RESUMO
Glioblastoma (GBM) is a deadly cancer in which cancer stem cells (CSCs) sustain tumor growth and contribute to therapeutic resistance. Protein arginine methyltransferase 5 (PRMT5) has recently emerged as a promising target in GBM. Using two orthogonal-acting inhibitors of PRMT5 (GSK591 or LLY-283), we show that pharmacological inhibition of PRMT5 suppresses the growth of a cohort of 46 patient-derived GBM stem cell cultures, with the proneural subtype showing greater sensitivity. We show that PRMT5 inhibition causes widespread disruption of splicing across the transcriptome, particularly affecting cell cycle gene products. We identify a GBM splicing signature that correlates with the degree of response to PRMT5 inhibition. Importantly, we demonstrate that LLY-283 is brain-penetrant and significantly prolongs the survival of mice with orthotopic patient-derived xenografts. Collectively, our findings provide a rationale for the clinical development of brain penetrant PRMT5 inhibitors as treatment for GBM.
Assuntos
Antineoplásicos/farmacologia , Neoplasias Encefálicas/metabolismo , Glioblastoma/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Animais , Apoptose , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Descoberta de Drogas , Epigenômica , Feminino , Regulação Neoplásica da Expressão Gênica , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Glioblastoma/patologia , Humanos , Camundongos , Células-Tronco Neoplásicas/metabolismo , Proteína-Arginina N-Metiltransferases/efeitos dos fármacos , Proteína-Arginina N-Metiltransferases/genética , Splicing de RNA , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Protein arginine methyltransferase 5 (PRMT5) is a type II arginine methyltransferase that catalyzes the formation of symmetric dimethylarginine in a number of nuclear and cytoplasmic proteins. Although the cellular functions of PRMT5 have not been fully unraveled, it has been implicated in a number of cellular processes like RNA processing, signal transduction, and transcriptional regulation. PRMT5 is ubiquitously expressed in most tissues and its expression has been shown to be elevated in several cancers including breast cancer, gastric cancer, glioblastoma, and lymphoma. Here, we describe the identification and characterization of a novel and selective PRMT5 inhibitor with potent in vitro and in vivo activity. Compound 1 (also called LLY-283) inhibited PRMT5 enzymatic activity in vitro and in cells with IC50 of 22 ± 3 and 25 ± 1 nM, respectively, while its diastereomer, compound 2 (also called LLY-284), was much less active. Compound 1 also showed antitumor activity in mouse xenografts when dosed orally and can serve as an excellent probe molecule for understanding the biological function of PRMT5 in normal and cancer cells.
RESUMO
MDM4 is a promising target for cancer therapy, as it is undetectable in most normal adult tissues but often upregulated in cancer cells to dampen p53 tumor-suppressor function. The mechanisms that underlie MDM4 upregulation in cancer cells are largely unknown. Here, we have shown that this key oncogenic event mainly depends on a specific alternative splicing switch. We determined that while a nonsense-mediated, decay-targeted isoform of MDM4 (MDM4-S) is produced in normal adult tissues as a result of exon 6 skipping, enhanced exon 6 inclusion leads to expression of full-length MDM4 in a large number of human cancers. Although this alternative splicing event is likely regulated by multiple splicing factors, we identified the SRSF3 oncoprotein as a key enhancer of exon 6 inclusion. In multiple human melanoma cell lines and in melanoma patient-derived xenograft (PDX) mouse models, antisense oligonucleotide-mediated (ASO-mediated) skipping of exon 6 decreased MDM4 abundance, inhibited melanoma growth, and enhanced sensitivity to MAPK-targeting therapeutics. Additionally, ASO-based MDM4 targeting reduced diffuse large B cell lymphoma PDX growth. As full-length MDM4 is enhanced in multiple human tumors, our data indicate that this strategy is applicable to a wide range of tumor types. We conclude that enhanced MDM4 exon 6 inclusion is a common oncogenic event and has potential as a clinically compatible therapeutic target.
Assuntos
Éxons , Melanoma/terapia , Proteínas Nucleares/genética , Oligonucleotídeos Antissenso/farmacologia , Proteínas Proto-Oncogênicas/genética , Animais , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células , Humanos , Melanoma/patologia , Camundongos , Proteínas de Ligação a RNA/fisiologia , Fatores de Processamento de Serina-Arginina , Proteína Supressora de Tumor p53/fisiologiaRESUMO
The tight control of gene expression at the level of both transcription and post-transcriptional RNA processing is essential for mammalian development. We here investigate the role of protein arginine methyltransferase 5 (PRMT5), a putative splicing regulator and transcriptional cofactor, in mammalian development. We demonstrate that selective deletion of PRMT5 in neural stem/progenitor cells (NPCs) leads to postnatal death in mice. At the molecular level, the absence of PRMT5 results in reduced methylation of Sm proteins, aberrant constitutive splicing, and the alternative splicing of specific mRNAs with weak 5' donor sites. Intriguingly, the products of these mRNAs are, among others, several proteins regulating cell cycle progression. We identify Mdm4 as one of these key mRNAs that senses the defects in the spliceosomal machinery and transduces the signal to activate the p53 response, providing a mechanistic explanation of the phenotype observed in vivo. Our data demonstrate that PRMT5 is a master regulator of splicing in mammals and uncover a new role for the Mdm4 pre-mRNA, which could be exploited for anti-cancer therapy.
Assuntos
Processamento Alternativo/genética , Proteínas Metiltransferases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Precursores de RNA/metabolismo , Processamento Pós-Transcricional do RNA/genética , Spliceossomos/patologia , Ubiquitina-Proteína Ligases/metabolismo , Animais , Linhagem Celular Tumoral , Células Cultivadas , Sistema Nervoso Central/patologia , Genes p53/genética , Células HCT116 , Células HEK293 , Homeostase/genética , Humanos , Proteínas de Filamentos Intermediários/genética , Proteínas de Filamentos Intermediários/metabolismo , Estimativa de Kaplan-Meier , Camundongos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Nestina , Ligação Proteica , Proteínas Metiltransferases/deficiência , Proteínas Metiltransferases/genética , Proteína-Arginina N-Metiltransferases , Proteínas Proto-Oncogênicas/genética , Precursores de RNA/genética , Transdução de Sinais , Spliceossomos/genética , Spliceossomos/metabolismo , Ubiquitina-Proteína Ligases/genéticaRESUMO
Protein arginine methyltransferases (PRMTs) play important roles in several cellular processes, including signaling, gene regulation, and transport of proteins and nucleic acids, to impact growth, differentiation, proliferation, and development. PRMT5 symmetrically di-methylates the two-terminal ω-guanidino nitrogens of arginine residues on substrate proteins. PRMT5 acts as part of a multimeric complex in concert with a variety of partner proteins that regulate its function and specificity. A core component of these complexes is the WD40 protein MEP50/WDR77/p44, which mediates interactions with binding partners and substrates. We have determined the crystal structure of human PRMT5 in complex with MEP50 (methylosome protein 50), bound to an S-adenosylmethionine analog and a peptide substrate derived from histone H4. The structure of the surprising hetero-octameric complex reveals the close interaction between the seven-bladed ß-propeller MEP50 and the N-terminal domain of PRMT5, and delineates the structural elements of substrate recognition.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Proteína-Arginina N-Metiltransferases/química , Domínio Catalítico , Cristalografia por Raios X , Dimerização , Humanos , Modelos Moleculares , Conformação ProteicaRESUMO
Ligand efficient fragments binding to PDK1 were identified by an NMR fragment-based screening approach. Computational modeling of the fragments bound to the active site led to the design and synthesis of a series of novel 6,7-disubstituted thienopyrimidin-4-one compounds, with low micromolar inhibitory activity against PDK1 in a biochemical enzyme assay.
Assuntos
Antineoplásicos/síntese química , Inibidores de Proteínas Quinases/síntese química , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Pirimidinonas/síntese química , Proteínas Quinases Dependentes de 3-Fosfoinositídeo , Animais , Antineoplásicos/farmacologia , Domínio Catalítico , Simulação por Computador , Desenho de Fármacos , Humanos , Cinética , Ligantes , Espectroscopia de Ressonância Magnética , Camundongos , Modelos Moleculares , Ligação Proteica , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/metabolismo , Pirimidinonas/farmacologiaRESUMO
A series of 2-anilino substituted 4-aryl-8H-purines were prepared as potent inhibitors of PDK1, a serine-threonine kinase thought to play a role in the PI3K/Akt signaling pathway, a key mediator of cancer cell growth, survival and tumorigenesis. The synthesis, SAR and ADME properties of this series of compounds are discussed culminating in the discovery of compound 6 which possessed sub-micromolar cell proliferation activity and 65% oral bioavailability in mice.
Assuntos
Compostos de Anilina/química , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Purinas/química , Bibliotecas de Moléculas Pequenas/química , Compostos de Anilina/farmacologia , Animais , Linhagem Celular Tumoral , Células Cultivadas , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Humanos , Concentração Inibidora 50 , Camundongos , Estrutura Molecular , Purinas/farmacologia , Piruvato Desidrogenase Quinase de Transferência de Acetil , Bibliotecas de Moléculas Pequenas/farmacologia , SolubilidadeRESUMO
A series of 3-(1,2-disubstituted-1H-benzimidazol-5-yl)-N-hydroxyacrylamides (1) were designed and synthesized as HDAC inhibitors. Extensive SARs have been established for in vitro potency (HDAC1 enzyme and COLO 205 cellular IC(50)), liver microsomal stability (t(1/2)), cytochrome P450 inhibitory (3A4 IC(50)), and clogP, among others. These parameters were fine-tuned by carefully adjusting the substituents at positions 1 and 2 of the benzimidazole ring. After comprehensive in vitro and in vivo profiling of the selected compounds, SB939 (3) was identified as a preclinical development candidate. 3 is a potent pan-HDAC inhibitor with excellent druglike properties, is highly efficacious in in vivo tumor models (HCT-116, PC-3, A2780, MV4-11, Ramos), and has high and dose-proportional oral exposures and very good ADME, safety, and pharmaceutical properties. When orally dosed to tumor-bearing mice, 3 is enriched in tumor tissue which may contribute to its potent antitumor activity and prolonged duration of action. 3 is currently being tested in phase I and phase II clinical trials.
Assuntos
Antineoplásicos/síntese química , Benzimidazóis/síntese química , Inibidores de Histona Desacetilases/síntese química , Administração Oral , Animais , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Benzimidazóis/farmacocinética , Benzimidazóis/farmacologia , Disponibilidade Biológica , Linhagem Celular Tumoral , Cães , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Histona Desacetilase 1/antagonistas & inibidores , Inibidores de Histona Desacetilases/farmacocinética , Inibidores de Histona Desacetilases/farmacologia , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Microssomos Hepáticos/metabolismo , Transplante de Neoplasias , Relação Quantitativa Estrutura-Atividade , Ratos , Ratos Wistar , EstereoisomerismoRESUMO
Thirty-six novel acylurea connected straight chain hydroxamates were designed and synthesized. Structure-activity relationships (SAR) were established for the length of linear chain linker and substitutions on the benzoylurea group. Compounds 5g, 5i, 5n, and 19 showed 10-20-fold enhanced HDAC1 potency compared to SAHA. In general, the cellular potency pIC(50) (COLO205) correlates with enzymatic potency pIC(50) (HDAC1). Compound 5b (SB207), a structurally simple and close analogue to SAHA, is more potent against HDAC1 and HDAC6 compared to the latter. As a representative example of this series, good in vitro enzymatic and cellular potency plus an excellent pharmacokinetic profile has translated into better efficacy than SAHA in both prostate cancer (PC3) and colon cancer (HCT116) xenograft models.
Assuntos
Inibidores de Histona Desacetilases/síntese química , Inibidores de Histona Desacetilases/farmacologia , Ácidos Hidroxâmicos/farmacologia , Ureia/farmacologia , Animais , Linhagem Celular Tumoral , Inibidores de Histona Desacetilases/química , Humanos , Ácidos Hidroxâmicos/química , Cinética , Camundongos , Relação Estrutura-Atividade , Transplante HeterólogoRESUMO
A series of alkenyl indazoles were synthesized and evaluated in Aurora kinase enzyme assays. Several promising leads were optimized for selectivity towards Aurora B. Excellent binding affinity and good selectivity were achieved with optimized compounds in isolated Aurora subfamily assays.
Assuntos
Inibidores de Proteínas Quinases/síntese química , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Aurora Quinases , Avaliação Pré-Clínica de Medicamentos , Modelos Moleculares , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/metabolismoRESUMO
Although clinical responses in liquid tumors and certain lymphomas have been reported, the clinical efficacy of histone deacetylase inhibitors in solid tumors has been limited. This may be in part due to the poor pharmacokinetic of these drugs, resulting in inadequate tumor concentrations of the drug. SB939 is a new hydroxamic acid based histone deacetylase inhibitor with improved physicochemical, pharmaceutical, and pharmacokinetic properties. In vitro, SB939 inhibits class I, II, and IV HDACs, with no effects on other zinc binding enzymes, and shows significant antiproliferative activity against a wide variety of tumor cell lines. It has very favorable pharmacokinetic properties after oral dosing in mice, with >4-fold increased bioavailability and 3.3-fold increased half-life over suberoylanilide hydroxamic acid (SAHA). In contrast to SAHA, SB939 accumulates in tumor tissue and induces a sustained inhibition of histone acetylation in tumor tissue. These excellent pharmacokinetic properties translated into a dose-dependent antitumor efficacy in a xenograft model of human colorectal cancer (HCT-116), with a tumor growth inhibition of 94% versus 48% for SAHA (both at maximum tolerated dose), and was also effective when given in different intermittent schedules. Furthermore, in APC(min) mice, a genetic mouse model of early-stage colon cancer, SB939 inhibited adenoma formation, hemocult scores, and increased hematocrit values more effectively than 5-fluorouracil. Emerging clinical data from phase I trials in cancer patients indicate that the pharmacokinetic and pharmacologic advantages of SB939 are translated to the clinic. The efficacy of SB939 reported here in two very different models of colorectal cancer warrants further investigation in patients.
Assuntos
Neoplasias Colorretais/tratamento farmacológico , Inibidores de Histona Desacetilases/administração & dosagem , Inibidores de Histona Desacetilases/farmacocinética , Ácidos Hidroxâmicos/administração & dosagem , Ácidos Hidroxâmicos/farmacocinética , Administração Oral , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacocinética , Disponibilidade Biológica , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Feminino , Células HCT116 , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Neoplasias/patologia , Resultado do Tratamento , Células Tumorais Cultivadas , Vorinostat , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
A series of N-hydroxy-1,2-disubstituted-1H-benzimidazol-5-yl acrylamides were designed and synthesized as novel HDAC inhibitors. General SAR has been established for the substituents at positions 1 and 2, as well as the importance of the ethylene group and its attachment to position 5. Optimized compounds are much more potent than SAHA in both enzymatic and cellular assays. A representative compound, 23 (SB639), has demonstrated antitumor activity in a colon cancer xenograft model.
Assuntos
Acrilamidas/síntese química , Antineoplásicos/síntese química , Benzimidazóis/síntese química , Inibidores de Histona Desacetilases , Acrilamidas/administração & dosagem , Acrilamidas/farmacologia , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Benzimidazóis/administração & dosagem , Benzimidazóis/farmacologia , Linhagem Celular Tumoral , Cristalografia por Raios X , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Feminino , Células HCT116 , Histona Desacetilases/metabolismo , Humanos , Camundongos , Camundongos Nus , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The Aurora family of serine/threonine kinases are mitotic regulators involved in centrosome duplication, formation of the bipolar mitotic spindle and the alignment of the chromosomes along the spindle. These proteins are frequently overexpressed in tumor cells as compared to normal cells and are therefore potential therapeutic oncology targets. An Aurora A high throughput screen revealed a promising sub-micromolar indazole-benzimidazole lead. Modification of the benzimidazole portion of the lead to a C2 linker with a phenyl ring was proposed to achieve novelty. Docking revealed that a conjugated linker was optimal and the resulting compounds were equipotent with the lead. Further structure-guided optimization of substituents on the 5 & 6 position of the indazole led to single digit nanomolar potency. The homology between the Aurora A & Aurora B kinase domains is 71% but their binding sites only differ at residues 212 & 217 (Aurora A numbering). However interactions with only the latter residue may be used for obtaining selectivity. An analysis of published Aurora A and Aurora B X-ray structures reveals subtle differences in the shape of the binding sites. This was exploited by introduction of appropriately sized substituents in the 4 & 6 position of the indazole leading to Aurora B selective inhibitors. Finally we calculate the conformational energy penalty of the putative bioactive conformation of our inhibitors and show that this property correlates well with the Aurora A binding affinity.
Assuntos
Inibidores de Proteínas Quinases/química , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Aurora Quinases , Modelos Moleculares , Estrutura Molecular , Inibidores de Proteínas Quinases/farmacologiaRESUMO
BACKGROUND: The mitotic checkpoint prevents the onset of anaphase before all chromosomes are attached to spindle microtubules. The checkpoint is thought to act by the catalytic generation at unattached kinetochores of a diffusible "wait signal" that prevents anaphase. Mad2 and Cdc20, two candidate proteins for components of a diffusible wait signal, have previously been shown to be recruited to and rapidly released from unattached kinetochores. RESULTS: Fluorescence recovery after photobleaching demonstrated that Mad1, Bub1, and a portion of Mad2, all essential mitotic-checkpoint components, are stably bound elements of unattached kinetochores (as are structural centromere components such as Centromere protein C [CENP-C]). After microtubule attachment, Mad1 and Mad2 are released from kinetochores and relocalize to spindle poles, whereas Bub1 remains at kinetochores. CONCLUSIONS: A long residence time at kinetochores identifies Bub1, Mad1, and a portion of Mad2 as part of a catalytic platform that recruits, activates, and releases a diffusible wait signal that is partly composed of the rapidly exchanging portion of Mad2. The release of Mad1 and Mad2, but not Bub1, from kinetochores upon attachment separates the elements of this "catalytic platform" and thereby silences generation of the anaphase inhibitor despite continued rapid cycling of Mad2 at spindle poles.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Genes cdc/fisiologia , Cinetocoros/metabolismo , Marsupiais , Fosfoproteínas/metabolismo , Proteínas Repressoras/metabolismo , Transdução de Sinais , Animais , Proteínas de Ciclo Celular , Células Cultivadas , DNA Complementar/metabolismo , Recuperação de Fluorescência Após Fotodegradação , Imunofluorescência , Vetores Genéticos , Células HeLa , Humanos , Proteínas Mad2 , Masculino , Proteínas Nucleares , Testes de Precipitina , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases , Retroviridae , Fuso Acromático/metabolismoRESUMO
Centromere-associated protein-E (CENP-E) is an essential mitotic kinesin that is required for efficient, stable microtubule capture at kinetochores. It also directly binds to BubR1, a kinetochore-associated kinase implicated in the mitotic checkpoint, the major cell cycle control pathway in which unattached kinetochores prevent anaphase onset. Here, we show that single unattached kinetochores depleted of CENP-E cannot block entry into anaphase, resulting in aneuploidy in 25% of divisions in primary mouse fibroblasts in vitro and in 95% of regenerating hepatocytes in vivo. Without CENP-E, diminished levels of BubR1 are recruited to kinetochores and BubR1 kinase activity remains at basal levels. CENP-E binds to and directly stimulates the kinase activity of purified BubR1 in vitro. Thus, CENP-E is required for enhancing recruitment of its binding partner BubR1 to each unattached kinetochore and for stimulating BubR1 kinase activity, implicating it as an essential amplifier of a basal mitotic checkpoint signal.