Development and Characterization of Novel Molecular Probes for Ca2+/Calmodulin-Dependent Protein Kinase Kinase, Derived from STO-609.
Development and Characterization of Novel Molecular Probes for Ca2+/Calmodulin-Dependent Protein Kinase Kinase, Derived from STO-609.
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源自 STO-609 的 Ca2/钙调蛋白依赖性蛋白激酶的新型分子探针的开发和表征。
DOI：
10.1021/acs.biochem.0c00149
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发表时间：
2020
期刊：
影响因子：
2.9
通讯作者：
and Tokumitsu H.
中科院分区：
文献类型：
--
作者：
Ohtsuka S;Ozeki Y;Fujiwara M;Miyagawa T;Kanayama N;Magari M;Hatano N;Suizu F;Ishikawa T;and Tokumitsu H.
钙/钙调蛋白依赖的蛋白激酶(CaMKK)激活包括CaMKI、CaMKIV和5‘AMP激活的蛋白激酶(AMPK)在内的多种多功能蛋白激酶，从而调节多种钙依赖的细胞过程，包括神经元、代谢和病理生理途径。我们开发并表征了一种新型的PAN-CaMKK抑制剂TIM-063(2-hydroxy-3-nitro-7H-benzo[de]benzo[4，5]imidazo[2，1-a]isoquinolin-7-one)(来源于STO-609(7H-benzimidazo[2，1-a]benz[de]isoquinoline-7-one-3-carboxylic酸)和一种非活性类似物(TIM-062)作为分子探针来分析CaMKK介导的细胞反应。与STO-609不同，TIM-063在体外对CaMKK亚型(CaMKKα和CaMKKβ)具有相似的抑制活性(对CaMKKβ的Ki=0.35μM，对CaMKKα的Ki=0.2μM)。两个缺少硝基(TIM-062)和羟基(TIM-064)的TIM-063类似物完全削弱了CaMKK抑制活性，表明这两个取代基是TIM-063抑制CaMKK活性所必需的。酶分析表明，TIM-063是一种直接针对CaMKK催化结构域的ATP竞争性抑制物，类似于STO-609。TIM-063抑制离子霉素诱导的外源性CaMKI、CaMKIV和内源性AMPKα的磷酸化，其IC50值为0.3COSKM，并抑制CaMKK亚型介导的∼-7细胞μ的磷酸化。因此，TIM-063，而不是非活性类似物(TIM-062)，显示出细胞通透性和抑制细胞内CaMKK活性的能力。综上所述，这些结果表明，TIM-063可能是精确分析CaMKK介导的信号通路的有用工具，并可能成为开发治疗CaMKK相关疾病的治疗药物的一种有前途的先导化合物。
Ca2+/calmodulin-dependent protein kinase kinase (CaMKK) activates particular multifunctional kinases, including CaMKI, CaMKIV, and 5′AMP-activated protein kinase (AMPK), resulting in the regulation of various Ca2+-dependent cellular processes, including neuronal, metabolic, and pathophysiological pathways. We developed and characterized a novel pan-CaMKK inhibitor, TIM-063 (2-hydroxy-3-nitro-7H-benzo[de]benzo[4,5]imidazo[2,1-a]isoquinolin-7-one) derived from STO-609 (7H-benzimidazo[2,1-a]benz[de]isoquinoline-7-one-3-carboxylic acid), and an inactive analogue (TIM-062) as molecular probes for the analysis of CaMKK-mediated cellular responses. Unlike STO-609, TIM-063 had an inhibitory activity against CaMKK isoforms (CaMKKα and CaMKKβ) with a similar potency (Ki= 0.35 μM for CaMKKα, andKi= 0.2 μM for CaMKKβ)in vitro. Two TIM-063 analogues lacking a nitro group (TIM-062) or a hydroxy group (TIM-064) completely impaired CaMKK inhibitory activities, indicating that both substituents are necessary for the CaMKK inhibitory activity of TIM-063. Enzymatic analysis revealed that TIM-063 is an ATP-competitive inhibitor that directly targets the catalytic domain of CaMKK, similar to STO-609. TIM-063 suppressed the ionomycin-induced phosphorylation of exogenously expressed CaMKI, CaMKIV, and endogenous AMPKα in HeLa cells with an IC50of ∼0.3 μM, and it suppressed CaMKK isoform-mediated CaMKIV phosphorylation in transfected COS-7 cells. Thus, TIM-063, but not the inactive analogue (TIM-062), displayed cell permeability and the ability to inhibit CaMKK activity in cells. Taken together, these results indicate that TIM-063 could be a useful tool for the precise analysis of CaMKK-mediated signaling pathways and may be a promising lead compound for the development of therapeutic agents for the treatment of CaMKK-related diseases.