An inhibitor of protein kinase D
IC50: Selective protein kinase D (PKD) antagonist with the IC50 of 0.182, 0.280, 0.227, >10, 15.3, 20.3, 40.5 and >50 μM for PKD1, PKD2, PKD3, PKC, CAK, PLK1, CAMKIIα and Akt respectively.
CID755673, benzoxoloazepinolone, is the first identified cell-active small molecule PKD antagonist. It inhibits the activity of PKD1 with an IC50 of 182 nM and demonstrates highest selectivity to PKD1 when compared with AKT, PLK1, CAK, CAMKIIα, PKD2 and PKD3. Moreover, it was not competitive with ATP for enzyme inhibition. [1]
In vitro: In cell based assays, CID755673 dose-dependently suppressed PKD1 activation induced by phorbol ester endogenous in LNCaP cells. It was also reported to reverse biological actions of PKD1 including class IIa histone deacetylase 5 nuclear exclusion, vesicular stomatitis, virus glycoprotein delivery from the Golgi to the plasma membrane as well as the ilimaquinone-induced Golgi fragmentation. Moreover, CID755673 suppressed prostate cancer cell proliferation, cell migration, and invasion. [1]
In vivo: Experimental models of acute pancreatitis were developed to study the effect of CID755673 on acute pancreatitis in vivo. Results demonstrated that this compound suppressed PKD1/2 and therefore significantly offset necrosis and severity of pancreatitis. [2]
Clinical trial: So far, no clinical trial has been conducted.
参考文献:
[1]Sharlow ER, Giridhar KV, LaValle CR, Chen J, Leimgruber S, Barrett R, Altamirano KB, Wipf P, Lazo JS and Wang QJ.? Potent and selective disruption of protein kinase D functionality by a benzoxoloazepinolone. J Biol Chem. 2008 Nov. 283(48): 3351246.
[2]Yuan JZ, Liu YN, Tan TY, Guha S, Gukovsky I, Gukovskaya A and Pandol SJ.? Protein kinase D regulates cell death pathways in experimental pancreatitis. Front Physiol. 2012 Mar. 3: DOI: 10.3389/fphys.2012.00060.
Kinase experiment: | The radiometric kinase assay is carried out by coincubating 0.5 μCi of [γ-32P]ATP, 20 μM ATP, 50 ng of purified recombinant human PKD (PKD1, PKD2, and PKD3) or CAMKIIα proteins, and 2.5 μg of Syntide-2 in 50 μL of kinase buffer that contains 50 mM Tris-HCl, pH 7.5, 4 mM MgCl2, 10 mM β-mercaptoethanol. The reaction is carried out under conditions that the initial rate is within the linear kinetic range[1]. |
Cell experiment: | The wound-induced migration is triggered by scraping the cells with a plastic pipette tip, and the wound is imaged immediately. The DU145 cells are then are treated with or without CID755673 at different concentrations. The wound is imaged immediately (0 h) and at different intervals with an inverted phase-contrast microscope with a ×10 objective. At the end of the assay, cells are fixed with methanol and stained with crystal violet for a final image[1]. |
Animal experiment: | Mice: For acute inhibitor studies, C57BL6 mice are administered a single dose of vehicle (5% DMSO in PBS, pH 7.4), or the selective PKD inhibitor CID755673 at 1 or 10mg/kg body weight. Mice are killed one or four hr later and heart collected for later analysis. For chronic inhibitor experiments, 8-week old db/db mice receives vehicle or CID755673 at 1 or 10mg/kg bodyweight for 16 days, by daily intraperitoneal (i.p.) injection[2]. |
参考文献: [1]. Sharlow ER, et al. Potent and selective disruption of protein kinase D functionality by a benzoxoloazepinolone. J Biol Chem. 2008 Nov 28;283(48):33516-26. | |
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