Forest Research Institute
The Why & Wherefore:Drug Targets
Nhut DiepSenior Medicinal Research Scientist
1. Cell Structure2. Cell Membrane3. Drug Targets 4. Intermolecular Bonding Forces
a. Electrostatic or ionic bondb. Hydrogen bonds c. Van der Waals interactionsd. Dipole-dipole/Ion-dipole/Induced dipole interactions
5. Desolvation penalties6. Hydrophobic interactions7. Drug Targets - Cell Membrane Lipids 8. Drug Targets – Carbohydrates
PROTEINS AS DRUG TARGETS:PROTEINS AS DRUG TARGETS:RECEPTORSRECEPTORS
THE WHY & THE WHEREFORE:THE WHY & THE WHEREFORE:DRUG TARGETSDRUG TARGETS
ContentsContents
1. Structure and function of receptors 6. Competitive (reversible) antagonistsa. Chemical Messengers 7. Non competitive (irreversible) antagonistsb. Mechanism 8. Non competitive (reversible) allosteric antagonists
3. The binding site 9. Antagonists by umbrella effect4. Messenger binding 10. Agonists
a. Introductionb. Bonding forces
5. Overall process of receptor/messenger interaction6. Signal transduction
a. Control of ion channels b. Activation of signal proteins
c. Activation of enzyme active site
PROTEINS AS DRUG TARGETS:PROTEINS AS DRUG TARGETS:EnzymesEnzymes
1. Structure and function of enzymes2. The active site3. Substrate binding
a. Induced fitb. Bonding forces
4. Catalysis mechanismsa. Acid/base catalysisb. Nucleophilic residues
5. Overall process of enzyme catalysis6. Competive (reversible) inhibitors7. Non competitive (irreversible) inhibitors 8. Non competitive (reversible) allosteric inhibitors
DRUG TARGETSDRUG TARGETS
1. Cell Structure1. Cell Structure
Human, animal and plant cells are eukaryotic cellsHuman, animal and plant cells are eukaryotic cells
The nucleus contains the genetic blueprint for life (DNA)The nucleus contains the genetic blueprint for life (DNA)
The fluid contents of the cell are known as the cytoplasmThe fluid contents of the cell are known as the cytoplasm
Structures within the cell are known as organellesStructures within the cell are known as organelles
Mitochondria are the source of energy productionMitochondria are the source of energy production
Ribosomes are the cell’s protein ‘factories’Ribosomes are the cell’s protein ‘factories’
Rough endoplasmic reticulum is the location for protein Rough endoplasmic reticulum is the location for protein synthesissynthesis
PhospholipidBilayer
ExteriorHigh [Na+]
InteriorHigh [K+]
2. Cell Membrane2. Cell Membrane
Proteins
PolarHeadGroup
Hydrophobic Tails
2. Cell Membrane2. Cell Membrane
PolarHeadGroup
Hydrophobic Tails
CHCH2 CH2
O O
O
P OO
O
CH2CH2NMe3
O O
PolarHeadGroup
Hydrophobic Tails
C HC H 2 C H 2
O O
O
P OO
O
C H 2 C H 2 N M e 3
O O
2. Cell Membrane2. Cell Membrane
2. Cell Membrane2. Cell Membrane
The cell membrane is made up of a phospholipid bilayerThe cell membrane is made up of a phospholipid bilayer
The hydrophobic tails interact with each other by van der Waals The hydrophobic tails interact with each other by van der Waals interactions and are hidden from the aqueous mediainteractions and are hidden from the aqueous media
The polar head groups interact with water at the inner and outer The polar head groups interact with water at the inner and outer surfaces of the membranesurfaces of the membrane
The cell membrane provides a hydrophobic barrier around the The cell membrane provides a hydrophobic barrier around the cell, preventing the passage of water and polar moleculescell, preventing the passage of water and polar molecules
Proteins are present, floating in the cell membraneProteins are present, floating in the cell membrane
Some act as ion channels and carrier proteinsSome act as ion channels and carrier proteins
LipidsLipidsCell membrane lipidsCell membrane lipids
ProteinsProteins ReceptorsReceptorsEnzymesEnzymesCarrier proteinsCarrier proteinsStructural proteins (tubulin)Structural proteins (tubulin)
Nucleic acidsNucleic acids
DNADNARNARNA
CarbohydratesCarbohydratesCell surface carbohydratesCell surface carbohydratesAntigens and recognition moleculesAntigens and recognition molecules
3. Drug targets3. Drug targets
3. Drug targets3. Drug targets
Drug targets are large molecules - macromoleculesDrug targets are large molecules - macromolecules
Drugs are generally much smaller than their targetsDrugs are generally much smaller than their targets
Drugs interact with their targets by binding to binding sitesDrugs interact with their targets by binding to binding sites
Binding sites are typically hydrophobic pockets on the surface of Binding sites are typically hydrophobic pockets on the surface of macromoleculesmacromolecules
Binding interactions typically involve intermolecular bondsBinding interactions typically involve intermolecular bonds
Most drugs are in equilibrium between being bound and Most drugs are in equilibrium between being bound and unbound to their targetunbound to their target
Functional groups on the drug are involved in binding Functional groups on the drug are involved in binding interactions and are called binding groupsinteractions and are called binding groups
Specific regions within the binding site that are involved in Specific regions within the binding site that are involved in binding interactions are called binding regionsbinding interactions are called binding regions
Macromolecular target
Drug
Unbound drug
Macromolecular target
Drug
Bound drug
Bindingsite
Drug
Binding site
Binding regions
Binding groups
Intermolecular bonds
3. Drug targets3. Drug targets
3. Drug targets3. Drug targets
Binding interactions usually result in an induced fit where the Binding interactions usually result in an induced fit where the
binding site changes shape to accommodate the drugbinding site changes shape to accommodate the drug
The induced fit may also alter the overall shape of the drug targetThe induced fit may also alter the overall shape of the drug target
Important to the pharmacological effect of the drugImportant to the pharmacological effect of the drug
4. 4. Intermolecular bonding forcesIntermolecular bonding forces
4.1 Electrostatic or ionic bond4.1 Electrostatic or ionic bond• Strongest of the intermolecular bonds (20-40 kJ molStrongest of the intermolecular bonds (20-40 kJ mol-1-1))• Takes place between groups of opposite chargeTakes place between groups of opposite charge• The strength of the ionic interaction is inversely proportional to The strength of the ionic interaction is inversely proportional to
the distance between the two charged groupsthe distance between the two charged groups• Stronger interactions occur in hydrophobic environmentsStronger interactions occur in hydrophobic environments• The strength of interaction drops off less rapidly with distance The strength of interaction drops off less rapidly with distance
than with other forms of intermolecular interactionsthan with other forms of intermolecular interactions• Ionic bonds are the most important initial interactions as a drug Ionic bonds are the most important initial interactions as a drug
enters the binding siteenters the binding site
DrugO
O H3N TargetDrug NH3
TargetO
O
4. Intermolecular bonding forces4. Intermolecular bonding forces
4.2 Hydrogen bonds4.2 Hydrogen bonds
X HDrug
Y TargetDrug X
TargetHY++
- ---
HBD HBA HBA HBD
• Vary in strengthVary in strength• Weaker than electrostatic interactions but stronger than van Weaker than electrostatic interactions but stronger than van
der Waals interactionsder Waals interactions• A hydrogen bond takes place between an electron deficient A hydrogen bond takes place between an electron deficient
hydrogen and an electron rich heteroatom (N or O) hydrogen and an electron rich heteroatom (N or O) • The electron deficient hydrogen is usually attached to a The electron deficient hydrogen is usually attached to a
heteroatom (O or N)heteroatom (O or N)• The electron deficient hydrogen is called a hydrogen bond The electron deficient hydrogen is called a hydrogen bond
donordonor• The electron rich heteroatom is called a hydrogen bond The electron rich heteroatom is called a hydrogen bond
acceptoracceptor
4. Intermolecular bonding forces4. Intermolecular bonding forces
4.2 Hydrogen bonds4.2 Hydrogen bonds
YX H YX H
Hybridisedorbital
Hybridisedorbital
1sorbital
HBAHBAHBDHBD
• The interaction involves orbitals and is directionalThe interaction involves orbitals and is directional
• Optimum orientation is where the X-H bond points directly 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 to the lone pair on Y such that the angle between X, H and Y
is 180is 180oo
4. Intermolecular bonding forces4. Intermolecular bonding forces
4.2 Hydrogen bonds4.2 Hydrogen bonds
• Examples of strong hydrogen bond acceptors Examples of strong hydrogen bond acceptors - carboxylate ion, phosphate ion, tertiary amine - carboxylate ion, phosphate ion, tertiary amine
• Examples of moderate hydrogen bond acceptorsExamples of moderate hydrogen bond acceptors- carboxylic acid, amide oxygen, ketone, ester, ether, alcohol- carboxylic acid, amide oxygen, ketone, ester, ether, alcohol
• Examples of poor hydrogen bond acceptors Examples of poor hydrogen bond acceptors - sulfur, fluorine, chlorine, aromatic ring, amide nitrogen, - sulfur, fluorine, chlorine, aromatic ring, amide nitrogen,
aromatic aminearomatic amine
• Example of good hydrogen bond donorsExample of good hydrogen bond donors- Quaternary ammonium ion- Quaternary ammonium ion
4. Intermolecular bonding forces4. Intermolecular bonding forces
4.3 Van der Waals interactions4.3 Van der Waals interactions
Binding site
DRUG
- +
• Very weak interactions (2-4 kJmolVery weak interactions (2-4 kJmol-1-1))• Occur between hydrophobic regions of the drug and the targetOccur between hydrophobic regions of the drug and the target• Due to transient areas of high and low electron densities Due to transient areas of high and low electron densities
leading to temporary dipoles leading to temporary dipoles • Interactions drop off rapidly with distanceInteractions drop off rapidly with distance• Drug must be close to the binding region for interactions to Drug must be close to the binding region for interactions to
occuroccur• The overall contribution of van der Waals interactions can be The overall contribution of van der Waals interactions can be
crucial to bindingcrucial to binding
+ -
Hydrophobic regions
Transient dipole on drug+ -
van der Waals interaction
4. Intermolecular bonding forces4. Intermolecular bonding forces
4.4 Dipole-dipole interactions4.4 Dipole-dipole interactions
• Can occur if the drug and the binding site have dipole Can occur if the drug and the binding site have dipole momentsmoments
• Dipoles align with each other as the drug enters the binding Dipoles align with each other as the drug enters the binding sitesite
• Dipole alignment orientates the molecule in the binding siteDipole alignment orientates the molecule in the binding site• Orientation is beneficial if other binding groups are positioned Orientation is beneficial if other binding groups are positioned
correctly with respect to the corresponding binding regionscorrectly with respect to the corresponding binding regions• Orientation is detrimental if the binding groups are not Orientation is detrimental if the binding groups are not
positioned correctly with respect to corresponding binding positioned correctly with respect to corresponding binding regionsregions
• The strength of the interaction decreases with distance more The strength of the interaction decreases with distance more quickly than with electrostatic interactions, but less quickly quickly than with electrostatic interactions, but less quickly than with van der Waals interactionsthan with van der Waals interactions
4. Intermolecular bonding forces4. Intermolecular bonding forces
4.4 Ion-dipole interactions4.4 Ion-dipole interactions
• Occur where the charge on one molecule interacts with the Occur where the charge on one molecule interacts with the dipole moment of anotherdipole moment of another
• Stronger than a dipole-dipole interactionStronger than a dipole-dipole interaction
• Strength of interaction falls off less rapidly with distance than Strength of interaction falls off less rapidly with distance than for a dipole-dipole interactionfor a dipole-dipole interaction
C
O
O
Binding siteBinding site
RR
CCRR OO
H3N
Binding siteBinding site
RR
CCRR OO
4. Intermolecular bonding forces4. Intermolecular bonding forces
4.4 Induced dipole interactions4.4 Induced dipole interactions
• Occur where the charge on one molecule induces a dipole on Occur where the charge on one molecule induces a dipole on anotheranother
• Occurs between a quaternary ammonium ion and an Occurs between a quaternary ammonium ion and an aromatic ringaromatic ring
Binding siteBinding site
RR NN RR33
RC
R
O
OH
HH H
O
H
H
O
H
H
O
OH
Binding site
Desolvation - Energy penalty Binding - Energy gain
OH
RC
R
O
Binding site
RC
R
O
OH
Binding site
5. Desolvation penalties5. Desolvation penalties
• Polar regions of a drug and its target are solvated prior to Polar regions of a drug and its target are solvated prior to interactioninteraction
• Desolvation is necessary and requires energyDesolvation is necessary and requires energy
• The energy gained by drug-target interactions must be The energy gained by drug-target interactions must be greater than the energy required for desolvationgreater than the energy required for desolvation
Unstructured waterUnstructured waterIncrease in entropyIncrease in entropy
DrugDrugDRUGDRUG
Structured water layer Structured water layer round hydrophobic regionsround hydrophobic regions
HydrophobicHydrophobic regionsregionsWaterWater
Binding siteBinding site Binding siteBinding site
DrugDrugDRUGDRUG
BindingBinding
6. Hydrophobic interactions6. Hydrophobic interactions
• Hydrophobic regions of a drug and its target are not solvatedHydrophobic regions of a drug and its target are not solvated
• Water molecules interact with each other and form an Water molecules interact with each other and form an ordered layer next to hydrophobic regions - negative entropyordered layer next to hydrophobic regions - negative entropy
• Interactions between the hydrophobic interactions of a drug Interactions between the hydrophobic interactions of a drug and its target ‘free up’ the ordered water molecules and its target ‘free up’ the ordered water molecules
• Results in an increase in entropyResults in an increase in entropy
• Beneficial to binding energyBeneficial to binding energy
Hydrophobic regionHydrophobic region
Drugs acting on cell membrane lipids - Drugs acting on cell membrane lipids - Anaesthetics and some antibioticsAnaesthetics and some antibiotics
Action of amphotericin B (antifungal agent)Action of amphotericin B (antifungal agent) - builds tunnels through membrane and drains cell- builds tunnels through membrane and drains cell
7. Drug Targets - Cell Membrane Lipids7. Drug Targets - Cell Membrane Lipids
HydrophilicHydrophilicHydrophilicHydrophilic
HydrophilicHydrophilic
OOH
HO
OHHOOC
OH
O
Me
OH
OHMe
OH O OH
Me
O
O
HONH2
HO
Me
H
Polar tunnel formedPolar tunnel formedEscape route for ionsEscape route for ions
CELLMEMBRANE
TUNNEL
HO
HO
HO
HO
HO
HO
HO
HO2C CO2H
Sugar
OH OH
Sugar
HO
HO
HO
HO
HO
HO
HO
OH
OH
OH
OH
OH
OH
OH
Sugar
OH OH
Sugar
HO2C CO2H
OH
OH
OH
OH
OH
OH
OH
7. Drug Targets - Cell Membrane Lipids7. Drug Targets - Cell Membrane Lipids
8. Drug Targets - Carbohydrates8. Drug Targets - Carbohydrates
• Carbohydrates play important roles in cell recognition, Carbohydrates play important roles in cell recognition, regulation and growthregulation and growth
• Potential targets for the treatment of bacterial and viral Potential targets for the treatment of bacterial and viral infection, cancer and autoimmune diseaseinfection, cancer and autoimmune disease
• Carbohydrates act as antigensCarbohydrates act as antigens
Carbohydrate 'tag'
CellCellmembranemembrane
7. Drug Targets - Carbohydrates7. Drug Targets - Carbohydrates
Ceramide 'anchor'Carbohydrate 'tag'
O
OHO
O
OH
HO
OH
(CH2)12CH3
HN (CH2)16CH3
O
Ceramide unit
SUGARS
Carbohydrates
OH
(CH2)12CH3HO
NH2
Sphingosine
HO (CH2)16CH3
O
Fatty Acid (e.g. Stearic acid)O
OHHO
RO
OH
HO
Carbohydrate (R=various carbohydrate structures)
PROTEINS AS DRUG TARGETS:PROTEINS AS DRUG TARGETS:RECEPTORSRECEPTORS
1. Structure and function of receptors1. Structure and function of receptors
• Globular proteins acting as a cell’s ‘letter boxes’Globular proteins acting as a cell’s ‘letter boxes’
• Located mostly in the cell membraneLocated mostly in the cell membrane
• Receive messages from chemical messengers coming from Receive messages from chemical messengers coming from other cellsother cells
• Transmit a message into the cell leading to a cellular effectTransmit a message into the cell leading to a cellular effect
• Different receptors specific for different chemical messengersDifferent receptors specific for different chemical messengers
• Each cell has a range of receptors in the cell membrane Each cell has a range of receptors in the cell membrane making it responsive to different chemical messengersmaking it responsive to different chemical messengers
Cell
Nerve
Messenger
Signal
Receptor
Nerve
NucleusCell
Response
1. Structure and function of receptors1. Structure and function of receptors
Chemical MessengersChemical Messengers
NeurotransmittersNeurotransmitters: Chemicals released from nerve endings which : Chemicals released from nerve endings which travel across a nerve synapse to bind with receptors on target cells, travel across a nerve synapse to bind with receptors on target cells, such as muscle cells or another nerve. Usually short lived and such as muscle cells or another nerve. Usually short lived and responsible for messages between individual cellsresponsible for messages between individual cells
HormonesHormones: Chemicals released from cells or glands and which : Chemicals released from cells or glands and which travel some distance to bind with receptors on target cells travel some distance to bind with receptors on target cells throughout the bodythroughout the body
• Chemical messengers ‘switch on’ receptors without Chemical messengers ‘switch on’ receptors without undergoing a reaction undergoing a reaction
1. Structure and function of receptors1. Structure and function of receptors
Nerve 1
Nerve 2Hormone
Bloodsupply
Neurotransmitters
1. Structure and function of receptors1. Structure and function of receptors
Mechanism Mechanism
• Receptors contain a binding site (hollow or cleft in the Receptors contain a binding site (hollow or cleft in the receptor surface) that is recognised by the chemical receptor surface) that is recognised by the chemical messengermessenger
• Binding of the messenger involves intermolecular bondsBinding of the messenger involves intermolecular bonds
• Binding results in an induced fit of the receptor proteinBinding results in an induced fit of the receptor protein
• Change in receptor shape results in a ‘domino’ effectChange in receptor shape results in a ‘domino’ effect
• Domino effect is known as Signal Transduction, leading to a Domino effect is known as Signal Transduction, leading to a chemical signal being received inside the cell chemical signal being received inside the cell
• Chemical messenger does not enter the cell. It departs the Chemical messenger does not enter the cell. It departs the receptor unchanged and is not permanently boundreceptor unchanged and is not permanently bound
1. Structure and function of receptors1. Structure and function of receptors
Mechanism Mechanism
CellMembrane
Cell
Receptor
Messenger
message
Induced fit
Cell
Receptor
Messenger
Message
Cell
Messenger
Receptor
1. Structure and function of receptors1. Structure and function of receptors
ENZYME
2. The binding site2. The binding site
• A hydrophobic hollow or cleft on the receptor surface - A hydrophobic hollow or cleft on the receptor surface - equivalent to the active site of an enzymeequivalent to the active site of an enzyme
• Accepts and binds a chemical messengerAccepts and binds a chemical messenger
• Contains amino acids which bind Contains amino acids which bind the messengerthe messenger
• No reaction or catalysis takes placeNo reaction or catalysis takes place
Binding siteBinding site
3. Messenger binding3. Messenger binding
• Binding site is nearly the correct shape for the messengerBinding site is nearly the correct shape for the messenger
• Binding alters the shape of the receptor (induced fit)Binding alters the shape of the receptor (induced fit)
• Altered receptor shape leads to further effects - signal Altered receptor shape leads to further effects - signal transductiontransduction
3.1 Introduction3.1 Introduction
MessengerMessenger
Induced fitInduced fit
MM
• IonicIonic• H-bondingH-bonding• van der Waalsvan der Waals
3.2 Bonding forces3.2 Bonding forces
Example:Example:
Receptor
Binding site
vdwvdwinteractioninteraction
ionicionicbondbond
H-bondH-bond
PheSer
OH
Asp
CO2
3. Messenger binding3. Messenger binding
3. Substrate binding3. Substrate binding
• Induced fit - Binding site alters shape to maximise Induced fit - Binding site alters shape to maximise intermolecular bondingintermolecular bonding
3.2 Bonding forces3.2 Bonding forces
Intermolecular bonds not optimum length for
maximum binding strength
Intermolecular bond lengths optimised
Phe
SerO
H
Asp
CO2 Induced Fit
Phe
Ser
OH
Asp
CO2
4. Overall process of receptor/messenger interaction4. Overall process of receptor/messenger interaction
MM
MM
EERR
• Binding interactions must be:Binding interactions must be: - strong enough to hold the messenger sufficiently long for signal - strong enough to hold the messenger sufficiently long for signal
transduction to take placetransduction to take place - weak enough to allow the messenger to depart - weak enough to allow the messenger to depart • Implies a fine balanceImplies a fine balance• Drug design - designing molecules with stronger binding Drug design - designing molecules with stronger binding
interactions results in drugs that block the binding site - interactions results in drugs that block the binding site - antagonistsantagonists
RR
MM
EERR
Signal transductionSignal transduction
5. Signal transduction5. Signal transduction
5.1 Control of ion channels5.1 Control of ion channels
• Receptor protein is part of an ion channel protein complexReceptor protein is part of an ion channel protein complex
• Receptor binds a messenger leading to an induced fitReceptor binds a messenger leading to an induced fit
• Ion channel is opened or closedIon channel is opened or closed
• Ion channels are specific for specific ions (NaIon channels are specific for specific ions (Na++, Ca, Ca2+2+, Cl, Cl--, K, K++))
• Ions flow across cell membrane down concentration gradientIons flow across cell membrane down concentration gradient
• Polarises or depolarises nerve membranesPolarises or depolarises nerve membranes
• Activates or deactivates enzyme catalysed reactions within Activates or deactivates enzyme catalysed reactions within cellcell
5. Signal transduction5. Signal transduction
Hydrophilictunnel
Cellmembrane
5.1 Control of ion channels5.1 Control of ion channels
Cellmembrane
Five glycoprotein subunitstraversing cell membrane
Messenger
Cellmembrane
Receptor
Inducedfit
‘Gating’(ion channel opens)
Cationic ion channels for KCationic ion channels for K++, Na, Na++, Ca, Ca2+2+ (e.g. nicotinic) = excitatory (e.g. nicotinic) = excitatoryAnionic ion channels for ClAnionic ion channels for Cl-- (e.g. GABA (e.g. GABAAA) = inhibitory) = inhibitory
Bindingsite
5.1 Control of ion channels5.1 Control of ion channels
5. Signal transduction5. Signal transduction
5.1 Control of ion channels:5.1 Control of ion channels:
Induced fit and opening
of ion channel
IONCHANNEL
(open)
Cell
Cellmembrane
MESSENGER
Ionchannel
Ionchannel
Cellmembrane
IONCHANNEL
(closed)
Cell
RECEPTORBINDING
SITE
Lock Gate
Ionchannel
Ionchannel
Cellmembrane
Cellmembrane
MESSENGER
5. Signal transduction5. Signal transduction
5.2 Activation of signal proteins5.2 Activation of signal proteins• Receptor binds a messenger leading to an induced fitReceptor binds a messenger leading to an induced fit• Opens a binding site for a signal protein (G-protein)Opens a binding site for a signal protein (G-protein)• G-Protein binds, is destabilised then splitG-Protein binds, is destabilised then split
messenger
G-proteinsplit
inducedfit
closed open
5. Signal transduction5. Signal transduction
5.2 Activation of signal proteins5.2 Activation of signal proteins• G-Protein subunit activates membrane bound enzymeG-Protein subunit activates membrane bound enzyme
Binds to allosteric binding siteBinds to allosteric binding siteInduced fit results in opening of active siteInduced fit results in opening of active site
• Intracellular reaction catalysedIntracellular reaction catalysed
active site(closed)
active site(open)
Enzyme
Intracellular reaction
Enzyme
5. Signal transduction5. Signal transduction
5.3 Activation of enzyme active site5.3 Activation of enzyme active site• Protein serves dual role - receptor plus enzymeProtein serves dual role - receptor plus enzyme• Receptor binds messenger leading to an induced fitReceptor binds messenger leading to an induced fit• Protein changes shape and opens active siteProtein changes shape and opens active site• Reaction catalysed within cellReaction catalysed within cell
closed
messenger
inducedfit
active site open
intracellular reaction
closed
messenger
5. Signal transduction5. Signal transduction
6. Competitive (reversible) antagonists6. Competitive (reversible) antagonists
• Antagonist binds reversibly to the binding site Antagonist binds reversibly to the binding site • Intermolecular bonds involved in bindingIntermolecular bonds involved in binding• Different induced fit means receptor is not activatedDifferent induced fit means receptor is not activated• No reaction takes place on antagonistNo reaction takes place on antagonist• Level of antagonism depends on strength of antagonist Level of antagonism depends on strength of antagonist
binding and concentrationbinding and concentration• Messenger is blocked from the binding site Messenger is blocked from the binding site • Increasing the messenger concentration reverses antagonismIncreasing the messenger concentration reverses antagonism
AnAn
EERR
MM
AnAn
RR
7. Non competitive (irreversible) antagonists7. Non competitive (irreversible) antagonists
• Antagonist binds irreversibly to the binding siteAntagonist binds irreversibly to the binding site• Different induced fit means that the receptor is not activated Different induced fit means that the receptor is not activated • Covalent bond is formed between the drug and the receptorCovalent bond is formed between the drug and the receptor• Messenger is blocked from the binding site Messenger is blocked from the binding site • Increasing messenger concentration does not reverse Increasing messenger concentration does not reverse
antagonismantagonism
X
OH OH
X
O
Covalent Bond
Irreversible antagonism
8. Non competitive (reversible) allosteric antagonists8. Non competitive (reversible) allosteric antagonists
• Antagonist binds reversibly to an allosteric site Antagonist binds reversibly to an allosteric site • Intermolecular bonds formed between antagonist and binding Intermolecular bonds formed between antagonist and binding
sitesite• Induced fit alters the shape of the receptorInduced fit alters the shape of the receptor• Binding site is distorted and is not recognised by the messengerBinding site is distorted and is not recognised by the messenger• Increasing messenger concentration does not reverse Increasing messenger concentration does not reverse
antagonismantagonism
ACTIVE SITE (open)
ENZYMEReceptor
AllostericAllostericsitesite
Binding siteBinding site
(open)ENZYMEReceptor
Inducedfit
Binding siteBinding siteunrecognisableunrecognisable
Antagonist
9. Antagonists by umbrella effect9. Antagonists by umbrella effect
• Antagonist binds reversibly to a neighbouring binding site Antagonist binds reversibly to a neighbouring binding site • Intermolecular bonds formed between antagonist and Intermolecular bonds formed between antagonist and
binding sitebinding site• Antagonist overlaps with the messenger binding siteAntagonist overlaps with the messenger binding site• Messenger is blocked from the binding siteMessenger is blocked from the binding site
Antagonist
Binding sitefor antagonist
Binding sitefor messenger
messenger
Receptor Receptor
10. Agonists10. Agonists
• Agonist binds reversibly to the binding site Agonist binds reversibly to the binding site • Similar intermolecular bonds formed as to natural messengerSimilar intermolecular bonds formed as to natural messenger• Induced fit alters the shape of the receptor in the same way as Induced fit alters the shape of the receptor in the same way as
the normal messengerthe normal messenger• Receptor is activatedReceptor is activated• Agonists are often similar in structure to the natural Agonists are often similar in structure to the natural
messengermessenger
EE
AgonistAgonist
RR EE
AgonistAgonist
RR
Signal transductionSignal transduction
AgonistAgonist
RR
Induced fitInduced fit
PROTEINS AS DRUG TARGETS:PROTEINS AS DRUG TARGETS:ENZYMESENZYMES
1. Structure and function of enzymes1. Structure and function of enzymes
• Globular proteins acting as the body’s catalystsGlobular proteins acting as the body’s catalysts• Speed up time for reaction to reach equilibriumSpeed up time for reaction to reach equilibrium• Lower the activation energy of a reactionLower the activation energy of a reaction
Example:Example:
LDH = Lactate dehydrogenase (enzyme)NADH2 = Nicotinamide adenosine dinucleotide (reducing agent & cofactor)Pyruvic acid = Substrate
LDH
Pyruvic acid Lactic acid
H3CC
CO
O HO
C OCH3C
H
NADH2 NAD+++
OH OH
Lowering the activation energy of reactionLowering the activation energy of reaction
Act. energy
Transition state
WITHOUT ENZYME
Product
Startingmaterial
Energy
WITH ENZYME
Product
Startingmaterial
Energy
∆G
Newtransition
state
∆G
Act. energy
• Enzymes lower the activation energy of a reaction but Enzymes lower the activation energy of a reaction but G remains the G remains the samesame
1. Structure and function of enzymes1. Structure and function of enzymes
Methods of enzyme catalysisMethods of enzyme catalysis
• Provide a reaction surface (the active site)Provide a reaction surface (the active site)
• Provide a suitable environment (hydrophobic)Provide a suitable environment (hydrophobic)
• Bring reactants togetherBring reactants together
• Position reactants correctly for reactionPosition reactants correctly for reaction
• Weaken bonds in the reactantsWeaken bonds in the reactants
• Provide acid / base catalysisProvide acid / base catalysis
• Provide nucleophilesProvide nucleophiles
1. Structure and function of enzymes1. Structure and function of enzymes
2. The active site2. The active site
• Hydrophobic hollow or cleft on the enzyme surfaceHydrophobic hollow or cleft on the enzyme surface
• Accepts reactants (substrates and cofactors)Accepts reactants (substrates and cofactors)
• Contains amino acids whichContains amino acids which - bind reactants (substrates and cofactors)- bind reactants (substrates and cofactors) - catalyse the reaction- catalyse the reaction
ENZYME
Active siteActive site
3. Substrate binding3. Substrate binding
• Active site is nearly the correct shape for the substrateActive site is nearly the correct shape for the substrate• Binding alters the shape of the enzyme (induced fit)Binding alters the shape of the enzyme (induced fit)• Binding will strain bonds in the substrateBinding will strain bonds in the substrate• Binding involves intermolecular bonds between functional groups in the Binding involves intermolecular bonds between functional groups in the
substrate and functional groups in the active sitesubstrate and functional groups in the active site
3.1 Induced fit3.1 Induced fit
Induced fitInduced fit
SubstrateSubstrate
SS
• IonicIonic• H-bondingH-bonding• van der Waalsvan der Waals
3.2 Bonding forces3.2 Bonding forces
Example:Example:
SS
Enzyme
Active site
vdwvdwinteractioninteraction
ionicionicbondbond
H-bondH-bond
Phe
Ser
OH
Asp
CO2
3. Substrate binding3. Substrate binding
van der Waals
H-Bond
Ionic
H3CC
C
O
O
O
• IonicIonic• H-bondingH-bonding• van der Waalsvan der Waals
3.2 Bonding forces3.2 Bonding forces
Example:Example: Binding of pyruvic acid in LDH Binding of pyruvic acid in LDH
O
H
H3N
H-Bond
Ionicbond
Possible interactions vdw-interactions
H3CC
C
O
O
O
3. Substrate binding3. Substrate binding
• Induced fit - Active site alters shape to maximise intermolecular bondingInduced fit - Active site alters shape to maximise intermolecular bonding
3.2 Bonding forces3.2 Bonding forces
Intermolecular bonds not optimum length for maximum bonding
Intermolecular bond lengths optimisedSusceptible bonds in substrate strainedSusceptible bonds in substrate more easily broken
SS Phe
SerO
H
Asp
CO2 Induced fit
SSPhe
Ser
OH
Asp
CO2
3. Substrate binding3. Substrate binding
Example:Example: Binding of pyruvic acid in LDH Binding of pyruvic acid in LDH
O
H
H3N
H3CC
C
O
O
O
O
O
O
3. Substrate binding3. Substrate binding
Example:Example: Binding of pyruvic acid in LDH Binding of pyruvic acid in LDH
O
H
H3N
pi bondpi bondweakenedweakened
H3CC
C
O
O
O
3. Substrate binding3. Substrate binding
4. Catalysis mechanisms4. Catalysis mechanisms
• HistidineHistidine
4.1 Acid/base catalysis4.1 Acid/base catalysis
4.2 Nucleophilic residues4.2 Nucleophilic residues
NNH
+H
-H NNH
H
Non-ionisedNon-ionisedActs as a basic catalystActs as a basic catalyst(proton 'sink')(proton 'sink')
IonisedIonisedActs as an acid catalystActs as an acid catalyst(proton source)(proton source)
H3N CO2
OH
H
L-SerineL-Serine
H3N CO2
SH
H
L-CysteineL-Cysteine
OH
Ser
X
Substrate
O
Ser
H2O
OH
Ser
HO Product
Serine acting as a nucleophileSerine acting as a nucleophile
4. Catalysis mechanisms4. Catalysis mechanisms
5. Overall process of enzyme catalysis5. Overall process of enzyme catalysis
SS
EE
ES
PP
EE
EP
PP
EE
E + P
EE
SS
E + S
EE
• Binding interactions must be;Binding interactions must be; - strong enough to hold the substrate sufficiently long for the reaction to - strong enough to hold the substrate sufficiently long for the reaction to
occuroccur - weak enough to allow the product to depart - weak enough to allow the product to depart • Implies a fine balanceImplies a fine balance• Drug design - designing molecules with stronger binding interactions results Drug design - designing molecules with stronger binding interactions results
in enzyme inhibitors which block the active sitein enzyme inhibitors which block the active site
6. Competitive (reversible) inhibitors6. Competitive (reversible) inhibitors
• Inhibitor binds reversibly to the active site Inhibitor binds reversibly to the active site • Intermolecular bonds are involved in bindingIntermolecular bonds are involved in binding• No reaction takes place on the inhibitorNo reaction takes place on the inhibitor• Inhibition depends on the strength of inhibitor binding and inhibitor Inhibition depends on the strength of inhibitor binding and inhibitor
concentrationconcentration• Substrate is blocked from the active site Substrate is blocked from the active site • Increasing substrate concentration reverses inhibitionIncreasing substrate concentration reverses inhibition• Inhibitor likely to be similar in structure to the substrateInhibitor likely to be similar in structure to the substrate
II
EEEE
SS
II
EE
7. Non competitive (irreversible) inhibitors7. Non competitive (irreversible) inhibitors
• Inhibitor binds irreversibly to the active site Inhibitor binds irreversibly to the active site • Covalent bond formed between the drug and the enzymeCovalent bond formed between the drug and the enzyme• Substrate is blocked from the active site Substrate is blocked from the active site • Increasing substrate concentration does not reverse inhibitionIncreasing substrate concentration does not reverse inhibition• Inhibitor likely to be similar in structure to the substrateInhibitor likely to be similar in structure to the substrate
X
OH OH
X
O
Covalent Bond
Irreversible inhibition
ACTIVE SITE (open)
ENZYMEEnzyme
8. Non competitive (reversible) allosteric inhibitors8. Non competitive (reversible) allosteric inhibitors
• Inhibitor binds reversibly to the allosteric site Inhibitor binds reversibly to the allosteric site • Intermolecular bonds are formedIntermolecular bonds are formed• Induced fit alters the shape of the enzymeInduced fit alters the shape of the enzyme• Active site is distorted and is not recognised by the substrateActive site is distorted and is not recognised by the substrate• Increasing substrate concentration does not reverse inhibitionIncreasing substrate concentration does not reverse inhibition• Inhibitor is not similar in structure to the substrateInhibitor is not similar in structure to the substrate
AllostericAllostericsitesite
Active siteActive site
(open)ENZYMEEnzyme
Inducedfit
Active siteActive siteunrecognisableunrecognisable
Allostericinhibitor
8. Non competitive (reversible) allosteric inhibitors8. Non competitive (reversible) allosteric inhibitors
• Enzymes with allosteric sites often at start of biosynthetic pathwaysEnzymes with allosteric sites often at start of biosynthetic pathways• Enzyme is controlled by the final product of the pathwayEnzyme is controlled by the final product of the pathway• Final product binds to the allosteric site and switches off enzymeFinal product binds to the allosteric site and switches off enzyme• Inhibitor may have a similar structure to the final productInhibitor may have a similar structure to the final product
P’’’P’’P’
Biosynthetic pathway
Feedback controlInhibition
PPSS
(open)ENZYMEEnzyme