RESUMO
Essential oils (EOs) are increasingly consumed as food supplements. The few published recommended doses available generally lack details both on the methodology used and concentration limits for substances of concern, including genotoxic carcinogens. We propose a tiered approach based on the toxicological evaluation of maximized concentrations of each constituent present in the EO investigated. The genotoxic potential of each constituent is assessed using literature data or QSAR analyses. Genotoxic constituents are evaluated according to the methodology provided in the ICHM7 guideline. A Toxicological Reference Value (TRV) is associated to each non-genotoxic constituent, using one of the following methodologies (decision-tree successive steps): extraction from recognized databases or clinical studies, application of adequate safety factors to NOAELs established in animal studies, read-across analyses and when none was possible, TTC of Cramer classes. An EO recommended dose is considered safe when the safety margin (ratio between TRV and systemic exposure) for all constituents is all at least equal to 1. In conclusion, this methodology has proven to be robust to establish safe recommended doses for EOs used as food supplements, consistent with those publicly available, and avoiding unnecessary dedicated new animal testing.
Assuntos
Suplementos Nutricionais/toxicidade , Óleos Voláteis/toxicidade , Animais , Simulação por Computador , Feminino , Inocuidade dos Alimentos/métodos , Humanos , Masculino , Camundongos , Óleos Voláteis/administração & dosagem , Ratos , Testes de Toxicidade/métodosRESUMO
Five new quinolizidine alkaloids were isolated from the leaves of Cylicomorpha solmstii (Urb.) Urb. (Caricaceae) and named cylicomorphins A-E (1-5). They all are ester derivatives of the same basic quinolizidine skeleton bearing hydroxy, methyl, and ethanoic acid substituents. Their structures were mainly established by NMR spectroscopy, and the absolute configuration is proposed on the basis of VCD data and Mosher ester derivatization. Compound 5 displayed cytotoxicity in the 10 µM range against an HCT-116 cell line.
Assuntos
Alcaloides/farmacologia , Antineoplásicos Fitogênicos/farmacologia , Caricaceae/química , Quinolizidinas/farmacologia , Alcaloides/isolamento & purificação , Antineoplásicos Fitogênicos/isolamento & purificação , Camarões , Células HCT116 , Humanos , Estrutura Molecular , Compostos Fitoquímicos/isolamento & purificação , Compostos Fitoquímicos/farmacologia , Folhas de Planta/química , Quinolizidinas/isolamento & purificaçãoRESUMO
Aberrant DNA hypermethylation of promoter of tumor suppressor genes is commonly observed in cancer, and its inhibition by small molecules is promising for their reactivation. Here we designed bisubstrate analogues-based inhibitors, by mimicking each substrate, the S-adenosyl-l-methionine and the deoxycytidine, and linking them together. This approach resulted in quinazoline-quinoline derivatives as potent inhibitors of DNMT3A and DNMT1, some showing certain isoform selectivity. We highlighted the importance of (i) the nature and rigidity of the linker between the two moieties for inhibition, as (ii) the presence of the nitrogen on the quinoline group, and (iii) of a hydrophobic group on the quinazoline. The most potent inhibitors induced demethylation of CDKN2A promoter in colon carcinoma HCT116 cells and its reactivation after 7 days of treatment. Furthermore, in a leukemia cell model system, we found a correlation between demethylation of the promoter induced by the treatment, chromatin opening at the promoter, and the reactivation of a reporter gene.
Assuntos
DNA (Citosina-5-)-Metiltransferases/antagonistas & inibidores , Desenho de Fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Neoplasias/enzimologia , Linhagem Celular Tumoral , DNA (Citosina-5-)-Metiltransferase 1 , Metilação de DNA , DNA Metiltransferase 3A , Genes Supressores de Tumor , Humanos , Neoplasias/patologia , Especificidade por SubstratoRESUMO
DNA methylation is the most studied epigenetic event. Since the methylation profile of the genome is widely modified in cancer cells, DNA methyltransferases are the target of new anticancer therapies. Nucleosidic inhibitors suffer from toxicity and chemical stability, while non-nucleosidic inhibitors lack potency. Here, we found a novel DNMT inhibitor scaffold by enzymatic screening and structure-activity relationship studies. The optimization studies led to an inhibitor containing three fragments: a gallate frame, a hydrazone linker and a benzothiazole moiety. Interestingly, the compound inhibits DNMT3A with micromolar potency (EC50 = 1.6 µM) and does not inhibit DNMT1; this DNMT3A selectivity is supported by a docking study. Finally, the compound reactivates a reporter gene in leukemia KG-1 cells.
Assuntos
Antineoplásicos/farmacologia , DNA (Citosina-5-)-Metiltransferases/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Ácido Gálico/farmacologia , Hidrazonas/farmacologia , Neoplasias/tratamento farmacológico , Antineoplásicos/síntese química , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , DNA (Citosina-5-)-Metiltransferases/metabolismo , DNA Metiltransferase 3A , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Ácido Gálico/química , Humanos , Hidrazonas/síntese química , Hidrazonas/química , Neoplasias/metabolismo , Relação Estrutura-AtividadeRESUMO
A screening program aimed at discovering novel anticancer agents based on natural products led to the selection of koningic acid (KA), known as a potent inhibitor of glycolysis. A method was set up to produce this fungal sesquiterpene lactone in large quantities by fermentation, thus allowing (i) an extensive analysis of its anticancer potential in vitro and in vivo and (ii) the semi-synthesis of analogues to delineate structure-activity relationships. KA was characterized as a potent, but non-selective cytotoxic agent, active under both normoxic and hypoxic conditions and inactive in the A549 lung cancer xenograft model. According to our SAR, the acidic group could be replaced to keep bioactivity but an intact epoxide is essential.
Assuntos
Antineoplásicos/síntese química , Neoplasias Pulmonares/tratamento farmacológico , Animais , Antineoplásicos/isolamento & purificação , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Hipóxia Celular , Linhagem Celular Tumoral , Fermentação , Glicólise/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Nus , Sesquiterpenos/síntese química , Sesquiterpenos/isolamento & purificação , Sesquiterpenos/farmacocinética , Sesquiterpenos/farmacologia , Relação Estrutura-Atividade , Trichoderma/química , Trichoderma/metabolismo , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Quinoline derivative SGI-1027 (N-(4-(2-amino-6-methylpyrimidin-4-ylamino)phenyl)-4-(quinolin-4-ylamino)benzamide) was first described in 2009 as a potent inhibitor of DNA methyltransferase (DNMT) 1, 3A and 3B. Based on molecular modeling studies, performed using the crystal structure of Haemophilus haemolyticus cytosine-5 DNA methyltransferase (MHhaI C5 DNMT), which suggested that the quinoline and the aminopyridimine moieties of SGI-1027 are important for interaction with the substrates and protein, we designed and synthesized 25 derivatives. Among them, four compoundsnamely the derivatives 12, 16, 31 and 32exhibited activities comparable to that of the parent compound. Further evaluation revealed that these compounds were more potent against human DNMT3A than against human DNMT1 and induced the re-expression of a reporter gene, controlled by a methylated cytomegalovirus (CMV) promoter, in leukemia KG-1 cells. These compounds possessed cytotoxicity against leukemia KG-1 cells in the micromolar range, comparable with the cytotoxicity of the reference compound, SGI-1027. Structureactivity relationships were elucidated from the results. First, the presence of a methylene or carbonyl group to conjugate the quinoline moiety decreased the activity. Second, the size and nature of the aromatic or heterocycle subsitutents effects inhibition activity: tricyclic moieties, such as acridine, were found to decrease activity, while bicyclic substituents, such as quinoline, were well tolerated. The best combination was found to be a bicyclic substituent on one side of the compound, and a one-ring moiety on the other side. Finally, the orientation of the central amide bond was found to have little effect on the biological activity. This study provides new insights in to the structure-activity relationships of SGI-1027 and its derivative.
Assuntos
Aminoquinolinas/síntese química , Aminoquinolinas/farmacologia , Metilação de DNA/efeitos dos fármacos , Desenho de Fármacos , Pirimidinas/síntese química , Pirimidinas/farmacologia , Quinolinas/química , Aminoquinolinas/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/antagonistas & inibidores , DNA (Citosina-5-)-Metiltransferases/metabolismo , DNA Metiltransferase 3A , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Pirimidinas/química , Relação Estrutura-AtividadeRESUMO
Six dichapetalins named dichapetalins N-S were isolated from Dichapetalum mombuttense, Dichapetalum zenkeri and Dichapetalum leucosia. They were accompanied in the same plants by the known dichapetalins A, B, C, I, L and M. The structures of the compounds were elucidated by 1D and 2D NMR experiments and mass spectrometry. They all possessed the dammarane skeleton substituted at position C-3 by a C6-C2 unit forming a 2-phenylpyran moiety. All contained a lactone ring in the side chain except dichapetalins O, Q and R, in which this ring was replaced by a lactol. Dichapetalin Q and R were also the first dichapetalins bearing a tertiary methyl and a double bond instead of the cyclopropane of the dammaranes. All these compounds were assayed against cancer cell lines HCT116 and WM 266-4 and displayed cytotoxic and anti-proliferative activities in the 10(-6) to 10(-8)M range.
Assuntos
Antineoplásicos Fitogênicos/química , Magnoliopsida/química , Extratos Vegetais/química , Raízes de Plantas/química , Antineoplásicos Fitogênicos/isolamento & purificação , Antineoplásicos Fitogênicos/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Células HCT116 , Células HL-60 , Compostos Heterocíclicos de 4 ou mais Anéis/química , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Humanos , Espectroscopia de Ressonância Magnética/métodos , Magnoliopsida/classificação , Estrutura Molecular , Extratos Vegetais/isolamento & purificação , Extratos Vegetais/farmacologia , Especificidade da Espécie , Compostos de Espiro/química , Compostos de Espiro/farmacologia , Relação Estrutura-AtividadeRESUMO
The interesting pharmacological properties of neoboutomellerones 1 and 2 were the basis for the assembly of a small library of analogues consisting of natural products isolated from the plant Neoboutonia melleri and of semisynthetic derivatives. As the two enone systems (C23-C24a and C1-C3) and the two hydroxyls groups (C22 and C26) of neoboutomellerones are required for activity, modifications were focused on these functional groups. Biological evaluation by using a cellular assay for proteasome activity provided clues regarding the mechanism of action of these natural products and synthetic derivatives. Certain neoboutomellerone derivatives inhibited the proliferation of human WM-266-4 melanoma tumor cells at submicromolar concentration and warrant evaluation as anticancer agents.
Assuntos
Antineoplásicos/síntese química , Produtos Biológicos/química , Inibidores de Proteassoma , Triterpenos/síntese química , Ubiquitina/antagonistas & inibidores , Antineoplásicos/química , Antineoplásicos/toxicidade , Linhagem Celular Tumoral , Euphorbiaceae/química , Humanos , Complexo de Endopeptidases do Proteassoma/metabolismo , Transdução de Sinais/efeitos dos fármacos , Triterpenos/química , Triterpenos/toxicidade , Ubiquitina/metabolismoRESUMO
Thirty new cycloartane derivatives (1-3, 5-12, 14-32) have been isolated from the leaves of Neoboutonia melleri. Their novelty stems from the loss of one of the C-4 methyl groups (1-3, 5-12, 14-25, and 32) and from the presence of an "extra" carbon atom in the side chain (1-3, 5-12, 14-20, 26-29, and 30-32). Furthermore, compound 32 possesses a rare triterpene skeleton with the cyclopropane ring fused onto C-1 and C-10, instead of C-9 and C-10. The structures were determined by spectrometric means, chemical correlations, and X-ray crystallography of derivative 1c. The substitution pattern in ring A, with a cyclopropyl ring conjugated with an α,ß-unsaturated carbonyl moiety, confers to the molecule a particular reactivity, giving rise to a formal inversion of the stereochemistry of the cyclopropane ring under UV irradiation. These compounds showed an interesting level of activity on the proteasome pathway, thus motivating their evaluation as possible anticancer agents. The large number of isolated compounds permitted a structure-activity relationship analysis, which showed that the presence of the two enone functions was a requirement for the activity.
Assuntos
Euphorbiaceae/química , Inibidores de Proteassoma , Triterpenos/isolamento & purificação , Triterpenos/farmacologia , Camarões , Estrutura Molecular , Folhas de Planta/química , Caules de Planta/química , Relação Estrutura-Atividade , Triterpenos/químicaRESUMO
Six carvotanacetone derivatives (1- 6), amongst which four new compounds (1- 4), were isolated from the aerial parts of Sphaeranthus ukambensis Vatke & O. Hoffm. The structures of the molecules were elucidated by complementary spectroscopic methods, and their biological properties were investigated using human DLD-1 colon cancer cells engineered to stably express a 4 ubiquitin-luciferase (4 Ub-Luc) reporter protein. Five of the isolated carvotanacetone derivatives (2- 6) were found to inhibit the proliferation of the colon cancer cells and interfere with the ubiquitin-proteasome pathway, with potencies in a micromolar range.
Assuntos
Asteraceae/química , Extratos Vegetais/química , Inibidores de Proteases/farmacologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Terpenos/farmacologia , Ubiquitina/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Quênia , Componentes Aéreos da Planta/química , Terpenos/química , Terpenos/isolamento & purificaçãoRESUMO
The ubiquitin-proteasome pathway plays a critical role in the degradation of proteins involved in tumor growth and has therefore become a target for cancer therapy. In order to discover novel inhibitors of this pathway, a cellular assay reporter of proteasome activity was established. Human DLD-1 colon cancer cells were engineered to express a 4 ubiquitin-luciferase (DLD-1 4Ub-Luc) reporter protein, rapidly degraded via the ubiquitin-proteasome pathway and designed DLD-1 4Ub-Luc cells. Following treatment with reference proteasome inhibitors, the 4Ub-Luc protein accumulated in DLD-1 4Ub-Luc cells and a 80-fold increase in luciferase-produced bioluminescence signal was measured, as compared to untreated cells. The screening of over 30,000 compounds using this DLD-1 4Ub-Luc assay led to the identification of physalin B as a novel inhibitor of the ubiquitin-proteasome pathway. Indeed, physalin B induced an increase in bioluminescence from DLD-1 4Ub-Luc cells, at concentrations also producing an accumulation of ubiquitinated proteins and inhibiting TNFalpha-induced NF-kappaB activation. Physalin B did not inhibit catalytic activities of purified proteasome and interfered with cellular proteasomal catalytic activities at 4- to 8-fold higher concentrations than that required to induce significant increase in bioluminescence and accumulation of ubiquitinated proteins in DLD-1 4Ub-Luc cells. Furthermore, physalin B proved to be cytotoxic, triggered apoptosis in DLD-1 4Ub-Luc cells and induced the proapoptotic protein NOXA, characteristic of the proteasome signaling pathway. Therefore, the use of the DLD-1 4Ub-Luc assay allowed the identification of a novel inhibitor of the ubiquitin-proteasome pathway that might interfere with proteasome functions in a different way from reference proteasome inhibitors.
Assuntos
Apoptose/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Secoesteroides/farmacologia , Ubiquitina/metabolismo , Proteínas Reguladoras de Apoptose/genética , Linhagem Celular Tumoral , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Extratos Vegetais/química , Inibidores de Proteases/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , Transdução de SinaisRESUMO
To discover original inhibitors of the ubiquitin-proteasome pathway, the authors have developed a cell-based bioluminescent assay and used it to screen collections of plant extracts and chemical compounds. They first established a DLD-1 human colon cancer cell line that stably expresses a 4Ubiquitin-Luciferase (4Ub-Luc) reporter protein, efficiently targeted to the ubiquitin-proteasome degradation pathway. The assay was then adapted to 96- and 384-well plate formats and calibrated with reference proteasome inhibitors. Assay robustness was carefully assessed, particularly cell toxicity, and the statistical Z factor value was calculated to 0.83, demonstrating a good performance level of the assay. A total of 18,239 molecules and 15,744 plant extracts and fractions thereof were screened for their capacity to increase the luciferase activity in DLD-1 4Ub-Luc cells, and 21 molecules and 66 extracts inhibiting the ubiquitin-proteasome pathway were identified. The fractionation of an active methanol extract of Physalis angulata L. aerial parts was performed to isolate 2 secosteroids known as physalin B and C. In a cell-based Western blot assay, the ubiquitinated protein accumulation was confirmed after a physalin treatment confirming the accuracy of the screening process. The method reported here thus provides a robust approach to identify novel ubiquitin-proteasome pathway inhibitors in large collections of chemical compounds and natural products.