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methods of receptor characterization, theories of receptors occupancy, scatchard's plot, schild's plot
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Biological and mathematical interpolation of receptor in
tissue
Dr AMIT RELHAN
A MACROMOLECULAR PROTEIN WHICH
BINDS TO SPECIFIC FUNCTIONAL GROUPS OF ENDOGENOUS SUBSTANCE OR A CHEMICAL SUBSTANCE.
DEFINITION OF RECEPTOR
Dose response curve
Maximal ceiling effect Position of curve (EC50 & pD2) Slope of curve
Claude Bernard- curare- injected frog skin –
progressive dimunition of motor reflex ( electric stimulus to muscle – contraction)
Curare acting on neither muscle nor nerve- NMJ
Paul Ehrlich –preferrential accumulation of Pb in CNS
Differential staining of tissue using dyes Arsenicals for T. pallidum- some sort of
selectivity for parasite (magic bullet)
Concept of receptor
Failure of arsenical in trypanosomes – lack of
binding Agents cannot act unless they are bound Selectivity of binding
J.N.Langley –autonomic transmission & NM
communiation Frog gastrocnemius- nicotine- contraction Blocked by curare ( even after degeneration of
nerve) Direct stimulation – contrction Both curare & nicotine act on same substance Receptive substance ∞ conc. of drug (potency) & its affinity to
receptive substance
Theories of drug receptor interactions
Studied antagonism b/w Ach & atropine in various
muscle preparation
Effect of drug is proportional to the fraction of the
receptor occupied by the drug
Maximum effects are produced when all receptors
are occupied Drug receptor complex breakdown at rate
proportional to rate of complex formed Saturability, Reversbility ,Dynamic equilibrium
Classical receptor theory by Clark(1937)
Based on law of mass action(isotherm equation -
langumuir) k1
[L] + [R] < ===[LR] k2
rate of association = K1[L][R] rate of dissociation = K2 [LR] At equilibrium, rate of association=rate of
dissociation
K1[L][R]= K2[LR] K1 /K2=[LR]/[L][R]=Ka –equilibrium association
constant 1/Ka= Kd=[L][R]/[LR] Kd –equilibrium dissociation constant
Equilibrium Dissociation constant (Kd).
However, it is not possible to measure [R], So, Rtot = [R] + [LR] and [R] = Rtot -[LR]
Scatchard equation
Scatchard plot
The Kd is the same for a given receptor and
drug combination in any tissue, in any species (as long as the receptor is the same)
The Kd can therefore be used to identify an unknown receptor
The Kd can be used to quantitatively compare the affinity of different drugs on the same receptor
Studied-Ach induced contraction of frog rectus
Ms. Ach induced inhibition of electrically
stimulated frog ventricle Slope of log concentration- response curve Linear correlation b/w occupancy &response
Slope of log conc.- response curve--- steeper
than predicted from mass action equation. Sometime even supramaximal conc.—not able
to elicit maximal contractile response. Dualism of homologus series of quaternary
ammonium salt in muscle preparation• butyl-/lower member of series-full contraction• Hexyl/heptyl-higher member-weak contraction• Applied simultaneosly with butyl - antagonism
Shortcomings of Clark theory
log
Webb (1950)- when the cholinesterase of isolated rabbit
auricles is blocked with physostigmine, the slope of the acetylcholine log-concentration-effect curve is about 10 times steeper than on normal auricles
Studied Dual behavior of phenylethylamine –in
cat BP experiments Agonistic and antagonistic effect –single
recptor Introduced intrinsic activity(IA)- ability of a
drug to elicit effect Effect ,E=α [LR] For max response –maximal occupancy not
required
Ariens theory
Explained concept of partial agonism
Not able to explain steeper log dose response relationship than expected from equation
Alquist (1948) Concentration- response curve of tissues or
organs of different receptor systems obtained rank order of potency was "adrenaline >
noradrenaline > α-methyl noradrenaline > isoprenaline" in promoting contraction of blood vessel- α-adrenoceptors
the rank order was "isoprenaline > adrenaline > α-methyl noradrenaline > norepinephrine" in the heart-β-adrenoceptors
Clark equation– conc. Of drug & conc of drug
receptor complex formed Tabulated slopes of log conc response curve in
literature- steeper than predicted Ach & histamine on guinea pig ileum- greater
response than predicted from receptor occupancy
Response not linearly ∞ to fractional receptor occupancy- only small fraction- max effect –receptor reserve
Stephenson theory
Concept of efficacy- capacity to start response Response=f. (stimulus)=f.(e.y) e=efficacy , y = fractional receptor
occupancy Explained dual behavior of homologus series Lower /butyl-high efficacy- agonist Higher/ hexyl-low efficacy- partial agonist Affinity but no efficacy- antagonist
Nickerson 1956- 1% histamine receptor occupancy
– max response in guiena pig ileum Furchgott 1955- studied antagonism by β
haloalkylamine on effect of adenaline - shift of only half log unit.
Goldstein 1974-studies on receptor antagonism β haloalkylamine- irrevesible antagonism of
histamine & catecholamine recpeptor Low dose- only rightward shift of drc(spare
receptor) High dose- both rightward shift & ↓max effect
Spare receptor
Receptors are said to be ‘spare’ for a given pharmacologicalresponse when the maximal response can be elicited by anagonist at a concentration that not result in occupancy of thefull complement of available receptors
Spare receptorEmax
Log Concentration
Res
pone
s(%
) Agonist alone
Agonist with noncompetitive antagonist in presence of
spare receptor
Agonist with noncompetitive antagonist in absence of
spare receptor
Studied competititive antagonism of
adrenaline by ergotamine on rabbit uterus. concepts of ‘dose-ratio’ Schild regresssion analysis –pA2 value & Kb DR-1=[B]/Kb Gaddum equation Log(DR-1)=log[B]-logKb Schild equation
Schild & Gaddum
Excitation by agonist (eg.nicotine)—block function Effect of agonist –fade with time Excitation ∞ rate of drug receptor interaction than
no. of receptor occupied
Agonists dissociates rapidly ,Kd-high
Antagonists slowly, Kd-low
Explained Persistent effect of an antagonist on a tissue
Explained tachypylaxis
Paton theory(1961)
Receptors exist in discrete conformational states Hill –O2 binding to Hb- steeper curve MWC model-1965 2 conformational state of receptor- equilibrium in
absence of ligand Ligand binding –displacement of equilibrium to state
having higher affinity The extent to which the equilibrium is shifted toward
the active state is determined by the relative affinity of the drug for the two conformations
Concept of coopertivity
Allosteric theory
The binding of a ligand to a macromolecule is
often enhanced if there are already other ligands present on the same macromolecule (this is known as cooperative binding). The Hill coefficient
Log(θ/1-θ)=nlog[L] - log Kd θ – fraction of occupied site where ligand can bind n >1 - Positively cooperative binding n<1- Negatively cooperative binding n=1- Non cooperative binding
Hill langmuier equation
No explanation about constitutive active
receptor Not able to explain about GPCR effect coupling
Black & Leff et al- mathematical model Diff. in relative potency order of ligands in tissues
with different recp. Reserve Receptor in diff. conformational state due to allostery [LR]=[Rtot][L]/KA+[L] Rectanguler hyperbola equation Concept of transducer ratio, τ =[Rtot]/Ke τ- efficiency by which occupancy transduced to
response
Operational model of agonism(1983)
Effect, E=Em[LR]n/Ke+[LR]n n>1 steep curve n<1 shallow curve n=1 linear relation
No insight into [LR] to E –linking event ,but provide τ- quantaive measurement of effect.
Leff & Hall et al. 2000 Difference in potency order for single receptor
interacting with different G protein Difference signal trasduction output from
same receptor To isolate pathway-pertusis toxin sensitive
Gi/Go coupled signal or G protein selective disrupting peptide
3 state model /ternary complex
Seifert 2002- inverse agonist concept Inverse agonist stabilize receptor in inactive
conformation IA-0 to -1
Agonist independent /constitutive activity
Methods of Characterization of Receptors
1. On basis of Pharmacological Responses
2. Radioligand binding studies
3. Molecular Cloning techniques
4. Analysis of biochemical pathway linked to
receptor activation
On Basis of Pharmacological Responses
a) Relative potency (Affinity) measurements of a
series of Agonists
b) Determination of Affinity or Dissociation
constant of Antagonists
c) Isomeric activity ratio of agonists
Relative potency(affinity) measurements of a series of
agonists.
Alquist (1948) Furchgott(1967)observed similar potency series adrenaline > nor-adrenaline > phenylephrine>
isoprenaline By calculating correlation coefficients of two
systems
e.g.sympathomimetics-
bronchodilatation/vasodepression-0.96 (similar)
Cardiac stimulation-bronchodilation-0.31(different)
Acetylcholine (ACh): One drug with different affinities for two different receptors
(adapted from Clark, 1933)
Muscarinic receptorsEC50 = apparent Kd ~ 3 x 10-8 M, pD2 ~7.5
Nicotinic receptorEC50 = apparent Kd ~ 3 x 10-6 M, pD2 ~5.5
Different affinities of related agonist drugs for the same receptor: Different potencies
(adapted from Ariëns et al., 1964)
Determination of affinity or dissociation constant of
antagonist-p(A2) or p(KB)
Schild plot-Different tissue with similar receptors- same value
e.g. acetylcholine –atropine in frog heart ,chick amnion ,mammalian intestine
A Schild plot -compares the reciprocal of the dose ratio versus the log of the antagonist concentration
Intercept on absicca- pA2 = log Kd, which represents the affinity of the competitive antagonist
pA2 (log molar concentration of antagonist producing a 2fold shift of the concentration response curve.
Different
pA2
values (affinities)for different receptors of some clinically
useful drugs:The basis of therapeutic selectivity
ISOMETRIC ACTIVITY RATIO
IAR=Antilog (negative molar EC50 of L-isomer minusEC5O of D-isomer)
High ratio – specific interaction eg-Isoprenaline (L) -35 times potent than (D)
Similarity of ratio to Enantiomorphs- similar receptors
Experimental Condition for characterization
RESPONSE-Should be1.Solely due to action on one type of receptors 2.Not be due to release of other active
substance3.Concentration of free drug –at steady level4.Proper control –to any change in sensitivity of
agonist5.Sufficient time-for antagonist to act in
equilibrium.
USE OF RADIOLABELLED LIGANDS
USE OF RADIOLABELLED LIGANDSa) Direct binding studies
Eg labelled bungarotoxin binds specifically and irreversibly to cholinergic receptors
Phenoxybenzamine –an irreversible alpha blocker .
Relative proportion of B1 and B2 receptor 4:1-Heart 2:1cerebral cortex 1:3 lungs
Scatchard Plots
Kd and R tot cannot be measured directly. plot the ratio of bound/unbound drug,[LR]/[L]
versus bound drug[LR]. intercept of the line with the abscissa -total
number of receptors available(Bmax)or R tot. Kd (the dissociation constant) from the
negative reciprocal of the slope of the line
b) Indirect radioligand binding/Competition binding
The affinity of un-labeled compounds –by competition binding. - - displacement from the binding site.
The concentration of un-labeled ligand which displaces half of the tagged is interpolated from the curve and refered to as the IC50 value.
The IC50 value -used to estimate the affinity
of the unlabeled ligand -Ki values. Rank order Ki values are a type of fingerprint
for a receptor subtype. Comparison between EC50 values (rank order
potency) and Ki/KD values (rank order affinity)
If Bmax remains unchanged and the slope of the lines decreases with increasing concentrations of the compound, the displacement is competitive. Unchanged slope and decreased Bmax indicate that the displacement is noncompetitive
The lower the IC50 or Kd, the higher the affinity
Ki value
Ki, the inhibitory (or affinity) constant of the displacer compound
when the displacement is noncompetitive (Ki = IC50)
for a competitive displacement -Cheng-Prusoff equation
Ki = IC50/(1 + [L]/Kd) [L]= concentration of the radioactive ligand.
Peroutka & Snyder (1979) 5HT1 – high affinity for [3H]5HT 5HT2 – low affinity for [3H]5HT, but high affinity for [3H] spiperone
Allosteric interaction
The affinity shift, -the ratio of radioligand affinity in the presence (KApp) to that obtained in the absence (KA) of each concentration of antagonist.
A plot of log (affinity shift1) versus log [antagonist] should yield a straight line
slope of 1 for a competitive interaction, curvilinear plot for an allosteric interaction.
Protean Agonism
After Proteus, the Greek god
Ligands act as partial agonists in quiescent silent systems
As inverse agonists in systems that show a high level of constitutive activity.
Agonist produces an active conformation of lower efficacy than a totally active conformation
Molecular Cloning
Heterogeneity of receptor, distinct sequences and
tissue distribution
Receptor-labelling tech. made it possible to extract
and purify receptor material
Firstly this approach used on Nicotinic ACh recp. in
1970
Transgenic & receptor knockout mice- subtypes of
receptor
Sequence homology of receptor Alpha1a,1b,1d – 70% Alpha 1 & 2 – only30%
Analysis of biochemical pathway linked to receptor
activation
Images of cAMP Transients in Cultured Aplysia Sensory Neurons.
The cell was loaded with a fluorophore that would allow for the quantification of cAMP concentrations within the cell.
A: Free cAMP in the resting cell is < 5 X 10-8 M.B: Stimulation with serotonin, activates adenylate cyclase increasing cytoplasmic cAMP to ~ 1 X 10-6 M (red), especially within fine processes with a high surface to volume ratio. Thurs, within 20 sec of stimulation, the intracellular [cAMP] increased ~ 20-fold.
Greengard et al. – nigrostriatal cAMP for
dopamine receptor identification D1 - increase cAMP D2 – decrease cAMP
Thank you