background results & outlook method
TRANSCRIPT
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: [email protected]
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