Blockade of inflammatory responses by a small-molecule inhibitor of the Rac activator DOCK2

Article abstract of DOI:10.1016/j.chembiol.2012.03.008

Tissue infiltration of activated lymphocytes is a hallmark of transplant rejection and organ-specific autoimmune diseases. Migration and activation of lymphocytes depend on DOCK2, an atypical Rac activator predominantly expressed in hematopoietic cells. Although DOCK2 does not contain Dbl homology domain typically found in guanine nucleotide exchange factors, DOCK2 mediates the GTP-GDP exchange reaction for Rac through its DHR-2 domain. Here, we have identified 4-[3′-(2″-chlorophenyl)-2′-propen-1′-ylidene] -1-phenyl-3,5-pyrazolidinedione (CPYPP) as a small-molecule inhibitor of DOCK2. CPYPP bound to DOCK2 DHR-2 domain in a reversible manner and inhibited its catalytic activity in vitro. When lymphocytes were treated with CPYPP, both chemokine receptor- and antigen receptor-mediated Rac activation were blocked, resulting in marked reduction of chemotactic response and T cell activation. These results provide a rational of and a chemical scaffold for development of the DOCK2-targeting immunosuppressant.

Full text of DOI:10.1016/j.chembiol.2012.03.008

Chemistry & Biology
Article
Blockade of Inflammatory Responses
by a Small-Molecule Inhibitor
of the Rac Activator DOCK2
Akihiko Nishikimi,1
,2,5,10
Takehito Uruno,
1,10
Xuefeng Duan, Qinhong Cao, Yuji Okamura, Takashi Saitoh, Nae Saito,
2
1
6
3
7
6
6
9
5,9
4
1,5
Shunsuke Sakaoka, Yao Du, Atsushi Suenaga, Mutsuko Kukimoto-Niino, Kei Miyano, Kazuhito Gotoh,
7
1,2,5
1,2,5
4
9
Takayoshi Okabe, Fumiyuki Sanematsu,
Yoshihiko Tanaka,
Hideki Sumimoto, Teruki Honma,
5
,8,9
Tetsuo Nagano, Daisuke Kohda, Motomu Kanai, and Yoshinori Fukui1,2,5,*
6,7
2,3
6
Shigeyuki Yokoyama,
1
Division of Immunogenetics, Department of Immunobiology and Neuroscience, Medical Institute of Bioregulation
2Research Center for Advanced Immunology
3Division of Structural Biology, Medical Institute of Bioregulation
4Department of Biochemistry, Graduate School of Medical Sciences
Kyushu University, Fukuoka 812-8582, Japan
5Japan Science and Technology Agency, Core Research for Evolutional Science and Technology (CREST), Tokyo 102-0075, Japan
6Graduate School of Pharmaceutical Sciences
7Open Innovation Center for Drug Discovery
8Department of Biophysics and Biochemistry, Graduate School of Science
The University of Tokyo, Tokyo 113-0033, Japan
9RIKEN Systems and Structural Biology Center, Yokohama 230-0045, Japan
10These authors contributed equally to this work
*Correspondence: fukui@bioreg.kyushu-u.ac.jp
DOI 10.1016/j.chembiol.2012.03.008
SUMMARY
cellular responses, including the chemokine-dependent migra-
tion of lymphocytes, the recognition by T cell receptors (TCRs)
Tissue infiltration of activated lymphocytes is a of allo- or self-peptides bound to major histocompatibility
hallmark of transplant rejection and organ-specific complex (MHC) molecules, the engagement of costimulation
and adhesion molecules with their ligands, and the activation
of multiple intracellular signal transduction pathways that lead
autoimmune diseases. Migration and activation of
lymphocytes depend on DOCK2, an atypical Rac
activator predominantly expressed in hematopoietic
cells. Although DOCK2 does not contain Dbl
homology domain typically found in guanine nucleo-
tide exchange factors, DOCK2 mediates the GTP-
to the release of cytokines that are key to lymphocyte expansion
and tissue destruction. Thus far, TCR signaling pathways have
been the major targets for drug discovery, and the resultant
calcineurin inhibitors, cyclosporine and FK506, successfully
controlled allograft rejection in clinical and experimental trans-
plantation (Denton et al., 1999). More recently, FTY720, which
acts as a functional antagonist for sphingosine 1-phosphate
GDP exchange reaction for Rac through its DHR-2
0
00
domain. Here, we have identified 4-[3 -(2 -chloro-
0
0
phenyl)-2 -propen-1 -ylidene]-1-phenyl-3,5-pyrazoli- receptor and inhibits lymphocyte egress from the secondary
dinedione (CPYPP) as a small-molecule inhibitor of lymphoid organs, has been developed as a novel immunomod-
DOCK2. CPYPP bound to DOCK2 DHR-2 domain ulatory drug (Brinkmann et al., 2010; Mandala et al., 2002).
However, because both migration and activation of lymphocytes
in a reversible manner and inhibited its catalytic
activity in vitro. When lymphocytes were treated
with CPYPP, both chemokine receptor- and antigen
receptor-mediated Rac activation were blocked, re-
sulting in marked reduction of chemotactic response
and T cell activation. These results provide a rational
of and a chemical scaffold for development of the
DOCK2-targeting immunosuppressant.
requires remodeling of the actin cytoskeleton (Dustin and
Cooper, 2000; Vicente-Manzanares and S a´ nchez-Madrid,
2004), inhibition of the cytoskeletal reorganization in lympho-
cytes may be an alternative approach to control transplant
rejection and organ-specific autoimmune diseases.
Rac is the small GTPase that regulates membrane polarization
and cytoskeletal dynamics in varied biological settings (Hall,
1998). Like other small GTPases, Rac cycles between GDP-
bound inactive and GTP-bound active states, and stimulus-
induced formation of the active Rac is mediated by guanine
nucleotide exchange factors (GEFs). The critical role of Rac
activation in lymphocyte migration has been well established
INTRODUCTION
Tissue infiltration of activated lymphocytes is a hallmark of (Faroudi et al., 2010; Tybulewicz and Henderson, 2009). More-
transplant rejection and organ-specific autoimmune diseases over, the activation of Rac has been implicated in immunological
(
Gerard and Rollins, 2001; Luster et al., 2005). This process synapse formation, a large-scale molecular movement at the
involves a complex cascade of molecular interactions and interface between T cells and antigen presenting cells (APCs),
488 Chemistry & Biology 19, 488–497, April 20, 2012 ª2012 Elsevier Ltd All rights reserved

Chemistry & Biology
An Inhibitor that Blocks Rac Activation by DOCK2
lymphocytes with CPYPP blocked DOCK2-mediated Rac acti-
vation and inflammatory responses in vitro and in vivo. Our
results thus provide a rational of and a chemical scaffold for
development of the DOCK2-targeting immunosuppressant.
RESULTS
Identification of CPYPP as a Small-Molecule Inhibitor
of DOCK2
DOCK2 binds to Rac through its DHR-2 domain and mediates
GTP–GDP exchange reaction (Brugnera et al., 2002; C oˆ t e´
and Vuori, 2002). Therefore, this interaction would be the first
target to block DOCK2-mediated inflammatory responses. We
screened 9,392 chemical compounds in terms of their ability to
DHR-2
inhibit interaction between DOCK2
and Rac1 (see Table
Figure 1. Synthesis of CPYPP (1) and Its Geometrically Pure Isomers
ꢀ
Reagents and conditions: a, Et
3
N, THF, –10 C, 68%, 3:1 inseparable mixture;
0
S1 available online), and identified 131 candidates including
CPYPP (compound 1; Figures 1 and 2A). CPYPP inhibited the
DHR-2
b, 1 M NaOH in EtOH, room temperature, 58%; c, 3-(2 -Chlorophenyl)prope-
nal, proline (10 mol %), THF, room temperature, 70%; d, Wash with CHCl
3
,
GEF activity of DOCK2
for Rac1 in a dose-dependent
90%; e, SiO medium-pressure column with AcOEt/hexanes = 2/5 elution. The
2
manner, with a half-maximal inhibitory concentration (IC50) of
2.8 ± 2.4 mM (Figure 2B). This inhibitory activity was indepen-
dent on length of preincubation time, ranging from 2 min to
0 min (Figure S2). In addition, CPYPP was nontoxic when
synthesis and purification of CPYPP and its geometrically pure isomers (12, 13)
are described in Supplemental Experimental Procedures in detail.
See also Figure S1.
2
3
–
–
applied to spleen cells or thymoma cells (BW5147a b ) at
which is considered to be critical for sustained T cell activation 100 mM for 3 hr or 3 days, respectively (Figure S2). Then, CPYPP
Dustin and Cooper, 2000; Villalba et al., 2001; Yu et al., 2001). was selected and synthesized in gram-scales for further evalua-
Therefore, Rac activation would be a target to manipulate tion (Figures 1 and S1).
lymphocyte functions. However, Rac is composed of three iso- Recent structure of DOCK2
forms, and the ubiquitous expression and redundancy of Rac karni et al., 2011) indicates that the DHR-2 domain of DOCK2
isoforms preclude using Rac itself as a drug target. is also organized into three lobes (lobes A, B, and C), as in
(
DHR-2
complexed with Rac (Kul-
DOCK2 is a member of the CDM family of proteins (Caeno- DOCK9 DHR-2 (Yang et al., 2009). Deletion of the amino acid
rhabditis elegans CED-5, mammals DOCK180, and Drosophila sequence corresponding to the lobe A (residues 1,195–1,335)
DHR-2
melanogaster Myoblast City) and is predominantly expressed in DOCK2 did not affect its GEF activity in vitro (Figure 2B),
in hematopoietic cells (Reif and Cyster, 2002). Although indicating that Rac binding site and catalytic center are gener-
DOCK2 does not contain the Dbl homology (DH) domain and ated entirely from lobes B and C. As expected, Rac1 activation
DHR-2DlobeA
the Pleckstrin homology (PH) domain typically found in GEFs, by this mutant (DOCK2
) was also inhibited by CPYPP
DOCK2 catalyzes the GTP–GDP exchange reaction for Rac by in vitro, although the IC50 value was a little bit higher than that
DHR-2
means of its DHR-2 (DOCK homology region 2; also known as for wild-type DOCK2
(Figure 2B). To determine whether
Docker and CZH2) domain (Brugnera et al., 2002; C oˆ t e´ and CPYPP binds to DHR-2 or Rac1, we utilized saturation transfer
Vuori, 2002; Meller et al., 2002). DOCK2 is essential for chemo- difference (STD)-nuclear magnetic resonance (NMR) spectros-
kine receptor- and antigen receptor-mediated Rac activation, copy (Meyer and Peters, 2003), which allows detection of weak
and plays a critical role in migration and activation of lympho- ligand binding by revealing the spatial proximity between ligand
cytes (Fukui et al., 2001; Nombela-Arrieta et al., 2004; Nom- protons and protein protons. Protein concentration used was
bela-Arrieta et al., 2007; Sanui et al., 2003; Shulman et al., 1 mM, and the ligands were added in 100-fold molar excess.
2
1
006). Consistent with the critical roles of DOCK2 in lymphocyte Therefore, only the ligand H signals were observed in the STD
1
functions, DOCK2 deficiency enables long-term survival of spectra, and the protein H signals were invisible with the
cardiac allografts (Jiang et al., 2005) and abrogates development assistance of the suppressive effect of the T1r filter in the
of autoimmune diabetes in NOD mice (our unpublished observa- NMR pulse sequence. By irradiation of the methyl region of
1
tion), a model for human type I diabetes (Anderson and Blue- the spectrum, all of the H signals of CPYPP showed the same
stone, 2005). Thus, DOCK2 may serve as a molecular target degree of reduction in intensity (average, –23%) in the presence
DHR-2DlobeA
for controlling immune-related disorders.
Here, we report identification and characterization of a difference was observed when CPYPP was mixed with Rac1
of the DOCK2
(Figure 2C). In contrast, no significant
0
00
0
0
small-molecule, 4-[3 -(2 -chlorophenyl)-2 -propen-1 -ylidene]-1- (Figure 2C). Thus, CPYPP inhibits Rac GEF activity of DOCK2
phenyl-3,5-pyrazolidinedione (CPYPP). We found that CPYPP by binding to the DHR-2 domain.
DHR-2
binds to the DOCK2 DHR-2 domain (DOCK2
) in a reversible
DOCK proteins are classified into four subfamilies (DOCK-A,
manner and inhibits its catalytic activity. Although overexpres- -B, -C, and -D) based on their structures and substrate speci-
sion of DOCK2, Tiam1, or Trio induced Rac activation in human ficity (C oˆ t e´ and Vuori, 2002). For example, DOCK180 and
embryonic kidney 293T (HEK293T) cells, CPYPP suppressed DOCK5, as well as DOCK2, belong to DOCK-A subfamily and
DOCK2-mediated Rac activation, without affecting Rac activa- act as Rac GEFs. On the other hand, DOCK9, a member of
tion by the classical GEFs. More importantly, treatment of DOCK-D subfamily, catalyzes Cdc42 activation through its
Chemistry & Biology 19, 488–497, April 20, 2012 ª2012 Elsevier Ltd All rights reserved 489

Chemistry & Biology
An Inhibitor that Blocks Rac Activation by DOCK2
Figure 2. CPYPP Inhibits Rac GEF Activity
of DOCK2 through Interaction with DHR-2
Domain
(
A) Structure of CPYPP. Ha-Hl correspond to the
1
H spectra in (C).
B) Concentration-dependent inhibition by CPYPP
DHR-2
(
of in vitro GEF activity of DOCK2
with or
without lobe A. Dose response curves and IC50
values (mean ± SD of three independent experi-
ments) are shown in lower panels.
(
C) The STD-NMR analysis showing that CPYPP
DHR-2
binds to DOCK2
1
expanded region of the 1D H NMR spectra of
, but not to Rac. The
CPYPP with (+SAT) or without (–SAT) saturation of
DHR-2DlobeA
the protein signals of DOCK2
or Rac1.
The impurity peak is denoted by x.
(
D) Comparison of inhibitory activity of CPYPP
among DOCK family proteins. The DHR-2 domain
lacking lobe A was prepared from each molecule
and analyzed for its GEF activity toward Rac
(
(
DOCK2, DOCK180, and DOCK5) or Cdc42
DOCK9) in the presence of various concentra-
tions of CPYPP. Graph shows IC50 values (mean ±
SD of three independent experiments) for each
protein. **p < 0.01.
See also Figure S2 and Table S1.
DHR-2 domain (Meller et al., 2002). To address the specificity of
The importance of the electron-deficient double bonds in
CPYPP, we compared the effect of CPYPP on the GEF activity of CPYPP may raise the possibility that its inhibitory effect is
these DOCK proteins in vitro. CPYPP inhibited the Rac GEF mediated by covalent adducts via Michael addition. To address
DHR-2DlobeA
DHR-2DlobeA
activity of DOCK180
IC50 value comparable to that for DOCK2
However, CPYPP was much less effective in inhibition of based instrument. Although compound 7 with fluoride at the
and DOCK5
with the this possibility, we analyzed the binding of CPYPP to
DHR-2DlobeA
DHR-2DlobeA
(Figure 2D). DOCK2
using a surface plasmon resonance (SPR)-
DHR-2DlobeA
DHR-2DlobeA
DOCK9
These results indicate that, although the inhibitory effect of CPYPP bound to DOCK2
CPYPP is not specific to DOCK2, CPYPP is able to discriminate ure 3D). In addition, we found that DOCK2
-mediated Cdc42 activation (Figure 2D). ortho-position did not show any binding to DOCK2
,
DHR-2DlobeA
in a reversible manner (Fig-
DHR-2
treated with
the structural difference among DOCK subfamily members.
CPYPP normally retained the GEF activity after removable of
CPYPP by gel filtration (Figure S3). These results indicate that
inhibition by CPYPP is reversible.
Structural Features of CPYPP
To determine the structure of CPYPP required for its inhibitory
CPYPP and all the analogs exist as hardly separable equilib-
effect, we synthesized CPYPP analogs (Figures 3A and S1) rium mixtures (1:1 to 1.6:1) of two geometrical isomers at the
0
and compared their effects on the Rac GEF activity of C4=C1 double bond (Figure 4A). Cis isomer 12 is less soluble
DHR-2
DOCK2
in vitro. When the double bonds were saturated to various organic solvents than trans isomer 13, allowing for
through hydrogenation in compound 2, the inhibitory activity isolation of cis isomer 12 as a pure solid form by washing
was markedly decreased (Figure 3B). In contrast, both a mixture with chloroform for several times. On the other
compound 3 with one double bond and compound 4 with three hand, trans isomer 13 was isolated by medium-pressure silica
double bonds inhibited DOCK2-mediated Rac activation with gel column chromatography. The configuration of 12 and 13
IC50 values comparable to that of CPYPP with two double bonds was determined from the comparison of the two coupling
3
3
0
(
Figure 3C). Thus, at least one double bond is essential for the constants,
J
C3-H1
0
and
J across the C4=C1 double
0
C5-H1
inhibitory activity of CPYPP. Another important feature of CPYPP bond (Figure S4). Isolated 12 and 13 readily equilibrated each
00
would be a chloride substituent at the ortho (2 )-position of an other when dissolved in solution (such as DMSO, methanol,
aryl group on the conjugated double bonds. We found that the and acetone) at ambient temperature, giving a 1:1 mixture of
inhibitory activity was significantly suppressed in nonsubstituted two isomers. Even so, isolated 12 and 13 were quickly sub-
compound 5 and compounds 6–9 with hydroxy, fluoride, jected to the Rac GEF activity assay to obtain preliminary infor-
bromide, and methyl groups, respectively, at the ortho-position mation about the double bond geometry-activity relationship.
(
Figure 3C). However, the position of the chloride group on the As a result, cis isomer 12 exhibited slightly higher activity
aromatic ring did not critically affect the inhibitory activity than CPYPP (1:1 mixture of 12 and 13), whereas trans isomer
Figures 3A and 3C; see compounds 10 and 11). 13 did not show any inhibitory activity (Figure 4B). These results
(
490 Chemistry & Biology 19, 488–497, April 20, 2012 ª2012 Elsevier Ltd All rights reserved

Chemistry & Biology
An Inhibitor that Blocks Rac Activation by DOCK2
Figure 3. Structure-Activity Relationship of CPYPP
(
A) CPYPP analogs used in this study (2–11).
DHR-2
(
B) Importance of the conjugated double bonds and the chloride substituent for the inhibitory activity of CPYPP. Rac GEF activity of DOCK2
was analyzed
in vitro in the presence of compounds 2, 3, or 7.
DHR-2
(
(
C) Comparison of IC50 values for DOCK2
-mediated Rac GEF activity among CPYPP analogs.
DHR-2DlobeA
D) SPR-based analysis showing that CPYPP, but not compound 7, binds to DOCK2
in a reversible manner. The graph indicates the response levels
normalized by dividing the measured response units by the molecular weight. Data are mean ± SD of three independent experiments. **p < 0.01.
See also Figure S3.
suggest that the inhibitory activity of CPYPP resides in cis compounds 4 and 6, the IC50 values were well correlated to
isomer.
the degrees of inhibition of DOCK2-mediated Rac activation in
HEK293T cells (Figure 5A). On the other hand, treatment with
CPYPP did not significantly inhibit Rac activation by Tiam1 and
CPYPP Is a Cell-Active Inhibitor of DOCK2
To examine whether CPYPP is a cell-active inhibitor, we ex- Trio under the same experimental condition (Figure 5B). These
pressed DOCK2 in HEK293T cells, and analyzed Rac activation results indicate that CPYPP is able to distinguish DOCK2 from
in the presence or absence of CPYPP. Although overexpression the Dbl-like GEFs and inhibit DOCK2-mediated Rac activation
of DOCK2 induced Rac activation in HEK293T cells, this activa- in cells.
tion was markedly suppressed by treating the cells with CPYPP
(
1) at 100 mM for 1 hr before assay (Figure 5A). Similar inhibition Inhibition by CPYPP of Lymphocyte Migration
was obtained when HEK293T cells were treated with CPYPP To examine whether CPYPP inhibits DOCK2-dependent
analogs such as 3, 10, or 11 (Figure 5A), all of which exhibited lymphocyte functions, we first analyzed the effect of CPYPP
IC50 values <30 mM in vitro (Figure 3C). Indeed, except for on chemokine-induced Rac activation. As previously reported
Chemistry & Biology 19, 488–497, April 20, 2012 ª2012 Elsevier Ltd All rights reserved 491

Chemistry & Biology
An Inhibitor that Blocks Rac Activation by DOCK2
Figure 4. Inhibitory Activity of CPYPP Resides in cis Isomer
(A) Structure of cis (12) and trans (13) isomers of CPYPP.
(B) The effect of cis and trans isomers on in vitro Rac GEF activity. Upon
isolation, cis and trans isomers of CPYPP were quickly subjected to in vitro
GEF assays.
See also Figure S4.
(
Fukui et al., 2001), chemokine-induced Rac activation was
–
/–
severely impaired in DOCK2 deficient (DOCK2 ) T and B cells,
indicating that this process is largely dependent on DOCK2
Figure 5. CPYPP Is a Cell-Active Inhibitor of DOCK2
(
Figures 6A and 6B). Although vehicle (DMSO) alone did not
(
A) Comparison of inhibitory activity of CPYPP and its analogs for DOCK2-
show any inhibitory effect, pretreatment of T cells or B cells
with CPYPP at 100 mM for 1 hr markedly suppressed Rac activa-
tion in response to CCL21 or CXCL13, respectively (Figures 6A
and 6B). Consistent with this finding, treatment with CPYPP
effectively inhibited migratory response of T cells and B cells to
these chemokines in a dose-dependent manner (Figures 6C
and 6D). Lymphocytes also express DOCK5 (our unpublished
mediated Rac activation in HEK293T cells. The correlation coefficient between
IC50 values and the percentage inhibition (mean of three independent exper-
iments) was 0.80. Compounds 4 and 6, and inactive compounds (2, 7, and 9)
that are excluded from the calculation are expressed as open circles.
(B) Selective inhibition by CPYPP of DOCK2-mediated Rac activation in
HEK293T cells. Results are expressed as the ratio of GTP-bound Rac to
total Rac after normalization of the value of DMSO-treated cells to an arbitrary
unit 1 for each GEF. Data are mean ± SD of four independent experiments.
–
/–
observation). When CPYPP was applied to DOCK2 lympho-
cytes, their migration defects were further augmented (Figures
*p < 0.05.
6
C and 6D), probably because of the cross-reactivity of CPYPP
to DOCK5.
To examine whether the effect of CPYPP could be extended reduced the percentage of the migrated T cells to <25% of the
control level (Figure 6E). Similar, but less remarkable effect,
to in vivo situation, we first tested administration route and
dosage of CPYPP by measuring its plasma concentration using was observed when T cell homing to the spleen was analyzed
liquid chromatography-tandem mass spectrometry (LC-MS/ (Figure 6E). On the other hand, intravenous injection of 0.05 mg
of CPYPP did not show any inhibitory effect on lymphocyte
MS). When 2.5 mg/kg of CPYPP was administrated intrave-
nously, the plasma concentration of CPYPP was only 2.4 mM homing in vivo (Figure 6E).
at 30 min (Figure S5). However, by intraperitoneally injecting
2
50 mg/kg of CPYPP into mice, the plasma concentration of Inhibition by CPYPP of Leukocyte Activation
CPYPP reached to 11.3 mM at 30 min and 10.9 mM at 1 hr, Having found that CPYPP inhibits migratory response of lympho-
respectively (Figure S5). With this dose and administration cytes, we next examined the effect of CPYPP on T cell activation.
route, we examined the effect of CPYPP on lymphocyte DOCK2 is a major Rac GEF acting downstream of TCRs (Sanui
homing in vivo. For this purpose, we adoptively transferred et al., 2003). Consistent with this, TCR-mediated Rac activation
spleen cells from mice that had been made by a ‘‘knock-in’’ was almost completely lost in T cells treated with CPYPP at
+
strategy to express endogenous DOCK2 as a fusion protein 100 mM for 1 hr (Figure 7A). CD4 T cells from C57BL/6 mice
with green fluorescent protein (GFP) (Nishikimi et al., 2009). vigorously proliferated when cultured for 3 days with APCs
Without pretreatment with CPYPP, 0.19% of the injected from B10.BR mice (Figure 7B). However, this mixed lymphocyte
+
T cells, on an average, migrated to the peripheral lymph nodes reaction (MLR) was blocked by cultivating CD4 T cells and
of the recipient mice (Figure 6E). However, intraperitoneal injec- APCs in the presence of CPYPP at the concentration of
tion of CPYPP (5 mg per mouse) 1 hr before adoptive transfer 12.5 mM (Figure 7B). This assay was performed in complete
492 Chemistry & Biology 19, 488–497, April 20, 2012 ª2012 Elsevier Ltd All rights reserved

Chemistry & Biology
An Inhibitor that Blocks Rac Activation by DOCK2
Figure 6. CPYPP Blocks Lymphocyte
Migration In Vitro and In Vivo
(
A and B) The effect of CPYPP on Rac activation in
–
/–
WT and DOCK2 T cells (A) or B cells (B) stimu-
lated with CCL21 or CXCL13, respectively.
Results are expressed as the ratio of GTP-bound
Rac to total Rac after normalization of the 15 s (A)
or 20 s (B) value of DMSO-treated cells to an
arbitrary unit 1. Data are mean ± SD of three
independent experiments.
(
C and D) Inhibition by CPYPP of migratory
–
/–
response of WT and DOCK2 T cells (C) or B cells
D) to CCL21 or CXCL13, respectively. NC, no
(
chemokine stimulation. Data are mean ± SD of
triplicate samples and are representative of three
independent experiments.
(
E) Suppression of lymphocyte homing to
secondary lymphoid organs in mice treated with
CPYPP. Numbers in dot plots indicate the
+
+
percentage of GFP cells in CD3 T cells. In
graphs, results are expressed as the percentage
of migrated T cells to total injected T cells. Data are
mean ± SEM of mice treated with CPYPP intra-
peritoneally (i.p.; 5 mg per mouse; n = 9) or intra-
venously (i.v.; 0.05 mg per mouse; n = 4), and are
representative of two independent experiments.
(
A–E) *p < 0.05; **p < 0.01.
See also Figure S5.
tion has been implicated in the pathogen-
esis of certain autoimmune diseases
such as psoriasis and systemic lupus
erythematosus (Blanco et al., 2001;
Nestle et al., 2005). Because DOCK2–
Rac axis is critical in pDCs for TLR-medi-
ated induction of type I IFNs, but not
inflammatory cytokines (Gotoh et al.,
2010), we also examined the effect of
CPYPP on pDC activation. When pDCs
were stimulated with A-type CpG DNA,
a TLR9 ligand, activated Rac was readily
detected (Figure 7C). However, treatment
with CPYPP severely impaired CpG-A-
induced Rac activation in pDCs (Fig-
ure 7C), resulting in loss of IFN-a induction
without affecting IL-12p40 levels (Fig-
ure 7D). Collectively, these results indi-
RPMI1640 medium supplemented with 10% fetal calf serum cate that treatment with CPYPP faithfully reproduces DOCK2-
FCS), whereas the effect of CPYPP on lymphocyte migration deficient phenotypes in lymphocytes and pDCs.
was examined in RPMI1640 medium containing 0.5% bovine
(
serum albumin (BSA). Because CPYPP was more effective in DISCUSSION
MLR than in migration assay (see Figure 6C), the efficiency of
CPYPP uptake by lymphocytes might be different between these Rac is an important signaling molecule that controls diverse
culture conditions. Indeed, we found that the intracellular cellular functions, including migration and activation of lympho-
content of CPYPP was much higher when splenocytes were cytes (Faroudi et al., 2010; Hall, 1998; Tybulewicz and Hender-
incubated with CPYPP in complete RPMI1640 medium supple- son, 2009; Villalba et al., 2001; Yu et al., 2001). Thus far, several
mented with 10% FCS (Figure S6).
chemical inhibitors acting at different steps of Rac signaling
By recognizing nucleic acid ligands through TLR7 and TLR9, have been identified. For example, NSC23766 or EHT 1864
pDCs produce not only inflammatory cytokines, but also large inhibits Rac activation by binding to Rac to compete with the
amounts of type I IFNs (Gilliet et al., 2008). This type I IFN induc- GEF binding or to destabilize the bound nucleotide, respectively
Chemistry & Biology 19, 488–497, April 20, 2012 ª2012 Elsevier Ltd All rights reserved 493

Chemistry & Biology
An Inhibitor that Blocks Rac Activation by DOCK2
Figure 7. CPYPP Blocks Activation of T
Cells and pDCs
(
A) Inhibition by CPYPP of Rac activation in T cells
stimulated with anti-CD3ε antibody. Results are
expressed as the ratio of GTP-bound Rac to total
Rac after normalization of the 0.5-min value of
DMSO-treated cells to an arbitrary unit 1. Data are
mean ± SD of three independent experiments.
(
B) Inhibition by CPYPP of MLR between C57BL/6
and B10.BR mice. Data are mean ± SD of triplicate
samples and are representative of three indepen-
dent experiments.
(
C) Inhibition by CPYPP of Rac activation in pDCs
stimulated with CpG-A. Results are expressed as
the ratio of GTP-bound Rac to total Rac after
normalization of the 1-min value of DMSO-treated
cells to an arbitrary unit 1. Data are mean ± SD of
three independent experiments.
(D)InhibitionbyCPYPPofTLR9–mediatedtypeIIFN
induction in pDCs. Data are mean ± SD of triplicate
samples and are representative of three indepen-
dent experiments. (A–D) *p < 0.05; **p < 0.01.
See also Figure S6.
(
Gao et al., 2004; Shutes et al., 2007). On the other hand, IPA-3 is the critical importance of the electron-deficient double bonds
an inhibitor targeting p21-activated kinase (PAK), a downstream in the compounds, conjugated to the two carbonyl groups of
effector of Rac (Deacon et al., 2008). However, because Rac and the pyrozolidinedione part, for their binding and inhibitory activ-
PAK isoforms are expressed in many cell-types, they are not ities. This result suggests the existence of critical hydrophobic
ideal targets to control immune responses. Unlike these mole- interactions between the inhibitor’s electron-deficient p-orbitals
DHR-2
cules, DOCK2 expression is limited to hematopoietic cells (Fukui and the binding pocket of DOCK2
. Additionally, the molec-
et al., 2001). More importantly, DOCK2 is a Rac activator ular flatness of compounds 1, 3, and 4, compared with 2 contain-
3
essential for migration and activation of lymphocytes (Fukui ing sp carbons, would also contribute to their binding ability. On
et al., 2001; Nombela-Arrieta et al., 2004, 2007; Sanui et al., the other hand, effects of the substituents on the aromatic ring
2
003; Shulman et al., 2006), and its deficiency has been demon- at the conjugated diene moiety on the inhibitory activity are
strated to suppress allograft rejection and autoimmune disease more difficult to rationalize. An ortho-substituent with an appro-
development (Jiang et al., 2005). Therefore, DOCK2 may serve priate size and electron-withdrawing ability (such as o-chloride
as a better therapeutic target for controlling immune-related containing 1) seems to be beneficial for the inhibitory activity. If
disorders.
a chloride substituent exists, however, its position on the
Here, we have identified CPYPP as a small-molecule inhibitor aromatic ring is not very critical (1, 10, and 11). This result may
DHR-2
of DOCK2. CPYPP bound to DOCK2
GEF activity with IC50 of 22.8 mM. Unfortunately, CPYPP was inhibitors’ p-orbitals for their interaction with DOCK2
and inhibited its Rac also support the importance of the electronic characteristics of
DHR-2
.
We also elucidated the cis-geometry of C4=C1 double bond
0
not specific to DOCK2 and showed cross-reactivity to other
DOCK-A members, DOCK5 and DOCK180. However, CPYPP in CPYPP as an additional structural requirement for the inhibi-
inhibited DOCK2-mediated Rac activation, without affecting tory activity. Because the double bond geometry was labile
Rac activation by Tiam1 and Trio. Thus, CPYPP is a selective and readily equilibrated each other in a solution state, pure
inhibitor that can distinguish DOCK2 from the classical GEFs. isomers were hardly separable. However, we fortunately found
Although CPYPP is a potentially reactive Michael acceptor, that the active cis-isomer 12 could be conveniently isolated in
the SPR-based analysis indicated that CPYPP binds to a gram scale through crystallization. The olefin geometry is
DHR-2
DOCK2
in a reversible manner. Indeed, when CPYPP was stable in a solid state. In addition, trans-isomer 13 was isolated
treated with 2-mercaptoethanol (1 equivalent) in the assay in a sub-mg scale by silica gel column chromatography.
buffers in the presence of BSA for 1 hr, CPYPP was recovered Comparison of the inhibitory activity between 12 and 13 demon-
quantitatively in intact form without Michael reaction (our un- strated that the inhibitory activity resides in the cis-isomer 12.
DHR-2
differentiates bent (cis) or linear (trans)
published observation). In addition, we confirmed that the Therefore, DOCK2
DHR-2
DOCK2
treated with CPYPP is functionally reversible. global structure of CPYPP.
Therefore, inhibitory activity of CPYPP is not due to covalent
modification of DOCK2.
Although calcineurin inhibitors such as cyclosporine and
FK506 have been successfully used to prevent ‘‘unwanted’’
CPYPP and its analogs can be readily synthesized in practical immune responses, development of novel immunosuppressants
scales. The synthetic route includes convergent Knoevenagel with different actions is required to reduce their side effects. In
condensation between aldehydes and pyrazolidinediones, this respect, DOCK2 also would be a good drug target, because
allowing for rapid supply of structurally diverse analogs of DOCK2 deficiency has been demonstrated to synergistically
CPYPP. Current structure–activity relationship studies clarified act with FK506 in controlling transplant rejection (Jiang et al.,
494 Chemistry & Biology 19, 488–497, April 20, 2012 ª2012 Elsevier Ltd All rights reserved

Chemistry & Biology
An Inhibitor that Blocks Rac Activation by DOCK2
2
005). Consistent with the role of DOCK2 as a master regulator
Plasmids
For expression of DOCK2, Tiam1, or Trio DH-D1/PH-D1 domain in mammalian
cells, their cDNAs were amplified by PCR with primers encoding hemagglutinin
for Rac activation in lymphocytes, treatment with CPYPP almost
completely abolished chemokine receptor- and TCR-mediated
Rac activation in lymphocytes, resulting in marked reduction
of chemotactic response and T cell activation. In addition,
we found that TLR9-mediated IFN-a induction is selectively
inhibited in pDCs treated with CPYPP. These results indicate
that CPYPP is able to block effectively DOCK2-mediated
inflammatory responses. Therefore, pharmacological inhibition
of DOCK2 could be an attractive therapeutic strategy for con-
trolling immune-related disorders, such as autoimmune
diseases and transplant rejection, caused by tissue infiltration
of activated lymphocytes. Design and synthesis of geometrically
constrained molecules with the electronic and structural pro-
perties identified in this study will lead to development of more
potent and more selective DOCK2 inhibitors that can be used
as a drug lead.
(
HA) tag, and cloned into pCI (Promega) or pcDNA-His/max (Invitrogen). The
pET-SUMO vector was created by subcloning SUMO cDNA into pET-28a
DHR-2
DHR-2DlobeA
vector (Novagen). The genes encoding DOCK2
DHR-2DlobeA
, DOCK2
DHR-2DlobeA
,
DHR-2DlobeA
DOCK180
, DOCK5
, and DOCK9
were cloned
into the pET-SUMO to express fusion proteins with His tag at their N terminus,
which were used for in vitro GEF assays, STD-NMR assays, and SPR-based
DHR-2
binding assays. For screening of chemical compounds, DOCK2
with
trigger factor (TF) at the N terminus and streptavidin binding protein (SBP) at
the C terminus was created using pET-22b vector. The gene encoding Rac1
or Cdc42 was cloned into pGEX-6P-1 (GE Healthcare) to express gluta-
thione-S-transferase (GST)-fusion protein.
Screening of Chemical Library
An ELISA was employed to screen for chemical compounds that can inhibit
DHR-2
interaction between DOCK2
and Rac. Briefly, 96-well polystyrene plates
were coated overnight with streptavidin (250 ng per well) dissolved in 50 mM
carbonate buffer (pH 9.6). Each well was blocked with 150 ml of 5% skim
milk in TBST (20 mM Tris-HCl, 150 mM NaCl, 0.1% Tween-20, pH7.5) at
SIGNIFICANCE
room temperature for 1 hr. After the wells were rinsed with TBST, recombinant
DHR-2
TF-DOCK2
-SBP (50 ml, 60 mg/ml in TBST) was added to each well and
incubated at room temperature for 1 hr. The wells were then rinsed with
TBST supplemented with 1 mM EDTA. To each well, the library component
or vehicle (50 ml, 200 mM in TBST/1 mM EDTA/2% DMSO) was added and incu-
bated at room temperature for 1 hr. After incubation, GST-Rac1 or GST-Cdc42
(50 ml, 50 mg/ml in TBST/1 mM EDTA) was added to each well and incubated
for 1 hr at room temperature. The wells were then rinsed with 150 ml of TBST/
DOCK2, a mammalian homolog of Caenorhabditis elegans
CED-5 and Drosophila melanogaster Myoblast city, is an
atypical Rac activator predominantly expressed in hemato-
poietic cells. Although DOCK2 does not contain Dbl
homology domain typically found in guanine nucleotide
exchange factors (GEFs), DOCK2 mediates the GTP-GDP
exchange reaction for Rac through its DHR-2 domain.
DOCK2 plays a key role in migration and activation of
lymphocytes, and its deficiency suppresses allograft
rejection and autoimmune disease development in mice.
Therefore, DOCK2 may serve as a molecular target for
controlling immune-related disorders. Here, we have identi-
1
mM EDTA. Horseradish peroxidase-conjugated anti-GST antibody (50 ml, in
TBST/1 mM EDTA) wad added to each well and incubated for 1 hr at room
temperature. After the wells were rinsed with TBST/1 mM EDTA, peroxidase
substrate (50 ml, 0.4 mg/ml o-phenylenediamine and 0.01% H
2 2
O in 0.1 M
citrate buffer, pH 5.0) was added to each well and incubated for 30–60 min
at room temperature. The absorbance was measured at 450 nm with a Multis-
kan Bichromatic plate reader (Labsystems) and compensated by subtracting
‘‘blank’’ absorbance of the well into which GST-Cdc42 was added. Percent
inhibition was calculated as follows: [(optical density with vehicle alone) –
0
00
0
0
fied 4-[3 -(2 -chlorophenyl)-2 -propen-1 -ylidene]-1-phenyl-
,5-pyrazolidinedione (CPYPP) as a small-molecule inhibitor
(
optical density with sample)]/(optical density with vehicle alone) 3 100.
Compounds were regarded as potential hits if they had an inhibitory effect
40%.
3
of DOCK2. CPYPP bound to DOCK2 DHR-2 domain in a
reversible manner and inhibited its catalytic activity, without
affecting Rac activation by the classical GEFs. The struc-
ture–activity relationship study revealed the importance of
the electron-deficient double bonds, a chloride substituent
on the aromatic ring, and the cis-geometry for inhibitory
activity of CPYPP. When lymphocytes were treated with
CPYPP, both chemokine receptor- and antigen receptor-
mediated Rac activation were blocked, resulting in marked
reduction of chemotactic response and T cell activation.
These results suggest that pharmacological inhibition of
DOCK2 could be a therapeutic strategy for controlling
immune-related disorders, and provide a rational of and
a chemical scaffold for development of the DOCK2-target-
ing immunosuppressant.
>
Mice
–
/–
DOCK2 and DOCK2-GFP knock-in mice have been described previously
(Fukui et al., 2001; Nishikimi et al., 2009). All mice were maintained under
specific pathogen-free conditions in the animal facility of Kyushu University.
Animal protocols were approved by the committee of Ethics on Animal
Experiment, Faculty of Medical Sciences, Kyushu University.
In Vitro GEF Assays
Recombinant GEF protein diluted in exchange buffer (20 mM MES-NaOH,
2
150 mM NaCl, 10 mM MgCl , 0.2 mg/ml BSA, 20 mM GDP, pH 7.0) was
incubated with chemical compounds or DMSO (vehicle) for 2–30 min in the
dark at room temperature. Unbound CPYPP was removed using a spin column
gel filtration (Edge Bio) at this stage when the functional reversibility of
DHR-2
DOCK2
was analyzed. Recombinant Rac or Cdc42 (15 mM) was loaded
with GDP in the same reaction buffer on ice for 30 min. GDP-loaded G-proteins
100 ml) were allowed to equilibrate in exchange buffer supplemented with N-
methylanthraniloyl (mant)-GTP (3.6 mM; Jena Bioscience) or BODIPY-FL-GTP
3.6 mM; Invitrogen) for 8 min. After equilibration, pretreated GEF (50 ml) was
(
EXPERIMENTAL PROCEDURES
(
Chemical Compounds
added to the mixtures and the change of mant-GTP fluorescence (excitation =
360 nm, emission = 440 nm) or BODIPY-FL fluorescence (excitation = 488 nm,
A total of 9,392 chemical compounds used for the initial screening were ob-
tained from Open Innovation Center for Drug Discovery at The University of
Tokyo, Japan. The synthesis and purification of CPYPP and its analogs are
described in Supplemental Experimental Procedures in detail. Compounds
were dissolved in DMSO, and diluted with appropriate buffers immediately
before use. The percentage of DMSO used for cellular assays or in vitro GEF
assay was 0.2% or 1%, respectively.
ꢀ
emission = 514 nm) was monitored at 30 C using a XS-N spectrofluorimeter
(Molecular Devices). For IC50 determination, the initial slope for each reaction
was determined using a curve fitting function of the GraphPad Prism 5
program (GraphPad Software), and plotted as a function of compound
concentrations. The slope in the absence of compound was set as ‘‘100%
activity.’’
Chemistry & Biology 19, 488–497, April 20, 2012 ª2012 Elsevier Ltd All rights reserved 495

Chemistry & Biology
An Inhibitor that Blocks Rac Activation by DOCK2
3
concentrations of CPYPP or DMSO for 84 hr, and [ H]-thymidine (0.037
NMR
All NMR spectra were recorded at 298 K on a Bruker Avance 600-MHz
spectrometer equipped with a TXI cryoprobe. NMR samples were prepared
MBq) was added during the final 16 hr of the culture. Cells were harvested
on glass filter paper, and the incorporated radioactivity was measured with
a liquid scintillation counter.
2
2
in 0.4 ml of 99.9%
H
2
O buffer containing 10 mM Tris-d8- HCl, pH 8.0, and
. Protein concentrations were 1 mM. CPYPP was dissolved in
9.96% DMSO-d6 at 2 mg/ml, and added to the protein samples at a final
10 mM MgCl
2
9
Measurement of Cytokine Production
concentration of 100 mM. STD NMR spectra were acquired with the pulse
sequence as described (Mayer and Meyer, 2001). The on-resonance irradia-
tion of the protein methyl signal region was set at a chemical shift of ꢁ0.2
ppm, and the off-resonance irradiation was set at ꢁ30 ppm, where no protein
signals were present, with a total saturation time of 2 s. The suppression of
Flt3 ligand-induced BM-derived pDCs were stimulated with CpG-A (3 mM)
for 24 hr in the presence of CPYPP or DMSO. Concentrations of IFN-a (R&D
Systems) and IL-12p40 (Thermo Fisher Scientific) in cell-culture supernatants
were measured by ELISA kit.
1
2
the residual H HO signal was achieved by the WATERGATE technique. The
STD effect was verified by efficient cancellation of the impurity peaks.
In Vivo Homing Assays
8
Spleen cells (1 3 10 ) isolated from DOCK2-GFP knock-in mice were trans-
ferred intravenously into C57BL/6 mice that had been treated intraperitoneally
with 100 ml of CPYPP (50 mg/ml in DMSO) or intravenously with 50 ml of CPYPP
(1 mg/ml in DMSO) 1 hr before the transfer. Mice were sacrificed 1 hr later, and
cells were prepared from the spleen and the peripheral lymph nodes. Cells
were then stained for CD3ε, and the percentages of migrated T cells were
calculated.
SPR-Based Binding Assays
SPR-based binding assays were performed using a Biacore T200 (GE Health-
DHR-2DlobeA
care). His-SUMO-DOCK2
was immobilized on NTA Sensor Chip
(GE Healthcare) with a covalent coupling of His-tagged protein to nitrilotriace-
tic acid (NTA) biosensor surface (Willard and Siderovski, 2006). NTA Sensor
Chip was washed with 0.35 M EDTA for 60 s (10 ml/min) followed by extra
wash with a buffer (10 mM HEPES, pH 7.4; 150 mM NaCl, 50 mM EDTA,
0.1% Tween 20). Then the chip was activated with 500 mM NiCl
2
for 60 s
Measurement of Plasma Concentration of CPYPP
(
10 ml/min) followed by extra wash with 3 mM EDTA. Finally, His-SUMO-
The plasma concentration of CPYPP was determined using LC-MS/MS
(Prominence 2000 series liquid chromatography system, Shimadzu, coupled
to an API-3000 mass spectrometer, Applied Biosystems). A CPYPP standard
curve using 5 ml of serially diluted CPYPP in DMSO into 50 ml of plasma (final
concentration range from 0.01 to 30 mg/ml) was used to determine CPYPP
concentration in plasma. Following protein precipitation by acetonitrile con-
taining 0.1 mg/ml of diazepam (internal standard), samples were cleared by
DHR-2DlobeA
DOCK2
Coupling Kit, GE Healthcare) after injection of EDC (1-ethyl-3(3-dimethylami-
nopropyl)-carbodiimide hydrochloride)/NHS (10 ml/min, min) and His-
(10 ml/min, 7 min), and deactivation with ethanol amine
10 ml/min, 7 min). We could capture significant amount of His-SUMO-
was immobilized on the chip by amine coupling (Amine
7
DHR-2
SUMO-DOCK2
(
DHR-2DlobeA
DOCK2
(ꢂ3,000 RU) for affinity experiments. SPR measurements
ꢀ
were carried out with a running buffer (10 mM HEPES, pH 7.4; 150 mM
NaCl, 50 mM EDTA, 0.1% Tween 20, and 8% DMSO). For this purpose,
compounds were diluted with the running buffer to keep DMSO concentration
at 8%. Then the sample was injected for 60 s (contact phase), and 180 s
of buffer flow (dissociation phase) was followed. The flow rate was set at
centrifugation (15 min, 3,500 rpm, 4 C) and fractionated on a Capcell PAK
ꢀ
C18 MG column (3 mm, 2.0 3 35 mm; Shiseido) at 50 C using a 0.1% formic
acid/water to 0.1% formic acid/acetonitrile gradient with a flow rate of
0.3 ml/min. Detection of CPYPP and diazepam was carried out by positive
electrospray ionization mode with transitions of m/z 325–164 and m/z
285–193, respectively. The ratio of chromatographic peak areas of CPYPP
to diazepam was used to calculate the plasma CPYPP concentration and
expressed as mM by unit conversion.
3
0 ml/min. For extra wash, 50% DMSO was used. The binding response
DHR-2DlobeA
(RU) of compounds to His-SUMO-DOCK2
was determined with
the Biacore T200 evaluation software.
Cell Preparation
Measurement of Intracellular CPYPP Content
pDCs were prepared from bone marrow (BM) cells using Flt3 ligand as
described previously (Gotoh et al., 2010). T cells and B cells were isolated
from spleen and lymph nodes using Pan-T cell isolation kit and B cell isolation
An oil filtration assay was applied to determine relative intracellular accumula-
tion of CPYPP by using Transporter Suspension Assay Kit (BD Biosciences).
Microcentrifuge tubes from the kit were conditioned by addition of 100 ml of
+
kit (both from Myltenii Biotec), respectively. CD4 T cells were isolated by
2
N NaOH solution followed by layering filtration oil onto the NaOH solution.
7
magnetic sorting with Dynabead Mouse CD4 followed by treatment with
DETACHaBEAD Mouse CD4 (both from Dynal). Cell viability was determined
with CytoTox 96 nonradioactive cytotoxicity assay (Promega) according to
the manufacture’s instructions.
Spleen cells of BALB/c mice (1 3 10 /ml) were incubated in RPMI1640
medium supplemented with 0.5% BSA or 10% FCS in the presence or
ꢀ
absence of 100 mM CPYPP. After 1 or 5 hr of incubation at 37 C, the spleno-
cyte suspension in each culture media was loaded onto the oil layer and
centrifuged (12,000 rpm, 30 s) to pellet cells in the NaOH layer, enabling quick
wash and splenocyte collection. Splenocyte suspension in NaOH were taken
off by scissoring microcentrifuge tube and aspirating the remaining oil, then
Rac Activation Assays
Before assays, cells were stimulated with CCL21 (1 mg/ml), CXCL13 (1 mg/ml),
anti-CD3ε antibody (145-2c11; 10 mg/ml) followed by crosslinking with anti-
hamster IgG (10 mg/ml), or CpG-A (3 mM). Aliquots of the cell extracts were
kept for total lysate controls, and the remaining extracts were incubated
ꢀ
incubated at 37 C for 10 min for solubilization. These samples were neutralized
by HCl and diluted by acetonitrile with 0.1 mg/ml of diazepam. Samples were
injected into the LC-MS/MS system to detect peak areas of CPYPP and diaz-
epam. The ratio of peak areas of CPYPP to diazepam was used to compare
intracellular content of CPYPP.
ꢀ
with GST-fusion Rac-binding domain of PAK1 at 4 C for 60 min. The bound
proteins and the same amounts of total lysates were analyzed by SDS-
PAGE, and blots were probed with anti-Rac antibody (23A8, Millipore).
Statistical Analysis
Chemotaxis Assays
Statistical analyses were performed using one-way analysis of variance
Transwell chemotaxis assays were performed using CCL21 (300 ng/ml) or
CXCL13 (2 mg/ml) as a chemoattractant for T cells or B cells, respectively. After
(
ANOVA) followed by Dunnett’s test (Figures 2D, 5B, 6C, 6D, 7B, and 7D) or
two-tailed Student’s t test (Figures 3D, 6A, 6B, 6E, 7A, and 7C). Analyses
were done with GraphPad Prism 5 software.
ꢀ
incubation for 2 hr at 37 C, spleen cells migrated to the lower chamber were
collected, and stained for Thy1.2 and B220. The percentage of migrated cells
+
+
was calculated by dividing the number of Thy1.2 cells (T cells) or B220 cells
B cells) in the lower chamber by the number of input cells.
(
SUPPLEMENTAL INFORMATION
MLR
Supplemental Information includes six figures, one table, and Supplemental
Experimental Procedures and can be found with this article online at doi:10.
1016/j.chembiol.2012.03.008.
5
T cells (3 3 10 ) from C57BL/6 mice were cultured with irradiated spleen
6
cells (1 3 10 ) from B10.BR or C57BL/6 mice in the presence of various
496 Chemistry & Biology 19, 488–497, April 20, 2012 ª2012 Elsevier Ltd All rights reserved

Chemistry & Biology
An Inhibitor that Blocks Rac Activation by DOCK2
ACKNOWLEDGMENTS
lator of the actin cytoskeleton in lymphocytes, suppresses cardiac allograft
rejection. J. Exp. Med. 202, 1121–1130.
We thank A. Inayoshi and M. Sanematsu for technical assistance. This work
was supported by grants for Targeted Proteins Research Program from the
Ministry of Education, Culture, Sports, Science and Technology of Japan,
Grants-in-Aid for Scientific Research from the Japan Society for the promotion
of Science, CREST program of Japan Science and Technology Agency, the
Kanae Foundation, the Mochida Memorial Foundation, and the Tokyo
Biochemical Research Foundation.
Kulkarni, K., Yang, J., Zhang, Z., and Barford, D. (2011). Multiple factors confer
specific Cdc42 and Rac protein activation by dedicator of cytokinesis (DOCK)
nucleotide exchange factors. J. Biol. Chem. 286, 25341–25351.
Luster, A.D., Alon, R., and von Andrian, U.H. (2005). Immune cell migration in
inflammation: present and future therapeutic targets. Nat. Immunol. 6, 1182–
1
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Mandala, S., Hajdu, R., Bergstrom, J., Quackenbush, E., Xie, J., Milligan, J.,
Thornton, R., Shei, G.-J., Card, D., Keohane, C., et al. (2002). Alteration of
lymphocyte trafficking by sphingosine-1-phosphate receptor agonists.
Science 296, 346–349.
Received: September 19, 2011
Revised: February 17, 2012
Accepted: March 1, 2012
Published: April 19, 2012
Mayer, M., and Meyer, B. (2001). Group epitope mapping by saturation trans-
fer difference NMR to identify segments of a ligand in direct contact with
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