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Updated: Mar 9, 2026
A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells
Published on: July 3, 2013
A triple exon-skipping luciferase reporter assay identifies a new CLK inhibitor pharmacophore
Yihui Shi1, Jaehyeon Park1, Chandraiah Lagisetti1
1Division of Biosciences, SRI International, 333 Ravenswood Ave., Menlo Park, CA 94025, USA.
Abstract:
The splicing of pre-mRNA is a critical process in normal cells and is deregulated in cancer. Compounds that modulate this process have recently been shown to target a specific vulnerability in tumors. We have developed a novel cell-based assay that specifically activates luciferase in cells exposed to SF3B1 targeted compounds, such as sudemycin D6. This assay was used to screen a combined collection of approved drugs and bioactive compounds. This screening approach identified several active hits, the most potent of which were CGP-74514A and aminopurvalanol A, both have been reported to be cyclin-dependent kinases (CDKs) inhibitors. We found that these compounds, and their analogs, show significant cdc2-like kinase (CLK) inhibition and clear structure-activity relationships (SAR) at CLKs. We prepared a set of analogs and were able to 'dial out' the CDK activity and simultaneously developed CLK inhibitors with low nanomolar activity. Thus, we have demonstrated the utility of our exon-skipping assay and identified new molecules that exhibit potency and selectivity for CLK, as well as some structurally related dual CLK/CDK inhibitors.
Insights
Researchers developed a novel assay to screen for compounds targeting pre-messenger RNA (mRNA) splicing, a process often deregulated in cancer. This screening identified potent inhibitors of Cyclin-dependent kinases (CDKs) and Cyclin-dependent kinase-like kinases (CLKs).
Area of Science:
- Biochemistry
- Molecular Biology
- Cancer Research
Background:
- Pre-messenger RNA (mRNA) splicing is crucial for normal cellular function but is frequently dysregulated in cancer.
- Targeting aberrant splicing pathways presents a promising strategy for cancer therapy.
- Specific vulnerabilities in tumors related to splicing are being explored for therapeutic intervention.
Purpose of the Study:
- To develop and validate a novel cell-based assay for screening compounds that modulate pre-mRNA splicing.
- To identify novel inhibitors of splicing-related kinases, specifically Cyclin-dependent kinase-like kinases (CLKs) and Cyclin-dependent kinases (CDKs).
- To discover potent and selective CLK inhibitors with potential therapeutic applications in cancer.
Main Methods:
- Development of a luciferase-based cell assay to detect compounds targeting SF3B1, a key splicing factor.
- Screening of a diverse compound library, including approved drugs and bioactive molecules.
- Structure-activity relationship (SAR) studies and analog synthesis to optimize inhibitor potency and selectivity.
Main Results:
- The assay successfully identified compounds that modulate splicing, including known Cyclin-dependent kinase (CDK) inhibitors.
- CGP-74514A and aminopurvalanol A were identified as potent hits, exhibiting significant Cyclin-dependent kinase-like kinase (CLK) inhibition.
- Analogs were synthesized, leading to the development of highly potent and selective CLK inhibitors, with some dual CLK/CDK inhibitors also identified.
Conclusions:
- The developed exon-skipping assay is a valuable tool for identifying splicing modulators.
- New potent and selective CLK inhibitors were discovered, offering potential for cancer therapy.
- The study highlights the utility of targeting splicing pathways and identified dual CLK/CDK inhibitors with potential therapeutic relevance.
