Page 77 - I. Chemical biology and drug discovery

JS-003 as a novel therapeutic natural compound for type 2
diabetes mellitus

1
J.S KIM , Sang J. Chung 1
1. Sungkyunkwan University, College of pharmacy, SUWON, KOREA

BACKGROUND AIM
In recent society, there are many metabolic diseases including PTP1B, PTP-MEG2, PTP1D, PTPRS and PTPRF are
diabetes which is representative of chronical diseases consist of acknowledged for phosphatase by which have negative effects
two types: Type 1 diabetes and Type 2 diabetes. in terms of Type on type 2 diabetes. JS-003 is sorted as a strong inhibitor of
2 diabetes, It stems from the disability of glucose-uptake in cells these phosphatases according to our lab database containing
related to insulin receptors as well as glucose transporter. Cell inhibition ability of 658 natural compounds in human-derived
signaling induced by phosphorylation and de-phosphorylation phosphatases. We conducted on research to determine
have a strong effect on expressing and translocation of these inhibition percentage which represents inhibition ability of JS-
receptor and transporters. In this aspect, phosphatases play a 003 in these phosphatases followed by experiments to probe
significant role in type 2 diabetes. JS-003 has a strong inhibition the efficacy of JS-003 for improving glucose-uptake ability in
effect on several phosphatases is known for deteriorating mammalian cells.
diabetes.
METHODS
Enzyme Kinetics 6,8-difluoro-4-methylumbelliferyl phosphate(DiFMUP) is used as substrate of purified phosphatase: PTP1B, PTP-MEG2, PTP1D, PTPRS, PTPRF. Phosphatases is diluted in
reaction buffer which is consist of 20 mM Bis-tris pH 6.0(PTP1B, PTP1D) or pH 7.0(PTP-MEG2, PTPRS, PTPRF), 150 mM NaCl, 2.5 mM dithiothreitol (DTT), 0.01% Tripton X-100 and Various
concentration of DiFMUP from 6.25 μM to 800 μM in a 96 well-plate. Victor X4 Multi label plate reader is operated to quantify fluorescence intensities for 10 min (Excitation/Emission = 355/460).
Phosphatases is added in reaction buffer with JS-003 to estimate inhibition ability of compound in these enzymes.
Glucose Uptake Assay C2C12 muscle cells were culture in DMEM high glucose with 20% FBS and 1% of 100x Penicillin/Streptomycin Solution. After 100% confluency, media was change to
DMEM high glucose containing 10% Horse Serum, 1 μM insulin and 1% antibiotics solutions around 3-4 days to completing differentiation of cells. Differentiated cells were cultured in DMEM low
glucose for 12 hours and media subsequently changed to DMEM Glucose-Free media with JS-003. For glucose uptake assay, the fluorescent agent, 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-
yl)amino]-2deoxyglucose (2-NBDG), was treated for 30 min. After 30 min, cells were washed with phosphate buffered saline (PBS). Fluorescence was measured by Victor X4 Multi label plate
reader (Excitation/Emission = 485/535).
Western Blotting For protein extraction, Lysis buffer containing 25 mM HEPES, 150 mM NaCl, 1% Tripton X-100, 10% glycerol, 5 mM EDTA, 10mM NaF, 2 mM Na 3 VO 4 and protein inhibitor
cocktail was treated into the wells with cells. protein was separated according to size using gel electrophoresis and transferred to a polyvinylidene fluoride membrane using wet transfer system.
For detecting phosphorylated AMPK, total AMPK, phosphorylated AKT and phosphorylated AKT, antibodies of these proteins were treated in membrane for 24 hours at 4°C. After 24 hours,
membrane was briefly rinsed with TBST buffer (Tris-Buffered Saline, 0.1% Tween) and treated anti-rabbit-IgG-horseradish peroxidase-conjugated secondary antibody. This complex were
detected by ECL method
RESULTS

Table 1. K M value of phosphatases and Table 1. shows that JS- Figure 1 . Cell Cytotoxicity Test of JS-003 in
Inhibition ability of compound according to the 003 downregulate several
concentration phosphatases known for C2C12 Cells
deteriorating T2DM.

Figure 1. JS-003 does not
have any cytotoxicity in
(Inhibition percent) C2C12 Cells treated for
48h after differentiation
Figure 2 . Glucose Uptake Figure 3. Western Blotting in C2C12 Cells
assay in C2C12 Cells. a. b. Figure 3 a.
JS-003 does not improve
Phosphorylated Akt form in
compound itself and co-treated with
insulin. It means that JS-003 is not
related to insulin signaling pathway

Figure 3 b.
Js-003 improve phosphorylated
AMPK. It shows that JS-003 induce
Figure 2. represents that JS-003 better glucose uptake ability in the
treated C2C12 cells have better ability cell via ampk induced signaling
pathway
of glucose uptake compared to DMSO
and insulin treated cells
CONCLUSION REFERENCES ACKNOWLEDGEMENTS

In summary, JS-003 strongly inhibits several Esteban.N;Gurzov;WilliamJ.;Stanley;ThomasC.Brodni This research was supported by the Bio & Medical
enzymes negatively effect on T2DM. cki;HelenE.Thomas.Protein tyrosine phosphatases: Technology Development Program of the National Research
Glucose uptake assay and Western Blotting molecular switches in metabolism and diabetes. Cell Foundation (NRF) funded by the Korean government (MSIT)
(NRF-2012M3A9C4048775 and NRF-2017M3A9C8031995)
Press. 2015,26,30-39
results are consistent with result of
enzymatic assay. Taken together, all results
powerfully represent that JS-003 is a Contact information
potential therapeutic candidate for T2DM. Email: sjchung@skku.edu