LCB 03-0110, a novel pan-discoidin domain receptor/c-Src family tyrosine kinase inhibitor, suppresses scar formation by inhibiting fibroblast and macrophage activation

Article abstract of DOI:10.1124/jpet.111.187328

Wound healing generally induces an inflammatory response associated with tissue fibrosis in which activated macrophage and myofibroblast cells are primarily involved. Although this is known to be the underlying mechanism for scarring and various fibrotic pathologies, no effective intervention is currently available. We identified (3-(2-(3-(morpholinomethyl)phenyl)thieno[3,2- b]pyridin- 7-ylamino)phenol (LCB 03-0110), a thienopyridine derivative, as a potent inhibitor of discoidin domain receptor family tyrosine kinases and discovered that this compound strongly inhibits several tyrosine kinases, including the c-Src family, spleen tyrosine kinase, Bruton's tyrosine kinase, and vascular endothelial growth factor receptor 2, which are important for immune cell signaling and inflammatory reactions. LCB 03-0110 suppressed the proliferation and migration of primary dermal fibroblasts induced by transforming growth factor β1 and type I collagen, and this result correlated with the inhibition ability of the compound against enhanced expression of α-smooth muscle actin and activation of Akt1 and focal adhesion kinase. In J774A.1 macrophage cells activated by lipopolysaccharide LCB 03-0110 inhibited cell migration and nitric oxide, inducible nitric-oxide synthase, cyclooxygenase 2, and tumor necrosis factor-α synthesis. LCB 03-0110 applied topically to full excisional wounds on rabbit ears suppressed the accumulation of myofibroblast and macrophage cells in the healing wound and reduced hypertrophic scar formation after wound closing, without delaying the wound closing process. Taken together, the pharmacological activities of LCB 03-0110 suggest that it could be an effective agent for suppressing fibroinflammation by simultaneously targeting activated fibroblasts and macrophages. Copyright

Full text of DOI:10.1124/jpet.111.187328

Supplemental material to this article can be found at:
http://jpet.aspetjournals.org/content/suppl/2011/11/29/jpet.111.187328.DC1.html
1521-0103/12/3403-510–519$25.00
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 2012 by The American Society for Pharmacology and Experimental Therapeutics
JPET 340:510–519, 2012
Vol. 340, No. 3
187328/3747789
LCB 03-0110, a Novel Pan-Discoidin Domain Receptor/c-Src
Family Tyrosine Kinase Inhibitor, Suppresses Scar Formation
□
S
by Inhibiting Fibroblast and Macrophage Activation
Xiaoyan Sun, Trong Nhat Phan, Seung Hee Jung, Sun Young Kim, Jong Un Cho,
Hyangsook Lee, Sung Ho Woo, Tae Kyo Park, and Beom-Seok Yang
Biomedical Research Institute, Korea Institute of Science and Technology, Seoul, Korea (X.S., T.N.P., S.H.J., B.-S.Y.);
University of Science and Technology, Daejeon, Korea (X.S., T.N.P., B.-S.Y.); and LegoChem Biosciences, Inc., Daejeon, Korea
(S.Y.K., J.U.C., H.L., S.H.W., T.K.P.)
Received August 31, 2011; accepted November 28, 2011
ABSTRACT
Wound healing generally induces an inflammatory response as- correlated with the inhibition ability of the compound against en-
sociated with tissue fibrosis in which activated macrophage and hanced expression of ␣-smooth muscle actin and activation of
myofibroblast cells are primarily involved. Although this is known Akt1 and focal adhesion kinase. In J774A.1 macrophage cells
to be the underlying mechanism for scarring and various fibrotic activated by lipopolysaccharide LCB 03-0110 inhibited cell migra-
pathologies, no effective intervention is currently available. We tion and nitric oxide, inducible nitric-oxide synthase, cyclooxygen-
identified (3-(2-(3-(morpholinomethyl)phenyl)thieno[3,2-b]pyridin- ase 2, and tumor necrosis factor-␣ synthesis. LCB 03-0110 ap-
7-ylamino)phenol (LCB 03-0110), a thienopyridine derivative, as a plied topically to full excisional wounds on rabbit ears suppressed
potent inhibitor of discoidin domain receptor family tyrosine ki- the accumulation of myofibroblast and macrophage cells in the
nases and discovered that this compound strongly inhibits several healing wound and reduced hypertrophic scar formation after
tyrosine kinases, including the c-Src family, spleen tyrosine ki- wound closing, without delaying the wound closing process.
nase, Bruton’s tyrosine kinase, and vascular endothelial growth Taken together, the pharmacological activities of LCB 03-0110
factor receptor 2, which are important for immune cell signaling suggest that it could be an effective agent for suppressing fibro-
and inflammatory reactions. LCB 03-0110 suppressed the prolif- inflammation by simultaneously targeting activated fibroblasts and
eration and migration of primary dermal fibroblasts induced by macrophages.
transforming growth factor ␤1 and type I collagen, and this result
organs despite the differences in the type of insult and the
organ involved. The wound healing process starts with in-
Introduction
The biological mechanism of wound healing is remarkably
flammatory responses followed by the generation of new tis-
similar in almost all tissues (Gurtner et al., 2008). For ex-
sue. Inflammatory cells such as macrophages and neutro-
ample, the sequence of events during wound healing after a
phils infiltrate the wound area and become activated because
skin injury caused by cutting or burn are similar to that
they are necessary for the defense against infection by invad-
concerning injuries of the liver, lung, kidney, brain, and other
ing microbes. Furthermore, these inflammatory cells secrete
chemicals and cytokines such as nitric oxide (NO), PDGF,
This study was supported by the Korea Institute of Science and Technology
[Grant 2V01550] and LegoChem Biosciences Inc. [Grant 2I20750].
Article, publication date, and citation information can be found at
http://jpet.aspetjournals.org.
http://dx.doi.org/10.1124/jpet.111.187328.
□S The online version of this article (available at http://jpet.aspetjournals.org)
contains supplemental material.
TGF-␤, and VEGF, which stimulate the growth of granula-
tion tissue and the recruitment and growth of endothelial
cells for angiogenesis. Studies have suggested that the acti-
vation of macrophage cells is responsible for tissue scarring
and fibrosis rather than for the regeneration of damaged
ABBREVIATIONS: NO, nitric oxide; iNOS, inducible nitric-oxide synthase; DDR, discoidin domain receptor; FAK, focal adhesion kinase; LPS,
lipopolysaccharide; TNF, tumor necrosis factor; COX, cyclooxygenase; PDGF, platelet-derived growth factor; TGF, transforming growth factor;
VEGF, vascular endothelial growth factor; Btk, Bruton’s tyrosine kinase; Syk, spleen tyrosine kinase; FLT, fms-like tyrosine kinase; Hck,
hematopoietic cell kinase; Lck, lymphocyte-specific protein tyrosine kinase; ECM, extracellular matrix; DMEM, Dulbecco’s modified Eagle’s
media; HEK, human embryonic kidney; FBS, fetal bovine serum; TBS, Tris-buffered saline; HPF, high-power field; RTK, receptor tyrosine kinase;
JNK, c-Jun NH2-terminal kinase; MAPK, mitogen-activated protein kinase; I␬B, inhibitor of nuclear factor-␬B; GAPDH, glyceraldehyde-3-
phosphate dehydrogenase; Eph, ephrin; LCB 03-0110, (3-(2-(3-(morpholinomethyl)phenyl)thieno[3,2-b]pyridin-7-ylamino)phenol.
510

A DDR/Src Family Kinase Inhibitor Suppressing Scar Formation
511
tissue (Martin et al., 2003; Duffield et al., 2005; Martin and Lck, and Lyn (Robinson et al., 2000). c-Src family proteins
Leibovich, 2005). Macrophage cells produce several cytokines are multifunctional: they are involved in various cellular
during wound healing (Hu¨bner et al., 1996), including PDGF, signaling pathways such as cell proliferation, migration, sur-
which was suggested to promote transformation of fibro- vival, and adhesion to the ECM (Gauld and Cambier, 2004).
blasts into proliferative and more contractile myofibroblast- In addition to their well established roles in tumorigenesis,
type cells (Mori et al., 2008). The transformed myofibroblast they perform a proinflammatory function by stimulating in-
cells secrete excessive extracellular matrix (ECM) proteins, tracellular signal transduction, leading to acute inflamma-
including fibrous collagen, that cause scar formation and tory responses. For example, Hck, Fgr, and Lyn are the
organ fibrosis. This fibrosis-associated inflammation, called predominant c-Src family tyrosine kinases that induce pro-
fibroinflammation, contributes to fibrotic pathologies in var- duction of multiple cytokines and chemokines such as TNF-␣,
ious tissues, for example, liver cirrhosis, kidney and lung interleukin-1, and interleukin-6 in macrophages upon acti-
fibrosis, and atherosclerosis, as well as skin disorders such as vation by lipopolysaccharide (LPS) (Cohen, 2002). Hck and
hypertrophic scars, keloids, and psoriasis (Nickoloff et al., Fgr are also involved in integrin-mediated cell signaling to
2006; Gurtner et al., 2008). Because a synergistic overacti- promote macrophage migration and attachment to the sites
vation of macrophage and fibrotic cells is a hallmark for of inflammation (Suen et al., 1999). Fgr and Lyn are associ-
fibroinflammation, the simultaneous suppression of the acti- ated with integrin-dependent activation and adhesion of neu-
vation of both cell types could be an effective way to inhibit trophils (Berton et al., 1994; Yan et al., 1995), and adhesion-
fibroinflammation.
dependent degranulation of neutrophils requires both Fgr
Discoidin domain receptors (DDRs) are members of the and Hck (Mo´csai et al., 1999). The Tec and Syk tyrosine
receptor tyrosine kinase (RTK) family, with two closely re- kinase family also belong to the non-RTK family of proteins.
lated types, DDR1 and DDR2, that share 89% homology in Syk was reported to play a key role in the downstream
their tyrosine kinase domain (Vogel et al., 2006). They bind signaling of the B-cell receptor in B cells (Hutchcroft et al.,
to native forms of various collagens for their activating li- 1991; Yamada et al., 1993), FCεIR receptor in mast cells, and
gands (Shrivastava et al., 1997; Vogel et al., 1997). DDR1 FC␥RIIA receptor in macrophages, monocytes, and platelets
promotes the accumulation of macrophages in atherosclerotic (Turner et al., 2000). Btk, one of the five Tec family members,
sites (Franco et al., 2009). DDR1 null mice showed a reduced is expressed in B cells, mast cells, and macrophages and is
accumulation of macrophage cells with a decreased inflam- involved in the signaling of these cells by being activated
matory response and a suppression of fibrosis in mouse mod- downstream of the c-Src and Syk families (Conley et al.,
els of atherosclerosis (Franco et al., 2008), renal disorder 2009). For these reasons, c-Src, Btk, and Syk family kinases
(Flamant et al., 2006), and lung fibrosis (Avivi-Green et al., have been suggested as promising targets in the development
2006). Activation of DDR2 in fibroblast cells induced cell of anti-inflammatory agents (Bradshaw, 2010).
proliferation, migration, and remodeling of the ECM with the
In this work, we identified (3-(2-(3-(morpholinomethyl)phe-
induction of matrix metalloproteases (Olaso et al., 2002), and nyl)thieno[3,2-b]pyridin-7-ylamino)phenol (LCB 03-0110), a po-
its increased expression was associated with the activation tent small-molecule inhibitor against the DDR family and c-Src
and proliferation of hepatic stellate cells in liver cirrhosis tyrosine kinase family as well as other tyrosine kinases such as
(Olaso et al., 2001), chondrocytes in osteoarthritis (Xu et al., Btk and Syk. Because DDR and c-Src family tyrosine kinases
2005), synovial fibroblast cells in rheumatoid arthritis (Wang are reported as important mediators for the activation of fibro-
et al., 2002), and vascular smooth muscle cells in atheroscle- blasts and macrophages, respectively, we evaluated LCB 03-
rosis (Ferri et al., 2004). The activity of DDR2 was suggested 0110 as a novel antifibroinflammatory agent both at the cellu-
to be necessary for epithelial-mesenchymal transition of lar level and in an animal model of skin wound healing.
HK-2 renal epithelial cells (Walsh et al., 2011). Chemical
proteomics approaches and an inhibition study identified
imatinib, nilotinib, and dasatinib as potent small-molecule
inhibitors against the discoidin domain receptor tyrosine ki-
Materials and Methods
Materials
N-(3-(4-methoxybenzyloxy)phenyl)-2-(3-(morpholinomethyl)
phenyl)thieno[3,2-b]pyridin-7-amine. A solution consisting of
3-(7-(3-(4-methoxybenzyloxy)phenylamino)thieno[3,2-b]pyridin-2-
yl)benzaldehyde (compound A; Fig. 1) (30 mg, 0.06 mmol) and mor-
pholine (64 ␮l, 0.61 mmol) in dichloromethane (1 ml) was stirred at
room temperature for 20 min. The reaction was treated sequentially
with sodium acetate (16 mg, 0.19 mmol) and NaBH(OAc)3 (82 mg,
0.38 mmol) and stirred at room temperature for an additional 5 h.
The reaction was diluted with dichloromethane (30 ml) and satu-
rated ammonium chloride solution (30 ml). The organic layer was
nase family (Bantscheff et al., 2007; Rix et al., 2007; Day et
al., 2008). These three chemicals were originally developed
for the inhibitors against Bcr-Abl tyrosine kinase, and they
are now being used for patients with chronic myeloid leu-
kemia. Of them, imatinib and nilotinib are more or less
selective by inhibiting a few tyrosine kinases, whereas
dasatinib is known to inhibit more than a dozen tyrosine
kinases.
c-Src family is a non-RTK family that consists of eight
kinases: Fgr, Fyn, Src, Yes, B-cell lymphocyte kinase, Hck, dried over anhydrous sodium sulfate, filtered, and concentrated un-
Fig. 1. Synthesis and chemical structure
of LCB 03-0110. Left, compound A. Cen-
ter, compound B. Right, LCB 03-0110..

512
Sun et al.
der vacuum. The residue was purified by flash column chromatog- diqui et al., 2009). One million J774A.1 cells in 12-well plates were
raphy (dichloromethane/methanol 25:1) to give N-(3-(4-methoxy-
benzyloxy)phenyl)-2-(3-(N-morpholinomethyl)phenyl)thieno[3,
treated with LCB 03-0110 in DMEM containing 1% FBS 30 min
before stimulation with LPS (100 ng/ml). To prepare primary dermal
2-b]pyridin-7-amine (17 mg, 48%) (compound B; Fig. 1). 1H-NMR fibroblasts from 8-week-old BALB/c mice (Taconic Farms, German-
(400 MHz, CDCl3); ␦ 8.20 (d, J ϭ 5.6 Hz, 1H), 7.79 (s, 1H), 7.69 (s, town, NY), a dorsal section was cut into small pieces and washed
1H), 7.57 (s, 1H), 7.37 to 7.28 (m, 4H), 6.94 to 6.83 (m, 6H), 6.19 (s, with sterile phosphate-buffered saline before being treated with 650
1H), 5.01 (s, 2H), 3.80 (s, 3H), 3.74 to 3.73 (m, 3H), 3.52 to 3.50 (m, U/ml collagenase in DMEM for 2 h at 37°C. The undigested tissue
4H), 2.49 to 2.48 (m, 3H); liquid chromatography/mass spectrometry: was removed by filtering, and filtered cells were precipitated by
538 (MHϩ).
centrifugation for 5 min at 1500 rpm and cultured. A half-million
LCB 03-0110. A solution of compound B (15 mg, 0.03 mmol) in dry dermal fibroblasts were cultured to a confluent monolayer overnight
dichloromethane (1 ml) under a nitrogen atmosphere at room tem- in a six-well dish with or without a coating with 200 ␮g/ml rat tail
perature was treated with 4 N HCl in dioxane (1 ml). After stirring type I collagen (Sigma). Then, the medium was switched to DMEM
for 15 h at room temperature, the reaction was concentrated. The containing 0.5% FBS before treatment with LCB 03-0110 in the
residue was redissolved in a mixture of dichloromethane-methanol- presence or absence of 5 ng/ml TGF-␤1 (R&D Systems).
ether. Concentration and drying under vacuum gave (3-(2-(3-(mor-
Western Blot Analysis
pholinomethyl)phenyl)thieno[3,2-b]pyridin-7-ylamino) phenol (14
mg, 99%) as a bright yellow solid. 1H-NMR (600 MHz, acetone-d6); ␦
8.37 (d, J ϭ 6.0 Hz, 1H), 8.08 (brs, 1H), 8.02 (s, 1H), 7.77 to 7.73 (m,
2H), 7.57 t, J ϭ 7.8 Hz, 1H), 7.33 (t, J ϭ 8.4 Hz, 1H), 7.29 to 7.26 (m,
1H), 7.02 to 7.01 (m, 1H), 6.93 to 6.91 (m, 3H), 4.52 (s, 2H), 4.07 to
4.03 (m, 4H), 3.59 to 3.55 (m, 4H); liquid chromatography/mass
spectrometry: 418 (MHϩ). More detailed description for the synthe-
sis of LCB 03-0110 was published previously (Yang et al., 2010).
Antibodies against Akt1, phospho-Akt (pSer473), p38, phospho-
p38 (pThr180/pTyr182), I␬B-␣, phospho-I␬B-␣ (pSer32), stress-acti-
vated protein kinase/JNK, phospho-stress-activated protein kinase/
JNK (pThr183/pTyr185), p44/p42 MAPK, phospho-p44/p42 MAPK
(pThr202/pTyr204), and GAPDH were obtained from Cell Signaling
Technology (Danvers, MA). Antibodies against iNOS, DDR1, and
␤-actin were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
Antibodies of COX-2 and DDR2 were from Millipore Corporation
(Billerica, MA) and R&D Systems (Minneapolis, MN), respectively.
Antibodies against FAK and its phosphor-specific antibodies
(pTyr397, pTyr407, pTyr576, pTyr577, and pTyr861) were from Bio-
source International (Camarillo, CA). Antibodies of ␣-smooth muscle
actin and F4/80 were from Sigma (St. Louis, MO) and Abcam plc
(Cambridge, UK), respectively.
Equal amounts of the total cell lysates were subjected to 7 or 10%
SDS-polyacrylamide gel electrophoresis and blotted to polyvi-
nylidene difluoride membranes (Millipore Corporation). The mem-
brane was blocked with 5% skim milk in TBS buffer before being
incubated with primary antibodies dissolved in 5% skim milk solu-
tion in TBS overnight at 4°C. The resulting membrane was washed
five times with TBS and applied with horseradish peroxidase-conju-
gated secondary antibody. A chemiluminescence detection kit (GE
Healthcare, Chalfont St. Giles, Buckinghamshire, UK) was used for
signal detection.
Measurement of TNF-␣ and NO
The amount of TNF-␣ in the cell culture medium was analyzed
with an enzyme-linked immunosorbent assay kit purchased from
R&D Systems and used according to the manufacturer’s recommen-
dations. Nitrite accumulation, an indicator of NO synthesis, was
measured in the culture medium by Griess reaction (Green et al.,
1982). In brief, 100 ␮l of cell culture medium was mixed with 100 ␮l
of Griess reagent (Sigma) and incubated at room temperature for 15
min. Absorbance at 540 nm was determined, and nitrite concentra-
tion was calculated from a sodium nitrite standard curve.
In Vitro Inhibition Assay against Kinases
Cell Migration and Invasion Assay
In vitro kinase inhibition assay against recombinant activated
DDR2 tyrosine kinase and nonactivated DDR2 kinase was per-
formed as described previously (Yang et al., 2005). In brief, the
inhibition assays were performed by using 20 ␮l of a reaction mix-
ture containing 1 ␮l of each inhibitor in DMSO, 4 ␮g of histone H2B
as a peptide substrate, and 10 to 20 ng of the purified form of either
activated or nonactivated recombinant DDR2 tyrosine kinase do-
main protein in 20 mM Tris-HCl, pH 8.0, 5 mM MgCl2, 0.5 mM
dithiothreitol, 0.01 mM ATP, and 0.1 ␮Ci of [32P]ATP. After 15 min
of incubation at 30°C, the reaction was stopped by adding a half
volume of 30% phosphoric acid. The reaction mixture was spotted on
P81 cellulose paper (Millipore Corporation) and washed five times
using 20 mM Tris-HCl, pH 8.0, containing 1 mM EDTA and 0.1 M
NaCl. The radioactivity in each spot was quantitated with BAS
32P-image analyzer (Fuji Film, Tokyo, Japan). Other kinase inhibi-
tion assays were carried out by the Reaction Biology Corporation
(Malvern, PA) using the HotSpot assay platform at 10 ␮M ATP
concentration.
Migration assay of mouse dermal fibroblast was carried out by
using the wound healing method on the confluent cell monolayer.
Twelve-well plates were coated or not with 200 ␮g/ml type I collagen
(Sigma) and added with 200,000 mouse dermal fibroblast cells. After
overnight incubation, a scratch wound was made on the cell mono-
layer, and the medium was replaced with DMEM containing 0.5%
FBS. Cells were treated with LCB 03-0110 and incubated for an
additional 24 h before being fixed with formalin. J774A.1 macro-
phage invasion assay was conducted by using Transwell cell culture
chambers (24 wells, 8-mm pore size; Corning Life Sciences, Lowell,
MA). In brief, 100,000 J774A.1 cells in 100 ␮l of DMEM containing
1% FBS were plated into the upper chamber, which was precoated
with 50 ␮l of 50 ␮g/ml collagen IV in sterile water (Sigma); then, cells
were treated by LPS (Sigma) with or without LCB 03-0110. One
milliliter of serum-free DMEM containing 20 ng/ml monocyte che-
motactic protein-1 (R&D Systems) was added to the lower chamber.
After 24 h of incubation at 37°C in the cell culture incubator, non-
invaded cells on the upper side of the membrane were removed with
a cotton swab, and invaded cells were fixed and stained with 1%
crystal violet.
Cell Cultures and Treatments
All cells were cultured using DMEM (Invitrogen, Carlsbad, CA)
with 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 U/ml
streptomycin (Invitrogen) at 37°C in a 5% CO2 humidified incubator.
Human embryonic kidney (HEK) 293 and J774A.1 murine macro-
Rabbit Ear Wound Healing Model and Estimation of
Hypertrophic Scar Formation
New Zealand white female rabbits (Taconic Farms) weighing 2.5
phage cell lines were obtained from the American Type Culture to 3.5 kg were anesthetized with ketamine (60 mg/kg) and xylazine (5
Collection (Manassas, VA). HEK293 cells stably expressing either mg/kg), and four full-thickness wounds of 6-mm diameter were made
human DDR1 or DDR2 and the autophosphorylation of the receptors over the ventral surface down to the bare cartilage in each ear by
induced by type I collagen treatment were described previously (Sid- using a 6-mm biopsy punch (Stiefel, Wachtersbach, Germany) and a

A DDR/Src Family Kinase Inhibitor Suppressing Scar Formation
microsurgical technique. One hundred fifty microliters of 0.1% LCB Statistical Analysis
513
03-0110 dissolved in 0.9% saline containing 10% polyethylene glycol
400 and 10% ethanol were applied topically to the wound sites every
3 days, whereas the carrier solution only was used for the nontreated
control group. All wounds were covered by occlusive DuoDERM
polyurethane dressing (ConvaTec, Stillmann, NJ) after treatment.
Twenty-one days after wounding, the animals were sacrificed, and
the scar tissue was excised by using an 8-mm punch biopsy followed
by fixation in 10% formalin to obtain paraffin-embedded tissue.
Slices (6 ␮m) were deparaffinized with xylene and stained with
hematoxylin and eosin. Digital images were taken at 100ϫ ampli-
tude under a light microscope. Hypertrophic scar formation was
evaluated by histomorphometric analysis following a previously de-
scribed procedure (Morris et al., 1997). Each digital image was pro-
cessed with Image-Pro Plus (Adobe Photoshop; Adobe Systems, San
Jose, CA) to measure the total area of the new scar, which included
the original tissue area before wounding and the elevated scar region
over it. The original tissue area before wounding was estimated by
measuring the thickness of adjacent unwounded dermis. The scar
elevation index for each hypertrophic scar was derived by calculating
the ratio of the total area of the new scar to the estimated original
tissue area. The animal experiment was approved by the Institu-
tional Ethics Committee for Animal Care of Korea Institution of
Science and Technology and followed the Guide for the Care and Use
of Laboratory Animals (Institute of Laboratory Animal Resources,
1996) as adopted and promulgated by the National Institutes of
Health.
Data were analyzed and plotted on graphs by using SigmaPlot
software (Systat Software Inc., San Jose, CA). Statistical analysis for
comparisons between two groups was performed by using an un-
paired Student’s t test. Statistical analysis for estimating a signifi-
cant difference in the wound closing rate between wounds of the LCB
03-0110-treated group and nontreated control group was performed
by using repeated-measures analysis of variance. A p value of Ͻ0.05
was considered statistically significant.
Results
LCB 03-0110. We aimed to discover a small-molecule in-
hibitor against DDR tyrosine kinase 2. Using the activated
form of DDR2 cytosolic kinase domain protein, we searched
our in-house compound library for the inhibitor. Optimiza-
tion of a hit compound by medicinal chemistry efforts pro-
duced a novel compound, LCB 03-0110 (Fig. 1). This com-
pound showed a potent inhibition against the activated
tyrosine kinase activity of DDR2 with IC50 values (95% con-
fidence interval) of 6 nM (4–9 nM), whereas it gave IC50
values (95% confidence interval) of 145 nM (128–164 nM)
against the nonactivated form of DDR2 tyrosine kinase, in-
dicating that the compound is more inhibitory against the
activated form of DDR2 tyrosine kinase (Fig. 2A). The inhi-
bition kinetics experiment of LCB 03-0110 against the acti-
vated DDR2 tyrosine kinase indicated that inhibition occurs
in an ATP-competitive manner (Fig. 2B). Using HEK293 cells
engineered to overexpress either DDR1b (HEK293-DDR1b)
or DDR2 (HEK293-DDR2), we analyzed the inhibition of
collagen-induced DDR1b or DDR2 receptor autophosphory-
lation by LCB 03-0110. LCB 03-0110 could suppress the
induced autophosphorylation of DDR1 and DDR2 similarly
with an IC50 of approximately 164 and 171 nM, respectively
(Fig. 2, C and D). These results suggest that LCB 03-0110
inhibits both DDR1 and DDR2 tyrosine kinase activity with
almost the same potency. DDR1 and DDR2 were reported to
share 89% homology in their tyrosine kinase domain (Vogel
Immunohistochemistry
For immunohistological evaluation of dermal tissue, 5-␮m-thick
slices of tissue section were deparaffinized with xylene and rehy-
drated, treated with 3% H2O2 for 5 min, and blocked with 1% normal
sera. The slides were then incubated with antibodies of ␣-smooth
muscle actin (1:400 dilution) or F4/80 (1:100 dilution), followed by
incubation with goat anti-mouse antibody conjugated with horserad-
ish peroxidase (1:200 dilution; GenDEPOT, Baker, TX). The signals
on the tissues were developed with DABϩ Substrate-Chromogen
(Dako, Carpinteria, CA) according to the manufacturer’s instruc-
tions. Thereafter, counterstaining was performed with hematoxylin,
and all sections were photographed under a microscope.
Fig. 2. LCB03-0110 inhibits the tyrosine
kinase activity of DDR1 and DDR2. A,
dose-dependent inhibition against the in
vitro activity of activated (E) and nonac-
tivated DDR2 tyrosine kinase (F) by LCB
03-0110. Data were fitted on a sigmoid
curve by using SigmaPlot software (Sys-
tat Software Inc.). B, reciprocal plotting
of DDR2 kinase activity in the presence of
various concentrations of LCB 03-0110,
using the least-square method. LCB 03-
0110 concentrations: no inhibitor (F), 1.1
nM (E), 3.3 nM (Œ), 10.0 nM (‚), and 30.0
nM (f). C and D, type 1 collagen-induced
autotyrosine phosphorylation of DDR1
(C) and DDR2 (D) expressed in HEK293
cells and their inhibition by LCB 03-0110.
Tyrosine phosphorylation in DDR protein
was detected by Western blotting with
phosphotyrosine antibody and quanti-
tated by densitometry for estimating IC50
values. GAPDH was used as the equal
loading control.

514
Sun et al.
et al., 2006). In a kinase panel inhibition assay against 60 serine 32 at I␬B-␣. However, LCB 03-0110 treatment could
kinases with 10 ␮M concentration of LCB 03-0110, the com- not prevent the decrease of the I␬B-␣ level by LPS at all, but
pound produced more than 90% inhibition against 20 ty- instead even further reduced the I␬B-␣ level at 10 ␮M con-
rosine kinases: all eight Src family kinases, Btk, Syk, Tie2, centration. This was correlated with an observation that LCB
FLT1, FLT3, FLT4, EphA3, EphB4, VEGF receptor 2, mis- 03-0110 did not significantly prevent the LPS induced phos-
shapen/niks-related kinase 1, c-Abl, and RET (rearranged phorylation of I␬B-␣ at serine 32 (Fig. 3B). These data show
during transfection) (see Supplemental Table 1). This sug- that LCB 03-0110 does not modulate the pathways involved
gests that LCB 03-0110 is a pan tyrosine kinase inhibitor. in the phosphorylation of MAPK and I␬B in the activation of
Therefore, we further estimated IC50 values of 21 more ty- macrophages. Taken together, these results suggest that
rosine kinases, and the values are shown with that of acti- LCB 03-0110 could suppress macrophage activation by inhib-
vated form of DDR2 in Table 1. We chose these 21 kinases iting the signaling pathways leading to the activation of p38
with the ones inhibited strongly by LCB 03-0110 as described and JNK. Next, we examined whether LCB 03-0110 could
in Supplemental Table 1 and ones to help us guess its selec- inhibit the enhanced production of inflammatory mediators
tivity. It is noteworthy that it was confirmed that LCB 03- when macrophage cells were activated. The induction of
0110 is able to potently inhibit tyrosine kinases that play iNOS and COX-2 by treating J774A.1 macrophage cells with
important roles in the signaling of various immune cells, for LPS was suppressed considerably by LCB 03-0110 at IC50
instance, c-Src family proteins, Btk, and Syk, in addition to values of approximately 240 and 190 nM, respectively (Fig.
its strong inhibition against the DDR tyrosine kinase family. 3C). In addition, LCB 03-0110 inhibited the induction of NO
Inhibition of Macrophage and Fibroblast Activation and TNF-␣ at IC50 values of approximately 750 nM and 1.99
In Vitro. Because LCB 03-0110 showed potent inhibition ␮M, respectively, in the activated macrophage cells (Fig. 3, D
against the DDR tyrosine kinase family as well as c-Src and E). One of the important characteristics for macrophages
family tyrosine kinases, and these two family kinases are to participate in inflammatory reactions is an enhanced mi-
involved in cellular signaling pathways for the activation of gratory capability. J774A.1 macrophage cells activated by
macrophage and fibroblast cells, we were interested in test- LPS showed an elevated migration through type VI collagen
ing whether this compound could have an antifibroinflam- matrix when the migration was induced by monocyte che-
matory activity by inhibiting the activation of both macro- motactic protein-1. However, treatment with LCB 03-0110
phage and fibroblast cells.
inhibited the migration at an IC50 of approximately 1.05 ␮M
To test whether this chemical could suppress the activation (Fig. 3F). Taken together, it is clear that LCB 03-0110 can
of macrophages, we stimulated J774A.1 macrophage cells inhibit the inflammatory activities of activated macrophage
with LPS in the presence of LCB 03-0110 for 1 h and exam- cells.
ined alterations in the activation of cellular molecules in-
Next, we tested whether LCB 03-0110 could also suppress
volved in the activation of macrophage cells. The activation of the activation of fibroblasts. When fibroblast cells become
macrophage cells by LPS induced the activating phosphory- activated into myofibroblast-type cells during wound healing,
lation of p38 and JNK. However, LCB 03-0110 potently sup- they show an enhanced expression of ␣-smooth muscle actin
pressed the induction of these proteins at IC50 values of and an increased capacity for migration. To activate primary
approximately 140 and 160 nM for p38 and JNK, respec- dermal fibroblasts in an in vitro cell culture, we treated the
tively, although it did not alter the cellular amount of these cells with TGF-␤1 in culture plates with or without type I
two proteins (Fig. 3A). These results suggest that LCB 03- collagen coating. TGF-␤1 treatment considerably induced the
0110 inhibits the pathways for the activation of p38 and expression of ␣-smooth muscle actin, whereas plating the
JNK. In contrast, LCB 03-0110 had an almost negligible cells on the collagen-coated dish only slightly increased
effect on the activation of MAPK even at 10 ␮M concentration the expression. The highest induction of ␣-smooth muscle
(Fig. 3B). On the other hand, the activation by LPS reduced actin was observed to be approximately 16-fold when TGF-␤1
the amount of cellular I␬B-␣ protein level, and this reduction was applied to cells in the collagen-coated plate. However,
was associated with the increase of the phosphorylation at this induction was abolished by LCB 03-0110 at an IC50 of
approximately 170 nM (Fig. 4A). In addition, we examined
TABLE 1
the activation of signaling mediators such as FAK and Akt1.
TGF-␤1 significantly induced the activating phosphorylation
of FAK at tyrosines 397, 407, 577, and 861, but relatively
IC50 values of LCB 03-0110 for the inhibition of protein tyrosine
kinases
Kinase
IC50
Kinase
IC50
slightly at tyrosine 576. On the other hand, plating cells on
the collagen-coated surface slightly increased the phosphor-
ylation at all five tyrosine residues. It is noteworthy that
treatment with TGF-␤1 of dermal fibroblast cells plated on
the collagen-coated plate showed a markedly enhanced phos-
phorylation at two tyrosines (residues 576 and 861) among
the five phosphorylated tyrosines, suggesting that TGF-␤1
and collagen signaling synergistically induce the phosphory-
lation of these two residues. However, it is noteworthy that
LCB 03-0110 treatment of cells exposed to TGF-␤1 and type
I collagen showed a marked inhibition preferentially in the
phosphorylation of tyrosine 861, to give approximately 50%
reduction at 1 ␮M and an almost complete abolishment at 3
␮M, whereas more or less suppression of phosphorylation
nM
nM
DDR2
c-Src
Lck
Lyn
Fyn
Yes
6.0
1.3
Syk
25.2
1732
4.6
5.1
26.1
273
ZAP70
VEGFR2
EphA3
FLT3
21.6
4.3
2.3
2.1
EGFR
IR
Fgr
3.7
10,750
69
Hck
Blk
Btk
Itk
4.4
PDGFR␣
PDGFR␤
c-Kit
50.7
17.7
Ͼ10,000
409
591.7
FGFR1
827.8
Blk, B-cell lymphocyte kinase; Itk, IL2-inducible T-cell kinase; ZAP70, ␨-chain-
associated protein kinase 70; VEGFR2, VEGF receptor 2; EGFR, endothelial growth
factor receptor; IR, insulin receptor; PDGFR, PDGFR receptor; FGFR, fibroblast
growth factor receptor.

A DDR/Src Family Kinase Inhibitor Suppressing Scar Formation
515
Fig. 3. LCB 03-0110 suppresses the activation of J774A.1 macrophage cells. A to C, after treating J774A.1 cells with LPS and LCB 03-0110 for 1 h
(A and B) or 18 h (C), Western blot analysis was performed by using the total cell lysate. The degree of the induced phosphorylation of each protein
and its inhibition by LCB 03-0110 was quantitated by densitometry for estimating IC50 values. ␤-Actin was used as the equal loading control. ERK,
extracellular signal-regulated kinase. D and E, nitrite accumulation assay (D) and enzyme-linked immunosorbent assay for TNF-␣ expression (E) were
performed by using culture media of cells treated with LPS and LCB 03-0110 for 18 h, and the average values with S.D. from three independent
experiments are shown as a bar graph. F, J774A.1 cells treated (ϩ) or not (Ϫ) with LPS were allowed to invade through type IV collagen gel in the
presence of LCB 03-0110. A total of 10 digital images of high-power fields (HPFs) at 400ϫ magnification for invaded cells were obtained at each
condition from three independent experiments. The average values with S.D. of invaded cells/HPF are shown as a bar graph.
was observed in the other four tyrosines (Fig. 4B). This result LCB 03-0110. In this study, we found that the activating
suggests that LCB 03-0110 can inhibit FAK activation in the phosphorylation of Akt1 at threonine 371 was not induced by
activated fibroblasts mainly by inhibiting cellular signaling TGF-␤1 treatment and was even suppressed slightly by the
to stimulate the phosphorylation of its tyrosine 861. We also presence of type I collagen. However, it is noteworthy that
studied the activation of Akt1 in primary dermal fibroblasts the simultaneous stimulation of cells with TGF-␤1 and type
treated with TGF-␤1 and type I collagen and its inhibition by I collagen considerably enhanced its phosphorylation and

516
Sun et al.
LCB 03-0110 treatment abolished this enhancement almost
completely at 3 ␮M (Fig. 4B). Taken together, these results
show that LCB 03-0110 can suppress the activation of
␣-smooth muscle actin, FAK, and Akt1 in fibroblast cells.
Because these three proteins are involved in the activation of
fibroblast cells, these results suggest that LCB 03-0110 could
suppress fibroblast activation. To support this conclusion, we
tested whether LCB 03-0110 could suppress the ability of
fibroblast cells to acquire an increased capacity for migra-
tion. We observed that dermal fibroblast cells exhibited a
significantly increased migration when plated on the type I
collagen-coated surface compared with the noncoated surface
of the culture dish. However, LCB 03-0110 treatment inhib-
ited the collagen-induced migration of dermal fibroblast cells
at IC50 of 194 nM (Fig. 4C).
Suppression of Macrophage and Fibroblast Activa-
tion in a Rabbit Ear Wound Healing Model. Upon ob-
serving that LCB 03-0110 could suppress the activation of
both fibroblasts and macrophages in the in vitro cell culture
experiment, we wanted to test whether this compound could
also inhibit the activation of the two cell types in an animal
model of wound healing. We generated full excisional wounds
on rabbit ears and applied the compound topically on the
wound sites. After 2 days, we performed an immunohisto-
chemical examination of the population of activated myofi-
broblast cells and the accumulation of macrophage cells in
the area of regenerating tissue in the wound site. A 0.1%
solution of LCB 03-0110 treatment significantly reduced the
number of ␣-smooth muscle actin-positive myofibroblast cells
by 57.7% and the F4/80 positive macrophage cells by 37.7%,
indicating that LCB 03-0110 suppressed the accumulation
of macrophage and myofibroblast cells in the wound site by
inhibiting their activation (Fig. 5). This result suggests
that LCB 03-0110 can suppress the activation of fibro-
blasts and macrophages in an animal model of wound
healing, which is consistent with the results from the cell
culture experiment.
Suppression of Hypertrophic Scar Formation in the
Rabbit Ear Wound Healing Model. Fibroinflammation
during skin wound healing results in a hypertrophic scar
after wound closing. The elevation of the scar over the orig-
inal skin tissue reflects the intensity of scar formation. Using
a full excisional wound healing model on rabbit ears, we
attempted to estimate how much potently LCB 03-0110 could
reduce scar formation by measuring the scar elevation index
of the scar formed after wound closing. When we applied
0.1% solution of LCB 03-0110 topically into the wound site
every 3 days after the generation of wound, this compound
significantly prevented hypertrophic scar formation by re-
ducing the scar elevation index by 38.5% compared with that
in the control group treated with only the carrier solution
(Fig. 6, top). Considering that scar formation resulted from
fibroinflammation by activated macrophage and myofibro-
blast cells, this result is consistent with the conclusion that
LCB 03-0110 exhibits antifibroinflammatory activity by sup-
pressing the activation of fibroblasts and macrophages. In
this wound healing experiment, we observed that treatment
with LCB 03-0110 did not delay wound closing but seems to
have stimulated it, as shown by the wound closing rate ob-
tained by monitoring the reduction of the unclosed wound
area (Fig. 6, bottom). This suggests that LCB 03-0110 does
not interfere with the regeneration of the epidermal layer,
Fig. 4. LCB 03-0110 suppresses the activation of dermal fibroblast cells.
A and B, primary dermal fibroblast cells plated on the control 12-well
plates (Ϫ) or on plates coated with type I collagen (ϩ), using DMEM
containing 0.5% FBS, were treated with 5 ng/ml TGF-␤ and LCB 03-0110
for 48 h. The total cell lysate was subjected to Western blot analysis.
␤-Actin was used as the equal loading control. C, the migratory ability of
the dermal fibroblast cells plated either on type I collagen-coated (ϩ) or
noncoated (Ϫ) wells was estimated by incubating the cells for 24 h in the
presence of LCB 03-0110 after generating a scratch wound in the conflu-
ent monolayer culture. A total of six digital images of HPFs at 400ϫ
magnification for cells that migrated into the scratched area were ob-
tained at each condition from three independent assays. Each bar repre-
sents the mean with S.D. of the counted cells per HPF.

A DDR/Src Family Kinase Inhibitor Suppressing Scar Formation
517
Fig. 5. LCB 03-0110 inhibits the accumu-
lation of macrophage and myofibroblast
cells in the wound site. Either 0.1% LCB
03-0110 or the carrier solution as a control
was applied topically to full excisional
wounds generated on the ears of New Zea-
land white rabbits with occlusive dressing.
After 2 days, a 5-␮m slice of the wound
tissue section was immunostained. A, left, a
representative image of immunostaining of
macrophage cells in the regenerating deep
dermis with the F4/80 antibody. Right, the
bar graph shows the mean (S.D.) of the
number of positively stained cells per HPF
at 400ϫ magnification from 12 digital im-
ages in the n ϭ 4 experiment. LCB 03-0110
significantly reduced the number of macro-
phage cells (p Ͻ 0.01). B, left, a representa-
tive image of immunostained myofibroblast
cells in the regenerating deep dermis with
antibody against ␣-smooth muscle actin.
Right, the bar graph represents the mean
with S.D. of the number of positively
stained cells/HPF at 400ϫ magnification
from 12 digital images in the n ϭ 4 experi-
ment. LCB 03-0110 significantly reduced
the number of myofibroblast cells (p Ͻ
0.01).
which is responsible for wound closing, whereas it acts on formation and activations of macrophage and fibroblast
fibroblast and macrophage cells with suppressing their
activation.
would be based on its nature as a multikinase inhibitor.
Among these kinases, we propose that its inhibitory activity
against DDR family and c-Src family tyrosine kinases might
have a major role in the suppression of the activation of both
cell types. This argument can be supported by the fact that
DDR family proteins have been reported to be involved in the
activation of fibroblasts (Olaso et al., 2002) and macrophages
(Franco et al., 2009), and c-Src family tyrosine kinases such
as Hck, Fgr, and Lyn were shown to be involved in cell
signaling to activate macrophages (Cohen, 2002).
Although LCB 03-0110 is a pan kinase inhibitor, it seems
to work with a certain degree of specificity in modulating
cellular signaling in activated macrophage and fibroblast
cells. LCB 03-0110 inhibited p38 and JNK activation,
whereas it has no influence on the pathways that induce
MAPK and I␬b phosphorylation although all four pathways
are associated with the activation of macrophage by LPS
(Guha and Mackman, 2001). This might be a reason we
needed higher concentrations of LCB 03-0110 to inhibit the
production of NO and TNF-␣ and cell migration than those
required to suppress the activated p38 and JNK in J774A.1
macrophage cells stimulated by LPS. Furthermore, among
the pathways that lead to an activating phosphorylation at
tyrosine residues within the FAK protein in fibroblast cells,
LCB 03-0110 interferes preferentially with the pathway that
induces phosphorylation at tyrosine 861. The phosphoryla-
tion of tyrosine 861 of FAK was suggested to be involved in
Discussion
Whenever inner and outer bodily organs acquire injury
caused by various damaging stimuli, a preprogrammed
wound healing mechanism is immediately turned on as a
natural defense mechanism of the body. However, an inflam-
matory response during the process activates an undesirable
fibrotic pathology that results in scar formation and organ
fibrosis. Although it is suggested that activated macrophage
and myofibroblast cells are primarily involved in fibrosis-
associated inflammation during the wound healing process
(Martin et al., 2003; Gurtner et al., 2008; Wynn, 2008), to
date there have been few attempts to simultaneously sup-
press the activation of macrophage and fibroblast cells to
prevent scar formation and fibrosis. In this work, we demon-
strated that LCB 03-0110 can reduce fibroinflammation and
scar formation in a skin wound healing model, and this
function is tightly associated with the ability of the com-
pound to simultaneously suppress the activation of both mac-
rophage and fibroblast cells. This suggests that inhibiting the
activation of both macrophages and fibroblasts by a small
molecule, as we demonstrated here, could be an effective
strategy in developing an antifibroinflammatory agent.
LCB 03-0110 was identified as a pan kinase inhibitor be-
cause it inhibits various tyrosine kinases as shown in Table
1. We think that the activity of LCB 03-0110 to inhibit scar stimulating cell migration (Lim et al., 2004), and this pro-

518
Sun et al.
published yet to confirm this conclusion (Day et al., 2008).
However, when we applied dasatinib to our rabbit ear wound
healing model experiment, it severely interfered with the
wound closing process in contrast to LCB 03-0110, which
stimulated wound healing (X. Sun, unpublished data). We
think that a certain difference in the kinase inhibition profile
between LCB 03-0110 and dasatinib might be responsible for
this different activity concerning wound healing, despite hav-
ing a largely similar kinase inhibition profile, including their
potent inhibition activity against DDR and c-Src family ty-
rosine kinases.
Authorship Contributions
Participated in research design: Sun, Phan, Jung, Kim, Cho, Lee,
Woo, Park, and Yang.
Conducted experiments: Sun, Phan, Jung, Kim, Cho, Lee, and
Woo.
Performed data analysis: Sun, Phan, Jung, Kim, Cho, Lee, Woo,
Park, and Yang.
Wrote or contributed to the writing of the manuscript Sun, Phan,
Kim, Park, and Yang.
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Address correspondence to: Dr. Beom-Seok Yang, Biomedical Research
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Sungbuk-Ku, Seoul 136-791, Korea. E-mail: bsyang@kist.re.kr