Structure-activity relationship of isoform selective inhibitors of Rac1/1b GTPase nucleotide binding

Article abstract of DOI:10.1016/j.bmcl.2009.08.037

The synthesis of a series of berberine, phenantridine and isoquinoline derivatives was realized to explore their Rho GTPase nucleotide inhibitory activity. The compounds were evaluated in a nucleotide binding competition assay against Rac1, Rac1b, Cdc42 and in a cellular Rac GTPase activation assay. The insertion of 19 AA in the splice variant Rac1b is shown to be sufficient to introduce a conformational difference that allows compounds 4, 21, 22, and 26 to exhibit selective inhibition of Rac 1b over Rac1.

Full text of DOI:10.1016/j.bmcl.2009.08.037

Bioorganic & Medicinal Chemistry Letters 19 (2009) 5594–5598
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Structure–activity relationship of isoform selective inhibitors
of Rac1/1b GTPase nucleotide binding
*
Eric Beausoleil , Cédric Chauvignac, Thierry Taverne, Sandrine Lacombe, Laure Pognante, Bertrand Leblond,
Diego Pallares, Catherine De Oliveira, Florence Bachelot, Rachel Carton, Hélène Peillon, Séverine Coutadeur,
Virginie Picard, Nathalie Lambeng, Laurent Désiré, Fabien Schweighoffer
Exonhit Therapeutics, 65 Boulevard Massena, F-75013 Paris, France
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis of a series of berberine, phenantridine and isoquinoline derivatives was realized to explore
their Rho GTPase nucleotide inhibitory activity. The compounds were evaluated in a nucleotide binding
competition assay against Rac1, Rac1b, Cdc42 and in a cellular Rac GTPase activation assay. The insertion
of 19 AA in the splice variant Rac1b is shown to be sufficient to introduce a conformational difference that
allows compounds 4, 21, 22, and 26 to exhibit selective inhibition of Rac 1b over Rac1.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 2 July 2009
Revised 7 August 2009
Accepted 9 August 2009
Available online 13 August 2009
Keywords:
Rac
Alkaloid
GTP binding protein
Rho GTPase
Molecular docking
Alternative splicing is a fundamental process, which is respon-
sible for much of the tremendous transcriptome diversity that is
derived from a finite number of genomically encoded genes. The
alterations in mRNA structure that result can lead to profound ef-
fects on protein structure, function and ligand specificity. When
deregulated, alternative splicing can also be a driving force for
the onset and progression of disease.1a
invasion, activities required for tumor metastasis.4 Alternative
splicing variant Rac1b was identified and found to be overexpres-
sed in colorectal and breast tumors.5 Rac1b has transforming
GDP GTP
GEF
Among genes that exhibit alternative splicing, Rac1 belongs to
the Rho family of small GTP binding proteins and regulates diverse
cellular processes, including actin cytoskeleton organization,
membrane trafficking, proliferation, and gene expression.1b Like
Ras and other members of the Ras superfamily, the GTP binding
state of Rac1 is tightly controlled by guanine nucleotide exchange
factors (GEF) and GTPase-activating proteins (GAP). Rho-GDP dis-
sociation inhibitor (Rho-GDI) exerts additional regulation of Rho-
GTPases by sequestering the GDP-bound forms in the cytoplasm,
preventing their activation (Fig. 1).
Rac
GDP
Rac
GTP
GAP
Geranyl-geranyl group
Pi
Rac
GDP
Rac
GTP
Rho
GDI
Effector
Rac1 overexpression and altered function of Rac1-specific regu-
lators or effectors have been found in various diseases that include
several cancers2a,b and Alzheimer’s disease.2c Rac1 can promote
cellular transformation and is required for Ras-induced transfor-
mation.3 Rac1 also induces cell survival and, by modulating the
actin cytoskeleton, regulates cell adhesion as well as motility and
Biological
responses
Figure 1. Regulation of Rac activity. Rho-GTPases cycle between an active GTP-bound
and an inactive GDP-bound state. Rho-GDI (Rho-guanine nucleotide dissociation
inhibitor) sequesters GDP-bound Rac GTPase in the cytoplasm and inhibits the
spontaneous GDP–GTP exchange activity. Guanine nucleotide exchange factors (GEFs)
catalyse GDP release and GTP binding. The GTP-bound GTPase is then able to interact
with effectors to initiate downstream responses. GTPase-activating proteins (GAPs)
stimulate the low GTPase activity to promote the conversion of the GTP-bound to the
GDP-bound form, which terminates signal transduction. Pi: inorganic phosphate.
* Corresponding author. Tel.: +33 153947762; fax: +33 153947763.
E-mail address: eric.beausoleil@exonhit.com (E. Beausoleil).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.08.037

E. Beausoleil et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5594–5598
5595
In our a search for potent GTP binding inhibitors,2c,9a we initi-
ated a medicinal chemistry program designed to study and im-
prove the GTPase inhibitory activity of sanguinarine like alkaloid
natural products9b that were shown to possess GTPase inhibitory
activity.9c
( )-Canadine 2 was obtained after reduction of berberine 1. The
tetrahydroxy berberine analog 4 and demethylene berberine 5
were prepared from 1 by Lewis acid catalyzed ether deprotection.
Subsequent treatment of 4 with NaBH4 afforded tetrahydroxycan-
adine 3 (Scheme 1). The tetrahydroxyderivative 7 was obtained
from coralyne chloride 6 using BBr3.
R3
O
O
R4
a
N+
NH+
R2
Cl-
MeO
Cl-
R1
R1
OMe OMe
OMe
R2 R3 R4
OCH2O
OH OH OH
1
2 :
3 :
OH
b
R3
R4
We envisioned that phenantridine alkaloids (Formula IV) could
be regarded as analogs with an alternative connection between the
A and C rings of the berberine structure after rotation around their
closest junction bond. Therefore, we sought to determine if analogs
such as 3-aryl-isoquinolines (formula V) that can adopt the re-
stricted conformations of formula I–III and formula IV were able
to demonstrate higher inhibition in the nucleotide binding assay.
In order to obtain 3-aryl-6,7-dimethoxy-isoquinolines(11–20)
(Scheme 2), 3,4-dimethoxyphenylacetic acid 8 was esterified and
acylated to form a benzopyrylium perchlorate salt which upon
treatment with ammonia formed a 3-hydroxyisoquinoline9d that
was triflated to form 9. The triflate 9 was coupled to 3,4-meth-
ylenedioxyboronic acid using Suzuki conditions to form 11 fol-
lowed by an alkylation using iodomethane to form 12. Selective
methylenedioxy deprotection was performed on 11 and 12 using
BCl3 to form respectively 15 and 13 while tetra hydroxyl derivative
14 and 16 were obtained from a complete aryl ether deprotection
of 12 and 11.
c
N+
R2
Cl-
R1
R1 R2 R3 R4
OH OH OH OH
4
5
d
1
OH OH
OMe OMe
OH
OH
OMe
HO
HO
MeO
MeO
e
OMe
x.H2O
N+
N+
Cl-
Cl-
6
7
Scheme 1. Reagents and conditions: (a) NaBH4 (4 equiv), MeOH, rt, 3 h, 89%; (b)
BBr3 (8 equiv), dry CH2Cl2, reflux, 63%; (c) NaBH4 (4 equiv), MeOH, rt, 3 h, 71%; (d)
BCl3 (4 equiv) dry CH2Cl2, reflux, 52%; (e) (i) BBr3 (6 equiv), dry CH2Cl2, rt, 48 h; (ii)
2 N HCl, 99%.
To obtain isoquinolines 17, 19 and 20 the triflate 9 was coupled
in presence of Pd(dppf)Cl2CH2Cl2 with 3-acetylphenylboronic acid,
3,4-dichlorophenylboronic acid and 2-naphthaleneboronic acid
respectively and 17 was N-methylated with MeI to obtain 18.
The compounds were evaluated using a nucleotide binding
competition assay.10 The nucleotide binding activity was moni-
tored using the increase of the fluorescence intensity (FI) that
accompanies the incorporation of Bodipy-GTP into the GTPase.
All compounds were tested initially at 50 lM to identify the com-
pounds to be selected for additional characterization.
In the protoberberine series, we observed a general increase in
potency with the highest unsaturated compounds (Table 1.) We
found that the reduction of berberine 1 to ( )-canadine 2 resulted
characteristics when exogenously expressed in cultured cells6a
,
and its expression promotes cell cycle progression and resistance
to apoptosis.6b
In addition, Rac1b induces epithelial–mesenchymal transition
in mouse mammary carcinoma cells7 and facilitates tumour pro-
gression by activating the Wnt signaling pathway and decreasing
the adhesive properties of colorectal cancer cells.8a These observa-
tions provide a rationale for the use of Rac1 and Rac1b nucleotide
binding inhibitors for the treatment of cancers and Alzheimer’s dis-
ease2c with an alternative mode of action for small molecule
GTPase regulation that does not proceed through a GEF8b,c or a
GDI8d dependant mechanism.
R3
R2
R1
MeO
OTf
MeO
MeO
OH
R4
e
a,b,c,d
N+
N
O
R5
Cl-
MeO
10
8
9
R1 R2 R3 R4 R5
11
12
OMe OMe OCH2O
H
f
OCH2O
OMe OMe
OMe OMe OH OH
Me
g
13
14
Me
h
OH OH OH OH Me
g
OMe OMe OH OH
H
H
15
16
h
OH OH OH OH
O
H
OMe OMe
H
17
18
f
O
Me
H
OMe OMe
H
OMe OMe Cl Cl
OMe OMe
19
20
H
Scheme 2. Reagents and conditions: (a) H2SO4, MeOH, 91%, overnight; (b) Ac2O, HClO4 0 °C to rt 74%; (c) NH4OH, H2O, 99%; (d) PhN(SO2CF3)2, Et3N, DMSO, 82%; (e) 2 N aq
Na2CO3, Pd(dppf)Cl2CH2Cl2, ArB(OH)2, toluene, 80 °C, 42–62%; (f) MeI, CH3CN, 90 °C, 62–64%, Amberlite Clꢀ; (g) (i) BCl3, CH2Cl2, ꢀ78 °C to rt; (ii) 2 N HCl; (h) (i) BBr3, ꢀ78 °C to
rt, CH2Cl2; (ii) 2 N HCl.

5596
E. Beausoleil et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5594–5598
Table 1
Nucleotide binding inhibition assay
R3
R3
2
R4
3
R4
R3
R4
1
A
12
13
R2
R1
4
11
B
C
10
R2
D
9
R1
NH+
N+
N+
5
X
R2
X
X
8
6
R1 R5
R5
II
III
I
R3
R3
R4
R2
R1
R4
N+
IV
X
X
N
R2
R6
V
R1
R5
Compd
Formula
X
R1
R2
R3
R4
R5
R6
Rac1a
Rac1ba
cdc42a
2
3
24
1
25
5
4
I
I
I
II
II
II
II
III
III
IV
IV
IV
IV
V
V
V
V
Clꢀ
Clꢀ
Clꢀ
Clꢀ
Clꢀ
Clꢀ
Clꢀ
Clꢀ
Clꢀ
Clꢀ
Clꢀ
Clꢀ
Clꢀ
Clꢀ
Clꢀ
Clꢀ
Clꢀ
OMe
OH
OMe
OH
–OCH2O–
OH
–OCH2O–
–OCH2O–
OMe
OH
OH
OMe
OH
H
H
Bz
H
H
H
H
Me
Me
—
—
—
—
Me
Me
Me
Me
—
—
—
—
—
—
—
—
—
—
—
—
—
H
1
3
0
67
31
23 1
19
54
>100
44
72
>100
95
61
100
18
3
46
16
4
4
ND
9
8
8
31
>100
23
70
63
6
OH
70
23
14
15
39
6
3
2
2
2
9
4
OMe
OMe
OMe
OMe
OH
OMe
OH
–OCH2O–
OMe
OMe
OH
OMe
OMe
OMe
OMe
OH
2
2
2
OMe
OH
OH
OMe
OH
3
2
9
>100
6
7
OMe
OH
35
63
63
49
47
100
13
0
25
6
5
5
4
4
3
2
4
6
7
5
26
21
22
23
11
12
15
13
–OCH2O–
–OCH2O–
OH
OMe
OMe
OH
OMe
OMe
OMe
OMe
3
4
35 10
OH
OH
38
73
14
0.3 0.3
15
5
2
4
3
OH
OMe
OMe
OMe
OMe
–OCH2O–
–OCH2O–
OH
2
2
2
Me
H
Me
3
2
OH
OH
5
2
1
2
OH
2
O
17
18
19
20
V
V
V
V
Clꢀ
Clꢀ
Clꢀ
Clꢀ
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
H
H
Cl
Me
Me
Me
Me
H
16
20
9
3
3
26
30
41
51
2
4
9
6
5
7
2
O
Me
H
7
1
1
Cl
7
5
H
46
2
1
29
5
14
16
V
V
Clꢀ
Clꢀ
OH
OH
OH
OH
OH
OH
OH
OH
Me
Me
Me
H
23
18
37
29
12
15
1
3
a
Values are expressed as % of Bodipy-GTP binding inhibition and are means of at least three independent experiments. Standard error of the mean (SEM) is given next to
the sign.
in a significant loss of activity against all GTPases tested and that
The nucleotide binding inhibition IC50 was calculated for the
most active compounds and the inhibition of Rac GTPase activation
was evaluated in Rac1b overexpressing HEK293 cells using a Rac
specific antibody and the G-LISATM assay11 (Table 2).
For all three Rac1, Rac1b and Cdc42, the GDP IC50 values ob-
served (2.7 0.2, 1.8 0.1, 1.4 0.1 lM) in the nucleotide binding
assay were similar for all three GTPases. However, we found signif-
icant differences in all other substances identified. The compounds
displayed a general tendency to be more selective for Rac1b when
compared to Rac1 and Cdc42. In particular 22, 4, 26 and 21 inhib-
itory activities are 2.5, 3, 13 and >14-fold higher towards Rac1b
than Rac1.
coralyne 6,
a compound with added unsaturations and a
3,4,10,11-tetramethoxy-8-methyl substitution pattern, exhibited
a slightly higher inhibition than berberine 1. However, indepen-
dently of the number of unsaturations in formula I, II and III, con-
version of the methoxy or the methylenedioxy analog to
tetrahydroxy substituted structures 3, 4 and 7 led to a general in-
crease in nucleotide binding inhibition. The examination of the
phenantridine analog chelerythrine and sanguinarine (21 and 26,
formula IV) revealed that this class of alkaloids produced a much
higher inhibitory activity in general when compared to the berber-
ine series (formula I, II and III).
Compounds from the 3-aryl-isoquinoline series (formula V)
were all found to be much less active than their structurally re-
stricted analogues. Interestingly, in this series, compounds with a
fused phenyl ring 20, a 30,40-dihydroxy 15 or dichloro substitution
on the 3-aryl moiety 19 exhibited an increased nucleotide binding
inhibitory activity when compared to its 30,40-dimethoxy substi-
tuted 3-aryl analog 11.
In order to explore hypothesis that could explain the differences
observed, we analyzed the amino acid sequences exposed to the
GTP binding site in Rac1, Rac1b and Cdc42 and found a high degree
of sequence homology (Fig. 2).
However, the alternative splicing that leads to the production of
Rac1b is associated with a 19 AA insertion near the switch II do-
main. Even if the sequence variation is away from the binding site,

E. Beausoleil et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5594–5598
5597
Table 2
IC50 values for nucleotide binding inhibition assay and % inhibition of GTPase activation at 50
l
M
a
a
a
Compd
Rac1 IC50
2.7 0.2
(l
M)
Rac1b IC50
1.8 0.1
(
lM)
Cdc42 IC50
(l
M)
G-Lisab (%)
cLog Pc
cLog Dd pH 7.4
GDP
3
4
5
7
15
20
21
22
23
26
1.4 0.1
>100
—
<20
<20
—
—
34
8
5
68
3
1.23
ꢀ0.52
0.49
2.97
3.58
5.53
1.86
0.92
ꢀ0.09
1.79
ꢀ0.12
ꢀ0.55
0.48
0.45
3.34
5.51
1.86
0.68
ꢀ0.34
1.79
3
2.7 0.4
6
1
63 10
38 14
104 13
31
13
37
4
4
6
>100
35
<20
61
2
6
26
3
>100
>100
>100
58
3
62 18
<20
40 10
39
>100
7
1
54
8
22
8
73
22
32
8
2
1
9.3 0.6
8.6 1.3
4.6 0.4
<20
58
4
50
3
a
b
c
Values are expressed in lM as IC50 of Bodipy-GTP binding inhibition and are means of at least three independent experiments.
Standard error of the mean (SEM) is given next to the sign.
Values calculated using Pallas 3.0 with Prolog P 6.0 Module.
Values calculated using Pallas 3.0 with Prolog D Module at pH 7.4.
d
this insertion is sufficient to produce a large conformational differ-
ence in the switch I domain that moves residues 31 to 35 and gen-
erates a much larger nucleotide binding pocket in Rac1b.12 When
molecular docking experiments were performed using Molegro
Virtual Docker13 with 21 and Rac1b, we observed a binding mode
that allows the ligand to bury the methylene dioxy group where
the guanine group of GDP is located in the published nucleotide
bond Rac1b structure12 and exposes the dimethoxy substituents
in the direction of the switch I domain (Fig. 3, Rac1b). In the bind-
ing pocket, the best pose suggests an hydrogen bonding interaction
between both backbone amide hydrogens of Ala178 and Thr134
main is in a closed conformation that prohibits the ligand to access
this binding pocket. Alternatively, the ligand could engage in a
binding mode perpendicular to the one observed in Rac1b closer
to the phosphate binding pocket (Fig. 3, Rac1). Since this binding
mode does not take advantage of the deep guanine binding pocket,
it is possible that this leads to an energetically unfavored state that
confers the selectivity observed for 21.
Using the cellular GTPase activation assay, we found that com-
pounds 5, 15, 21 and 26 inhibit Rac activation (35–61%). However
compounds 3, 4, 7, 20 and 23 that are active in the nucleotide bind-
ing assay were found inactive in the cellular assay. Since cellular
penetration is an additional requirement to inhibit Rac activation,
it is expected that compounds with a poor cellular penetration pro-
file are found inactive in a cellular assay. To further support this
hypothesis, a cLog P and cLog D analysis revealed that compounds
3, 4, and 23 that are inactive in the cellular assay also exhibit neg-
ative cLog D values indicating a strong tendency to for these mole-
cules to partition preferentially in water (Table 2). However, the
cLog D data can’t provide a rationale for compound 7 and 20 since
they were both found to have positive cLog D (0.45 and 5.53).
and one oxygen of the methylene dioxy group of 21, a
p–p edge
face interaction between Phe28 and the phenantridine moiety
and finally favorable interaction with the hydrophobic environ-
ment created by Leu179 the methylene groups of Lys135, Cys18
and Phe28 (Fig. 4). When the docking procedure was applied to
21 and Rac114 a totally different binding mode was observed. In
this binding mode, the ligand cannot interact with the Rac1 amino
acids exposed to the guanine group of GDP because the switch I do-
1213 14 15 16 1718 313233 34 35
E Y
I P T
Rac1
G A V G K T C
121314 15 16 1718 313233 34 35
E Y
I P T
Rac1b G A V G K T C
5960 116118 119
158 159160
Rac1 A G
K
D
L
K
D
L
5960 135137 138
177
178179
Rac1b A G
K
D
L
K
D
L
Figure 2. Residues in blue are located 5 Å away from the nucleotide in the Rac114
and Rac1b12 X-ray co-crystal structure.
Figure 3. Docking results obtained with chelerytrine 21 in Rac1 and Rac1b using
Molegro Virtual Docker13 represented using the electrostatic protein surface in the
binding site. The surfaces of Rac1 and Rac1b are colored by electrostatic potential
with red and blue for negative and positive potential, respectively.
Figure 4. Orientation of the first ranked pose of 21 in Rac1b using MolegroVirtual
Docker13 (displayed following the atom type color codes and thin stick). For the
sake of clarity, only a few amino acid residues of Rac1b GTP binding site are
represented with the hydrogen bonds highlighted with a green dashed line.

5598
E. Beausoleil et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5594–5598
that described Berberine activity was released (Tsang, C.M.; Lau, E.P.; Di, K.;
Cheung, P.Y.; Hau P.M.; Ching Y.P.; Wong Y.C.; Cheung, A.L.; Wan, T.S.; Tong, Y.;
Tsao, S.W.; Feng, Y. Int. J. Mol. Med. 2009, 24, 131) and confirmed our initial
observation previously disclosed in Leblond, B.; Beausoleil, E.; Chauvignac, C.;
Taverne, T.; Picard V.; De Oliveira C.; Schweighoffer F.; EP20070301230, 2008.;
(c) Vollmer, W. Appl. Microbiol. Biotechnol. 2006, 73, 37; (d) Kanojia, R. M.; Press,
J. B.; Lever, O. W., Jr.; Williams, L.; McNally, J. J.; Tobia, A. J.; Falotico, R.; Moore,
J. B., Jr. J. Med. Chem. 1988, 31, 1363.
Nevertheless, we can’t exclude that it is possible that in some case
the ligand binding maintain the GTPase in an active conformation.
The results obtained demonstrate that the isoquinoline 15 and
phenantridine derivatives 21, 22, 26 are competitive inhibitors of
Bodipy-GTP nucleotide binding, inhibit Rac activation in a cellular
model and show selective inhibition of Rac1b over Rac1 and Cdc42.
Although compound 26 and 21 were shown to interact with
other proteins,15 Protein Kinase C16 and oligonucleotide targets,17
this study provides important structural features required for the
development of isoform selective Rac1b nucleotide binding
inhibitors.
10. Guanine nucleotide competition assay: bacterially-expressed GST-Rac1, His6-
Tiam1 (DH/PH domains), GST-Rac1b and GST-Cdc42 were prepared in 50 mM
Tris pH 8.6, 50 mM NaCl, 1 mM MgCl2 and 10% glycerol and cDNAs were kindly
provided by Dr. Channing J. Der. The guanine competition assay was monitored
as the increase in relative fluorescence of
a fluorescent GTP analog, the
BODIPY-GTP (BODIPY FL GTP; invitrogen), upon binding. The guanine
nucleotide exchange factor Tiam1 was used to stimulate the binding of the
nucleotide to Rac1, whereas no GEF was needed for Rac1b and Cdc42 (intrinsic
References and notes
activity). In our experiments, 2
Tiam1 were incubated in buffer containing 20 mM Tris pH 8.6, 50 mM NaCl and
1 mM MgCl2 for 10 min at 20 °C. After equilibrium, 2 M BODIPY-GTP with or
without 50 M of compound to be tested were added. Fluorescence was
lM of Rac1b, Cdc42 or Rac1 plus 1.3 lM of
l
1. (a) Cooper, T. A.; Wan, L.; Dreyfuss, G. Cell 2009, 136, 777; Villate, O.; Rastrojo,
A.; López-Díez, R.; Hernández-Torres, F.; Aguado, B. Infect. Disord. Drug Targets
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J. C. Bioassays 2005, 27, 602; (c) Désiré, L.; Bourdin, J.; Loiseau, N.; Peillon, H.;
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Lacombe, S.; Drouin, D.; Schweighoffer, F. J. Biol. Chem. 2005, 280, 37516.
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l
measured using a Fluoroscan (Thermo Scientific; kexc 485 nm, kem 538 nm)
before and after the addition of the fluorescent nucleotide and compound.
Maximal RFU value obtained at the end of the recording (40 min after BODIPY-
GTP addition) was used for comparison with appropriate control.
11. Rac1b activation assay: HEK293 cells overexpressing human Rac1b were
plated in 100 mm diameter dishes and grown for 5 days in MEM medium
supplemented with 10% FBS,
L-glutamine and antibiotics. Cells were then
treated with 50 M of compounds or the solvent for 5 minutes, and the GTP
l
loading of Rac1b was measured using a Rac Activation Assay Kit (Cytoskeleton)
according to manufacturer’s recommendations.
12. Fiegen, D.; Haeusler, L.-C.; Blumenstein, L.; Herbrand, U.; Dvorsky, R.; Vetter, I.
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