PIK-93 inhibits PI3Kγ and PI4KIIIβ, with IC50 values of 16 nM and 19 nM, respectively. PIK-93 also inhibits other members of PI3Ks, including PI3Kα, β, and δ, with IC50 values of 39 nM, 0.59 μM, and 0.12 μM, respectively. PIK-93 shows no obvious inhibitory effect against a panel of other kinases, even at a concentration of 10 μM[1]. In differentiated HL60 (dHL60) cells, PIK-93 (0.5 μM-1 μM) impairs consolidation and stability of the leading edge formed after treatment with uniform f-Met-Leu-Phe (fMLP). PIK-93 alters the localization, but not the amount, of the fMLP-dependent accumulation of total F-actin. In fMLP gradients, PIK-93 reduces the chemotactic index and triples the cells' turning frequency[2]. In COS-7 cells, PIK-93 (250 nM) effectively abrogates the accumulation of CERT-PH domain and FL-Cer in Golgi. PIK-93 of the same concentration also significantly inhibits the conversion of [3H]serine-labeled endogenous ceramide to sphingomyelin. These facts indicate a key role of PI4KIIIβ in ceramide transport between the ER and Golgi, as well as in the regulation of spingomyelin synthesis[3]. In T6.11 cells, PIK-93 (300 nM) reduces carbachol-induced translocation of TRPC6 to the plasma membrane and net Ca2+ entry[4]. A recent report shows that PIK-93 has anti-enterovirus effects, as revealed by its inhibition of both poliovirus (PV) and hepatitis C virus (HCV) replication, with EC50 values of 0.14 µM and 1.9 µM, respectively[5].
Kinase Assay
IC50 values are measured using a standard TLC assay for lipid kinase activity. Kinase reactions are performed by preparing areaction mixture containing kinase, PIK-93 (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 min 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 hours-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. Kinase activity is typically measured at 10-12 concentrations of PIK-93 representing two-fold dilutions from the highest concentration of 100 μM.
Cell Assay
For actin staining, dHL60 cells are preincubated in suspension with PIK-93 or vehicle for 40 min, centrifuged for 5 min at 2000 rpm at room temperature in a J6-B centrifuge, resuspended in mHBSS containing the respective agent at the same concentration, allowed to stick to fibronectin-covered coverslips, and subjected to stimulation with a uniform concentration of 100 nM f-Met-Leu-Phe (fMLP) for 3 min. Cells are fixed in 3.7% PFA and stained with 10 units/mL rhodamine-phalloidin for 15 min.
Animal Administration
References
[1]. Knight ZA, et al. A pharmacological map of the PI3-K family defines a role for p110alpha in insulin signaling. Cell. 2006 May 19;125(4):733-47.
[2]. Van Keymeulen A, et al. To stabilize neutrophil polarity, PIP3 and Cdc42 augment RhoA activity at the back as well as signals at the front. J Cell Biol. 2006 Jul 31;174(3):437-45.
[3]. Toth B, et al. Phosphatidylinositol 4-kinase IIIbeta regulates the transport of ceramide between the endoplasmic reticulum and Golgi. J Biol Chem. 2006 Nov 24;281(47):36369-77.
[4]. Monet M, et al. Involvement of phosphoinositide 3-kinase and PTEN protein in mechanism of activation of TRPC6 protein in vascular smooth muscle cells. J Biol Chem. 2012 May 18;287(21):17672-81.
[5]. 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.
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