RESUMO
p53-independent malignant cancer is still severe health problem of human beings. HIF-1 pathway is believed to play an important role in the survival and developing progress of such cancers. In the present study, with the aim to inhibit the proliferation of p53-independent malignant cells, we disclose the optimization of 6a, the starting compound which is discovered in the screening of in-house compound collection. The structure-activity relationship (SAR) is summarized. The most potent derivative 8d, inhibits the proliferation of both p53-null and p53-mutated cells through inhibition of HIF-1 pathway. Our findings here provide a new chemotype in designing potent anticancer agent especially against those p53-independent malignant tumors.
Assuntos
Antineoplásicos/farmacologia , Benzofuranos/farmacologia , Descoberta de Drogas , Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Proteína Supressora de Tumor p53/antagonistas & inibidores , Antineoplásicos/síntese química , Benzofuranos/síntese química , Benzofuranos/química , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Células HCT116 , Humanos , Células MCF-7 , Estrutura Molecular , Neoplasias/metabolismo , Neoplasias/patologia , Relação Estrutura-Atividade , Proteína Supressora de Tumor p53/deficiênciaRESUMO
Anorectal malformations (ARMs, congenital obstruction of the anal opening) are among the most common birth defects requiring surgical treatment (2-5/10 000 live-births) and carry significant chronic morbidity. ARMs present either as isolated or as part of the phenotypic spectrum of some chromosomal abnormalities or monogenic syndromes. The etiology is unknown. To assess the genetic contribution to ARMs, we investigated single-nucleotide polymorphisms and copy number variations (CNVs) at genome-wide scale. A total of 363 Han Chinese sporadic ARM patients and 4006 Han Chinese controls were included. Overall, we detected a 1.3-fold significant excess of rare CNVs in patients. Stratification of patients by presence/absence of other congenital anomalies showed that while syndromic ARM patients carried significantly longer rare duplications than controls (P = 0.049), non-syndromic patients were enriched with both rare deletions and duplications when compared with controls (P = 0.00031). Twelve chromosomal aberrations and 114 rare CNVs were observed in patients but not in 868 controls nor 11 943 healthy individuals from the Database of Genomic Variants. Importantly, these aberrations were observed in isolated ARM patients. Gene-based analysis revealed 79 genes interfered by CNVs in patients only. In particular, we identified a de novo DKK4 duplication. DKK4 is a member of the WNT signaling pathway which is involved in the development of the anorectal region. In mice, Wnt disruption results in ARMs. Our data suggest a role for rare CNVs not only in syndromic but also in isolated ARM patients and provide a list of plausible candidate genes for the disorder.
Assuntos
Anus Imperfurado/genética , Anus Imperfurado/fisiopatologia , Variações do Número de Cópias de DNA , Duplicação Gênica , Peptídeos e Proteínas de Sinalização Intercelular/genética , Animais , Malformações Anorretais , Povo Asiático , Aberrações Cromossômicas , Feminino , Dosagem de Genes , Regulação da Expressão Gênica , Estudo de Associação Genômica Ampla , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos ICR , Fenótipo , Polimorfismo de Nucleotídeo Único , Estudos Prospectivos , Via de Sinalização WntRESUMO
Hirschsprung disease (HSCR) is a congenital disorder characterized by aganglionosis of the distal intestine. To assess the contribution of copy number variants (CNVs) to HSCR, we analysed the data generated from our previous genome-wide association study on HSCR patients, whereby we identified NRG1 as a new HSCR susceptibility locus. Analysis of 129 Chinese patients and 331 ethnically matched controls showed that HSCR patients have a greater burden of rare CNVs (p = 1.50 × 10(-5)), particularly for those encompassing genes (p = 5.00 × 10(-6)). Our study identified 246 rare-genic CNVs exclusive to patients. Among those, we detected a NRG3 deletion (p = 1.64 × 10(-3)). Subsequent follow-up (96 additional patients and 220 controls) on NRG3 revealed 9 deletions (combined p = 3.36 × 10(-5)) and 2 de novo duplications among patients and two deletions among controls. Importantly, NRG3 is a paralog of NRG1. Stratification of patients by presence/absence of HSCR-associated syndromes showed that while syndromic-HSCR patients carried significantly longer CNVs than the non-syndromic or controls (p = 1.50 × 10(-5)), non-syndromic patients were enriched in CNV number when compared to controls (p = 4.00 × 10(-6)) or the syndromic counterpart. Our results suggest a role for NRG3 in HSCR etiology and provide insights into the relative contribution of structural variants in both syndromic and non-syndromic HSCR. This would be the first genome-wide catalog of copy number variants identified in HSCR.
Assuntos
Variações do Número de Cópias de DNA/genética , Deleção de Genes , Doença de Hirschsprung/genética , Neurregulinas/genética , Feminino , Frequência do Gene , Estudo de Associação Genômica Ampla , Genótipo , Haplótipos , Humanos , Masculino , Polimorfismo de Nucleotídeo ÚnicoRESUMO
Hirschsprung's disease (HSCR), or aganglionic megacolon, is a congenital disorder characterized by the absence of enteric ganglia in variable portions of the distal intestine. RET is a well-established susceptibility locus, although existing evidence strongly suggests additional loci contributing to sporadic HSCR. To identify these additional genetic loci, we carried out a genome-wide association study using the Affymetrix 500K marker set. We successfully genotyped 293,836 SNPs in 181 Chinese subjects with sporadic HSCR and 346 ethnically matched control subjects. The SNPs most associated with HSCR were genotyped in an independent set of 190 HSCR and 510 control subjects. Aside from SNPs in RET, the strongest overall associations in plausible candidate genes were found for 2 SNPs located in intron 1 of the neuregulin1 gene (NRG1) on 8p12, with rs16879552 and rs7835688 yielding odds ratios of 1.68 [CI(95%):(1.40, 2.00), P = 1.80 x 10(-8)] and 1.98 [CI(95%):(1.59, 2.47), P = 1.12 x 10(-9)], respectively, for the heterozygous risk genotypes under an additive model. There was also a significant interaction between RET and NRG1 (P = 0.0095), increasing the odds ratio 2.3-fold to 19.53 for the RET rs2435357 risk genotype (TT) in the presence of the NRG1 rs7835688 heterozygote, indicating that NRG1 is a modifier of HSRC penetrance. Our highly significant association findings are backed-up by the important role of NRG1 as regulator of the development of the enteric ganglia precursors. The identification of NRG1 as an additional HSCR susceptibility locus not only opens unique fields of investigation into the mechanisms underlying the HSCR pathology, but also the mechanisms by which a discrete number of loci interact with each other to cause disease.
Assuntos
Predisposição Genética para Doença , Genoma Humano , Doença de Hirschsprung/genética , Proteínas do Tecido Nervoso/genética , Feminino , Marcadores Genéticos , Estudo de Associação Genômica Ampla , Humanos , Masculino , Neuregulina-1 , Polimorfismo de Nucleotídeo Único , Proteínas Proto-Oncogênicas c-ret/genéticaRESUMO
Hirschsprung's disease (HSCR) is a congenital disorder in which ganglion cells are absent in variable portions of the lower digestive tract according to which patients are classified. The RET gene is the major HSCR gene, although reduced penetrance of RET mutations and variable expression of HSCR phenotype indicates that more than one gene is required. An unidentified RET-dependent modifier on 3p21 appears to be necessary for transmission of the short HSCR (S-HSCR) phenotype. We investigated 6 Mb of the 3p21 region on a quest for the HSCR-susceptibility locus. Fifty-eight S-HSCR case-parent trios were genotyped using Sequenom technology for 214 tag single nucleotide polymorphisms (SNPs) distributed along 6 Mb of the 3p21 region. A five-marker haplotype, spanning a 118 kb gene-rich region, was found to be overtransmitted to affected offspring. The associated haplotype encompasses three genes involved in neurological phenotypes. Importantly, this association was replicated in an independent sample of 172 S-HSCR cases and 153 unrelated controls. Ranking markers by proximity to candidate genes or by expected functional consequences could be used in follow-up studies to finally pinpoint this HSCR locus.
Assuntos
Mapeamento Cromossômico , Cromossomos Humanos Par 3/genética , Doença de Hirschsprung/genética , Povo Asiático/genética , Estudos de Casos e Controles , Família , Feminino , Marcadores Genéticos , Predisposição Genética para Doença , Haplótipos , Humanos , Desequilíbrio de Ligação/genética , Masculino , Polimorfismo de Nucleotídeo Único/genética , Controle de QualidadeRESUMO
VACTERL acronym is assigned to a non-random association of malformations in humans with poorly known etiology. It is comprised of vertebral defects (V), anal atresia (A), cardiac anomaly (C), tracheoesophageal fistula with esophageal atresia (TE), renal dysplasia (R) and limb lesions (L). Here, we report on, for the first time, a female patient with VACTERL association with a 21 base-pair deletion in the exon 1 triplet repeats of HOXD13, a sonic hedgehog (SHH) downstream target. Our data provide the first piece of clinical evidence of the implication of the SHH pathway in VACTERL. Moreover, HOXD13 may not only be implicated in limb malformations but also in the development of gut and genitourinary structures, as predicted from the mouse models.
Assuntos
Anormalidades Múltiplas/genética , Proteínas de Homeodomínio/genética , Mutação/genética , Fatores de Transcrição/genética , Anormalidades Múltiplas/diagnóstico por imagem , Adolescente , Sequência de Aminoácidos , Anus Imperfurado/complicações , Anus Imperfurado/genética , Sequência de Bases , Análise Mutacional de DNA , Atresia Esofágica/complicações , Atresia Esofágica/genética , Feminino , Cardiopatias Congênitas/complicações , Cardiopatias Congênitas/genética , Humanos , Deformidades Congênitas dos Membros/complicações , Deformidades Congênitas dos Membros/diagnóstico por imagem , Deformidades Congênitas dos Membros/genética , Dados de Sequência Molecular , Radiografia , Síndrome , Fístula Traqueoesofágica/complicações , Fístula Traqueoesofágica/genéticaRESUMO
BACKGROUND: Anorectal malformations (congenital absence of the anal opening) are among the most common pediatric surgical problems and carry a significant chronic morbidity. METHODS: Direct sequencing was used to screen 88 anorectal malformations patients for mutations and polymorphisms in SHH and GLI3. These genes were chosen according to the phenotype presented by mutant mice and their expression patterns. RESULTS: We report on 10 GLI3 variants (IVS3+141C>G, T183A, IVS4+124T>C, IVS7+17G>A, IVS8+1 G>C, N503N, P941P, P998L, A1005A, A1039A) and four SHH mutation/variants (IVS1-49C>T, IVS2+111A>C, L214L, G290D). CONCLUSIONS: These variants are not over-represented in the healthy population and most are predicted to be benign. This study conveys the problematic assessment of the pathogenic role in disease of rare point mutations and variants.
Assuntos
Canal Anal/anormalidades , Análise Mutacional de DNA , Proteínas Hedgehog/genética , Fatores de Transcrição Kruppel-Like/genética , Proteínas do Tecido Nervoso/genética , Reto/anormalidades , Adulto , Idoso , Substituição de Aminoácidos/genética , Criança , Pré-Escolar , Feminino , Variação Genética , Humanos , Masculino , Pessoa de Meia-Idade , Mutação Puntual , Polimorfismo de Nucleotídeo Único/genética , Valor Preditivo dos Testes , Síndrome , Proteína Gli3 com Dedos de ZincoRESUMO
Directly disrupting the Keap1-Nrf2 protein-protein interaction (PPI) has emerged as an attractive way to activate Nrf2, and Keap1-Nrf2 PPI inhibitors have been proposed as potential agents to relieve inflammatory and oxidative stress diseases. In this work, we investigated the diacetic moiety around the potent Keap1-Nrf2 PPI inhibitor DDO1018 (2), which was reported by our group previously. Exploration of bioisosteric replacements afforded the ditetrazole analog 7, which maintains the potent PPI inhibition activity (IC50 = 15.8 nM) in an in vitro fluorescence polarization assay. Physicochemical property determination demonstrated that ditetrazole replacement can improve the drug-like property, including elevation of pK a, log D, and transcellular permeability. Additionally, 7 is more efficacious than 2 on inducing the expression of Nrf2-dependent gene products in cells. This study provides an alternative way to replace the diacetic moiety and occupy the polar subpockets in Keap1, which can benefit the subsequent development of Keap1-Nrf2 PPI inhibitor.
RESUMO
Protein-protein interactions (PPIs) as drug targets have been gaining growing interest, though developing drug-like small molecule PPI inhibitors remains challenging. Peptide PPI inhibitors, which can provide informative data on the PPI interface, are good starting points to develop small molecule modulators. Computational methods combining molecular dynamics simulations and binding energy calculations could give both the structural and the energetic perspective of peptide PPI inhibitors. Herein, we set up a computational workflow to investigate Keap1-Nrf2 peptide PPI inhibitors and predict the activity of novel sequences. Furthermore, we applied this method to investigate p62 peptides as PPI inhibitors of Keap1-Nrf2 and explored the activity change induced by the phosphorylation of serine. Our results showed that because of the unfavorable solvation effects, the binding affinity of the phosphorylated p62 peptide is lower than the Nrf2 ETGE peptide. Our research results not only provide a useful method to investigate the Keap1-Nrf2 peptide inhibitors, but also give a good example to show how to incorporate computational methods into the study of peptide PPI inhibitors. Besides, applying this method to p62 peptides provides a detailed explanation for the expression of cytoprotective Nrf2 targets induced by p62 phosphorylation, which may benefit the further study of the crosstalk between the Keap1-Nrf2 pathway and p62-mediated selective autophagy.
Assuntos
Descoberta de Drogas , Proteína 1 Associada a ECH Semelhante a Kelch/química , Modelos Moleculares , Fator 2 Relacionado a NF-E2/química , Peptídeos/química , Relação Quantitativa Estrutura-Atividade , Sequência de Aminoácidos , Sítios de Ligação , Relação Dose-Resposta a Droga , Desenho de Fármacos , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Conformação Molecular , Fator 2 Relacionado a NF-E2/metabolismo , Peptídeos/metabolismo , Peptídeos/farmacologia , Fosforilação , Ligação Proteica/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacosRESUMO
Ulcerative colitis (UC) is a chronic relapsing-remitting form of inflammatory bowel disease (IBD) that increases the risk of colorectal cancer, the third most common malignancy in humans. Oxidative stress is a risk factor for the development of UC. The Keap1-Nrf2-ARE pathway is one of the most important defensive mechanisms against oxidative and/or electrophilic stresses. In this study, we identified CPUY192018 as a potent small-molecule inhibitor of the Keap1-Nrf2 PPI, investigated the cyto-protective effects of CPUY192018 on the NCM460 colonic cells and evaluated whether treatment with the inhibitor of the Keap1-Nrf2 PPI exerts protection on an established experimental model of UC induced by dextran sodium sulfate (DSS). Our study clearly demonstrated that CPUY192018 had a cytoprotective effect against DSS in both NCM460 cells and mouse colon via the activation of Nrf2 signaling. These results suggested that activation of Nrf2 by directly inhibiting the Keap1-Nrf2 PPI may be beneficial as a treatment for UC.
Assuntos
Colite/tratamento farmacológico , Colo/citologia , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Bibliotecas de Moléculas Pequenas/administração & dosagem , Animais , Linhagem Celular , Colite/induzido quimicamente , Colite/metabolismo , Colo/efeitos dos fármacos , Sulfato de Dextrana/efeitos adversos , Modelos Animais de Doenças , Células Hep G2 , Humanos , Camundongos , Ligação Proteica/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologiaRESUMO
Directly disrupting the Keap1-Nrf2 protein-protein interaction (PPI) is an effective way to activate Nrf2. Using the potent Keap1-Nrf2 PPI inhibitor that was reported by our group, we conducted a preliminary investigation of the structure-activity and structure-property relationships of the ring systems to improve the drug-like properties. Compound 18e, which bore p-acetamido substituents on the side chain phenyl rings, was the best choice for balancing PPI inhibition activity, physicochemical properties, and cellular Nrf2 activity. Cell-based experiments with 18e showed that the Keap1-Nrf2 PPI inhibitor can activate Nrf2 and induce the expression of Nrf2 downstream proteins in an Nrf2-dependent manner. An exploratory in vivo experiment was carried out to further evaluate the anti-inflammatory effects of 18e in a LPS-challenged mouse model. The primary results indicated that 18e could reduce the level of circulating pro-inflammatory cytokines induced by LPS and relieve the inflammatory response.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Fator 2 Relacionado a NF-E2/antagonistas & inibidores , Ativação Metabólica/efeitos dos fármacos , Animais , Anti-Inflamatórios não Esteroides/síntese química , Anti-Inflamatórios não Esteroides/farmacologia , Linhagem Celular , Citocinas/biossíntese , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteína 1 Associada a ECH Semelhante a Kelch , Lipopolissacarídeos/antagonistas & inibidores , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Ligação Proteica , RNA Interferente Pequeno/genética , Relação Estrutura-AtividadeRESUMO
Parkinson's disease (PD) is the second most common neurodegenerative disorder characterized by the accumulation of protein aggregates (namely Lewy bodies) in dopaminergic neurons in the substantia nigra region of the brain. Alpha-synuclein (α-syn) is the major component of Lewy bodies in PD patients, and impairment of the autophagy-lysosomal system has been linked to its accumulation. In our previous study, we identified an oxindole alkaloid Corynoxine B (Cory B), isolated from Uncaria rhynchophylla (Miq.) Jacks (Gouteng in Chinese), as a Beclin-1-dependent autophagy inducer. In this work, we show that Cory, an enantiomer of Cory B, also induces autophagy in different neuronal cell lines, including N2a and SHSY-5Y cells, which is paralleled with increased lysosomal enzyme cathepsin D. In vivo, Cory promotes the formation of autophagosomes in the fat bodies of Drosophila. By inducing autophagy, Cory promotes the clearance of wild-type and A53T α-syn in inducible PC12 cells. Interestingly, different from its enantiomer Cory B, Cory induces autophagy through the Akt/mTOR pathway as evidenced by the reduction in the levels of phospho-Akt, phospho-mTOR and phospho-p70 S6 Kinase. Collectively, our findings provide experimental evidence for developing Cory as a new autophagy enhancer from Chinese herbal medicine, which may have potential application in the prevention or treatment of PD.
Assuntos
Alcaloides/farmacologia , Autofagia/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina-Treonina Quinases TOR/metabolismo , alfa-Sinucleína/metabolismo , Animais , Animais Geneticamente Modificados , Autofagia/fisiologia , Linhagem Celular Tumoral , Drosophila , Humanos , Alcaloides Indólicos , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/fisiologiaRESUMO
PURPOSE: The combination of partial absence of the sacrum, anorectal anomalies, and presacral mass constitutes Currarino syndrome (CS), which is associated with mutations in MNX1 motor neuron and pancreas homeobox 1 (previously HLXB9). Here, we report on the MNX1 mutations found in a family segregating CS and in 3 sporadic CS patients, as well as on the clinical characteristics of the affected individuals. METHODS: MNX1 mutations were identified by direct sequencing the coding regions, intron/exon boundaries of MNX1 in 5 CS Japanese family members and 3 Chinese sporadic cases and their parents. RESULTS: There were 2 novel (P18PfsX37, R243W) and 2 previously described (W288G and IVS2 + 1G > A) mutations. These mutations were not found in 198 control individuals and are predicted to impair the functioning of the MNX1 protein. CONCLUSIONS: The variability of the CS phenotype among related or unrelated patients bearing the same mutation advocates for differences in the genetic background of each individual and invokes the implication of additional CS susceptibility genes.