methods of receptor characterization, theories of receptors occupancy, scatchard's plot, schild's plot

1. Dr AMIT RELHAN

2. 
 A MACROMOLECULAR PROTEIN WHICH BINDS
TO SPECIFIC FUNCTIONAL GROUPS OF
ENDOGENOUS SUBSTANCE OR A CHEMICAL
SUBSTANCE.
DEFINITION OF RECEPTOR

3. 
Dose response curve
 Maximal ceiling effect
 Position of curve (EC50 & pD2)
 Slope of curve

4. 

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

8. 
Theories of drug receptor
interactions

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

11. 
 K1[L][R]= K2[LR]
 K1 /K2=[LR]/[L][R]=Ka –equilibrium association
constant
 1/Ka= Kd=[L][R]/[LR]
 Kd –equilibrium dissociation constant

12. 
Equilibrium Dissociation constant (Kd).
However, it is not possible to measure [R],
So, Rtot = [R] + [LR] and [R] = Rtot -[LR]
Scatchard equation

13. 
Scatchard plot

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

16. 

17. 

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

19. 
log

20. 

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

26. 

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

31. 

32. 

33. 

34. 

35. 

36. 

37. 

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

45. 

46. 

47. 
 Seifert 2002- inverse agonist concept
 Inverse agonist stabilize receptor in inactive
conformation
 IA-0 to -1
Agonist independent
/constitutive activity

48. 

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.

56. 

57. 

58. 
Different
pA2
values
(affinities)
for different
receptors
of some
clinically
useful
drugs:
The basis of
therapeutic
selectivity

59. 

60. 

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.

63. 
USE OF RADIOLABELLED
LIGANDS

64. 
RADIOLABELLED
LIGANDS
a) 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

65. 

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.

71. 

72. 
 Peroutka & Snyder (1979)
 5HT1 – high affinity for [3H]5HT
 5HT2 – low affinity for [3H]5HT, but
high affinity for [3H] spiperone

73. 

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.

75. 

76. 

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

79. 
 Sequence homology of receptor
 Alpha1a,1b,1d – 70%
 Alpha 1 & 2 – only30%

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.

82. 
 Greengard et al. – nigrostriatal cAMP for dopamine
receptor identification
 D1 - increase cAMP
 D2 – decrease cAMP

83. 