Potential therapeutic application of small molecule with sulfonamide for chondrogenic differentiation and articular cartilage repair

Article abstract of DOI:10.1016/j.bmcl.2016.08.069

The restoration of damaged articular cartilage is a long-pursued goal in regenerative medicine. Chondrocyte-specific differentiation of mesenchymal stem cells (MSCs) may be an effective means of repairing damaged cartilage. We identified small molecule 6

Full text of DOI:10.1016/j.bmcl.2016.08.069

Accepted Manuscript
Potential therapeutic application of small molecule with sulfonamide for chon-
drogenic differentiation and articular cartilage repair
Eunhyun Choi, Jiyun Lee, Seahyoung Lee, Byeong-Wook Song, Hyang-Hee
Seo, Min-Ji Cha, Soyeon Lim, Chulho Lee, Suk-Won Song, Gyoonhee Han, Ki-
Chul Hwang
PII:
S0960-894X(16)30902-7
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.08.069
BMCL 24195
Reference:
Bioorganic & Medicinal Chemistry Letters
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Revised Date:
Accepted Date:
27 June 2016
18 August 2016
20 August 2016
Please cite this article as: Choi, E., Lee, J., Lee, S., Song, B-W., Seo, H-H., Cha, M-J., Lim, S., Lee, C., Song, S-
W., Han, G., Hwang, K-C., Potential therapeutic application of small molecule with sulfonamide for chondrogenic
differentiation and articular cartilage repair, Bioorganic & Medicinal Chemistry Letters (2016), doi: http://
dx.doi.org/10.1016/j.bmcl.2016.08.069
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Bioorganic & Medicinal Chemistry Letters
Potential therapeutic application of small molecule with sulfonamide for
chondrogenic differentiation and articular cartilage repair
Eunhyun Choia,b,†, Jiyun Leec,†, Seahyoung Leea,b,†, Byeong-Wook Songa,d, Hyang-Hee Seoc, Min-Ji Chaa,d,
Soyeon Lima,b, Chulho Leee,f, Suk-Won Songg, Gyoonhee Hane,f,, and Ki-Chul Hwanga,b,
a Catholic Kwandong University International St. Mary’s Hospital, Seo-go, Incheon Metropolitan City 22711, Republic of Korea
b Institute for Bio-Medical Convergence, College of Medicine, Catholic Kwandong University, Gangneung-si, Gangwon-do 25601, Republic of Korea
c Brain Korea 21 PLUS Project for Medical Science, Yonsei University, Seodamun-gu, Seoul 03722, Republic of Korea
d Institute for Integrative Medicine, College of Medicine, Catholic Kwandong University, Gangneung, Gangwon-do 25601, Republic of Korea
e Department of Integrated OMICS for Biomedical Sciences (WCU Program), Yonsei University, Seodamun-gu, Seoul 03719, Republic of Korea
f Translational Research Center for Protein Function Control, Department of Biotechnology, Yonsei University, Seodamun-gu, Seoul 03719, Republic of Korea
g Department of Cardiovascular Surgery, Gangnam Severance Hospital, Yonsei University College of Medicine, Gangnam-gu, Seoul 06273,, Republic of Korea
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
The restoration of damaged articular cartilage is a long-pursued goal in regenerative medicine.
Chondrocyte-specific differentiation of mesenchymal stem cells (MSCs) may be an effective
means of repairing damaged cartilage. We identified small molecule 6 with sulfonamide as an
agent that promotes specific chondrogenic differentiation of human adipose-derived MSCs
(hASCs). Unlike other chondrogenic differentiation media composed of various defined
components, simply adding compound 6 into culture medium was sufficient to induce
chondrogenesis in this study. In an animal osteoarthritis model, both the small molecule 6 and
the 6-treated hASCs exhibited enhanced recovery of injured articular cartilage. This work
provides new insight into MSC differentiation induced by small molecules and potential new
therapeutic approaches for articular cartilage injury.
Keywords:
Chondrogenesis
Mesenchymal stem cells
Osteoarthritis
Sulfonamide
Regenerative medicine
2009 Elsevier Ltd. All rights reserved.
Chondrogenesis is a primary process in skeletal development
and is regulated by numerous growth factors and a network of
transcription factors.1,2 Chondrocytes are a major element of
articular cartilage, a simple and dense connective tissue that has
the potential to maintain the cushion between bones and joints.3
Articular cartilage injuries cause severe pain, the loss of
chondrocytes and their function, and an irreversible debilitation.
However, damaged articular cartilage is incapable of self-
recovery.4 As temporary expedients, exercise, non-steroidal anti-
inflammatory drugs (NSAIDs), intra-articular hyaluronic acid
injection, and replacement with artificial joints are used for
treatment and pain relief.5 For more effective and successful
treatment, the fields of regenerative medicine and tissue
engineering have utilized chondrocytes and/or mesenchymal
stem cells (MSCs).6 To date, strategies using druggable small
molecules in adult stem cell therapies have focused on
identifying molecules for more specific and selective control of
handful of studies in which small molecules induced potent and
specific chondrogenic differentiation of MSCs have been
published.8-10 Therefore, the identification of a small molecule
that promotes chondrogenesis is important for the development
of a novel therapeutic agent to treat damaged articular cartilage.
Herein, we report the identification and characterization of a
small molecule containing sulfonamide that promotes direct
human adipose-derived MSC (hASC) differentiation into
chondrocytes.
In our previous study, we demonstrated that protein kinases
play crucial roles in modulating stem cell fate. In that particular
study, we screened our house protein kinase inhibitor library
composed of six major subfamilies, and the results indicated that
a protein kinase A (PKA) inhibitor H-89 (N-[2-(p-bromo-
cinnamylamino) ethyl]-5-isoquinolinesulfonamide • 2HCl, Fig.
1) had the potential to induce chondrogenic differentiation in
MSCs.11 In the present study, we further validated that a chemical
compound can be used to induce chondrogenesis in MSCs.
MSC proliferation and differentiation.7 Nevertheless, only a
———
 Corresponding author. Tel.: +82-2-2123-2882; fax: +82-2-362-7265; e-mail: gyoonhee@yonsei.ac.kr (G. Han)
 Corresponding author. Tel.: +82-32-290-3883; fax: +82-32-290-2774; e-mail: kchwang@cku.ac.kr (K.C. Hwang)
† These authors contributed equally to this manuscript and are co-first authors.

that chondrogenic activity was more influenced by R1
substituents than R2 substituents. Although the number of
analogues exhibited good aggrecan-inducing ability, we focused
on compound 6 (Fig. 1) because it exhibited the highest aggrecan
expression at 1 μM and was an appropriate chondrogenic inducer
of hASCs based on SAR analysis.
Figure 1. Structure of H-89 and compound 6.
Lineage-specific differentiation of hASCs in the presence of
compound 6 was further confirmed by alcian blue staining. The
intensity of alcian blue staining increased approximately 3-fold in
the hASCs treated with compound 6 for 11 days (Fig. 2b).
Additionally, aggrecan expression was dose-dependently
In an effort to identify a novel synthetic small molecule with
chondrogenic potential, we designed and synthesized a series of
sulfonamide analogues based on the structure of H-89. The
synthetic procedure of sulfonamide analogues involved a simple
and one-step reaction, the coupling of a sulfonyl chloride with a
primary or secondary amine,12-14 and these analogues have two
substituents, R1 and R2. The R1 substituents consisted of
quinoline, naphthyl, biphenyl, 4-tert-butyl, or 4-methylbenzene,
and the R2 substituents were composed of benzene, naphthyl, or
cyclohexyl with substituents (Scheme 1 and all synthetic
products can be found in Supplementary data). Generally,
sulfonamide compounds are well kwon for antibiotics,15,16 and
then among our synthesized compounds, some compounds
reported that they have potential medicinal applications including
antibacterial,17 antiviral,18 and antitumor activity.19,20 However,
chondrogenic properties of sulfonamides remain to be uncovered.
increased with compound
6 treatment (Fig. 2c). During
chondrogenic differentiation by compound 6, we observed a
time-dependent increase of ERK phosphorylation (Fig. 2d). The
inhibition of the ERK signaling pathway by a selective MEK
inhibitor U0126 suppressed aggrecan expression even in the
presence of compound 6 (Fig. 2c, d).
Figure 2. Compound 6 induces chondrocyte differentiation in hASCs. a) The
measurement of aggrecan contents as a chondrogenic marker at day 11; b)
Compound 6 upregulates aggrecan expression; c) Compound 6 upregulates
aggrecan expression in a dose-dependent manner, but ERK inhibitor (U0126)
inhibits aggrecan expression; d) The chondrogenic effect of compound 6 is
mediated by the ERK pathway.
Scheme 1. General synthesis of sulfonamide derivatives.
To examine the chondrogenic activity of the synthesized
sulfonamide analogues, hASCs were cultured in a complete
growth medium with 1 μM or 10 μM of sulfonamide analogues
for 11 days. The expression of aggrecan, a chondrogenesis
marker, was determined by sandwich enzyme-linked
immunosorbent assay (ELISA) and was used to evaluate the
chondrogenic potential of analogues. The aggrecan expression
analyses indicated that six analogues (6-11) increased aggrecan
expression approximately 2-fold compared with the control at
both concentrations used (1 μM and 10 μM). Other analogues
failed to induce the expression of aggrecan at the lower
concentration used (Fig. 2a).
To evaluate the effects of compound 6-treated hASCs and
compound 6 alone in vivo, we used the collagenase type II-
induced chronic joint injury (osteoarthritis; OA) model.21,22 Knee
joints from each group (intra-articular injection of 1) hASCs, 2)
hASCs treated with compound 6 or 3) compound 6 in 50 µl of
PBS on days 7 and 21) were harvested and prepared for
sectioning and immunohistochemical analysis 6 weeks later.
The sections were strained with safranin O/Fast Green for
detecting sulfated glycosaminoglycans (GAGs). The thickness of
articular cartilage significantly decreased in the OA group
compared with the normal group (Fig. 3a, b). However, thickness
recovered in the group injected with compound 6-treated hASCs
as well as in the group injected with compound 6 alone (Fig. 3d,
e). In addition, the proteoglycan contents were increased in the
compound 6-treated hASCs-injected group compared with the
OA and hASCs-injected group (Fig. 3b-d). The compound 6
alone-injected group exhibited similar recovery effects to that of
the compound 6-treated hASCs-injected group (Fig. 3e). The
results of safranin O/Fast Green staining are also correlated with
aggrecan immunohistochemistry. Compared with normal
Using sandwich ELISA, a structure-activity relationship
(SAR) analysis was conducted. Quinoline was the most active,
whereas 4-methylbenzen was less active than other substituents
in R1 (5 vs. 7). Neither having substituent (2-hydroxyethyl)
cyclohexyl in R2 position (23-25), having a long carbon chain
between sulfonamide and R2 (27-29), nor introducing N-ethyl in
sulfonamide (31 and 32) affected aggrecan expression at 1 μM
and 10 μM. In the case of naphthalenol in R2, the position of
hydroxyl influenced the activity; 4-hydroxy (11) > 5-hydroxy (4)
> 7-hydroxy (5) and hydroxy- and chloro- (15), fluoro- (18) or
nitro- (22) substituted benzene were more active than only
hydroxy-substituted benzene (3) in R2 at 10 μM. This suggested

articular cartilage, the OA model exhibited low aggrecan
expression levels (Fig. 4a, b), whereas the hASC injection group
exhibited slightly increased levels of aggrecan (Fig. 4c) and
treatment with compound 6-induced chondrogenic-differentiated
hASCs dramatically increased aggrecan expression (Fig. 4d)
compared with the OA group. Notably, the compound 6 injection
group also exhibited higher expression levels of aggrecan (Fig.
4e) relative to the OA group. These results implied that
compound 6 induces naïve chondrocyte properties in hASCs and
raises the possibility that both compound 6-treated hASCs and
compound 6 itself impart protective effects against the damaged
articular cartilage.
in vivo chondrogenic capacity and protective efficacy of cartilage
damage by compound 6, we suggest that compound 6 is a new
potential disease-modifying OA drug candidate.31 Moreover, in
this study, we provide new insights and strategies in regenerative
medicine and novel therapeutic methods for the treatment of
degenerative diseases, including cartilage defects.
Figure 3. Chondrogenically differentiated-hASCs by compound 6.
Compound 6 promotes cartilage repair in collagenase II-induced osteoarthritis
models in rats. a) Naïve rat knee joints; b) 6 weeks after intra-articular
injection of collagenase II on day 1 and day 4; c) 4 x 105 hASCs; d) 4 x 105
cells from chondrogenically differentiated hASCs; e) 10 µM compound 6 on
days 7 and 21. Scale bars=250 µm.
Stem cell-based approaches for the treatment of OA have
focused on cellular regenerative properties, including the
proliferation and differentiation of chondrocytes. However, these
studies exhibit some limitations, such as poor cell survival and
regeneration potential.23 Furthermore, the major chondrogenic
agent TGF-β can cause nonspecific differentiation and undesired
phenotype changes, such as hypertrophic maturation and
osteophyte formation, which leads to the defunctionalization of
joints and is unsuitable for cartilage regeneration.24-26 To
overcome these limitations, in the current study, we focused on
compound 6 with a sulfonamide moiety as a therapeutic reagent
that has chondrogenic activity and confirmed that it restores
articular cartilage in an OA rat model. Although many signaling
pathways are related to chondrogenesis27 and there is controversy
regarding whether the MEK/ERK signaling cascade is necessary
for chondrogenic differentiation,28 we confirmed that the
chondrogenic differentiation mechanism of compound 6 is
associated with an increase in the ERK phosphorylation state.
Certainly, undifferentiated hASCs can partially repair cartilage
degradation; however, we observed that chondrogenically
differentiated hASCs by compound 6 could be more easily
appropriated for cartilage regeneration. Perhaps the
chondrogenically differentiated states of hASCs have
characteristics that are more similar to those of naïve
chondrocytes, such as active cellular signaling molecules and
biological properties.29,30 Future studies are still necessary to
elucidate the in vivo mechanism of action of compound 6-
induced chondrogenically differentiated hASCs on impeding
cartilage degradation. However, based on the overall in vitro and
Figure 4. Articular cartilage of knees treated with chondrogenically
differentiated hASCs by compound 6 or compound 6 alone exhibited
increased expression of aggrecan relative to the OA group. a) Naïve rat knee
joints; b) 6 weeks after intra-articular injection of collagenase II on day 1 and
day 4; c) 4 x 105 cells of hASCs; d) 4 x 105 cells of chondrogenically
differentiated hASCs with compound 6; e) 10 μM compound 6 on days 7 and
21. Aggrecan: green; Cell nuclei: DAPI, blue. Scale bar=200 μm.
Acknowledgments
This research was supported by a Korea Science and
Engineering Foundation grant funded by the Korean government
(MEST)
(NRF-2015M3A9E6029519,
NRF-
2015M3A9E6029407, and NRF- 2015M3A9E6029598), research
fund of Catholic Kwandong University International St. Mary's
Hospital (CKURF-201407080001),
a
grant from the
Translational Research Center for Protein Function Control
(NRF-2009-0083522), the Ministry of Science, ICT and Future
Planning (NRF-2013M3A6A4072536), the Basic Science

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Potential therapeutic application of small
molecule with sulfonamide for chondrogenic
differentiation and articular cartilage repair
Leave this area blank for abstract info.
Eunhyun Choi, Jiyun Lee, Seahyoung Lee, Byeong-Wook Song, Hyang-Hee Seo, Min-Ji Cha, Soyeon Lim,
Chulho Lee, Suk-Won Song, Gyoonhee Han, and Ki-Chul Hwang