Mul parametric Measurements of RTK Ac vi es to Determine Pharmacological Profi les of Compounds.

Jan P. Wintgens1, Sven P. Wichert1, Johanna Zach1, Sabrina Galinski, Moritz J. Rossner1,2, Michael C. Wehr1*

1 Molecular and Behavioral Neurobiology, Department of Psychiatry, Ludwig Maximilians University of Munich, Germany;2 Department of Neurogene cs, Max Planck Ins tute of Experimental Medicine, Gö ngen, Germany

*correspondence: mwehr@med.lmu.de

Receptor tyrosine kinases (RTKs) are transmembrane receptors important for cell-to-cell communica on. In humans, the RTK family comprises 58 members providing a great variety for cellular signalling. RTK signallnig has also been linked to various diseases, making them a key target for drug discovery. For example, the ERBB4 receptor is implicated in schizophrenia (SZ), as Neuregulin1-ERBB4 pathway ac vity is suggested to modulate posi ve, nega ve and cogni ve symptoms of schizophrenia (Yin, 2013; Falkai et al., 2015). We aim at profi ling ac vi es of ERBB4, its closely and distantly related RTKs to assess compound specifi ci es and ac vi es on these drug targets.

Background

1.Genome-Wide Assocai on studies (GWAS) Iden fy RTK-related Risk Genes in Schizophrenia

4. Single RTK Assays: Dimerisa on, Adapter Binding and SH2 Domain Binding 6. Experimental Outlook5. EXTassay Workfl ow

3. EXTassays for Assessing Target Specifi ci es2. Compound Screening Approaches and RTK Targets for Assessing Specifi city

The results show the applicability of the split TEV assays. Using single assays, RTK dimerisa on, adapter binding and clustered SH2 domain binding can be monitored robustly. Currently, we are applying the EXTassay technique to generate a comprehensive profi le of ERBB, FGFR, and INSR ac vi es. In future, we are aiming at adding TRK, EPHR, and VEGFR families to the panel to extend the RTK profi ler. This setup will enable us to predict compound ac ons within one measurement, highligh ng desired eff ects (ON target) from unwanted side eff ects (OFF target). Using this profi ling RTK assay, we hope to pave the way for new treatment strategies, with a focus on schizophrenia.

Results & Outlook

The split TEV technique is a robust and fl exible molecular tool to measure protein-protein interac ons and receptor ac vi es in living cells (Wehr et al., 2006). Using this method in a drug screening approach, we assess compound ac ons on closely and distantly related RTKs to discriminate between ON and OFF target eff ects. In par cular, we are addressing ac vi es of the ERBB, FGFR, TRK, EPHR, VEGFR and insulin receptor families, to establish an RTK profi le for compounds screened. Further, we assay RTK ac vi es using a modular SH2 domain-based interac on readout, increasing robustness and fl exibility of the assay.

Method

(a) Manha an Plot of SZ risk genes. The x-axis is chromosomal posi on and the y-axis is the signifi cance of associa on (−log10(P) Ripke et al. (Nature, 2014). (b) RTKs FGFR1 and ERBB4 present in SZ risk genes derived from Ripke et al.(signifi cance: 5e-8). (c) Direct RTK adapters NCK1 and PTPRF are SZ risk genes. (d) The SZ risk pathway NRG1-ERBB4.

All assays are based on the split TEV protein-protein interac on detec on technique. (a) ERBB4 dimerisa on scheme. (b) ERBB receptor adapter interac on scheme (c) Full-lenght GRB2 protein containing an SH2 domain (top) and an ar fi cially clustered SH2 domain adapter protein. (d-i) Dose response curves for indicated combina ons.

(a) Summary of all RTKs and corresponding adapters selected to obtain substan al RTK profi le ac vi es. ERBB family, including EGFR, ac vi es can be monitored robustly. INSR and FGFR1-3 are currently added to the RTK profi ler; future work includes TRK, EPHR, and VEGFR families.S muli for receptor families are shown on the le . Most adapters (right panel) are applicable, the SRC adapter has not been tested yet. All ar fi cially clustered SH2 domain adapter proteins are func onal.

(a)The EXTassay technique is used to monitor various RTK ac vi es under diverse assay condi ons within one measurement. Mul ple single transfec- ons of RTK assays are mixed and then split into diff erent experiment setups

to address various compound ac ons simultaneously. (b) Example RTK profi ler, including a GPCR (HTR2A) control. Cell Pooling and EXT read-out have been tested. FGFR3, fi broblast growth factor receptor 3, INSR, insulin receptor.

(a) EXTassays to monitor target specifi ci es. The single assay is based on the split TEV technique. When applied in a mul -parametric assay setup (EXTassay), compound candidates can be measured on various targets (receptor ac vi es) and at various condi ons. (b) Sample EXTassay setup and experimantal fl ow-chart shown for 3 RTKs. For the EXTassay analysis, the RNA is extracted, decoded (RT-PCR, PCR ampl.), and analysed using NGS technology.

(a) The ERBB4-PIK3R1 split TEV assay monitors NRG1-ERBB4 signalling. (b) Dose-response assay using EGF-like domain as s mulus. (c) Dose-response assay using the pan-ERBB inhibitor lapa nib as inhibitor. (d) Human kinase tree showing the RTK families to cover within an RTK target specifi city assay.

NE

DC

BA

NE

DC

BA

NE

DC

BA

X targetsY conditions

1 measurement effects (ON target) side effects

(OFF target)

RTK No 3RTK No 1 RTK No 2

Concentration

Act

ivity

EXT barcode 1

EXT barcode 2

EXT barcode 3

RNA isolationRT-PCR of EXTs

& PCR amplification

6 2

1

1

326

1

1745 8

58

NGS Analysis

b

a

NRG1

Schizophrenia risk pathway

ERBB4

PIK3

a

b c

d

Interfering compound

56656 1 56691 2(2)

FGFR1(ERBB4)

RTKs

SZ risk genes pValue 1e-6

SZ risk genes pValue 5e-8

RTK AdaptersSZ risk genes pValue 5e-8

NCK1PTPRF

b

c

Fluc10xUAS

GV

etvS

GV

CTEVNTEV

ERBB4

PI3KR1

a

d

ERBB receptor family FGFR family NGF receptor family Insulin receptor family VEGFR family EPH receptor family PDGFR receptor family

EGF-like domain (NRG1)

IC50: 5.0614 [μM]lapatinib [μM]

activ

ity[%

]

0.01 1 1000

20

40

60

80

100

EC50: 0.1581 ng/mlEGFld [ng/ml]

activ

ity[%

]

0.01 1 1000

20

40

60

80

100

a

b

4-4

ERBB dimers

ERBB activities (adapter assays)

PIK3R1

GRB2SHC1

ERBB4 - ERBB4

ERBB2/3 - PIK3R1

IC50: 2.4373 [μM]lapatinib [μM]

activ

ity[%

]

0.0001 0.01 1 1000

20

40

60

80

100

EC50: 4.0362 ng/mlEGFld [ng/ml]

activ

ity[%

]

0.0001 0.01 1 1000

20

40

60

80

100

EC50: 0.031 ng/mlEGFld [ng/ml]

activ

ity[%

]

0.0001 0.01 1 1000

20

40

60

80

100

EC50: 0.296 ng/mlEGFld [ng/ml]

activ

ity[%

]

0.0001 0.01 1 1000

20

40

60

80

100

IC50: 4.3071 [μM]lapatinib [μM]

activ

ity[%

]

0.0001 0.01 1 1000

20

40

60

80

100GRB2

SH2(GRB2)

1-1 2-3

XX EGFR - GRB2

ERBB4 - ERBB4

c ERBB activities (SH2 Domain assays)

ERBB4 - SH2(GRB2) ERBB4 - SH2(GRB2)

SH2 SH2SH2

aa 303

SH2SH3 SH3

aa 217

f i

e h

d g

EC50: 0.5555 ng/mlEGF [ng/ml]

activ

ity[%

]

0 0.001 0.1 10 10000

20

40

60

80

100

a

b

Unstimulated

Unstimulated

Condition 1 Condition 2 Condition 3

10xUASEXT 1

10xUAS EXT 2

10xUAS EXT 3

10xUASEXT 4

10xUASEXT 5

10xUASEXT 6

10xUASEXT 7

10xUASEXT 8

10xUAS EXT 9

singletransfections

mix & divide

experimentalconditions

sample setup: 10 RTKs

(EGFR, ERBB2/3/4, FGFR1/2/3, NTRK1/2/3) 6 stimuli

(EGFld, EGF, FGF, NGF, BDNF, Ctrl) 8 concentrations 2 biol. replicates 3 EXT replicates

= 2880 data points

EGFld

EGF

Insulin

aFGF

FGFR3EGFR

ERBB2/3ERBB4

INSR

HTR2A

Serotonin

N Conditions

1 Measurement

6 targets

6 + N conditions

a

ERBB 2-4

EGFR

INSR

FGFR 1-3

TRK A-C

EPHA 1/4

VEGFR 1-3

Receptors

Adapters

GRB2

SHC1

PIK3R1

PLCG1/2

SRC

IRS1

SH2 Domains

EGFld

EGF

Insulin

aFGF

NGF

Ephrin-A

VEGF A/D