannabinol (THC)

O O OH

O

O URB-597 UCM-707 O H N O NH2 N H O O Chapter 10

Intermediate metabolism, diabetes, and atherosclerosis Intermediate metabolism, diabetes, and atherosclerosis

Genetic enzyme defects rare; comparatively little effort spent on targeted drug development; only a few defects can be treated with drugs Gout more common; multiple causes, similar manifestation and treatment Diabetes mellitus very common; treatment with insulin injections (types 1 and 2) and oral antidiabetics (type 2) Atherosclerosis exceedingly common; drug therapy targets underlying metabolic conditions, other risk factors, and consequences of advanced disease Degradation of phenylalanine and tyrosine

Phenylalanine Tyrosine Hydroxyphenylpyruvate Homogentisate

HO O HO O HO O O + BH α-Ketoglutarate O 2 4 2 HO O H2N H2N O OH

H2O + BH2 Glutamate CO2 1 2 3 OH OH OH

O2 Fumarylacetoacetate 4 O O O O HO O O O HO O O OH N O 5 O O H2O O 6 OH F F O O O F O Maleylacetoacetate HO HO OH NTBC Acetoacetate Fumarate Phenylketonuria (PKU)

◮ Homozygous defect of phenylalanine hydroxylase ◮ Frequency: 1 newborn among 10,000 in Caucasians; lower frequency in other races ◮ Possible heterozygote advantage: reduced fetal susceptibility to ochratoxin A ◮ Symptomatic excess of phenylalanine manifest only after birth; intrauterine development normal ◮ Cognitive and neurological deficits, probably due to cerebral serotonin deficit ◮ Treated with phenylalanine-restricted diet ◮ Some cases due to reduced affinity of enzyme for cofactor tetrahydrobiopterin

(BH4), can be treated with high dosages of BH4

DOPA transport Ochratoxin A inhibits phenylalanyl-tRNA synthetase

Phe-tRNA synthetase

ATP AMP + PPi

OH OH

NH2 OH Cl O

NH2 OH O H N O O O OH O Tyrosinemia

◮ Homozygous defect of fumarylacetoacetate hydrolase ◮ Prevalence: 1 in 100,000 people worldwide; 1 in 1,850 in the Saguenay region (Quebec) ◮ Fumarylacetoacetate and preceding metabolites back up ◮ Fumaryl- and maleylacetoacetate react with glutathione and other cellular nucleophiles, causing liver toxicity, cirrhosis, carcinoma ◮ The drug NTBC inhibits p-hydroxyphenylpyruvate dioxygenase, intercepting the degradative pathway upstream of the toxic metabolites pathway glutamine The urea cycle in context 1

NH3 2 carbamylphosphate 3 citrulline

4 ornithine α-ketoglutarate aspartate

11 9 argininosuccinate

oxaloacetate 5 fumarate P-5-C glutamate malate 10 8 7

arginine 6 urea Alternate pathway therapy in urea cycle enzyme defects

phenylbutanoic acid

enzymes of β-oxidation

phenylacetyl-CoA

glutamine N-phenylacetyltransferase

glutamine N-phenylacetyl-glutamine

urea cycle defect CoA-SH

urea urine

Gln conjugation Overview of purine degradation

Adenosine Inosine Hypoxanthine

NH2 H2O NH3 O P ribose- P O N N N N N N

N N N N N N HO O HO O H H H

O2 OH OH OH OH

H2O2 O H2O O

N NH N NH O N N 2 N NH2 N O Uric acid H NH3 H H O Guanine Xanthine H N NH O H O 2 2 N N O H H Adenosine deaminase deficiency causes dysregulation of DNA synthesis

Ribonucleotide reductase dI

Adenosine deaminase

ADP dADP dATP dA dAMP

GDP dGDP dGTP dGMP DNA CDP dCDP dCTP dCMP

UDP dUDP dTTP dTMP Therapy of adenosine deaminase deficiency

1. Bone marrow transplant 2. Ex vivo gene therapy 3. Enzyme replacement therapy—PEGylated bovine ADA 4. Experimental in vitro approach: Inhibition of salvage kinases Gout

◮ Genetic or dietary factors promote increased production or retention of uric acid ◮ Uric acid has low solubility, and excess levels form crystalline deposits, preferentially in joints and soft tissue ◮ Urate crystals promote inflammation and lead to arthritis that is painful and destructive Transport of uric acid in the kidneys

Interstitial fluid / Tubule epithelial cell Tubule lumen blood plasma (nascent urine)

urate urate ? URAT1 organic acid organic acid

urate urate ATP? MRP4 Dietary factors that promote gout

◮ Overly purine-rich food ◮ Drugs that contain purines: dideoxyadenosine ◮ Alcoholic beverages—but not all kinds: beer yes, wine no ◮ Anorexia nervosa (!) ◮ Drugs that interfere with uric acid excretion: pyrazinamide, salicylic acid ◮ Excessive fructose or sucrose Drugs that may promote gout by promoting tubular reuptake of urate

O OH O NH2 O OH O OH

HO N N N N N N

O

Salicylic acid Pyrazinamide Pyrazinoate 5-Hydroxypyrazinoate High dietary fructose promotes gout

fructokinase aldolase B fructose fructose-1- P glyceraldehyde

dihydroxyacetone- P ATP ADP glyceraldehyde-3- P

1,3-bis- P- 3- P -glycerate Pi PG-kinase glycerate

adenylate NADH+H+ NAD+ kinase purine degradation 1/2 urate 1/2 AMP 1/2 ATP Drugs used in the treatment of gout

O Br

HN O OH O

N O Br NH O N N O H O O O

Allopurinol Colchicine Benzbromarone Complementary effects of allopurinol and “rasburicase”

Adenine Hypoxanthine

Allopurinol

Guanine Xanthine

Urine

Uric acid

Rasburicase

Allantoin Lysosomal storage diseases

◮ Acidic hydrolases in lysosomes break down many cellular macromolecules, including lipids and mucopolysaccharides ◮ Enzyme defects cause accumulation of undegraded substrates, often in liver, spleen, and bone marrow, leading to organ enlargement and loss of function ◮ Some enzyme defects can be treated with enzyme substitution therapy Biochemistry of Gaucher disease

AB C HO OH 100

HO O β-Glucosidase Native NeuNAc HO O H N Gal Gal HO O Partially GlcNAc GlcNAc deglycosylated

Enzyme in plasma (%) 10 Man Man 0 5 10 15 20 25 30 Time after injection (min) Man

GlcNAc D HO OH

GlcNAc Fuc HO N

Enzyme protein HO Diabetes mellitus

◮ Lack of insulin activity due to

◮ destruction of pancreatic island β cell (type 1) ◮ loss of insulin sensitivity in peripheral organs (type 2) ◮ excessive levels of hormones antagonistic to insulin

◮ Blood glucose accumulates and causes acute and chronic pathology ◮ Treated with insulin substitution (type 1 and 2) and oral drugs (type 2) KATP channels in pancreatic β cells regulate insulin secretion

Glucose Insulin

Ca2+ Ca2+ Glucose

Membrane ADP + P Insulin i ATP potential K+ K+

ATP ATP Sulfonylurea receptor H2O, CO2 Oral antidiabetic drugs

Rosiglitazone O Tolbutamide O H NH S N N N S H O O N O O

CH2OH CH3 CH2OH CH2OH CH2OH O O O O OH OH OH OH OH H2N N N HO N O O O OH NH NH OH H OH OH OH OH 2

Acarbose Metformin

KATP channels Hypothetical mode of action of metformin

NAD+ Lactate

AMP-activated 4 protein kinase AMP ATP

NADH Pyruvate 3 1 Metformin – 1 2 e 2 O2 ADP ADP

+ + NAD 2 H Pi 2

+ + H H2O H ATP Atherosclerosis

◮ Degenerative and inflammatory disease of the arteries ◮ Promoted by hypercholesterolemia, hypertension, diabetes, smoking ◮ Damaged arteries subject to chronic or acute obstruction, hemorrhage ◮ Most common cause of death, ahead of all cancers and leukemias combined ◮ Treatment strategies address cholesterol levels, blood pressure, blood coagulation Appearance of atherosclerotic lesions

A B

Normal artery Foam cells in early lesion

C D

Detritus, fibrosis in advanced lesion High-grade stenosis, thrombus Development of an atherosclerotic lesion

Endothelial cell Native LDL

Macrophage Modified LDL

Foam cell Cholesterol crystals

Thrombocyte

ABCD Transport and metabolism of cholesterol

Small intestine Liver Macrophage / foam cell

Carbohydrates Glucose

scavenger receptor Triglycerides Acetyl-CoA Modified LDL Cholesterol

HMG-CoA ROS, enzymes HMG-CoA reductase

LDL receptor Cholesterol Cholesterol LDL Cholesterol

CETP

Bile acids HDL

Peripheral cell Intestinal cholesterol uptake Inhibitors of intestinal cholesterol uptake

Cholesterol Sitosterol Ezetimibe F

O

F N OH

HO HO F F F F F OH F O NH S O N N O N H S S N H

Lomitapide CP-113,818 Inhibition of HMG-CoA reductase with statins

Mevastatin Atorvastatin

O O HN O

O N HO O HO

HO OH F OH O HO OH O Mevalonate Overview of blood coagulation

Exposed collagen in vascular lesion Tissue factor GPIa-IIa GPVI

Coagulation factors

TXA, ADP

Prothrombinase

TP, P2Y receptors Platelets

PAR Thrombin Prothrombin

GPIIA/IIIB Fibrinogen

Fibrin Plasminogen Plasmin

Fibrin fragments tPA Strategies for inhibiting thrombocyte activity

◮ Low-dose acetylsalicylic acid ◮ Inhibitors of P2Y receptors (e.g. ticlopidine) ◮ Inhibitors of thromboxane A synthase and thromboxane receptors (e.g. ramatroban) Inhibition of plasmatic blood coagulation with warfarin

−O −O O −O O

O O OH Bromadiolone CO2, O2 O OH

OH H2O O

O OH O R R OH O Warfarin O O Br Vitamin K Warfarin

NADP+ + GSSG NADPH + 2 GSH Chapter 11

Chemotherapy of infectious diseases Diversity of infectious pathogens

◮ Bacteria ◮ Fungi ◮ Parasites—eukaryotes other than fungi

◮ Protozoa—unicellular ◮ Metazoa—multicellular

◮ Viruses The tree of life, slightly pruned

Giardia lamblia Leishmania major Plasmodium falciparum Arabidopsis thaliana Gallus gallus x y Anopheles gambiae Caenorhabditis elegans Saccharomyces cerevisiae Pyrococcus furiosus Staphylococcus aureus Streptomyces avermitilis Mycobacterium tuberculosis Escherichia coli Drug targets for antimicrobial therapy

◮ Macromolecules that occur in the cells of the pathogen but not within the human host. Examples:

◮ the bacterial cell wall (penicillin) ◮ de novo synthesis of folic acid (sulfonamides)

◮ Macromolecules that occur in both humans and the pathogen but are structurally divergent. Examples:

◮ ribosomes (chloramphenicol) ◮ dihydrofolate reductase (trimethoprim) ◮ DNA topoisomerase (ciprofloxacin) Structures of folic acid and of three inhibitors of dihydrofolate reductase

Folic acid O OH Trimethoprim O OH NH O N 2 H O N O N N N n H H2N N O H2N N N H O O OH O OH Cl NH2 N H N O N N N

H2N N N H2N N NH2

Methotrexate Pyrimethamine Microbial resistance mechanisms

◮ Mechanisms affecting the target:

◮ Structural alteration / mutation ◮ Compensatory overexpression

◮ Mechanisms affecting the drug:

◮ Reduced uptake ◮ Active extrusion ◮ Enzymatic inactivation Overview of antibacterial chemotherapy

◮ Targets

◮ Cell wall ◮ Ribosomes ◮ Enzymes related to cell division ◮ Intermediate metabolism

◮ Antibiotic resistance

◮ Bacteria have short generation times—fast de novo evolution of resistance ◮ Resistance genes exist in producers of antibiotics—can spread to pathogenic bacteria by gene transfer Gene transfer mechanisms in bacteria

◮ Transformation: cellular uptake of naked DNA ◮ Conjugation: plasmid-encoded active transfer between bacterial cells ◮ Transduction: gene transfer mediated by bacteriophages ◮ Transposons: transfer of genes between carrier DNA molecules (chromosomes, plasmids) Bacterial cell wall structure

Porin MDR system Porin

LPS LTA Outer membrane Mycolic Outer acids membrane Arabino- galactan Murein

Inner membrane

Gram-negative Gram-positive Mycobacteria Action mechanism of isoniazid (INH)

NAD+ N KatG N N O O O O HN 3H, 2N NH2 NH2

N O O

O −O P O OH OH ... NH2 ACP O − N NADH O P O N

N InhA O O N NAD+ O OH OH ... ACP

Isoniazid metabolism Outline of bacterial murein synthesis Fosfomycin mimics both phosphoenolpyruvate and glycerophosphate

O− O− O−

O P OH O P OH O P OH

H2C O O O H3C HO O O− OH

Fosfomycin PEP Glycerophosphate Cycloserine inhibits alanine racemase and d-alanine ligase

d-cycloserine

2 l-Ala 2 d-Ala d-Ala-d-Ala

H N O O O OH O OH

H2N H2N H2N

d-cycloserine d-alanine l-alanine The lipopeptide laspartomycin sequesters undecaprenol phosphate

Asp Gly Gly

Asp D-Thr

Gly Ile/Val

D-Pip Pro Dab-NH

Asp

O β-Lactam antibiotics resemble the substrate of the transpeptidase reaction

R d d H -alanyl- -alanine N O H N O H O HO

Penicillin G H N S O N O O HO Reactions of penicillin G with transpeptidase and β-lactamase

Transpeptidase H H N N S S O O O N O N H O Transpeptidase–serine–O HO O HO β-Lactamase

H H N S H2O N S O O O N O O N O H H β-Lactamase–serine–O HO β-Lactamase OH HO Structures of β-lactam antibiotics (1)

Penicillin G AmpicillinHO Ticarcillin NH O 2 S H H H N N N S S S O O O N N N O O O O O O HO HO HO

O Methicillin Imipenem Clavulanic acid NH OH 2 H N O N S O OH O S N N N O O O O OH OH HO O O Structures of β-lactam antibiotics (2)

Cefotaxime Ceftobiprole N O N N OH S S H H N N N N H2N S H2N S O O N O N N O O NH O O HO O HO O

HO O O Moxalactam Aztreonam HO O N O O S O N H H N N N S H2N N O O N O HO N O OH N O S O O− Inactivation of SHV-1 β-lactamase by clavulanic acid

Ser70-OH O O OH

O N O OH H+ Ser70 H O O OH N OH O O O N Ser70 OH O O OH

Ser130 H2O O O O Ser130 Ser130 OH O O O OH O O O O Ser70 H N Ser70 2 O O OH Ser70 Ser70 Ser130-OH O OH

H2O Vancomycin sequesters the substrate of the transpeptidase reaction Vancomycin can be modified to overcome bacterial resistance

Vancomycin Vancomycin amidine derivative

Cl R Cl Cl R Cl O O O O

HO OH HO OH O O O O O O N O O N O O H H N R H H N R O N O N N N H N N H N N −O H O H R H −O H N H R H H

OH O H O− OH O O− H H HO OH N N HO OH N O R N O R N O H H R O R O

d-Ala—d-Ala d-Ala––d-Lac Antibiotics that inhibit ribosomal protein synthesis

◮ Aminoglycosides ◮ Tetracyclines ◮ Macrolides ◮ Chloramphenicol ◮ Puromycin ◮ ... Chloramphenicol lodges into the peptidyl-transfer site of the ribosome

nascent Protein

mRNA Chloramphenicol in the peptidyl transferase site of the ribosome

G2044 Chloramphenicol A2430

Cl OH H U2483 N Cl O O OH N O

U2485 Structure and action mechanism of puromycin

NH2 NH2 N N N N N N N

N N N N N N HO HO HO O O O

O tRNA O tRNA HN OH

O O O

NH2 NH NH2 OH O Peptide

Aminoacyl-tRNA Peptidyl-tRNA Puromycin Diverse inhibitors of bacterial macromolecular synthesis

OH

O Fusidic acid (Elongation factor 2) HO O

O O O OH N HO O N N

O O O NH F OH OH OH O O O OH N N HN HO

Ciprofloxacin (DNA gyrase) Rifampicin (mRNA transcription) Structure of the potassium ionophore valinomycin

H O N O HN O O O O O NH O O O O HN O O O O O NH O N O H Daptomycin permeabilizes membranes containing phosphatidylglycerol

l-Asp d -Ala Gly Val/K+ 100 PC/PG l d + -Asp -Ser 75 K + 50 Na l-Orn l-MeGlu 25 choline

Gly l-Kyn Fluorescence (A.U.) 0 Thr-O 0 1 2 3 4 5

l-Asp + 100 PC/no PG Val/K d-Asn 75 l-Trp 50 O 25 K+ Fluorescence (A.U.) 0 0 1 2 3 4 5 Time (min) Structures of polymyxin B and lipopolysaccharide

HO OH OH HO O O OH OH HO − O O O Polymyxin B O Lipopolysaccharide O− O HO − O NH2 O H H P O O O H2N N N HO HN O O O O O N H NH2 O NH HO O HN O H N O O O NH O HO O O OH O NH O NH O P O− O NH O O O HO H2N NH NH2 O O NH HO O A model of the polymyxin B–LPS complex Drug targets in antifungal chemotherapy

◮ ergosterol in cell membranes (amphotericin B) ◮ ergosterol synthesis (ketoconazole; terbinafine) ◮ thymidylate synthase (5-fluorocytosine) ◮ 1,3-β-glucan synthesis (echinocandins) Structure and action mode of amphotericin B

HO OH HO O O NH2

HO O O OH

OH OH

OH HO

OH 20 OH ergosterol

O 10 O

release (%) cholesterol + K 0 HO 0 10 20 30 40 Amphotericin B Ergosterol Sterol content (%)

drug delivery Other antifungal drugs

Caspofungin N

H N 2 H N OH Terbinafine O HO N O H N H2N N H O O HN OH HO NH NH2 O O O H O H F 2 F HO N N HN HN

OH O OH O N O N NH3 Flucytosine 5-Fluorouracil HO

ketoconazole Antiprotozoal drugs

Nitazoxanide Metronidazole Chloroquine Artemisinin

N Cl O O OH O N O O S N N N NH O O O N O O NH

O N

O Miltefosine O− O P +N O O

tree Hemozoin inside malaria parasites inside red blood cells Chloroquine inhibits formation of β-hematin

CQ = 0 80

60 CQ = 50µM

40

20 CQ = 100µM -hematin formed (%) β 0 0 100 200 300 400 Time (min) Action mechanism and selective toxicity of nitroimidazoles

Ferredoxin–Fe2+ Ferredoxin–Fe3+ Reductive radical formation (aerobes and anaerobes)

⊖ • ⊖ O ⊕ O O O N N

HO N HO N DNA damage (anaerobes) N N

• Oxygen–mediated radical O– O 2 2 detoxification (aerobes)

H2O2 Trypanosomes in a blood smear Melarsene oxide binds trypanothione

O

HO

NH2 H2N O HN O N H O N N NH2 N N H OH O SH As N O H NH2 HN O NH O O SH H H N N N N H H S O O HN O H N As NH2 NH N S O HO H O H 2 H2N N N N N O H NH2 O NH O

NH2 OH

O Leishmania in bone marrow The life cycle of influenza virus

1 7 8

2

6 H+ 3

5 4

Hemagglutinin M2 protein RNA polymerase Neuraminidase Amantadine and rimantadine block the M2 proton channel

NH2

Amantadine

NH2

Rimantadine Oseltamivir inhibits influenzavirus neuraminidase

OH HN O HN O

OH O NH2 HO OH O

R O OH O O O

Neuraminic acid residue Oseltamivir Inhibition of HIV fusion with target cells by the peptide enfuvirtide

HIV envelope 1 2 3

gp41 gp120

CD4 Cell membrane Inhibitors of virus genome replication

O NH2 NH2 N N N N N N N O N O N N O H O P O OH O O O O O O O HO O P OH O Didanosine Cidofovir Tenofovir disoproxil OH O O N H S NH2 N O O− S O O N N −O P − N O O N N

Foscarnet Nevirapine Bay 57-1293 Activation of acyclovir

Aciclovir Deoxyguanosine O O N NH N NH HO N HO N O N NH2 O N NH2

viral kinase cellular kinases OH

O O N NH N NH P O N P O N O N NH2 O N NH2

cellular kinases OH

O O N NH N NH P P P O N P P P O N O N NH2 O N NH2

viral DNA cellular DNA OH polymerase polymerases

viral DNA cellular DNA cellular DNA Function of virus proteases Structure of HIV protease, with the inhibitor saquinavir bound Chapter 12

Cancer chemotherapy Some terminology

◮ malignant tumour / cancer: A tumour that

◮ grows without regard for anatomical boundaries ◮ typically also metastasizes

◮ benign tumour: a tumour that does neither ◮ Carcinoma: a malignant tumour derived from an epithelial tissue

◮ colon, lung, kidney carcinomas ◮ hepatocellular carcinoma (liver)

◮ Sarcoma: a malignant tumour derived from a non-epithelial tissue

◮ lipo-, myo-, fibro-, osteosarcoma

◮ Leukemia: a malignancy of white blood cell precursors

◮ Lymphatic leukemia (related to lymphocyte precursors) ◮ myeloic leukemia (related to granulocyte or monocyte precursos) ◮ Rare: Erythremia/Erythroleukemia Cancer therapy

Forms of therapy

◮ Surgery ◮ Radiation ◮ Chemotherapy

Criteria for therapy selection

◮ Benign or malignant tumor ◮ Tissue of origin, histological variant of tumor ◮ Stage of tumor—early and localized vs. advanced and disseminated Cellular pathways that control proliferation and apoptosis

Growth factors, adhesion molecules Fas-ligand, TNF

Receptor tyrosine kinases, GPCRs Death receptors Cell membrane

PIP3-kinases Adapter proteins

Proliferation AKT Caspases Apoptosis

Microtubule Bcl family Cytochrome C Mitochondria disruption

MDM2 p53 ATM, ATR, ARF

Cell cycle arrest, DNA repair DNA damage Nucleus Dysregulation of growth in tumor cells

Normal body cells

◮ grow or persist only when stimulated by growth factors ◮ undergo apoptosis when deprived of growth factor stimulation

Tumor cells contain mutations that

◮ create surrogate growth stimuli

◮ constitutively active growth factor receptors ◮ autocrine secretion of growth factors

◮ disrupt activation of apoptosis downstream of growth factor deprivation ◮ but also make tumor cells more susceptible to some apoptotic stimuli than normal cells Leukemic (CML) cells are more susceptible to tyrosine kinase inhibitors

100 Normal cells

10

CML cells Colonies (% of control) 1 0 5 10 15 20 AG 957 (µM)

apoptosis pathways A mutant of caspase 9 inhibits apoptosis in response to anticancer drugs

60 Mutant caspase 9 Wild type

40

Apoptosis (%) 20

0 Control Etoposide AG 957

apoptosis pathways Knock-down of p53 promotes survival of cells treated with an inhibitor of MDM2

100

75 p53 siRNA

50

Control Cell survival (%) 25

0 0 0.01 0.05 0.1 0.5 1

RITA (µM)

apoptosis pathways Disruption of microtubules with taxol induces apoptosis, which is inhibited by Bcl-2

80 Bcl−2 overexpressed

60

40

Bcl−2 normally expressed 20 Hypodiploid cells (%)

0 0 0.1 0.5 1 10

Taxol (µM)

apoptosis pathways The cell cycle and its checkpoints

G2 phase arrest M phase arrest (DNA damage) (defective spindle)

S phase arrest G1 phase arrest (unreplicated DNA) (DNA damage) Progressive cell aneuploidy in a recurring tumor

800 300 initial cancer recurrent cancer 600 200

400

Cell count 100 200

0 0 0 50 100 150 200 250 0 50 100 150 200 250

Fluorescence intensity (DNA content) Fluorescence intensity (DNA content) Cell type-specific anticancer drugs

◮ Hormones and growth factors: interferon-α in hairy cell leukemia ◮ Hormone antagonists: most significant with breast and prostate cancer ◮ Tissue-specific prodrug activation: mitotane in adrenal gland tumors ◮ Tissue-specific accumulation of radioactive iodine: thyroid cancer Sexual hormones and receptor antagonists

O N

OH

4-Hydroxytamoxifen

mifepristone Aromatase and two of its inhibitors

OH Exemestane NADPH + O2 HO C D O

A B

O H2O O

Testosterone O

CH2 NADPH + O2

2 H O 2 N N

Estradiol OH

NADPH + O O 2 N N N HO O Letrozole HCOOH + H2O The anticancer prodrug mitotane is selectively activated in the adrenal cortex

R−NH2 H Cl Cl CYP11B O Cl O N R Cl Cl Cl

HCl

Cl Cl Cl Anticancer drugs that target specific oncoproteins

◮ α-Retinoic acid in promyelocyte leukemia ◮ Imatinib in chronic myeloic leukemia; other tyrosine kinase inhibitors in various tumors ◮ Monoclonal antibodies against growth factor receptors on the cell surface, for example Her2/neu in breast cancer A chromosomal translocation causes promyelocytic leukemia The mutant PML-RARA receptor blocks promyelocyte differentiation Retinoic acid therapy restores promyelocyte differentiation

R

Dysfunctional PML-RARA/RXR t(15;17) R receptor dimer

O R R OH Binding of RA induces Retinoic acid PML-RARA breakdown

17 R R

RXR recruits RARA expressed from R R intact copy of chromosome 17 Chronic myeloid leukemia

◮ Caused by reciprocal translocation between chromosomes 9 and 22 (“Philadelphia chromosome”) ◮ Translocation produces a chimeric, constitutively active protein tyrosine kinase (Bcr-Abl) that drives the proliferation of myeloid precursor cells ◮ Treatment with imatinib or other tyrosine kinase inhibitors controls, but usually does not eradicate leukemic cells ◮ After several years, CML typically ends in a “blast crisis”, which resembles an acute myeloid leukemia Imatinib bound to its target enzyme c-Abl kinase

N N

HN O

NH

N N

N Cytotoxic anticancer drugs

◮ Antimetabolites ◮ Inhibitors of DNA topoisomerase ◮ Proteasome inhibitors ◮ Inhibitors of mitosis ◮ DNA-alkylating and other DNA-damaging agents Dual action mode of 5-fluorouracil

O O O

F CH3 N N N

O N O N O N

Uridine 5-F-Uridine 5-F-d-Uridine d-Thymidine

5-F-dUDP

dUMP 5-F-dUMP 5-F-dUTP dTTP

Thymidylate Mutagenesis synthase

dTMP DNA Catalytic mechanism of thymidylate synthase

N,N′-methylene-tetrahydrofolate dTMP dihydrofolate H O H2N N N H CH3 NH2 H2N N N HN HN ⊖ Enzyme N S HN O N N O N R dR O N R + H H

H H H R H2N N N H2N N N H2N N N HN HN R HN ⊕ HN HN N N N H H+ O CH2 N R O O H H O CH2 O O CH2 + NH2 HN H NH2 HN NH HN 3 Enzyme ⊖S O N S Enzyme O N S Enzyme O N dR dR dR

dUMP covalent intermediate Mutagenesis through mispairing of the 5-FU iminol tautomer

Adenine Guanine

N H N dR N N dR N O H H N N O N N O H H F F N N N H H O N O N dR dR

5-FU, amide form 5-FU, iminol form Thymine and various halogen analogues

Fluorouracil Bromouracil Iodouracil Thymine Blockade of dihydrofolate reductase also inhibits thymidylate synthesis

NADPH+H+ NADP+ DHFR H H H2N N N H2N N N

HN HN N N H O HN R O HN R

dTMP serine

TS SHMT H H2N N N dUMP glycine HN N

O H2C N R Inhibitors of dihydrofolate reductase

Folic acid O OH Trimetrexate O O OH O O N NH2 H N O N N N n N N O H H

H2N N N H2N N H

MethotrexateO OH Pemetrexed O OH O O

NH2 N O N H H N N N O HN O

H2N N N OH H2N N N OH H Structure of cytosine arabinoside (araC)

NH2 NH2 NH2

N N N

HO N O HO N O HO N O O O O HO

OH OH OH OH

Cytidine Deoxycytidine Cytosine arabinoside (araC) Metabolic activation and inactivation of araC

MDR ENT

dC deaminase araU araC

5 ′-nucleotidase dC kinase

dCMP deaminase dNMP, dNDP kinases araUMP araCMP araCTP Overexpression of 5′-nucleotidase in leukemic cells correlates with reduced survival rates

100

75

no 5-NT overexpression

50

25 Patients surviving (%) 5-NT overexpression

0 0 12 24 36 48 60

Time (months) Action mode of araCTP Function of DNA topoisomerases DNA topoisomerase inhibitors

O O OH N O O OH HO N O O O O N O O OH HO O O O Topotecan (topo I) Etoposide (topo II) Topotecan bound to topoisomerase I and DNA Bortezomib inhibits the proteasome

HO OH B N NH H N O N O Vinblastine inhibits tubulin polymerization

OH N

N N H H O OH O N O O HO O O Alkylating anticancer drugs

Mechlorethamine Cisplatin Streptozotocin HO N NH2 O Cl OH HO O Pt2+ Cl O Cl Cl HO OH N H N 2 O S O HN N H2N O O O N P O H O N N N H O N N O S O Cl Cl Cl Cl Cl Cl O

Cyclophosphamide Melphalan Busulfan BCNU Reaction of mechlorethamine with DNA

O

Cl Cl Cl ⊕ N H N N N

N Cl– N NH2 dR

Cl OH N OH N OH ⊕ ⊕ ⊕ N N N N N N

N N N H2N N N NH2 N NH2 dR dR dR BCNU decay and adduct formation

O H N N N Cl Cl HO O N N Cl N Cl C O – O N2,OH

N ⊕ R—NH2 HN Cl

N H2N N dR

R OH H Cl N NH ⊕ N Cl N O N H2N N dR Reaction of daunorubicin with DNA

Fe2+ O O OH R

O

O O OH O O

OH NH dR N N NH

NH N

O Chapter 13

Ribonucleic acids as drugs and drug targets Antibiotics that inhibit the bacterial ribosome

Bacterial ribosome (70S)

50S subunit 30S subunit

5S rRNA (120 nucleotides) 16S rRNA (1540 nucleotides) 23S rRNA (2900 nucleotides) 21 proteins 34 proteins

Peptidyl transfer Blasticidin S Aminoacyl site Paromomycin Chloramphenicol Tetracycline Tiamulin Tobramycin Virginiamycin M Streptomycin Viomycin Exit tunnel Erythromycin Some aminoglycoside antibiotics

NH2 Ribostamycin

O HO Streptomycin HO H2N NH2 O H2N HO NH2 NH Tobramycin O OH O HN HO OH NH2 HO NH HO O OH OH O H O NH NH2 O HN H N 2 O NH2 HO O OH OH O

O HO HO O OH HO NH NH2 HO Paromomycin in the ribosomal aminoacyl acceptor site

R N A1408 N G1491 N R H N N 2 N N NH2

NH OH H N HO N O H O− H HO R O P O R O O O O

H O NH2 H N O O H OH O OH OH R N N N H H H N H N O N O O H N H 2 NH R O P O R G1494 O− N O U1495 R Inactivation of kanamycin by resistance enzymes

HO H2N O OH ATP OH NH2 ATP O O HO O

OH OH PPi HO ADP NH2 NH2

HO HO H2N H2N O OH O OH OH NH2 OH NH2 O O HO O acetyl-CoA O O HO O

O OH O OH HO − HO CoA-SH O P O NH2 NH2 NH2 NH2 O−

NH2 H3C O N N HO − HN O O P O OH OH NH O N N 2 O O HO O

OH OH OH OH HO NH2 NH2 Amikacin, a semisynthetic derivative of kanamycin

HO HO H2N H2N O OH O OH OH NH2 OH NH2 O O HO O O O HO O

OH OH OH OH OH HO HO NH2 NH2 NH2 HN NH2 O kanamycin amikacin Interactions of chloramphenicol with RNA in the peptidyl transferase site of the ribosome

G2044 Chloramphenicol A2430

Cl OH H U2483 N Cl O O OH N O

U2485 The RNA component of human telomerase

P6

Antisense 3′- C A CG AAAA CG A GGGGCGCG-5′ Sense 5′- G U GC UUUU GC U CCCCGCGC-3′

P5

P4.2 Folded RNA GC UUU CGCGC segment U G U C P8 G C P4.1 C U C

P7 P4 P3 P1 3′ P2

P2b ′ 5 Unusual nucleotide linkages in synthetic oligonucleotides

ABCD

R′O Base R′O Base R′O Base R′NH

O O O Base N O OH O O O O O O P S− O P O− O P O− O NH Base Base Base O O O Base O O O N

R′O OH R′O O O O R′O O NH′R The HIV transactivation-responsive region (TAR)

GG O NH2 U G C A H C G NH2 NH2 N NH2 G C H2N A U H2N U G C NH C U A U G C NH2 A U C G C G N A G C A U O A A U G N G C G U U A C G S U G C G U A OH C U A N G C N G C C U C N N 5′– G CACU CG UU AA CG A A NH GUGAGUUCCGUUCG U C A A A N UU C AA ′ H –3 Blocking premature translational termination with PTC124 (ataluren)

Ribosomes Regular stop codon

Nascent proteins Functional protein Mutant stop codon

Incomplete protein PTC124

Functional protein Ataluren in cystic fibrosis

Phase 1 Phase 2 10

5 pathological range 0

-5

-10 normal range -15 Nasal potential difference (mV) -20 0 14 28 42 Time (days) RNA interference

dsRNA

Dicer

siRNA

RISC

mRNA

mRNA cleavage blocked translation The SELEX process for generating RNA aptamers

RNA library Washing

Column binding

amplified selected RNA Elution

in vitro transcription

cDNA weak binders RT-PCR avid binders Chapter 14

Drug delivery Protecting drugs from gastric acid through prodrug formation

H O O O O H H N S N S

O N O N O O OH OH O O Carbenicillin Carindacillin Optimizing a drug structure for bilayer permeation

H HO HO N N O OH N N N N Cl Cl N N

EXP7711 Losartan Trapping an estradiol prodrug inside the brain

Periphery BBB Brain

Estradiol-17-dihydro- trigonelline O O O O N N

HO HO Oxidation

O O O O ⊕ ⊕ N N

HO HO

Esterase OH O Elimination HO ⊕ N HO Succinylsulfathiazole, a prodrug designed for reduced absorption

O O O− H Succinyl-sulfathiazole N S N H N O O S

H2O Bacterial esterase (colon, slow) succinate

O H Sulfathiazole N S NH2 N O S acetyl-CoA N-Acetyltransferase (liver, fast) CoA-SH

O Acetyl-sulfathiazole H H (poorly soluble) N S N N O O S Inhibition of DOPA decarboxylase in the periphery improves l-DOPA uptake into the brain

HO O BBB

NH2

l-DOPA l-DOPA HO O OH H HO N

NH2 DOPA DOPA decarboxylase decarboxylase HO OH NH2 Carbidopa

Dopamine Dopamine HO OH Gludopa, a prodrug for selective release of dopamine in the kidneys

H2N O

OH

O H2O O NH O NH2 NH2

HO HO

Glutamate CO2

DOPA HO OH γ-GT HO OH decarboxylase HO OH Protecting drugs from gastric acid through acrylate copolymer coating

H CH3 CH3 H ... C CH2 C CH2 C CH2 C ...

C O C O C O C O

OH O OH O

CH 3 C2H5 n Site-selective delivery of BCNU

Carmustine (BCNU) O N H N N Cl Cl O

Gliadel®

O O O O O O O O O O O 80 20 1,3-Bis(carboxyphenoxy)propane Decanedioic acid Cyclodextrins: Structure and use in drug delivery

OH O

HO O O OH O OH HO OH OH OH O HO OH HO O O OH O HO HO OH OH HO O O HO O OH OH HO HO OH O O O HO HO Solubilization of hydrophobic drugs with surfactants Liposomes as drug delivery vehicles

Hydrophilic cargo drug Hydrophobic cargo drug PEG surface modification Amphotericin B, ergosterol, and cholesterol

HO OH HO O O NH2

HO O O OH

OH OH OH

OH HO

OH

OH

O O

HO

Amphotericin B Ergosterol Cholesterol Liposomal vs. deoxycholate-solubilized amphotericin B in a mouse infection model

100 uninfected control liposomal AmB (15 mg/kg/day) 80

AmB in deoxycholate (1 mg/kg/day) 60 Survivors (%) 40 infected control (no treatment)

0 2 4 6 8 10 12 Days after infection Liposomes and the Enhanced Permeability and Retention (EPR) effect Humanized antibodies

Monoclonal mouse Human antibody without “Humanized” hybrid antitumor antibody antitumor specificity antitumor antibody A calicheamicin-antibody conjugate

O Anti-CD33 antibody Linker DNA-reactive O O moiety NH

HO O O H N O O H N N H S S

O I O S O O N H O O O OH HO O O HO O OH O N O O DNA-binding oligosaccharide moiety Activation of calicheamicins

O O R H⊕ R 2 G—SS—R1 2

HO HO

O O ⊖ sugar S sugar S

S R1 ⊖ G S O O

R2 R2

• HO HO diradical • intermediate O O S S sugar sugar DNA cleavage by activated calicheamicins

P P

O O • H H Base • Base H O O O2 H+ • O O

P P P

O P H Base P HO O O O⊖ O O H H Base ⊖ Base O O O O P

– O O G−S G−S−OH P P Aggregation of insulin delays its uptake into the circulation

Hexamer Dimer Monomer

Capillary wall Structure of the insulin hexamer

Dimer 1

Gly B23

Glu B21

Pro B28

Dimer 2 Dimer 3 The Viadur® implant Chapter 15

Drug discovery Stages of drug discovery

◮ Target molecule

◮ selection ◮ validation

◮ Candidate compounds

◮ acquisition ◮ screening Chemical structures of subtype-selective glutamate receptor ligands

O O HO O O HO O O HO O OH OH N OH OH H NH

H2N H O H2N H NH

l-Glutamate AMPA Kainate NMDA

− O O O O OH P O NH2 HO O O HN N OH H O H2N H OH

OH O H2N H

ACPD Quisqualate l-AP4 Sources of candidate compounds

◮ Synthetic libraries ◮ Natural compounds ◮ Semisynthesis ◮ Gene technology Combinatorial synthesis: the Ugi reaction

R1 OH R2 O R5

+ NH2 + + N O R3 R4 C

R2 O

R1 N R5 N R3 R4 H O Semisynthesis of penicillins

6-Aminopenicillanic acid Semisynthetic penicillin

R R H H H H N N 2 S X S O O

O O O O HO HX HO

O NH2 F N

R = Cl HO

Penicillin G Amoxicillin Flucloxacillin Semisynthesis of cephalosporins

N N O H2N H H H H2N S S N S O O S N O O NH O N HO O HO O O O 7-Aminocephalosporanic acid Ceftriaxone Biosynthesis of polyketides

M1

S O M R1 1

M2 M2 M2 M2 M2 CoA S AT S KS S KR S DH S ER S O O O O O O R2 R2 R2 R2 R2 R2 HOOC CoA HOOC CO2 O HO R1 R1 R1 R1

KS KS KS M3 ... CoA S AT S KS ... O O R3 R3 HOOC CoA HOOC CO2 Structure of native 6-deoxyerythronolide B synthase

ER DH KR KR KR KR KR AT ACP KS AT ACP KS AT ACP KS AT ACP KS AT ACP KS AT ACP KS AT ACP TE

S S S S S S S O O O O O O O

HO HO O HO HO

HO HO O HO

HO HO O

O HO HO O

HO HO

OH HO

O OH TE O OH Two compounds produced by engineered variants of 6-deoxyerythronolide B synthase

O O O

OH OH

O OH O OH O OH

O OH O OH O O

6-Deoxyerythronolide B Engineered 6-deoxyerythronolide B analogues Compound screening

Experimental approach Typical applications

Activity assays on purified Enzymes target proteins Cell-based activity assays GPCRs, ion channels Computational screening Targets with available 3D structure

Phenotypic screening Cytotoxic activity Peptide deformylase and Met aminopeptidase

O R O R H 1 H 3 H N N Bacterial N N H H translation product O O R2 O

S H O Peptide 2 deformylase HCOO–

O R1 O R3 ⊕ H Bacterial intermediate/ H3N N N N eukaryotic translation H H O R2 O product

S

H O Methionine 2 aminopeptidase methionine R O R 1 H 3 ⊕ N H3N N Mature protein H O R2 O In vitro screening of Met aminopeptidase inhibitors

Apoenzyme purified from cells is inactive

Co2+ Mn2+ Fe2+ Enzyme is activated by various metals

Inhibitors screened O on enzymes in vitro O NH OH OH HN O O OH S N Cl

S Measurement of antibacterial activity A non-covalent yet irreversible enzyme inhibitor

O O O O N N P H H O−

NH2 HN HN O O

O O OH O OH

Carboxypeptidase A peptide Cbz–Phe–Val–Phe substrate (Phe–Val–Phe) phosphonate analog A generic assay for GPCR activation

Antibody tagged with Ligand pH-sensitive dye

GPCR

β-Arrestin

Endocytosis

Fluorescent signal H+ H+ H+ Laser beam ATP A cell-based fluorescence assay of membrane depolarization

Laser Acceptor signal Laser Donor signal A fluorescence assay of Ca++ influx

O O Fura-2-AM + Ca2+ O

O O O O O 2+ N - Ca O O O O O O O N Fluorescence signal

O 250 350 450 Excitation wavelength (nm) O O N O− O O O O O O O N HO O O O− O O O N

O 5 H2O Fura-2 O− N O O 5 CH3COOH + 5 CH2O OH An in silico docking experiment

N N

N

O NH

N starting conformation N N H

docked poses Electrostatic potential mapped onto the electronic density for acetaminophen

H

O O H

N H H H Hypothetical pharmacophore for inhibitors of ATP:L-Methionine S-Adenosyltransferase