Inhibitors of CLK Protein Kinases Suppress Cell Growth and Induce Apoptosis by Modulating Pre-mRNA Splicing

Abstract

Accumulating evidence has demonstrated the importance of alternative splicing in various physiological processes, including the development of different diseases. CDC-like kinases (CLKs) and serine-arginine protein kinases (SRPKs) are components of the splicing machinery that are crucial for exon selection. The discovery of small molecule inhibitors against these kinases is of significant value, not only to delineate the molecular mechanisms of splicing, but also to identify potential therapeutic opportunities. Here we describe a series of small molecules that inhibit CLKs and SRPKs and thereby modulate pre-mRNA splicing. Treatment with these small molecules (Cpd-1, Cpd-2, or Cpd-3) significantly reduced the levels of endogenous phosphorylated SR proteins and caused enlargement of nuclear speckles in MDA-MB-468 cells. Additionally, the compounds resulted in splicing alterations of RPS6KB1 (S6K), and subsequent depletion of S6K protein. Interestingly, the activity of compounds selective for CLKs was well correlated with the activity for modulating S6K splicing as well as growth inhibition of cancer cells. A comprehensive mRNA sequencing approach revealed that the inhibitors induced splicing alterations and protein depletion for multiple genes, including those involved in growth and survival pathways such as S6K, EGFR, EIF3D, and PARP. Fluorescence pulse-chase labeling analyses demonstrated that isoforms with premature termination codons generated after treatment with the CLK inhibitors were degraded much faster than canonical mRNAs. Taken together, these results suggest that CLK inhibitors exhibit growth suppression and apoptosis induction through splicing alterations in genes involved in growth and survival. These small molecule inhibitors may be valuable tools for elucidating the molecular machinery of splicing and for the potential development of a novel class of antitumor agents.

A triple exon-skipping luciferase reporter assay identifies a new CLK inhibitor pharmacophore

“…17 These results, taken together with the recent observations of pronounced modulation of splicing (through inhibition of the CLK mediated phosphorylation of SR proteins 17 ) by compounds such as Araki compound-2 (see Figure 1), 18 strongly suggested that CLK inhibition was responsible for the alternative splicing effects seen with compounds 1 and 2 ; though splicing modulation has not been recognized as an activity of 1 or 2 , to our knowledge. In order to explore the CLKs as possible targets, and to better understand the structure-activity relationships (SAR), additional new structurally related analogs along with the associated CLK biochemical inhibition data were clearly needed so decided to evaluate the most potent of these compounds (compound 1 ) for CLK activity.…”

“…However, since it known that CLK inhibition can lead to modulation of pre-mRNA splicing, 18 and since we observed that the weakly cell-active, but selective, CLK inhibitor KH-CB19 shows activity in the MDM2-Luc reporter assay, we decided to explore the activity of 1 (and our active analogs) in regard to their biochemical inhibition of CLK 1-4 (see Table 2). The results summarized in Table 2 clearly show that CGP-74514A ( 1 ), which has been considered a selective CDK inhibitor 46 actually also shows significant inhibition of CLK 1, 2, and 4 and so can be considered a dual CLK/CDK inhibitor.…”

“…Several groups have independently discovered new diverse small molecule structural classes that effect pre-mRNA splicing, using a range of screening strategies (see Figure 1). These compounds include TG-003, 16 KH-CB19, 17 Araki Cpd-2, 18 and Madrasin 19 (see Figure 1). Compounds KH-CB19 and the Araki Cpd-2 have been reported to be highly selective inhibitors of the of cdc2-like kinase (CLK) family, 17, 18 while the molecular target of Madrasin has not been reported.…”

CLK-dependent exon recognition and conjoined gene formation revealed with a novel small molecule inhibitor

“…1a), by chemical modifications of the structure of previously identified tool compounds 22 from an extensive high throughput chemical library screen against CLK2 protein (Supplementary Tables 1, 2). The structure–activity relationship (Supplementary Fig.…”

Molecular Pathways: Understanding and Targeting Mutant Spliceosomal Proteins

“…SR proteins have conserved arginine- and serine-rich domains, which are subject to phosphorylation by multiple kinases, including the SR protein kinases and the CDC2-like kinases. Although the role of phosphorylation of these domains remains to be clarified, modulation of SR protein phosphorylation clearly impacts splicing (68, 69). Characterization of the effects of these new classes of compounds on splicing and potential effects on spliceosomal-mutant malignancies may represent novel therapeutic approaches for conquering malignancies with aberrant spliceosomal catalysis.…”