Small molecule inhibitors that target components of the spliceosome have great potential as tools to probe splicing mechanism and dissect splicing regulatory networks in cells. These compounds also hold promise as drug leads for diseases in which splicing regulation plays a critical role, including many cancers. Because the spliceosome is a complicated and dynamic macromolecular machine comprised of many RNA and protein components, a variety of compounds that interfere with different aspects of spliceosome assembly is needed to probe its function. By screening chemical libraries with high-throughput splicing assays, several labs have added to the collection of splicing inhibitors, although the mechanistic insight into splicing yielded from the initial compound hits is somewhat limited so far. In contrast, SF3B1 inhibitors stand out as a great example of what can be accomplished with small molecule tools. This group of compounds were first discovered as natural products that are cytotoxic to cancer cells, and then later shown to target the core spliceosome protein SF3B1. The inhibitors have since been used to uncover details of SF3B1 mechanism in the spliceosome and its impact on gene expression in cells. Continuing structure activity relationship analysis of the compounds is also making progress in identifying chemical features key to their function, which is critical in understanding the mechanism of SF3B1 inhibition. The knowledge is also important for the design of analogs with new and useful features for both splicing researchers and clinicians hoping to exploit splicing as pressure point to target in cancer therapy.
The protein SF3B1 is a core component of the spliceosome, the large ribonucleoprotein complex responsible for pre-mRNA splicing. Interest in SF3B1 intensified when tumor exome sequencing revealed frequent specific SF3B1 mutations in a variety of neoplasia and when SF3B1 was identified as the target of three different cancer cell growth inhibitors. A better mechanistic understanding of SF3B1's role in splicing is required to capitalize on these discoveries. Using the inhibitor compounds, we probed SF3B1 function in the spliceosome in an in vitro splicing system. Formerly, the inhibitors were shown to block early steps of spliceosome assembly, consistent with a previously determined role of SF3B1 in intron recognition. We now report that SF3B1 inhibitors also interfere with later events in the spliceosome cycle, including exon ligation. These observations are consistent with a requirement for SF3B1 throughout the splicing process. Additional experiments aimed at understanding how three structurally distinct molecules produce nearly identical effects on splicing revealed that inactive analogs of each compound interchangeably compete with the active inhibitors to restore splicing. The competition indicates that all three types of compounds interact with the same site on SF3B1 and likely interfere with its function by the same mechanism, supporting a shared pharmacophore model. It also suggests that SF3B1 inhibition does not result from binding alone, but is consistent with a model in which the compounds affect a conformational change in the protein. Together, our studies reveal new mechanistic insight into SF3B1 as a principal player in the spliceosome and as a target of inhibitor compounds.
Background: Pladienolide B is a complex natural product that potently inhibits pre-mRNA splicing. Results: The same molecular features of pladienolide B are required for the drug's effects on cell growth, morphology, and splicing. Conclusion: Simplified synthesis and modification of active pladienolide B is possible. Significance: Pladienolide B analogs can be used to study the relationship between splicing and cancer cell function.
Mitochondrial pre-messenger RNAs in kinetoplastid protozoa are substrates of uridylate-specific RNA editing. RNA editing converts non-functional pre-mRNAs into translatable molecules and can generate protein diversity by alternative editing. Although several editing complexes have been described, their structure and relationship is unknown. Here, we report the isolation of functionally active RNA editing complexes by a multistep purification procedure. We show that the endogenous isolates contain two subpopulations of B20S and B35-40S and present the three-dimensional structures of both complexes by electron microscopy. The B35-40S complexes consist of a platform density packed against a semispherical element. The B20S complexes are composed of two subdomains connected by an interface. The two particles are structurally related, and we show that RNA binding is a main determinant for the interconversion of the two complexes. The B20S editosomes contain an RNA-binding site, which binds gRNA, pre-mRNA and gRNA/pre-mRNA hybrid molecules with nanomolar affinity. Variability analysis indicates that subsets of complexes lack or possess additional domains, suggesting binding sites for components. Together, a picture of the RNA editing machinery is provided.
FR901464 (1) and spliceostatin A (2) are potent inhibitors of spliceosomes. These compounds have shown remarkable anticancer activity against multiple human cancer cell lines. Herein, we describe efficient, enantioselective syntheses of FR901464, spliceostatin A, six corresponding diastereomers and an evaluation of their splicing activity. Syntheses of spliceostatin A and FR901464 were carried out in the longest linear sequence of 9 and 10 steps, respectively. To construct the highly functionalized tetrahydropyran A-ring, we utilized CBS reduction, Achmatowicz rearrangement, Michael addition, and reductive amination as key steps. The remarkable diastereoselectivity of the Michael addition was specifically demonstrated with different substrates under various reaction conditions. The side chain B was prepared from an optically active alcohol, followed by acetylation and hydrogenation over Lindlar’s catalyst. The other densely functionalized tetrahydropyran C-ring was derived from readily available (R)-isopropylidene glyceraldehyde through a route featuring 1,2-addition, cyclic ketalization, and regioselective epoxidation. These fragments were coupled together at a late stage through amidation and cross-metathesis in a convergent manner. Six key diastereomers were then synthesized to probe the importance of specific stereochemical features of FR901464 and spliceostatin A, with respect to their in vitro splicing activity.
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