Slides of my thesis defence on 14th of July 2015. As part of my PhD thesis, I analyzed β₂-adrenoceptor-mediated signal transduction. The β₂-adrenoceptor is pathophysiologically relevant e.g. in case of bronchial asthma. My studies add to the receptor-pharmacological concept of functional selectivity. Functional selective (or biased ) ligands may be exploited to achieve highly selective drug effects and, thus, improved drug safety with less adverse effects. Further, my studies included a collaboration with allergy geneticists. Here we linked genomic and pharmacological profiling of the β₂-adrenoceptor in 60 volunteers. Such pharmacogenomical data may help to stratify treatment groups and create a basis for a more effective, personalized asthma therapy. (German title: "Molekulare, physiologische und pathophysiologische Analyse des β₂-Adrenorezeptors")

1. 1
Molekulare, physiologische und
pathophysiologische Analyse
des β2-Adrenorezeptors
Rezeptorpharmakologie / Pharmakogenetik
In vitro / Ex vivo
Funktionelle Selektivität / Individuelle Arzneimittelreaktion
Disputation
Michael T. Reinartz

2. 2
Warm-Up: Receptor Pharmacology
Two states?

3. 3
Warm-Up: Receptor Pharmacology
Multiple states!Two states?

4. 4
Warm-Up: Pharmacogenetics
One patient group?

5. 5
Warm-Up: Pharmacogenetics
Individual drug-response!One patient group?
responders
with adverse
effects
responders
only
adverse
effect
non
responders

6. 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

7. 8
Fight-or-Flight reaction via adrenergic receptors
Epinephrine activates adrenergic receptors

8. 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

9. 10
Fight-or-Flight reaction via β2-adrenergic receptors
Selective activation of β2AR-specific effects

10. 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

11. 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

12. 13
Ligand-specific pharmacology
of 14 β2AR-ligands
Reinartz MT et al., Naunyn Schmiedebergs Arch Pharmacol. 388:1 (2015)
β2AR-
specific
information
Part 1

13. 14
β2AR ligands can be functionally selective
Ligand classification
Simmons MA Mol Interv 5:3 (2005)

14. 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

15. 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

16. 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)

17. 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

18. 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

19. 20
Fenoterol
A β2AR-selective sympathomimeticum
CH3
OH
NHOH
OH
OH
* *
NH
OH
OH
OH
(R)-EPI

20. 21
Fenoterol
A β2AR-selective sympathomimeticum
CH3
OH
NHOH
OH
OH
* *
(R)-EPI
NH
OH
OH
OH
 yields β2AR-selectivity
Aminoalkyl-tail

21. 22
3 Fenoterol derivatives + 4 stereoisomers = 12 ligands
Modifications at aminoalkyl-tail + differences in chirality
CH3
OH
NHOH
OH
OH
CH3
O
NHOH
OH
OH
CH3
O
NHOH
OH
OH
* *
* *
* *
1
2
3
 yields β2AR-selectivity
 derivatization
 hydroxy-benzyl (1)
 methoxy-benzyl (2)
 methoxy-naphthyl (3)
Aminoalkyl-tail

22. 23
3 Fenoterol derivatives + 4 stereoisomers = 12 ligands
Modifications at aminoalkyl-tail + differences in chirality
CH3
OH
NHOH
OH
OH
CH3
O
NHOH
OH
OH
CH3
O
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

23. 24
Extensive pharmacological profiling
In vitro, in cell and ex vivo
 Gi-GTPase
 Gs-GTPase
 AC
 Binding
β2AR-Gxα- fusion proteins

24. 25
Extensive pharmacological profiling
In vitro, in cell and ex vivo
 Gi-GTPase
 Gs-GTPase
 AC
 Binding
β2AR-Gxα- fusion proteins
 β-arrestin-2 recruitment
HEK293-cells expressing β2AR
Takakura H et al., ACS Chem Biol 7:5 (2012)

25. 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-Gxα- 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

26. 27
Six functional assays
RESULTS PART 1

27. 28
Comparison of concentration-response data
OH OO
Gs- and Gi-coupling are influenced by aminoalkyl-tail and stereochemistry

28. 29
Comparison of concentration-response data
OH OO
Gs- and Gi-coupling are influenced by aminoalkyl-tail and stereochemistry

29. 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) = Gs - Gi
Comparison of two pathways

30. 31
Bias analysis: Gs- vs. Gi-coupling at β2AR
OH OO

31. 32
Bias analysis: Gs- vs. Gi-coupling at β2AR
 Gs-bias of
 (S,S')-methoxy-
fenoterol
 (R,S')-
methoxy-
naphthyl-
fenoterol
OH OO

32. 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
OH OO

33. 34
Summary as functional “fingerprints”
Pairwise comparison of six assays per ligand
ΔΔlog
A
B
0 vs.
ΔΔlog
A
B
0 vs.
ΔΔlog
A
B
0 vs.
ΔΔlog
A
B
0 vs.
ΔΔlog
A
B
0 vs.
ΔΔlog
A
B
0 vs.
ΔΔlog
A
B
0 vs.
ΔΔlog
A
B
0 vs.
ΔΔlog
A
B
0 vs.
ΔΔlog
A
B
0 vs.
ΔΔlog
A
B
0 vs.
ΔΔlog
A
B
0 vs.
ΔΔlog
A
B
0 vs.
ΔΔlog
A
B
0 vs.
ΔΔlog
A
B
0 vs.
Six β2AR assays
15 pair-wise comparisons
Single fingerprint per ligand

34. 35
Heatmap of 14 functional “fingerprints”
Sums up 84 concentration-response curves (with n ≥ 3)
Heatmap to compare
14 ligand fingerprints

35. 36
Results from the heatmap analysis
 Gs-bias (red) for most
fenoterol ligands

36. 37
Results from the heatmap analysis
 Gs-bias (red) for most
fenoterol ligands
 reference and
unmodified
(R,R')-fenoterol
mostly balanced

37. 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
> >
> >
> >
> >
> >
> >
> >

38. 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

39. 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

40. 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
CH3
O
NHOH
OH
OH
*
S

41. 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

42. 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

43. 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
CH3
O
NHOH
OH
OH
*
S

44. 45
Individual β2AR-drug-response
of 60 volunteers
Reinartz MT et al., submitted to Allergy
Subject-
specific
information
Part 2

45. 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
othersgenetic (ADRB2, ...) up to 70 %
Severe asthma

46. 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 geneSevere asthma
http://www.pharmgkb.org/gene/PA39
othersgenetic (ADRB2, ...) up to 70 %

47. 48
Connecting β2AR SNPs and ex vivo responsiveness
Study with DNA and neutrophils of 60 volunteers

48. 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

49. 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
CH
3
CH
3
NH
OH
OH
OH
*
CH
3 OH
NHOH
OH
OH
*
*
CH
3
CH
3
CH
3
NH
OH
OH
OH
*
CH
3 O
CH
3
NH
OH
NH
O
OH
*
*
(R,R’)-FEN
(R)-SAL
(R,R’)-FORM

50. 51
RESULTS PART 2
Ex vivo assay and genetic data

51. 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
Gly16Arg
this 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

52. 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

53. 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

54. 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

55. 56
Precise drugs and personalized treatment
Outlook
Improved
therapy

56. 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

57. 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

58. 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

59. 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

60. 61
Appendix

61. 62
Model of Multiple Receptor Conformations
A complex composed melody and NOT only a single tune

62. 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

63. 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

64. 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

65. 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 β2AR with Gs
4 5 6 7 8 9
4
5
6
7
8
9
10
pEC50,GTPase = 3.9 + 0.521  pKi,low,DHA
R² = 0.56
pKi,low,DHA,control
pEC50,GTPase
4 5 6 7 8 9
4
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
pEC50,GTPase

66. 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 9
4
5
6
7
8
9
10
pEC50,GTPase = 3.9 + 0.521  pKi,low,DHA
R² = 0.56
pKi,low,DHA,control
pEC50,GTPase
4 5 6 7 8 9
4
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
pEC50,GTPase
 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

67. 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 9
4
5
6
7
8
9
10
pEC50,GTPase = 3.9 + 0.521  pKi,low,DHA
R² = 0.56
pKi,low,DHA,control
pEC50,GTPase
4 5 6 7 8 9
4
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
pEC50,GTPase
 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

68. 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
CH3
OH
NHOH
OH
OH
* *

69. 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
CH3
OH
NHOH
OH
OH
* *
O

70. 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
CH3
OH
NHOH
OH
OH
* *
O

71. 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
CH3
OH
NHOH
OH
OH
* *
O

72. 73
EX VIVO ASSAY IN NEUTROPHILS OF 60
VOLUNTEERS
Supplemental Results: Study

73. 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

74. 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

75. 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

76. 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)

77. 78
Standardization of response values of 4 ligands
Substraction of the ligand-group mean
Apple and Oranges ?
(difficult to pool & compare)
1110987
pIC50,ROS
(R)-ISO (R,R)-FEN (R)-SAL (R,R)-FORM

78. 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)
1110987
pIC50,ROS
(R)-ISO (R,R)-FEN (R)-SAL (R,R)-FORM
-2-1012
(R)-ISO

79. 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

80. 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

81. 82
Sanger Sequencing of ADRB2 Exon
Analysis of known and unknown SNPs

82. 83
Study on Gly16Arg influencing β2AR-agonist therapy
Israel et al., Am J Respir Crit Care Med 162:75 (2000)

83. 84
METHODS
Appendix

84. 85
G-protein cycle at 7TMR-Gs fusion proteins
Binding-, GTPase- and AC-assays

85. 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

86. 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

87. 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

88. 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

89. 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

90. 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

91. 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

92. 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

93. 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

94. 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

95. 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

96. 97
DATA-ANALYSIS
Appendix

97. 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/KA) → agonist – (R)-EPI
1st Step: Fitting the
transducer coefficient
2nd Step:
Normalization to reference
ΔΔlog(τ/KA) = Gs - Gi
3rd Step:
Comparison
agonist β2AR KA,Gsα agonist-β2AR effectorGsα signalGsα
τGsαlog(τGsα/KA,Gsα)
modulator conduit guest
transducer coefficient

98. 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)
GTPaseactivityofb2AR-Gi2
(equimolarresponse)
 endogenous ligand epinephrine
activates Gi and Gs to similar extent
→ reference compound

99. 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)
GTPaseactivityofb2AR-Gi2
(equimolarresponse)
 endogenous ligand epinephrine
activates Gi and Gs to similar extent
→ reference compound
 relatively more bending towards x-
axis depicts Gs bias

100. 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)
GTPaseactivityofb2AR-Gi2
(equimolarresponse)
 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

101. 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)
GTPaseactivityofb2AR-Gi2
(equimolarresponse)
 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

102. 110
Bronchial Asthma
Bronchodilatative Effect

103. 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)

104. 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)

105. 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)

106. 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

107. 115
Acute Heartfailure
Cardio-protective Effect

108. 116
GPCR Network, The Scripps Research Institute.

109. 117
- +
not studied
Institute of Pharmacology
Roland Seifert
Effects of fenoterol stereoiomers on ERK activation
Differential phosphoprofiles
(R,R’)-Fenoterol (S,R’)-Fenoterol

110. 118
control 1 µM (R,R)-Fenoterol 10 µM (S,R)-Fenoterol
Effects of fenoterol stereoiomers on ERK activation
Phosphoprofiler in HL-60 promyelocytes