3
.
Gotoh, C.; Hong, Y. H.; Iga, T.; Hishikawa, D.; Suzuki, Y.; Song, S. H.;
Choi, K. C.; Adachi, T.; Hirasawa, A.; Tsujimoto, G.; Sasaki, S.; Roh, S.
G. Biochem Biophys Res Commu. 2007, 354, 591.
Matsumura, S.; Mizushige, T.; Yoneda, T.; Iwanaga, T.; Tsuzuki, S.;
Inoue, K.; Fushiki, T. Biomed. Res. 2007, 28, 49.
Hudson, B. D.; Shimpukade, B.; Mackenzie, A. E.; Butcher, A. J.; Pediani,
J. D.; Christiansen, E.; Heathcote, H.; Tobin, A. B.; Ulven, T.; Milligan,
Kimura, I. Int. J. Mol. Sci. 2016, 17, 450.
HBSS containing probenecid for 10 min. The test compounds were added
during the assay by a Flexstation 3.0 plate reader (Molecular Devices)
2
+
and intracellular Ca concentrations were measured as the difference
between 485/525 ratios before and after addition of the test compounds.
The Calcium influx assay on hGPR40-expressing CHO cells is similar
with hGPR120. EC50 values for each curve were calculated by Prism 5.0
software (GraphPad Software).
4
5
.
.
13. Sun, Q.; Hirasawa, A.; Hara, T.; Kimural, I.; Adachi, T.; Awaji, T.;
Ishiguro, M.; Suzuki, T.; Miyata, N.; Tsujimoto, G. Mol. Pharmacol.
2014, 289, 20345.
6
7
.
.
Briscoe, C. P.; Peat, A. J.; McKeown, S. C.; Corbett, D. F.; Goetz, A. S.;
Littleton, T. R.; McCoy, D. C.; Kenakin,T. P.; Andrews, J. L.; Ammala,
C.; Fornwald, J. A.; Ignar, D. M.; Jenkinson, S. Br. J. Pharmacol. 2006,
15. Milligan, G.; Alvarez-Curto, E.; Watterson, K. R.; Ulven, T.; Hudson, B.
D. Br. J. Pharmacol. 2015, 172, 3254.
1
48, 619.
8
9
.
.
Suzuki, T.; Igari, S.; Hirasawa, A. Hata, M.; Ishiguro, M.; Fujieda, H.;
Itoh, Y.; Hirano, T.; Nakagawa, H.; Ogura, M.; Makishima, M.;
Tsujimoto, G.; Miyata, N. J. Med. Chem. 2008, 51, 7640.
Shimpukade, B.; Hudson, B. D.; Hovgaard, C. K.; Milligan, G.; Ulven, T.
16. Oral Glucose Tolerance Test: Six-week-old male ICR mice were
purchased from Slac Laboratory Animal (Shanghai, China) and
maintained under
a 12:12 hr light-dark cycle condition. After
acclimatization one week, the mice were fasted for 16 h and randomly
divided into five groups (n=8). The fasting blood glucose (time −60 min)
was attained by ACCUCHEK (Roche) in the tail vein prior to the orally
administered 0.5% methylcellulose (control), 4 (10 mg/kg), and 6a (10,
30 and 100mg/kg). Then 60 min later, blood glucose was measured as 0
min, and all the mice were gavaged with 2.5 g/kg glucose. Next, the
blood glucose was measured at time 30, 60, 90, 120 min post glucose
challenge. The experimental procedure was permitted by the Shanghai
Institute of Materia Medica Experimental Animal Care and Use
Committee (accreditation number 2017-06-WHY-15).
1
Madeira, M.; Akiyama, T. E.; Di Salvo, J.; Suzuki, T.; Wang, N.; Truong,
Q.; Gilbert, E.; Zhou, D.; Verras, A.; Kirkland, M.; Pachanski, M.;
Powles, M.; Yin, W.; Ujjainwalla, F.; Venkatraman, S.; Edmondson, S. D.
ACS Med. Chem. Lett. 2017, 8, 96.
1
1. Kim, Y. K.; Park, S. Y.; Joo, H. W.; Choi, E. S. LG Life Sciences Ltd., S.
Korea W. O. Patent 2014209034, Dec. 31, 2014.
2. Calcium Influx Activity Assay: The method for calcium influx activity
assay as described previously with some modifications. Briefly, chinese
hamster ovary (CHO) cells, stably expressing human GPR120, were
1
Supplementary Material
seeded in 96-well plates (Corning) and cultured in 5% CO
2
at 37 °C for
2
4 h, then the cells were incubated in Hank’s Balanced Salt Solution
Supplementary data 1: Synthetic procedure and Bioanalysis
methods.
Supplementary data 2: NMR and HRMS.
(HBSS) containing 20mM HEPES, calcium indicator Fluo 4-AM dye
(
3μM), and probenecid (2.5 mM) at 37 °C. After 60 min incubation, the
cells were washed three times using HBSS buffer and maintained in
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