pladienolide-b and herboxidiene

The spliceosome mediates precursor mRNA splicing in eukaryotes, including the model organism Saccharomyces cerevisiae (yeast). Despite decades of study, no chemical inhibitors of yeast splicing in vivo are available. We have developed a system to efficiently inhibit splicing and block proliferation in living yeast cells using compounds that target the human spliceosome protein SF3B1. Potent inhibition is observed in yeast expressing a chimeric protein containing portions of human SF3B1. However, only a single point mutation in the yeast homolog of SF3B1 is needed for selective inhibition of splicing by pladienolide B, herboxidiene, or meayamycin in liquid culture. Mutations that enable inhibition also improve splicing of branch sites containing mismatches between the intron and small nuclear RNA-suggesting a link between inhibitor sensitivity and usage of weak branch sites in humans. This approach provides powerful new tools for manipulating splicing in live yeast and studies of spliceosome inhibitors.

Topics: Amino Acid Sequence; Epoxy Compounds; Fatty Alcohols; Humans; Macrolides; Mutagenesis; Phosphoproteins; Pyrans; Ribonucleoprotein, U2 Small Nuclear; RNA Precursors; RNA Splicing; RNA Splicing Factors; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Sequence Alignment; Small Molecule Libraries

Constitutive and alternative splicing of pre-mRNAs from multiexonic genes controls the diversity of the proteome; these precisely regulated processes also fine-tune responses to cues related to growth, development, and stresses. Small-molecule inhibitors that perturb splicing provide invaluable tools for use as chemical probes to uncover the molecular underpinnings of splicing regulation and as potential anticancer compounds.. Here, we show that herboxidiene (GEX1A) inhibits both constitutive and alternative splicing. Moreover, GEX1A activates genome-wide transcriptional patterns involved in abiotic stress responses in plants. GEX1A treatment -activated ABA-inducible promoters, and led to stomatal closure. Interestingly, GEX1A and pladienolide B (PB) elicited similar cellular changes, including alterations in the patterns of transcription and splicing, suggesting that these compounds might target the same spliceosome complex in plant cells.. Our study establishes GEX1A as a potent splicing inhibitor in plants that can be used to probe the assembly, dynamics, and molecular functions of the spliceosome and to study the interplay between splicing stress and abiotic stresses, as well as having potential biotechnological applications.

Topics: Abscisic Acid; Arabidopsis; Arabidopsis Proteins; Epoxy Compounds; Fatty Alcohols; Gene Expression Regulation, Plant; Germination; Macrolides; Promoter Regions, Genetic; Pyrans; RNA Precursors; RNA Splicing; RNA, Plant; Seeds; Serine-Arginine Splicing Factors; Stress, Physiological; Transcriptome

Pladienolide, herboxidiene and spliceostatin have been identified as splicing modulators that target SF3B1 in the SF3b subcomplex. Here we report that PHF5A, another component of this subcomplex, is also targeted by these compounds. Mutations in PHF5A-Y36, SF3B1-K1071, SF3B1-R1074 and SF3B1-V1078 confer resistance to these modulators, suggesting a common interaction site. RNA-seq analysis reveals that PHF5A-Y36C has minimal effect on basal splicing but inhibits the global action of splicing modulators. Moreover, PHF5A-Y36C alters splicing modulator-induced intron-retention/exon-skipping profile, which correlates with the differential GC content between adjacent introns and exons. We determine the crystal structure of human PHF5A demonstrating that Y36 is located on a highly conserved surface. Analysis of the cryo-EM spliceosome B

Topics: Adenosine; Alternative Splicing; Carrier Proteins; Cell Proliferation; Cell Survival; Cryoelectron Microscopy; Crystallography, X-Ray; Epoxy Compounds; Exons; Fatty Alcohols; HCT116 Cells; Humans; Introns; Macrolides; Mass Spectrometry; Mutagenesis, Site-Directed; Mutation; Myeloid Cell Leukemia Sequence 1 Protein; Phosphoproteins; Protein Binding; Protein Conformation; Pyrans; Recombinant Proteins; RNA Interference; RNA Splicing Factors; RNA-Binding Proteins; Sequence Analysis, RNA; Spiro Compounds; Spliceosomes; Trans-Activators

Small-molecule splice modulators have recently been recognized for their clinical potential for diverse cancers. This, combined with their use as tools to study the importance of splice-regulated events and their association with disease, continues to fuel the discovery of new splice modulators. One of the key challenges found in the current class of materials arises from their instability, where rapid metabolic degradation can lead to off-target responses. We now describe the preparation of bench-stable splice modulators by adapting carbohydrate motifs as a central scaffold to provide rapid access to potent splice modulators.

Topics: Alternative Splicing; Antineoplastic Agents; Carbohydrates; Drug Screening Assays, Antitumor; Epoxy Compounds; Fatty Alcohols; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Macrolides; Models, Molecular; Pyrans; Spliceosomes

Herboxidiene is a natural product that has previously been shown to exhibit antitumor activity by targeting the spliceosome. This activity makes herboxidiene a valuable starting point for the development of anticancer drugs. Here, we report an improved enantioselective synthesis of herboxidiene and the first report of its biologically active totally synthetic analog: 6-norherboxidiene. The synthesis of the tetrahydropyran moiety utilizes the novel application of inverse electron-demand Diels-Alder chemistry and the Ferrier-type rearrangement as key steps. We report, for the first time, cytotoxicity IC50s for synthetic herboxidiene and analogs in human tumor cell lines. We have also demonstrated that synthetic herboxidiene and its analogs can potently modulate the alternate splicing of MDM-2 pre-mRNA.

Topics: Alternative Splicing; Antineoplastic Agents; Cell Line, Tumor; Cell Survival; Cycloaddition Reaction; Drug Design; Epoxy Compounds; Fatty Alcohols; Humans; Inhibitory Concentration 50; Macrolides; Models, Molecular; Molecular Structure; Proto-Oncogene Proteins c-mdm2; Pyrans; RNA Precursors