patenten

1
The MAPPIT ToolboxMAmmalian Protein-Protein
Interaction Trapstrategies to analyze molecular
interactions in intact cellsRoscoffSeptember
23, 2008
2
JAK-STAT signalling
type I cytokine receptor
extracellular
intracellular
Y Y Y
Y Y Y
Y
JAK
JAK
JAK kinase
Y
3
JAK-STAT signalling
cytokine
4
JAK-STAT signalling
5
JAK-STAT signalling
P
Y Y Y
Y Y Y
P
STAT
6
JAK-STAT signalling
P
Y Y Y
Y Y Y
P
cytoplasm
nucleus
STAT responsive promoter
7
MAPPIT
cytokine receptor
F F F
F F F
Y
JAK2
JAK2
Y
leptin receptor (YgtF)
Eyckerman et al. (2001) Nat. Cell Biol.
8
MAPPIT
ligand
F F F
F F F
Y
JAK2
JAK2
Y
Eyckerman et al. (2001) Nat. Cell Biol.
9
MAPPIT
ligand
F F F
F F F
Y
JAK2
JAK2
Y
Eyckerman et al. (2001) Nat. Cell Biol.
10
MAPPIT
ligand
F F F
F F F
Y
JAK2
JAK2
Y
bait
Eyckerman et al. (2001) Nat. Cell Biol.
11
MAPPIT
ligand
F F F
F F F
Y
JAK2
JAK2
Y
Y Y Y Y
gp130
prey
Eyckerman et al. (2001) Nat. Cell Biol.
12
MAPPIT
Eyckerman et al. (2001) Nat. Cell Biol.
13
MAPPIT
ligand
F F F
F F F
Y
JAK2
JAK2
Y
Y Y Y Y
STAT3
Eyckerman et al. (2001) Nat. Cell Biol.
14
MAPPIT
ligand
F F F
F F F
Y
JAK2
JAK2
Y
P
Y Y Y Y
P
P
rPAP1 promoter
reporter gene
Eyckerman et al. (2001) Nat. Cell Biol.
15
MAPPIT
ligand
F F F
F F F
Y
JAK2
JAK2
Y
Y Y Y Y
P
rPAP1 promoter
reporter gene
Eyckerman et al. (2001) Nat. Cell Biol.
16
MAPPIT example
Lemmens et al., Nucl. Ac. Res., 31(14)e75
(2003) Montoye et al., Blood, 105, 4264-4271
(2005) Lavens et al., J. Cell Sci., 119,
2214-2224 (2006) Montoye et al., FEBS Lett. 580,
3301-3307 (2006) Piessevaux et al., J. Biol.
Chem. 281, 32953-66 (2006) Lavens et al.,
Biochem. J., 401, 257-267 (2007) Ulrichts et al.,
FEBS Lett., 581, 629-636 (2007) Erkeland et al.,
Oncogene, 26, 1984-1995 (2007) Uyttendaele et
al., Mol. Endocrinol., 21, 2821-2831
(2007) Wauman et al., Mol. Endocrinol., 22,
965-977 (2008) Ulrichts et al., Immunol. Lett.,
115, 141-148 (2008) Piessevaux et al., J. Biol.
Chem., 283, 21334-21346 (2008) Pattyn et al., J.
Virol. Methods 153, 7-15 (2008) Ulrichts et al.,
Methods Mol. Biol., in press.
17
Interactome mapping

• Large-scale programs to map complete interactomes
  of an organism
• ? collaboration with Marc Vidal group (DFCI,
  Harvard U, Boston)
• systematic matrix-based Y2H analysis
• Yu et al., High quality binary protein
  interaction map of the yeast interactome network
• Science (2008)
• Simonis et al., Empirically-controlled
  mapping of the C. elegans protein-protein
  interactome network
• Nat. Methods, revised manuscript under review
• Venkatesan et al., An empirical framework
  for binary interactome mapping,
• Nat. Methods, revised manuscript under review
• Critical issues coverage (false negatives)
• accuracy (false positives)

18
MAPPIT performance
Braun et al., Nature Methods, revised manuscript
under review
19
ArrayMAPPIT screening assay
human ORFeome collection
activation
MAPPIT prey collection
luciferase read-out
20
Screening for Skp1 interaction partners
10-fold induction
Lievens et al., Array MAPPIT high-throughput
interactome analysis in mammalian cells, J.
Proteome Res., under revision
21
MAPPIT summary

• Operates in intact human cells close to normal
  physiological context
• High sensitivity and specificity
• Easy to perform, simple readout
• Automation Runs on 96 and 384 well format on
  Tecan Evo platform
• ? Interactome mapping (20.000 PPIs/year)
• ? Drug screening

22
Example HIV RT dimerisation
p51/p660.3µM p66/p664.0µM p51/p51230µM
23
Efavirenz stabilizes the HIV RT dimer
24
Efavirenz stabilizes the HIV RT dimer
25
Example Apobec3G Vif interaction
26
Example Apobec3G Vif interaction
27
Example Apobec3G Vif interaction
28
Example Apobec3G Vif interaction
29
Growing list of disrupter compounds (smPPIIs)

• Compounds 1-4 LFA-1 ICAM1 (2002)
• Compound 2 i-NOS i-NOS (2002)
• Madindoline A Gp130 Gp130 (2002)
• Dihydrodipyrazolopyridinone B7-1
  CD28 (2002)
• BMS-488043 HIVgp120 CD4 (2003)
• L-series b-aminoketones TRb SRC2 (2003)
• Compound 10058 Myc Max (2003)
• Adamanolol ESX Sur-2 (2003)
• SP-4206 IL2R IL2 (2004)
• Nutlins MDM2 p53 (2004)
• PKF115-584 b-catenin Tcf4 (2004)
• AMD-070 HIVgp120 CXCR4 (2004)
• BMS-433771 RSV-HR-N RSV-HR-C (2004)
• 10506-2A Anthrax EF Calmodulin (2004)
• NSC23766 Rac1 GEFs TrioN, Tiam1 (2004)
• Polyphenylureas XIAP Caspases-3 and
  -7 (2004)
• ABT-737 Bcl2 Bak1 (2005)
• Maraviroc HIVgp120 CCR5 (2005)
• SP304 TNFa dimer TNFa (2005)

30
Reverse MAPPIT
EpoR
F F Y
F F Y
Y
JAK2
JAK2
Y
LR-FFY
? Y1138 functional STAT3 recruitment site
bait
Eyckerman et al. (2005) Nature Methods
31
Reverse MAPPIT
Epo
F F Y
F F Y
Y
JAK2
JAK2
Y
Eyckerman et al. (2005) Nature Methods
32
Reverse MAPPIT
Epo
P
F F Y
F F Y
Y
JAK2
JAK2
Y
P
P
P
rPAP1 promoter
luciferase
Eyckerman et al. (2005) Nature Methods
33
Reverse MAPPIT
Epo
F F Y
F F Y
Y
JAK2
JAK2
Y
phosphatase domain
prey
Eyckerman et al. (2005) Nature Methods
34
Reverse MAPPIT
Epo
F F Y
F F Y
Y
JAK2
JAK2
Y
protein/organic compound
Eyckerman et al. (2005) Nature Methods
35
ReverseMAPPIT example FK506 (ALK4-FKBP12)
nM FK506
36
ReverseMAPPIT example Nutlins (p53-MDM2)
37
MAPPIT screens

• Screens ongoing for
• HIV RTp51/RTp66
• HIV Apobec3G/Vif
• HIV LEDGF/INT
• HCV NS3/NS4A
• In collaboration with Tibotec (JJ group)

38
MASPIT
Epo
EpoR
F F F
F F F
Y
JAK2
JAK2
Y
LR-F3
DHFR
Caligiuri et al. (2006) Chem. Biol.
39
MASPIT
Epo
F F F
F F F
Y
JAK2
JAK2
Y
compound
PEG linker
methotrexate
Caligiuri et al. (2006) Chem. Biol.
40
MASPIT
Epo
F F F
F F F
Y
JAK2
JAK2
Y
Y Y Y Y
Caligiuri et al. (2006) Chem. Biol.
41
MASPIT
Epo
F F F
F F F
Y
JAK2
JAK2
Y
Y Y Y Y
Y Y Y Y
rPAP1 promoter
luciferase
Caligiuri et al. (2006) Chem. Biol.
42
MASPIT example PD173955

• Relative affinity measurement in intact cells
• MASPIT EC50 better reflects reality than IC50
  obtained with in vitro assays (e.g. compound
  uptake, interference of other proteins with
  binding)

43
MASPIT example camptothecin

• Camptothecin ? potent antitumor antibiotic (DNA
  topoisomerase-1 inhibitor)
• ? also suppresses HCV replication (target unknown)

44
MASPIT example

• Camptothecin

45
MASPIT example

• Camptothecin
• Topotecan (colon cancer)

46
MASPIT example

• Camptothecin
• Topotecan (colon cancer)
• Irinotecan (ovarian cancer)

47
MASPIT example

• Camptothecin
• Topotecan (colon cancer)
• Irinotecan (ovarian cancer)

protein X
48
ACKNOWLEDGMENTS

• Cytokine Receptor LaboratoryDelphine LavensIrma
  LemmensSam LievensFrank PeelmanJulie
  PiessevauxJosé Van der HeydenNele
  VanderroostAnnick Verhee
• Dana Farber Cancer Institute (CCSB)Marc
  VidalDave Hill
• Tibotec
• Sabine Hallenberger
• GPC-BiotechNikolai KleyMaureen Caligiuri