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1
Molekulare, physiologische und
pathophysiologische Analyse
des β2-Adrenorezeptors
Rezeptorpharmakologie / Pharmakogenetik
In vitro / Ex vivo
Funktionelle Selektivität / Individuelle Arzneimittelreaktion
Disputation
Michael T. Reinartz
2
Warm-Up: Receptor Pharmacology
Two states?
3
Warm-Up: Receptor Pharmacology
Multiple states! Two states?
4
Warm-Up: Pharmacogenetics
One patient group?
5
Warm-Up: Pharmacogenetics
Individual drug-response! One patient group?
responders
with adverse
effects
responders
only
adverse
effect
non
responders
6
precise drugs
and
personalized
treatment
Individual
β2AR-
drug-response
of
60 volunteers
Ligand-specific
pharmacology
of
14 β2AR-ligands
Contribution of my PhD research
β2AR- specific
information
Subject-specific
information
Improved
therapy
Part 1 Part 2 Outlook
8
Fight-or-Flight reaction via adrenergic receptors
Epinephrine activates adrenergic receptors
9
Fight-or-Flight reaction via adrenergic receptors
Epinephrine activates adrenergic receptors
cardiac output
respiratory rate
glycogenolysis
immune defense
digestion
„Fight-or-Flight“- Physiology
...
http://www.openclipart.org
10
Fight-or-Flight reaction via β2-adrenergic receptors
Selective activation of β2AR-specific effects
11
Fight-or-Flight reaction via β2-adrenergic receptors
Selective activation of β2AR-specific effects
cardiac output
broncho dilation
glycogenolysis
immune-suppression
digestion
... utilized for
...
http://www.openclipart.org
tocolysis
12
Broncho-dilative effect of β2AR-selective agonists
Pathology of bronchial asthma
relaxed
smooth
muscles
air trapped
in alveoli
tightened
smooth
muscles
normal airway asthmatic airway
during attack
asthmatic airway
wall inflamed
and
thickened
http://www.ocallergy.com
13
Ligand-specific pharmacology
of 14 β2AR-ligands
Reinartz MT et al., Naunyn Schmiedebergs Arch Pharmacol. 388:1 (2015)
β2AR- specific
information
Part 1
14
β2AR ligands can be functionally selective
Ligand classification
Simmons MA Mol Interv 5:3 (2005)
15
β2AR ligands can be functionally selective
Ligand classification
Gstimulatory Ginhibitory β-arrestin
β2AR agonists
(unbiased, Gs-biased, Gi-biased, β-arr-biased)
β2AR β2AR β2AR
Signal transduction pathways via β2AR
16
β2AR ligands can be functionally selective
Ligand classification
Gstimulatory Ginhibitory β-arrestin
β2AR agonists
(unbiased, Gs-biased, Gi-biased, β-arr-biased)
β2AR β2AR β2AR
Signal transduction pathways via β2AR
17
β2AR ligands can be functionally selective
Ligand classification
Functional selectivity or “ligand bias“ is the ligand-dependent selectivity for
certain signal transduction pathways in one and the same receptor.
This can be present when a receptor has several possible signal transduction pathways.
Gstimulatory Ginhibitory β-arrestin
β2AR agonists
(unbiased, Gs-biased, Gi-biased, β-arr-biased)
β2AR β2AR β2AR
Signal transduction pathways via β2AR
Simmons MA Mol Interv 5:3 (2005)
18
Relevance of functional selectivity via β2AR
Gstimulatory
AC
cAMP
Ginhibitory
AC MAPK
(fast) cAMP
β-arrestin
MAPK
(delayed)
other
signals
?
β2AR agonists (unbiased, Gs-biased, Gi-biased, β-arr-biased)
β2AR β2AR β2AR
airway smooth muscle
relaxation contractile sensitization desensitization
19
Relevance of functional selectivity via β2AR
Gstimulatory
AC
cAMP
Ginhibitory
AC MAPK
(fast) cAMP
β-arrestin
MAPK
(delayed)
other
signals
?
β2AR agonists (unbiased, Gs-biased, Gi-biased, β-arr-biased)
β2AR β2AR β2AR
immune cells
immune-suppression pro-inflammatory? immune-modulation?
desensitization
airway smooth muscles
relaxation contractile sensitization desensitization
20
Fenoterol A β2AR-selective sympathomimeticum
C H 3 O H
N H O H
O H
O H
* *
N H
O H
O H
O H
(R)-EPI
21
Fenoterol A β2AR-selective sympathomimeticum
C H 3 O H
N H O H
O H
O H
* *
(R)-EPI
N H
O H
O H
O H
yields β2AR-selectivity
Aminoalkyl-tail
22
3 Fenoterol derivatives + 4 stereoisomers = 12 ligands Modifications at aminoalkyl-tail + differences in chirality
CH3OH
NHOH
OH
OH
CH3O
NHOH
OH
OH
CH3O
NHOH
OH
OH
* *
* *
* *1
2
3
yields β2AR-selectivity
derivatization
hydroxy-benzyl (1)
methoxy-benzyl (2)
methoxy-naphthyl (3)
Aminoalkyl-tail
23
3 Fenoterol derivatives + 4 stereoisomers = 12 ligands Modifications at aminoalkyl-tail + differences in chirality
CH3OH
NHOH
OH
OH
CH3O
NHOH
OH
OH
CH3O
NHOH
OH
OH
* *
* *
* *1
2
3
two stereo-centers
(R,R’), (R,S’), (S,R’), (S,S’)
yields β2AR-selectivity
derivatization
hydroxy-benzyl (1)
methoxy-benzyl (2)
methoxy-naphthyl (3)
Aminoalkyl-tail
Chirality
24
Extensive pharmacological profiling In vitro, in cell and ex vivo
Gi-GTPase
Gs-GTPase
AC
Binding
β2AR-G
xα- fusion proteins
25
Extensive pharmacological profiling In vitro, in cell and ex vivo
Gi-GTPase
Gs-GTPase
AC
Binding
β2AR-G
xα- fusion proteins
β-arrestin-2 recruitment
HEK293-cells expressing β2AR
Takakura H et al., ACS Chem Biol 7:5 (2012)
26
Extensive pharmacological profiling In vitro, in cell and ex vivo
cAMP accumulation
inhibition of ROS production
(radical oxygen species)
Neutrophils expressing β2AR
Gi-GTPase
Gs-GTPase
AC
Binding
β2AR-G
xα- fusion proteins
β-arrestin-2 recruitment
HEK293-cells expressing β2AR
Takakura H et al., ACS Chem Biol 7:5 (2012)
AC
NOX
IP3
DAG
ROS
PKC
FPR
b2AR
Gi b
b
cAMP
PIP2
Gs
27
Six functional assays
RESULTS PART 1
28
Comparison of concentration-response data
O H
O O
Gs- and Gi-coupling are influenced by aminoalkyl-tail and stereochemistry
29
Comparison of concentration-response data
O H
O O
Gs- and Gi-coupling are influenced by aminoalkyl-tail and stereochemistry
30
Quantification of receptor activation & ligand bias
log(τ/KA)Gsα
→ each agonist
log(τ/KA)Giα
→ each agonist
Δlog(τ/KA) → agonist – (R)-EPI
Condensing efficacy and potency to log(τ/KA)
Normalization cancels system bias
ΔΔlog(τ/KA) = G
s - G
i
Comparison of two pathways
31
Bias analysis: Gs- vs. Gi-coupling at β2AR
O H
O O
32
Bias analysis: Gs- vs. Gi-coupling at β2AR
Gs-bias of
(S,S')-methoxy-
fenoterol
(R,S')-
methoxy-
naphthyl-
fenoterol
O H
O O
33
Bias analysis: Gs- vs. Gi-coupling at β2AR
Gs-bias of
(S,S')-methoxy-
fenoterol
(R,S')-
methoxy-
naphthyl-
fenoterol
extreme Gs-bias
not quantifiable
no detectable
Gi-activation by
(S,S')-3
O H
O O
34
Summary as functional “fingerprints” Pairwise comparison of six assays per ligand
ΔΔ
log
A
B 0 vs.
ΔΔ
log
A
B 0 vs.
ΔΔ
log
A
B 0 vs.
ΔΔ
log
A
B 0 vs.
ΔΔ
log
A
B 0 vs.
ΔΔ
log
A
B 0 vs.
ΔΔ
log
A
B 0 vs.
ΔΔ
log
A
B 0 vs.
ΔΔ
log
A
B 0 vs.
ΔΔ
log
A
B 0 vs.
ΔΔ
log
A
B 0 vs.
ΔΔ
log
A
B 0 vs.
ΔΔ
log
A
B 0 vs.
ΔΔ
log
A
B 0 vs.
ΔΔ
log
A
B 0 vs.
Six β2AR assays
15 pair-wise comparisons
Single fingerprint per ligand
35
Heatmap of 14 functional “fingerprints” Sums up 84 concentration-response curves (with n ≥ 3)
Heatmap to compare 14 ligand fingerprints
36
Results from the heatmap analysis
Gs-bias (red) for most
fenoterol ligands
37
Results from the heatmap analysis
Gs-bias (red) for most
fenoterol ligands
reference and
unmodified
(R,R')-fenoterol
mostly balanced
38
Results from the heatmap analysis
Gs-bias (red) for most
fenoterol ligands
reference and
unmodified
(R,R')-fenoterol
mostly balanced
modification at
aminoalkyl-tail
increases Gs-bias
> >
> >
> >
> >
> >
> >
> >
39
Results from the heatmap analysis
Gs-bias (red) for most
fenoterol ligands
reference and
unmodified
(R,R')-fenoterol
mostly balanced
modification at
aminoalkyl-tail
increases Gs-bias
disfavored / silenced
Gi-coupling
β-arr-2 recruitment
40
Results from the heatmap analysis
Gs-bias (red) for most
fenoterol ligands
reference and
unmodified
(R,R')-fenoterol
mostly balanced
modification at
aminoalkyl-tail
increases Gs-bias
disfavored / silenced
Gi-coupling
β-arr-2 recruitment
(R,S')- and (S,S')-
methoxy-naphthyl-
fenoterol (3) are
strong / extreme Gs-
biased
41
Results from the heatmap analysis
Gs-bias (red) for most
fenoterol ligands
reference and
unmodified
(R,R')-fenoterol
mostly balanced
modification at
aminoalkyl-tail
increases Gs-bias
disfavored / silenced
Gi-coupling
β-arr-2 recruitment
(R,S')- and (S,S')-
methoxy-naphthyl-
fenoterol (3) are
strong / extreme Gs-
bias
Naphthyl-moiety AND (X,S‘)-chirality
Structure-bias relationship
C H 3 O
N H O H
O H
O H
* S
42
TM 4
TM 5
TM 6
TM 1 TM 2
TM 3
OH
N+
OH OH
OH
TM 7
site 1 site 2
adopted from Jozwiak, K et al., Chirality, 23 (2011)
Specific interactions crucial for Gi and β-arr-2 signaling
Orthosteric ligand binding site of β2AR
43
TM 4
TM 5
TM 6
TM 1 TM 2
TM 3
OH
N+
OH OH
OH
TM 7
site 1 site 2
interactions with transmembrane domain 7 (TM 7)
stabilisation of an inactive conformation
selective β-arrestin-2 activation
Structure Bias Relationship
Specific interactions crucial for Gi and β-arr-2 signaling
OMe
adopted from Jozwiak, K et al., Chirality, 23 (2011)
Orthosteric ligand binding site of β2AR
44
confirmation of β2AR functional selectivity
aminoalkyl-derivatization and stereochemistry of
fenoterol modify Gi and β-arr-2 signalling
insights into Gs-biased β2AR agonism
Value for Gs-biased drug development
Conclusions – β2AR-specific information
β2AR- specific
information
Part 1
C H 3 O
N H O H
O H
O H
* S
45
Individual β2AR-drug-response
of 60 volunteers
Reinartz MT et al., submitted to Allergy
Subject-specific
information
Part 2
46
Genetic factors determine asthma therapy success
Determination of therapy success
difficult to treat
increased exacerbation rate
persistent symptoms
hospitalization
more emergencies
reduced life-quality
http://www.pharmgkb.org/gene/PA39
others genetic (ADRB2, ...) up to 70 %
Severe asthma
47
Genetic factors determine asthma therapy success
Determination of therapy success
difficult to treat
increased exacerbation rate
persistent symptoms
hospitalization
more emergencies
reduced life-quality
Mutations in receptor gene Severe asthma
http://www.pharmgkb.org/gene/PA39
others genetic (ADRB2, ...) up to 70 %
48
Connecting β2AR SNPs and ex vivo responsiveness
Study with DNA and neutrophils of 60 volunteers
49
Induced sputum
from chronic asthmatics
Neutrophils are pathophysiologically relevant
macrophages 40%
neutrophils 40%
epithelial cells 13%
eosinophils 6%
lymphocytes 1%
Woodruff PG et al., J Allergy Clin Immunol, 2001, 108, 753-758
bacteria
formylpeptides
neutrophilic granulocytes
hypersecretion remodelling
airway constrictions
infection
β2AR
β2AR
AC
NOX
IP3
DAG
ROS
PKC
FPR
b2AR
Gi b
b
cAMP
PIP2
Gs
Ex vivo model for β2AR signalling
50
Clinically-relevant sympathomimetics
(R)-Isoproterenol
used as reference in vitro
(R,R’)-Fenoterol
sometimes for acute asthma
(R)-Salbutamol
relief for acute asthma
(R,R’)-Formoterol
control of moderate, chronic asthma
Four prototypical β2AR-agonists
(R)-ISO
C H 3
C H 3
N H
O H
O H
O H
*
C H 3 O H
N H O H
O H
O H
* *
C H 3
C H 3
C H 3
N H
O H
O H
O H
*
C H 3 O
C H 3
N H
O H
N H
O
O H
* *
(R,R’)-FEN
(R)-SAL
(R,R’)-FORM
51
RESULTS PART 2
Ex vivo assay and genetic data
52
“Caucasian“ background
comparable to HAPMAP-CEPH
healthy volunteers recruited at MHH
22 – 56 yrs old
Cohort characteristics (n=60)
60 healthy volunteers – background check
0 0,2 0,4 0,6
Thr283Ser
Thr164Ile
Gln27Glu
Gly16Argthis study
HAPMAP-CEPH
/ MAF 0% 50% 100%
asthma
atopic
smoking
male
yes
no
HAPMAP-CEPH: haplotype map of the human genome, CEPH (809
“Caucasian” individuals); MAF: minor allele frequency
53
No Influence of relevant assay parameter
no relevant confounding effect on
formylpeptide (fMLP)-induced ROS (radical oxygen species) production
β2AR-mediated inhibition of fMLP-induced ROS-production
One-way ANOVA; p-value > 0.05 = n.s.
Statistical testing for difference between sub-populations
sex asthma atopy smoking age
54
multivariate regression analysis:
description of
dependent variable (responsiveness)
using
influencing factors (ligand, SNPs)
stepwise improvement of model
Influence of Glycine-16-Arginine Polymorphism
small decrease if carrying Arg-allele (p < 0.002)
0.146 on the logarithmic concentration scale
SNP as possible marker or cause for decreased responsiveness
in silico analysis suggests deleterious effect of non-synonymous amino acid exchange
on biological function
Influence on pooled responsiveness
Multivariate regression detects decrease in responsiveness
estimated influence: -0.40 ± 0.13
55
Conclusions – Subject-specific information
ROS-inhibition assay in
neutrophils is
a sensitive and robust ex vivo
test model for individual
β2AR responsiveness
not influence by sex, age,
smoking, atopy, or asthma
Gly16Arg SNP
as a genetic marker for
decreased
β2AR responsiveness
Inter-individual variability of β2AR responsiveness
Subject-specific
Information
Part 2
AC
NOX
IP3
DAG
ROS
PKC
FPR
b2AR
Gi b
b
cAMP
PIP2
Gs
56
Precise drugs and personalized treatment
Outlook
Improved
therapy
57
β2AR-specific information
Gs-biased ligands based on fenoterol scaffold
more precise therapy (e. g. bronchial asthma)
improved tools to research 7TMR functional selectivity
Perspectives
more structural information on 7TMR-ligand-signalling-protein
complexes needed
insights from new techniques looking at receptor
conformations
rational medicinal-chemical design of biased ligands
increased complexity of drug development
Challenges
Outlook
Improved
therapy
58
Patient-specific information
ex vivo ROS assay results as parameter in clinical studies on
β2AR-pharmacogenetics
e.g. with neutrophils isolated from patients
stratification of patient groups
Perspectives
further verification, that SNPs influence responsiveness
more and more individual data ((epi-)genomic, microbiomic,
life-style (wearables), ...)
employment of “BigData”-analyses for personalized medicine
Challenges
Outlook
Improved
therapy
59
Thank You Ideas, advice, assistance, collaboration, material & any support
... and to all study-participants!!
Prof. M. Gaestel (Physiological Chemistry)
Prof. E. Ponimaskin (Neurophysiology)
Prof. R. Seifert (Pharmacology)
Dr. C. Happle
Prof. M. Kabesch
Dr. M. Wetzke
(Clinic for Paediatric Pneumology and Neonatology)
Pharmacogenetic Studies
A. Garbe (ZFA Metabolomics)
S. Kälble (Pharmacology)
Prof. V. Kaever (ZFA Metabolomics)
Technical Assistance / Analyses
Review & Supervision
Dr. I. R. Wainer (National Institute on Aging)
Dr. A. Schnapp (Boehringer Ingelheim)
Enantiopure Ligands
T. Littmann (Pharmacology)
Prof. T. Ozawa (University of Tokyo)
β-arrestin data & cell line
Prof. S. Dove (University of Regensburg)
Prof. A. Koch (Biometry)
R. Scherer (Biometry)
Statistical Advice
60
Questions and Answers
β2-AR
functional selectivity
in vitro, in cell, ex vivo
bias quantification
Receptor Pharmacology Pharmacogenetics
Inter-individual variability
ex vivo
study with 60 volunteers
Naunyn Schmiedebergs Arch
Pharmacol. 2015 May;388(5):517-24
Naunyn Schmiedebergs Arch
Pharmacol. 2015 Jan;388(1):51-65
PLoS One. 2013 May 31;8(5):e64556
Review on β2AR Functional Selectivity:
BIOspektrum, 2014 March; 20(2):130-
135
Submitted to Allergy
Rasmussen et al., Nature 2011 Jan.; 469:175-180
61
Appendix
62
Model of Multiple Receptor Conformations A complex composed melody and NOT only a single tune
63
Interaction of fenoterol stereoisomers with β2-
adrenoceptor-Gsα fusion proteins:
antagonist and agonist competition binding
Reinartz, MT et al., Naunyn Schmiedebergs Arch Pharmacol. 388:5 (2015)
β2AR- specific
information
Part 1B
64
β2AR-Gs fusion protein – binding assays Competition with radioactive labeled agonist or antagonist
analysis of the binding affinity of ligands using β2AR-Gs fusion protein
displacement of fenoterol ligands by radio-actively labeled ligands
[3H]-DHA AND [3H]-(R,R‘)-Methoxynaphthyl-Fenoterol (2)
Analysis of ternary complex (ligand + receptor + G-protein)
with AND without GTP
Concentration-dependent inhibition of radio-ligand binding
65
12 Fenoterol ligands – pick-locking tool to probe the binding site
Seifert, R & Dove, S, Mol Pharmacol, 2009, 75
detektei-schutzdienst-shop.de, wikihow.com/Pick-a-Lock
D113
S203
S204
S207
F290
C191
Y316
H93
W109
F193
Molecular Modeling mit
(R,R') und (S,R')-Fenoterol
66
Association of
ligand binding to the receptor ([3H]DHA or [3H](R,R‘)-2)
activation of proximal downstream signalling (GTPase)
Potency vs. Affinity
Gs-GTPase vs. competition binding A closer look at the coupling of β
2AR with Gs
4 5 6 7 8 94
5
6
7
8
9
10
pEC50,GTPase = 3.9 + 0.521 pKi,low,DHA
R² = 0.56
pKi,low,DHA,control
pE
C5
0,G
TP
ase
4 5 6 7 8 94
5
6
7
8
9
10
pEC50,GTPase = 2.1 + 0.719 pKi,low,(R,R')-2
R² = 0.77
pKi,low,(R,R')-2
pE
C5
0,G
TP
ase
67
Association of
ligand binding to the receptor ([3H]DHA or [3H](R,R‘)-2)
activation of proximal downstream signalling (GTPase)
Potency vs. Affinity
Gs-GTPase vs. competition binding [3H]-(R,R‘)-Methoxy-Fenoterol probes active conformation
4 5 6 7 8 94
5
6
7
8
9
10
pEC50,GTPase = 3.9 + 0.521 pKi,low,DHA
R² = 0.56
pKi,low,DHA,control
pE
C5
0,G
TP
ase
4 5 6 7 8 94
5
6
7
8
9
10
pEC50,GTPase = 2.1 + 0.719 pKi,low,(R,R')-2
R² = 0.77
pKi,low,(R,R')-2
pE
C5
0,G
TP
ase agonist as radio-ligand
reflects active
receptor
conformation
fenoterol derivative as
radio-ligand
structural-similar to
the other „fenoterols“
Higher Association for
pEC50 vs. pKi,low,(R,R‘)-2
68
Association of
ligand binding to the receptor ([3H]DHA or [3H](R,R‘)-2)
activation of proximal downstream signalling (GTPase)
Potency vs. Affinity
Gs-GTPase vs. competition binding Information on structure-(bias)/activity-relationship
4 5 6 7 8 94
5
6
7
8
9
10
pEC50,GTPase = 3.9 + 0.521 pKi,low,DHA
R² = 0.56
pKi,low,DHA,control
pE
C5
0,G
TP
ase
4 5 6 7 8 94
5
6
7
8
9
10
pEC50,GTPase = 2.1 + 0.719 pKi,low,(R,R')-2
R² = 0.77
pKi,low,(R,R')-2
pE
C5
0,G
TP
ase agonist as radio-ligand
reflects active
receptor
conformation
fenoterol derivative as
radio-ligand
structural-similar to
the other „fenoterols“
„clustering“ by
stereoconfiguration
antagonist as radio
ligand
(R,S‘)- and (S,S‘)-3
step-out
Higher Association for
pEC50 vs. pKi,low,(R,R‘)-2
69
Binding assays (antagonist and agonist)
Functional assays (GTPase and AC)
agonist competition at β2AR-Gs
fusion proteins fits in silico data best
(not shown)
supports medicinal-chemical
rationale (Wainer et al.)
Insights into stereoselective
interaction & functional selectivity Various Levels in the G-protein cycle:
binding, transmission, effector
Taken together – Binding Assays Adds to functional data and published analyses
O H
C H 3 O H
N H O H
O H
O H
* *
70
Binding assays (antagonist and agonist)
Functional assays (GTPase and AC)
agonist competition at β2AR-Gs
fusion proteins fits in silico data best
(not shown)
supports medicinal-chemical
rationale (Wainer et al.)
Insights into stereoselective
interaction & functional selectivity Various Levels in the G-protein cycle:
binding, transmission, effector
Taken together – Binding Assays Adds to functional data and published analyses
O H
C H 3 O H
N H O H
O H
O H
* * O
71
Binding assays (antagonist and agonist)
Functional assays (GTPase and AC)
agonist competition at β2AR-Gs
fusion proteins fits in silico data best
(not shown)
supports medicinal-chemical
rationale (Wainer et al.)
Insights into stereoselective
interaction & functional selectivity Various Levels in the G-protein cycle:
binding, transmission, effector
Taken together – Binding Assays Adds to functional data and published analyses
O H
C H 3 O H
N H O H
O H
O H
* *
O
72
Binding assays (antagonist and agonist)
Functional assays (GTPase and AC)
agonist competition at β2AR-Gs
fusion proteins fits in silico data best
(not shown)
supports medicinal-chemical
rationale (Wainer et al.)
(R,S‘)- and (S,S‘)-3 depict unique
dissociation of binding and GTPase
activity
supports (own) functional
analyses
Insights into stereoselective
interaction & functional selectivity Various Levels in the G-protein cycle:
binding, transmission, effector
Taken together – Binding Assays Adds to functional data and published analyses
O H
C H 3 O H
N H O H
O H
O H
* *
O
73
EX VIVO ASSAY IN NEUTROPHILS OF 60
VOLUNTEERS
Supplemental Results: Study
74
Study with neutrophils of 60 volunteers Connecting β2AR SNPs and ex vivo responsiveness
96-well format
1 x 105 cells/well, just 4-8 ml of blood needed
4 hrs from blood collection to assay completion
ROS assay and pharmacological analysis
60 volunteers
180 96-well-plate assays
240 concentration-response
curves
Numbers
75
healthy volunteers recruited at MHH
„caucasian“ background
Cohort characteristics (n=60)
Sixty Volunteers – Background check Receptor polymorphisms (SNP) vs. β2AR pharmacology
0% 50% 100%
asthma
atopic
smoking
male
yes
no
0 0,2 0,4 0,6
Thr283Ser
Thr164Ile
Gln27Glu
Gly16Arg
this study
HAPMAP-CEPH
/ MAF
Cell isolation & ex vivo „performance“
neutrophils eosinophils basophils
0
1000
2000
3000
4000
granulocytes count / mL blood
76
Influence of factors despite SNPs No significant difference in responsiveness
One-way ANOVA; p-value > 0.05 = n.s.
possible confounders are excluded from the model or have no significant effect
comparing β2AR-responsiveness from fitting concentration-response data
standardized
pooled for all four ligands β2AR-agonists
Statistical modeling for difference between sub-populations
77
Ligand-specific responsiveness (potency) 60 volunteers ~ 180 Assays
concentration-response data yielded pIC50 values on the β2AR-mediated ROS-inhibition
Formoterol (FORM) most potent, Salbutamol (SAL) least potent
looking at individual -> ligand-specific responsiveness (color-coded)
78
Standardization of response values of 4 ligands Substraction of the ligand-group mean
Apple and Oranges ?
(difficult to pool & compare)
11
10
9
8
7
p
IC 5
0,R
OS
(R)-ISO (R,R)-FEN (R)-SAL (R,R)-FORM
79
Standardization of response values of 4 ligands Substraction of the ligand-group mean
Standardized responsiveness
(values directly comparable)
Apple and Oranges ?
(difficult to pool & compare)
11
10
9
8
7
p
IC 5
0,R
OS
(R)-ISO (R,R)-FEN (R)-SAL (R,R)-FORM
-2
-1
0
1
2
(R)-ISO
80
Low-concentration or high-concentration responders Correlation of ligand-specific responsiveness
significant correlation between the seperate pIC50 values
ISO vs. FEN, ISO vs. SAL, ISO vs. FORM
allowed pooling of values
researching the association with the genetic background (ADRB2 gene)
Ligand-independency of inter-individual variability
81
ADRB2 exon Analysis of known and unknown SNPs
-468 -406 -367
-376 -262
-47 -26
+66 +659
+46
+79 +252 +491
+523
+1053
+1098
+1239
+1268 +1269
+1275
+1277
+1278
+1629
+1678
ADRB2
Chr. 5
82
Sanger Sequencing of ADRB2 Exon Analysis of known and unknown SNPs
83
Study on Gly16Arg influencing β2AR-agonist therapy
Israel et al., Am J Respir Crit Care Med 162:75 (2000)
84
METHODS
Appendix
85
G-protein cycle at 7TMR-Gs fusion proteins Binding-, GTPase- and AC-assays
86
Extraction of recombinant β2AR from insect cells Sf9 cells – a well-established baculoviral expression system
Sf9 cells are suitable to culture and
prepare human recombinant receptors
3 x 106 cells/mL are inoculated with high-
titer virus encoding β2AR-Gxα fusion
protein
harvesting of cells after 48 h
(very-late phase of infection)
washing, lysis, homogenization and
sedimentation
membrane pellets are resuspended in
binding buffer and stored at -80°C
Infection and Membrane Preparation
87
β2AR-Gs/-Gi fusion protein - GTPase Assay Turn-over at the mediator of the G-Protein cycle
Sf9 membranes expressing β2AR-Gsα or β2AR-Giα
activation of the coupled Gxα
GTPase activity produces GDP and anorganic phosphate
radiometric analysis of [γ-32P]GTP turnover (20 min at 25°C)
concentration-response data
GTPase
+H2O
88
β2AR-Gs/-Gi fusion protein - GTPase Assay Radiometric analysis using [γ-32P]GTP
GTPase
+H2O
+ GDP
in 50 mM Tris/HCl buffer
100 µM adenosine-5‘-[b,g-imido]triphosphate
100 nM GTP
100 µM ATP
1 mM MgCl2
100 µM EDTA
0.2% BSA
5 mM creatine phosphate and 0.4 µg creatine kinase
Buffered reaction mixture with a regenerative component
89
β2AR-Gs fusion protein – Adenylyl Cyclase Assay Production of the 2nd messenger cAMP
Sf9 membranes expressing β2AR-Gsα
activation of endogenous AC
cAMP production
radiometric analysis of [α-32P]ATP turn-over (20 min at 37 °C)
concentration-response data
90
β2AR-Gs fusion protein – AC Assay Radiometric analysis of cAMP generation
AC
high-speed centrifugation
single-column, gravity-driven seperation
Al2O3 packing restrains [α-32P]ATP
[32P]cAMP eluted to scintillation vials (0.1 M NH4-AcO)
followed by liquid scintillation counting (Cerenkov)
Chromatographic separation of [α-32P]ATP and [32P]cAMP
+ PPi
91
β2AR-Gs fusion protein – AC Assay Turn-over of [α-32P]ATP (20 min @ 37°C)
AC
+ PPi
in binding buffer
10 µM GTP
40 µM [α-32P]ATP
0.1 mM cAMP
2.7 mM mono(cyclohexyl)ammonium phosphoenolpyruvate
0.125 IU pyruvate kinase and 1 IU myokinase
Buffered reaction mixture with a regenerative component
92
β-arrestin-2 recruitment assays Analysis of G-protein-independent signalling
stimulation of seeded cells @ 37°C
stopped after 10 min by addition of detection reagent
reading luminescence counts (2s/well)
HEK293-cells expressing β2AR
Takakura et al.
complementation of luciferase fragments
CHO-cells expressing β2AR
DiscoverX PathHunter®
complementation of β-gal fragments
93
β-arrestin-2 recruitment assays Commercial buy-in vs. academic collaboration
own data (DiscoverX, N=1) comparable to data by Timo Littmann (Takakura Assay, N=3)
decision against costly assay ready kit (~ 400 € per 96 well plate)
future publication (Littmann et al) on β-arr-1 vs. β-arr-2 recruitment in preparation
includes comparison of assays (no difference)
Comparison of DiscoverX PathHunter® and Takakura Assay
94
Neutrophils from human whole blood (EDTA) Gentle cell isolation without pre-activation
Ficoll seperating solution
density 1.077 g/mL
30 min @ 400 x g seperates into layers of
plasma
lymphocytes (white)
erythrocytes / granulocytes (red)
followed by
selective lysis of erythrocytes (ddH2O)
washing (PBS)
resuspension in cold PBS
> 98% viable neutrophils
Density Gradient Centrifugation
Q: Nature Protocols
95
cAMP production in human neutrophils The second messenger in a cellular context
stimulation of 5 x 105 cells/tube
in 100 µL PBS
with 1 mM CaCl2, 100 µM IBMX
10 min @ 37°C
stopped @ 95°C
addition of 100 µL eluent A
3/97 MeOH/H20, 50 mM NH4OAc, 0.1%
HOAc
100 ng tenofovir/mL (internal standard)
hand-over to the Core Facility
Metabolomics
quantification by reversed-phase HPLC
mass spectrometry
cAMP extraction
AC
b2AR b
Gi b
cAMP
Gs
b2AR
96
O2.- + Ferricytochrome C (Fe(III)) → O2 + Ferrocytochrome C (Fe(II))
reduction alters absorbance at 550 nm
isosbestic point at 557 nm (no change)
A550(t=30 min) - A550(t=0) = ΔA550
concentration-response data
Inhibition of ROS production in human neutrophils A robust assay of pathophysiologically-relevant signalling
stimulation of 105 cells/well
in a coated 96-well plate
PBS with
1 CaCl2, 100 µM ferricytochrome c,
0.3 µg/mL cytochalasin b
30 min @ 37°C
absorbance at 550 nm (A550)
Colormetric cytochrome c assay
AC
NOX
IP3
DAG
ROS
PKC
FPR
b2AR
Gi b
b
cAMP
PIP2
Gs
97
DATA-ANALYSIS
Appendix
98
Gs vs. Gi coupling at β2AR – quantitatively Condensing efficacy and potency to the transducer coefficient
log(τ/KA)Gsα
→ each agonist
log(τ/KA)Giα
→ each agonist Δlog(τ/K
A) → agonist – (R)-EPI
1st Step: Fitting the
transducer coefficient
2nd Step:
Normalization to reference
ΔΔlog(τ/KA) = G
s - G
i
3rd Step:
Comparison
agonist β2AR KA,Gsα agonist-β2AR effectorGsα signalGsα
τGsα log(τGsα/KA,Gsα)
modulator conduit guest
transducer coefficient
100
Gs vs. Gi coupling at β2AR – bias plot (still qualitatively) Epinephrine and ISO as reference
0 50 100
0
50
100(R)-EPI
(R)-ISO
GTPase activity of b2AR-Gs
(equimolar response)
GT
Pa
se
ac
tiv
ity o
fb
2A
R-G
i2
(eq
uim
ola
r re
sp
on
se
)
endogenous ligand epinephrine
activates Gi and Gs to similar extent
→ reference compound
101
Gs vs. Gi coupling at β2AR – bias plot (still qualitatively) (R,R')-stereoisomers more Gs-biased as reference
0 50 100
0
50
100(R)-EPI
(R)-ISO
(R,R')-1
(R,R')-2
(R,R')-3
GTPase activity of b2AR-Gs
(equimolar response)
GT
Pa
se
ac
tiv
ity o
fb
2A
R-G
i2
(eq
uim
ola
r re
sp
on
se
)
endogenous ligand epinephrine
activates Gi and Gs to similar extent
→ reference compound
relatively more bending towards x-
axis depicts Gs bias
103
Gs vs. Gi coupling at β2AR Tendency to Gs clearly dominates – no Gi-bias
0 50 100
0
50
100(R)-EPI
(R)-ISO
(R,R')-1
(R,S')-1
(S,R')-1
(S,S')-1
(R,R')-2
(R,S')-2
(S,R')-2
(S,S')-2(R,R')-3
(R,S')-3
(S,R')-3(S,S')-3
GTPase activity of b2AR-Gs
(equimolar response)
GT
Pa
se
ac
tiv
ity o
fb
2A
R-G
i2
(eq
uim
ola
r re
sp
on
se
)
endogenous ligand epinephrine
activates Gi and Gs to similar extent
→ reference compound
relatively more bending towards x-
axis depicts Gs bias
no curve „near“ y-axis = no Gi-
biased ligand
104
Gs vs. Gi coupling at β2AR – bias plot Bending curves towards x-axis depict Gs bias
0 50 100
0
50
100(R)-EPI
(R)-ISO
(S,S')-2
(R,S')-3
(S,S')-3
GTPase activity of b2AR-Gs
(equimolar response)
GT
Pa
se
ac
tiv
ity o
fb
2A
R-G
i2
(eq
uim
ola
r re
sp
on
se
)
endogenous ligand epinephrine
activates Gi and Gs to similar extent
→ reference compound
relatively more bending towards x-
axis depicts Gs bias
no curve „near“ y-axis = no Gi-
biased ligand
(S,S')-Methoxy and (R,S')-
Methoxy-naphthyl-fenoterol partial
agonists at Gi (< 50%)
strong Gs-bias
(S,S')-Methoxy-naphthyl-fenoterol
extremely Gs-biased
no Gi-GTPase activity at all
110
Bronchial Asthma Bronchodilatative Effect
111
Structure-biological techniques
analysis of single structure elementslemente
energy landscape
mechanical flexibility
conformative variability
single-molecule force spectroscopy
Zocher et al Chem Soc 42:19 (2013)
112
Structure-biological techniques
analysis of different receptor conformations
agonist / antagonist bound ..
detects chemical shifts -> distance between 1H- / 13C- / 15N-labeled residues
Solid state nuclear magnetic resonance
Bokoch et al Nature 463 (2010)
113
Structure-biological techniques
analysis of different receptor conformations
agonist / antagonist bound ..
detects chemical shifts -> distance between 1H- / 13C- / 15N-labeled residues
Solid state nuclear magnetic resonance
Bokoch et al Nature 463 (2010)
114
Functional Selectivity via β2-Adrenoreceptor
Relevance of signalling during chronic heart failure
Gs
AC
cAMP ↑
Gi
AC MAPK
(fast) cAMP ↓
β-arrestin
MAPK
(delayed)
other
signals?
PTX
β2AR agonists
(unbiased, Gs-biased, Gi-biased, β-arr-biased)
β2AR β2AR β2AR
immune cell
immunosuppression pro-inflammatory? immune-modulation?
desensitization
cardiomyocyte
contractile support cardio-protective
compromised contractile support
cardio-protective
cardiac remodeling
airway smooth muscle
relaxation contractile sensitization desensitization
openclipart.org & http://hsc.uwe.ac.uk/rcp/rs-rt-bronchioles.aspx
115
Acute Heartfailure Cardio-protective Effect
116
GPCR Network, The Scripps Research Institute.
117
- +
not studied
Institute of Pharmacology
Roland Seifert
8.10.2012
Effects of fenoterol stereoiomers on ERK activation Differential phosphoprofiles
(R,R’)-Fenoterol (S,R’)-Fenoterol
118
control 1 µM (R,R)-Fenoterol 10 µM (S,R)-Fenoterol
Effects of fenoterol stereoiomers on ERK activation Phosphoprofiler in HL-60 promyelocytes
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