Nienberg Christian, Garmann Claudia, Gratz Andreas, Bollacke Andre, Götz Claudia, Jose Joachim
Institut für Pharmazeutische und Medizinische Chemie, PharmaCampus, Westfälische Wilhelms-Universität Münster, Correnstraße 48, D-48149 Münster, Germany.
Medizinische Biochemie und Molekularbiologie, Universität des Saarlandes, Kirrberger Str., Geb. 44, D-66421 Homburg, Germany.
Pharmaceuticals (Basel). 2017 Jan 5;10(1):6. doi: 10.3390/ph10010006.
Human protein kinase CK2 has emerged as promising target for the treatment of neoplastic diseases. The vast majority of kinase inhibitors known today target the ATP binding site, which is highly conserved among kinases and hence leads to limited selectivity. In order to identify non-ATP competitive inhibitors, a 12-mer peptide library of 6 × 10⁵ variants was displayed on the surface of by autodisplay. Screening of this peptide library on variants with affinity to CK2 was performed by fluorophore-conjugated CK2 and subsequent flow cytometry. Single cell sorting of CK2-bound yielded new peptide variants, which were tested on inhibition of CK2 by a CE-based assay. Peptide B2 (DCRGLIVMIKLH) was the most potent inhibitor of both, CK2 holoenzyme and the catalytic CK2α subunit (IC = 0.8 µM). Using different ATP concentrations and different substrate concentrations for IC determination, B2 was shown to be neither ATP- nor substrate competitive. By microscale thermophoresis (MST) the K value of B2 with CK2α was determined to be 2.16 µM, whereas no binding of B2 to CK2β-subunit was detectable. To our surprise, besides inhibition of enzymatic activity, B2 also disturbed the interaction of CK2α with CK2β at higher concentrations (≥25 µM).
人类蛋白激酶CK2已成为治疗肿瘤疾病的一个有前景的靶点。当今已知的绝大多数激酶抑制剂靶向ATP结合位点，该位点在激酶中高度保守，因此导致选择性有限。为了鉴定非ATP竞争性抑制剂，通过自展示在 的表面展示了一个由6×10⁵个变体组成的12聚体肽库。通过荧光团偶联的CK2和随后的流式细胞术对该肽库进行针对与CK2有亲和力的变体的筛选。对结合CK2的 进行单细胞分选产生了新的肽变体，通过基于毛细管电泳的分析对其抑制CK2的能力进行测试。肽B2（DCRGLIVMIKLH）是CK2全酶和催化性CK2α亚基二者最有效的抑制剂（IC = 0.8 μM）。在测定IC时使用不同的ATP浓度和不同的底物浓度，结果表明B2既不是ATP竞争性也不是底物竞争性的。通过微量热泳动（MST）测定B2与CK2α的K值为2.16 μM，而未检测到B2与CK2β亚基的结合。令我们惊讶的是，除了抑制酶活性外，B2在较高浓度（≥25 μM）时还会干扰CK2α与CK2β的相互作用。
