Identification of Small Molecule Inhibitors of Pre-mRNA Splicing

Pawellek, Andrea; McElroy, Stuart P.; Samatov, Timur R.; Mitchell, Lee; Woodland, Andrew; Ryder, Ursula; Gray, David W.; Lührmann, Reinhard; Lamond, Angus I.

doi:10.1074/jbc.m114.590976

Cited by 63 publications

(44 citation statements)

References 50 publications

(73 reference statements)

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“…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.…”

Section: Introductionmentioning

confidence: 99%

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

Shi

Park

Lagisetti

et al. 2017

Bioorganic & Medicinal Chemistry Letters

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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.

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Section: Introductionmentioning

confidence: 99%

“…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. 19 …”

Section: Introductionmentioning

confidence: 99%

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

Shi

Park

Lagisetti

et al. 2017

Bioorganic & Medicinal Chemistry Letters

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“…Many types of RNAP II transcripts controlled by ARS2 likely contribute to the cell cycle phenotype of altering ARS2 levels. Indeed, microRNA (28), mRNA splicing (29,30), and replication-dependent histones are all important for cell cycle progression (13,34). However, the distinctive phenotype of the high-BrdU-containing cells in early S phase points to defective chromatin packaging as a consequence of histone deficiency as a major contributor for this unique population within the overexpressing cells.…”

Section: Discussionmentioning

confidence: 99%

“…2G). Although we cannot rule out contributions to the cell cycle phenotype from other types of RNA processed by ARS2 with known roles in the cell cycle (28)(29)(30), histone deficiency during S phase and the inability to package chromatin caused by improper processing of histone RNAs are likely major contributors to the S phase arrest associated with high levels of ARS2 overexpression.…”

Section: Ars2 Is Required For Cell Cycle Progressionmentioning

confidence: 99%

Mutagenesis of ARS2 Domains To Assess Possible Roles in Cell Cycle Progression and MicroRNA and Replication-Dependent Histone mRNA Biogenesis

O’Sullivan

Christie

Pienaar

et al. 2015

Molecular and Cellular Biology

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ARS2 is a regulator of RNA polymerase II transcript processing through its role in the maturation of distinct nuclear cap-binding complex (CBC)-controlled RNA families. In this study, we examined ARS2 domain function in transcript processing. Structural modeling based on the plant ARS2 orthologue, SERRATE, revealed 2 previously uncharacterized domains in mammalian ARS2: an N-terminal domain of unknown function (DUF3546), which is also present in SERRATE, and an RNA recognition motif (RRM) that is present in metazoan ARS2 but not in plants. Both the DUF3546 and zinc finger domain (ZnF) were required for association with microRNA and replication-dependent histone mRNA. Mutations in the ZnF disrupted interaction with FLASH, a key component in histone pre-mRNA processing. Mutations targeting the Mid domain implicated it in DROSHA interaction and microRNA biogenesis. The unstructured C terminus was required for interaction with the CBC protein CBP20, while the RRM was required for cell cycle progression and for binding to FLASH. Together, our results support a bridging model in which ARS2 plays a central role in RNA recognition and processing through multiple protein and RNA interactions. The generation of mature RNA in the nucleus is a highly coordinated process that requires distinct complexes for the biogenesis of different RNA families. For RNA polymerase II (RNAP II) transcripts, a critical step is the addition of a 7-methylguanosine (m7G) cap to the nascent transcript (1), which is subsequently bound by CBP20 and CBP80 of the nuclear cap-binding complex (CBC) (2). CBC-controlled transcripts include mRNA, microRNA (miRNA), replication-dependent histone (RDH) mRNA, small nucleolar RNA (snoRNA), and small nuclear RNA (snRNA), each with its own unique processing requirements. Binding of the CBC to the m7G cap of these RNAs occurs cotranscriptionally, protects the transcripts from degradation, and plays a central role in recruiting the appropriate machinery for processing different RNA families (3-8). However, exactly how distinct RNA families are differentially recognized to allow for correct processing complex formation is not fully understood. Recently, a protein called ARS2 (or SRRT in humans) has been shown to be part of the CBC, and it plays an important role in the maturation of several distinct RNA families (8, 9).Clues to how ARS2 participates in recruiting different RNA processing machineries are beginning to emerge from biochemical and structural studies. ARS2 interacts directly with the assembled CBP20/80 cap complex to form a tertiary complex termed CBCA (9). One hypothesis is that ARS2 bridges the CBCA to the appropriate processing machinery by interacting with both protein and RNA elements. This hypothesis is based on studies of the ARS2 plant orthologue SERRATE in miRNA biogenesis. Both the Arg/Pro-rich N terminus and zinc finger (ZnF) domain of SERRATE are required for primary miRNA (primiRNA) binding (10, 11). Additionally, SERRATE, through its N terminus and ZnF, has been shown to bind directly to...

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“…Madrasin was isolated from a screen of PM5 pre-mRNA splicing [56] against a library of 71,504 small molecules and also shown to inhibit splicing of endogenous genes in both HeLa and HEK293 cells [57]. Furthermore, a high-throughput screen using a luciferase splicing reporter identified clotrimazole, flunarizine, and chlorhexidine [46, 58] as splicing modulators in cultured HeLa cells.…”

Section: Discovery Of Small Molecule Splicing Modulatorsmentioning

confidence: 99%

Small Molecule Modulators of Pre-mRNA Splicing in Cancer Therapy

Salton

Misteli

2016

Trends in Molecular Medicine

134

128

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Pre-mRNA splicing is a fundamental process in mammalian gene expression and alternative RNA splicing plays a considerable role in generating protein diversity. RNA splicing events are key to the pathology of numerous diseases, including cancers. Some tumors are molecularly addicted to specific RNA splicing isoforms making interference with pre-mRNA processing a viable therapeutic strategy. Several RNA splicing modulators have been recently characterized showing promise in pre-clinical studies. While the targets of most splicing modulators are constitutive RNA processing components, with undesirable side effects, selectivity for individual splicing events has been observed. Given the high prevalence of splicing defects in cancer, small molecule modulators of RNA processing represent a novel therapeutic strategy in cancer treatment. Here, we review their reported effects, potential mechanisms, and limitations.

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Identification of Small Molecule Inhibitors of Pre-mRNA Splicing

Cited by 63 publications

References 50 publications

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

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

Mutagenesis of ARS2 Domains To Assess Possible Roles in Cell Cycle Progression and MicroRNA and Replication-Dependent Histone mRNA Biogenesis

Small Molecule Modulators of Pre-mRNA Splicing in Cancer Therapy

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