Structural Basis for the Selective Inhibition of Cdc2-Like Kinases by CX-4945.

Joo Youn Lee,Ji-Sook Yun,Hang-Suk Chun,Jeong Ho Chang,Woo-Keun Kim,Hyeonseok Jin,Sungchan Cho

doi:10.1155/2019/6125068

Abstract

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.

Highlights

The serine/arginine-rich protein (SR protein) is a wellconserved trans-acting protein that plays an important role in the splicing regulation of eukaryotic genes [1]
In order to elucidate the selective inhibitory effect of CX-4945 on Cdc2-like kinases (CLKs), we determined the structures of CLK1, CLK2, and CLK3 in complex with CX-4945
Extensive structural analysis performed in this study revealed that CLK1, CLK2, and CLK3 shared structural similarities as well

Summary

Introduction

The serine/arginine-rich protein (SR protein) is a wellconserved trans-acting protein that plays an important role in the splicing regulation of eukaryotic genes [1]. As the SR protein is phosphorylated, the splicingpromoting and -inhibitory factors are recruited to the phosphorylated RS domains through the protein-protein interaction and control the selective splicing of the pre-mRNA [4]. SR proteins are phosphorylated mainly by cdc2-like kinase (CLK) and serine/argininerich protein kinase (SRPK) [6]. They are phosphorylated at the serines on the N-terminal region of the RS domain mostly by SRPK1 in the cytoplasm and transported into the nucleus by a carrier protein such as transportin-SR2 [7]. In the SR protein regulation, SRPK1 and CLK1 seem to partition their activities for SR protein phosphorylation based on Ser-Pro versus Arg-Ser placement rather than on N- and C-terminal preferences along the RS domain [8, 9]

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