Kinase inhibition assays
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The consumption of ATP is coupled via the pyruvate kinase/lactic dehydrogenase enzyme pair to the oxidation of NADH, which can be monitored through the decrease in absorption at 340 nm. Reactions contained 100 mM Tris (pH 8), 10 mM MgCl2, 2.2 mM ATP, 1 mM phosphoenolpyruvate, 0.6 mg/mL NADH, 75 units/mL pyruvate kinase, 105 units/mL lactate dehydrogenase, and 0.5 mM substrate peptide (sequence: EAIYAAPFAKKK). Reactions (75 μL) were started by adding sufficient kinase to bring the reactions to 30 nM kinase concentration and the decrease in absorbance was monitored over 30 mins at 30 °C in a microtiter plate spectrophotometer. Inhibitory constants were obtained through addition of 3.75 μL VX-680 in 100% DMSO or DMSO alone. Ki values were calculated as follows, Ki = IC50 / (1 + [S]/Kd), where [S] = [ATP] = 2.2 mM, and Kd (of ATP to Abl) = 70 μM. These values were calculated assuming a Kd (ATP) of 70 μM for wild type and H396P Abl kinase domain.
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References:
[1]. Young MA, Shah NP, Chao LH, Seeliger M, Milanov ZV, Biggs WH 3rd, Treiber DK, Patel HK, Zarrinkar PP, Lockhart DJ, Sawyers CL, Kuriyan J. Structure of the kinase domain of an imatinib-resistant Abl mutant in complex with the Aurora kinase inhibitor VX-680. Cancer Res. 2006 Jan 15;66(2):1007-14.
[2]. Arlot-Bonnemains Y, Baldini E, Martin B, Delcros JG, Toller M, Curcio F, Ambesi-Impiombato FS, D'Armiento M, Ulisse S. Effects of the Aurora kinase inhibitor VX-680 on anaplastic thyroid cancer-derived cell lines. Endocr Relat Cancer. 2008 Jun;15(2):559-68.
[3]. Harrington EA, Bebbington D, Moore J, Rasmussen RK, Ajose-Adeogun AO, Nakayama T, Graham JA, Demur C, Hercend T, Diu-Hercend A, Su M, Golec JM, Miller KM. VX-680, a potent and selective small-molecule inhibitor of the Aurora kinases, suppresses tumor growth in vivo. Nat Med. 2004 Mar;10(3):262-7. Epub 2004 Feb 22.
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