RESUMO
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.
Assuntos
Fosfoproteínas/antagonistas & inibidores , Precursores de RNA/genética , Splicing de RNA , RNA Mensageiro/genética , Ribonucleoproteína Nuclear Pequena U2/antagonistas & inibidores , Trifosfato de Adenosina/metabolismo , Sítios de Ligação , Éxons , Humanos , Fatores de Processamento de RNARESUMO
The spliceosome is a dynamic complex of five structural RNAs and dozens of proteins, which assemble together to remove introns from nascent eukaryotic gene transcripts in a process called splicing. Small molecules that target different components of the spliceosome represent valuable research tools to investigate this complicated macromolecular machine. However, the current collection of spliceosome inhibitors is very limited. To expand the toolkit we used a high-throughput in vitro splicing assay to screen a collection of pre-fractions of natural compounds derived from marine bacteria for splicing inhibition. Further fractionation of initial hits generated individual peaks of splicing inhibitors that interfere with different stages of spliceosome assembly. With additional characterization of individual peaks, we identified N-palmitoyl-l-leucine as a new splicing inhibitor that blocks a late stage of spliceosome assembly. Structure-activity relationship analysis of the compound revealed that length of carbon chain is important for activity in splicing, as well as for effects on the cytological profile of cells in culture. Together these results demonstrate that our combination of in vitro splicing analysis with complex natural product libraries is a powerful strategy for identifying new small molecule tools with which to probe different aspects of spliceosome assembly and function.
Assuntos
Produtos Biológicos/farmacologia , Leucina/análogos & derivados , Splicing de RNA/efeitos dos fármacos , Spliceossomos/efeitos dos fármacos , Produtos Biológicos/química , Produtos Biológicos/isolamento & purificação , Células HeLa , Ensaios de Triagem em Larga Escala , Humanos , Íntrons , Leucina/química , Leucina/isolamento & purificação , Leucina/farmacologia , RNA Mensageiro/química , RNA Mensageiro/genética , Spliceossomos/metabolismo , Relação Estrutura-AtividadeRESUMO
Herboxidiene is a potent inhibitor of spliceosomes. It exhibits excellent anticancer activity against multiple human cancer cell lines. Herein, we describe an enantioselective synthesis of a desmethyl derivative and the corresponding carba-derivatives of herboxidiene. The synthesis involved Suzuki coupling of a vinyl iodide with boronate as the key reaction. For the synthesis of carba-derivatives, the corresponding optically active cyclohexane-1,3-dicarbonyl derivatives were synthesized using an enantioselective desymmetrization of meso-anhydride. The biological properties of these derivatives were evaluated in an in vitro splicing assay.
Assuntos
Desenho de Fármacos , Álcoois Graxos/síntese química , Álcoois Graxos/farmacologia , Piranos/síntese química , Piranos/farmacologia , Splicing de RNA/efeitos dos fármacos , Ácidos Borônicos/química , Técnicas de Química Sintética , Álcoois Graxos/química , Concentração Inibidora 50 , Piranos/química , EstereoisomerismoRESUMO
Pladienolide B (PB) is a potent cancer cell growth inhibitor that targets the SF3B1 subunit of the spliceosome. There is considerable interest in the compound as a potential chemotherapeutic, as well as a tool to study SF3B1 function in splicing and cancer development. The molecular structure of PB, a bacterial natural product, contains a 12-member macrolide ring with an extended epoxide-containing side chain. Using a novel concise enantioselective synthesis, we created a series of PB structural analogs and the structurally related compound herboxidiene. We show that two methyl groups in the PB side chain, as well as a feature of the macrolide ring shared with herboxidiene, are required for splicing inhibition in vitro. Unexpectedly, we find that the epoxy group contributes only modestly to PB potency and is not absolutely necessary for activity. The orientations of at least two chiral centers off the macrolide ring have no effect on PB activity. Importantly, the ability of analogs to inhibit splicing in vitro directly correlated with their effects in a series of cellular assays. Those effects likely arise from inhibition of some, but not all, endogenous splicing events in cells, as previously reported for the structurally distinct SF3B1 inhibitor spliceostatin A. Together, our data support the idea that the impact of PB on cells is derived from its ability to impair the function of SF3B1 in splicing and also demonstrate that simplification of the PB scaffold is feasible.
Assuntos
Antineoplásicos/farmacologia , Compostos de Epóxi/química , Macrolídeos/química , Proteínas de Neoplasias/antagonistas & inibidores , Neoplasias/metabolismo , Fosfoproteínas/antagonistas & inibidores , Splicing de RNA/efeitos dos fármacos , Ribonucleoproteína Nuclear Pequena U2/antagonistas & inibidores , Antineoplásicos/química , Células HeLa , Humanos , Proteínas de Neoplasias/química , Proteínas de Neoplasias/metabolismo , Neoplasias/tratamento farmacológico , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Piranos/química , Piranos/farmacologia , Fatores de Processamento de RNA , Ribonucleoproteína Nuclear Pequena U2/química , Ribonucleoproteína Nuclear Pequena U2/metabolismo , Compostos de Espiro/química , Compostos de Espiro/farmacologiaRESUMO
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.
Assuntos
Piranos/química , Piranos/síntese química , Piranos/farmacologia , Compostos de Espiro/química , Compostos de Espiro/síntese química , Compostos de Espiro/farmacologia , Alcenos/química , Catálise , Linhagem Celular Tumoral , Humanos , Estrutura Molecular , Splicing de RNA , EstereoisomerismoRESUMO
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 approximately 20S and approximately 35-40S and present the three-dimensional structures of both complexes by electron microscopy. The approximately 35-40S complexes consist of a platform density packed against a semispherical element. The approximately 20S 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 approximately 20S 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.
Assuntos
Edição de RNA , RNA de Protozoário/metabolismo , Trypanosoma/metabolismo , Animais , Microscopia Crioeletrônica , Modelos Biológicos , Modelos Moleculares , Proteínas de Protozoários/química , Proteínas de Protozoários/isolamento & purificação , Proteínas de Protozoários/ultraestrutura , RNA de Protozoário/química , RNA de Protozoário/isolamento & purificação , RNA de Protozoário/ultraestrutura , Trypanosoma/ultraestrutura , UltracentrifugaçãoRESUMO
Pre-mRNA splicing is a key controller of human gene expression. Disturbances in splicing due to mutation lead to dysregulated protein expression and contribute to a substantial fraction of human disease. Several classes of splicing modulator compounds (SMCs) have been recently identified and establish that pre-mRNA splicing represents a target for therapy. We describe herein the identification of BPN-15477, a SMC that restores correct splicing of ELP1 exon 20. Using transcriptome sequencing from treated fibroblast cells and a machine learning approach, we identify BPN-15477 responsive sequence signatures. We then leverage this model to discover 155 human disease genes harboring ClinVar mutations predicted to alter pre-mRNA splicing as targets for BPN-15477. Splicing assays confirm successful correction of splicing defects caused by mutations in CFTR, LIPA, MLH1 and MAPT. Subsequent validations in two disease-relevant cellular models demonstrate that BPN-15477 increases functional protein, confirming the clinical potential of our predictions.
Assuntos
Aprendizado Profundo , Marcação de Genes/métodos , Splicing de RNA , Animais , Biologia Computacional , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Éxons , Células HEK293 , Humanos , Camundongos , Camundongos Transgênicos , Proteína 1 Homóloga a MutL/genética , Mutação , Fenetilaminas/administração & dosagem , Piridazinas/administração & dosagem , Esterol Esterase/genética , Transcriptoma , Proteínas tau/genéticaRESUMO
Huntington's disease (HD) is a hereditary neurodegenerative disorder caused by expansion of cytosine-adenine-guanine (CAG) trinucleotide repeats in the huntingtin (HTT) gene. Consequently, the mutant protein is ubiquitously expressed and drives pathogenesis of HD through a toxic gain-of-function mechanism. Animal models of HD have demonstrated that reducing huntingtin (HTT) protein levels alleviates motor and neuropathological abnormalities. Investigational drugs aim to reduce HTT levels by repressing HTT transcription, stability or translation. These drugs require invasive procedures to reach the central nervous system (CNS) and do not achieve broad CNS distribution. Here, we describe the identification of orally bioavailable small molecules with broad distribution throughout the CNS, which lower HTT expression consistently throughout the CNS and periphery through selective modulation of pre-messenger RNA splicing. These compounds act by promoting the inclusion of a pseudoexon containing a premature termination codon (stop-codon psiExon), leading to HTT mRNA degradation and reduction of HTT levels.
Assuntos
Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Doença de Huntington/tratamento farmacológico , Doença de Huntington/genética , Splicing de RNA , Bibliotecas de Moléculas Pequenas/administração & dosagem , Animais , Sistema Nervoso Central/efeitos dos fármacos , Sistema Nervoso Central/metabolismo , Modelos Animais de Doenças , Humanos , Doença de Huntington/metabolismo , Camundongos , Splicing de RNA/efeitos dos fármacos , Estabilidade de RNA/efeitos dos fármacos , Expansão das Repetições de Trinucleotídeos/efeitos dos fármacosRESUMO
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.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Precursores de RNA/genética , Splicing de RNA/genética , Bibliotecas de Moléculas Pequenas/farmacologia , Spliceossomos/genética , Animais , HumanosRESUMO
An enantioselective total synthesis of GEX1Q1 has been accomplished in a convergent manner. The C-5 asymmetric center has now been assigned through synthesis. GEX1Q1 displayed slightly better spliceosome inhibitory activity over its C-5 epimer. The salient features of this synthesis include an asymmetric hetero-Diels-Alder reaction to construct the tetrahydropyran ring and a Suzuki cross-coupling to assemble the key segments.
Assuntos
Álcoois Graxos/síntese química , Piranos/síntese química , Reação de Cicloadição , Álcoois Graxos/química , Álcoois Graxos/farmacologia , Macrolídeos , Estrutura Molecular , Piranos/química , Piranos/farmacologia , Spliceossomos , EstereoisomerismoRESUMO
An enantioselective total synthesis of spliceostatin E has been accomplished. The δ-lactone unit A was constructed from readily available (R)-glycidyl alcohol using a ring-closing olefin metathesis as the key reaction. A cross-metathesis of ring A containing δ-lactone and the functionalized tetrahydropyran B-ring provided spliceostatin E. Our biological evaluation of synthetic spliceostatin E revealed that it does not inhibit splicing in vitro and does not impact speckle morphology in cells. Spliceostatin E was reported to possess potent antitumor activity.
Assuntos
Antineoplásicos/síntese química , Lactonas/síntese química , Lactonas/farmacologia , Piranos/síntese química , Piranos/farmacologia , Pironas/síntese química , Pironas/farmacologia , Compostos de Espiro/síntese química , Compostos de Espiro/farmacologia , Antineoplásicos/química , Antineoplásicos/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais , Células HeLa , Humanos , Lactonas/química , Estrutura Molecular , Piranos/química , Pironas/química , Compostos de Espiro/química , EstereoisomerismoRESUMO
The spliceosome is the macromolecular machine responsible for pre-mRNA splicing, an essential step in eukaryotic gene expression. During splicing, myriad subunits join and leave the spliceosome as it works on the pre-mRNA substrate. Strikingly, there are very few small molecules known to interact with the spliceosome. Splicing inhibitors are needed to capture transient spliceosome conformations and probe important functional components. Such compounds may also have chemotherapeutic applications, as links between splicing and cancer are increasingly uncovered. To identify new splicing inhibitors, we developed a high-throughput assay for in vitro splicing using a reverse transcription followed by quantitative PCR readout. In a pilot screen of 3080 compounds, we identified three small molecules that inhibit splicing in HeLa extract by interfering with different stages of human spliceosome assembly. Two of the compounds similarly affect spliceosomes in yeast extracts, suggesting selective targeting of conserved components. By examining related molecules, we identified chemical features required for the activity of two of the splicing inhibitors. In addition to verifying our assay procedure and paving the way to larger screens, these studies establish new compounds as chemical probes for investigating the splicing machinery.
Assuntos
Ensaios de Triagem em Larga Escala , Precursores de RNA/antagonistas & inibidores , Splicing de RNA/efeitos dos fármacos , Saccharomyces cerevisiae/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Spliceossomos/efeitos dos fármacos , Células HeLa , Humanos , Reação em Cadeia da Polimerase , Precursores de RNA/química , Precursores de RNA/metabolismo , Transcrição Reversa , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Bibliotecas de Moléculas Pequenas/química , Spliceossomos/química , Spliceossomos/metabolismo , Relação Estrutura-AtividadeRESUMO
Spliceosome assembly requires several structural rearrangements to position the components of the catalytic core. Many of these rearrangements involve successive strengthening and weakening of different RNA:RNA and RNA:proteins interactions within the complex. To gain insight into the organization of the catalytic core of the spliceosome arrested between the two steps of splicing chemistry (C complex), we investigated the effects of exposing C complex to low concentrations of urea. We find that in the presence of 3M urea C complex separates into at least three sub-complexes. One sub-complex contains the 5'exon, another contains the intron-lariat intermediate, and U2/U5/U6 snRNAs likely comprise a third sub-complex. We purified the intron-lariat intermediate sub-complex and identified several proteins, including U2 snRNP and PRP19 complex (NTC) components. The data from our study indicate that U2 snRNP proteins in C complex are more stably associated with the lariat-intron intermediate than the U2 snRNA. The results also suggest a set of candidate proteins that hold the lariat-intron intermediate together in C complex. This information is critical for further interpreting the complex architecture of the mammalian spliceosome.