RESUMEN
Few genetically dominant mutations involved in human disease have been fully explained at the molecular level. In cases where the mutant gene encodes a transcription factor, the dominant-negative mode of action of the mutant protein is particularly poorly understood. Here, we studied the genome-wide mechanism underlying a dominant-negative form of the SOX18 transcription factor (SOX18RaOp) responsible for both the classical mouse mutant Ragged Opossum and the human genetic disorder Hypotrichosis-lymphedema-telangiectasia-renal defect syndrome. Combining three single-molecule imaging assays in living cells together with genomics and proteomics analysis, we found that SOX18RaOp disrupts the system through an accumulation of molecular interferences which impair several functional properties of the wild-type SOX18 protein, including its target gene selection process. The dominant-negative effect is further amplified by poisoning the interactome of its wild-type counterpart, which perturbs regulatory nodes such as SOX7 and MEF2C. Our findings explain in unprecedented detail the multi-layered process that underpins the molecular aetiology of dominant-negative transcription factor function.
Asunto(s)
Glomerulonefritis/genética , Hipotricosis/genética , Linfedema/genética , Factores de Transcripción SOXF/genética , Telangiectasia/genética , Transcripción Genética , Animales , Células COS , Chlorocebus aethiops , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Redes Reguladoras de Genes , Genes Reporteros , Glomerulonefritis/metabolismo , Glomerulonefritis/patología , Células HeLa , Células Endoteliales de la Vena Umbilical Humana , Humanos , Hipotricosis/metabolismo , Hipotricosis/patología , Luciferasas/genética , Luciferasas/metabolismo , Linfedema/metabolismo , Linfedema/patología , Factores de Transcripción MEF2/genética , Factores de Transcripción MEF2/metabolismo , Ratones , Mutación , Factores de Transcripción SOXF/metabolismo , Imagen Individual de Molécula , Telangiectasia/metabolismo , Telangiectasia/patologíaRESUMEN
Biophysical parameters that govern transcription factors activity are binding locations across the genome, dwelling time at these regulatory elements and specific protein-protein interactions. Most molecular strategies used to develop small compounds that block transcription factors activity have been based on biochemistry and cell biology methods that that do not take into consideration these key biophysical features. Here, we review the advance in the field of transcription factor biology and describe how their interactome and transcriptional regulation on a genome wide scale have been deciphered. We suggest that this new knowledge has the potential to be used to implement innovative research drug discovery program.
Asunto(s)
Redes Reguladoras de Genes , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Animales , Humanos , Unión Proteica , Mapas de Interacción de ProteínasRESUMEN
An Australian estuarine isolate of Penicillium sp. MST-MF667 yielded 3 tetrapeptides named the bilaids with an unusual alternating LDLD chirality. Given their resemblance to known short peptide opioid agonists, we elucidated that they were weak (Ki low micromolar) µ-opioid agonists, which led to the design of bilorphin, a potent and selective µ-opioid receptor (MOPr) agonist (Ki 1.1 nM). In sharp contrast to all-natural product opioid peptides that efficaciously recruit ß-arrestin, bilorphin is G protein biased, weakly phosphorylating the MOPr and marginally recruiting ß-arrestin, with no receptor internalization. Importantly, bilorphin exhibits a similar G protein bias to oliceridine, a small nonpeptide with improved overdose safety. Molecular dynamics simulations of bilorphin and the strongly arrestin-biased endomorphin-2 with the MOPr indicate distinct receptor interactions and receptor conformations that could underlie their large differences in bias. Whereas bilorphin is systemically inactive, a glycosylated analog, bilactorphin, is orally active with similar in vivo potency to morphine. Bilorphin is both a unique molecular tool that enhances understanding of MOPr biased signaling and a promising lead in the development of next generation analgesics.
Asunto(s)
Analgésicos Opioides/farmacología , Proteínas Fúngicas/farmacología , Oligopéptidos/farmacología , Penicillium/química , Receptores Opioides mu/agonistas , Analgésicos Opioides/química , Animales , Sitios de Unión , Línea Celular Tumoral , Proteínas Fúngicas/química , Células HEK293 , Humanos , Ratones , Simulación del Acoplamiento Molecular , Oligopéptidos/química , Unión Proteica , Receptores Opioides mu/química , Receptores Opioides mu/metabolismoRESUMEN
During embryogenesis, vascular development relies on a handful of transcription factors that instruct cell fate in a distinct sub-population of the endothelium (1). The SOXF proteins that comprise SOX7, 17 and 18, are molecular switches modulating arterio-venous and lymphatic endothelial differentiation (2,3). Here, we show that, in the SOX-F family, only SOX18 has the ability to switch between a monomeric and a dimeric form. We characterized the SOX18 dimer in binding assays in vitro, and using a split-GFP reporter assay in a zebrafish model system in vivo. We show that SOX18 dimerization is driven by a novel motif located in the vicinity of the C-terminus of the DNA binding region. Insertion of this motif in a SOX7 monomer forced its assembly into a dimer. Genome-wide analysis of SOX18 binding locations on the chromatin revealed enrichment for a SOX dimer binding motif, correlating with genes with a strong endothelial signature. Using a SOX18 small molecule inhibitor that disrupts dimerization, we revealed that dimerization is important for transcription. Overall, we show that dimerization is a specific feature of SOX18 that enables the recruitment of key endothelial transcription factors, and refines the selectivity of the binding to discrete genomic locations assigned to endothelial specific genes.
Asunto(s)
Factores de Transcripción SOXF/química , Secuencias de Aminoácidos , Animales , Técnicas Biosensibles , Proteínas de Unión al ADN/química , Células Endoteliales/metabolismo , Endotelio/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas Fluorescentes Verdes/química , Humanos , Ratones , Mutación , Sistemas de Lectura Abierta , Dominios Proteicos , Multimerización de Proteína , Pez Cebra , Proteínas de Pez Cebra/químicaRESUMEN
Vascular endothelial growth factor-D (VEGFD) is a potent pro-lymphangiogenic molecule during tumor growth and is considered a key therapeutic target to modulate metastasis. Despite roles in pathological neo-lymphangiogenesis, the characterization of an endogenous role for VEGFD in vascular development has remained elusive. Here, we used zebrafish to assay for genetic interactions between the Vegf/Vegf-receptor pathway and SoxF transcription factors and identified a specific interaction between Vegfd and Sox18. Double knockdown zebrafish embryos for Sox18/Vegfd and Sox7/Vegfd exhibit defects in arteriovenous differentiation. Supporting this observation, we found that Sox18/Vegfd double but not single knockout mice displayed dramatic vascular development defects. We find that VEGFD-mitogen-activated protein kinase kinase-extracellular signal-regulated kinase signaling modulates SOX18-mediated transcription, functioning at least in part by enhancing nuclear concentration and transcriptional activity in vascular endothelial cells. This work suggests that VEGFD-mediated pathologies include or involve an underlying dysregulation of SOXF-mediated transcriptional networks.
Asunto(s)
Vasos Sanguíneos/embriología , Neovascularización Fisiológica/genética , Factores de Transcripción SOXF/metabolismo , Factor D de Crecimiento Endotelial Vascular/fisiología , Proteínas de Pez Cebra/metabolismo , Pez Cebra/embriología , Animales , Animales Modificados Genéticamente , Embrión de Mamíferos , Embrión no Mamífero , Femenino , Regulación del Desarrollo de la Expresión Génica , Redes Reguladoras de Genes/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Factores de Transcripción SOXF/genética , Pez Cebra/genética , Proteínas de Pez Cebra/genéticaRESUMEN
This review discusses successful strategies and potential pitfalls to assembling a natural product-based library suitable for high-throughput screening. Specific extraction methods for plants, microorganisms, and marine invertebrates are detailed, along with methods for generating a fractionated sub-library. The best methods to store, maintain and prepare the library for screening are addressed, as well as recommendations on how to develop a robust high-throughput assay. Finally, the logistics of moving from an assay hit to pure bioactive compound are discussed.
Asunto(s)
Productos Biológicos , Descubrimiento de Drogas/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Bibliotecas de Moléculas Pequeñas , Animales , Productos Biológicos/uso terapéutico , Humanos , Invertebrados/química , Extractos Vegetales/uso terapéutico , Plantas/químicaRESUMEN
Bioassay guided fractionation of three southern Australian marine sponges of the genus Psammocinia, selected for their ability to modulate glycine-gated chloride channel receptors (GlyRs), yielded the rare marine sesterterpenes (-)-ircinianin (1) and (-)-ircinianin sulfate (2), along with the new biosynthetically related metabolites (-)-ircinianin lactam A (3), (-)-ircinianin lactam A sulfate (4), (-)-oxoircinianin (5), (-)-oxoircinianin lactam A (6) and (-)-ircinianin lactone A (7). Acetylation of 1 returned (-)-ircinianin acetate (8). Whole cell patch-clamp electrophysiology on 1-8 established 3 as an exceptionally potent and selective α3 GlyR potentiator, and 6 as a selective α1 GlyR potentiator. The discovery and characterization of sesterterpenes 1-8, and in particular the glycinyl-lactams 3 and 6, provide valuable new insights into GlyR pharmacology. These insights have the potential to inform and inspire the development of new molecular tools to probe GlyR distribution and function, and therapeutics to treat a wide array of GlyR mediated diseases and disorders.
Asunto(s)
Organismos Acuáticos/química , Glicina/química , Lactamas/química , Lactamas/farmacología , Poríferos/química , Receptores de Glicina/metabolismo , Sesterterpenos/química , Animales , Productos Biológicos/química , Productos Biológicos/aislamiento & purificación , Productos Biológicos/farmacología , Células HEK293 , Humanos , Lactamas/aislamiento & purificaciónRESUMEN
Chemical analysis of a specimen of the sponge Ianthella cf. flabelliformis returned two new sesquiterpene glycinyl lactams, ianthellalactams A (1) and B (2), the known sponge sesquiterpene dictyodendrillin (3) and its ethanolysis artifact ethyl dictyodendrillin (4), and five known sponge indole alkaloids, aplysinopsin (5), 8E-3'-deimino-3'-oxoaplysinopsin (6), 8Z-3'-deimino-3'-oxoaplysinopsin (7), dihydroaplysinopsin (8) and tubastrindole B (9). The equilibrated mixture 6/7 exhibited glycine-gated chloride channel receptor (GlyR) antagonist activity with a bias towards α3 over α1 GlyR, while tubastrindole B (9) exhibited a bias towards α1 over α3 GlyR. At low- to sub-micromolar concentrations, 9 was also a selective potentiator of α1 GlyR, with no effect on α3 GlyR-a pharmacology that could prove useful in the treatment of movement disorders such as spasticity and hyperekplexia. Our investigations into the GlyR modulatory properties of 1-9 were further supported by the synthesis of a number of structurally related indole alkaloids.
Asunto(s)
Alcaloides/química , Canales de Cloruro/antagonistas & inhibidores , Poríferos/química , Receptores de Glicina/metabolismo , Alcaloides/farmacología , Animales , Australia , Canales de Cloruro/química , Espectroscopía de Resonancia Magnética , Estructura Molecular , Unión Proteica/efectos de los fármacos , Receptores de Glicina/químicaRESUMEN
The SOX transcription factor family is pivotal in controlling aspects of development. To identify genotype-phenotype relationships of SOX proteins, we performed a non-biased study of SOX using 1890 open-reading frame and 6667 amino acid sequences in combination with structural dynamics to interpret 3999 gnomAD, 485 ClinVar, 1174 Geno2MP, and 4313 COSMIC human variants. We identified, within the HMG (High Mobility Group)- box, twenty-seven amino acids with changes in multiple SOX proteins annotated to clinical pathologies. These sites were screened through Geno2MP medical phenotypes, revealing novel SOX15 R104G associated with musculature abnormality and SOX8 R159G with intellectual disability. Within gnomAD, SOX18 E137K (rs201931544), found within the HMG box of ~0.8% of Latinx individuals, is associated with seizures and neurological complications, potentially through blood-brain barrier alterations. A total of 56 highly conserved variants were found at sites outside the HMG-box, including several within the SOX2 HMG-box-flanking region with neurological associations, several in the SOX9 dimerization region associated with Campomelic Dysplasia, SOX14 K88R (rs199932938) flanking the HMG box associated with cardiovascular complications within European populations, and SOX7 A379V (rs143587868) within an SOXF conserved far C-terminal domain heterozygous in 0.716% of African individuals with associated eye phenotypes. This SOX data compilation builds a robust genotype-to-phenotype association for a gene family through more robust ortholog data integration.
Asunto(s)
Proteínas del Grupo de Alta Movilidad , Factores de Transcripción SOX , Humanos , Proteínas del Grupo de Alta Movilidad/química , Proteínas del Grupo de Alta Movilidad/genética , Proteínas del Grupo de Alta Movilidad/metabolismo , Factores de Transcripción SOX/genética , Secuencia de Aminoácidos , Dimerización , Genotipo , Factores de Transcripción SOXF/genética , Factores de Transcripción SOXF/metabolismo , Factores de Transcripción SOXB2/genética , Factores de Transcripción SOXB2/metabolismo , Factores de Transcripción SOXE/genéticaRESUMEN
Propranolol and atenolol, current therapies for problematic infantile hemangioma (IH), are composed of R(+) and S(-) enantiomers: the R(+) enantiomer is largely devoid of beta blocker activity. We investigated the effect of R(+) enantiomers of propranolol and atenolol on the formation of IH-like blood vessels from hemangioma stem cells (HemSCs) in a murine xenograft model. Both R(+) enantiomers inhibited HemSC vessel formation in vivo. In vitro, similar to R(+) propranolol, both atenolol and its R(+) enantiomer inhibited HemSC to endothelial cell differentiation. As our previous work implicated the transcription factor sex-determining region Y (SRY) box transcription factor 18 (SOX18) in propranolol-mediated inhibition of HemSC to endothelial differentiation, we tested in parallel a known SOX18 small-molecule inhibitor (Sm4) and show that this compound inhibited HemSC vessel formation in vivo with efficacy similar to that seen with the R(+) enantiomers. We next examined how R(+) propranolol alters SOX18 transcriptional activity. Using a suite of biochemical, biophysical, and quantitative molecular imaging assays, we show that R(+) propranolol directly interfered with SOX18 target gene trans-activation, disrupted SOX18-chromatin binding dynamics, and reduced SOX18 dimer formation. We propose that the R(+) enantiomers of widely used beta blockers could be repurposed to increase the efficiency of current IH treatment and lower adverse associated side effects.
Asunto(s)
Atenolol/farmacología , Hemangioma , Células Madre Neoplásicas/metabolismo , Neovascularización Patológica , Propranolol/farmacología , Animales , Hemangioma/irrigación sanguínea , Hemangioma/tratamiento farmacológico , Hemangioma/metabolismo , Humanos , Ratones , Neovascularización Patológica/tratamiento farmacológico , Neovascularización Patológica/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
A bioassay-guided search for inhibitors of lipid droplet formation in a deep-water southern Australian marine sponge, Spongia (Heterofibria) sp., yielded six new compounds, fatty acids heterofibrins A1 (1) and B1 (4), along with related monolactyl and dilactyl esters, heterofibrins A2 (2), B2 (5), A3 (3) and B3 (6). Heterofibrin structures were assigned on the basis of detailed spectroscopic analysis, with comparison to chiral synthetic model compounds. All heterofibrins possess a diyne-ene moiety, while the monolactyl and dilactyl moiety featured in selected heterofibrins is unprecedented in the natural products literature. SAR by co-metabolite studies on the heterofibrins confirmed them to be non-cytotoxic, with the carboxylic acids 1 and 4 inhibiting lipid droplet formation in A431 fibroblast cell lines. Such inhibitors have potential application in the management of obesity, diabetes and atherosclerosis
Asunto(s)
Citoplasma/efectos de los fármacos , Citoplasma/metabolismo , Ácidos Grasos/farmacología , Poríferos/química , Animales , Bacterias/efectos de los fármacos , Línea Celular Tumoral , Cromatografía Líquida de Alta Presión , Ácidos Grasos/química , Ácidos Grasos/aislamiento & purificación , Ácidos Grasos/toxicidad , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Hongos/efectos de los fármacos , Espectroscopía de Resonancia MagnéticaRESUMEN
Screening an extract library of >2500 southern Australian and Antarctic marine invertebrates and algae for modulators of glycine receptor (GlyR) chloride channels identified three Irciniidae sponges that yielded new examples of a rare class of glycinyl lactam sesterterpene, ircinialactam A, 8-hydroxyircinialactam A, 8-hydroxyircinialactam B, ircinialactam C, ent-ircinialactam C and ircinialactam D. Structure-activity relationship (SAR) investigations revealed a new pharmacophore with potent and subunit selective modulatory properties against alpha1 and alpha3 GlyR isoforms. Such GlyR modulators have potential application as pharmacological tools, and as leads for the development of GlyR targeting therapeutics to treat chronic inflammatory pain, epilepsy, spasticity and hyperekplexia.
Asunto(s)
Alcaloides Indólicos/química , Lactamas/química , Receptores de Glicina/metabolismo , Animales , Australia , Línea Celular , Humanos , Alcaloides Indólicos/aislamiento & purificación , Alcaloides Indólicos/farmacología , Lactamas/aislamiento & purificación , Lactamas/farmacología , Poríferos/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Receptores de Glicina/química , Relación Estructura-ActividadRESUMEN
Propranolol is an approved non-selective ß-adrenergic blocker that is first line therapy for infantile hemangioma. Despite the clinical benefit of propranolol therapy in hemangioma, the mechanistic understanding of what drives this outcome is limited. Here, we report successful treatment of pericardial edema with propranolol in a patient with Hypotrichosis-Lymphedema-Telangiectasia and Renal (HLTRS) syndrome, caused by a mutation in SOX18. Using a mouse pre-clinical model of HLTRS, we show that propranolol treatment rescues its corneal neo-vascularisation phenotype. Dissection of the molecular mechanism identified the R(+)-propranolol enantiomer as a small molecule inhibitor of the SOX18 transcription factor, independent of any anti-adrenergic effect. Lastly, in a patient-derived in vitro model of infantile hemangioma and pre-clinical model of HLTRS we demonstrate the therapeutic potential of the R(+) enantiomer. Our work emphasizes the importance of SOX18 etiological role in vascular neoplasms, and suggests R(+)-propranolol repurposing to numerous indications ranging from vascular diseases to metastatic cancer.
Asunto(s)
Antagonistas Adrenérgicos beta/farmacología , Inhibidores Enzimáticos/farmacología , Hemangioma/tratamiento farmacológico , Hipotricosis/tratamiento farmacológico , Linfedema/tratamiento farmacológico , Propranolol/farmacología , Factores de Transcripción SOXF/antagonistas & inhibidores , Telangiectasia/tratamiento farmacológico , Antagonistas Adrenérgicos beta/administración & dosificación , Animales , Modelos Animales de Enfermedad , Inhibidores Enzimáticos/administración & dosificación , Humanos , Ratones , Modelos Teóricos , Propranolol/administración & dosificaciónRESUMEN
Antibodies are routinely used to study the activity of transcription factors, using various in vitro and in vivo approaches such as electrophoretic mobility shift assay, enzyme-linked immunosorbent assay, genome-wide method analysis coupled with next generation sequencing, or mass spectrometry. More recently, a new application for antibodies has emerged as crystallisation scaffolds for difficult to crystallise proteins, such as transcription factors. Only in a few rare cases, antibodies have been used to modulate the activity of transcription factors, and there is a real gap in our knowledge on how to efficiently design antibodies to interfere with transcription. The molecular function of transcription factors is underpinned by complex networks of protein-protein interaction and in theory, setting aside intra-cellular delivery challenges, developing antibody-based approaches to modulate transcription factor activity appears a viable option. Here, we demonstrate that antibodies or an antibody single-chain variable region fragments are powerful molecular tools to unravel complex protein-DNA and protein-protein binding mechanisms. In this study, we focus on the molecular mode of action of the transcription factor SOX18, a key modulator of endothelial cell fate during development, as well as an attractive target in certain pathophysiological conditions such as solid cancer metastasis. The engineered antibody we designed inhibits SOX18 transcriptional activity, by interfering specifically with an 8-amino-acid motif in the C-terminal region directly adjacent to α-Helix 3 of SOX18 HMG domain, thereby disrupting protein-protein interaction. This new approach establishes a framework to guide the study of transcription factors interactomes using antibodies as molecular handles.
Asunto(s)
Factores de Transcripción SOXF/análisis , Factores de Transcripción SOXF/química , Anticuerpos de Cadena Única , HumanosRESUMEN
Pharmacological modulation of transcription factors (TFs) has only met little success over the past four decades. This is mostly due to standard drug discovery approaches centered on blocking protein/DNA binding or interfering with post-translational modifications. Recent advances in the field of TF biology have revealed a central role of protein-protein interaction in their mode of action. In an attempt to modulate the activity of SOX18 TF, a known regulator of vascular growth in development and disease, we screened a marine extract library for potential small-molecule inhibitors. We identified two compounds, which inspired a series of synthetic SOX18 inhibitors, able to interfere with the SOX18 HMG DNA-binding domain, and to disrupt HMG-dependent protein-protein interaction with RBPJ. These compounds also perturbed SOX18 transcriptional activity in a cell-based reporter gene system. This approach may prove useful in developing a new class of anti-angiogenic compounds based on the inhibition of TF activity.
Asunto(s)
Factores de Transcripción SOXF/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/química , Animales , Sitios de Unión , Productos Biológicos/química , Productos Biológicos/metabolismo , Productos Biológicos/farmacología , Células COS , Chlorocebus aethiops , ADN/química , ADN/metabolismo , Diseño de Fármacos , Genes Reporteros , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/química , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/metabolismo , Concentración 50 Inhibidora , Ratones , Conformación de Ácido Nucleico , Unión Proteica , Mapas de Interacción de Proteínas , Estructura Terciaria de Proteína , Factores de Transcripción SOXF/genética , Factores de Transcripción SOXF/metabolismo , Ácido Salicílico/química , Ácido Salicílico/metabolismo , Ácido Salicílico/farmacología , Bibliotecas de Moléculas Pequeñas/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Relación Estructura-Actividad , Activación Transcripcional/efectos de los fármacosRESUMEN
Pharmacological targeting of transcription factors holds great promise for the development of new therapeutics, but strategies based on blockade of DNA binding, nuclear shuttling, or individual protein partner recruitment have yielded limited success to date. Transcription factors typically engage in complex interaction networks, likely masking the effects of specifically inhibiting single protein-protein interactions. Here, we used a combination of genomic, proteomic and biophysical methods to discover a suite of protein-protein interactions involving the SOX18 transcription factor, a known regulator of vascular development and disease. We describe a small-molecule that is able to disrupt a discrete subset of SOX18-dependent interactions. This compound selectively suppressed SOX18 transcriptional outputs in vitro and interfered with vascular development in zebrafish larvae. In a mouse pre-clinical model of breast cancer, treatment with this inhibitor significantly improved survival by reducing tumour vascular density and metastatic spread. Our studies validate an interactome-based molecular strategy to interfere with transcription factor activity, for the development of novel disease therapeutics.
Asunto(s)
Antineoplásicos/metabolismo , Neoplasias de la Mama/prevención & control , Factores de Transcripción SOXF/antagonistas & inhibidores , Transcripción Genética/efectos de los fármacos , Animales , Fenómenos Biofísicos , Vasos Sanguíneos/embriología , Modelos Animales de Enfermedad , Genómica , Ratones , Proteómica , Resultado del Tratamiento , Pez Cebra/embriología , Proteínas de Pez Cebra/antagonistas & inhibidoresRESUMEN
Cylindrospermopsin (CYN) is a cyanobacterial toxin found in drinking-water sources world wide. It was the likely cause of human poisonings in Australia and possibly Brazil. Although CYN itself is a potent protein synthesis inhibitor, its acute toxicity appears to be mediated by cytochrome p-450 (CYP450)-generated metabolites. CYN also induces genotoxic effects both in vitro and in vivo, and preliminary evidence suggests that tumors are generated by oral exposure to CYN. To understand the role of CYP450-activated CYN metabolites on in vitro genotoxicity, this study quantified the process in primary mouse hepatocytes using the COMET assay in both the presence and absence of CYP450 inhibitors known to block acute CYN cytotoxicity. CYN was cytotoxic at concentrations above 0.1 microM (EC50 = 0.5 microM) but produced significant increases in Comet tail length, area, and tail moment at 0.05 microM and above; hence genotoxicity is unlikely to be secondary to metabolic disruption due to toxicity. The CYP450 inhibitors omeprazole (100 microM) and SKF525A (50 microM) completely inhibited the genotoxicity induced by CYN. The toxin also inhibits production of glutathione (GSH), a finding confirmed in this study. This could potentiate cytotoxicity, and by implication genotoxicity, via reduced reactive oxygen species (ROS) quenching. The lipid peroxidation marker, malondialdehyde (MDA) was quantified in CYN-treated cells, and the effect of the reduced glutathione (GSSG) reductase (GSSG-rd.) inhibitor 1,3-bis(chloroethyl)-l-nitrosourea (BCNU) on both MDA production and lactate dehydrogenase (LDH) leakage was examined. MDA levels were not elevated by CYN treatment, and block of GSH regeneration by BCNU did not affect lipid peroxidation or cytotoxicity. It therefore seems likely that CYP450-derived metabolites are responsible for both the acute cytotoxicity and genotoxicity induced by CYN.
Asunto(s)
Sistema Enzimático del Citocromo P-450/fisiología , Hepatocitos/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Uracilo/análogos & derivados , Uracilo/toxicidad , Alcaloides , Animales , Toxinas Bacterianas , Ensayo Cometa , Toxinas de Cianobacterias , Inhibidores Enzimáticos del Citocromo P-450 , Hepatocitos/enzimología , Peroxidación de Lípido/efectos de los fármacos , Masculino , Ratones , Pruebas de Mutagenicidad , Estrés Oxidativo/genética , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Much research on transcription factor biology and their genetic pathways has been undertaken over the last 30 years, especially in the field of developmental biology and cancer. Yet, very little is known about the molecular modalities of highly dynamic interactions between transcription factors, genomic DNA, and protein partners. Methodological breakthroughs such as RNA-seq (RNA-sequencing), ChIP-seq (chromatin immunoprecipitation sequencing), RIME (rapid immunoprecipitation mass spectrometry of endogenous proteins), and single-molecule imaging will dramatically accelerate the discovery rate of their molecular mode of action in the next few years. From a pharmacological viewpoint, conventional methods used to target transcription factor activity with molecules mimicking endogenous ligands fail to achieve high specificity and are limited by a lack of identification of new molecular targets. Protein-protein interactions are likely to represent one of the next major classes of therapeutic targets. Transcription factors, known to act mostly via protein-protein interaction, may well be at the forefront of this type of drug development. One hurdle in this field remains the difficulty to collate structural data into meaningful information for rational drug design. Another hurdle is the lack of chemical libraries meeting the structural requirements of protein-protein interaction disruption. As more attempts at modulating transcription factor activity are undertaken, valuable knowledge will be accumulated on the modality of action required to modulate transcription and how these findings can be applied to developing transcription factor drugs. Key discoveries will spawn into new therapeutic approaches not only as anticancer targets but also for other indications, such as those with an inflammatory component including neurodegenerative disorders, diabetes, and chronic liver and kidney diseases.
RESUMEN
Elevated levels of reactive alpha,beta-unsaturated aldehydes (e.g. malondialdehyde, 4-hydroxynonenal and acrolein) in the affected tissues of various degenerative conditions suggest these substances are active propagators of the disease process. One experimental approach to attenuating damage by these intermediates employs 'aldehyde-sequestering drugs' as sacrificial nucleophiles, thereby sparing cell macromolecules and perhaps slowing disease progression. Drugs with demonstrated trapping activity toward lipid-derived aldehydes include various amine compounds such as aminoguanidine, carnosine and pyridoxamine. We have focused on identifying scavengers of acrolein, perhaps the most toxic aldehyde formed during lipid peroxidation cascades. Various phthalazine compounds (hydralazine and dihydralazine) were found to trap acrolein readily, forming hydrazone derivatives in a rapid Schiff-type reaction. These compounds strongly protect against acrolein-mediated toxicity in isolated hepatocytes.