why & wherefore drug targets
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why & wherefore drug targets Presentation Transcript

  • 1. Forest Research Institute The Why & Wherefore: Drug Targets Nhut Diep Senior Medicinal Research Scientist
  • 2. 1. Cell Structure 2. Cell Membrane 3. Drug Targets 4. Intermolecular Bonding Forces a. Electrostatic or ionic bond b. Hydrogen bonds c. Van der Waals interactions d. Dipole-dipole/Ion-dipole/Induced dipole interactions 5. Desolvation penalties 6. Hydrophobic interactions 7. Drug Targets - Cell Membrane Lipids 8. Drug Targets – Carbohydrates PROTEINS AS DRUG TARGETS: RECEPTORS THE WHY & THE WHEREFORE: DRUG TARGETS Contents
    • Structure and function of receptors 6. Competitive (reversible) antagonists
    • a. Chemical Messengers 7. Non competitive (irreversible) antagonists
    • b. Mechanism 8. Non competitive (reversible) allosteric antagonists
    • The binding site 9. Antagonists by umbrella effect
    • Messenger binding 10. Agonists
    • a. Introduction
    • b. Bonding forces
    • Overall process of receptor/messenger interaction
    • Signal transduction
    • a. Control of ion channels
    • b. Activation of signal proteins
    • c. Activation of enzyme active site
  • 3. PROTEINS AS DRUG TARGETS: Enzymes 1 . Structure and function of enzymes 2. The active site 3. Substrate binding a. Induced fit b. Bonding forces 4. Catalysis mechanisms a. Acid/base catalysis b. Nucleophilic residues 5. Overall process of enzyme catalysis 6. Competive (reversible) inhibitors 7. Non competitive (irreversible) inhibitors 8. Non competitive (reversible) allosteric inhibitors
  • 4. DRUG TARGETS
  • 5. 1. Cell Structure Human, animal and plant cells are eukaryotic cells The nucleus contains the genetic blueprint for life (DNA) The fluid contents of the cell are known as the cytoplasm Structures within the cell are known as organelles Mitochondria are the source of energy production Ribosomes are the cell’s protein ‘factories’ Rough endoplasmic reticulum is the location for protein synthesis
  • 6. 2. Cell Membrane Phospholipid Bilayer Exterior High [Na + ] Interior High [K + ] Proteins
  • 7. 2. Cell Membrane Polar Head Group Hydrophobic Tails C H C H 2 C H 2 O O O P O O O C H 2 C H 2 N M e 3 O O
  • 8. 2. Cell Membrane Polar Head Group Hydrophobic Tails C H C H 2 C H 2 O O O P O O O C H 2 C H 2 N M e 3 O O
  • 9. 2. Cell Membrane The cell membrane is made up of a phospholipid bilayer The hydrophobic tails interact with each other by van der Waals interactions and are hidden from the aqueous media The polar head groups interact with water at the inner and outer surfaces of the membrane The cell membrane provides a hydrophobic barrier around the cell, preventing the passage of water and polar molecules Proteins are present, floating in the cell membrane Some act as ion channels and carrier proteins
  • 10. Lipids Cell membrane lipids Proteins Receptors Enzymes Carrier proteins Structural proteins (tubulin) Nucleic acids DNA RNA Carbohydrates Cell surface carbohydrates Antigens and recognition molecules 3. Drug targets
  • 11. 3. Drug targets Drug targets are large molecules - macromolecules Drugs are generally much smaller than their targets Drugs interact with their targets by binding to binding sites Binding sites are typically hydrophobic pockets on the surface of macromolecules Binding interactions typically involve intermolecular bonds Most drugs are in equilibrium between being bound and unbound to their target Functional groups on the drug are involved in binding interactions and are called binding groups Specific regions within the binding site that are involved in binding interactions are called binding regions
  • 12. Unbound drug 3. Drug targets Macromolecular target Drug Macromolecular target Drug Bound drug Binding site Drug Binding site Binding regions Binding groups Intermolecular bonds
  • 13. 3. Drug targets Binding interactions usually result in an induced fit where the binding site changes shape to accommodate the drug The induced fit may also alter the overall shape of the drug target Important to the pharmacological effect of the drug
  • 14. 4. Intermolecular bonding forces
    • 4.1 Electrostatic or ionic bond
    • Strongest of the intermolecular bonds (20-40 kJ mol -1 )
    • Takes place between groups of opposite charge
    • The strength of the ionic interaction is inversely proportional to the distance between the two charged groups
    • Stronger interactions occur in hydrophobic environments
    • The strength of interaction drops off less rapidly with distance than with other forms of intermolecular interactions
    • Ionic bonds are the most important initial interactions as a drug enters the binding site
  • 15. 4. Intermolecular bonding forces 4.2 Hydrogen bonds
    • Vary in strength
    • Weaker than electrostatic interactions but stronger than van der Waals interactions
    • A hydrogen bond takes place between an electron deficient hydrogen and an electron rich heteroatom (N or O)
    • The electron deficient hydrogen is usually attached to a heteroatom (O or N)
    • The electron deficient hydrogen is called a hydrogen bond donor
    • The electron rich heteroatom is called a hydrogen bond acceptor
  • 16. 4. Intermolecular bonding forces 4.2 Hydrogen bonds
    • The interaction involves orbitals and is directional
    • Optimum orientation is where the X-H bond points directly to the lone pair on Y such that the angle between X, H and Y is 180 o
    HBA HBD
  • 17. 4. Intermolecular bonding forces 4.2 Hydrogen bonds
    • Examples of strong hydrogen bond acceptors
      • - carboxylate ion, phosphate ion, tertiary amine
    • Examples of moderate hydrogen bond acceptors
      • - carboxylic acid, amide oxygen, ketone, ester, ether, alcohol
    • Examples of poor hydrogen bond acceptors
      • - sulfur, fluorine, chlorine, aromatic ring, amide nitrogen, aromatic amine
    • Example of good hydrogen bond donors
      • - Quaternary ammonium ion
  • 18. 4. Intermolecular bonding forces 4.3 Van der Waals interactions DRUG
    • Very weak interactions (2-4 kJmol -1 )
    • Occur between hydrophobic regions of the drug and the target
    • Due to transient areas of high and low electron densities leading to temporary dipoles
    • Interactions drop off rapidly with distance
    • Drug must be close to the binding region for interactions to occur
    • The overall contribution of van der Waals interactions can be crucial to binding
    Binding site  -  +  +  - Hydrophobic regions Transient dipole on drug  +  - van der Waals interaction
  • 19. 4. Intermolecular bonding forces 4.4 Dipole-dipole interactions
    • Can occur if the drug and the binding site have dipole moments
    • Dipoles align with each other as the drug enters the binding site
    • Dipole alignment orientates the molecule in the binding site
    • Orientation is beneficial if other binding groups are positioned correctly with respect to the corresponding binding regions
    • Orientation is detrimental if the binding groups are not positioned correctly with respect to corresponding binding regions
    • The strength of the interaction decreases with distance more quickly than with electrostatic interactions, but less quickly than with van der Waals interactions
  • 20. 4. Intermolecular bonding forces
    • 4.4 Ion-dipole interactions
    • Occur where the charge on one molecule interacts with the dipole moment of another
    • Stronger than a dipole-dipole interaction
    • Strength of interaction falls off less rapidly with distance than for a dipole-dipole interaction
    Binding site   R C R O Binding site   R C R O
  • 21. 4. Intermolecular bonding forces
    • 4.4 Induced dipole interactions
    • Occur where the charge on one molecule induces a dipole on another
    • Occurs between a quaternary ammonium ion and an aromatic ring
    Binding site R N R 3   
  • 22. Desolvation - Energy penalty Binding - Energy gain 5. Desolvation penalties
    • Polar regions of a drug and its target are solvated prior to interaction
    • Desolvation is necessary and requires energy
    • The energy gained by drug-target interactions must be greater than the energy required for desolvation
    R C R O O H H H H O H H O H H O O H Binding site O H R C R O Binding site R C R O O H Binding site
  • 23. Unstructured water Increase in entropy Structured water layer round hydrophobic regions 6. Hydrophobic interactions
    • Hydrophobic regions of a drug and its target are not solvated
    • Water molecules interact with each other and form an ordered layer next to hydrophobic regions - negative entropy
    • Interactions between the hydrophobic interactions of a drug and its target ‘free up’ the ordered water molecules
    • Results in an increase in entropy
    • Beneficial to binding energy
    Drug DRUG Hydrophobic regions Water Binding site Binding site Drug DRUG Binding
  • 24. Drugs acting on cell membrane lipids - Anaesthetics and some antibiotics Action of amphotericin B (antifungal agent) - builds tunnels through membrane and drains cell 7. Drug Targets - Cell Membrane Lipids Hydrophobic region Hydrophilic Hydrophilic Hydrophilic
  • 25. Polar tunnel formed Escape route for ions 7. Drug Targets - Cell Membrane Lipids
  • 26. 8. Drug Targets - Carbohydrates
    • Carbohydrates play important roles in cell recognition, regulation and growth
    • Potential targets for the treatment of bacterial and viral infection, cancer and autoimmune disease
    • Carbohydrates act as antigens
    Cell membrane
  • 27. 7. Drug Targets - Carbohydrates
  • 28. PROTEINS AS DRUG TARGETS: RECEPTORS
  • 29. 1. Structure and function of receptors
    • Globular proteins acting as a cell’s ‘letter boxes’
    • Located mostly in the cell membrane
    • Receive messages from chemical messengers coming from other cells
    • Transmit a message into the cell leading to a cellular effect
    • Different receptors specific for different chemical messengers
    • Each cell has a range of receptors in the cell membrane making it responsive to different chemical messengers
  • 30. 1. Structure and function of receptors Cell Nerve Messenger Signal Receptor Nerve Nucleus Cell Response
  • 31. Chemical Messengers Neurotransmitters : Chemicals released from nerve endings which travel across a nerve synapse to bind with receptors on target cells, such as muscle cells or another nerve. Usually short lived and responsible for messages between individual cells Hormones : Chemicals released from cells or glands and which travel some distance to bind with receptors on target cells throughout the body
    • Chemical messengers ‘switch on’ receptors without undergoing a reaction
    1. Structure and function of receptors
  • 32. Nerve 1 Nerve 2 Blood supply Neurotransmitters 1. Structure and function of receptors Hormone
  • 33. Mechanism
    • Receptors contain a binding site (hollow or cleft in the receptor surface) that is recognised by the chemical messenger
    • Binding of the messenger involves intermolecular bonds
    • Binding results in an induced fit of the receptor protein
    • Change in receptor shape results in a ‘domino’ effect
    • Domino effect is known as Signal Transduction, leading to a chemical signal being received inside the cell
    • Chemical messenger does not enter the cell. It departs the receptor unchanged and is not permanently bound
    1. Structure and function of receptors
  • 34. Mechanism Cell Receptor 1. Structure and function of receptors Cell Membrane Messenger message Induced fit Cell Receptor Messenger Message Cell Messenger Receptor
  • 35. 2. The binding site
    • A hydrophobic hollow or cleft on the receptor surface - equivalent to the active site of an enzyme
    • Accepts and binds a chemical messenger
    • Contains amino acids which bind the messenger
    • No reaction or catalysis takes place
    ENZYME Binding site Binding site
  • 36. 3. Messenger binding
    • Binding site is nearly the correct shape for the messenger
    • Binding alters the shape of the receptor (induced fit)
    • Altered receptor shape leads to further effects - signal transduction
    3.1 Introduction Messenger Induced fit M
  • 37.
    • Ionic
    • H-bonding
    • van der Waals
    3.2 Bonding forces Example: 3. Messenger binding Receptor Binding site vdw interaction ionic bond H-bond Phe Ser O H Asp CO 2
  • 38. 3. Substrate binding
    • Induced fit - Binding site alters shape to maximise intermolecular bonding
    3.2 Bonding forces Intermolecular bonds not optimum length for maximum binding strength Intermolecular bond lengths optimised Phe Ser O H Asp CO 2 Induced Fit Phe Ser O H Asp CO 2
  • 39. 4. Overall process of receptor/messenger interaction
    • Binding interactions must be:
    • - strong enough to hold the messenger sufficiently long for signal
    • transduction to take place
    • - weak enough to allow the messenger to depart
    • Implies a fine balance
    • Drug design - designing molecules with stronger binding interactions results in drugs that block the binding site - antagonists
    M M E R R M E R Signal transduction
  • 40. 5. Signal transduction
    • 5.1 Control of ion channels
    • Receptor protein is part of an ion channel protein complex
    • Receptor binds a messenger leading to an induced fit
    • Ion channel is opened or closed
    • Ion channels are specific for specific ions (Na + , Ca 2+ , Cl - , K + )
    • Ions flow across cell membrane down concentration gradient
    • Polarises or depolarises nerve membranes
    • Activates or deactivates enzyme catalysed reactions within cell
  • 41. 5. Signal transduction 5.1 Control of ion channels Hydrophilic tunnel Cell membrane
  • 42. Five glycoprotein subunits traversing cell membrane Cationic ion channels for K + , Na + , Ca 2+ (e.g. nicotinic) = excitatory Anionic ion channels for Cl - (e.g. GABA A ) = inhibitory 5.1 Control of ion channels 5. Signal transduction Cell membrane Messenger Cell membrane Receptor Induced fit ‘ Gating’ (ion channel opens) Binding site
  • 43. 5.1 Control of ion channels: 5. Signal transduction Induced fit and opening of ion channel ION CHANNEL (open) Cell Cell membrane MESSENGER Ion channel Ion channel Cell membrane ION CHANNEL (closed) Cell RECEPTOR BINDING SITE Lock Gate Ion channel Ion channel Cell membrane Cell membrane MESSENGER
  • 44.
    • 5.2 Activation of signal proteins
    • Receptor binds a messenger leading to an induced fit
    • Opens a binding site for a signal protein (G-protein)
    • G-Protein binds, is destabilised then split
    5. Signal transduction messenger G-protein split induced fit closed open
  • 45.
    • 5.2 Activation of signal proteins
    • G-Protein subunit activates membrane bound enzyme
      • Binds to allosteric binding site
      • Induced fit results in opening of active site
    • Intracellular reaction catalysed
    active site (closed) active site (open) 5. Signal transduction Enzyme Intracellular reaction Enzyme
  • 46.
    • 5.3 Activation of enzyme active site
    • Protein serves dual role - receptor plus enzyme
    • Receptor binds messenger leading to an induced fit
    • Protein changes shape and opens active site
    • Reaction catalysed within cell
    active site open 5. Signal transduction closed messenger induced fit intracellular reaction closed messenger
  • 47. 6. Competitive (reversible) antagonists
    • Antagonist binds reversibly to the binding site
    • Intermolecular bonds involved in binding
    • Different induced fit means receptor is not activated
    • No reaction takes place on antagonist
    • Level of antagonism depends on strength of antagonist binding and concentration
    • Messenger is blocked from the binding site
    • Increasing the messenger concentration reverses antagonism
    An E R M An R
  • 48. 7. Non competitive (irreversible) antagonists
    • Antagonist binds irreversibly to the binding site
    • Different induced fit means that the receptor is not activated
    • Covalent bond is formed between the drug and the receptor
    • Messenger is blocked from the binding site
    • Increasing messenger concentration does not reverse antagonism
    X OH OH X O Covalent Bond Irreversible antagonism
  • 49. 8. Non competitive (reversible) allosteric antagonists
    • Antagonist binds reversibly to an allosteric site
    • Intermolecular bonds formed between antagonist and binding site
    • Induced fit alters the shape of the receptor
    • Binding site is distorted and is not recognised by the messenger
    • Increasing messenger concentration does not reverse antagonism
    ACTIVE SITE (open) ENZYME Receptor Allosteric site Binding site (open) ENZYME Receptor Induced fit Binding site unrecognisable Antagonist
  • 50. 9. Antagonists by umbrella effect
    • Antagonist binds reversibly to a neighbouring binding site
    • Intermolecular bonds formed between antagonist and binding site
    • Antagonist overlaps with the messenger binding site
    • Messenger is blocked from the binding site
    Antagonist Binding site for antagonist Binding site for messenger messenger Receptor Receptor
  • 51. 10. Agonists
    • Agonist binds reversibly to the binding site
    • Similar intermolecular bonds formed as to natural messenger
    • Induced fit alters the shape of the receptor in the same way as the normal messenger
    • Receptor is activated
    • Agonists are often similar in structure to the natural messenger
    E Agonist R E Agonist R Signal transduction Agonist R Induced fit
  • 52. PROTEINS AS DRUG TARGETS: ENZYMES
  • 53. 1. Structure and function of enzymes
    • Globular proteins acting as the body’s catalysts
    • Speed up time for reaction to reach equilibrium
    • Lower the activation energy of a reaction
    Example: LDH = Lactate dehydrogenase (enzyme) NADH 2 = Nicotinamide adenosine dinucleotide (reducing agent & cofactor) Pyruvic acid = Substrate
  • 54. Lowering the activation energy of reaction
    • Enzymes lower the activation energy of a reaction but  G remains the same
    1. Structure and function of enzymes Act. energy Transition state WITHOUT ENZYME Product Starting material Energy WITH ENZYME Product Starting material Energy ∆ G New transition state ∆ G Act. energy
  • 55. Methods of enzyme catalysis
    • Provide a reaction surface (the active site)
    • Provide a suitable environment (hydrophobic)
    • Bring reactants together
    • Position reactants correctly for reaction
    • Weaken bonds in the reactants
    • Provide acid / base catalysis
    • Provide nucleophiles
    1. Structure and function of enzymes
  • 56. 2. The active site
    • Hydrophobic hollow or cleft on the enzyme surface
    • Accepts reactants (substrates and cofactors)
    • Contains amino acids which
      • - bind reactants (substrates and cofactors)
      • - catalyse the reaction
    ENZYME Active site Active site
  • 57. 3. Substrate binding
    • Active site is nearly the correct shape for the substrate
    • Binding alters the shape of the enzyme (induced fit)
    • Binding will strain bonds in the substrate
    • Binding involves intermolecular bonds between functional groups in the substrate and functional groups in the active site
    3.1 Induced fit Induced fit Substrate S
  • 58.
    • Ionic
    • H-bonding
    • van der Waals
    3.2 Bonding forces Example: 3. Substrate binding S Enzyme Active site vdw interaction ionic bond H-bond Phe Ser O H Asp CO 2
  • 59.
    • Ionic
    • H-bonding
    • van der Waals
    3.2 Bonding forces Example: Binding of pyruvic acid in LDH 3. Substrate binding van der Waals H-Bond Ionic O H H 3 N H-Bond Ionic bond Possible interactions vdw-interactions
  • 60.
    • Induced fit - Active site alters shape to maximise intermolecular bonding
    3.2 Bonding forces Intermolecular bonds not optimum length for maximum bonding Intermolecular bond lengths optimised Susceptible bonds in substrate strained Susceptible bonds in substrate more easily broken 3. Substrate binding S Phe Ser O H Asp CO 2 Induced fit S Phe Ser O H Asp CO 2
  • 61. Example: Binding of pyruvic acid in LDH O H H 3 N 3. Substrate binding O O O
  • 62. Example: Binding of pyruvic acid in LDH O H H 3 N 3. Substrate binding pi bond weakened
  • 63. 4. Catalysis mechanisms
    • Histidine
    4.1 Acid/base catalysis 4.2 Nucleophilic residues Non-ionised Acts as a basic catalyst (proton 'sink') Ionised Acts as an acid catalyst (proton source) L-Serine L-Cysteine
  • 64. Serine acting as a nucleophile 4. Catalysis mechanisms
  • 65. 5. Overall process of enzyme catalysis
    • Binding interactions must be;
    • - strong enough to hold the substrate sufficiently long for the reaction to occur
    • - weak enough to allow the product to depart
    • Implies a fine balance
    • Drug design - designing molecules with stronger binding interactions results in enzyme inhibitors which block the active site
    S E ES P E EP P E E + P E S E + S E
  • 66. 6. Competitive (reversible) inhibitors
    • Inhibitor binds reversibly to the active site
    • Intermolecular bonds are involved in binding
    • No reaction takes place on the inhibitor
    • Inhibition depends on the strength of inhibitor binding and inhibitor concentration
    • Substrate is blocked from the active site
    • Increasing substrate concentration reverses inhibition
    • Inhibitor likely to be similar in structure to the substrate
    I E E S I E
  • 67. 7. Non competitive (irreversible) inhibitors
    • Inhibitor binds irreversibly to the active site
    • Covalent bond formed between the drug and the enzyme
    • Substrate is blocked from the active site
    • Increasing substrate concentration does not reverse inhibition
    • Inhibitor likely to be similar in structure to the substrate
    X OH OH X O Covalent Bond Irreversible inhibition
  • 68. 8. Non competitive (reversible) allosteric inhibitors
    • 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 recognised by the substrate
    • Increasing substrate concentration does not reverse inhibition
    • Inhibitor is not similar in structure to the substrate
    ACTIVE SITE (open) ENZYME Enzyme Allosteric site Active site (open) ENZYME Enzyme Induced fit Active site unrecognisable Allosteric inhibitor
  • 69. 8. Non competitive (reversible) allosteric inhibitors
    • Enzymes with allosteric sites often at start of biosynthetic pathways
    • Enzyme is controlled by the final product of the pathway
    • Final product binds to the allosteric site and switches off enzyme
    • Inhibitor may have a similar structure to the final product
    Inhibition P’’’ P’’ P’ Biosynthetic pathway Feedback control P S (open) ENZYME Enzyme
  • 70.