MOLECULAR TARGETS FOR DRUG ACTION (and other topics – a review before the final test) Prof. M. Kršiak Department of Pharmacology, Third Faculty of Medicine, Charles University in Prague Charles University in Prague, Third Faculty of Medicine Cycle II, Subject: General Pharmacology

FOUR MAJOR TARGETS FOR DRUGS: 1. RECEPTORS 2. ION CHANNELS 3. CARRIER MOLECULES 4. ENZYMES Molecular Targets For Drug Action

Cellular RECEPTORS Cell Membrane Intracellular - Receptors linked to gene transcription(nuclear receptors) Channel-linked receptors G-protein-coupled receptors Proteinkinase-linked receptors 1. RECEPTORS

Dopamine receptors D 1-5 (type D 1,5, type D 2,3,4 ) They differ in localization (occur mostly in the CNS, post- or pre- synaptically), they differ in mechanisms of transduction (some are coupled with Gs, some with Gi, some act via adenylyl cyclase, some via phospholipase C, or via ion channels – K, Ca) Synthesis of dopamine: tyrosine → L-DOPA →dopamine → noradrenalin →adrenaline Decarboxylase: L-DOPA→dopamine Elimination of dopamine: extracellulary(in the synaptic cleft): transport protein (reuptakes DA from synapt.cleft to the presynaptic nerve ending) COMT catechol-O-methyl transferase intracellulary: MAO monoamino oxidase DOPAMINERGIC SYSTEM Clinical potency of antipsychotics correlates with their affinity for D2 receptors Decarboxylase inhibitors in combination with levodopa → antiparkinsonics COMT inhibitors→ antiparkinsonics Inhibitors of MAO (IMAO) → antidepressants Inhibitors of DA, NA, 5-HT reuptake → antidepressants

Downloaded from: StudentConsult (on 28 October :42 PM) © 2005 Elsevier Ac, nucleus accumbens; Am, amygdaloid nucleus; C, cerebellum; Hip, hippocampus; Hyp, hypothalamus; P, pituitary gland; SN, substantia nigra; Sep, septum; Str, corpus striatum; VTA, ventral tegmental area; Reward system Chemoreceptor trigger zone MAJOR DOPAMINERGIC PATHWAYS/SYSTEMS IN CNS

PHARMACOLOGY OF MAJOR DOPAMINERGIC SYSTEMS IN CNS System Clinically most important drugs/ effects * Note Mesocortical, mesolimbic ↓antipsychotics→ antipsychotic effect ↑ e.g.. levodopa → psychosis Nigrostriatal ↓ antipsychotics → extrapyramidal adverse effects ↑antiparkinsonics (dopaminergic) Tuberohypophyseal↓ antipsychotics → hyperprolactinemia ↑ e.g.bromocriptine → therapy of hyperprolactinemia Reward system (nc. accumbens) ↑addictive drugs e.g. metamphetamine, morphine, nicotine, etc. Vomiting centre Chemoreceptor trigger zone in medulla, area postrema ↓ antiemetics → inhibition of nausea, vomiting - metoclopramide, domperidon ↑ e.g. apomorphine → vomiting ↓ inhibition, ↑ stimulation * Additional neuromediator systems may participate in these effects (e.g. serotonergic, glutamatergic systems in antipsychotic effects, cholinergic system in antiparkinsonic, antiemetic effects, etc.)

Antipsychotics D1D1 D2D2 alfa 1 H1H1 mAch 5- HT 2A Notes 1st generation chlorpromazine EPS, increased prolactin, hypotension, antimuscarinic effects haloperidol ± + As chlorpromazine but fewer antimuscarinic effects 2nd generation (atypical) clozapine Risk of agranulocytosis! Regular blood counts required. Weight gain. No EPS olanzapine Weight gain. Without risk of agranulocytosis, No EPS risperidone Weight gain. Significant risk of EPS sulpiride Increased prolactin (gynaecomastia) quetiapine Weight gain. No EPS aripiprazole PA ++-++Fewer side effects [“Third generation?“- dopamine stabilizers] EPS=extrapyramidal side effects, PA = partial agonist

Figure 45.1 Correlation between the clinical potency and affinity for dopamine D2 receptors among antipsychotic drugs. Clinical potency is expressed as the daily dose used in treating schizophrenia, and binding activity is expressed as the concentration needed to produce 50% inhibition of haloperidol binding. (From Seeman P et al Nature 361: 717.) Downloaded from: StudentConsult (on 15 December :54 AM) © 2005 Elsevier Correlation between the clinical potency and affinity for dopamine D2 receptors among antipsychotic drugs.

DRUG TREATMENT OF PARKINSON‘S DISEASE Normal extrapyramidal system: Nigrostriatal dopaminergic neurons inhibit cholinergic neurones in striatum Parkinson‘s disease: Death of nigrostriatal dopaminergic neurons → disinhibition of cholinergic neurons The aim of pharmacotherapy is, therefore, to enhance the dopaminergic transmission and to reduce the cholinergic transmision

Dopaminergic antiparkinsonics: Levodopa (+ inhibitors of dekarboxylase in the periphery:carbidopa, benserazid) IMAO (selegiline) Agonists of dopamine (ropinirol, pramipexol) Other: amantadine, inhibitors of COMT Anticholinergic antiparkinsonics: biperiden ANTIPARKINSONICS

ANTIDOPAMINERGIC ANTIEMETICS: metoclopramide, domperidone Also gastroprokinetic effect common adverse reactions: extrapyramidal - akathisia, dystonia

Serotonin receptors 14 subtypes (!) in 7 classes (5-HT 1-7 ) Almost all are metabotropic: They differ in localization (occur mostly in the CNS, post- or pre-synaptically), but also in the periphery. They differ in mechanisms of transduction (are coupled with various G proteins, some act via adenylyl cyclase, some via phospholipase C, or via ion channels –Ca) Only 5-HT3 receptors are ionotropic Synthesis of serotonin/5- hydroxytryptamine(5-HT): tryptofan → 5-hydroxytryptofan →5-hydroxytryptamine Elimination of serotonin: extracellular (in synaptic cleft): transport protein (reuptakes 5-HT back in the nerve terminal) intracelular: MAO monoamino oxidase Inhibitors of MAO (IMAO) → antidepressants Reuptake inhibitors of 5-HT → SSRI and some other antidepressants SEROTO(NI)NERGIC SYSTEM

Downloaded from: StudentConsult (on 28 October :26 AM) © 2005 Elsevier MAJOR SEROTONERGIC PATHWAYS/SYSTEMS IN CNS:

FUNCTION OF SEROTONERGIC SYSTEM IN THE BRAIN: regulation of emotion (e.g. depression, anxiety), sleep, body temperature, eating, sexual functions, pain, perception (halucinations), nausea- vomiting IN THE PERIPHERY: ↑ peristalsis in the GIT, vasoconstriction, ↑↓ BP, ↑platelet agregation

CLINICALLY IMPORTANT DRUGS ACTING VIA SEROTONERGIC SYSTEM: TRIPTANS (5-HT1D agonists)- e.g. sumatriptan – ANTIMIGRAINE DRUGS SSRI (selective serotonin reuptake inhibitors) e.g. fluoxetin, citalopram, sertralin, effective as ANTIDEPRESSANTS and in ANXIETY DISORDERS Some other antidepressant can also inhibit reuptake of seotonin IMAO (inhibitors of MAO) – ANTIDEPRESSANTS e.g.. moclobemide „SETRONS“ (5-HT 3 antagonists)- e.g. ondansetron – ANTIEMETICS SDA ( serotonin dopamine antagonists) atypic antipsychotics e.g. risperidone

Histamine receptors, H 1,H 2, H 3, (H4) All are metabotropic They occur in the brain and in the periphery Synthesis, elimination of histamine – not utilized in applied pharmacology HISTAMINERGIC SYSTEM Drugs producing release of histamine – morphine, atracurium

IN THE BRAIN: H 1 –↑ vigility, H 3 – presynaptic ↓ release of neuromediators H1 antagonists 1. generation → sedation, drowseness, e.g. promethazine, antiemetics – dimenhydrinate in motion sickness IN THE PERIPHERY: H1 – mast cells, vasodilatation, ↑ capilar permeability, alergic reactions (itching, urticaria, allergic rhinitis), bronchoconstriction H2 – parietal cell in stomach mucose (↑ sekretion HCl) H1 antagonists – drugs for allergic rhinitis, urticaria - H1 antagonists 2. generation (nonsedating) - cetirizin H2 antagonists – drugs for peptic ulcer disease – ranitidine, famotidine H3 antagonist betahistine→ vasodilatation in the inner ear – antivertigo drug ( Méniere‘s disease) CLINICALLY IMPORTANT DRUGS ACTING VIA HISTAMINERGIC SYSTEM:

FOUR MAJOR TARGETS FOR DRUGS: 1. RECEPTORS 2. ION CHANNELS 3. CARRIER MOLECULES 4. ENZYMES Molecular Targets For Drug Action

ION CHANNELS VOLTAGE-DEPENDENT CHANNELS LIGAND-GATED CHANNELS Extracellular ligands Calcium channels Sodium channels GABA-gated Cl- channels Nicotinic receptor NMDA receptor Intracellular ligands ATP-sensitive potassium channels

VOLTAGE-DEPENDENT CHANNELS Calcium channels - Ca++ flows into cells, necessary for contraction of cardiac and smooth muscles, blocked by CALCIUM CHANNEL BLOCKERS : amlodipine, verapamil –used in hypertension, angina pectoris, dysrytmias Sodium channels - Na+ flows into cells, necessary for propagation of action potentials in excitable cells, blocked by LOCAL ANAESTHETICS : procaine, lidocaine, articaine, bupivacaine, some Antiepileptics: phenytoin, some Antidysrhytmics : lidocaine

LIGAND-GATED CHANNELS Extracellular ligands GABA-gated Cl- channels –Benzodiazepines as modulators (ANXIOLYTICS) –, diazepam, alprazolam, midazolam GABA A receptor Benzodiazep. receptor

LIGAND-GATED CHANNELS Extracellular ligands Nicotinic receptor NEUROMUSCULAR-BLOCKING DRUGS Non-depolarising blocking agents, e.g. atracurium act as competitive antagonists at the nicotinic receptors of the motor endplate act by activating nicotinic receptors and thus causing persistent depolarisation of the motor endplate Depolarising blocking agents - suxamethonium

to Ca 2+, as well as to other cations, so activation of NMDA receptors is particularly effective in promoting Ca 2+ entry. LIGAND-GATED CHANNELS Extracellular ligands NMDA (N-methyl-D-aspartate) receptor glutamate receptor Activation of NMDA receptors results in the opening of an ion channel It requires co-activation by two ligands: glutamate and either d-serine or glycine NMDA receptor antagonist – ketamine (General anaesthetic – intravenous) produces 'dissociative' anaesthesia, in which the patient may remain conscious although amnesic and insensitive to pain. Sometimes psychotomimetic effects

LIGAND-GATED CHANNELS Intracellular ligands ATP-sensitive potassium channels (K ATP channels) In the presence of increased levels of ATP, or by action of sulfonylureas (Antidiabetics ) e.g. glimepiride the K ATP channels close, causing the membrane potential of the cell to depolarize, thus promoting insulin release The K ATP channels in pancreatic beta cells when open, allow potassium ions to flow out the cell. K+K+ K+K+ ATP See also Fig Golan et al. 2012, p. 528

FOUR MAJOR TARGETS FOR DRUGS: 1. RECEPTORS 2. ION CHANNELS 3. CARRIER MOLECULES 4. ENZYMES Molecular Targets For Drug Action

3. CARRIER MOLECULES „pumps“ sodium pump - Na + /K + ATPase, „pumps“ Na + from the cell, inhibited by cardiac glycosides proton pump - H + /K + ATPase, „pumps“ H + from the cell, proton pump inhibitors transporters transporters for noradrenaline, serotonine inhibited by most antidepressants (RUI, TCA, SSRI etc)

TRANSPORTERS „Pumps“ Transport proteins transporters for noradrenaline (NA), serotonin(5-HT), dopamine (DI) P-glycoprotein (P-gp) sodium pump proton pump

sodium pump - Na + /K + ATPase, „ pumps“ Na + from the cell. This is inhibited by c ardiac glycosides - digoxin – which lowers extrusion of Ca ++ from cardiac muscle -> the intracellular concentration of Ca ++ is increased -> force of cardiac muscle contraction is increased proton pump - H + /K + ATPase, „ pumps“ H + from the cell in the stomach mucosa – increased production of HCl, inhibited by, Proton pump inhibitors omeprazol used in peptic ulcer „Pumps“

ION CHANNELS VOLTAGE-GATED CHANNELS LIGAND-GATED CHANNELS Extracellular ligands Calcium channels Sodium channels GABA-gated Cl- channels Nicotinic receptor NMDA receptor Intracellular ligands ATP-sensitive potassium channels CALCIUM CHANNEL BLOCKERS LOCAL ANAESTHETICS Summary : ANXIOLYTICS - Benzodiazepines NEUROMUSCULAR-BLOCKING DRUGS INTRAVENOUS ANAESTHETIC - ketamine ANTIDIABETICS -sulfonylureas

Transporters for noradrenaline, serotonine, dopamine inhibited by most Antidepressants – Reuptake inhibitors (RUI), TCA, SSRI etc) Transport proteins

NERVE ENDING (presynaptic) SYNAPTIC CLEFT POSTSYNAPTIC NEURON ↓ ELIMINATION by MAO moklobemid ↓ REUPTAKE imipramin Almost all antidepressants increase supply of monoamine transmitters at postsynaptic receptors

P-glycoprotein It is an efflux pump capable of transporting a wide range of compounds from the intracellular space into the extracellular matrix. Intestinal P-glycoprotein reduces effective drug absorption by actively transporting drugs back into the intestinal lumen. P- glycoprotein in the liver and kidneys promotes excretion of drugs from the blood stream into the bile and urine, respectively. In addition, P-glycoprotein is present at the blood– brain barrier, where it reduces drug access to the CNS. P-glycoprotein can be induced and inhibited by other drugs Transport proteins

Inhibition of P-glycoprotein [and CYP3A4] Grapefruit juice inhibits P-glycoprotein [and CYP3A4] GRAPEFRUIT-DRUG INTERACTIONS The P-gp and CYP3A4 are located in the enterocytes ( intestinal absorptive cells) → first-pass effect Grapefruit juice by inhibition of P-glycoprotein [and CYP3A4] can markedly increase the bioavailability and toxicity of some drugs, particularly (most hazardous) in: amiodarone (arrythmias) simvastatin, lovastatin (rhabdomyolysis)

TRANSPORTERS „Pumps“ Transport proteins transporters for noradrenaline (NA), serotonin(5-HT), dopamine (DI) P-glycoprotein (P-gp) sodium pump proton pump CARDIAC GLYCOSIDES -digoxin PROTON PUMP INHIBITORS - omeprazol ANTIDEPRESSANTS- Reuptake Inhibitors GRAPEFRUIT-DRUG INTERACTIONS Summary :

FOUR MAJOR TARGETS FOR DRUGS: 1. RECEPTORS 2. ION CHANNELS 3. CARRIER MOLECULES 4. ENZYMES Molecular Targets For Drug Action

Other drug-enzymes interactions Enzyme inhibition by drugs

EnzymesInhibitors Therapeutic groups, indications Cyclo-oxygenaseaspirin, ibuprofen, diclofenac Antiinflammatory and antirheumatic agents, analgesics Monoamine oxidasemoclobemide Antidepressants Acetylcholinesteraseneostigmine, rivastigmin Parasympathomimetics, Anti-dementia- drugs Angiotensin-converting enzyme enalapril, ramipril Antihypertensives HMG-CoA reductasesimvastatin, atorvastatin Lipid modifying agents; (hypercholesterolaemia) XanthinoxidaseallopurinolDrugs inhibiting uric acid production Phosphodiesterase type VsildenafilDrugs used in erectile dysfunction Dihydrofolate reductase trimethoprim Antimicrobial agents methotrexateAntimetabolites, folic acid analogues NeuroamidaseoseltamivirAntivirals ( influenza virus) Thymidine kinaseaciclovirAntivirals (Herpes virus) HIV proteasesaquinavirAntivirals (HIV), protease inhibitors Many drugs are targeted on enzymes and mostly act by inhibiting them:

Drugs can inhibit enzymes reversibly (usually a competitive inhibition by non-covalent binding) or irreversibly (enzyme is usually changed chemically by covalent binding) An enzyme inhibitor is a molecule which binds to enzymes and decreases their activity Irreversible inhibitors usually react with the enzyme and change it chemically (e.g. via covalent bond formation). These inhibitors modify key amino acid residues needed for enzymatic activity (e.g. aspirin, acting on cyclo-oxygenase) Competitive inhibition is a form of enzyme inhibition where binding of the inhibitor to the active site on the enzyme prevents binding of the substrate and vice versa. Often, the drug molecule is a substrate analogue (e.g. captopril, acting on angiotensin-converting enzyme)

The active site of angiotensin-converting enzyme. [A] Binding of angiotensin I. [B] Binding of the inhibitor captopril, which is an analogue of the terminal dipeptide of angiotensin I. Downloaded from: StudentConsult (on 6 November :30 PM) © 2005 Elsevier Reversible competitive inhibition of enzyme (inhibition of ACE by captopril)::

Irreversible non-competitive inhibition of enzyme (inhibition of COX-1 or COX-2 by aspirin): This makes aspirin different from other NSAIDs (such as diclofenac and ibuprofen, which are reversible inhibitors). Aspirin acetylates serine residue in the active site of the COX enzyme

Irreversible inhibition of enzyme: Recovery is possible only by synthesis of a new enzyme

Those of importance in the metabolism of psychotropic drugs are CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4, the last being responsible for the metabolism of more than 90% of psychotropic drugs that undergo hepatic biotransformation. Cytochrome P450 (CYP) enzymes Many psychotropic drugs have a high affinity for one particular CYP enzyme but most are oxidised by more than one Drug - cytochrome P450 interactions The most important enzymes involved in drug interactions are members of the cytochrome P450 (CYP) system that are responsible for many of the phase 1 biotransformations of drugs. These metabolic transformations, such as oxidation, reduction and hydrolysis, produce a molecule that is suitable for conjugation.

Genetic polymorphism The CYP enzymes that demonstrate pharmacogenetic polymorphism include CYP2C9, CYP2C19 and CYP2D6. In clinical practice, the polymorphism produces distinct phenotypes, described as poor metabolisers, extensive metabolisers (the most common type) and ultra-rapid metabolisers. Genetic effects:

CYP enzymes can be induced or inhibited by drugs or other biological substances, with a consequent change in their ability to metabolise drugs that are normally substrates for those enzymes. Drug effects:

Enzymatic induction enzymatic induction can cause a decrease as well as an increase in the drug’s effect The onset and offset of enzyme induction take place gradually, usually over 7–10 days The most important are inducers of CYP3A4 and include carbamazepine, phenobarbital, phenytoin, rifampicin and St John’s wort (Hypericum perforatum). An example of an interaction in psychiatric practice is the reduced efficacy of haloperidol (or alprazolam) when carbamazepine is started, resulting from induction of CYP3A4.

Enzymatic inhibition enzymatic inhibition can cause an increase as well as a decrease in the drug’s effect Most hazardous drug interactions involve inhibition of enzyme systems, which increases plasma concentrations of the drugs involved, in turn leading to an increased risk of toxic effects. Inhibition of CYP enzymes is the most common mechanism that produces serious and potentially life-threatening drug interactions Inhibition is usually due to a competitive action at the enzyme’s binding site. Therefore, in contrast to enzyme induction, the onset and offset of inhibition are dependent on the plasma level of the inhibiting drug

4. ENZYMES sites of action of about 30% of drugs degradating cGMP stops the virus from chemically cutting ties with its host cell

G-protein coupled receptors membr. Voltage gated - Calcium chan. - Sodium chan. „pumps“ - sodium - proton transporters cardiac glykosides PP inhibitors lok. anaesthetetics Calcium ch. blockers about 45% of drugs,e.g. beta-blockers antidepressants ACE inhibitors, IMAO perif. muscle relaxants Examples of drugs:: Proteinkinase-linked receptors c. intracelul. Ligand-gated, G-prot.,… ACE, MAO, COX, HMG-CoA reductase Channel-linked receptors 1. RECEPTORS 2. ION CHANNELS 3. CARRIER MOLECULES Molecular mechanisms of drug effects - summary FOUR MAJOR TARGETS FOR DRUGS: 4. ENZYMES imatinib