Receptor Theory:
Evaluating my therapeutic?
Goals and Objectives
• Sites of Drug Action / Drug Targets
• Drug Properties
– Agonists and Antagonists
– Partial and Inverse agonists
– Efficacy
– Potency
• Dose-Response Curves
Joey Barnett, Ph.D.
Sites of Drug Action
Physical Actions
Receptor-mediated Actions
fecal softener
laxatives
osmotic diuretics
membrane and intracellular
targets
Chemical Actions
antacids
chelators
resins
Enzymatic Actions
digestive
thrombolytic
anti-cancer
Nonreceptor-mediated Actions
Disinfectants
volatile anesthetics
• Therapeutic Index & Margin of Safety
Most Agents work via Receptors
What is a receptor?
It is a bifunctional molecule
―Recognizes and binds reversibly and with
extraordinary specificity
―Initiates a functional consequence
i.e. the receptor does something with the
information that it is occupied
Receptors represent a diverse array of structural
and functional entities.
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Definitions
Receptor Theory 101
Agonist Drug which binds to the same receptor as the endogenous
compound and produces the same type of signal as the endogenous
hormone/neurotransmitter.
Enzyme Kinetics:
Antagonist Drug which binds to the same receptor as the endogenous
compound and inhibits the signal produced by the activating
hormone/neurotransmitter.
Substrate (S) + enzyme (E)
Partial Agonist Drug which when maximally bound to the receptor causes a
sub-maximal response.
Inverse Agonist (Negative Antagonist) Drugs which cause a decrease in
basal receptor activity i.e. in the absence of agonist.
Affinity A measure of the strength of interaction between a receptor and its
cognate ligand; KD is defined as the concentration at which half of the receptor
is occupied.
Efficacy A measure of the efficiency in which a bound ligand activates its
target receptor’s signal transduction/biological response.
ES → E + Product (P)
Agonist (A) + Receptor (R)
AR →→ effects
Agonist is a Drug that elicits a biological effect.
The agonist does not get modified, or changed, by
interaction with the receptor
Potency An overall measure of the ability of a ligand to activate its target
receptor. Related to the EC50 ;the concentration at which a half maximal
effect is achieved.
Clark - ”Simple Occupation Theory”
Assumptions of A. J. Clark:
Law of Mass Action
k1
D+R
DR
k2
k 2 [D][R]
KD = =
[DR]
k1
D = drug
R = receptor
DR = receptor/drug complex
R + DR = RTOT
k1 = on rate
k2 = off rate
KD equilibrium dissociation constant KD = k2 / k1 units are M
KA equilibrium association constant KA = k1 / k2 units are M-1
KD = 1/ KA
Responses to drugs result from formation of reversible complexes with
receptors.
One molecule of drug occupies one receptor.
The response to a drug is proportional to the number of receptors occupied.*
A maximum response to a drug occurs when all receptors are occupied
(clearly not always true).*
The total amount of drug bound to receptors is negligible relative to the total
amount of drug present.
The concentration of DR is affected by both [D] and [R]!
Therefore receptor density will affect the dose response.
The more receptors are present, the more leftward shifted will be the dose
response curve.
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Two of Clark’s assumptions do not hold
Receptor‐mediated effects demonstrate a graded
dose‐response relationship
Percent
Maximal Response
Spare Receptors
A
B
100
100
50
50
0
0
EC50
[A]
Log [A]
A maximum response to a drug occurs when all receptors are occupied
(clearly not always true).*
Graded Dose Response Curve
The response to a drug is proportional to the number of receptors
occupied.*
Threshold, Slope, and Maxima
Depiction of agonist receptor interaction
response = ƒ[agonist][receptor]KA•e
where KA = equilibrium affinity constant and e =
efficiency of receptor-effector coupling leading
to biological response
In various diseases
response is observed.
a change in
Classic endocrine disorders: due to a change in agonist availability
Change in KA = consequence of receptor mutations, post-translational modifications
(especially phosphorylation)
Changes in e = typically due to post-translational modifications mediated by downstream
kinases, phosphatases, or changes in expression levels of downstream proteins
Changes in R = mutation; perturbation of expression or turnover by other biological
processes
(20-80% is essentially linear)
Key Concepts
Receptor activation can be described with mathematical
models.
Receptor activation is not linear with ligand binding.
Usually relatively low receptor occupancy is required to
elicit relatively high response. I.e., 25% activation takes
place at less than 25% occupancy.
Receptor mediated activity is affected by the inherent
properties of the ligand, the number of receptors present
and the characteristics of the signal transduction
machinery.
Numerous biological processes reflect changes in signal transduction with
development, disease, aging, etc., it has been necessary to obtain quantitative data
on RECEPTOR OCCUPANCY and SIGNALING EFFICIENCY
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Definitions of therapeutic drugs
What dose response curves look like
for these various types of drugs….
1. Agonists ‐ “doer” drugs
‐ Mimic the actions of endogenous agents
‐ Activate receptors
‐ Both “full” and partial agonists
200
150
Simple Model
D●Ri
D●Ra
antagonist
100
Classically, have no effect of their own, other than to block the receptors, so agonists can’t bind ( null, competitive antagonists)
….but there are other antagonist mechanisms that we now understand
Ra
partial agonist
Level of Response
(arbitrary units)
2. Antagonists‐ Blocker drugs
Ri
agonist
50
inverse agonist
Log [Drug]
Quantifying Drug Effects
D + R DR → → effect
200
agonist
150
Level of Response
(arbitrary units)
100
Ra >>>Ri
partial agonist
Ra >Ri
antagonist
Ra=Ri
50
inverse agonist
Ri >>>Ra
1 2
1) Affinity – describes “tightness” of interaction between a receptor and drug
2) Efficacy – describes the “strength” or “extent” of the pharmacological effect
3) Potency – in clinical settings, reflects both 1 and 2.
Log [Drug]
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Potency & Efficacy
Potency & Efficacy
Potency
Potency
Efficacy
EC50
Potency & Efficacy
Driving home the difference between potency and efficacy
B
Efficacy
Response
A
D
Log [Drug]
C
Equal efficacy - curves A & B
Equal efficacy - curves C & D
Equal potency –
curves A & D
curves B & C
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[Response] Relationships Define
Receptor Specificity
Maximal
effect
Decreasing Potency
Decreasing Potency
100
100
50
50
op
e
variability
sl
Intensity of effect
Variability among individuals manifests itself in multiple aspects of dose‐response curves
potency
EC50
Log [Agonist]
EC50
Log [Agonist]
concentration
Fixed dose-varying magnitudes
Varying doses required to get a specific response
Smooth Muscle Contraction
Cardiac Chronotropy & Inotropy
100
100
Percent
Maximal Response
Percent
Maximal Response
Alpha- & Beta-adrenergic receptors:
Ahlquist’s landmark hypothesis of a
single Mediator with two receptors
50
Blood Pressure Effects
50
NE EPI ISO
Iso EPI NE
Log [Agonist]
Log [Agonist]
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Which agonists act at the same site?
Irreversible Blockade
Estimation of Dissociation Constants of Agonists
For the following derivations and approach, the following assumptions are made:
the agonist elicits a response of only one type in the preparation and by the
reacting with only one type of receptor.
the population of this type of receptor is uniform with respect to KD both
before and after the irreversible inactivation of a fraction of the receptors, E is
the same function of S and S has the same relationship to [DR].
When E is measured, [D] is essentially equal in the region of receptors and in
the bathing medium (no accessibility problems).
A negligible fraction of D is bound to receptors.
When E is measured, a steady state exists which is governed by mass action
law relating to [DR] / [Rt] to [D] and Kd.
there is no desensitization of R after interaction and dissociation with D.
Inactivation of receptors by an agent such as dibenamine is completely
irreversible for all practical purposes.
Using dibenamine as an irreversible blocking agent, in rabbit aortic strips,
Furchgott demonstrated that in rabbit aortic strips, cross-protection occurred
among epinephrine (epi), norepinephrine (norepi) and isoproterenol (iso). This was
taken as evidence that they all acted on the same receptor. None of the
catecholamines (epi, norepi, iso) protected against inactivation of receptors for
histamine, acetylcholine or serotonin, and none of the latter protect among
themselves or towards catecholamines.
Stephenson’s concept of spare receptors is essential to understanding the
approach, as is the use of irreversible receptor antagonists to demonstrate the
existence of spare receptors.
Based on these findings, Furchgott deduced the existence of four receptor
subtypes in rabbit aorta, all of which could mediate contraction.
Furchgott conceptualized protection experiments as either “self protection” or
“cross protection”:
⎛ DR ⎞
E
= f (S) = f ⎜e
⎟
⎝ RTOT ⎠
E max
When spare receptors are present, a straightforward determination of KD by
equating it with EC50 is impossible. If only a small fraction of receptors must be
occupied to produce a maximum response, the EC50 will be less than KD. The
approach of Stephenson and Furchgott is to inactivate a fraction of the receptors
with an irreversible antagonist and then to mathematically treat dose response
curves before and after receptor inactivation to estimate both KD and the fraction of
receptors inactivated (or remaining activated).
See Furchgott, R., J Pharm Exper Ther 111:265, 1954.
Estimation of Dissociation Constants of Agonists
Receptor classification by antagonists
Classification of receptors by antagonists is perhaps the most common method
for receptor classification
Determination of antagonist KD on tissues
Schild Analysis of Antagonist binding.
General Principles
Competitive antagonist acting on the same receptor will have the same KD
regardless of the agonist which is displaced.
D' 1 D' (1 − q)
= +
D q
KD * q
or
D
D'
=q
D + KD
D' +KD
Identical receptors in different tissues or experimental preparations should
display the same dissociation constant for the same competitive antagonist.
Theory/Calculations
This is for strictly competitive, reversible antagonists. The ability to shift the
dose-response curve is influenced by the antagonist affinity (KDB) for the
receptor and the antagonist concentration ([B]).
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Schlid Analysis
Antagonists
– Competitive
pA2 = -logKDB
Non-competitive Antagonists
also exist
Most common pharmacological antagonists are competitive antagonists
Non-competitive antagonism also
can be due to allosteric effects
• Non-competitive antagonism can be due to
pseudo-irreversibility of antagonist binding
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Allosteric Modifiers can also potentiate,or
sensitize, the response
Quantal Response Curves
Therapeutic Index describes relationship
desired biological effect and toxicity
• Dose-dependent
• Therapeutic ratio or index
– Toxic (Lethal) dose50
Effective dose50
• Margin of safety
– Toxic (Lethal) dose1
Effective dose99
• Ratios less helpful when
non- parallel curves
80
• Used when not able to do full dose‐response curve
Cumulative
Frequency
Distribution
60
40
Frequency
Distribution
• Y axis can be 20
5
7
10
– Cumulative response or – Frequency distribution
20
Concentration (mg/ml)
Therapeutic Index versus Margin of Safety
Percent of
Subjects responding
Note the opportunity for drug effect at
very low [agonist/endogenous agent]
when a Positive allosteric modifier is
present
Number Responding
100
100
100
Drug A
Therapeutic
Toxic
Therapeutic
Toxic
50
50
0
Drug B
1
3
10
30 100
0
1
3
10
30 100
300
Dose-log scale
Therapeutic Index =
Margin of Safety =
toxic dose 50
therapeutic dose50
toxic dose 1
therapeutic dose99
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References
Cell Surface Receptors: a short course on theory and methods. Lee Limbird,
Springer, Third edition 2004
Receptor Theory:
Evaluating my therapeutic?
Goodman and Gilman: The Pharmacological Basis of Therapeutics. Chapter
2-Mechanisms of Drug Action and Relationship between Drug Concentration
and Effect. EM Ross & TP Kenakin. Hardman and Limbird, eds. 10th edition,
2001.
Joey Barnett, Ph.D.
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