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Biochemistry 230
Receptor Binding
Tracy Handel
3246 PSB
Oct 28, 2008 Receptors have two major properties: Binding and Transduction
Binding: To a first approximation, obeys laws of thermodynamics.
Typically stereoselective, saturable, reversible.
Transduction: The second property of a receptor is that the binding of
an agonist must be transduced into some kind of functional response
(biological or physiological).
Different receptor types are linked to effector systems either directly or
through simple or more-complex intermediate signal amplification
systems. Some examples are:
Ligand-gated ion channels nicotinic Ach receptorsSingle-transmembrane receptors RTKs like insulin or EGF receptors7-transmembrane GPCRs opioid receptors
Soluble steroid hormones estrogen receptor
Drews, J. Drug discovery:A historical perspective. Science287(2000)1960-1964.
Drug Receptors
Basic Binding Equilibrium Plotting methods Competitive and non-competitive
relationships
Agonism and Antagonism Receptor Theory
Spare receptors
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kon = # of binding events/time (Rate of association) =[ligand] [receptor] kon = M
-1 min-1
koff= # of dissociation events/time (Rate ofdissociation) = [ligand receptor] koff= min
-1
Binding occurs when ligand and receptor collide withthe proper orientation and energy.
Interaction is reversible. Rate of formation [L] + [R] or dissociation [LR]
depends solely on the number of receptors, the
concentration of ligand, and the rate constants konand koff.
kon/k1
[ligand] + [receptor] [ligand receptor]
koff/k2
Basic Binding Equilibrium
At equilibrium, the rate of formation equals that ofdissociation so that:
[L] [R] kon = [LR] koff
KD = koff/kon = [L][R]
[LR]*this ratio is the equilibrium dissociation constant or KD.
G= -RTlnK
Basic Binding Equilibrium
KD is expressed in molar units (M/L) and expresses theaffinity of a drug for a particular receptor.KD is an inverse measure of receptor affinity.KD = [L] which produces 50% receptor occupancy
BasIC Binding Equation
P + L P-Lkon
koff
Kd=
[P][L]
[PL]
[P]o= [P]+ [PL]
[P]o [PL]= [P]
[PL]=[P]o[L]o
[L]o +Kd
Kd= [L]
o
[P]
[PL]
If [L]o >> [P]o , [L]o = [L]
Kd=L
o
Po[ ] PL[ ][ ][ ]PL[ ]
=
LoPo[ ]
PL[ ]
Lo
[PL]
[P]o
=
[L]o
[L]o+K
d
Fraction Bound Protein =
[PL]
[P]o
Know this
Know thismeasure this
Calculate this
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Receptor Fractional Occupancy (F.O)
F.O. =
Use the following numbers:
[L] = KD= 50% F.O.
[L] = 0.5 KD = 33% F.O.
[L] = 10x KD = 90.9%+ F.O.
[L] = 0= 0% F.O.
100
50
0
Ligand Concentration
FractionalOccupancy
[PL]
[P]o=
[L]o
[L]o +Kd
The amount of drug bound at any time issolely determined by:
the number of receptors the concentration of ligand added the affinity of the drug for its receptor.
Binding of drug to receptor is essentially the same asdrug to enzyme as defined by theMichelis-Menten
equation.
BINDING VS KINETIC EQUATIONS
[PL]
[P]o
[L]o
Nonlinear Plots
Kd
[PL]
[P]o
[L]o
Semilog Plots
0 5 100
0.5
1
1010.1
Kd
0
0.5
1
Can compare Ligands
with Many log differences
in affinity on SemiLog Plots
1/[PL]/[Po]
1/[L]
-1/Kd
Double Reciprocal Plot (linearization)
[PL]
[P]o
=
[L]o
[L]o+K
dLike
Lineweaver-Burke Plot for
Enzyme Kinetics!
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Classical Scatchard Plot (Another Linearization)
Kd =
[P][L]
[PL]
[PL] =[P][L]
Kd
=
[P]o [PL]( )[L]K
d
[PL]
[L]=
[P]o
Kd
[PL]
Kd
[PL]
[L]
[PL]
Bound
Free
Bound
1
Kd
Scatchard with n Independent, Equivalent Binding Sites
[PL]
[P]o= n
[L]
[L]+Kd
[PL]/[P]o
[L]=
n
Kd
[PL]/[P]o
Kd
# of sites
[PL]/[P]o
[L]
[PL]/[P]o
1
Kd
n
fraction bound
free
fraction bound
Multiple Site Binding Models
P + 2L P-L2
[PL2]
[P]o
[L]o
Both Sites
First Site (High Affinity)
Second Site (Low Affinity)
Independent, nonequivalent binding sites
Bound
Free
Free
Both Sites
Nonlinearity in the Scatchard plot is also observed withmixture of different receptor subtypes
Biphasic scatchard plot seenwhen Kds for two sites differ by10-fold or more
Cooperativity in Ligand Binding
P + nL P-LnKd
Y =[L]
n
[L]n+Kd
n = 1: noncooperativity
n < 1: negative cooperativity
n > 1: positive cooperativity
0
0.2
0.4
0.6
0.8
1
0 1 2 3 4 5 6
Nonlinear Plot
N = 2
N = 1
N = 0.5
Y
SemiLog Plot
Y
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Receptor occupancy =[RL]
[Rtotal]
[L]
[L] + K=
Ligand concentration
Cant Always Measure Direct Binding When Studying Receptors.
Often Measure Some Response that is a Function of Agonist
Binding to Receptor. Efficacy term Defines Response with Binding
/max
[L]Receptor response =
[]
[max] [L] + K=
= 1 for simplicityfor now
DERIVATION OF COMPETITIVE INHIBITION
Competitive Inhibition
I BINDS AT SAME SPOT AS L
[RL]
[Rtotal]=
[L]
[L] + K[I]
KI1 +
[L]
[L] +K=
Max Bound/Max Response unchanged. Can always overcome inhibition
with more agonistPARALLEL SHIFTS
/max =
K K K
0 = [I] < [I] < [I]
/max
1/[L]
-1/Kd
Double Reciprocal Plot of Competitive Inhibition
-I
+I
-1/K
[PL]/[Po]
/max
1
1
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The Schild plot
Linearization of Competitive Binding Data
[I]
KI[A] = [A] 1 +
[I]
KI= 1 +
[A]
[A]= Dose ratio = K
Dose ratio 1 = [I] / KI
log(Dose ratio - 1) = log([I]) log(KI)
y = x - b
A and A: concentration of
agonist in the absence and
presence of I, respectively
required to get
the same response /max
K
When Does ratio =2 then I= Ki (because log 1=0)
[A] [A] [A]
/max
The Schild plot
log(doseratio1)
-log([I])
-log(KI)
Noncompetititve Inhibition
Inhibitor binds at different site
than agonist, does not affect Kd,
but LRI and RI are non-functional
so reduces /max. Binding of agonist
and inhibitor dont affect each other
0 = [I] < [I] < [I] < [I]
Kd
/max
max
' = 1
1+[I]
Ki
max
= 1
1+[I]
Ki
[L]
[L]+KD
when [I]=Ki
max
= 0.5
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[PL]/[Po]
1/[L]
-1/Kd
Double Reciprocal Plot of NonCompetitive Inhibition
-I
+I
/max
1
1
Competitive antagonists
.
Bind to the same site as theendogenous ligand or agonist.
Can be over come! Their presence produces a right-ward
shift in both the binding and dose-response curves.
No change in max. Similar dose-response curve shapes
indicates the presence of a
competitive agonist (competing forthe same binding sites).
A = agonist aloneB = antagonist (one concentration)
A+B = agonist + antagonist
surmountableKi
Kd response
Non-competitive antagonis
Does not prevent formation of the DRcomplex, but impairs the conformation
change which triggers a response.
Bind to a site different than the agonistbinding site
Cannot be overcome by adding moreagonist
max is reduced but EC50 (Kd) remains thesame.
Dose-response curves will havedifferent shapes indicating different
binding sites.
response
Ki Ki
Kd
Kd
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Not every ligand is radiolabeledWhatto do?????
Some Practical Issues
Competition binding assays
Allows one to determine a rough estimate of anunlabeled ligands affinity for a receptor.
Introduction into the incubation mixture of a non-radioactive drug (e.g. drug B) that also binds to R will
result in less of R being available for binding with D*,
thus reducing the amount of [D*R] that forms. This
second drug essentially competes with D* foroccupation of R. Increasing concentrations of B result
in decreasing amounts of [D * R] being formed.
Method: Single concentration of labeled ligand Multiple (log-scale) concentrations of the unlabeled/
competing ligand.
Competition binding assays The concentration of inhibitor which displaces 50% of the
radiolabeled ligand is known as the IC50for that drug.
IC50 cannot be viewed as the KD of the inhibitor because it isjust an estimate.
Ki = the equilibrium inhibitor dissociation constant. It is the concentration of the competing ligand that would
bind to 50% of sites in the absence of the radioligand.
Ki can only be determined after the IC50 is known. Uses the equation of Cheng and Prusoff.
Ki
= IC50
1 + [radiolabeled ligand]
Kd (radiolabeled ligand)
Example: Find Ki of morphine in a preparation with3H-diprenorphine.
IC50
= 100 nM Ki= 25 nM
[L] = 3 nM (labeled)KD = 1 nM (of labeled L)
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Drug dose-effect relationships
(Receptor=Effector)Drug
ReceptorDrug Effector2nd Messenger
Examples:
Enzymes observed effect: enzyme activity Ion channels observed effect: ion conductivity
Examples:
Enzymes observed effect: some physiological / clinicalreadout (acetylsalicylic acid / cyclo-oxygenase / pain relief)
Alpha-adrenergic receptor IP3 Ca++ increasedmuscle contraction
Drug dose-effect relationships
Receptor = effector
Occupancy
Effect
log [Drug]
Receptor
occupancy
(%)
Observed
effect (%)
Downstream Receptor Activation: What Can Measure Upon
GPCR Activation
GTPS binding (the most direct measure of GPCR activation;
detect with radioactive GTP35S) non-hydrolyzeable analogue
cAMP Levels (Direct Detection, or couple to a reporter gene
(GFP) linked through CREB)
Calcium Flux (detect with Calcium Sensitive Dyes)
Phosphorylation of Proteins (detect with Antibodies)
Production of New Proteins (detect with Antibodies)
Internalization of Receptor (visualize with GFP-fusions)
Migration......other
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( )[ ][ ]
[ ][ ]
+=
==
LK
Lf
R
LRfSf
Dt
max
The Response of a Receptor Involves an Occupation Term and
an Efficacy Term Affinity: The tenacity by which a drug binds to its
receptor.
Efficacy: Relative maximal effect of a drug Full agonist = 1 (*equal to the endogenous
ligand)
Antagonist = 0 Partial agonist = 0~1 (*produces less than the
maximal response, but with maximal binding to
receptors.)
Potency: ability of a drug to cause a measuredfunctional change. Related to affinity but related tofunctional readout rather than binding.
DEFINITIONS
Agonist alone
antagonist or
inverse agonist
alone
Log10 [Ligand]
/max
Definitions: Agonist, Inverse Agonist, Antagonist
Response of a Receptor with No Basal Activity
Definitions: Constitutively Active Receptors
Simple Two State Model of Receptor Activation. The bottom shows aconstitutively active receptor
R R* response
R R* response
L + R LR* response
A lot of mutations dispersed throughout receptors cause increased constitutiveactivity
Most GPCRs have some basal activity
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Definitions: Constitutive Activity, Agonist Inverse Agonist, Antagonist
Full
agonist
alone
Inverse
agonist
antagonist alone
Log10 [Ligand]
/max
constitutive
activity
of receptor
alone
Response of a Receptor with Constitutive Activity
Definitions
Drug potency and efficacy
log [Drug]
Observedeffect
Efficacy:
effect at
saturating
concentration
potency: 1 / EC50EC50
Red more Potent than Blue, Blue has higher efficacy
( )[ ][ ]
[ ][ ]
+=
==
LK
Lf
R
LRfSf
Dt
max
67
Need to Understand the Physical Basis for Efficacy
Effector
enzymes
channels
. . .
Odorants,
Tastes
Generic GPCR Signaling
Small Molecules
Peptides
Nucleosides, nucleotides
Amino acids, amines, Ach
Lipid derived (PGs, LTs,LPA, S1P, ...)
Proteins
TSH, LH, FSH, hCG
Thrombin
VIP, Glucagon, PTH
C5a, chemokines
Adapted from Bockaert J & Pin JP
EMBO J18:1723-29 [1999]
G-protein
Response: All kinds of changes in the cell
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Structure of rhodopsin (top, in the
membrane) and the approximate
orientation of a heterotrimeric G
protein (bottom). The subunits are
highligted as follows: G (purple),
GDP (red), G (green), G
(yellow).
Coupling of a GPCR to Heterotrimeric G Proteins: A
Docked Model Based on Rhodopsin Structure and a G protein Complex
DRYBox is on IC Loop 2*
*
Extracellular View of Helices
in Rhodopsin (based on structure)
Extracellular View of -adrenergic rece
with bound epinephrine/adrenaline(cartoon from Gomperts text on Signal Transduction)
Proposed Conformational Changes Upon Agonist Binding
Ligand Binding on EC side
causes inward movement
of helices on EC side
and outward movement on
IC side
Data suggest that
TM VI and VII move
away from TMIII, opening
up IC side of GPCR and
exposing the DRY box
to G proteins
View from IC side
Flourescent Label Here
Why??
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Effect of Agonists and Partial Agonists on 2AR
Fluorescence Intensity (Conformation) and GTP S
binding (Activation)
Detection of a ConformationalTransition that Tracks with Activation
Efficacy:
ISO & EPI: full agonists
SAL & DOB: partial agonists
Alprenolol is an antagonis
Swaminath JBC 2004
Sequential Binding...
biphasic: fast and slow;
slow dependent on
presence of chiral -
hydroxy
Conformational Changes in B2AR detected by Fluorescence
biphasic: slow dependent
on chirality of-hydroxy
Nepi: full
Dop: partial
Swaminath JBC 2004
Sequential Binding...
Conformational Changes in B2AR detected by Fluorescence
biphasic: slow dependent
on presence of chiral -
hydroxy; magnitude and
rate dependent on alkyl
substituent
fast rate dependent on
catechol hydroxyls
Cat: weak partial ag
Dop: partial ag
Funtionally, slower conformational changes ~~ correlate with
efficient agonist induced receptor activation/internalization
G-protein activation &
internalization
G-protein activation
G-protein activation &
small internalization
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Agonist binding to B2AR
Lock and Key Model: Bindi
Preformed Site
Sequential Binding Model
Different Conformations and
Rates of Conformational
Change Influence Signaling
Response
all have
same
Kd
Kd ~ 100-fold
lower
Understanding Efficacy in B2AR
( )[ ][ ]
[ ][ ]
+=
==
LK
LfR
LRfSf
Dt
max