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

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.

Cdc-Like Kinases (CLKs): Biology, Chemical Probes, and Therapeutic Potential

“…The structures of CLKs contain catalytic domains with β-strands and α-helixes distributed between the N- and C-regions. The N-lobe contains three β-strands followed by α-helix and additional two β-strands ( Figure 3 ) [ 39 , 45 ].…”

“…The tertiary structures of all CLKs are quite similar ( Figure 4 ), nevertheless the structure of CLK3 contains some additional motifs ( Figure 2 ), as described below [ 39 , 45 , 46 ].…”

“…CLK3 contains a small insert between stands β6 and β9 in the kinase core that interacts with a hydrophobic pocket near the hinge region that connects the N- and C-lobes [ 50 ]. The C-lobe of CLK3 is formed by three conserved regions: the LAMMER motif, mitogen-activated insertion, the MAPK-like insertion containing an α8 (residues 419–427), and β-hairpin insertion ( Figure 2 and Figure 5 ) [ 45 , 50 ].…”

“…Also, CLK3 possesses one Lys248 residue in the entrance of the active site pocket ( Figure 5 ), which modifies the electrostatic surface charge distribution therein—this might be unfavorable for the entry of some CLK inhibitors [ 45 , 46 ].…”

“… Overlay of X-ray crystal structures: CLK1 (yellow, ligand:12h, PDB: 6I5H) [ 47 ], CLK2 (green, PDB: 3NR9) [ 43 ], CLK3 (blue, PDB: 6YTW [ 48 ], CLK4 (pink, ligand: CX4945, PDB: 6FYV) [ 45 , 49 ]. …”