Department of Biology Education, Kyungpook National University, 80 Daehak-ro, Buk-gu, Daegu 41566, Republic of Korea.
Biosystem Research Group, Korea Institute of Toxicology, 141 Gajeong-ro, Yuseong-gu, Daejeon 34114, Republic of Korea.
Biomed Res Int. 2019 Aug 18;2019:6125068. doi: 10.1155/2019/6125068. eCollection 2019.
Cdc2-like kinases (CLKs) play a crucial role in the alternative splicing of eukaryotic pre-mRNAs through the phosphorylation of serine/arginine-rich proteins (SR proteins). Dysregulation of this processes is linked with various diseases including cancers, neurodegenerative diseases, and many genetic diseases. Thus, CLKs have been regarded to have a potential as a therapeutic target and significant efforts have been exerted to discover an effective inhibitor. In particular, the small molecule CX-4945, originally identified as an inhibitor of casein kinase 2 (CK2), was further revealed to have a strong CLK-inhibitory activity. Four isoforms of CLKs (CLK1, CLK2, CLK3, and CLK4) can be inhibited by CX-4945, with the highest inhibitory effect on CLK2. This study aimed to elucidate the structural basis of the selective inhibitory effect of CX-4945 on different isoforms of CLKs. We determined the crystal structures of CLK1, CLK2, and CLK3 in complex with CX-4945 at resolutions of 2.4 Å, 2.8 Å, and 2.6 Å, respectively. Comparative analysis revealed that CX-4945 was bound in the same active site pocket of the CLKs with similar interacting networks. Intriguingly, the active sites of CLK/CX-4945 complex structures had different sizes and electrostatic surface charge distributions. The active site of CLK1 was somewhat narrow and contained a negatively charged patch. CLK3 had a protruded Lys248 residue in the entrance of the active site pocket. In addition, Ala319, equivalent to Val324 (CLK1) and Val326 (CLK2), contributed to the weak hydrophobic interactions with the benzonaphthyridine ring of CX-4945. In contrast, the charge distribution pattern of CLK2 was the weakest, favoring its interactions with benzonaphthyridine ring. Thus, the relatively strong binding affinities of CX-4945 with CLK2 are consistent with its strong inhibitory effect defined in the previous study. These results may provide insights into structure-based drug discovery processes.
CDC2 样激酶 (CLKs) 通过磷酸化丝氨酸/精氨酸丰富蛋白 (SR 蛋白) 在真核前体 mRNA 的可变剪接中发挥关键作用。这个过程的失调与各种疾病有关，包括癌症、神经退行性疾病和许多遗传疾病。因此，CLKs 被认为具有作为治疗靶点的潜力，并且已经做出了巨大的努力来发现有效的抑制剂。特别是，最初被鉴定为酪蛋白激酶 2 (CK2) 抑制剂的小分子 CX-4945，进一步被揭示具有很强的 CLK 抑制活性。CLKs 的四个同工型 (CLK1、CLK2、CLK3 和 CLK4) 都可以被 CX-4945 抑制，对 CLK2 的抑制作用最强。本研究旨在阐明 CX-4945 对不同 CLK 同工型的选择性抑制作用的结构基础。我们分别以 2.4Å、2.8Å 和 2.6Å 的分辨率确定了 CLK1、CLK2 和 CLK3 与 CX-4945 复合物的晶体结构。比较分析表明，CX-4945 结合在 CLKs 的相同活性口袋中，具有相似的相互作用网络。有趣的是，CLK/CX-4945 复合物结构的活性位点具有不同的大小和静电表面电荷分布。CLK1 的活性位点有些狭窄，并且包含带负电荷的补丁。CLK3 的活性口袋入口处有一个突出的 Lys248 残基。此外，Ala319（相当于 CLK1 的 Val324 和 CLK2 的 Val326）有助于与 CX-4945 的苯并萘啶环进行弱疏水相互作用。相比之下，CLK2 的电荷分布模式最弱，有利于与苯并萘啶环相互作用。因此，CX-4945 与 CLK2 较强的结合亲和力与其在之前研究中定义的强抑制作用一致。这些结果可能为基于结构的药物发现过程提供见解。
