RESUMO
BACKGROUND: The symptoms of Clostridium difficile infection are mediated primarily by two toxins, TcdA and TcdB, the expression of which is governed by a multitude of factors including nutrient availability, growth phase and cell stress. Several global regulators have been implicated in the regulation of toxin expression, such as CcpA and CodY. RESULTS: During attempts to insertionally inactivate a putative secondary cell wall polysaccharide synthesis gene, we obtained several mutants containing off-target insertions. One mutant displayed an unusual branched colony morphology and was investigated further. Marker recovery revealed an insertion in mfd, a gene encoding a transcription-coupled repair factor. The mfd mutant exhibited pleiotropic effects, in particular increased expression of both toxin A and B (TcdA and TcdB) compared to the parental strain. Western blotting and cellular cytotoxicity assays revealed increased expression across all time points over a 24 h period, with inactivation of mfd resulting in at least a 10 fold increase in cell cytotoxicity. qRT-PCR demonstrated the upregulation of both toxins occurred on a transcriptional level. All effects of the mfd mutation were complemented by a plasmid-encoded copy of mfd, showing the effects are not due to polar effects of the intron insertion or to second site mutations. CONCLUSIONS: This study adds Mfd to the repertoire of factors involved in regulation of toxin expression in Clostridium difficile. Mfd is known to remove RNA polymerase molecules from transcriptional sites where it has stalled due to repressor action, preventing transcriptional read through. The consistently high levels of toxin in the C. difficile mfd mutant indicate this process is inefficient leading to transcriptional de-repression.
Assuntos
Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , Clostridioides difficile/genética , Enterotoxinas/genética , Mutagênese Insercional/genética , Fatores de Transcrição/genética , Animais , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/toxicidade , Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/toxicidade , Sobrevivência Celular/efeitos dos fármacos , Parede Celular/genética , Chlorocebus aethiops , Clostridioides difficile/metabolismo , Clostridioides difficile/fisiologia , Enterotoxinas/metabolismo , Enterotoxinas/toxicidade , Regulação Bacteriana da Expressão Gênica , Células HT29 , Humanos , Mutagênese Insercional/imunologia , Transcrição Gênica , Regulação para Cima , Células VeroRESUMO
Various bis-benzimidazole derivatives have been reported to possess activity against Gram-positive pathogens. No mechanism of action has been elucidated to fully account for the antibacterial activity of this class of compounds. A group of symmetric bis-benzimidazoles (BBZ) designed as anticancer agents have previously been shown to possess moderate antiproliferative activity. We sought to assess the antibacterial activity and mechanism of action of BBZ compounds against Staphylococcus aureus. Antibacterial activities were assessed by determination of minimal inhibitory concentrations (MICs), time-kill curves, and scanning electron microscopy. Transcriptional responses to BBZ treatment were determined using whole genome microarrays. Activities against bacterial type II topoisomerases were investigated using in vitro supercoiling, decatenation, DNA binding, and DNA cleavage inhibition assays. MICs for EMRSA-16 were between 0.03 and 0.5 µg/mL. The compounds showed concentration-dependent bactericidal activity and induced cell swelling and lysis. Transcriptional responses to BBZ were consistent with topoisomerase inhibition and DNA damage. A subset of BBZ compounds inhibited S. aureus DNA gyrase supercoiling activity with IC(50) values in the range of 5-10 µM. This inhibition was subsequently shown to operate through both inhibition of binding of DNA gyrase to DNA and accumulation of single-stranded DNA breaks. We conclude that BBZ compounds are potent anti-staphylococcal agents and operate at least in part through DNA gyrase inhibition, leading to the accumulation of single-stranded DNA breaks, and by preventing the binding of gyrase to DNA.
Assuntos
Antibacterianos/farmacologia , Benzimidazóis/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/enzimologia , Inibidores da Topoisomerase II , Antibacterianos/química , Benzimidazóis/química , Quebras de DNA de Cadeia Simples/efeitos dos fármacos , DNA Girase/metabolismo , DNA Topoisomerase IV/antagonistas & inibidores , DNA Topoisomerase IV/metabolismo , DNA Bacteriano/metabolismo , Humanos , Testes de Sensibilidade Microbiana , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/enzimologiaRESUMO
A series of naphthalene derivatives with disubstituted triazole side-arms have been assembled by click chemistry. Lead compounds show a high level of selectivity for renal, osteo- and Ewing's sarcomas that express the HIF-1α transcription factor. They also interact selectively with the quadruplex DNAs located in the promoter of the HIF genes and it is suggested that the mechanism of action involves inhibition of transcription by drug-mediated quadruplex stabilization in these regions.
Assuntos
Antineoplásicos/farmacologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/antagonistas & inibidores , Quadruplex G/efeitos dos fármacos , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Neoplasias Renais/tratamento farmacológico , Osteossarcoma/tratamento farmacológico , Antineoplásicos/síntese química , Antineoplásicos/química , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neoplasias Renais/metabolismo , Neoplasias Renais/patologia , Ligantes , Estrutura Molecular , Osteossarcoma/metabolismo , Osteossarcoma/patologia , Regiões Promotoras Genéticas/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Relação Estrutura-AtividadeRESUMO
Academic drug discovery is playing an increasingly important role in the identification of new therapies for a wide range of diseases. There is no one model that guarantees success. We describe here a drug discovery story where chance, the ability to capitalise on chance, and the assembling of a range of expertise, have all played important roles in the discovery and subsequent development of an antibiotic chemotype based on the bis-benzimidazole scaffold, with potency against a number of current therapeutically challenging diseases. One compound in this class, SMT19969, has recently entered Phase 2 human clinical trials for the treatment of Clostridium difficile infections.
RESUMO
The hypoxia-inducible transcription factor (HIF) co-ordinates the response of tumours to low oxygen by stimulating genes involved in metabolism and angiogenesis. HIF pathway activation is associated with decreased progression-free survival and increased mortality; compounds that target this pathway are potential agents for the treatment of a range of solid tumour malignancies. Renal cancers are likely to be particularly sensitive to inhibition of the HIF pathway since ~80% show constitutive activation of HIF. We have previously described the di-substituted naphthalene derivative, CL67, which binds to a G-quadruplex higher-order structure in the HIF promoter sequence in vitro. We show here that CL67 blocks HIF expression leading to inhibition of HIF-transactivation and down-regulation of downstream target genes and proteins in renal carcinoma cell lines and in a mouse xenograft model of renal cancer. This inhibition is independent of pathways that control HIF abundance through oxygen-dependant degradation and oxygen dependant HIF sub-unit expression.
Assuntos
Carcinoma de Células Renais/tratamento farmacológico , Hipóxia Celular/efeitos dos fármacos , Quadruplex G/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Neoplasias Renais/tratamento farmacológico , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Western Blotting , Carcinógenos/farmacologia , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/patologia , Feminino , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Técnicas Imunoenzimáticas , Neoplasias Renais/genética , Neoplasias Renais/patologia , Camundongos , Camundongos Nus , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Elementos de Resposta/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ativação Transcricional/efeitos dos fármacos , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Tetra-substituted naphthalene diimide (ND) derivatives with positively charged termini are potent stabilizers of human telomeric and gene promoter DNA quadruplexes and inhibit the growth of human cancer cells in vitro and in vivo. The present study reports the enhancement of the pharmacological properties of earlier ND compounds using structure-based design. Crystal structures of three complexes with human telomeric intramolecular quadruplexes demonstrate that two of the four strongly basic N-methyl-piperazine groups can be replaced by less basic morpholine groups with no loss of intermolecular interactions in the grooves of the quadruplex. The new compounds retain high affinity to human telomeric quadruplex DNA but are 10-fold more potent against the MIA PaCa-2 pancreatic cancer cell line, with IC50 values of ~10 nM. The lead compound induces cellular senescence but does not inhibit telomerase activity at the nanomolar dosage levels required for inhibition of cellular proliferation. Gene array qPCR analysis of MIA PaCa-2 cells treated with the lead compound revealed significant dose-dependent modulation of a distinct subset of genes, including strong induction of DNA damage responsive genes CDKN1A, DDIT3, GADD45A/G, and PPM1D, and repression of genes involved in telomere maintenance, including hPOT1 and PARP1.
Assuntos
Antineoplásicos/uso terapêutico , Desenho de Fármacos , Quadruplex G , Imidas/uso terapêutico , Naftalenos/uso terapêutico , Neoplasias Pancreáticas/tratamento farmacológico , Telômero/efeitos dos fármacos , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Avaliação Pré-Clínica de Medicamentos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Imidas/farmacologia , Ligantes , Modelos Moleculares , Naftalenos/farmacologia , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologiaRESUMO
The ECTOPICALLY PARTING CELLS 1 (EPC1) gene encodes a putative retaining glycosyltransferase of the GT64 family, and epc1-1 mutant plants have a severely dwarfed phenotype. A new mutant allele of this gene, epc1-2, has been isolated. Reduced cell adhesion that has previously been reported for the epc1-1 mutant was not observed for either the epc1-1 or epc1-2 mutants grown in our conditions, suggesting that EPC1 does not affect cell adhesion but is involved in some other process affecting plant growth and development. It is shown that the epc1-2 mutant exhibits hypersensitivity to the phytohormone abscisic acid in germination and root elongation assays, however it shows an unaltered response to gibberellin, epi-brassinosteroid, auxin, or ethylene. An EPC1:YFP fusion protein is localized to small motile structures within the cytosol that are similar in size and number to the Golgi apparatus. Analysis of cell wall pectins revealed that levels of beta-(1,4)-galactan in the epc1-2 mutant are reduced by 50%, whilst other pectic polysaccharides (homogalacturonan, arabinan, and rhamnogalacturonan II) are unchanged.