Intrinsic Relative Activity (RAIntrinsic Relative Activity (RA ii) of an ) of an

Agonist: Understanding Agonist-Agonist: Understanding Agonist-Receptor Behavior Simply from Agonist Receptor Behavior Simply from Agonist

Concentration-Response CurvesConcentration-Response Curves

John A TranJohn A TranPh.D. Dissertation DefensePh.D. Dissertation Defense

October 24October 24thth, 2008, 2008

Receptor TheoryReceptor Theory

Stimulus – Agonist Stimulus – Agonist binding and receptor binding and receptor activationactivation

Transducer – Transducer – downstream signaling downstream signaling mechanismsmechanisms

Response – result of Response – result of receptor activation receptor activation that is measuredthat is measured

Characterization of Agonist ActivityCharacterization of Agonist Activity

ECEC5050 – concentration – concentration

of agonist that elicits of agonist that elicits half-maximal half-maximal responseresponse

EEmaxmax – maximal – maximal

responseresponse

50

max

ECx

Exresponse

n

n

Affinity (1/Affinity (1/KKXX)– ability )– ability

of the agonist to bind of the agonist to bind the agonistthe agonist

Efficacy (Efficacy () – ability of ) – ability of the agonist to turn on the agonist to turn on the receptorthe receptor

Response

Stimulus

Transducer

Transducer function unknown

XRstimulus

x

T

Kx

xRXR

50

max

ECx

Exresponse

n

n

Furchgott AnalysisFurchgott Analysis

Partial inactivation of receptors by irreverisble Partial inactivation of receptors by irreverisble antagonistantagonist– Comparison of equiactive concentrations of the agonist before Comparison of equiactive concentrations of the agonist before

and after receptor alkylation.and after receptor alkylation.

x

T

x

T

Kx

qRxtransducerresponsetransducer

Kx

xR

'

'

(Furchgott, 1966; Furchgott and Bursztyn, 1967)

q = fraction of receptors remaing after receptor inactivation with irreversible antagonist

x’ = concentration of agonist after receptor inactivation that results in the same response as x

x

T

x

T

Kx

qRxtransducerresponsetransducer

Kx

xR

'

'

xx Kx

qx

Kx

x

'

'

')1(

'

xqK

qKxLogxLog

x

x

(Ehlert, 1987)

Nonlinear regression

(Ehlert, 1987)

')1(

'

xqK

qKxLogxLog

x

x

Log [x’]

Log

[x]

Determination of Affinity

Control curve

Curve after alkylation

xKx

x

100occupancy %

Determine relative efficacy of test agonist relative to standard agonist – /s

(Ehlert, 1987)

2 % 4 % 80 %

Control curve

Curve after alkylation

Furchgott AnalysisFurchgott Analysis

Allows the estimation of affinity and efficacy Allows the estimation of affinity and efficacy using experimental manipulation of the responseusing experimental manipulation of the response

What can you get from a What can you get from a concentration-response curve?concentration-response curve?

RARAii Analysis Analysis

RARAii is the product of observed affinity and intrinisic is the product of observed affinity and intrinisic efficacy of the agonist of interest (efficacy of the agonist of interest (YY) relative to a ) relative to a standard agonist (standard agonist (XX))

RARAii is a measure of affinity for the active state of the is a measure of affinity for the active state of the receptorreceptor

XX

YYi K

KRA

/1

/1

X

Y

YofstimulustransducerresponsetransducerXofstimulus

x

Tx

KX

RX

y

Ty

KY

RY

x

Tx

KX

RX

y

Ty

KY

RY

YofstimulustransducerresponsetransducerXofstimulus

y

Ty

x

Tx

KY

RY

KX

RX

x

yq

x

y

K

Kp

x

x

pKqY

qYKLogXLog

)1()(

xx

yyi K

K

p

qRA

)/1(

)/1(

What is the product of affinity and What is the product of affinity and efficacy?efficacy?

Relative measure of microscopic affinity constant for the active state of the receptor

The RAThe RAii is a relative measure of the affinity for is a relative measure of the affinity for

the active state of the receptorthe active state of the receptor

XX

YYi K

KRA

/1

/1

X

Y

Y

X

XR*

YR*

HypothesisHypothesis

In a biological system, the RAIn a biological system, the RA ii is equivalent to is equivalent to

the product of observed affinity and intrinsic the product of observed affinity and intrinsic efficacy for the agonist of interest (efficacy for the agonist of interest (YY) relative to ) relative to a standard agonist (a standard agonist (XX))

XX

YYi K

KRA

/1

/1

AimsAims

1.1. Compare RACompare RAii with product of affinity and with product of affinity and

efficacyefficacy

2.2. Compare RACompare RAii value between systems value between systems

3.3. Determine relative contribution of Determine relative contribution of receptor subtypes for eliciting a response receptor subtypes for eliciting a response using modified RAusing modified RAii analysis analysis

Aim 1Aim 1

RARAii = product of observed affinity and intrinisic = product of observed affinity and intrinisic efficacy of test agonist relative standard agonistefficacy of test agonist relative standard agonist

Comparison of observed affinity and intrinisic Comparison of observed affinity and intrinisic efficacy values procured individually through efficacy values procured individually through Furchgott analysis with RAFurchgott analysis with RA ii

– Furchgott analysis – irreversible antagonismFurchgott analysis – irreversible antagonism

– RARAii analysis – of simple agonist concentration- analysis – of simple agonist concentration-response curvesresponse curves

CHO-MCHO-M22 cells cells

(Tran et al, submitted)

')1(

'

xqK

qKxLogxLog

x

x

xKx

x

100occupancy %

KOxo-M = 0.29 MKCarb = 4.3 M

55.0

Carb

MOxo

RARAii = product of affinity and = product of affinity and

efficacyefficacy

(Tran et al, submitted)

Mouse ileum, “MMouse ileum, “M22 KO” KO”

(Tran et al, submitted)

Mouse ileum, “MMouse ileum, “M22 KO” KO”

(Tran et al, submitted)

Aim 1 SummaryAim 1 Summary

RARAii values agree with the product of values agree with the product of

observed affinity and intrinsic efficacy for observed affinity and intrinsic efficacy for the test agonist relative to the standardthe test agonist relative to the standard

Aim 2Aim 2

Compare RACompare RAii values between systems values between systems– CHO cells vs tissueCHO cells vs tissue

The RAThe RAii is inherent property of the active state of is inherent property of the active state of the receptor that causes it associate with its the receptor that causes it associate with its coupling protein (e.g., G protein).coupling protein (e.g., G protein).

Therefore RATherefore RAii values should be consistent values should be consistent between different systems that signal through between different systems that signal through the same G protein.the same G protein.

G1 G1G2

RAi values are equivalentRAi values not necessarily equivalent

MM22 receptor receptor

Guinea Pig Left AtriumCHO-M2 cells

(Tran and Ehlert, in preparation)

MM22 RA RAii comparison comparison

(Tran and Ehlert, in preparation)

MM33 receptor receptor

(Ehlert et al., 1999)

(Tran et al, submitted)

Mouse ileum, “M2 KO”CHO-M3

MM33 receptor receptor

(Tran et al, submitted)

Characterization of McN-A-343Characterization of McN-A-343

(Tran et al, submitted)

Mouse ileum, “M2 KO”

M2/M3 KO

M2 KO

M2 KO

(Tran et al, submitted)

Higher activity of McN-A-343 in MHigher activity of McN-A-343 in M22 KO ileum is a KO ileum is a mixed response that may include Mmixed response that may include M11 receptor receptor activationactivation

(Tran et al, submitted)

11 receptor receptor

(Tran and Ehlert, in preparation)

CHO-1 cells

Rat Left Atrium

1

3(-)

(+)

Contraction

X

Unknown dobutamine effectUnknown dobutamine effect

Aim 2 SummaryAim 2 Summary

The RAThe RAii is consistent between systems and is consistent between systems and responses measured signaling from the same G responses measured signaling from the same G protein pathwayprotein pathway

The unique effects of McN-A-343 and The unique effects of McN-A-343 and dobutamine are likely due to effects in addition dobutamine are likely due to effects in addition to binding the receptor-G protein pathway under to binding the receptor-G protein pathway under studystudy

Aim 3Aim 3

Determination of the contribution of various Determination of the contribution of various receptor subtypes to the response measured in receptor subtypes to the response measured in knockout studies using a modified RAknockout studies using a modified RA ii– Relative Contribution (Relative Contribution (RCRC) analysis) analysis

The contribution of MThe contribution of M11, M, M22 and M and M33 receptors to receptors to oxotremorine-M-induced phosphoinositide oxotremorine-M-induced phosphoinositide hydrolysis in the mouse urinary bladder and hydrolysis in the mouse urinary bladder and ileumileum– MM22 KO KO– MM33 KO KO– MM22/M/M33 KO KO

R2 R1

Assumptions:

1. Receptors can each mediate the response measured individually

2. Agonist has same affinity for all the receptors involved

MM11, M, M33 – mediate – mediate

phosphoinositide hydrolysis phosphoinositide hydrolysis through Gthrough Gqq

MM22 – may mediate – may mediate

phosphoinositide hydrolysis phosphoinositide hydrolysis through Gthrough Gii

Oxotremorine-M has similar Oxotremorine-M has similar affinities for all the receptorsaffinities for all the receptors

RCRC analysis analysis

RC1 = 1 – RC2

150max

501max2

KOWT

WTKO

ECE

ECERC

R2 R1

stimulus Total

stimulus R22 RC

(Tran et al., 2006)

M3 receptor is solely responsible for response

Mouse Urinary BladderMouse Urinary Bladder

Mouse IleumMouse Ileum

Complex Result with possible involvement of M2 in addition to M3 as well as another receptor

(Tran et al., 2006)

MM22/M/M33 KO KO

(Tran et al., 2006)

MM11 MM22 MM33 MM44 MM55

6.26.2 7.37.3 6.16.1 6.06.0 5.35.3

7.87.8 6.06.0 6.66.6 7.27.2 6.66.6

Binding affinities (pKd)

Antagonist affinities (pKB)

AF-DX 116 – 6.42 ± 0.42

Pirenzepine – 8.04 ± 0.47

Esqueda et al, 1996

M1 receptor mediated response

AF-DX 116

Pirenzepine

MM22/M/M33 KO KO

(Tran et al., 2006)

M1 receptor exists in the smooth muscle

M1, RC1 = 15 % (M2/M3 KO)

M3, RC3 = 80% (M3 KO)

M2, RC2 = 1-(RC3 + RC23) = 5%

(Tran et al., 2006)

Aim 3 ConclusionsAim 3 Conclusions

The The RCRC analysis was used to estimate the analysis was used to estimate the contributions of the Mcontributions of the M11, M, M22 and M and M33

receptors to the oxotremorine-M-induced receptors to the oxotremorine-M-induced phosphoinositide hydrolysisphosphoinositide hydrolysis

Evidence is shown for a MEvidence is shown for a M11 and M and M22

phosphoinositide response in ileum phosphoinositide response in ileum

SummarySummary

The RAThe RAi i is a relative measure is a relative measure of microscopic affinity of microscopic affinity constants of the active state of constants of the active state of the receptorthe receptor

The RAThe RAii is the product of is the product of affinity and efficacy of the test affinity and efficacy of the test agonist relative to a standard agonist relative to a standard agonistagonist

The The RCRC analysis can be used analysis can be used in knockout studies to in knockout studies to determine the contribution of determine the contribution of various receptor subtypes to various receptor subtypes to the response measuredthe response measured

XX

YYi K

KRA

/1

/1

150max

501max2

KOWT

WTKO

ECE

ECERC

AcknowledgementsAcknowledgements

Frederick Ehlert, Ph.D.Frederick Ehlert, Ph.D.

Kelvin Gee, Ph.D.Kelvin Gee, Ph.D.

Frances Leslie, Ph.D.Frances Leslie, Ph.D.

Ralph Purdy, Ph.D.Ralph Purdy, Ph.D.

Qun-Yong Zhou, Ph.D.Qun-Yong Zhou, Ph.D.

Alan Goldin, M.D.,Ph.D.Alan Goldin, M.D.,Ph.D.

Ehlert LabEhlert Lab– Michael Griffin, Ph.D.Michael Griffin, Ph.D.– Hinako Suga, Ph.D.Hinako Suga, Ph.D.– Katherine Figueroa, Ph.D.Katherine Figueroa, Ph.D.– Kirk PakKirk Pak– Alexander ChangAlexander Chang

Department of Department of PharmacologyPharmacology

FamilyFamily