Drug Receptor Interactions

Figure 42
Drug receptor Interactions - 2

Prof Graham Ladds
Dept Pharmacology
grl30@cam.ac.uk
Figure 43
Drug receptor interactions - the topics
1. Concept of “receptor”
2. Mechanism of drug receptor interactions
3. Agonism and antagonism
4. Quantifying drug receptor interactions
Receptor Families
1. Intracellular receptors.
2. Receptors that are ion channels.
3. Receptors with intrinsic enzyme activity.
4. Receptors linked to protein kinases.
5. Receptors coupled to target proteins via a G protein.
Intracellular signalling - Second messengers.
Figure 44
Quantifying drug receptor interactions…
Rang and Dale 9th edition

Figure 45
Rang and Dale 9th edition

Figure 46
Quantitation of drug/receptor interactions
k+1
D + R DR
k-1
forward rate = k+1[D][R]
backward rate = k-1[DR]
• At equilibrium:
Þ forward rate equals backward rate
Þ k+1[D][R] = k-1[DR]
Þ k+1 = Ka = [DR]
k-1 [D][R]
• Ka is the affinity constant and has units of M-1

1 = Kd
K1a
• Kd is the (equilibrium) dissociation constant

Rang and Dale 9th edition and has units of M
Figure 47
k+1
D + R DR
k-1
• At equilibrium:
Þ k+1[D][R] = k-1[DR]
Þ k+1 = Ka = [DR]
k-1 [D][R]

1 = Kd
K1a

Figure 48
k+1
D + R DR
k-1
• At equilibrium:
Þ k+1[D][R] = k-1[DR]
Þ k+1 = Ka = [DR]
k-1 [D][R]
a massive number - 1000 ~ 10^9.
Ka is essentially a ratio of [product]:[reactants] because drugs gotta bind

e.g. mols/mg
1 = Kd
K1a Kd is essentially a ratio of [reactants]:[products]

Figure 49
BUT how many receptors are actually associated with the drug?
Receptor occupancy
• Suppose a fraction a of receptors combines

with drug
D + R DR
(1-a) a
Then Ka = [DR]
[D][R]
Þ Ka = a
[D](1 - a)
Þ[D]Ka- [D]Kaa = a
Þ a (1 + [D]Ka) = [D]Ka
Þ a = [D]Ka
1 + [D]Ka
Figure 50
Receptor occupancy

with drug
D + R DR
(1-a) a
Then Ka = [DR]
[D][R]
Þ Ka = a
[D](1 - a)
Þ[D]Ka- [D]Kaa = a
Þ a (1 + [D]Ka) = [D]Ka
Þ a = [D]Ka
1 + [D]Ka
Figure 51
Receptor occupancy

with drug
D + R DR
(1-a) a
Then Ka = [DR]
[D][R]
Þ Ka = a
[D](1 - a)
Þ[D]Ka- [D]Kaa = a
Þ a (1 + [D]Ka) = [D]Ka
Þ a = [D]Ka
1 + [D]Ka
[D][R]
Figure 52
Þ
BUT how many receptors are actually associated with
Ka the
= a
drug?
[D](1 - a)
Receptor occupancy
Þ[D]Ka- [D]Kaa = a
• Suppose a fraction a of receptors combines Þ a (1 + [D]Ka) = [D]Ka

with drug
Þ a = [D]Ka
D + R DR 1 + [D]Ka
(1-a) a
Then Ka = [DR] 50 percent associated
[D][R] WHEN a = 0.5, [D]Ka = 1
Þ Ka = a
Þ [D] = 1 = Kd
[D](1 - a)
Ka
Þ[D]Ka- [D]Kaa = a
Kd - conc. when 50% of
of receptors are occupied.
Þ a (1 + [D]Ka) = [D]Ka ≠EC50 -
- EC50 only applies to agonists
- amplification
Þ a = [D]Ka
1 + [D]Ka
Figure 53
The absolute numbers of receptors in a tissue
• Fractional occupancy is only a percentage of the total receptors.
• It is not an absolute number.
• Are there any ways we can obtain an absolute number??Agonism and antagonism
• The actions of a drug are related to
Isolated organ experiments
concentration
e.g. Guinea-pig ileum
Amplification
• Magnus preparation 100
Agonist + Receptor
% ¯
Drug-receptor 50
complex
Response
¯
Conformational change
¯ 0
Second messenger system0 activated50 100 1 2 3
[Agonist] log [Agonist]
¯
Second messenger
• Log generated
concentration-response curves are used
¯
• Pseudo-linear at midpoint
[Ca2+]i
Contraction
to ACh
1 min
Kd is NOT EC50
¯ • Competitive antagonists give parallel shift
Þ Can accommodate large shifts
Actin/myosin activated of an agonist
¯
100
Muscle contracts
ACh ACh ACh ACh ACh Agonist
alone
%
Þ Kdiss is not the EC50 for50an agonist Agonist
Figure 54
Radioligand binding is the answer
• Allows direct analysis of the Drug - Receptor interact
• A radiolabelled drug enables us the essentially ‘count’ receptors
• Common isotopes are:
3H - tritium
125I Radio-iodine (good for proteins)
Figure 55
Radioligand binding
• Allows direct observation of drug-receptor
interactions 3H - tritium
• A radiolabelled drug ("radioligand") is used to
'count' receptors
Radiolabelled Drug
How to access the

Receptors bound receptors?
• Common isotopes used:
3H - tritium
125I - radio-iodine
• Iodine is particularly useful to label peptide

ligands
Figure 56
Radioligand binding
• Allows direct observation of drug-receptor
interactions 3H - tritium
Separating bound from free radioligand
• A radiolabelled drug ("radioligand") is used to
'count' receptors centrifugation
Radiolabelled Drug
must be in high concentrations so that it reaches equilibria
Wash pellet
then count
radioactivity
Centrifuge
How to access the

Receptors bound receptors?
membrane
receptor
• Common isotopes used: Filter drug
(glass fibre)
3H - tritium
125I - radio-iodine
We must separate the unbound ‘free’ label
• Iodine is particularly useful to label peptide
ligands from that bound to the receptor Wash filter
vacuum pump
The unbound fraction (D*) is in excess compare then count
radioactivity
to the D*R complex

Figure 57
Specific vs non-specific binding
• How do we discriminate between the specific and non-specific binding?
• Radioligands like sticking to things non-specifically
• e.g. plastic,
Specific membranes
and non-specific etc
binding
• They will do this ± presence of receptor
• Only ligand molecules marked are

specifically bound properties of specific bindingn and nonspecific binding is different:
i.e. to the receptor site gives a way to remove nonspecific binding
• SPECIFIC BINDING: high affinity

low capacity
NON-SPECIFIC BINDING :
low affinity
high capacity
or the ligand simply partitions into the
membrane
Figure 58
• If we add excess non-radioligand we can block specific binding.
Text
Figure 59
• If we add excess non-radioligand we can block specific binding.
• Anything else must be non-specific and it is linear with concentration.
How do you control for the amounts of Binding curves

radiolabelled/nonlabelled ligands?
Bound
radioligand
Total binding
Bmax
Specific binding
Bmax
Non-specific binding
2
[radioligand]
Kd
Kdiss
You can measure the amount of nonspecific binding

Figure 60
Obtaining parameters from the binding
• As with M&M kinetics, for Emax and Km Total number of receptors bound
Bmax (maximum binding or RT) is difficult to estimate from the curve
• If Bmax is inaccurate Ka and Kd will be inaccurate. Since they are related.
• Kd = the concentration of a drug that occupies 50% of the total receptors.
Binding curves
Bound
radioligand
Total binding
Bmax
Specific binding
Bmax
2
[radioligand]
Kdiss
Kd Km in michaelis-menten curve (is it always MM?)

Receptor occupan
Figure 61
Obtaining parameters from the binding • Suppose a fraction a of recepto
with drug
• As with M&M kinetics, for Emax and Km
Bmax (maximum binding or RT) is difficult to estimate from D +theRcurve DR
• If Bmax is inaccurate Ka and Kd will be inaccurate. Since they are related.(1-a) a
• Kd = the concentration of a drug that occupies 50% of the totalThen

Binding curves receptors.
Ka = [DR]
[D][R]
Þ Ka = a
Bound [D](1 - a)
radioligand
Total binding Þ[D]Ka- [D]Kaa = a
Bmax
Specific binding Þ a (1 + [D]Ka) = [D]Ka
Bmax
2
Þ
Remember a = [D]Ka
1 + [D]Ka
[radioligand] WHEN a = 0.5, [D]Ka = 1

Kdiss
Kd Þ [D] = 1 = Kd
Ka
Figure 62
Two methods can help us……
• Estimates are easier when using data that is linear - Re: M & M kinetics..
Figure 63
• 1. Double reciprocal plot…
Binding curves
a = B = [D]K1a
Bmax 1 + [D]K1a
Þ Bmax = 1 + [D]K1a
B [D]K1a
Þ Bmax = 1 +1
B [D]K1a
• Plot 1 versus 1
B [D]
Þ Double reciprocal plot
1/B
1/Bmax
- K1 0
-Ka 1/[D]
Figure 64
Binding curves
Binding curves a = B = [D]Ka
1
a = B = [D]K1a Bmax 1 + [D]K1a
Bmax 1 + [D]K1a
Þ B + B[D]Ka
1 = Bmax[D]K1
a
Þ B + BKa = BmaxKa
B [D]K1a 1 1
[D]
Þ Bmax = 1 +1
Þ B = BmaxKa
1 - BK1a
B [D]K1a
[D]
• Plot 1 versus 1
B [D] • Plot B versus B
[D]
Þ Scatchard plot
N.B. [D] strictly represents free drug
1/B
B/[D]
1/Bmax
-Ka
slope = -K1
- K1 0
-Ka 1/[D]
0 B
Bmax
Figure 65
Binding curves
Binding curves a = B = [D]Ka
1
a = B = [D]K1a Bmax 1 + [D]K1a
Bmax 1 + [D]K1a
Þ B + B[D]Ka
1 = Bmax[D]K1
a
Þ B + BKa = BmaxKa
B [D]K1a 1 1
[D]
Þ Bmax = 1 +1
Þ B = BmaxKa
1 - BK1a
B [D]K1a
[D]
• Plot 1 versus 1
[D]
Þ Scatchard plot
1/B
B/[D]
1/Bmax
-Ka
slope = -K1
- K1 0
-Ka 1/[D]
0 B
Bmax
Figure 66
• 1. Double reciprocal plot… MUST KNOW
• 2. Scatchard plot… Binding curves
Binding curves a = B = [D]K1
a = B
Bmax
=
Key Assumptions:
[D]K1
1 + [D]K1
Þ
Bmax
B + B[D]K1 = Bmax[D]K1
1 + [D]K1
Þ Bmax
B 1. Drug is in excess to RT
= 1 + [D]K1
[D]K1 Þ B + BK1
[D]
= BmaxK1
2. Reaction is in equilibrium
Þ Bmax = 1 +1
Þ B = BmaxK1 - BK1
B [D]K1
[D]
• Plot 1 versus
3. Reversible reaction
1
[D]
4. One binding site Þ
N.B.
Scatchard plot
[D] strictly represents free drug
1/B
B/[D]
1/Bmax
-Ka
slope = -K1
- K1 0
-Ka 1/[D]
0 B
Bmax
Figure 67
Binding with Binding withtwo
more than twosites
receptor populations
• The Scatchard plot is well-suited to
• When we have more than one site the linear relationship changes…
demonstrating the presence of > 1 binding
• The Scatchard plot is well-suited to demonstrate this presence of > 1 site.
sites for a ligand
irl its a curve because there is no single “cutoff point” for 2nd site binding
B/[D]
high affinity site
low affinity site
0 B
Bmax1
Bmax1 + Bmax2
two binding sites
Cooperativity - exploring multiple binding sites Figure 68
Study of channel activity

Patch Clamping
Neher & Sakmann - 1978
glass electrode
polished tip
cell
NAChR in
membrane
patch
gigaohm
(109 W) seal
membrane
cytosol
Cooperativity - exploring multiple binding sites Figure 69
Patch clamp of Na+ channels

Study of channel activity • • • • • • • opening frequency
Patch Clamping similar to oxygen binding
to haemoglobin - cooperativity. closed
Neher & Sakmann - 1978 2nd binding site binding gets easier 1 open
once 1st is bound
glass electrode NOTE: not a log graph
current
2 open
polished tip
cell
• Ion flux = 104 Na+ ions ms-1
Agonist Mean channel open time
Acetylcholine 1.0 ms
Carbachol 0.3 ms
NAChR in
membrane Suberyldicholine 1.7 ms
patch
gigaohm
Expected
(109 W) seal
Frequency
membrane of channel Observed
opening
cytosol
Concentration of ACh
100 ACh on
NAChR
Cooperativity - exploring multiple binding
Response sites steep slope Figure 70
1:1 binding
50
Remember O2 binding to Haemoglobin Shallow slope
0
1 3 5
The Hill plot log [Agonist]
• Concentration-response curves can have • Replotting these as a Hill plot

different slopes can show how many binding sites there are (lvl of coop).
~2 log ACh on positive negative
100 ACh on units
NAChR NAChR coop coop
e.g. CO2 binding
steep slope nH > 1
Response log to haemogl.
(
1:1 binding
)
R
nH < 1 nH = 1 one to one
50 Rmax - R
shallow slope
Shallow slope
0
1 3 5
1 3 5
log [Drug]
log [Agonist]
• nH ~2 for ACh on NAChR
• Replotting these as a Hill plot
ACh on
NAChR
nH > 1
log
( R
)
Rmax - R
nH < 1
shallow slope
nH = 1
Generating radioligands for all drugs is not feasible Figure 71
• It is expensive and often not possible to generate radioligands of all drugs
• How can we use one single radioligand to tell us about other drugs binding?
• We can use competition binding of radioligand with non-labelled ones.
• Two types of competition - homologous or heterologous. COMPETITION BINDING
• Homologous - competing ligand the same as radioligand. finding specific binding
• Heterologous - competing ligand is different to radioligand.
COLD ANALOGUE
Competition heterologous binding curves Figure 72
Radioligand binding
• Inhibit binding of fixed concentration of
radioligand (L) with increasing concentrations
of unlabelled ligand (U)
• L gives an amount of binding X in the
absence of U
• The concentration of U inhibiting half of
the specific binding is the IC50
X Systematically increase the concentration of the unlabelled ligand that

competes with the labelled ligand.
Binding
of Specific binding
X/2
labelled
drug IC50
0
log [Unlabelled drug]
IMPORTANT
X is not the same as Bmax; it can be any degree
of occupancy between 0 and 100% normally 40-60% — WHY (maybe sigmoidal curve)?
Radioligand binding:
Competition heterologous binding Figure
curvescompetition experiments 73
Radioligand binding
• Inhibit binding of fixed concentration of SAY 40% of receptors are initially occupied by L ( )
absence of U
TO REDUCE occupancy by L to 20% (i.e.by 50%),
• The concentration of U inhibiting half of need to occupy 50% with U ( )
Binding aU = [U]KU
1 + [U]KU + [L]KL
of Specific binding
X/2
labelled WHEN [U] = IC50, aU = 0.5
drug IC50 Þ 0.5 + 0.5(IC50)KU + 0.5[L]KL = IC50.KU

0
Non-specific binding Þ 0.5 + 0.5[L]KL = 0.5(IC50)KU
Þ 1 + [L]KL = IC50.KU
Þ KU = 1 + [L]KL
IMPORTANT IC50
of occupancy between 0 and 100%
Competition heterologous binding Figure
curvescompetition experiments 74
Radioligand binding
• Inhibit binding of fixed concentration of SAY 40% of receptors are initially occupied by L ( )
absence of U
TO REDUCE occupancy by L to 20% (i.e.by 50%),
• The concentration of U inhibiting half of need to occupy 50% with U ( )
X
this is when IC50 happens
Binding aU = [U]KU
1 + [U]KU + [L]KL
of Specific binding
X/2 two ligands in the denom
labelled WHEN [U] = IC50, aU = 0.5
drug IC50 Þ 0.5 + 0.5(IC50)KU + 0.5[L]KL = IC50.KU

0
Non-specific binding Þ 0.5 + 0.5[L]KL = 0.5(IC50)KU
Þ 1 + [L]KL = IC50.KU
Þ KU = 1 + [L]KL
IMPORTANT IC50
Cheng-Prusoff eqn
aU = [U]KU
Þ 1 + [L]KL = IC50.KU 1 + [U]KU + [L]KL
Homologous competition binding Figure 75
Radioligand binding Þ WHEN KU = 1a+
[U] = IC50, = 0.5L
U [L]K
• Inhibit binding of fixed concentration of Þ 0.5 + 0.5(IC50)KU IC50

+ 0.5[L]KL = IC50.KU
radioligand (L) with increasing concentrations Þ 0.5 + 0.5[L]KL = 0.5(IC50)KU
of unlabelled ligand (U) Þ 1 + [L]KL = IC50.KU
• L gives an amount of binding X in the linearising is easy: just make the Y axis IC50
absence of U Þ IC50 = 1 + [L]KL
competition experiments
KU
• The concentration of U inhibiting half of PLOT IC50 versus [L]
X IC50
Binding slope = KL/
KU
of Specific binding
X/2
intercept = 1/
labelled KU
drug IC50 0 [Radioligand]

0
WHEN U and L are the same drug
log [Unlabelled drug] THEN KL = KU = K
Þ 1 + [L]K = IC50.K
IMPORTANT
Þ K(IC50 - [L]) = 1
of occupancy between 0 and 100% Þ K = 1
(IC50 - [L])
Alternative to radioligand binding Figure 76
radioligand no longer used
Using fluorescent ligands

Measured using BRET
lower nonspecificity, and safer without radioactivity.
Fluor. ligand
interacts with luciferase
conjugated receptor.
how does
this not change
the behaviour
of the receptor?
The relationships between Affinity and Efficacy Figure 77
• We have spent a lot of time calculating affinity of a drug to a target.

• How does this relate to the effect it produces????
Text

Pre-1960’s Receptor
occupancy
view BAD Response
EC50 = KD

But now we response

occupancy
How does
clearly some
know this funny

amplification
going on
here
this arise?
EC50 KD
EC50 = Kd
Figure 80
Irreversible antagonist provide the answer
• As mentioned before cell systems contain amplification.
• Thus Kd and EC50 are NOT the same. Why?
• How can we explore this???
Isolated organ experiments acetylcholine
e.g. Guinea-pig ileum
Amplification
Agonist + Receptor
reactive
¯
chlorethyl
Drug-receptor complex
¯ benzilycholine mustard
Conformational change
¯
Second messenger system activated
¯
Second messenger generated
¯
[Ca2+]i
¯ irreversible
Actin/myosin activated occlusion of ligand-
¯ binding site
Muscle contracts
Þ Kdiss is not the EC50 for an agonist

Figure 81
Irreversible binding of an antagonist uncovers ‘spare receptors’
• The assumptions was irreversible binding immediately reduces the response.
acetylcholine
reactive
chlorethyl
benzilycholine mustard
cy
number (5˃1)
an
decreasing
up
se
receptor
on
c
Oc
sp
Re
Predicted
Decreased maximal responses
5
1.0
4
irreversible
Response
3 occlusion of ligand-
0.5 binding site
2
1
0.0
-3 -2 Unchanged
-1 0 EC50 1 2 3
log [Agonist]
Figure 82
• However this was not the case - there was ‘reserve receptors’…
acetylcholine
in reality there are “spare receptors” — response moves
to the right at low concentrations of the irreversible receptors.
e.g. concentration
reactive
can respond to multiple signals? would
the body just ignore the new signals then?
chlorethyl
number (8˃1)
irreversible antagonist basically should take out receptors.
decreasing
receptor
cy
number (5˃1)
an
decreasing
up
se
receptor
Observed
on
c
Oc
sp
Re
maximal responses
Predicted
5 8 7 6 5 4
1.0
4
irreversible 3
Response
3 occlusion of ligand-
2
0.5 binding site
2
1
1
0.0 EC50: 8 7 6 5-1

-3 -2 Unchanged
-1 0 EC50 1 2 3
log [Agonist]
Figure 83

• However this was not the case - there was ‘reserve receptors’…
• Before the benzilycholine mustard reduced the response it needed to
occupy excess receptors that had not been predicted to exist.
number (8˃1)
decreasing
receptor
cy
number (5˃1)
an
decreasing
up
se
receptor
Observed
on
c
Oc
sp
Re
maximal responses
Predicted
5 8 7 6 5 4
1.0
4
3
Response
3
2
0.5
2
1
1
0.0 EC50: 8 7 6 5-1

-3 -2 Unchanged
-1 0 EC50 1 2 3
log [Agonist]
Spare receptors or receptor reserve Figure 84
• At each step we have amplification of the response.

• Agonist binding leads to multiple steps all with amplification.
Isolated organ experiments
[receptor-agonist] [2]
e.g. Guinea-pig ileum 1. Agonist
Agonist + Receptor
Amplification
[agonist] [1]
[active G protein] [3]

Drug-receptor complex 2. Receptor-agonist
¯
Conformational change [receptor-agonist] [2]
[phospholipase C] [4]
3. Active G protein
Second messenger system activated
¯ [active G protein] [3]
Second messenger generated

¯ 4. Phospholipase C
[IP3 ] [5]
[Ca2+]i 5. IP3
¯ 6. IP3 receptor [phospholipase C] [4]
7. Ca2+
Actin/myosin activated 8. Ca2+-calmodulin
on
racti
9. Active MLCK
ing
¯ 10. MLCK phosphorylation
bind
cont
11. Contraction
Muscle contracts
log [agonist]
Drug receptor interactions - summary of L2 Figure 85
1. Quantifying
Receptordrug receptor interactions - occupancy
occupancy

with drug Binding curves
D + R DR
(1-a) a
Bound
radioligand
Then Ka = [DR] Total binding
[D][R] Bmax
Specific binding
Þ Ka = a
[D](1 - a) Bmax
2
Þ[D]Ka- [D]Kaa = a
Þ a (1 + [D]Ka) = [D]Ka
[radioligand]
Kdiss
Þ a = [D]Ka
1 + [D]Ka Kd
1 max 1
Drug receptor interactions

Þ B + BK1 = -Bmax
summary
K1 of L2 Figure 86
[D]
Þ B = BmaxK1 - BK1
2. Scatchard plot to[D]measure Ka and Bmax
• Plot B versus B
[D]
Þ Scatchard plot
B/[D]
-Ka
slope = -K 1
0 B
Bmax
• Inhibit binding of fixed concentration of
radioligand (L) withRadioligand binding:
increasing concentrations Figure 87
Drug receptor
of unlabelled (U)interactions
competition
ligand experiments - summary of L2
absence
SAY of
40%U of receptors are initially occupied by L ( )
3.• The
Cheng-Prusoff eqn half
concentration of U inhibiting forofcompetitive binding
competition experiments
TOXREDUCE occupancy by L to 20% (i.e.by 50%), PLOT IC50 versus [L]
need to occupy 50% with U ( )
Binding
of Specific binding
X/2
IC50
labelled
IC50 slope = KL/
drug aU = [U]KU KU
0 1 + [U]KU + [L]KL
Non-specific binding intercept = 1/
KU
WHEN [U] = IC50, aU = 0.5
log [Unlabelled drug] 0 [Radioligand]
Þ 0.5 + 0.5(IC50)KU + 0.5[L]KL = IC50.KU
IMPORTANT
Þ 0.5 + 0.5[L]KL = 0.5(IC50)KU
WHEN U and L are the same drug
X is not Þ
the1 same
+ [L]KLas Bmax; Uit can be any degree
= IC50.K
of occupancy between 0 and 100% THEN KL = KU = K
Þ KU = 1 + [L]KL Þ 1 + [L]K = IC50.K
IC50 Þ K(IC50 - [L]) = 1
Þ K = 1
(IC50 - [L])
Drug receptor interactions - summary of L2 Figure 88
4. Spare receptors acetylcholine
reactive
chlorethyl
number (8˃1)
decreasing
receptor
Observed
response
occupancy irreversible
maximal responses
occlusion
8 of7ligand-
6 5 4
binding site
3
2
1
EC50 KD EC50: 8 7 6 5-1

• The concentration of U inhibiting half of
Example exam questions from L2
the specific binding
TO REDUCE is thebyIC50
occupancy L Figure
to 20% (i.e.by
need to occupy 50% with U ( )
89 50%)
9. In a radioligand binding assay, the a1-adrenoceptor antagonist prazosin displaced the
specific binding of 1.2 x 10-7 M [3H]phenylephrine in a concentration-dependent manner, X
with an IC50 of 1 x 10-9 M.
The affinity constant of [3H]phenylephrine is 0.5 x 108 M-1, which of the following is the Binding
correct affinity constant for prazosin?
of aU =Specific [U]K
binding
U
a) 5.0 x 108 M-1 X/2
b) 7.0 x 108 M-1 1 + [U]KU + [L]KL
c) 4.0 x 109 M-1 labelled
d) 5.0 x 109 M-1
WHEN IC50
[U] = IC50, aU = 0.5
e) 7.0 x 109 M-1
drug
10. Benzilylcholine mustard (BCM) irreversibly alkylates the muscarinic receptor and was used 0
to provide early evidence for the existence of ‘spare receptors’. It is now appreciated that ‘spare
9. In aor
radioligand binding assay, a1-adrenoceptor
Þ 0.5 + 0.5(IC50)KU Non-specific
+ 0.5[L]KL = IC50.K
binding
U
receptors’ a ‘receptor reserve’ are the
a feature antagonist
of many receptor prazosin displaced the
systems.
specific binding of [3H-]phenylephrine in a concentration dependent manner such that a plot
of the ICof50the
Which for following
prazosin against the concentration
statements [3H-]phenylephrine
about spareofreceptors is correct? was linear and the Þ 0.5 + 0.5[L]KL = 0.5(IC50)KU
slope was 0.2. The affinity constant of [3H-]phenylephrine is 0.5 x 108 M-1. log [Unlabelled drug]
Which
a) of the
All full following
agonists is the
have the correct affinity constant
same proportion forreceptors
of spare prazosin? in a particular tissue
Þ 1 + [L]KL = IC50.KU
b) In systems
a) 1.0 x 108 M-1with spare receptors, radioligand binding detects two distinct populations of
b) 2.5 x 108 M-1
receptors
c) 4.0 108 M-1 of spare receptors increases the sensitivity of tissues to agonists
Thexpresence IMPORTANT
d) 1.0 x 109 M-1 of spare receptors for a particular full agonist is constant, irrespective of the
d) The proportion Þ KU = 1 + [L]KL
e) 2.5 x 109 M-1
tissue X is not the same as Bmax; itIC50
can be any degree
e) When the receptor reserve is progressively removed using an irreversible antagonist, the
sensitivity of the tissue to agonist (i.e. the EC50) is initially unaffected
10. Benzilylcholine mustard (BCM) irreversibly alkylates the muscarinic receptor and
was used to provide early evidence for the existence of ‘spare receptors’. Radioligand binding:
Which of the following best describes the effect of progressive alkylation of cholinergic
11. Sulphonylurea
receptors by BCM ondrugs, such as glibenclamide,
the responses of the guinea pigare usedtoasagonists?
ileum oral hypoglycaemic agents. competition experiments
Which of the following best describes the action of sulphonylureas?
a) The maximum response to acetylcholine decreases gradually as the EC50 shifts to the right PLOT IC50 versus [L]
b) They
a) The maximum response
are commonly to acetylcholine
used to treat Typefalls gradually, but the EC50 remains constant
I diabetes
c) The maximum response to acetylcholine
+ falls
b) They bind within the pore of the K ATP channel immediately to a constant level
d) The maximum response to acetylcholine initially remains unaffected but eventually begins
c) They cause hyperpolarisation of pancreatic β-cells
to fall
d) They cause the K+ATP channel to close always attainable at very high agonist
e) The maximum response to acetylcholine is
e) They have the opposite effect on the K+ATP channel to that of ATP itself
concentrations IC50
slope = KL/
11. The ‘dose ratio’ method allows the determination of antagonist affinity constants. The KU
contractions of a section of guinea pig ileum in response to histamine were measured in the
absence and presence of the histamine H1 receptor antagonist mepyramine. The EC50 for
histamine in the absence of mepyramine was 1.1 x 10-6 M. In the presence of 1.6 x 10-8 M intercept = 1/
mepyramine, the EC50 for histamine was 9.9 x 10-6 M. [Question 12 starts on the next page] KU
What is the affinity constant for mepyramine?
0 [Radioligand]
a) 2.0 x 108 M-1
b) 5.0 x 108 M-1
8 -1

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Figure 42

Drug receptor Interactions - 2

Rang and Dale 9th edition

Rang and Dale 9th edition

• Ka is the affinity constant and has units of M-1

• Kd is the (equilibrium) dissociation constant

• Ka is the affinity constant and has units of M-1

• Kd is the (equilibrium) dissociation constant

• Ka is the affinity constant and has units of M-1

• Kd is the (equilibrium) dissociation constant

• Suppose a fraction a of receptors combines

• Suppose a fraction a of receptors combines

• Suppose a fraction a of receptors combines

• Suppose a fraction a of receptors combines Þ a (1 + [D]Ka) = [D]Ka

Then Ka = [DR] 50 percent associated

[D][R] WHEN a = 0.5, [D]Ka = 1

How to access the

• Iodine is particularly useful to label peptide

How to access the

to the D*R complex

• Only ligand molecules marked are

• SPECIFIC BINDING: high affinity

How do you control for the amounts of Binding curves

You can measure the amount of nonspecific binding

Kd Km in michaelis-menten curve (is it always MM?)

• Kd = the concentration of a drug that occupies 50% of the totalThen

[radioligand] WHEN a = 0.5, [D]Ka = 1

Þ Double reciprocal plot

4. One binding site Þ

low affinity site

Study of channel activity

Neher & Sakmann - 1978

Patch clamp of Na+ channels

Agonist Mean channel open time

Remember O2 binding to Haemoglobin Shallow slope

• Concentration-response curves can have • Replotting these as a Hill plot

X Systematically increase the concentration of the unlabelled ligand that

log [Unlabelled drug]

drug IC50 Þ 0.5 + 0.5(IC50)KU + 0.5[L]KL = IC50.KU

labelled WHEN [U] = IC50, aU = 0.5

drug IC50 Þ 0.5 + 0.5(IC50)KU + 0.5[L]KL = IC50.KU

• Inhibit binding of fixed concentration of Þ 0.5 + 0.5(IC50)KU IC50

drug IC50 0 [Radioligand]

Using fluorescent ligands

• We have spent a lot of time calculating affinity of a drug to a target.

• We have spent a lot of time calculating affinity of a drug to a target.

• We have spent a lot of time calculating affinity of a drug to a target.

But now we response

know this funny

Þ Kdiss is not the EC50 for an agonist

0.0 EC50: 8 7 6 5-1

• The assumptions was irreversible binding immediately reduces the response.

0.0 EC50: 8 7 6 5-1

• At each step we have amplification of the response.

[active G protein] [3]

Second messenger generated

• Suppose a fraction a of receptors combines