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Desenvolvimento de novos inibidores da necroptose direcionados à RIPK1
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A morte celular é um processo fundamental na eliminação de células danificadas. Durante várias décadas, a apoptose foi considerada como a única forma de morte celular programada. Contudo, estudos recentes sugerem que a necrose pode ser altamente regulada, num processo designado por necroptose, o qual incorpora aspetos característicos da necrose e da apoptose. A necroptose, é uma via de morte celular regulada, alternativa e independente de caspases, sendo mediada pelas proteínas cinase que interagem com o recetor 1 (RIPK1) e com o recetor 3 (RIPK3) e a pseudocinase MLKL.
Após o estabelecimento da necroptose como uma forma alternativa de morte celular, alguns estudos já evidenciaram este mecanismo como o principal contribuinte na etiologia e/ou progressão de doenças humanas, nomeadamente, em pancreatite, doença isquémica e doenças neurodegenerativas, entre outras. Adicionalmente, a necroptose está também envolvida na regulação de vários tipos de cancros. Como tal, estratégias que tenham como alvo a necroptose, constituem oportunidades únicas para desenvolver novas terapias para o tratamento das doenças anteriormente referidas.
Apesar de terem já sido descritos vários compostos que inibem alguns dos elementos da via de sinalização da morte celular por necroptose, a grande maioria apresenta várias limitações a nível farmacológico, não existindo ainda nenhum inibidor da RIPK1/3 na prática clínica. O presente trabalho, focou-se na síntese de novos compostos derivados do hit compound AZ12651922, selecionado através de um screening fenotípico desenvolvido para a identificação de compostos com atividade inibitória contra marcadores da necroptose. Desta forma, este trabalho incidiu no desenvolvimento de novos inibidores da necroptose, avaliando o espaço químico em torno do anel 1,3-tiazol (núcleo central da molécula AZ12651922) de modo a revelar quais as características estruturais que controlam a potência e seletividade para a modulação da cinase RIP1.
Todos os compostos finais foram sintetizados com bons rendimentos e caracterizados através de RMN (protão e carbono) e espectrometria de massa. Para avaliar a atividade biológica dos compostos sintetizados, foram realizados estudos de toxicidade nas linhas celulares L929 (linha celular murina de fibrossarcoma) e HT29 (linha celular humana de adenocarcinoma colorretal). Para os compostos selecionados, foram determinados os EC50 (half maximal effective concentration) nos mesmos modelos celulares. Os resultados obtidos indicam que o composto selecionado (36) inibe a necroptose induzida pelo TNF-α em ambas as linhas celulares nas concentrações na ordem dos micromolares (EC50 (L929) = 15,68 μM e EC50 (HT29) = 4,99 μM), não apresentando toxicidade no intervalo de concentrações utilizado.
Cell death is a fundamental process in the elimination of damaged cells. For several decades, apoptosis was considered as the sole form of programmed cell death. However, recent studies suggest that necrosis can also be highly regulated, incorporating features of both necrosis and apoptosis in a process referred as necroptosis. Necroptosis is an alternative caspase-independent regulated cell death pathway and is mediated by the receptor-interacting protein kinase 1 (RIPK1) and receptor 3 (RIPK3) and the MLKL pseudokinase. The establishment of necroptosis as an alternative form of regulated cell death resulted in the publication of several studies implicating it as the main contributor in the etiology and/or progression of human diseases, such as pancreatitis, ischemic injury, and neurodegenerative diseases, among others. Additionally, several studies also indicate that necroptosis is also involved in the regulation of cancer. Therefore, targeting necroptosis may provide an unprecedented opportunity to develop novel therapeutic strategies for the aforementioned diseases. Although several compounds have been described as inhibitors of molecular mediators of necroptosis, the majority of them present pharmacological limitations, and no RIPK1/3 inhibitor is yet available for clinical use. The present work was focused on the synthesis of new analogues of the hit compound AZ12651922, selected from a phenotypic screening developed to identify novel small molecules with inhibitory activity against necroptosis markers. Thus, this work was focused on the development of new necroptosis inhibitors by evaluating the chemical space around the 1,3-thiazol ring (central core of the AZ12651922 molecule) and disclose which structural features control potency and selectivity against RIP1 kinase. All final compounds were synthesized with good yields and fully characterized by NMR (proton and carbon) and mass spectrometry. To evaluate the biological activity of the synthesized compounds, toxicity studies were performed on L929 (murine fibrosarcoma cell line) and HT29 (colorectal adenocarcinoma cell line) cell lines. To further characterize the activities of selected compounds, the half maximal effective concentration (EC50) was determined in the same cellular models. Our results show that the selected compound (36) inhibits TNF-α-induced necroptosis at micromolar concentrations (EC50 (L929) = 15,68 μM and EC50 (HT29) = 4,99 μM) and was not cytotoxic at the chosen range of concentrations.
Cell death is a fundamental process in the elimination of damaged cells. For several decades, apoptosis was considered as the sole form of programmed cell death. However, recent studies suggest that necrosis can also be highly regulated, incorporating features of both necrosis and apoptosis in a process referred as necroptosis. Necroptosis is an alternative caspase-independent regulated cell death pathway and is mediated by the receptor-interacting protein kinase 1 (RIPK1) and receptor 3 (RIPK3) and the MLKL pseudokinase. The establishment of necroptosis as an alternative form of regulated cell death resulted in the publication of several studies implicating it as the main contributor in the etiology and/or progression of human diseases, such as pancreatitis, ischemic injury, and neurodegenerative diseases, among others. Additionally, several studies also indicate that necroptosis is also involved in the regulation of cancer. Therefore, targeting necroptosis may provide an unprecedented opportunity to develop novel therapeutic strategies for the aforementioned diseases. Although several compounds have been described as inhibitors of molecular mediators of necroptosis, the majority of them present pharmacological limitations, and no RIPK1/3 inhibitor is yet available for clinical use. The present work was focused on the synthesis of new analogues of the hit compound AZ12651922, selected from a phenotypic screening developed to identify novel small molecules with inhibitory activity against necroptosis markers. Thus, this work was focused on the development of new necroptosis inhibitors by evaluating the chemical space around the 1,3-thiazol ring (central core of the AZ12651922 molecule) and disclose which structural features control potency and selectivity against RIP1 kinase. All final compounds were synthesized with good yields and fully characterized by NMR (proton and carbon) and mass spectrometry. To evaluate the biological activity of the synthesized compounds, toxicity studies were performed on L929 (murine fibrosarcoma cell line) and HT29 (colorectal adenocarcinoma cell line) cell lines. To further characterize the activities of selected compounds, the half maximal effective concentration (EC50) was determined in the same cellular models. Our results show that the selected compound (36) inhibits TNF-α-induced necroptosis at micromolar concentrations (EC50 (L929) = 15,68 μM and EC50 (HT29) = 4,99 μM) and was not cytotoxic at the chosen range of concentrations.
Descrição
Tese de mestrado, Química Medicinal e Biofarmacêutica, 2022, Universidade de Lisboa, Faculdade de Farmácia.
Palavras-chave
Morte celular Necroptose RIPK1 Inibidores 1,3-tiazol Teses de mestrado - 2022