Lecture 18-19 Drug Targets For Class PDF

Principles of Drug Action Drug Targets Enzymes and Receptors Medicinal Chemistry of Enzyme Inhibitors Lectures 18-19  Medicinal Chemistry Drug Physicochemical...

Principles of Drug Action Drug Targets Enzymes and Receptors Medicinal Chemistry of Enzyme Inhibitors Lectures 18-19  Medicinal Chemistry Drug Physicochemical Properties of Drugs-1 (Drug Target) Functional Group (FG) Acidity and Basicity of FG Enzymes Receptors Physicochemical Properties of Drugs-2 and 3 - Chirality - Salt Solubility  LEARNING OBJECTIVES Define enzyme and discuss the importance of enzyme as a drug target. Explain with examples of Reversible, Irreversible, and Transition- State Inhibitors. What are Allosteric Inhibitors and how do they bind to a site different than the active site? How to calculate IC50 from the enzyme inhibition curve?  Drug Targets Lipids Cell membrane lipids G-Protein Proteins Receptors 45% Hormones & (agonists, antagonists) Receptors Factors 11% Enzymes Enzymes (analogs of natural ligands) Transport proteins (agonist, antagonist) 28% (Inhibitors Structural proteins (tubulin) Reversible Irreversible) Unknown 7% Nuclear Nucleic acids receptors DNA Ion channels 5% DNA 2% (analogs of natural ligands) 2% antimetabolites (channel blockers) RNA (substrate analogs) Carbohydrates Cell surface carbohydrates Antigens and recognition molecules Drug targets Binding regions Drug Binding groups Intermolecular bonds Binding site Binding Drug site Drug Induced fit Macromolecular target Macromolecular target Unbound drug Bound drug  1. Receptor Specialized target macromolecule that binds a drug and mediates its pharmacological action Enzymes, nucleic acids, or specialized membrane-bound proteins Contain functional groups, which interact with complementary functional groups of the drug DRUG + RECEPTOR DRUG-RECEPTOR COMPLEX BIOLOGICAL RESPONSE  1. Receptor Receptor Messenger Induced fit Messenger Messenger Cell Membrane Receptor Receptor Receptor Cell Cell Cell Message Most receptors are located in the cell membrane Receptors are activated by chemical messengers (neurotransmitters or hormones). Binding and induced fit result in the “Domino” effect. (Also known as Signal Transduction) Chemical messenger does not enter the cell. It departs the receptor unchanged and is not permanently bound.  Definitions Receptor (also known as Target) – Is a macromolecule – Binds a molecule AND mediates some pharmacological action – (most general usage, in this case refers to all drug targets) Many, but not all, Receptors/targets are proteins Enzyme – Is a protein – Binds a ligand (usually called the substrate) AND – Catalyzes chemical reaction on that ligand to produce a product  More protein receptor types G-Protein receptor (often called simply receptor—don’t get confused!) – Is a protein – Binds a ligand (usually called agonist) AND – Transfers that signal across a membrane into the cell Nuclear receptor – Is a protein – Binds a ligand in the cytoplasm AND – Transfers a signal from the cytoplasm to the nucleus of the cell Ion channel (and other transport proteins) – Is a protein – Transports a molecule across/through a membrane cell Interaction with G-Protein-coupled receptor Largest and most diverse protein families in nature https://blog.addgene.org/gpcrs-how-do-they-work-and-how-do-we-study-them Nuclear Receptors Ion Channels https://www.khanacademy.org/science/ap-biology/cell-communication-and-cell-cycle/signal-transduction/a/signal-perception https://study.com/skill/practice/analyzing-the-interaction-of-ligands-ligand-gated-channels-questions.html  Non-protein receptor types Nucleic acid (DNA, RNA) – Is NOT a protein (rather, a nucleic acid polymer) – That normally is used as the template for replication and transcription Lipids (phospho-lipid membranes) – Is NOT a protein (rather, lipid) – That is responsible for the stability of the cell membrane Carbohydrates (glycoproteins, glycolipids) – Responsible for cell-cell (or protein-cell) recognition  2. Enzymes 1. Structure and function of Enzymes Special types of receptors Mostly soluble and found in the cytosol of cells Interact with substrates to form complexes, but, unlike other receptors, enzymes catalyze reactions, thereby forming the substrates into products that are released. Enzymatic reactions 1. Substrate approaches active site 2. Enzyme-substrate complex forms 3. Substrate transformed into products 4. Products released 5. Enzyme recycled  Enzyme mechanism Sucrase Example 1: glucose Sucrose + fructose  2. Enzymes Example 2: Protein Tyrosine Kinase (Phosphorylation!) Phenolic OH group Protein Tyrosine Kinase  2. Enzyme Properties Activation energy: Amount of energy needed to disrupt stable molecule so that reaction can take place Energy Energy Transition state New transition state Act. Act. energy energy Starting Starting ∆G material ∆G material Product Product WITHOUT ENZYME WITH ENZYME ∆G = -RT ln K Equilibrium constant = K = [Product]/[Reactant] Enzymes lower the activation energy of a reaction, but ∆G remains the same  2. Enzyme Methods of Enzyme Catalysis Properties Provides a reaction surface (the active site) Provides a suitable environment (hydrophobic) Brings reactants together Positions reactants correctly for reaction Weakens bonds in the reactants Stabilizes the transition state with intermolecular bonds Provides acid/base catalysis or Provides nucleophilic groups  2. Enzymes 2. The Active Site Active Site Hydrophobic hollow or cleft on the enzyme surface Accepts reactants (substrates and cofactors) What is the role of AAs at the binding site? Contains amino acids which - bind reactants (substrates and cofactors) - participate in the enzyme-catalysed reaction Active site Active site ENZYME 3. Substrate Binding 3. Substrate Binding Fischer’s Lock and Key Hypothesis: Both enzyme and substrate were seen as rigid structures, with the substrate (the key) fitting perfectly into the active sites (the lock). 3. Substrate Binding 3. Substrate Binding Koshland’s Theory of induced fit: It is proposed that the substrate is not quite the ideal shape for the active site, and that it forces the active site to change shape when it enters a kind of modulating Process. Substrate induces the active site to take up the ideal shape to accommodate it.  3. Substrate Binding 3. Substrate Binding 3.1 Induced fit 3.1 Induced fit Substrate S Induced fit Active site is nearly the correct shape for the substrate Binding alters the shape of the enzyme (induced fit) The binding process can strain bonds in the substrate Binding involves intermolecular bonds between functional groups in the substrate and functional groups in the active site  3. Substrate Binding 3. Substrate Binding 3.2 Binding forces 3.2. Bonding forces Ionic H-bonding van der Waals vdw interaction S H-bond Active site H ionic Phe Ser O bond CO2 Asp Enzyme  3. Substrate Binding 3.2 Binding forces Induced fit - Active site alters shape to maximize intermolecular bonding S S H Ser O Phe H Phe Ser O CO2 CO2 Induced Asp fit Asp Intermolecular bonds Intermolecular bond lengths not optimum length for optimized. Susceptible bonds maximum bonding in substrate strained. Susceptible bonds in substrate more easily broken.  3. Substrate Binding 3.2 Binding forces Example 3 - Binding of pyruvic acid in lactate dehydrogenase (LDH) O HO H O LDH O C C NADH + H3 C C H3 C C + NAD+ OH OH Pyruvic acid Lactic acid O O H-Bond H C O H3C C O C O O H3C C + H3N O Possible interactions vdw-interactions Ionic bond H-Bond van der Waals Ionic Example 4 - Binding of pyruvic acid in LDH 3. Substrate Binding 3.2 Binding forces O H O C O H3C C + H3N O  3. Substrate Binding 3.2 Binding forces Induced fit O p bond H weakened O C O H3C C + H3N O 4. Mechanism of Enzyme Catalysis There are a variety of mechanisms that the enzyme can utilize to catalyze the conversion of the substrate to product. The most common mechanisms are: – A. Nucleophile (Covalent) Catalysis – B. General Acid-Base Catalysis  Enzymes 4. Catalysis mechanisms 4. Catalysis 4.1. Nucleophilic residues H H H3N CO2 H3N CO2 L-Serine L-Cysteine OH SH 4.2. Acid/base catalysis Histidine +H+ NH NH -H+ N N H Non-ionized Ionized Acts as a basic catalyst Acts as an acid catalyst (proton 'sink') (proton source)  Enzymes 4.1. Nucleophilic (covalent) catalysis 4. Catalysis Serine acting as a nucleophile Substrate X Product HO H2O O OH OH Ser Ser Ser Some enzymes can use nucleophilic amino acid side-chains (X) in the active site to form covalent bonds to the substrate. In some cases, a second can react with this enzyme-substrate intermediate to generate the product.  4.2. Acid-Base Catalysis An enzyme can utilize acid and base catalysis simultaneously. The protonated base (BH+) is an acidic amino acid side chain and the free base (B:) is a basic residue Example: Chymotrypsin  Simultaneous acid/base & nucleophilic catalysis Histidine acting as base, Serine (O-) acting as (somewhat stronger) nucleophile NH + HN NH H2O N HO O OH OH Ser Ser Ser  Enzymes 4. Catalysis Example 5: Mechanism for chymotrypsin Catalytic triad of Serine, Histidine, and Aspartate :N H.. N :O H O O Ser His Asp Chymotrypsin  Enzymes 4. Catalysis Example 5: Mechanism for chymotrypsin (Continued) : O: C Protein NH Protein :N H.. N :O H O O Ser His Asp Chymotrypsin Example 5: Mechanism for chymotrypsin (Continued) :O: : Protein C NH Protein :N H N :O H O O Ser His Asp Chymotrypsin Example 5: Mechanism for chymotrypsin (Continued) (Watch arrows and charges) :O: : Protein C NH Protein + H H N N :O: O O Ser His Asp Chymotrypsin Example 5: Mechanism for chymotrypsin (Continued) : H H : O : Protein O : C :N H N :O :: O O Ser His Asp Chymotrypsin Example 5: Mechanism for chymotrypsin (Continued) :O: : H Protein O C H :N H N :O : O O Ser His Asp Chymotrypsin Example 5: Mechanism for chymotrypsin (Continued) :O: : H Protein O: : C H H N N :O : : O O Ser His Asp Chymotrypsin Example 5: Mechanism for chymotrypsin (Continued) :O: C Protein OH :N H.. N :O H O O Ser His Asp Chymotrypsin  Enzymes 4. Catalysis Example 5: Mechanism for chymotrypsin (Summary) :O: C protein NH protein.. :N N H :O H O O Ser His Asp.... :O : :O : H H C C protein O O : protein NH protein protein NH protein : : C : H :O :N N H :O : N N H :O : :N N H H O O O O O O Ser His Asp Ser His Asp Ser His Asp.. H.. protein OH :O: : O :.. C protein O.. H protein OH.. O C C H.. :O : :N N H :O : N N H : OH :N N H O O O O O O Ser His Asp Ser His Asp Ser His Asp  Cofactors Non-protein substances required for reaction – Red-Ox reactions: NAD/NADH, NADP/NADPH, Heme – Metal ion: Zn2+, Ca2+ – Donors: ATP donates –PO3- NH2 N N Already in high-energy state N HO HO HO N - O O O O P P P O O O O H H ATP HO OH  Enzymes 4. Catalysis Overall Process of Enzyme Catalysis (Summary) S P S P EE E E E E+S ES EP E+P Binding interactions must be strong enough to hold the substrate sufficiently long for the reaction to occur Interactions must be weak enough to allow the product to depart Interactions stabilize the transition state Designing molecules with stronger binding interactions results in enzyme inhibitors that block the active site  Medicinal Chemistry Drug Physicochemical Properties of Drugs-1 (Drug Target) Functional Group (FG) Acidity and Basicity of FG Enzymes Receptors Physicochemical Properties of Drugs-2 and 3 - Chirality - Salt Solubility  Enzymes 5. Why inhibit enzymes? 5. Inhibition Many diseases arise from a deficiency or excess of a specific metabolite in the body, from an infestation of a foreign organism, or from aberrant cell growth. The situation can be normalized by inhibiting a specific enzyme Any compound that slows down or blocks enzyme catalysis is an enzyme inhibitor Pharmacist Alert Enzyme targets for useful medications Antibacterial agents Dihydropteroate synthetase, transpeptidase Antiviral agents HIV reverse transcriptase, HIV protease, viral DNA polymerase Anti-inflammatory agents Cyclooxygenase Cholesterol lowering agents HMG-CoA reductase Antidepressants Monoamine oxidase Anticancer agents Tyrosine kinase, dihydrofolate reductase, thymidylate synthase, aromatase, etc Pharmacist Alert More Enzyme targets for useful medications Antihypertensive agents Renin, angiotensin converting enzyme Treatment of male erectile dysfunction Phosphodiesterase Anti-gout agents Xanthine oxidase Alzheimers disease Cholinesterases Diuretics Carbonic anhydrase  Enzymes 6. E Inhibitors 6. Enzyme Inhibitors (EIs) A. Reversible Enzyme Inhibitors B. Irreversible Enzyme Inhibitors C. Allosteric Inhibitors D. Transition State Analogs  6. EIs 6A. Reversible EIs 6A. Reversible Enzyme Inhibitors Any compound that slows down or blocks enzyme catalysis Inhibitor binds reversibly to the active site Enzyme is not available for catalysis when the inhibitor is bound Substrate is blocked from the active site Increasing substrate concentration reverses inhibition Inhibitor likely to be similar in structure to substrate, product, or cofactor S I I EE E  6. EIs 6A. Reversible EIs Competitive and Non-competitive inhibition Competitive Non-Competitive Inhibition Inhibition Sulfonamides Diuretics Kinase inhibitors  6. EIs Pharmacist Alert 6A. Reversible EIs Example: Reversible EI: Sulfa Drugs Pterin  Sulfa drugs Prontosil was converted by reduction to the active antibacterial agent, namely, p-aminobenzenesulfonamide (also called sulfanilamide). Sufanilamide inhibits folic acid biosynthesis by inhibiting dihyropteroate synthetase that catalyzes the synthesis of dihydrofolate from diphosphate and p-aminobenzoic acid. Because of the structural similarity of sulfanilamide to P- aminobenzoic acid, it is a potent competitive inhibitor of dihyropteroate synthetase. Coadministration of PABA and sulfanilamide prevents the antibacterial action of the drug (reversible inhibitor)  6. EIs B. Irreversible Inhibitors 6B. Irreversible EIs X Covalent Bond X OH OH O Irreversible inhibition Inhibitor binds irreversibly to the active site A covalent bond formed between the drug and the enzyme Substrate is blocked from the active site Increasing substrate concentration does not reverse the inhibition Inhibitor likely to be similar in structure to the substrate  6. EIs Pharmacist Alert 6C. Irreversible EIs Examples – Nerve gases, Penicillins, Disulfuram, Cephalosporins, Proton Pump Inhibitor, Orlistat Orlistat C6H13 C11H23 O O O C11H23 O O C6H13 NHCHO C11H23 O But NHCHO O C6H13 But O O OH O H O O O O But NHCHO Ser Ser Ser Pancreatic lipase Pancreatic lipase Pancreatic lipase Orlistat is an anti-obesity drug that inhibits pancreatic lipase The enzyme is blocked from digesting fats in the intestine Fatty acids and glycerol are less absorbed as a result Leads to reduced biosynthesis of fat in the body  6. EIs 6C Allosteric Inhibitors C. Allosteric Inhibitors Active site Active site unrecognisable Induced ACTIVE SITE fit (open) Enzyme ENZYME (open) Enzyme ENZYME Allosteric binding site Allosteric inhibitor Inhibitor binds reversibly to the allosteric site Intermolecular bonds are formed Induced fit alters the shape of the enzyme Active site is distorted and is not recognized by the substrate Increasing substrate concentration does not reverse the inhibition Inhibitor is not similar in structure to the substrate  6. EIs D. Transition-state Inhibitors 6D. Transition-state Is Drugs designed to mimic the transition state of an enzyme- catalyzed reaction Transition-state inhibitors are likely to bind more strongly than drugs mimicking the substrate or product Transition states are high energy, transient species and cannot be isolated or synthesized Drug design can be based on reaction intermediates which are closer in character to transition states than substrates or products Design a drug that mimics the stereochemistry and binding properties of the reaction intermediate, but is stable  6. EIs 6D. Transition-state Is Example 6: Renin inhibitors Inhibitor Angiotensin converting Renin enzyme (ACE) Angiotensinogen Angiotensin I Angiotensin II Renin inhibitors block synthesis of angiotensin I and II Angiotensin II constricts blood vessels and raises blood pressure Renin inhibitors act as antihypertensives (lower blood pressure)  6. EIs 6D. Transition-state Is Example 6: Renin inhibitors Reaction mechanism Tetrahedral intermediate R1 R1 R1 H Substrate H + N N H2N CO2H O O R2 R2 O R2 H H O H H H H H O O O O O O O O O O O O Asp Asp Asp Asp Asp Asp Renin Renin Renin Renin Two aspartyl residues involved in enzyme-catalyzed reaction Tetrahedral intermediate involved  6. EIs 6B. Transition-state Inhibitors Example 6: Renin inhibitors: Aliskiren MeO CHMe2 Protein H N MeO O O O Protein HO OH H2N N NH2 Reaction H Me Me intermediate OH CHMe2 Hydroxyethylene transition-state mimic Aliskiren contains a hydroxyethylene transition-state mimic Mimics the tetrahedral geometry of the reaction intermediate Mimics one of the hydroxyl groups (binding group) Stable - no leaving group present Other Transition-state inhibitors (Statins, Protease Inhibitors, ACE inhibitors)  7. IC50 7. IC50 IC50 = concentration of inhibitor required to reduce enzyme activity by 50%. IC50 is only measured at a single substrate concentration The IC50 is approximately 100 nM EC50 value The concentration of inhibitor required to reduce a particular cellular effect by 50% Uses Whole cell or whole tissue (cellular effect) Includes transport and various other in vivo parameters  Key Points Basic properties of enzymes, e.g. activation energy, enzyme theory, acid-base, covalent catalysis, IC50, an example of EIs. Competitive inhibitors bind to the active site and compete with either the substrate or cofactor Reversible inhibitors bind through intermolecular forces only Irreversible inhibitors make a covalent bond with enzymes (suicide!) Allosteric inhibitors bind to a site different than the active site. Alter the shape of the enzyme such that catalysis does not occur Transition State Analogs: Transition state analogs are molecules designed to mimic the high-energy transition state of a reaction. The activity of different enzyme inhibitors can be compared by measuring IC50.