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1.
Cell Chem Biol ; 28(8): 1145-1157.e6, 2021 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-33689684

RESUMO

Dysregulated pre-mRNA splicing is an emerging Achilles heel of cancers and myelodysplasias. To expand the currently limited portfolio of small-molecule drug leads, we screened for chemical modulators of the U2AF complex, which nucleates spliceosome assembly and is mutated in myelodysplasias. A hit compound specifically enhances RNA binding by a U2AF2 subunit. Remarkably, the compound inhibits splicing of representative substrates and stalls spliceosome assembly at the stage of U2AF function. Computational docking, together with structure-guided mutagenesis, indicates that the compound bridges the tandem U2AF2 RNA recognition motifs via hydrophobic and electrostatic moieties. Cells expressing a cancer-associated U2AF1 mutant are preferentially killed by treatment with the compound. Altogether, our results highlight the potential of trapping early spliceosome assembly as an effective pharmacological means to manipulate pre-mRNA splicing. By extension, we suggest that stabilizing assembly intermediates may offer a useful approach for small-molecule inhibition of macromolecular machines.


Assuntos
Precursores de RNA/efeitos dos fármacos , Splicing de RNA/efeitos dos fármacos , RNA Neoplásico/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Fator de Processamento U2AF/antagonistas & inibidores , Feminino , Células HEK293 , Humanos , Células K562 , Simulação de Acoplamento Molecular , Estrutura Molecular , Precursores de RNA/genética , Splicing de RNA/genética , RNA Neoplásico/genética , RNA Neoplásico/metabolismo , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/química , Fator de Processamento U2AF/genética , Fator de Processamento U2AF/metabolismo
2.
RNA Biol ; 16(10): 1346-1354, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31213125

RESUMO

Structural models of large and dynamic molecular complexes are appearing in increasing numbers, in large part because of recent technical advances in cryo-electron microscopy. However, the inherent complexity of such biological assemblies comprising dozens of moving parts often limits the resolution of structural models and leaves the puzzle as to how each functional configuration transitions to the next. Orthogonal biochemical information is crucial to understanding the molecular interactions that drive those rearrangements. We present a two-step method for chemical probing detected by tandem mass-spectrometry to globally assess the reactivity of lysine residues within purified macromolecular complexes. Because lysine side chains often balance the negative charge of RNA in ribonucleoprotein complexes, the method is especially useful for detecting changes in protein-RNA interactions. By probing the E. coli 30S ribosome subunit, we established that the reactivity pattern of lysine residues quantitatively reflects structure models derived from X-ray crystallography. We also used the strategy to assess differences in three conformations of purified human spliceosomes in the context of recent cryo-electron microscopy models. Our results demonstrate that the probing method yields powerful biochemical information that helps contextualize architectural rearrangements of intermediate resolution structures of macromolecular complexes, often solved in multiple conformations.


Assuntos
Lisina/química , Substâncias Macromoleculares/química , Modelos Moleculares , Conformação Molecular , Acetilação , Cristalografia por Raios X , Humanos , Peptídeos/química , RNA/química , Subunidades Ribossômicas Menores de Bactérias/metabolismo , Spliceossomos/metabolismo , Espectrometria de Massas em Tandem
3.
J Org Chem ; 83(9): 5187-5198, 2018 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-29696980

RESUMO

Thailanstatin A has been isolated recently from the fermentation broth of B. thailandensis MSMB43. We describe here an enantioselective convergent synthesis of thailanstatin A methyl ester and evaluation of its splicing activity. Synthesis of both highly functionalized tetrahydropyran rings were carried out from commercially available tri- O-acetyl-d-glucal as the key starting material. Our convergent synthesis involved the synthesis of both tetrahydropyran fragments in a highly stereoselective manner. The fragments were then coupled using cross-metathesis as the key step. The synthesis of the diene subunit included a highly stereoselective Claisen rearrangement, a Cu(I)-mediated conjugate addition of MeLi to set the C-14 methyl stereochemistry, a reductive amination reaction to install the C16-amine functionality, and a Wittig olefination reaction to incorporate the diene unit. The epoxy alcohol subunit was synthesized by a highly selective anomeric allylation, a Peterson olefination, and a vanadium catalyzed epoxidation that installed the epoxide stereoselectively. Cross-metathesis of the olefins provided the methyl ester derivative of thailanstatin A. We have carried out in vitro splicing studies of the methyl ester derivative, which proved to be a potent inhibitor of the spliceosome.


Assuntos
Ésteres/química , Piranos/síntese química , Piranos/farmacologia , Splicing de RNA/efeitos dos fármacos , Técnicas de Química Sintética , Piranos/química , Estereoisomerismo
4.
RNA ; 24(6): 769-777, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29487104

RESUMO

Prp8 is an essential protein that regulates spliceosome assembly and conformation during pre-mRNA splicing. Recent cryo-EM structures of the spliceosome model Prp8 as a scaffold for the spliceosome's catalytic U snRNA components. Using a new amino acid probing strategy, we identified a dynamic region in human Prp8 that is positioned to stabilize the pre-mRNA in the spliceosome active site through interactions with U5 snRNA. Mutagenesis of the identified Prp8 residues in yeast indicates a role in 5' splice site recognition. Genetic interactions with spliceosome proteins Isy1, which buttresses the intron branch point, and Snu114, a regulatory GTPase that directly contacts Prp8, further corroborate a role for the same Prp8 residues in substrate positioning and activation. Together the data suggest that adjustments in interactions between Prp8 and U5 snRNA help establish proper positioning of the pre-mRNA into the active site to enhance 5' splice site fidelity.


Assuntos
Precursores de RNA/genética , Sítios de Splice de RNA , RNA Nuclear Pequeno/genética , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteína Nuclear Pequena U4-U6/metabolismo , Ribonucleoproteína Nuclear Pequena U5/metabolismo , Saccharomyces cerevisiae/genética , Domínio Catalítico , Humanos , Proteínas de Ligação a RNA/genética , Ribonucleoproteína Nuclear Pequena U4-U6/genética , Ribonucleoproteína Nuclear Pequena U5/genética , Saccharomyces cerevisiae/metabolismo , Spliceossomos
5.
Org Lett ; 20(1): 96-99, 2018 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-29218995

RESUMO

An enantioselective total synthesis of spliceostatin G has been accomplished. The synthesis involved a Suzuki cross-coupling reaction as a key step. The functionalized tetrahydropyran ring was constructed from commercially available optically active tri-O-acetyl-d-glucal. Other key reactions include a highly stereoselective Claisen rearrangement, a Cu(I)-mediated 1,4 addition of MeLi to install the C8 methyl group, and a reductive amination to incorporate the C10 amine functionality of spliceostatin G. Biological evaluation of synthetic spliceostatin G and its methyl ester revealed that it does not inhibit splicing in vitro.


Assuntos
Ésteres/química , Aminação , Estrutura Molecular , Splicing de RNA , Compostos de Espiro , Estereoisomerismo
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