Screening de pequenas moléculas potenciais inibidoras da p38δ MAPK como estratégia para a busca de um novo tratamento para câncer de mama
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Data
2020-09
Autores
Orientador
Goettert, Márcia Inês
Banca
Bustamante Filho, Ivan Cunha
Ducati, Rodrigo Gay
Morrone, Fernanda Bueno
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Resumo
O câncer de mama é a neoplasia mais frequente entre as mulheres e a principal causa de morte relacionada ao câncer nas mulheres do mundo todo. Os principais tratamentos para essa doença, como a quimioterapia, radioterapia e cirurgia, podem apresentar diversos efeitos colaterais e não serem eficazes em estágios avançados da doença quando existem altas chances de metástase. Pequenas moléculas que atuam em alvos específicos de vias de sinalização que podem estar desreguladas no câncer como, por exemplo, a via p38 MAPK, estão sendo estudadas para a terapêutica antineoplásica e são alvos promissores para o desenvolvimento de novos fármacos mais seletivos. Visto que no câncer de mama o aumento da expressão da isoforma p38δ está relacionado com progressão do tumor e metástases, e visando a carência de inibidores específicos de p38δ, este estudo teve como primeiro objetivo identificar, por meio de um screening virtual, pequenas moléculas com potencial de inibir p38δ. Posteriormente, o objetivo foi avaliar o potencial antitumoral da molécula selecionada na linhagem de adenocarcinoma de mama humano MCF-7. As estruturas 3D da p38δ na sua forma ativa (DFG-in) e inativa (DFG-out) foram selecionadas do banco de dados PDB. Candidatos a ligantes foram selecionados após revisão na literatura e suas estruturas foram obtidas do banco de dados PubChem. O screening virtual indicou a Hipericina como a molécula de maior afinidade pela p38δ ativa, sendo assim, um candidato a inibidor da enzima p38δ. A viabilidade e proliferação de células MCF-7 foram determinadas após diferentes tratamentos com Hipericina por 24, 48 e 72 horas, através do ensaio de MTT e clonogênico, respectivamente. Entretanto, nesses ensaios a Hipericina não demonstrou ter atividade antitumoral na MCF-7. Visto que a Hipericina não apresentou a citotoxicidade esperada, a molécula PIT97 foi escolhida para ser estudada quanto ao seu potencial antitumoral in vitro e como uma possível molécula candidata ao tratamento do câncer de mama. As linhagens MCF-7 e de macrófagos murino RAW 264.7 foram tratadas com diferentes concentrações do PIT97 e a viabilidade celular foi avaliada após 48 e 72 horas, através do ensaio de MTT. A proliferação de células MCF-7 tratadas com PIT97 por 72 horas foi avaliada pelo ensaio clonogênico. Os resultados obtidos demonstraram redução da viabilidade da MCF-7 após o tratamento com o PIT97 por 48 e 72 horas, enquanto que na linhagem RAW 264.7 baixas concentrações da molécula (0,1 μM e 0,01 μM) não foram tóxicas. Além disso, foi demonstrado que o PIT97 não só atua a curto prazo na linhagem MCF-7, mas continua tendo efeitos citotóxicos a longo prazo. Desta forma, a molécula PIT97 possui potencial como molécula candidata e como alternativa terapêutica mais seletiva para o câncer de mama. Consequentemente, estudos serão realizados visando elucidar e compreender o mecanismo pelo qual a molécula exerce seu efeito.
Breast cancer is the most frequent neoplasm among women and the leading cause of cancer- related death in women worldwide. Treatments for this disease, such as chemotherapy, radiotherapy, and surgery, can display several side effects. Moreover, these treatments may not be effective in advanced stages of the disease when there are high chances of metastasis. Small molecules that act on specific targets of unregulated signaling pathways in cancer, such as p38 MAPK pathway, are being studied for antineoplastic therapy and are promising targets of selective new drugs. Since in breast cancer the increase of p38δ isoform expression is related to tumor progression and metastasis, and aiming the lack of specific p38δ inhibitors, this study aimed to identify, through a virtual screening, small molecules with potential to inhibit p38δ. Subsequently, the aim was evaluating the antitumor potential of the selected molecule in human breast adenocarcinoma MCF-7 cell line. p38δ 3D structures of in its active (DFG-in) and inactive (DFG-out) form were selected from the PDB database. Ligands candidates were selected after a literature review and their structures were obtained from PubChem database. The virtual screening indicated Hypericin as the molecule with the highest affinity for active p38δ, thus this molecule is a candidate for p38δ inhibitor. Viability and proliferation of MCF-7 cells were determined after different treatments with Hypericin for 24, 48 and 72 hours, through the MTT and clonogenic assays, respectively. However, in these assays Hypericin not demonstrated antitumor activity in MCF-7. Since Hypericin did not present the expected cytotoxicity, the PIT97 molecule was chosen to be studied for its antitumor potential in vitro and as a possible candidate molecule for the treatment of breast cancer. MCF-7 and murine macrophages RAW 264.7 cell lines were treated with different concentrations of PIT97 and cell viability was evaluated after 48 and 72 hours, through MTT assay. Proliferation of MCF-7 cells treated with PIT97 for 72 hours was evaluated by the clonogenic assay. The results revealed a reduction in MCF-7 viability after treatment with PIT97 for 48 and 72 hours, while in RAW 264.7 low concentrations of the molecule (0.1 μM and 0.01 μM) were nontoxic. Furthermore, PIT97 not only acts in the short term in MCF-7 cell line, but also have long-term cytotoxic effects. Thus, PIT97 has potential as a candidate molecule, and as a more selective therapeutic alternative for breast cancer. Consequently, studies will be carried out in order to elucidate and understand the mechanism by which the molecule acts.
Breast cancer is the most frequent neoplasm among women and the leading cause of cancer- related death in women worldwide. Treatments for this disease, such as chemotherapy, radiotherapy, and surgery, can display several side effects. Moreover, these treatments may not be effective in advanced stages of the disease when there are high chances of metastasis. Small molecules that act on specific targets of unregulated signaling pathways in cancer, such as p38 MAPK pathway, are being studied for antineoplastic therapy and are promising targets of selective new drugs. Since in breast cancer the increase of p38δ isoform expression is related to tumor progression and metastasis, and aiming the lack of specific p38δ inhibitors, this study aimed to identify, through a virtual screening, small molecules with potential to inhibit p38δ. Subsequently, the aim was evaluating the antitumor potential of the selected molecule in human breast adenocarcinoma MCF-7 cell line. p38δ 3D structures of in its active (DFG-in) and inactive (DFG-out) form were selected from the PDB database. Ligands candidates were selected after a literature review and their structures were obtained from PubChem database. The virtual screening indicated Hypericin as the molecule with the highest affinity for active p38δ, thus this molecule is a candidate for p38δ inhibitor. Viability and proliferation of MCF-7 cells were determined after different treatments with Hypericin for 24, 48 and 72 hours, through the MTT and clonogenic assays, respectively. However, in these assays Hypericin not demonstrated antitumor activity in MCF-7. Since Hypericin did not present the expected cytotoxicity, the PIT97 molecule was chosen to be studied for its antitumor potential in vitro and as a possible candidate molecule for the treatment of breast cancer. MCF-7 and murine macrophages RAW 264.7 cell lines were treated with different concentrations of PIT97 and cell viability was evaluated after 48 and 72 hours, through MTT assay. Proliferation of MCF-7 cells treated with PIT97 for 72 hours was evaluated by the clonogenic assay. The results revealed a reduction in MCF-7 viability after treatment with PIT97 for 48 and 72 hours, while in RAW 264.7 low concentrations of the molecule (0.1 μM and 0.01 μM) were nontoxic. Furthermore, PIT97 not only acts in the short term in MCF-7 cell line, but also have long-term cytotoxic effects. Thus, PIT97 has potential as a candidate molecule, and as a more selective therapeutic alternative for breast cancer. Consequently, studies will be carried out in order to elucidate and understand the mechanism by which the molecule acts.
Descrição
Palavras-chave
Câncer de mama; p38δ MAPK; Pequenas moléculas; Hipericina; Breast cancer; Small molecules; Hypericin
Citação
ANTON, Débora Bublitz. Screening de pequenas moléculas potenciais inibidoras da p38δ MAPK como estratégia para a busca de um novo tratamento para câncer de mama. 2020. Dissertação (Mestrado) – Curso de Biotecnologia, Universidade do Vale do Taquari - Univates, Lajeado, 15 set. 2020. Disponível em: http://hdl.handle.net/10737/2936.