Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 289, Issue 50, Pages 34683-34698Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M114.590976
Keywords
High Throughput Screening; RNA; RNA Splicing; Small Molecule; Spliceosome; DDD00107587; Madrasin
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Funding
- EU [LSHG-CT-2005-518238]
- Wellcome Trust [073980/Z/03/Z]
- GRE Strategic Award [097945/B/11/Z]
- Biological Chemistry and Drug Discovery Strategic Award [100476/Z/12/Z]
- Medical Research Council [MC_G0900864] Funding Source: researchfish
- MRC [MC_G0900864] Funding Source: UKRI
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Background: There is a need for new small molecule pre-mRNA splicing inhibitors as biotools. Results: High throughput screening resulted in the identification of small molecule splicing inhibitors that are active in vitro and in cells. Conclusion: New small molecules for studying pre-mRNA splicing in vitro and in cells are identified. Significance: Small drug-like molecules are identified that modulate splicing in vitro and in cells. Eukaryotic pre-mRNA splicing is an essential step in gene expression for all genes that contain introns. In contrast to transcription and translation, few well characterized chemical inhibitors are available with which to dissect the splicing process, particularly in cells. Therefore, the identification of specific small molecules that either inhibit or modify pre-mRNA splicing would be valuable for research and potentially also for therapeutic applications. We have screened a highly curated library of 71,504 drug-like small molecules using a high throughput in vitro splicing assay. This identified 10 new compounds that both inhibit pre-mRNA splicing in vitro and modify splicing of endogenous pre-mRNA in cells. One of these splicing modulators, DDD00107587 (termed madrasin, i.e. 2-((7methoxy-4-methylquinazolin-2-yl)amino)-5,6-dimethylpyrimidin-4(3H)-one RNAsplicing inhibitor), was studied in more detail. Madrasin interferes with the early stages of spliceosome assembly and stalls spliceosome assembly at the A complex. Madrasin is cytotoxic at higher concentrations, although at lower concentrations it induces cell cycle arrest, promotes a specific reorganization of subnuclear protein localization, and modulates splicing of multiple pre-mRNAs in both HeLa and HEK293 cells.
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