Assuntos
Antibióticos Antineoplásicos , Cardiotoxicidade , Doxorrubicina , Miócitos Cardíacos , Humanos , Doxorrubicina/efeitos adversos , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Antibióticos Antineoplásicos/efeitos adversos , Splicing de RNA/efeitos dos fármacos , Processamento Alternativo/efeitos dos fármacosRESUMO
The self-splicing group II introns are bacterial and organellar ancestors of the nuclear spliceosome and retro-transposable elements of pharmacological and biotechnological importance. Integrating enzymatic, crystallographic, and simulation studies, we demonstrate how these introns recognize small molecules through their conserved active site. These RNA-binding small molecules selectively inhibit the two steps of splicing by adopting distinctive poses at different stages of catalysis, and by preventing crucial active site conformational changes that are essential for splicing progression. Our data exemplify the enormous power of RNA binders to mechanistically probe vital cellular pathways. Most importantly, by proving that the evolutionarily-conserved RNA core of splicing machines can recognize small molecules specifically, our work provides a solid basis for the rational design of splicing modulators not only against bacterial and organellar introns, but also against the human spliceosome, which is a validated drug target for the treatment of congenital diseases and cancers.
Assuntos
Domínio Catalítico , Íntrons , Splicing de RNA , Spliceossomos , Splicing de RNA/efeitos dos fármacos , Spliceossomos/metabolismo , Spliceossomos/efeitos dos fármacos , Humanos , Íntrons/genética , Bibliotecas de Moléculas Pequenas/farmacologia , Bibliotecas de Moléculas Pequenas/químicaRESUMO
Antisense oligonucleotides (ASOs) are single stranded nucleic acids that target RNA. The US Food and Drug Administration has approved ASOs for several diseases. ASOs utilize three principal modes of action (MOA). The first MOA is initiated by base-pairing between the ASO and its target mRNA, followed by RNase H-dependent mRNA degradation. The second MOA is triggered by ASOs that occlude splice acceptor sites in pre-mRNAs leading to skipping of a mutation-bearing exon. The third MOA involves ASOs that sterically hinder mRNA function, often inhibiting translation. ASOs contain a variety of modifications to the sugar-phosphate backbone and bases that stabilize the ASO or render them resistant to RNase activity. RNase H-dependent ASOs include inotersen and eplontersen (for hereditary transthyretin amyloidosis), fomiversen (for opportunistic cytomegalovirus infection), mipomersen (for familial hypercholesterolemia), and tofersen [for amyotrophic lateral sclerosis (ALS)]. Splice modulating ASOs include nursinersen (for spinal muscular atrophy) and eteplirsen, golodirsen, viltolarsen, and casimersen (all for the treatment of Duchenne muscular dystrophy). In addition, a designer ASO, milasen, was used to treat a single individual afflicted with Batten disease. Since ASO design relies principally upon knowledge of mRNA sequence, the bench to bedside pipeline for ASOs is expedient compared with protein-directed drugs. [Graphical abstract available.].
Assuntos
Aprovação de Drogas , Oligonucleotídeos Antissenso , United States Food and Drug Administration , Humanos , Oligonucleotídeos Antissenso/uso terapêutico , Oligonucleotídeos Antissenso/farmacologia , Estados Unidos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Animais , Splicing de RNA/efeitos dos fármacosRESUMO
ABSTRACT: Provirus integration site for Moloney murine leukemia virus (PIM) family serine/threonine kinases perform protumorigenic functions in hematologic malignancies and solid tumors by phosphorylating substrates involved in tumor metabolism, cell survival, metastasis, inflammation, and immune cell invasion. However, a comprehensive understanding of PIM kinase functions is currently lacking. Multiple small-molecule PIM kinase inhibitors are currently being evaluated as cotherapeutics in patients with cancer. To further illuminate PIM kinase functions in cancer, we deeply profiled PIM1 substrates using the reverse in-gel kinase assay to identify downstream cellular processes targetable with small molecules. Pathway analyses of putative PIM substrates nominated RNA splicing and ribosomal RNA (rRNA) processing as PIM-regulated cellular processes. PIM inhibition elicited reproducible splicing changes in PIM-inhibitor-responsive acute myeloid leukemia (AML) cell lines. PIM inhibitors synergized with splicing modulators targeting splicing factor 3b subunit 1 (SF3B1) and serine-arginine protein kinase 1 (SRPK1) to kill AML cells. PIM inhibition also altered rRNA processing, and PIM inhibitors synergized with an RNA polymerase I inhibitor to kill AML cells and block AML tumor growth. These data demonstrate that deep kinase substrate knowledge can illuminate unappreciated kinase functions, nominating synergistic cotherapeutic strategies. This approach may expand the cotherapeutic armamentarium to overcome kinase inhibitor-resistant disease that limits durable responses in malignant disease.
Assuntos
Leucemia Mieloide Aguda , Inibidores de Proteínas Quinases , Proteínas Proto-Oncogênicas c-pim-1 , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Humanos , Proteínas Proto-Oncogênicas c-pim-1/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-pim-1/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Camundongos , Animais , Linhagem Celular Tumoral , Especificidade por Substrato , Splicing de RNA/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Hepatocellular carcinoma (HCC) is the main histological subtype of primary liver cancer and remains one of the most common solid malignancies globally. Ferroptosis was recently defined as an iron-catalyzed form of regulated necrosis. Because cancer cells exhibit higher iron requirements than noncancer cells, treatment with ferroptosis-inducing compounds may be a feasible strategy for cancer therapy. However, cancer cells develop acquired resistance to evade ferroptosis, and the mechanisms responsible for ferroptosis resistance are not fully clarified. In the current study, we reported that DDX39B was downregulated during sorafenib-induced ferroptosis in a dose- and time-dependent manner. Exogenous introduction of DDX39B ensured the survival of HCC cells upon exposure to sorafenib, while the opposite phenomenon was observed in DDX39B-silenced HCC cells. Mechanistically, we demonstrated that DDX39B increased GPX4 levels by promoting the splicing and cytoplasmic translocation of GPX4 pre-mRNA, which was sufficient to detoxify sorafenib-triggered excess lipid ROS production, lipid peroxidation accumulation, ferrous iron levels, and mitochondrial damage. Inhibition of DDX39B ATPase activity by CCT018159 repressed the splicing and cytoplasmic export of GPX4 pre-mRNA and synergistically assisted sorafenib-induced ferroptotic cell death in HCC cells. Taken together, our data uncover a novel role for DDX39B in ferroptosis resistance by modulating the maturation of GPX4 mRNA via a posttranscriptional approach and suggest that DDX39B inhibition may be a promising therapeutic strategy to enhance the sensitivity and vulnerability of HCC cells to sorafenib.
Assuntos
Antineoplásicos , Carcinoma Hepatocelular , RNA Helicases DEAD-box , Ferroptose , Neoplasias Hepáticas , Fosfolipídeo Hidroperóxido Glutationa Peroxidase , Precursores de RNA , Sorafenibe , Ferroptose/efeitos dos fármacos , Ferroptose/fisiologia , Sorafenibe/farmacologia , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/genética , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/patologia , RNA Helicases DEAD-box/metabolismo , RNA Helicases DEAD-box/genética , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/metabolismo , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/genética , Precursores de RNA/metabolismo , Precursores de RNA/genética , Antineoplásicos/farmacologia , Animais , Camundongos , Splicing de RNA/efeitos dos fármacos , Camundongos Nus , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Camundongos Endogâmicos BALB C , Masculino , Citoplasma/metabolismo , Citoplasma/efeitos dos fármacosRESUMO
Perturbation of protein phosphorylation represents an attractive approach to cancer treatment. Besides kinase inhibitors, protein phosphatase inhibitors have been shown to have anti-cancer activity. A prime example is the small molecule LB-100, an inhibitor of protein phosphatases 2A/5 (PP2A/PP5), enzymes that affect cellular physiology. LB-100 has proven effective in pre-clinical models in combination with immunotherapy, but the molecular underpinnings of this synergy remain understood poorly. We report here a sensitivity of the mRNA splicing machinery to phosphorylation changes in response to LB-100 in colorectal adenocarcinoma. We observe enrichment for differentially phosphorylated sites within cancer-critical splicing nodes of U2 snRNP, SRSF and hnRNP proteins. Altered phosphorylation endows LB-100-treated colorectal adenocarcinoma cells with differential splicing patterns. In PP2A-inhibited cells, over 1000 events of exon skipping and intron retention affect regulators of genomic integrity. Finally, we show that LB-100-evoked alternative splicing leads to neoantigens that are presented by MHC class 1 at the cell surface. Our findings provide a potential explanation for the pre-clinical and clinical observations that LB-100 sensitizes cancer cells to immune checkpoint blockade.
Assuntos
Neoplasias do Colo , Splicing de RNA , Humanos , Processamento Alternativo/efeitos dos fármacos , Antígenos de Neoplasias/metabolismo , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/imunologia , Linhagem Celular Tumoral , Neoplasias do Colo/genética , Neoplasias do Colo/imunologia , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/metabolismo , Inibidores Enzimáticos/farmacologia , Fosforilação , Proteína Fosfatase 2/metabolismo , Splicing de RNA/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Processamento de Serina-Arginina/metabolismo , Fatores de Processamento de Serina-Arginina/genética , Piperazinas/farmacologiaRESUMO
Pharmacological modulation of RNA splicing by small molecules is an emerging facet of drug discovery. In this context, the SMN2 splicing modifier SMN-C5 was used as a prototype to understand the mode of action of small molecule splicing modifiers and propose the concept of 5'-splice site bulge repair. In this study, we combined in vitro binding assays and structure determination by NMR spectroscopy to identify the binding modes of four other small molecule splicing modifiers that switch the splicing of either the SMN2 or the HTT gene. Here, we determined the solution structures of risdiplam, branaplam, SMN-CX and SMN-CY bound to the intermolecular RNA helix epitope containing an unpaired adenine within the G-2A-1G+1U+2 motif of the 5'-splice site. Despite notable differences in their scaffolds, risdiplam, SMN-CX, SMN-CY and branaplam contact the RNA epitope similarly to SMN-C5, suggesting that the 5'-splice site bulge repair mechanism can be generalised. These findings not only deepen our understanding of the chemical diversity of splicing modifiers that target A-1 bulged 5'-splice sites, but also identify common pharmacophores required for modulating 5'-splice site selection with small molecules.
Assuntos
Desenho de Fármacos , Sítios de Splice de RNA , Splicing de RNA , Humanos , Compostos Azo , Modelos Moleculares , Conformação de Ácido Nucleico , Pirimidinas , Splicing de RNA/efeitos dos fármacos , Proteína 2 de Sobrevivência do Neurônio Motor/genética , Proteína 2 de Sobrevivência do Neurônio Motor/metabolismoRESUMO
Alternative splicing is a co-transcriptional process that significantly contributes to the molecular landscape of the cell. It plays a multifaceted role in shaping gene transcription, protein diversity, and functional adaptability in response to environmental cues. Recent studies demonstrate that drugs of abuse have a profound impact on alternative splicing patterns within different brain regions. Drugs like alcohol and cocaine modify the expression of genes responsible for encoding splicing factors, thereby influencing alternative splicing of crucial genes involved in neurotransmission, neurogenesis, and neuroinflammation. Notable examples of these alterations include alcohol-induced changes in splicing factors such as HSPA6 and PCBP1, as well as cocaine's impact on PTBP1 and SRSF11. Beyond the immediate effects of drug exposure, recent research has shed light on the role of alternative splicing in contributing to the risk of substance use disorders (SUDs). This is exemplified by exon skipping events in key genes like ELOVL7, which can elevate the risk of alcohol use disorder. Lastly, drugs of abuse can induce splicing alterations through epigenetic modifications. For example, cocaine exposure leads to alterations in levels of trimethylated lysine 36 of histone H3, which exhibits a robust association with alternative splicing and serves as a reliable predictor for exon exclusion. In summary, alternative splicing has emerged as a critical player in the complex interplay between drugs of abuse and the brain, offering insights into the molecular underpinnings of SUDs.
Assuntos
Encéfalo , Transtornos Relacionados ao Uso de Substâncias , Humanos , Transtornos Relacionados ao Uso de Substâncias/genética , Transtornos Relacionados ao Uso de Substâncias/metabolismo , Encéfalo/metabolismo , Encéfalo/efeitos dos fármacos , Animais , Processamento Alternativo , Splicing de RNA/efeitos dos fármacosRESUMO
Stargardt disease type 1 (STGD1), the most common form of hereditary macular dystrophy, can be caused by biallelic combinations of over 2200 variants in the ABCA4 gene. This leads to reduced or absent ABCA4 protein activity, resulting in toxic metabolite accumulation in the retina and damage of the retinal pigment epithelium and photoreceptors. Approximately 21% of all ABCA4 variants that contribute to disease influence ABCA4 pre-mRNA splicing. This emphasizes the need for therapies to restore disrupted ABCA4 splicing and halt STGD1 progression. Previously, QR-1011, an antisense oligonucleotide (AON), successfully corrected splicing abnormalities and restored normal ABCA4 protein translation in human retinal organoids carrying the prevalent disease-causing variant c.5461-10T>C in ABCA4. Here, we investigated whether QR-1011 could also correct splicing in four less common non-canonical splice site (NCSS) variants flanking ABCA4 exon 39: c.5461-8T>G, c.5461-6T>C, c.5584+5G>A and c.5584+6T>C. We administered QR-1011 and three other AONs to midigene-transfected cells and demonstrate that QR-1011 had the most pronounced effect on splicing compared to the others. Moreover, QR-1011 significantly increased full-length ABCA4 transcript levels for c.5461-8T>G and c.5584+6T>C. Splicing restoration could not be achieved in the other two variants, suggesting their more severe effect on splicing. Overall, QR-1011, initially developed for a single ABCA4 variant, exhibited potent splice correction capabilities for two additional severe NCSS variants nearby. This suggests the possibility of a broader therapeutic impact of QR-1011 extending beyond its original target and highlights the potential for treating a larger population of STGD1 patients affected by multiple severe ABCA4 variants with a single AON.
Assuntos
Transportadores de Cassetes de Ligação de ATP , Oligodesoxirribonucleotídeos Antissenso , Organoides , Doença de Stargardt , Humanos , Transportadores de Cassetes de Ligação de ATP/genética , Éxons , Retina/citologia , Splicing de RNA/efeitos dos fármacos , Doença de Stargardt/tratamento farmacológico , Doença de Stargardt/genética , Oligodesoxirribonucleotídeos Antissenso/farmacologia , Organoides/efeitos dos fármacosRESUMO
No fully effective treatment has been developed since the discovery of Chagas' disease by Carlos Chagas in 1909. Since drug-resistant Trypanosoma cruzi strains are occurring and the current therapy is effectiveness in the acute phase but with various adverse side effects, more studies are needed to characterize the susceptibility of T. cruzi to new drugs. Many natural and/or synthetic substances showing trypanocidal activity have been used, even though they are not likely to be turned into clinically approved drugs. Originally, drug screening was performed using natural products, with only limited knowledge of the molecular mechanism involved in the development of diseases. Trans-splicing, which is unusual RNA processing reaction and occurs in nematodes and trypanosomes, implies the processing of polycistronic transcription units into individual mRNAs; a short transcript spliced leader (SL RNA) is trans-spliced to the acceptor pre-mRNA, giving origin to the mature mRNA. In the present study, permeable cells of T. cruzi epimastigote forms (Y, BOL and NCS strains) were treated to evaluate the interference of two drugs (hydroxymethylnitrofurazone - NFOH-121 and nitrofurazone) in the trans-splicing reaction using silver-stained PAGE analysis. Both drugs induced a significant reduction in RNA processing at concentrations from 5 to 12.5 æM. These data agreed with the biological findings, since the number of parasites decreased, especially with NFOH-121. This proposed methodology allows a rapid and cost-effective screening strategy for detecting drug interference in the trans-splicing mechanism of T. cruzi.