5.
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
6.
Failure of arsenical in trypanosomes – lack of
binding
Agents cannot act unless they are bound
Selectivity of binding
7.
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
9.
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)
10.
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
12.
Equilibrium Dissociation constant (Kd).
However, it is not possible to measure [R],
So, Rtot = [R] + [LR] and [R] = Rtot -[LR]
Scatchard equation
14.
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
15.
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
18.
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
21.
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
22.
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
23.
Explained concept of partial agonism
Not able to explain steeper log dose response
relationship than expected from equation
24.
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
25.
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
27.
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
28.
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
29. Receptors are said to be ‘spare’ for a given pharmacological
response when the maximal response can be elicited by an
agonist at a concentration that not result in occupancy of the
full complement of available receptors
Spare receptor
Emax
Log Concentration
Respones(%)
Agonist
alone
Agonist with
noncompetitive
antagonist in
presence of spare
receptor
Agonist with
noncompetitiv
e antagonist in
absence of
spare receptor
30.
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
38.
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)
39.
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
40.
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
41.
No explanation about constitutive active receptor
Not able to explain about GPCR effect coupling
42.
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)
43.
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.
44.
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
49.
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
50.
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
51.
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)
52.
Acetylcholine (ACh): One drug with different
affinities for two different receptors
(adapted from Clark, 1933)
Muscarinic receptors
EC50 = apparent Kd ~ 3 x 10-8
M, pD2 ~7.5
Nicotinic receptor
EC50 = apparent Kd ~ 3 x 10-6
M, pD2 ~5.5
53.
Different affinities of related agonist drugs for the same
receptor: Different potencies
(adapted from Ariëns et al., 1964)
54.
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
55. 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.
61.
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
62.
Experimental Condition
for characterization
RESPONSE-Should be
1.Solely due to action on one type of receptors
2.Not be due to release of other active substance
3.Concentration of free drug –at steady level
4.Proper control –to any change in sensitivity of agonist
5.Sufficient time-for antagonist to act in equilibrium.
66.
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
67.
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.
68.
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)
69.
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
70.
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.
74.
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.
77.
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
78.
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
81. Images of cAMP Transients in Cultured Aplysia Sensory Neurons.
The cell was loaded with a fluorophore that would allow for the quantification
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 cytoplas
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 intracellula
[cAMP] increased ~ 20-fold.
