Derivados 1,3,4-tiadiazóis mesoiônicos e indeno[1,2-b] indóis : citotoxicidade e efeitos sobre transportadores ABC

Orientador : Profª. Drª. Silvia Maria Suter Correia CadenaCoorientador : Prof. Dr. Attilio di PietroTese (doutorado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Ciências : Bioquímica. Defesa: Curitiba, 16/10/2015Inclui referências: fls.138-150Resumo: O carcinoma hepatocelular (CHC) possui uma alta taxa de mortalidade e apenas um quimioterápico, com eficiência limitada, está disponível para seu tratamento. Buscando potenciais drogas para o tratamento do CHC, neste estudo foram avaliados os efeitos de derivados 1,3,4-tiadiazóis mesoiônicos (MI-D, MI-J, MI-F e MI-2,4diF) em células de hepatocarcinoma humano (HepG2) e em hepatócitos de rato em cultura. A viabilidade das células HepG2, avaliada pelo método do MTT, foi reduzida em ~50% pelos derivados (25 ?M para MI-J, MI-F, MI-2,4diF e 50 ?M para MI-D - 24h). Esta citotoxicidade foi confirmada pelo aumento da atividade da enzima lactato desidrogenase (LDH) no meio extracelular, em ? 55, 24 e 16% após incubação com MI-D, MI-J e MI-F (25 ?M - 24 h), respectivamente. Nestas condições, análises por citometria de fluxo mostraram um aumento no número de células marcadas com anexina V e iodeto de propídio, de ?11, 76, 25 e 25 para MI-D, MI-J, MI-F e MI-2,4diF, respectivamente. O aumento na fragmentação do DNA também foi observado, em ?12, 9 e 8% para MI-J, MI-F e MI-2,4diF, respectivamente. Alterações morfológicas características de indução de apoptose foram observadas após incubação com todos os derivados (3h - 5 ?M). Nenhuma citotoxicidade foi observada quando células não-tumorais (hepatócitos de ratos) foram tratadas com os compostos na concentração de 25 ?M por 24 h. Neste estudo também se investigou a interação destes derivados com os principais transportadores responsáveis pela resistência aos tratamentos quimioterápicos: Pgp, ABCG2 e MRP1. MI-D, MI-4F e MI-2,4diF foram substratos de Pgp (RR= 2,4; 2,8 e 5,2), cuja atividade transportadora foi inibida por MI-J (? 13% - 30min). Todos os derivados inibiram tanto a atividade de ABCG2 (? 37, 21, 12 e 18 % para MI-D, MI-J, MI-4F e MI-2,4diF - 30min) quanto de MRP1 (? 8, 36, 10 e 20% para MI-D, MI-J, MI-4F e MI-2,4diF -30min). Ainda neste estudo, novos inibidores de ABCG2 foram desenvolvidos através de substituições apropriadas nos anéis do núcleo indeno[1,2-b]indol. Foram obtidos potentes inibidores de ABCG2 (4c, 4h, 4i, 4j, 4k - IC50= 0,21-0,49 ?M - N5 = fenetila) ou da caseína quinase II (CK2) (4a, 4p, 4e - IC50= 0,0025-0,36 ?M N5 = isopropila) quando o núcleo cetônico indeno[1,2-b]indol foi utilizado. Os derivados 4h, 4j, 4l e 4d apresentaram baixa citotoxicidade intrínseca (IG50 28-100 ?M), o que resultou em índices terapêuticos (IT) aceitáveis para utilização em modelos in vivo (IT= 98-430), entretanto, 4h, 4j, 4k não foram seletivos em relação à atividade inibitória em MRP1 (44, 87 e 86% - 10 ?M). A utilização de um núcleo fenólico indeno[1,2-b]indol resultou em inibidores com maior potência (5c, 5f, 5h- IC50= 0,15-0,16 ?M) e seletividade em CK2, Pgp e MRP1. Baixa citotoxicidade intrínseca (5c, 5f, 5h- IG50= 42-54 ?M) e alto IT (267-360) também foram encontrados. A partir dos resultados obtidos, conclui-se que derivados 1,3,4-tiadiazóis mesoiônicos e indeno[1,2-b]indóis fenólicos são candidatos promissores para futuras investigações in vivo visando o tratamento do CHC e a quimiosensibilização de tumores superexpressando ABCG2, respectivamente. Palavras-chave: Carcinoma hepatocelular. Células HepG2. Cultura primária de hepatócitos. Derivados 1,3,4-tiadiazóis mesoiônicos. Derivados indeno[1,2-b]indóis. Inibidores de ABCG2.Abstract: Hepatocellular carcinoma (HCC) has a high mortality rate and only one chemotherapy drug is available for its treatment, which has limited efficacy. Aiming to find potential drugs for HCC treatment, in this study the effects of 1,3,4-thiadiazolium mesoionic derivatives (MI-D, MI-J, MI-F and MI-2,4diF) were evaluated on human hepatocellular carcinoma cells (HepG2) and primary rat hepatocytes. The viability of HepG2 was reduced by ~ 50% after treatment with derivatives (25 ?M for MI-J, MI-F and MI-2,4diF and 50 ?M for MI-D- 24h), as shown by MTT assay. The cytotoxicity was confirmed by the increase of lactate dehydrogenase (LDH) in cells supernatant at 55, 24 and 16% for MI-J, MI-4F and MI-2,4diF, respectively (at 25 ?M after 24 h). Under same conditions, all compounds increased the number of cell doubly-stained with annexin V and PI (76% for MI-J, 25% for MI-4F and MI-2,4diF, and 11% for MI-D), as demonstrated by flow cytometry. DNA fragmentation was also observed upon MI-J, MI-4F and MI-2,4diF treatment (by 12%, 9% and 8%, respectively). Morphological features of apoptosis induction were observed after incubation with all derivatives (3h - 5 ?M). It was not observed cytotoxicity when non-tumor hepatocytes were treated with derivatives (25 ?M - 24h). This study also investigated the interaction of these derivatives with the main transporters related to chemotherapy resistance: Pgp, ABCG2 and MRP1. MI-D, MI-4F e MI-2,4diF were Pgp substrates (RR = 2.4; 2.8 and 5.2), and MI-J inhibited the transporter activity of Pgp by 13% (30 min). All derivatives inhibited both ABCG2 (by ? 37, 21, 12 and 18 % for MI-D, MI-J, MI-4F e MI-2,4diF - 30min) and MRP1(? 8, 36, 10 and 20% for MI-D, MI-J, MI-4F e MI-2,4diF - 30min) transporter activity. New ABCG2 inhibitors were developed through substitutions in indeno[1,2-b]indoles scaffold. Potent ABCG2 (4c, 4h, 4i, 4j, 4k - IC50= 0.21-0.49 ?M - N5 = phenethyl) or casein kinase II (CK2) (4a, 4p, 4e - IC50= 0.0025-0.36 ?M N5 = isopropyl) inhibitors were obtained with a cetonic indeno[1,2-b]indoles scaffold. The derivatives 4j, 4l and 4d showed low intrinsic cytotoxicity (IG50 28-100 ?M), resulting in therapeutic ratio (TR) suitable for use on in vivo models (TR = 98-430). However, the derivatives 4h, 4j, 4k were not selective to ABCG2, also inhibiting MRP1 activity (44, 87 e 86% - 10 ?M). The derivatives of phenolic indeno[1,2-b]indoles were more potent (5c, 5f, 5h- IC50= 0.15-0.16 ?M) and selective to ABCG2 than cetonic derivatives. Low intrinsic cytotoxicity (5c, 5f, 5h- IG50= 42-54 ?M) and high TR (267-360) were also observed. These results suggest that 1,3,4-thiadiazolium mesoionic derivatives and indeno[1,2-b]indoles are promising candidates for future investigations on in vivo, targeting HCC treatment and chemosensitization of tumors overexpressing ABCG2, respectively. Keywords: Hepatocellular carcinoma. HepG2 cells. Primary rat hepatocytes. 1,3,4-thiadiazolium mesoionic derivatives. Indeno[1,2-b]indoles derivatives. ABCG2 inhibitors

Efeitos da sidnona syd-1 sobre parâmetros relacionados ao estresse oxidativo e transição de permeabilidade mitocondriais

Resumo: Neste estudo avaliou-se o efeito do SYD-1 (3-[4-cloro-3-nitrofenil]-1,2,3- oxadiazolio-5-olato) sobre parametros de estresse oxidativo relacionados as funcoes mitocondriais, no sentido de contribuir para o esclarecimento dos mecanismos envolvidos na acao antitumoral do composto. Em mitocondrias isoladas de figado de rato, SYD-1 inibiu a lipoperoxidacao induzida por Fe3+/ADP-oxoglutarato (100% - 1,0 ƒÊmol.mg-1 de prot). Quando a lipoperoxidacao foi iniciada por radicais peroxil gerados a partir do azocomposto AAPH, a sidnona, na mesma concentracao, nao promoveu alteracao no perfil de peroxidacao ipidica. SYD-1 tambem demonstrou uma capacidade sequestradora de O2.- produzidos pelo sistema NADH/PSM (~14% - 1,0 ƒÊmol.mg-1 de prot.), enquanto que nenhuma alteracao foi observada sobre as atividades da enzimas superoxido dismutase (SOD) e catalase (CAT). Em relacao a glutationa redutase (Gred) e glutationa peroxidase (Gpx), SYD-1 promoveu reducoes de suas atividades em ~47 e 66% (1,0 ƒÊmol de SYD-1.mg-1 de prot.), respectivamente; efeito refletido na menor oxidacao de NADPH (~48% -1,0 ƒÊmol.mg-1 de prot.) durante o estresse oxidativo induzido por calcio. Na ausencia do ion, o mesoionico promoveu o aumento da oxidacao de NADPH em ~11% na mesma concentracao (1,0 ƒÊmol.mg-1 de prot.). Os efeitos da sidnona sobre o inchamento mitocondrial dependente da formacao/abertura do poro de transicao de permeabilidade (PTPM) tambem foram avaliados. SYD-1 (1,0 ƒÊmol.mg-1de prot.) inibiu a formacao/abertura do PTPM induzido por Ca2+/Pi em ~30%. Quando calcio e H2O2 ou calcio e diamida foram utilizados como indutores do inchamento mitocondrial, SYD-1 reduziu o fenomeno em ~12 e 78% (1,0ƒÊmol.mg-1 de prot.) respectivamente. O transporte mitocondrial de calcio foi analisado spectrofotometricamente, utilizando-se arsenazo III, onde SYD-1 promoveu aumento na captacao do ion. Em relacao a velocidade de efluxo, o mesoionico promoveu um aumento quando induzido pelo esacoplador FCCP e reducao quando induzido por vermelho de rutenio (VR). A partir destes resultados conclui-se que SYD-1 apresenta efeitos relacionados com acoes antioxidantes e prooxidantes, atuando de forma complexa sobre a bioenergetica mitocondrial e fenomenos associados a condicoes de estresse oxidativo

Candidate Molecule Selection Based on In Silico Predicted ADMET Properties of 12 Indenoindole Derivatives

International audienc

Effect of temperature acclimation on the liver antioxidant defence system of the Antarctic nototheniids Notothenia coriiceps and Notothenia rossii

Made available in DSpace on 2019-09-12T16:53:34Z (GMT). No. of bitstreams: 0 Previous issue date: 2014Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ)Instituto Nacional de Ciência e Tecnologia Antártico de Pesquisas Ambientais (INCT-APA)The aim of this study was to determine whether endemic Antarctic nototheniid fish are able to adjust their liver antioxidant defence system in response to the temperature increase. The activity of the superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione-S-transferase (GST) and glutathione reductase (GR) enzymes as well as the content of non-enzymatic oxidative stress markers such as reduced glutathione (GSH), lipid peroxidation (LPO) and protein carbonyl (PC) were measured in the liver of two Antarctic fish species, Notothenia rossii and Notothenia coriiceps after 1,3 and 6 days of exposure to temperatures of 0 C and 8 C. The GST activity showed a downregulation in N. rossii after 6 days of exposure to the increased temperature. The activity profiles of GST and GR in N. rossii and of GPx in N. coriiceps also changed as a consequence of heating to 8 C. The GSH content increased by heating to 8 C after 3 days in N. coriiceps and after 6 days in N. rossii. The content of malondialdehyde (MDA), a LPO marker, showed a negative modulation by the heating to 8 C in N. rossii after 3 days of exposure to temperatures. Present results show that heating to 8 C influenced the levels and profiles of the antioxidant enzymes and defences over time in the nototheniid fish N. rossii and N. coriiceps. (C) 2014 Elsevier Inc. All rights reserved.[Machado, Cintia; Zaleski, Tania; Krebsbach, Priscila; Rios, Flavia Sant'Anna; Donatti, Lucelia] Univ Fed Parana, Dept Cell Biol, Adapt Biol Lab, BR-80060000 Curitiba, Parana, Brazil[Rodrigues, Edson] Universidade de Taubaté (Unitau), Basic Biosci Inst, Sao Paulo, Brazil[Carvalho, Cleoni dos Santos] Univ Fed Sao Carlos, Dept Biol, Sorocaba, SP, Brazil[Suter Correia Cadena, Silvia Maria; Gozzi, Gustavo Jabor] Univ Fed Parana, Dept Biochem, Curitiba, Parana, Brazi

Candidate Molecule Selection Based on In Silico Predicted ADMET Properties of 12 Indenoindole Derivatives

International audienceFor considering future in vivo assays, it is necessary to investigate pharmacokinetic and toxicity profile of new chemical entities to select the best candidate(s) for further evaluations. Physicochemical parameters and ADMET (Absorption, Distribution, Metabolism, Elimination and Toxicity) properties of 12 indenoindole derivatives – identified as potent inhibitors of the ABCG2 protein - were predicted in silico with the Molinspiration and the ACD/Percepta softwares. The evaluation of mutagenicity and carcinogenicity was achieved by using the QSAR Toolbox software. Based on the exercise, i) two phenolic derivatives should not be metabolically activated by CYP enzymes according to the QSAR Toolbox software leading to a lower mutagenic risk, ii) compounds 2b, 2c could be excluded from further studies because of clastogenic risks and again compound 2c for a relatively low oral bioavailability, iii) one compound for its blood toxicity and five because for their pulmonary toxicity. Finally, six out of the 12 derivatives (1a, 1b, 2a, 2d, 2e and 2g), were predicted, in terms of ADMET properties, to be good candidates for further in vivo investigations

Phenolic indeno[1,2-b]indoles as ABCG2-selective potent and non-toxic inhibitors stimulating basal ATPase activity.

Ketonic indeno[1,2-b]indole-9,10-dione derivatives, initially designed as human casein kinase II (CK2) inhibitors, were recently shown to be converted into efficient inhibitors of drug efflux by the breast cancer resistance protein ABCG2 upon suited substitutions including a N (5)-phenethyl on C-ring and hydrophobic groups on D-ring. A series of ten phenolic and seven p-quinonic derivatives were synthesized and screened for inhibition of both CK2 and ABCG2 activities. The best phenolic inhibitors were about threefold more potent against ABCG2 than the corresponding ketonic derivatives, and showed low cytotoxicity. They were selective for ABCG2 over both P-glycoprotein and MRP1 (multidrug resistance protein 1), whereas the ketonic derivatives also interacted with MRP1, and they additionally displayed a lower interaction with CK2. Quite interestingly, they strongly stimulated ABCG2 ATPase activity, in contrast to ketonic derivatives, suggesting distinct binding sites. In contrast, the p-quinonic indenoindoles were cytotoxic and poor ABCG2 inhibitors, whereas a partial inhibition recovery could be reached upon hydrophobic substitutions on D-ring, similarly to the ketonic derivatives

Selective Cytotoxicity of 1,3,4-Thiadiazolium Mesoionic Derivatives on Hepatocarcinoma Cells (HepG2).

In this work, we evaluated the cytotoxicity of mesoionic 4-phenyl-5-(2-Y, 4-X or 4-X-cinnamoyl)-1,3,4-thiadiazolium-2-phenylamine chloride derivatives (MI-J: X=OH, Y=H; MI-D: X=NO2, Y=H; MI-4F: X=F, Y=H; MI-2,4diF: X=Y=F) on human hepatocellular carcinoma (HepG2), and non-tumor cells (rat hepatocytes) for comparison. MI-J, M-4F and MI-2,4diF reduced HepG2 viability by ~ 50% at 25 μM after 24-h treatment, whereas MI-D required a 50 μM concentration, as shown by 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. The cytotoxicity was confirmed with lactate dehydrogenase assay, of which activity was increased by 55, 24 and 16% for MI-J, MI-4F and MI-2,4diF respectively (at 25 μM after 24 h). To identify the death pathway related to cytotoxicity, the HepG2 cells treated by mesoionic compounds were labeled with both annexin V and PI, and analyzed by flow cytometry. All compounds increased the number of doubly-stained cells at 25 μM after 24 h: by 76% for MI-J, 25% for MI-4F and MI-2,4diF, and 11% for MI-D. It was also verified that increased DNA fragmentation occurred upon MI-J, MI-4F and MI-2,4diF treatments (by 12%, 9% and 8%, respectively, at 25 μM after 24 h). These compounds were only weakly, or not at all, transported by the main multidrug transporters, P-glycoprotein, ABCG2 and MRP1, and were able to slightly inhibit their drug-transport activity. It may be concluded that 1,3,4-thiadiazolium compounds, especially the hydroxy derivative MI-J, constitute promising candidates for future investigations on in-vivo treatment of hepatocellular carcinoma

Annexin V-FITC and propidium iodide staining of HepG2 treated with 1,3,4-thiadiazolium derivatives (the experimental conditions are described in the Materials and Methods section 2.5.3).

<p>The cells were seeded with or without 1,3,4-thiadiazolium derivatives at 25 μM for 18–24 h. Then, the cells were collected with trypsin and 10.000 events were analyzed by flow cytometry by FL2 and FL1 filters. (A) control, (B) MI-D, (C) MI-J, (D) MI-4F and (E) MI-2,4diF. The figures show representative dot-plot with the different cell populations: left-bottom = labeled cells; left-top = PI labeled; right-top = doubly labeled; right-bottom = annexin V labeled. The results were expressed as mean ± SD of three independents experiments.</p