role of receptors in drug design
DESCRIPTION
Receptor theories, types, receptor binding studiesTRANSCRIPT
WELCOME…
PREPARED BY,ROSHNI ANN BABYM.PHARM PART I -
PHARM.CHEMISTRY
RECEPTORS IN DRUG DESIGN
CONTENTS
CONCEPT OF RECEPTORSDEFINITIONMOLECULAR BIOLOGYRECEPTOR THEORIESRADIOLIGAND BINDING ASSAY
Concept of Receptors
First postulated by John N Langley (1878)-Established after his experiments using nicotine and curare analogues on muscle contraction.
-Langley concluded that a protoplasmic "receptive substance" must exist in which the two drugs compete for directly. He further added that the effect of combination of the receptive substance with competing drugs was determined by their comparative chemical affinities for the substance and relative dose.
Concept of receptors contd..
Further the term was introduced by Paul Ehrlich (1907)
-the compounds do not act unless bound.
-Demonstrated that stereoselectivity was imperative in drug-receptor signaling.
Definition
Receptor-is a protein molecule embedded either within
the cell membrane with a part of its structure facing the
outside of the cell or inside the cell. Occupation of
receptor may result in its activation leading to a cellular
response .
Receptors have two major properties:
Recognition and Transduction
Recognition: The receptor protein must exist in a conformational state that allows for recognition and binding of a compound and must satisfy the following criteria:SaturabilityReversibilityStereoselectivity Agonist specificity Tissue specificity
Transduction: The second property of a receptor is that the binding of an agonist must be transduced into some kind of functional response (biological or physiological).
Important Terms
Ligand: Any endogenous or exogenous chemical agent that binds to a receptor is known as a ligandBinding domain: The general region on a receptor where a ligand binds is known as binding domain.It is equivalent to enzyme active site but with no catalytic activity.Affinity: The ability of the drug to bind with receptorIntrinsic Activity: The ability of the drug to elicit pharmacological response
Agonist: The drug molecule possessing high affinity as well as high intrinsic activity
Antagonist:Drugs having high affinity but poor intrinsic activity
Partial Agonist: Drug with an affinity equal or less than that of agonist but with less intrinsic activity
Inverse agonist: Produces responses opposite to those of the agonist.
Signal Transduction: The mechanism by which any message carried by the ligand is translated through the receptor system into tissue response.
Receptors-Molecular Biology
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
Cell
Nerve
Messenger
Signal
Receptor
Nerve
NucleusCell
Response
Structure and function of receptors
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
Structure and function of receptors
Neurotransmitters relay signal
between a neuron and another cell
Classical Hormones are produced by the glands of the endocrine system, shown below
The major endocrine glands: (Male left, female right) 1 Pineal gland 2 Pituitary gland 3 Thyroid gland 4 Thymus 5 Adrenal gland 6 Pancreas 7 Ovary 8 Testes
Nerve 1
Nerve 2Hormone
Bloodsupply
Neurotransmitters
Structure and function of receptors
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
Structure and function of receptors
Chemical messenger do not undergo chemical
reaction. It fits into the binding site of receptor
protein,passes on it message and then leaves
unchanged. The messenger binds to the receptor and induces a
change in shape(conformational
change) which subsequently affects other
components of the cell membrane and leads to a
biological effect. The receptor then reforms its
original shape.
Difference B/W Enzyme and Chemical
Messenger
Mechanism
CellMembrane
Cell
Receptor
Messenger
message
Induced fit
Cell
Receptor
Messenger
Message
Cell
Messenger
Receptor
Structure and function of receptors
ENZYME
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
Binding siteBinding site
The binding site
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
• Ionic• covalent• H-bonding• hydrophobic• van der Waals
3.2 Bonding forces
Example:
Receptor
Binding site
vdwinteraction
ionicbond
H-bond
PheSer
OH
Asp
CO2
Messenger binding
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
SerOH
Asp
CO2 Induced Fit
Phe
SerOH
Asp
CO2
Letting Go
Overall process of receptor/messenger interaction
M
M
ER
• 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
R
M
ER
Signal transduction
Signal transduction
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+, Ca2+, Cl-, K+)
• Ions flow across cell membrane down concentration gradient
• Polarises or depolarises nerve membranes
• Activates or deactivates enzyme catalysed reactions within cell
Closed or Opened?
Signal transduction
Hydrophilictunnel
Cellmembrane
Control of ion channels
Opening the Door
Cellmembrane
Five glycoprotein subunitstraversing cell membrane
Messenger
Cellmembrane
Receptor
Inducedfit
‘Gating’(ion channel opens)
Cationic ion channels for K+, Na+, Ca2+ (e.g. nicotinic) = excitatoryAnionic ion channels for Cl- (e.g. GABAA) = inhibitory
Bindingsite
Control of ion channels
Signal transduction
Control of ion channels:
Induced fit and opening
of ion channel
IONCHANNEL(open)
Cell
Cellmembrane
MESSENGER
Ionchannel
Ionchannel
Cellmembrane
IONCHANNEL(closed)
Cell
RECEPTORBINDINGSITE
Lock Gate Ion
channelIon
channelCell
membraneCell
membrane
MESSENGER
Signal transduction
GABAA Receptor
P2X4 Receptor Ion Channel
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
messenger
G-proteinsplit
inducedfit
closed open
Signal transduction
Activation of signal proteins• G-Protein subunit activates membrane bound enzyme
Binds to allosteric binding siteInduced fit results in opening of active site
• Intracellular reaction catalysed
active site(closed)
active site(open)
Enzyme
Intracellular reaction
Enzyme
Signal transduction
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
closed
messenger
inducedfit
active site open
intracellular reaction
closed
messenger
Signal transduction
Intracellular Receptors
Competitive Antagonists
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
ER
M
An
R
Irreversible Antagonists
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
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)
ENZYMEReceptor
Allostericsite
Binding site
(open)ENZYMEReceptor
Inducedfit
Binding siteunrecognisable
Antagonist
The Umbrella Effect
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 sitefor antagonist
Binding sitefor messenger
messenger
Receptor Receptor
Agonists
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
Clonidine
Dexmedetomidine
RECEPTOR THEORIES
Clark’s Occupancy Theory
Based on Law of Mass Action [R] + [D] [DR] Response The intensity of the response at any time was proportional to the number of receptors occupied by the drug:the greater the number occupied, the greater the pharmacological effect.drawbacks:Can’t explain inverse agonistDoesnot rationalize partial agonist
Derivation
Response effect E ∞ [DR] 2A maximum response would be obtained when all the
receptors were occupied Maximum response effect Emax ∞ [RT] 3
Where RT is the total number of receptors.
Thus for a given dose of a drug the fraction of maximum response is given byFraction of the maximum response E = [DR] 4 Emax [RT]The dissociation of the drug receptor complex may be
represented as D-R D + R 5
Derivation contd..
Applying the law of mass actionKD = [D] [R] 6 KD - dissociation constant for
[DR] the drug receptor complex.But the total receptor concentration is
[RT] = [R] + [DR] 7Substituting 7 in 6 gives KD = [D]([RT]-[DR]) 8
[DR]
Derivation contd…
Rearranging eqnKD = [D][RT] – [D][DR] 9
[DR] [DR] Therefore KD =[D][RT] – [D] 10
[DR]
And KD + [D] = [D][RT] 11
[DR]KD + [D] = [RT] 12
[D] [DR]Substituting 12 in 4 E = [DR] = [D]Emax [RT KD+ [D]
Derivation contd…
The eqn shows that the relationship between E and molar drug concentration [D] is in the form of a rectangular hyperbola whereas that between E and log [D] is sigmodial.
Substituting the value of E\Emax = ½ in Equation 9 gives the relationship
KD = EC50 ;value of dissociation constant is a measure of affinity of the drug for the receptor
Modified occupancy theory
By Ariens and StephensonPointed out 2 terms: agonist ,antagonistAccording to this ,drug – receptor interacion
involves two stagesComplexation-affinityInitiatian of the biological effect-efficacyDrawbacksDoes not account for why 2 drug occupy the same
receptors can act differently.
Major Postulates
• receptor-ligand complex is reversible.• Association is a bimolecular process, while
dissociation is a monomolecular process.• All receptors of a given class are equivalent and binds
to ligand independently of one another.• Formation of receptor-ligand doesnot alter free ligand
concentration or affinity of receptor for ligand.• Response elicited by the number of receptors occupied.
Rate theory
They proposed that the most important factor determining drug action is the rate at which drug receptor combination takes place.
Pharmacological activity is a function of the rate of association and dissociation of the drug with the receptor and not the number of the occupied receptors.
Drawback:
Does not rationalise why the different types of compounds exhibit the characteristics that they do.
Rate theory represented by :
E= Veqφ
E = Effect produced
φ =Proportionality factor
e =Efficacy component
V =Velocity Rate theory explained is only on the basis of RL formationIf the dissociation rate constant is large the ligand will be an
agonistIf the dissociation rate constant is small, the ligand will be an
antagonist
The Two state Model
Receptors can exist in either an active or inactive state.The active state is known as the relaxed or R state and the inactive state is referred to as the tensed or T state.
Receptors in the R state can provide a stimulus but those in the T state are unable to produce a stimulus.
The two state model postulates that in the absence of any ligands a population of receptors of the same type will consist of an equilibrium mixture of receptors in the R and T states
k1 k1- rate of forward reaction
T R k2 - rate of reverse reaction
k2 k1> k2Increased R receptors-tissue response k2> k1Increased T receptors- no response
Induced fit theory of Enzymes
As the drug approaches the receptor a confirmation change occurs in the receptor to allow for effective binding.
The receptor need not necessarily exist in the appropriate conformation.As the drug approaches the receptor a conformational change is induced which orients the essential binding site towards the approaching ligand
Proposed by Koshland.
Macromolecular perturbation theoryCombination of induced fit and rate theories.Two types of conformation changes exist and
rate of their existence determines biological response.
Agonist produce the specific perturbation required for biological response
Antagonist produce a non-specific perturbation which fails to yield a biological response
No account on the activity of partial agonist
Activation Aggregation
Even in the absence of the drug, the receptors are in dynamic equilibrium between the active form (R0) which is responsible for biological response and inactive form(T0).
Agonist shift the equilibrium to active form and antagonist shift to the inactive form.
Accounts for the activity of inverse agonist.
Classification of receptors based on their mechanism
Ligand-gated ion channels – nicotinic Ach receptors transmembrane GPCRs – opioid receptorsKinase linked receptors•Nuclear receptors
Ligand gated ion channels
G-PROTEIN COUPLED RECEPTOR
Enzyme linked receptor
Nuclear receptors
Intracellular receptor.Interact with chemical messengers like steroids,
thyroid hormones.• Hydrophobic in nature eg:oestrogen receptor
Nuclear receptors contd..
Receptor types and subtypesreceptor type subtype egs of
agonistsegs of antagonists
Cholinergic
Nicotinic
Muscarinic
4 Subtypes
M1-M5 GI motility,Glaucoma
Nm blockers and muscle relaxantsPeptic ulcersMotion sickness
Adrenergic
α1, α2
β
α1 A, α1B α1D, α1A- Α2cβ 1 β2 β3 Antiasthmatics β Blockers
Dopamine
D1-D5 Parkinsons disease
Antidepressants
Histamine
H1-H3 Vasodilation Allergies,ulcers,sedatives
Opioid ,ORL-1 Analgesics Antidote to morphine OD.
5 HT 5HT1-5HT7
5HT1A-B 5HT1D-F5HT2A-C5HT5A-B
Antimigraine
GI motility,
Antiemetics
Ketanserin
Oestrogen
Contraception Breast Cancer
Radioligand binding assay
Radiolabelled ligand
CELLS OR TISSUES CONTAINING TARGET
receptor
UNBOUND LIGANDS ARE SEPARATED BY
WASHING,FILTRATION OR CENTRIFUGATION
EXTEND OF BINDING DETECTED BY MEASURING THE RADIOACTIVITY PRESENT IN THE CELLS/TISSUES
AFTER EQUILIBRIUM
tissue containing receptor.
radiolabelled ligand.
technique to separate bound from free ligand.
apparatus for determination of samples radioactivity.
Essential requirements
Equilibrium constant for bound versus unbound radioligand is defined by dissociation binding constant,Kd
[L]+[R] [LR](receptor ligand complex)
Kd = [L] X [R] eqn -1
[ LR][L] and [LR] can be found by measuring
radioactivity of unbound and bound ligand respectively.
total no: of receptors present,
R total = no: of receptors occupied by ligand[LR] + those that are unoccupied[R].
No: of receptors unoccupied by a ligand.
[R] = R total –[LR] eqn 2
Kd and R total can be determined from Schatchard plot.
R total
Slope-measure of radioligand affinity for receptor ie, -1/KdThus Kd can be determined.
SCHATCHARD PLOT
Competition binding assays
Allows one to determine a rough estimate of an unlabeled ligand’s affinity for a receptor.
Competitive or non-competitive.Introduction into the incubation mixture of a non-
radioactive drug (e.g. drug B) that also binds to R will result in less of R being available for binding with D*, thus reducing the amount of [D*R] that forms. This second drug essentially competes with D* for occupation of R. Increasing concentrations of B result in decreasing amounts of [D * R] being formed.
The inhibitory or affinity constant Ki for the test compound is the same as the IC50 value if non competitive interactions are involved
For compounds that are in competition with the radioligand for the binding site the inhibitory constant depends on the level of radioligand present and is given by
Ki = IC50
1+[L]tot/Kd
References
Graham. L. Patrick An Introduction to Medicinal Chemistry,3rd edn, Pg No:43-57
Gareth Thomas, Medicinal Chemistry An Introduction Pg No:287-326
Burgers Medicinal Chemistry and drug discovery; Vol II, VIth edn Pg No:323-345
http://en.wikipedia.org/wiki/receptorhttp://www.chem.ace.dk/www/weeknotes
Thank you