Modulating splicing with small molecular inhibitors of the spliceosome

Abstract

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. WIREs RNA 2017, 8:e1381. doi: 10.1002/wrna.1381 For further resources related to this article, please visit the WIREs website.

Figure 1. Spliceosome assembly pathway is blocked at different stages by splicing inhibitors

Simplified schematic of the step-wise assembly of a spliceosome on a model pre-mRNA substrate consisting of two exons flanking an intron. Key splicing sequences are highlighted for the topmost substrate: 5' splice site (5' SS), branch point sequence (BP) and 3' splice site (3'SS). In actuality, the U snRNPs (U1, U2, U4, U5 and U6) are dynamic complexes that each contains a structured RNA and several proteins. The SF3B1 subunit of U2 snRNP, which is a target of many inhibitors, is indicated. The U snRNPs form several RNA-RNA interactions with each other and the pre-mRNA to first recognize the splice sites in E and A complex, before rearranging in B complex to create the catalytically active C complex. After the 1^st and 2^nd steps of splicing chemistry take place, the spliceosome disassociates into released mRNA product and intron lariat complex. Not illustrated are the dozens of additional protein components of the spliceosome. The assembly transitions affected by select splicing inhibitors are also highlighted.

Figure 2. Splicing inhibitors may impact competition between splicing substrates in cells

Different splice sites in pre-mRNA substrates (indicated by differently shaded exons flanking introns) compete for the spliceosome (ssome). Competitiveness is determined by a combination of splice site (SS) sequence strength, the presence of positive alternative splicing factors (alt SF) and total amount of pre-mRNA transcript (indicated by pre-mRNA size). Under normal cellular conditions (left panel) the weakest competitors "lose" for splicing (not engaged by ssome), typically resulting in alternative splicing changes. With splicing inhibitors (red and white "no" sign) additional splice sites will lose for splicing (right panel), resulting in alternative splicing changes and intron retention. Splicing events required for a cells growth (golden pre-mRNAs) will drive the response to the splicing inhibitor. Importantly, the relative competitiveness of an intron will differ in different cell types and under different conditions because the presence of alternative splicing factors differ along with pre-mRNA transcript levels.

Figure 3. Graphical summary of structure activity relationship studies for two SF3B1 inhibitors

A. FR901464. Functional group changes (in black outlined boxes) are linked by lines to the corresponding positions in the FR901464 structure (circled). Blue arrows point to the position of bond orientations changes (in blue outlined boxes). The effect and relative magnitude of specific changes on the compound's activity is indicated by the open arrow direction and color as indicated in the inset box legend. B. Pladienolide B. Same as panel A.