Molecular, physiological and pathophysiological analysis of the β₂-adrenoreceptor

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



Gstimulatory Ginhibitory β-arrestin

β2AR agonists

(unbiased, Gs-biased, Gi-biased, β-arr-biased)

β2AR β2AR β2AR

Signal transduction pathways via β2AR

16




β2AR agonists


β2AR β2AR β2AR


17



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.


β2AR agonists


β2AR β2AR 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


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


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’)


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


Gi-GTPase

Gs-GTPase

AC

Binding

β2AR-G


β-arrestin-2 recruitment

HEK293-cells expressing β2AR

Takakura H et al., ACS Chem Biol 7:5 (2012)

26


cAMP accumulation

inhibition of ROS production

(radical oxygen species)

Neutrophils expressing β2AR

Gi-GTPase

Gs-GTPase

AC

Binding

β2AR-G


β-arrestin-2 recruitment


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


Gs-bias of

(S,S')-methoxy-

fenoterol

(R,S')-

methoxy-

naphthyl-

fenoterol

O H

O O

33


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



fenoterol ligands

reference and

unmodified

(R,R')-fenoterol

mostly balanced

38



fenoterol ligands

reference and

unmodified

(R,R')-fenoterol

mostly balanced

modification at

aminoalkyl-tail

increases Gs-bias

> >

> >

> >

> >

> >

> >

> >

39



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



fenoterol ligands

reference and

unmodified

(R,R')-fenoterol

mostly balanced

modification at

aminoalkyl-tail

increases Gs-bias


Gi-coupling


(R,S')- and (S,S')-

methoxy-naphthyl-

fenoterol (3) are

strong / extreme Gs-

biased

41



fenoterol ligands

reference and

unmodified

(R,R')-fenoterol

mostly balanced

modification at

aminoalkyl-tail

increases Gs-bias


Gi-coupling


(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




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


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


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




Gs-GTPase vs. competition binding Information on structure-(bias)/activity-relationship

4 5 6 7 8 94

5

6

7

8

9

10


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


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





(not shown)







O H

C H 3 O H

N H O H

O H

O H

* * O

71





(not shown)







O H

C H 3 O H

N H O H

O H

O H

* *

O

72





(not shown)



(R,S‘)- and (S,S‘)-3 depict unique

dissociation of binding and GTPase

activity

supports (own) functional

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)


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)


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


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



GT

Pa

se

ac

tiv

ity o

fb

2A

R-G

i2

(eq

uim

ola

r re

sp

on

se

)




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



GT

Pa

se

ac

tiv

ity o

fb

2A

R-G

i2

(eq

uim

ola

r re

sp

on

se

)






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



GT

Pa

se

ac

tiv

ity o

fb

2A

R-G

i2

(eq

uim

ola

r re

sp

on

se

)






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


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


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


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


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

Molecular, physiological and pathophysiological analysis of the β₂-adrenoreceptor

Science

Transcript of Molecular, physiological and pathophysiological analysis of the β₂-adrenoreceptor