Benz p23

Further refinements of Drug-Receptor Theory:

Nonlinear Relationship between Occupancy and Response:
(EC50 ≠ KD)
Although the Ariens’ model, which incorporated receptor transduction processes, could
account for the existence of full and partial agonists, it could not account for the results
of additional pharmacological experimentation:

In Clarke and Ariens’

models, the response Potency
and occupancy curves Fig 2-7
for a full agonist were ) % Max

) % Max
superimposable A B C

Intrinsic
Activity
100% response
Response (

Occupancy (
required 100%
occupancy

Stephenson was the first

to suggest that it might
be unreasonable to
[Agonist] M (log scale)
assume that response
would be linearly related
to receptor occupancy
He observed that the log
concentration-response
curves for several drug-
receptor systems had
slopes far steeper than that
predicted from the law of
mass action.

Stephenson
Brit. J. Pharmacol. 11, 379-393 (1956)
100

80

60

40 16
20

0
10-10 10-9 4.0x10-9M 10-8
2.5x10-10M
 P&T p74-76
CH3 +

CH3 N R
CH3

In addition, concentration-response data he collected on a homologous series

of alkyl trimethylammonium agonists acting on the guinea-pig ileum suggested
that the EC50 for contractile response and the KA for receptor occupancy were
not numerically identical for the most potent agonists in the series.
If 1-5 represent steps in a biochemical signal transduction sequence leading to the final
pharmacological response, then one of these steps could reach its maximal level with
only a small fraction of the available receptors occupied.
Benz p24-25
G-Protein AC ↑ cAMP ↑ PKA ↑ P-Pi
A+R AR 1 2 3 4 5 Response
To account for experimental observations such as these, Stephenson proposed that, in
some receptor systems, the controlling factor for a full biological response may not be
receptor occupancy but rather some intervening process subsequent to occupation.
the nonlinearity between occupancy and response was handled by introducing the
term Stimulus as being equal to the efficacy (e) times the occupancy (p).
[A]
S = e ∗ (5)
Stephenson [A] + K D

= e ∗ p (fractional occupancy)

EA [A]
Ariens’ = αA ∗
Emax [A] + EC50
= αA ∗ p (fractional occupancy)
 [A] Benz p25
S = e ∗ (5)
[A] + KD
= e ∗ p (fractional occupancy)

(6)
Kenakin
Molecular Pharmacology
Blackwell Science 1997
 (7) (5)

Substituting for S in terms of efficacy and

occupancy gives:

(8)
P&T p79
Benz p25
Concentration-response curves predicted by
Fig 2-18
this model of drug action are presented in the
Figure. The concentration-occupancy curve,
assuming an agonist KD = 10-6 M, is also
plotted to serve as a reference point.

If the KD is fixed at 10-6 M and the efficacy is progressively increased by factors of 10, it can
be seen that the EC50 gets progressively smaller as the efficacy of the agonist increases.

This reflects the fact that significant amplification of the initial drug-receptor occupation
signal can occur.
Clearly, in this model, the EC50, which is a measure of the agonist’s potency, and the KD,
which is a measure of the agonist’s affinity, are no longer numerically identical.

EC50 can be much less than the KD

Kenakin
Molecular Pharmacology
Blackwell Science 1997
Sequential Series of Inputs and Outputs Leading to Final Response

Kenakin
A Pharmacology Primer Kenakin
Elsevier Academic Press 2004 Molecular Pharmacology
Blackwell Science 1997

Relatively Small Amount of Receptor Occupancy Could Result in a Maximal Response if a

series of Signal Amplification Steps Separate Receptor Occupancy from the Final Effect
 Benz p 25

Threshold Phenomenon

In some receptor systems, the reverse situation may operate.

The EC50 could be greater than the KD if some process in the signal transduction
pathway had to achieve some threshold level before passing the signal on to
subsequent steps.
Systems that behave in this way would be said to display a threshold
phenomenon.
 In Enzymology

Vmax @ [E3]
100
90
80
70 Vmax @ [E2]
Velocity

60
50
40 Vmax @ [E1]
30
20
10

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

[ Substrate A ] (nM)
Intrinsic Activity= α, Efficacy = e, Intrinsic Efficacy = ε (Furchgott)
Benz p 25

Vmax + [S] e + [A]

v = S =
Km + [S] KD + [A]

Vmax + S e + [A]
[ET] [RT]
v = S =
Km + S KD + [A]

kcat + [S] ε + [A]

v = S =
Km + [S] KD + [A]

Furchgott’s “intrinsic efficacy” ε = Stephenson’s e/[RT]

Intrinsic efficacy ε = efficacy per drug receptor complex

 Benz p 25

Stephenson [6]

Tissue Dependent

[A]
Furchgott Response % Max = f ------------ • ε • [RT]
[A] + KD

Drug Dependent
 Drug-Receptor Signal Transduction Mechanisms

Benz p16

Recognition Transduction

Intrinsic
Occupancy
Activity

Response
Affinity f & RT
Efficacy
Agonist Receptor Complex Stimulated
Receptor

Occupancy Model
 Tissue Dependent

[A]
Response % Max = f ------------ • ε • [RT]
[A] + KD

Drug Dependent

α receptor
agonists
 Tissue 3 Tissue 2
Oxymetazoline

Response
Norepinephrine

Tissue 3
Tissue 1

Response
Tissue 2
Norepinephrine
Response

f & RT
Oxymetazoline

Tissue 1

Stimulus

Norepinephrine
KD
Oxymetazoline
Occupancy-Response Curves for three Calcitonin receptors transfected into three
different β agonists in CHO cells different cell types with different Responses
transfected with the β-adrenergic receptor as f(Occupancy) for Calcitonin
 Benz p25

“Efficacy” vs. “Intrinsic Activity”

• Not identical mathematically

• Both reflect drug’s ability to “Stimulate” Receptor
• Consider identical for our purposes
• Term “Efficacy” has clinical connotation
• Competitive Antagonists have no molecular efficacy
but may have clinical efficacy
 Benz p25

Concept of agonist signal amplification:

If a drug’s KD and EC50 can be measured independently, then the

ratio KD/EC50 will be a measure of the efficacy of the agonist.
This ratio can be used to compare the relative efficacies of a
series of agonists for the same receptor site.

How do we measure KD ???

Competitive Binding Assay:

Incubate fixed [L*] with [R] in the P&T p56

presence of various [I] of the
competitor (I) and then assay for
[L*R] as a function of [I].

Define IC50 as the [I] which

displaces 50% of the specific
binding L*R, thus gives direct
measure of the relative
potencies of a series of
competitors e.g A > B > C > D
[I]
IC50 depends on KD
and KI and [L*] and [I] IC50
for two simultaneous KI = 1 + [L*]
equilibria.
KD
 Relative
Efficacies

the concentration needed for half-maximal occupancy and that needed

 Two-State Model of Drug Action
Benz p26

Possible Biochemical Basis for Intrinsic Activity (Efficacy)

KT
A + RT ART “inactive”
[RT] KT
L L = ------- >> 1 Intrinsic Activity = ------- - 1
[RR] KR
A + RR ARR “active”
KR

•Receptor exists in 2 conformational forms (RT & RR) KT = 10-6 M vs. KR = 10-9 M
•active form (RR) coupled to Response
•inactive form (RT) predominates in absence of drug KT = 10-6 M vs. KR = 10-7 M
•drugs vary in ability to bind to each form
•intrinsic activity is function of relative affinity for each KT = KR = Competitive Antagonist
form
 Two-State Model of Drug Action

closed open

LM

c = [A]/KAR M = KAR/KAT Mc = [A]/KAT

0.0013
0.000013 0.00013
0.0048

0.017

0.034

0.063

n = 4 subunits L = [R]/[T] = 5.6 KAT = 100

KAR decreased so M has values in figure

D. Colquhoun in Drug Receptors A Symposium

Ed. H.P. Rang, Univ Park Press, 1973
Two-State Model Can Predict Existence of “Inverse” Agonists

Inverse Agonists have higher affinity for the Inactive Form

Operational Model of Black and Leff, Proc. R. Soc. Lond. B. 220, 141-162 (1983)

KA

τ = [Rt]/KE
τ is a term that quantifies the power of the agonist to produce
a response (efficacy) and the ability of the transduction
system to process the receptor stimulus into a response
 Cubic Ternary Complex Model for G-Protein Coupled Receptors

βKact[G]/KG(1 + αγδ[A]/KA)
ρ= ------------------------------------------------------------------------------------
[A]/KA(1+αKact+γ[G]/KG(1+αβδKact)) + [G]/KG(1+βKact) + Kact+1 Kenakin
A Pharmacology Primer
Elsevier Academic Press 2004
 Benz p28

Molecular Mechanisms of Tachyphylaxis or Tolerance

• Pharmacodynamic: e.g. desensitization,

down regulation
• Pharmacokinetic: e.g. enzyme induction
Nicotinic Acetylcholine Receptor (skeletal muscle) Benz p28

Katz & Thesleff Receptor Desensitization Model

R AR A2R A2 R*

K1 K2

K1’ K2’

[R’]
R’ AR’ A2R’ D= << 1
[R]

If K1’ & K2’ << K1 & K2 → desensitization

↓ Affinity of
β2 AR for GS

DRC shifts
to right
 Beta 2 Adrenergic Receptor (βAR)

Down Regulation

Opposite possible
Supersensitivity/Up Regulation

desensitization