반응 #304603

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penicillin

streptomycin

G418 disulfate

Lipofectamine

→

G418

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후처리

1. 1
   workup.ADDITIONintroduced to an expression vector

실험 절차

The in vitro S1P1 agonist action of the compound of the present invention was evaluated by the increase in the functional binding activity of GTP[γ-35S] to G-protein using the membrane of a human S1P1 expressing cell. A cDNA encoding a human S1P1 was cloned from a human colorectal cDNA library and introduced to an expression vector pcDNA3.1 to construct a S1P1-pcDNA3.1. Then, by Lipofectamine 2000 (GIBCO), the S1P1-pcDNA3.1 was transfected into a CHO cell, and cultured in a Ham's F-12 culture medium containing 10% fetal bovine serum, 100 U/mL penicillin, 100 μg/mL streptomycin, and 1 mg/mL G418 disulfate, to obtain a stable, G418-resistant strain. The cultured human S1P1 expressing cells were isolated in a 1 mM EDTA.2Na-containing PBS, and disrupted under ice-cooling by a homogenizer made of glass in a 1 mM Tris HCl (pH 7.4) buffer solution containing 0.1 mM EDTA and a protein inhibitor. It was centrifuged at 1,400×10 mM, and a supernatant was further centrifuged at 4° C. for 60 min at 100,000×g, and suspended in a 10 mM Tris HCl (pH 7.4) buffer solution containing 1 mM EDTA to purify the membrane. The obtained membrane (0.13 mg/mL) and 50 μM GTP[γ-35S] (NEN; inactive 1250 Ci/mmol) were reacted in a 20 mM HEPES (pH 7.0) buffer solution (total amount: 150 μL) containing 100 mM NaCl, 10 mM MgCl2, 0.1% fatty acid-free BSA, and 5 μM GDP for 1 hour together with the compound of the present invention (10−12 to 10−5 M), and then a membrane was recovered on a GF-C filter plate with a Cell Harvester (Packard, FilterMate). The filter plate was dried at 50° C. for 60 min, and Microscinti-o (Packard) was added thereto for measurement by a liquids scintillation counter for a microplate (Packard, TOP count). For evaluation of the human S1P1 agonist action of the compound of the present invention and the comparative compound, the percentages with the rate of a maximum reaction to make the GTP[γ-35S] bonds saturated in the presence of the compound being set at 100%, and the rate of the reaction of the GTP[γ-35S] bonds in the absence of the compound being set at 0% were used, a non-linear regression curve was plotted, and a concentration to cause an agonist action operating 50% of the maximum reaction was defined as an EC50 value (nM).

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