Chemical genetics strategies for identification of molecular targets
José Manuel Botubol Ares, Mª Jesús Durán-Peña, Rosario Hernández-Galán & *Isidro
G. Collado
Department of Organic Chemistry, Faculty of Science, University of Cadiz, Polígono
Río San Pedro s/n, 11510, Puerto Real (Cadiz), Spain.
e-mail: isidro.gonzalez@uca.es
Suppporting Information
Table of contents:
Table 1. Examples of affinity chromatography……………………………………......2-8
Table 2. Examples of fluorescense-based affinity……………………………………….9
Table 3. Examples of photoaffinity…………...………………………………………..10
Table 4. Examples of DARTS………………...…………………………………….11-12
Table 5. Examples of SPROX……………………………………………………….....13
Table 6. Examples of SILAC………………….…………………………………….....14
Table 7. Examples of iTRAQ…………………………………………………………..15
Table 8. Examples of Y3H……………………………………………………...…..16-17
Table 9: Examples of phage display…………………………………………......…18-19
Table 10. Examples of protein microarrays………………………………….……..…..20
Table 11. Examples of gene overexpression…..…………………………………….…21
Table 12. Examples of synthetic lethality……..…………………………………….....22
References……..……………………………………………..…………………......22-28
1
Table 1. Successful examples of target proteins identified by affinity chromatography
Small molecule
Activity
Antiproliferative
Hepatitis C
replication inhibitor
Target
References
CDK1 (cyclin-
Rosania et al.
dependent kinase 1)
2000
HCV NS5A
(hepatitis C virus
non-structural 5A)
Blocks insulin-
MFP-2
induced
(multifunctional
adipogenesis
protein 2)
Anticancer
Gao et al.
2010
Choi et al.
2006
Ornithine-δ-
Wang et al.
aminotransferase
2007
Inhibitor of mitotic
NQO1 (NADPH
spindle assembly
dehydrogenase,
quinone 1)
Wignall et al.
2004
Greenbaum et
Antiparasitic
Cathepsin B, H, L.
al. 2000 and
2002
Anticancer
Cytosolic malate
Oda et al.
dehydrogenase
2003
2
Table 1. Successful examples of target proteins identified by affinity chromatography
Small molecule
Activity
Target
References
Anticancer
Vimentin
Ermakova et
al. 2005
Antiinflammatory
Antitumor
Hsp 60 (Heat shock
Nagumo et al.
protein 60)
2005
Proteasomal β-
Meng et al.
catalytic subunit
1999a
LMP2 and LMP7
Antiinflammatory
LMP7, X, MECL1
Meng et al.
and Z catalytic
1999b
subunits of 20S
proteasome
Antitumor and
antiproliferative
ALDH-1 (aldehyde
Kaiser et al.
dehydrogenase 1),
2001; Schnier
glycogen
et al. 1999
phosphorylase
Anticancer
Carboxylesterase 1
Adam et al.
2003
3
Table 1. Successful examples of target proteins identified by affinity chromatography
Small molecule
Activity
Regulates DAF-2
insulin signaling in
C. Elegans
Target
References
GAPDH
(glyceraldehyde 3-
Min et al.
phosphate
2007
dehydrogenase)
Anticancer
RICK and GAK
Brehmer et al.
Kinases
2005
HSP90 (heat shock
Anticancer
protein 90)
Neckers 2003;
Whitesell et al.
1994
Proteasome subunit
Anticancer
α type 1 and
Hong et al.
subunit β type 4
2007
precursor
Glycogen synthase
kinase 3, MitogenAntitumor
activated protein
Wan et al.
kinase-1, p90
2004
ribosomal S6
kinase, β-tubulin
Cyclic AMPAnticancer
responsive elementbinding protein
Emami et al.
2004
4
Table 1. Successful examples of target proteins identified by affinity chromatography
Small molecule
Activity
Target
Antiinflammatory
Glyoxalase I
Anticancer
Rpd3
Anticancer
Antitumor
Induce melanin
synthesis
References
Sato et al.
2007
Kwon et al.
1998
SOD1 (superoxide
Somwa et al.
dismutase 1)
2011
CRM1
Prohibitin
Fukuda et al.
1997
Snyder et al.
2005
Dihydrofolate
Anticancer
reductase,
Uga et al.
Deoxycytidine
2006
kinase
Induce pigmentation
in albino murine
melanocytes
F1F0-ATP synthase
Williams et al.
2004
5
Table 1. Successful examples of target proteins identified by affinity chromatography
Small molecule
Activity
Target
Antiangiogenesis
Tubulin
Antibiotic and
immunosuppressant
Palmitoyltransferas
e subunits LCB1
and LCB2
Eukaryotic
Anticancer
translation initiation
factor 4A
References
Perez et al.
2002
Chen et al.
1999
Bordeleau et
al. 2005
GSK-3 (glycogen
synthase kinase 3),
Antitumor
mMDH
Knockaert et
(mitochondrial
al. 2002
malate
dehydrogenase)
Antiinflammatory
Redox factor 1
Chong et al.
2004
ARFGAP1 (ADPAnticancer
ribosylation factor
Zhang et al.
GTPase-activating
2007
protein 1)
Class Ia PI3Ks
Anticancer
(phosphatidylinositi
de 3-kinases)
Yang et al.
2007
6
Table 1. Successful examples of target proteins identified by affinity chromatography
Small molecule
Activity
Target
References
CDK5 (cyclindependent kinase5), ERK1 and
Anticancer
ERK2 (extracellular
signal-regulated
Bach et al.
2005
kinase 1 and 2) and
pyridoxalkinase
GAPDH
Antiproliferative
(glyceraldehyde 3-
Xing et al.
phosphate
2004
dehydrogenase)
ERK1 and RasGAP
Activator of stem
cell renewal
(Ras GTPase
activating protein)
Chen et al.
2006
Por1p (a channel
Suppressor of
protein in the outer
Butcher et al.
FK506
mitochondrial
2003
membrane)
Antitumor
Splicing factor 3b
Kaida et al.
2007
Lee et al.
Antiresorptive
Prohibitin
2010; Chang
et al. 2011
7
Table 1. Successful examples of target proteins identified by affinity chromatography
Small molecule
Activity
Target
References
Anticancer
Cereblon
Ito et al. 2010
Liu et al.
Antiangiogenic/angi
ostatic
Met-AP-2
1998; Sin et al.
(methionyl
1997;
aminopeptidase 2)
Yamaoka et al.
1993
Antitumor
Neurogenesis
HDAC (histone
Taunton et al.
deacetylase)
1996
GSK-3 (glycogen
Ding et al.
synthase kinase 3)
2003
BargagnaAntitumor and
antiangiogenesis
Vimentin
Mohan et al.
2007
8
Table 2. Successful examples of target proteins identified by fluorescence-based affinity
Small molecule
Activity
Target
Cytotoxic
Myosin
Cytotoxic
Myosin
Antitumoral
Antibiotic and
cytotoxic
Antibiotic and
cytotoxicity
Antitumor
References
Hughes et al.
2009a
Hughes et al.
2009a
Smo (smoothened
Chen et al.
protein)
2002
Actin
Hughes et al.
2009b
Hughes et al.
Actin
2009b
SF3b (splicing
Kotake et al.
factor 3b)
2007
9
Table 3. Successful examples of target proteins identified by photoaffinity
Small molecule
Activity
Target
Antiinflammatory
Sec61α
References
Mackinnon et
al. 2007
TACC3
(transforming acidic
Neurogenesis
inducer
coiled-coilcontaining protein
3)
TIP47 (tailAntitumor
interacting protein
Warashina et
al. 2006;
Wurdak et al.
2010
Jessen et al.
2005
47)
Immunosuppressant
Met-AP2
and
(methionyl
antimicrosporidial
aminopeptidase 2)
Griffith et al.
1997
Carboxylesterase
ES10, L-FABP
Anticancer
(liver fatty acid-
Mesange et al.
2002
binding protein),
epoxy hydrolase
10
Table 4. Successful examples of target proteins identified by DARTS
Small molecule
Activity
Antiinflammatory
Anticancer,
antitumor,
antiinflammatory
Immunosuppressant,
antiviral, cytotoxic
Anticancer
Immunosuppressant
Tumor promoter
Target
References
COX-2
Lomenick et
(cyclooxygenase 2)
al. 2009
mTORC1
(mammalian target
Lomenick et
of rapamycin
al. 2009
complex 1)
EF-1-alpha
(elongation factor 1-
Lomenick et
al. 2009
alpha 1)
TOR (target of
Lomenick et
rapamycin)
al. 2009
FKBP12 (FK506-
Lomenick et
binding protein)
al. 2009
PP2A (protein
Nishiya et al.
phosphatase 2)
2009
11
Table 4. Successful examples of target proteins identified by DARTS
Small molecule
Activity
Antibiotic, antitumor
Immunosuppressant
Target
References
Hsp90 (heat shock
Nishiya et al.
protein 90)
2009
mTOR (mammalian
Lomenick et
target of rapamycin)
al. 2009
TIF1 (intermediary
Antioxidant
transcriptional
factor 1)
SCF(Met30) (Skp1Anticancer
Cdc53-F-box
Lomenick et
al. 2009
Aghajan et al.
2010
protein Met30)
Sam68 (Src
Antibiotic
associated in
Nishiya et al.
2009
mitosis of 68 kDa)
12
Table 5. Successful examples of target proteins identified by SPROX
Small molecule
Activity
Antibacterial
Target
References
Bovine carbonic
West et al.
anyhdrase II
2008
Cyclophilin A,
UDP-glucose-4epimerase,
Immunosuppressant
carbamoylphosphate
synthetase,
West et al.
2010
glycogen synthase
among others
Cytosolic aldehyde
dehydrogenase,
40S, S9-A and 60S
ribosomal protein
translation
Antioxidant
elongation factor
DeArmond et
3A, nascent
al. 2011
polypeptideassociated complex
subunit alpha, , 2dehydropantoate 2reductase
13
Table 6. Successful examples of target proteins identified by SILAC
Small molecule
Activity
Target
References
Anticancer
Several kinases
Ong et al. 2009
Anticancer
Several kinases
Ong et al. 2009
Anticancer
Several kinases
Ong et al. 2009
Immunosuppressant
Several
immunphilins
Several
Immunosuppressant
immunphilins
Ong et al. 2009
Ong et al. 2009
14
Table 7. Successful examples of target proteins identified by iTRAQ
Small molecule
Activity
Target
References
BCR-ABL
(breakpoint cluster
region-abelson),
ARG (abelsonrelated gene),
Anticancer
DDR1 (discoidin
domain receptor
Bantscheff et
al. 2007
family, member 1),
FAK (focal
adhesion kinase)
and others kinases
BCR-ABL, ARG,
Anticancer
DDR1 and others
kinases
Bantscheff et
al. 2007
BCR-ABL, DDR1,
NQO2 (NADPH
Anticancer
dehydrogenase,
quinone 2) and
Bantscheff et
al. 2007
others kinases
15
Table 8. Successful examples of target proteins identified by Y3H
Small molecule
Activity
Target
References
SPL3, SPL4, SPL13
-
(squamosa promoter
Cottier et al.
binding protein-like
2011
3, 4 and 13)
Growth regulator in
higher plants
Epoxide hydrolase,
5-methylthioribose
kinase
Cottier et al.
2011
PP2C6
Antiinflammatory
(serine/threonine
Cottier et al.
protein phosphatase
2011
2C)
Immunosuppressant
FKBP12, Gcn4
Licitra et al.
(general control
1996; Marton
nonderepressible 4)
et al. 1998
A large number of
Anticancer
CDC/CDK and
Becker et al.
serine/threonine
2004
kinases
DHFR
Anticancer
(dihydrofolate
reductase)
Henthorn et al.
2002
16
Several kinases
(CDK1, CDK5,
CDK6, CLK3,
Anticancer
EPHB2, FLT4,
FYN, PAK4,
Becker et al.
2004
PCTK1, PCTK2,
RSK3 and YES)
CDK2, CSNK1D
and CSNK1E
Anticancer
(casein kinase I
Becker et al.
isoform delta and
2004
epsilon) and others
kinases
17
Table 9. Successful examples of target proteins identified by phage display
Small molecule
Activity
Target
Anticancer
Ca+2- calmodulin
References
Jung et al.
2009
Topoisomerase I,
HEP1 receptor
Anticancer
(human
Takakusagi et
Prostaglandin E
al. 2007
receptor 1),
carboxylesterase
Glyceraldehyde 3Activator of insulin
receptor
phosphate
dehydrogenase
Anticancer
Ca+2-calmodulin
Immunosuppressant
FKBP12
Kim et al.
2007
Jin et al. 2002
Sche et al.
1999
18
Table 9. Successful examples of target proteins identified by phage display
Small molecule
Activity
Target
References
hNopp140 (human
Anticancer
nucleolar
phosphoprotein
Shim et al.
2004
140)
hRPS25 (human
Anticancer
Piggott et al.
ribosomal protein
S25 gene)
2008
Aoki et al.
Bcl-2 (B-cell
lymphoma 2),
Anticancer
2007;
Neuronal Ca+2
Boehmerle et
sensor 1, nuclear
al. 2006; Rodi
transcription factor,
X-box binding 1.
et al. 1999 and
2001
Several breast
cancer-associated
antigen, breast
cancer resistance
Anticancer
Feng et al.
protein, deathassociated
2006
transcription factor,
and human cyclindependent kinase
19
Table 10. Successful examples of target proteins identified by protein microarrays
Small molecule
Activity
Target
References
Tep1p (yeast
homologue of the
mammalian protein
PTEN tumour
suppressor
(phosphatase and
Anticancer
tensin homologue
deleted on
Huang et al.
2004
chromosome 10))
and Nir1p
(component of the
TOR signaling
network)
Huang et al.
Anticancer
Tep1p and Nir1p
2004
Huang et al.
Anticancer
Tep1p and Nir1p
2004
Huang et al.
Anticancer
Tep1p and Nir1p
2004
Huang et al.
Anticancer
Tep1p and Nir1p
2004
20
Table 11. Successful examples of target proteins identified by gene overexpression
Small molecule
Activity
Target
References
3-hydroxy-3Rine et al.
Hypolipidemic
methyl-glutaryl1983
CoA
SRine et al.
Cytotoxic
adenosylmethionine
1983
synthetase
Mitochondrial
cytochrome-c
Launhardt et
Antifungal
oxidase and
al. 1998
lanosterol C-14
demethylase
ACC1 (acetyl-CoA
Vahlensieck et
carboxylase 1)
al. 1994
Antifungal
UDP-N-acetylAntifungal and
Rine et al.
glucosamine-1-P
antibacterial
1983
transferase
21
Table 12. Successful examples of target proteins identified by synthetic lethality
Small molecule
Activity
Anticancer
Target
rRNA processing
ribosome
References
Lum et al.
2004
Lum et al.
Vasodilator
Lanosterol synthase
2004
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