receptor theory: evaluating my therapeutic agent•the efficacy of coupling to signal transduction...
TRANSCRIPT
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Receptor Theory: Evaluating my therapeutic agent
Lee Limbird July 7, 2010
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Goals and ObjectivesGoals and Objectives• Sites of Drug Action / Drug Targets• Drug Properties
– Agonists and Antagonistsg g– Partial and Inverse agonists– Efficacy – Potency
• Dose-Response Curves• Impact of Amplifcation and so-called
‘spare receptors’
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Sites of Drug ActionPhysical Actions
fecal softenerlaxatives
Receptor-mediated Actions
membrane and intracellulartargets
osmotic diureticsg
Chemical Actions
antacidschelatorschelators
resins
Enzymatic Actions
digestiveNonreceptor-mediated Actions
Di i f t tg
thrombolyticanti-cancer
Disinfectantsvolatile anesthetics
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Most Agents work via ReceptorsMost Agents work via Receptors
What is a receptor?It is a bifunctional molecule―Recognizes and binds reversibly and with
extraordinary specificityextraordinary specificity―Initiates a functional consequence
i th t d thi ith thi.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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Receptor‐mediated effects demonstrate a gradeddose response relationshipdose‐response relationship
100se
A B
100
50 50
100
rcen
t l R
espo
ns
50
0
50
0
PeM
axim
al
EC50
[A] Log [A]
Graded Dose Response Curve(20-80% is essentially linear)
Threshold, Slope, and Maxima
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What dose response curves look likefor various types of drugsfor various types of drugs….
200 agonist
150Level of Response
(arbitrary units)
partial agonist
100(arbitrary units)
antagonist
50
inverse agonist
Log [Drug]
inverse agonist
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Agents at Receptor induce or stabilize conformations that activate response, blockconformations that activate response, block
occupancy, or suppress response( not a simple two-state model of on or off )
200 agonist
150Level of Response partial agonist
100
Level of Response (arbitrary units)
antagonist
50
Log [Drug]
inverse agonist
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Definitions for DrugsAgonist Drug which binds to the same receptor as theendogenous compound and produces the same type of signal
g
endogenous compound and produces the same type of signalas the endogenous hormone/neurotransmitter.
Antagonist Drug which binds to the same receptor as theendogenous compound and inhibits the signal produced bythe activating hormone/neurotransmitter.
Partial Agonist Drug which when maximally bound to thePartial Agonist Drug which when maximally bound to thereceptor causes a sub-maximal response.
Inverse Agonist (Negative Antagonist) Drugs which causeg ( g g ) ga decrease in basal receptor activity i.e. in the absence ofagonist.
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Receptor Theory 101p yAnalogy to Enzyme Kinetics:Substrate (S) + enzyme (E) ES → E + Product (P)
Drug- Receptor Interactions : Agonist (A) + Receptor (R) AR →→ effects
Th A i t hi h li it ff t d t tThe Agonist, which elicits effects, does not get modified, or changed, by interaction with the receptor
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Quantifying Drug EffectsQuantifying Drug Effects
D + R DR→→ effectD + R DR →→ effect
1 21 21) Affinity – describes “tightness” of
interaction between a receptor and druginteraction between a receptor and drug2) Efficacy – describes the “strength” or
“extent” of the pharmacological effectextent of the pharmacological effect3) Potency – in clinical settings, reflects both 1
and 2.and 2.
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Potency – reflected in the position of h dthe dose‐response curve
PotencyEC50
Response
50
Log [ Agonist]
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Efficacy reflected by the extent of ResponseResponse
Response
Effi
cacy
E
Log [ Agonist]
……. or the ratio of Kd to Kact
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Driving home the difference between d ffipotency and efficacy
Equal efficacy - curves A & BA B
Equal efficacy - curves C & DCD
espo
nse
Equal potency –curves A & D
Re
curves B & CLog [Drug]
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Disease, like native response, can result from changes in R, Effectors, or R‐E efficacy ofchanges in R, Effectors, or R E efficacy of
interactions
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Key Concepts …….
•Receptor activation can be described with mathematical models – model complexity increases with new data
• Response is not a linear relationship between ligand binding and activation ; there is AMPLIFICATION.
- Typically, only low R occupancy elicits full responseresponse
• Receptor mediated activity is affected by • the inherent properties of the ligand and its interaction• the inherent properties of the ligand, and its interaction with R ( induced or stabilized R conformations )• the Number of Receptors present•The efficacy of coupling to signal transduction machinery
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Amplification between occupancy and responsemeans there are so-called ‘ Spare’ Receptorsmeans there are so called Spare ReceptorsImplications for experimental interpretations due to ……
- impact of siRNA or chemical modification to reduce functional N b f tNumber of receptors- impact of overexpression of R by cDNA in cultured cellsor in transgenic animals
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R t ti ti b d ib d ith
Key Concepts Re-iterated•Receptor activation can be described with mathematical models.
• Receptor activation is not a linear relationship with ligand binding; there is AMPLIFICATION.g g;
• Typically, low R occupancy is required to elicit hi hhigh response.
• Receptor mediated activity is affected by• Receptor mediated activity is affected by •the inherent properties of the ligand• the number of receptors presentt e u be o ecepto s p ese t•The efficacy of coupling to signal transduction machinery
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How Receptors are ‘Defined’How Receptors are Defined
It’s all about specificity ….
• Order of agonist potency• Antagonist specificityAntagonist specificity
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[Response] Relationships Define Receptor SpecificityReceptor Specificity
100 100Decreasing Potency Decreasing Potency
50 50
EC50 Log [Agonist] EC50 Log [Agonist]
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α- & β-adrenergic receptors: Ahlquist’s landmark hypothesis of aAhlquist s landmark hypothesis of a single mediator with two receptors
100Smooth Muscle Contraction
e 100Cardiac Contraction ( and rate)
50cent
Res
pons
e
50ent
Res
pons
e
Perc
Max
imal
R 50
Perc
eM
axim
al R
Log [Agonist]
M
NE EPI ISO
Log [Agonist]
MIso EPI NE
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Specificity of AntagonistsSpecificity of Antagonists
Antagonists‐ Blocker drugsClassically, have no effect of their own, other than to block the receptors, so agonists can’t bind ( null,
titi t i t )competitive antagonists)
but there are other antagonist mechanisms that we….but there are other antagonist mechanisms that we now understand
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AntagonistsAntagonists
– CompetitiveCompetitive
Most common pharmacological antagonistsMost common pharmacological antagonists are competitive antagonists
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Non‐competitive Antagonistsl ialso exist
• Non‐competitive antagonism can be due to pseudo‐Non competitive antagonism can be due to pseudoirreversibility of antagonist binding
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Non‐competitive antagonism also can be d t ll t i ff tdue to allosteric effects
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Allosteric Modifiers can also potentiate,or sensitize the responsesensitize, the response
N t th t it f d ff t tNote the opportunity for drug effect at very low [agonist/endogenous agent] when a P iti ll t i difi i tPositive allosteric modifier is present
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Let’s talk In Vivo
• Human beings and their inherentHuman beings, and their inherent variability
• Animal studies- what agonist amplification f i l f i K lof signal means for measuring KD values
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Variability among individuals manifests itself in multiple aspects of dose response curvesin multiple aspects of dose‐response curves
Maximaleffect
effe
ct
variability
sity
of e
t
Inte
ns
concentration
potency
concentrationFixed dose-varying magnitudes Varying doses required to get a specific response
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Variability among individuals manifests itself in multiple aspects of dose response curvesin multiple aspects of dose‐response curves
Maximaleffect
effe
ct
variability
sity
of e
t
Inte
ns
concentration
potency
concentrationFixed dose-varying magnitudes Varying doses required to get a specific response
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Quantal Response CurvesQuantal Response Curves
d h bl d100 • Used when not able to do
full dose‐response 80
100Cumulative FrequencyDistributionin
g
curve60
Res
pond
i
• Y axis can be C l ti20
40 FrequencyDistribution
Num
ber R
– Cumulative response or
– Frequency distribution
20
75 10 2075 10 20
Concentration (mg/ml)
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Therapeutic Index describes relationship desired biological effect and toxicitydesired biological effect and toxicity
• Dose-dependentp• Therapeutic ratio or index
– Toxic (Lethal) dose50Toxic (Lethal) dose50
Effective dose50
• Margin of safetyMargin of safety– Toxic (Lethal) dose1
Effective doseEffective dose99
• Ratios less helpful when non- parallel curvesnon parallel curves
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Therapeutic Index versus Margin of Safety
100
ding
Drug A Drug B100
T iTh ti T iTh ti
50
rcen
t of
s re
spon
d
50
ToxicTherapeutic ToxicTherapeutic
0
Per
Subj
ects
01 3 10 30 100 300
01 3 10 30 100
Dose-log scale
0
toxic dose 50
therapeutic dose50Therapeutic Index =
toxic dose 1therapeutic dose99
Margin of Safety =
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How do investigators determine Kd values f i / i i i ?for agonists/antagonists in vivo ?
• Agonists:Agonists: – Remember, the dose for RESPONSE reflects not only BINDING but amplification to RESPONSEonly BINDING, but amplification to RESPONSE
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Estimation of Dissociation Constants ( Kd values)For Agonists From RESPONSE DATAFor Agonists From RESPONSE DATA
Since there are almost always ‘spare receptors’, musttreat cell tissue or animal agent to reduce functional [ R ]treat cell, tissue, or animal agent to reduce functional [ R ]
(classically: irreversible blocker/ currently: siRNA)
Certain assumptions must hold to do this determination….
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Estimation of Dissociation Constants of Agonists
DD + KD
= qD'
D' +KD
D'D
=1q+
D' (1 − q)KD * q or
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Estimation of Dissociation Constants of Agonists
Assumptions:
-agonist elicits a response of only one type by the reacting withonly one type of receptor in the preparation.
- When E is measured, [D] is essentially equal in the region ofreceptors and in the bathing medium (no accessibility problems).
-When E is measured, a steady state exists which is governed bymass action law relating to [DR] / [Rt] to [D] and Kd.
-there is no desensitization of R after interaction with D-there is no desensitization of R after interaction with D.
- Inactivation of receptors by agent completely irreversible, ie [ R]not changing during the course of analysisnot changing during the course of analysis
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Obtaining KD values for antagonists based on receptor response databased on receptor response data
- most common method for receptor classification when antagonist agents availableavailable
Schild Analysis of Antagonist Affinities at R
Ag + R Ag R
B RB + R BR
General Principles : Competitive antagonist acting on the same receptor will have the same p g g pKD regardless of the agonist activating R for which antagonist is competing ! .
Identical receptors in different tissues or experimental preparations should display the same dissociation constant for the same competitive antagonist.
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Schild Analysis
][]][[2
BRRB
kkKD ==
][1 BRk
pA = logKpA2 = -logKDB
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ReferencesCell Surface Receptors: A short course on theory and methods. Lee E. Limbird, Springer, Third edition 2004
I can provide an electronic version of this book for those interested
Goodman and Gilman: The Pharmacological Basis of Therapeutics Chapter 2Goodman 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.
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Receptor Theory:
Questions ? .