Role of receptors in drug design

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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


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


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


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


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


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


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


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

http://en.wikipedia.org/wiki/receptor

http://www.chem.ace.dk/www/weeknotes

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Role of receptors in drug design