Receptor Theory: Evaluating my therapeutic agent

Lee Limbird July 7, 2010

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’

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

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.

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

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

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

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.

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

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.

Potency – reflected in the position of h dthe dose‐response curve 

PotencyEC50

Response

50

Log [ Agonist]

Efficacy reflected by the extent of ResponseResponse

Response

Effi

cacy

E

Log [ Agonist]

……. or the ratio of Kd to Kact

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]

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

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

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

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

How Receptors are ‘Defined’How Receptors are Defined

It’s all about specificity ….

• Order of agonist potency• Antagonist specificityAntagonist specificity

[Response] Relationships Define Receptor SpecificityReceptor Specificity

100 100Decreasing Potency Decreasing Potency

50 50

EC50 Log [Agonist] EC50 Log [Agonist]

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

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

AntagonistsAntagonists

– CompetitiveCompetitive

Most common pharmacological antagonistsMost common pharmacological antagonists are competitive antagonists

Non‐competitive Antagonistsl ialso exist

• Non‐competitive antagonism can be due to pseudo‐Non competitive antagonism can be due to pseudoirreversibility of antagonist binding

Non‐competitive antagonism also can be d t ll t i ff tdue to allosteric effects

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

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

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

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

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)

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

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 =

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

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….

Estimation of Dissociation Constants of Agonists

DD + KD

= qD'

D' +KD

D'D

=1q+

D' (1 − q)KD * q or

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

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.

Schild Analysis

][]][[2

BRRB

kkKD ==

][1 BRk

pA = logKpA2 = -logKDB

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.

Receptor Theory:

Questions ? .