BF738735

Kinase experiment:

The PI4K in vitro activity assay is performed. Briefly, recombinant PI4KIIIβ or PI4KIIIα and their substrate, phosphatidylinositol (PI)-phosphatidylserine (PS), are diluted in buffer containing Triton X-100. The reaction is started by addition of a mixture of ATP and 0.25 μCi of [γ-33P]ATP. After 75 to 90 min of incubation at 30°C, the reaction is terminated by addition of phosphoric acid. The incorporated radioactivity is measured by using a TopCount NXT microplate scintillation counter. Data are converted to the percent inhibition relative to controls[1].

Cell experiment:

Buffalo green monkey (BGM) kidney cells, HeLa R19 cells, and HeLa Rh cells are grown at 37°C, 5% CO2 in minimal essential medium (MEM) supplemented with 10% fetal bovine serum, penicillin, and streptomycin. The assays to determine the 50% effective concentration (EC50) and 50% cytotoxic concentration (CC50) of BF738735 are performed. Briefly, cells are infected with 100 CCID50 for 2 h, after which the virus is removed and serial dilutions of BF738735 (0.01, 0.033, 0.1, 0.33, 1, 3.33, 10, 33.33 and 100 μM) are added. For determination of the CC50, serial dilutions of BF738735 are added to the cells. Following 3 to 4 days of incubation, the medium is replaced with CellTiter 96 AQueous One solution reagent. Optical densities at 490 nm are corrected for background absorbance, which is determined from wells that lack cells. The resulting values for untreated cells are set to 100%[1].

Animal experiment:

Mice[2]BF738735 is used to study the bioavailability and antiviral effect in vivo. BF738735 is administrated in mice, 1 mg/kg intravenously or 5 mg/kg orally to treat coxsackievirus serotype B4 (CVB4) induced pancreatitis.

References:

[1]. van der Schaar HM, et al. A novel, broad-spectrum inhibitor of enterovirus replication that targets host cell factor phosphatidylinositol 4-kinase IIIβ. Antimicrob Agents Chemother. 2013 Oct;57(10):4971-81.
[2]. V Saarnio. Antiviral Molecules of Enteroviruses. 13.1.2017.