Background
PIK-93 is a potent and novel inhibitor of p110γ (PI3Kγ) and PI4KIIIβ (IC50= 16 nM and 19 nM, respectively).
PI3K (phosphatidylinositol-4,5-bisphosphate 3-kinase) is a family of enzymes involved in cellular functions such as cell growth, proliferation, differentiation, motility, survival and intracellular trafficking, which in turn are involved in cancer. It plays a key role in PI3K/Akt/mTOR pathway.
Inhibition of PI3K via PIK-93 reduced the carbachol-activated translocation of TRPC6 to the plasma membrane and carbachol-activated net Ca(2+) entry into T6.11 cells. [1] PIK-93 also selectively inhibited the PI4KIIIβ enzyme, and siRNA-mediated down regulation of individual PI4-enzymes in COS-7 cells. [2] Furthermore, as a potent anti-PV compound, PIK93 targeted PI4Kβ to inhibit interaction of viral 3D polymerase and phosphatidylinositol 4-phosphate on the reorganized membrane vesicle for viral replication complex formation. [3] PIK93 also showed anti-PV with an EC50 of 0.14 μM for PV pseudovirus infection. [4]
参考文献:
[1] Monet M, Francoeur N, Boulay G.? 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.
[2] Tóth B, Balla A, Ma H 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.
[3] Hsu, N.? Y., et al. 2010. Viral reorganization of the secretory pathway generates distinct organelles for RNA replication.? Cell 141:799-811.
[4] Arita M, Kojima H, Nagano T 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.
Protocol
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Kinase experiment:
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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.
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Cell experiment:
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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.
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参考文献:
[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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