A small molecule inhibitor of α4 integrin-dependent cell migration

Article abstract of DOI:10.1016/j.bmc.2008.02.067

A small molecule inhibitor of α4 integrin-dependent cell migration was identified through a cell-based screen of small molecule libraries. Biochemical and cellular experiments suggest that this molecule functions by interacting with γ-parvin. This molecul

Full text of DOI:10.1016/j.bmc.2008.02.067

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 17 (2009) 977–980
A small molecule inhibitor of a4 integrin-dependent cell migration
Jongkook Lee,a, Jiyong Hong,a,b,à Tae-Gyu Nam,a Eric C. Peters,b Anthony P. Orth,b
Bernhard H. Geierstanger,b Lawrence E. Goldfinger,c Mark H. Ginsberg,c
Charles Y. Chob,* and Peter G. Schultza,b,
*
aDepartment of Chemistry and The Skaggs Institute for Chemical Biology, The Scripps Research Institute,
10550 North Torrey Pines Road, La Jolla, CA 92037, USA
bGenomics Institute of the Novartis Research Foundation, San Diego, CA 92121, USA
cDepartment of Medicine, University of California San Diego, La Jolla, CA 92093, USA
Received 13 November 2007; revised 19 February 2008; accepted 21 February 2008
Available online 26 February 2008
Abstract—A small molecule inhibitor of a4 integrin-dependent cell migration was identified through a cell-based screen of small
molecule libraries. Biochemical and cellular experiments suggest that this molecule functions by interacting with c-parvin. This mol-
ecule should serve as a useful tool to study a4 integrin signaling and may lead to new therapeutics for the treatment of autoimmune
diseases.
ꢀ 2008 Elsevier Ltd. All rights reserved.
1. Introduction
unbiased phenotypic screen for molecules that block
a4 integrin-dependent cell migration.
Integrins are a family of cell adhesion receptors com-
posed of an a and a b subunit which mediate cell adhe-
sion and cell migration by interacting with the
extracellular matrix. The a4 integrins (a4b1 and a4b7)
are essential for embryogenesis, hematopoiesis, and im-
mune responses and are of particular interest with re-
spect to leukocyte trafficking and activation.1 The
importance of a4 integrin signaling in various chronic
inflammatory diseases2 and tumor metastases3 has led
to the development of several a4 antagonists.4 These in-
clude antibodies that bind a4 integrins,5,6 peptidomimet-
ics that block integrin binding,7–9 and small molecules
that disrupt the a4 integrin–paxillin complex.10 Inhibi-
tion of a4 integrin signaling by targeting proteins down-
stream of a4 integrin may offer an alternative approach
for modulating a4 integrin-mediated leukocyte traffick-
ing, as well as provide new insights into integrin-depen-
dent signaling pathways. To this end we carried out an
2. Results and discussion
2.1. Cell-based screen of small molecule libraries
Cell migration was assayed using an automated 384-well
plate wound-healing assay11,12 with Chinese hamster
ovary cells that were engineered to express a4 integrin
(CHO-a4).13 CHO-a4 cells were plated into 384-well
plates coated with a segment of fibronectin (CS-1), a li-
gand for a4b1 integrin. Cells were grown to high density
and each well was mechanically scratched. The cells
were then treated with compounds from a library of
approximately 50,000 heterocycles which included
known drugs, pyrrolopyrimidines, thiazoles, oxazoles,
trisubstituted purines, disubstituted pyrimidines, and
thiazines14 at 37 ꢁC for 12 h (5 lM final concentration).
Cells were fixed and the nuclei were stained followed by
automated high throughput fluorescence microscopy.
Approximately 250 compounds were found to inhibit
the migration of CHO-a4 cells. To distinguish selective
inhibitors of a4 integrin-dependent cell migration (spe-
cifically a4 vs a5 integrin-dependent cell migration in
the case of CHO cells), the effects of these primary hits
on the migration of CHO-a4 cells in 384-well plates
coated with a 9–11 fragment of fibronectin (a ligand
*
Corresponding authors. Tel.: +1 858 784 9273; fax: +1 858 784
9440; e-mail addresses: ccho@gnf.org; schultz@scripps.edu
Present address: Center for Cancer Research, Drug Discovery
Division, Korea Research Institute of Chemical Technology, Daejeon
305-600, Republic of Korea.
à
Present address: Department of Chemistry, Duke University, Dur-
ham, NC 27708, USA.
0968-0896/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.02.067

978
J. Lee et al. / Bioorg. Med. Chem. 17 (2009) 977–980
Cl
HN
N
NH2
N
H
N
JK273
Figure 1. Structure of JK273.
for a5b1 integrin) were assayed. Among the primary hits
that did not inhibit a5 integrin-dependent cell migration
(see Supplementary Figs. 1 and 2), JK273 (Fig. 1)15 was
the most potent (and showed dose dependent behavior).
To verify JK273 activity on a4 integrin signaling in a
lymphocyte derived cell line, the effect of JK273 on the
motility of Jurkat T cells in a modified Boyden chamber
assay was determined and significant inhibitory activity
was found (IC50 0.5 lM). A preliminary structure–activ-
ity relationship (SAR) analysis revealed that the 3-chlor-
ophenylamino group is essential for activity, while the 4-
aminophenyl group can be replaced with other substi-
tuted phenyls without significant loss in activity (see
Supplementary Fig. 2).
2.2. The molecular mechanism of JK273
To investigate the molecular mechanism by which
JK273 blocks cell migration, we attempted to identify
the cellular target(s) of this compound by affinity chro-
matography (Fig. 2). An affinity matrix 2 was prepared
by coupling JK273 derivative 1 to Affi-Gel 10 through
the amino substituent by a polyethylene glycol linker
based on the above SAR analysis (see Supplementary
material). Matrix 2 was treated with Jurkat cell extracts,
and retained proteins were separated by SDS–PAGE.
To identify those proteins that bind specifically to
JK273, cell extracts were pre-incubated with JK273
(100 lM). JK273 effectively blocked the binding of
37 kDa and 41 kDa proteins to the affinity matrix,
which were identified by mass spectral analysis as c-par-
vin and adenosine kinase (ADK), respectively (see Sup-
plementary Tables 1 and 2). These results were verified
by Western blotting with anti-c-parvin and anti-ADK
antibodies (Fig. 2c). To confirm the involvement of
c-parvin and ADK in Jurkat cell migration, expression
of these proteins was independently knocked down with
interfering short-hairpin RNAs (shRNAs).16 Jurkat cells
were infected with viruses producing shRNAs specific
for c-parvin or ADK and their migration was again as-
sayed using a modified Boyden chamber assay. Down-
regulation of ADK with multiple shRNAs had only a
small effect on cell migration, suggesting that binding
of JK273 to ADK is not a major functional contributor
to the cell migration phenotype (data not shown).
Figure 2. (a) Structure of ligand 1 for affinity matrix. (b) Silver-stained
SDS–PAGE gel showing c-parvin and ADK proteins pulled down by
affinity matrix. Lane 1: cell extracts were treated with DMSO (1%);
lane 2: cell extracts were treated with JK273 (100 lM). (c) Confirma-
tion of c-parvin and ADK identity by Western blot analysis. Lane 1:
cell extracts were treated with DMSO (1%); lane 2: cell extracts were
treated with JK273 (100 lM). (d) Boyden chamber assay with Jurkat
cells and quantitative RT-PCR analysis of c-parvin expression after
infection with lentiviruses producing shRNAs targeting c-parvin for
84 h. An anti-a4 integrin antibody (clone HP2/1) was used as a positive
control for the inhibition of cell migration in the Boyden chamber
assay. GAPDH was used as an internal control for quantitative RT-
PCR analysis. Data are means SD.
We thus turned our attention to the role of the
JK273–c-parvin interaction in the inhibition of a4-
dependent cell migration. c-Parvin is a CH domain
containing protein that is highly expressed in leuko-
cytes, localized to focal adhesions, and a binding part-
ner with several integrin-signaling related proteins
including paxillin, integrin-linked kinase (ILK), a-acti-

J. Lee et al. / Bioorg. Med. Chem. 17 (2009) 977–980
979
nin, and ARHGEF6.16 Reduction of transcript levels
4.2. Automated cell migration assay
of c-parvin with RNA interference caused a corre-
sponding reduction of migration of Jurkat cells
(Fig. 2d); when c-parvin mRNA levels were reduced
by >75%, the degree of inhibition of cell migration
was similar to that from blocking a4 integrin function
with an inhibitory a4 integrin antibody. These results
are in agreement with recent studies by Yoshimi et al.
who further suggest that c-parvin plays an essential
role in cell spreading in leukocytes.17 To determine
if JK273 affects a4 integrin-dependent cell attachment,
U937 or Jurkat cells were stimulated with phorbol 12-
myristate 13-acetate (PMA) or stromal cell derived
factor 1-a (SDF1-a) to induce adhesion to fibronec-
tin-coated plastic.18 Treatment of a suspension of
U937 or Jurkat cells with JK273 reduced attachment
to fibronectin-coated surfaces by 40–80% (Supplemen-
tary Fig. 3), confirming that JK273 activity can phe-
nocopy c-parvin loss-of-function via RNAi. Taken
together, these data suggest that JK273 acts through
the alteration of c-parvin function. The detailed mech-
anism by which JK273 binds and inhibits c-parvin
activity and the JK273 specificity profile for other
parvins (not detected in Jurkat cells19) are currently
under investigation.
CHO-a4 cells were plated into 384-well assay plates
(12,000 cells/well) coated with CS-1 and allowed to
form monolayers at 37 ꢁC and 5% CO2 for 5 h.
After wounds were generated by an automated
wound-healing assay device,11 cells were treated with
a library of heterocycles (5 lM final concentration).
The cells were allowed to migrate for 12 h, fixed
with paraformaldehyde (4%), and stained with
DAPI. Each well of the 384-well plates was photo-
graphed with an IC100 (Beckman Coulter) to auto-
mate image capture. A 4· objective lens was used
to visualize a majority of the area of each well with-
in one field of view.
4.3. Transwell cell migration assay (modified Boyden
chamber assay)
Transwell plate inserts (Corning Costar, 6.5 mm
diameter, 3 lm pore size) were coated with CS-1
(10 lg/mL) in 0.1 M of NaHCO3 solution at 4 ꢁC
for 16 h and blocked with 1.5% of heat inactivated
BSA in PBS at room temperature for 0.5 h. Five
hundred microliters of serum free medium and
200 lL of a cell suspension (2 · 105 cells/well) were
added to the lower chamber and upper chamber,
respectively. SDF1-a (Sigma, 15 ng/mL) was added
to the lower chamber and the chambers were incu-
bated in a CO2 incubator at 37 ꢁC for 4.5 h. Mi-
grated cells (both in medium and adherent on the
bottom of the inserts) were collected and counted
using a hemacytometer. Cell dissociation buffer (Invit-
rogen/Gibco, 500 lL/well) was used to detach adher-
ent cells on the bottom of the inserts.
3. Conclusion
We have identified a selective small molecule inhibitor of
a4 integrin-dependent cell migration using a pathway
specific cellular screen. Biochemical experiments suggest
that JK273 binds to c-parvin, a component of focal
adhesions and previously untargeted class or proteins.
JK273 should serve as a useful chemical probe of a4
integrin signaling and may ultimately lead to the synthe-
sis of new therapeutics for the treatment of autoimmune
diseases.
4.4. Cell adhesion assay
Jurkat or U937 cells (2 · 104 cells/well, 1 mL) were pla-
ted into 12-well plates coated with fibronectin (BD Bio-
CoatTM) and treated with phorbol 12-myristate 13-
acetate (PMA, Sigma,100 nM) or SDF1-a (Sigma,
150 ng/mL) in the presence or absence of JK273 (1 or
5 lM) for 4.5 h. The wells were gently washed with
PBS twice and the adherent cells were fixed with formal-
dehyde (4%) in PBS. Three areas/well were selected ran-
domly and the adherent cells were counted under a
microscope.
4. Experimental
4.1. Cell culture
All culture media and supplements were purchased
from Invitrogen/Gibco unless otherwise specified. All
cells were purchased from American Type Culture
Collection (ATCC), and incubated at 37 ꢁC, in a
humidified atmosphere with 5% CO2. Chinese hamster
ovary cells expressing a4 integrin (CHO-a4)13 were
cultured in Dulbecco’s modified Eagle’s medium
(DMEM) supplemented with fetal bovine serum
(FBS, 10%) and non-essential amino acids (Sigma,
1%). Human T-cell leukemia Jurkat (Clone E6-1)
and human histiocytic lymphoma U937 cells were
maintained in RPMI 1640 medium with HEPES
(10 mM), L-glutamine (2 mM), and FBS (10%). Hu-
man embryonic kidney (HEK) 293T cells were cul-
tured in DMEM medium supplemented with L-
glutamine (2 mM) and FBS (10%). Additionally, all
media were supplemented with penicillin (50 U/mL)
and streptomycin (50 lg/mL).
4.5. Production of lentivirus
All recombinant lentiviruses were produced by tran-
sient transfection of 293T cells using Fugene
6
(Roche) according to manufacturer’s protocol. Briefly,
subconfluent 293T cells (30–40%) in 10 cm dish were
co-transfected with 10 lg of transfer plasmid contain-
ing the shRNA, packaging plasmids producing RRE
(6.5 lg) and REV (2.5 lg), and 3.5 lg of envelope vec-
tor producing VSVG. After 24 h, the medium was re-
placed with fresh medium, and 24 h later the
supernatants were harvested, filtered through 0.45 lm
pore-sized membranes.

980
J. Lee et al. / Bioorg. Med. Chem. 17 (2009) 977–980
4.6. RNA interference (RNAi) experiments
Supplementary data
Jurkat cells (2 · 106 cells/dish) were plated into 10 cm
dishes and polybrene (Sigma, 4 lg/mL) was added.
The cells were infected with lentiviruses producing shR-
NAs targeting c-parvin or adenosine kinase, incubated
for 84 h, and harvested for a transwell cell migration as-
say. Transcript levels of c-parvin and adenosine kinase
were measured by TaqMan quantitative RT-PCR.
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.bmc.
2008.02.067.
References and notes
1. Shimizu, Y.; Rose, D. M.; Ginsberg, M. H. Adv. Immunol.
1992, 72, 325.
Sequences of shRNAs targeting c-parvin
2. von Andrian, U. H.; Mackay, C. R. N. Engl. J. Med. 2000,
343, 1020.
3. Holzmann, B.; Gosslar, U.; Bittner, M. Curr. Top.
Microbiol. Immunol. 1998, 231, 125.
4. Jackson, D. Y. Curr. Pharm. Des. 2002, 8, 1229.
5. Podolsky, D. K.; Lobb, R. R.; King, N.; Benjamin, C. D.;
Pepinsky, B.; Sehgal, P.; deBeaumont, M. J. Clin. Invest.
1993, 92, 372.
6. Hesterberg, P. E.; Winsor-Hines, D.; Briskin, M. J.; Soler-
Ferran, D.; Merrill, C.; Mackay, C. R.; Newman, W.;
Ringler, D. J. Gastroenterology 1996, 111, 1373.
7. Fotouhi, N.; Joshi, P.; Fry, D.; Cook, C.; Tilley, J. W.;
Kaplan, G.; Hanglow, A.; Rowan, K.; Schwinge, V.;
Wolitzky, B. Bioorg. Med. Chem. Lett. 2000, 10, 1171.
8. Jackson, D. Y.; Quan, C.; Artis, D. R.; Rawson, T.;
Blackburn, B.; Struble, M.; Fitzgerald, G.; Chan, K.;
Mullins, S.; Burnier, J. P.; Fairbrother, W. J.; Clark, K.;
Bersini, M.; Chui, H.; Renz, M.; Jones, S.; Fong, S.
J. Med. Chem. 1997, 40, 3359.
9. Souers, A. J.; Virgilio, A. A.; Schurer, S. S.; Ellman, J. A.;
¨
Kogan, T. P.; West, H. E.; Ankener, W.; Vanderslice, P.
Bioorg. Med. Chem. Lett. 1998, 8, 2297.
10. Ambroise, Y.; Yaspan, B.; Ginsberg, M. H.; Boger, D. L.
Chem. Biol. 2002, 9, 1219.
shRNA#1: GGACGTCTTTGATGAATTA (sense
sequence)
shRNA#2: GGGCCTGTCTGTGCAGAAT (sense
sequence)
shRNA#3: GCTTCTTCCTGCACTTAAA (sense
sequence)
shRNA#4: GAAGGCTTCTTCCTGCACT (sense
sequence)
Sequences of shRNAs targeting ADK
shRNA#1: GCCACACAAAGCAGCAACA (sense
sequence)
shRNA#2: GGCTTTGAGACTAAAGACA (sense
sequence)
shRNA#3: GGTCCCTCATAGCTAATCT (sense
sequence)
4.7. TaqMan quantitative RT-PCR
Jurkat cells were harvested and total RNA was extracted
using Trizol (Invitrogen) and an RNeasy purification kit
(Qiagen). cDNAs of the total mRNAs were synthesized
using the cDNA Archive kit (Applied Biosystems), and
transcript levels of c-parvin were measured using Taq-
Man Universal PCR Master Mix (Applied Biosystems)
and TaqMan Gene Expression Assays (primers & probe
sets, Applied Biosystems) following the manufacturer’s
protocol. GAPDH was used as an internal control.
11. Collins, C. S.; Hong, J.; Sapinoso, L.; Zhou, Y.; Liu, Z.;
Micklash, K.; Schultz, P. G.; Hampton, G. M. Proc. Natl.
Acad. Sci. U.S.A. 2006, 103, 3775.
12. Yarrow, J. C.; Perlman, Z. E.; Westwood, N. J.; Mitch-
ison, T. J. BMC Biotechnol. 2004, 4, 21.
13. Liu, S.; Thomas, S. M.; Woodside, D. G.; Rose, D. M.;
Kiosses, W. B.; Pfaff, M.; Ginsberg, M. H. Nature 1999,
402, 676.
14. Ding, S.; Gray, N. S.; Wu, X.; Ding, Q.; Schultz, P. G.
J. Am. Chem. Soc. 2002, 124, 1594.
15. Trexler, P.; Bold, G.; Lang, M.; Frei, J. PCT Int. Appl.
WO 9807726.
16. Sandy, P.; Ventura, A.; Jacks, T. Biotechniques 2005, 39,
215.
Acknowledgments
This work was supported by the Novartis Research
Foundation and The Skaggs Institute of Chemical Biol-
ogy (P.G.S.), by the National Institutes of Health
(Grant AR 27214 to M.H.G) and partially by the Post-
doctoral Fellowship of Korea Research Foundation
(J.L., 04-12-07-1). We thank Ms. Myleen Medina
(Genomics Institute of the Novartis Research Founda-
tion) for providing us with both c-parvin and ADK
shRNA clones.
17. Yoshimi, R.; Yamaji, S.; Suzuki, A.; Mishima, W.;
Okamura, M.; Obana, T.; Matsuda, C.; Miwa, Y.; Ohno,
S.; Ishigatsubo, Y. J. Immunol. 2006, 176, 3611.
´
18. Maqueda, A.; Moyano, J. V.; Gutierrez-Lopez, M. D.;
Ovalle, S.; Rodrıeguez-Frade, J. M.; Caban˜as, C.; Garcia-
´
´
Pardo, A. J. Cell. Physiol. 2006, 207, 746.
19. Chu, H.; Thieessen, I.; Sixt, M.; La¨mmermann, T.;
Waisman, A.; Braun, A.; Noegel, A. A.; Fa¨ssler, R.
Mol. Cell. Biol. 2006, 26, 1817.