PI-103 Hydrochloride

IC₅₀ values are measured using either a standard thin-layer chromatography (TLC) assay for lipid kinase activity or a high-throughput membrane capture assay. Kinase reactions are performed by preparing a reaction mixture containing kinase, inhibitor (2% DMSO final concentration), buffer (25 mM HEPES, pH 7.4, 10 mM MgCl₂), and freshly sonicated phosphatidylinositol (100 μg/mL). Reactions are initiated by the addition of ATP containing 10 μCi of γ-32P-ATP to a final concentration 10 or 100 μM, and allowed to proceed for 20 minutes at room temperature. For TLC analysis, reactions are then terminated by the addition of 105 μL 1N HCl followed by 160 μL CHCl₃:MeOH (1:1). The biphasic mixture is vortexed, briefly centrifuged, and the organic phase transferred to a new tube using a gel loading pipette tip precoated with CHCl₃. This extract is spotted on TLC plates and developed for 3-4 hours in a 65:35 solution of n-propanol:1M acetic acid. The TLC plates are then dried, exposed to a phosphorimager screen, and quantitated. For each compound, kinase activity is typically measured at 10-12 inhibitor concentrations representing two-fold dilutions from the highest concentration tested (100 μM). For compounds showing significant activity, IC₅₀ determinations are repeated two to four times, and the reported value is the average of these independent measurements^[4].

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.

For proliferation assays, MOLM14, OCI-AmL3 and MV4-11 cells are cultured during 48 h at 10⁵ cells/mL, in triplicate, in 10% FCS, without or with 0.1 or 1 μM PI-103, and then pulsed 6 h with 1μCi (37 kBq) [³H]-thymidine. The amounts of radioactivity are determined after trichloracetic acid precipitation^[2].

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.

Mice^[3]
Five to six month old males of either FVB/N strain or nude BALB/c strain are injected subcutaneously with one million cells in PBS. When tumor reaches between 50 and 100 mm³, mice are treated with 10 mg/kg or 70 mg/kg of PI-103 and/or 50 mg/kg sorafenib by IP injection daily. Control mice are treated with the same volume of DMSO. Tumor size and mice weight is monitored every 2 days. When mice are sacrificed, tumors are dissected and processed^[3].

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.