AG1109对重组人蛋白激酶CK2全酶的抑制作用

背景与目的：蛋白激酶CK2是一种高度进化保守性的丝／苏氨酸激酶,它的活性在人的多种癌症是上调的。CK2可作为一种癌基因起作用,它的靶向抑制可用于肿瘤的治疗。为了寻找特异性CK2抑制剂,本文观察Tyrphostink和AG1109对重组人蛋白激酶CK2全酶的直接作用,以探讨其酶动力学机制。方法：利用基因工程克隆,表达和纯化而获得重组人蛋白激酶CK2α和β亚基,在体外等摩尔数混合构成有最大生物活性的重组CK2全酶,在不同条件下测定CK2的活性。CK2活性通过测定转移到CK2底物上的[γ-^32P]ATP或[γ-^32P]GTP的[^32P]放射活度来检测。结果：重组人CK2是一种Ca^2 ,cAMP和cGMP等第二信使非依赖性蛋白激酶,与天然CK2的性质一致。AG1109对重组人CK2全酶具有很强的抑制作用,其IC50为9.7μmol/L,抑制作用稍大于已知的CK2抑制剂5,6－二氯－1－β－呋喃糖苯并咪唑（DRB）和N－（2－氨乙基）－5－氯萘－1－硫胺（A3）。AG1109对重组人CK2的动力学研究表明,它与GTP呈以竞争性为主的混合型抑制作用,与酪蛋白呈非竞争性抑制作用。结论：AG1109是一种很强的重组人CK2抑制剂。重组人蛋白激酶CK2可作为一种较为简便地筛选和开发有效的CK2抑制剂的分子靶点。

Inhibitory effect of AG1109 on recombinant human protein kinase CK2 holoenzyme

BACKGROUND & OBJECTIVE: Protein kinase CK2 is an evolutionarily conserved serine/threonine kinase that is upregulated and can serve as an oncogene in many cancer cells. Targeted inhibition of CK2 could be useful in the treatment of many cancers. To search specific CK2 inhibitors we investigated the direct effect of tyrphostin AG1109 on recombinant human protein kinase CK2 holoenzyme and its kinetics. METHODS: Recombinant human protein kinase CK2 alpha and beta subunits were cloned and expressed by gene engineering, and purified to homogeneous. The two subunits were mixed at the same molar ratio and reconstituted CK2 holoenzyme, which exerted the maximum biological activity. The CK2 activity was assayed by detecting incorporation of 32P of [gamma-32P]ATP or [gamma-32P]GTP into the substrate in various conditions. RESULTS: The recombinant human CK2 was the second messengers (Ca2+, cAMP, and cGMP) independent protein kinase, the characterization and function of the reconstituted holoenzyme were consistent with those of native CK2. AG1109 strongly inhibited the holoenzyme activity of recombinant human protein kinase CK2 with an IC50 of 9.7 mumol/L, which was slightly more effective than 5, 6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) and N-(2-aminoethyl)-5-chloronaphthalene-1-sulfonamide (A3), which were both known CK2 special inhibitors. Kinetic studies of AG1109 on recombinant human CK2 showed that the inhibition was mixed (competitive is dominant) with GTP and noncompetitive with casein. CONCLUSIONS: AG1109 is an effective inhibitor of recombinant human protein kinase CK2 holoenzyme. The recombinant human protein kinase CK2 might be used as a molecular target for simpler screening and development of more effective inhibitors of CK2.