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