Kinase experiment:

The inhibitory effect of T-00127_HEV1 at a concentration of 10 μM on in vitro cellular protein kinase activities is assessed by kinase profiling with an ATP concentration near the Km for each kinase. Inhibitory effects of GW5074, AN-12-H5, and T-00127_HEV1 at a concentration of 10 μM on in vitro PI kinase activities are assessed by the SelectScreen kinase profiling service with an ATP concentration of 10 μM. For T-00127_HEV1, the 50% inhibitory concentration (IC50) for in vitro PI4KB activity is also measured with an ATP concentration of 10 μM[1].

Cell experiment:

RD cells (1.0×104 cells per well in 100 μL medium) in a 96-well plate are infected with PV1, EV71, or CVB3 at multiplicities of infection (MOI) of 10, 1.0, and 0.1 at 37℃ for 1 h in the absence of T-00127_HEV1. The cells are washed three times with 10% FCS-DMEM, followed by the addition of 100 μL of 10% FCS-DMEM containing 10 or 0 μM T-00127_HEV1. Cells are collected at 16 h p.i., and then viral RNA is extracted from the cells using a viral RNA purification kit. The number of copies of the viral genome is quantified using a real-time PCR system[1].

T-00127_HEV1 is a phosphatidylinositol 4-kinase III beta (PI4KB) inhibitor with an IC50 of 60 nM.

T-00127_HEV1 shows more potent anti-poliovirus (PV) activity (EC50 of 0.77 μM) than other candidate compounds (EC50 of 1.7 to 4.7 μM). GW5074 and T-00127_HEV1 almost completely inhibit PI4KB kinase activity at 10 μM (3% and 5% of residual activity, respectively), in contrast to AN-12-H5 (108% of activity [no inhibition])[1].

[1]. Arita M, et al. Phosphatidylinositol 4-kinase III beta is a target of enviroxime-like compounds for antipoliovirus activity. J Virol. 2011 Mar;85(5):2364-72.