Construção de bibliotecas de inibidores de serinoproteases em sistema phage display: seleção de inibidores para enzimas de Flavivírus e do mosquito Aedes aegypti
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Data
2023-02-09
Autores
Manzato, Verônica de Moraes [UNIFESP]
Orientadores
Tanaka, Aparecida Sadae [UNIFESP]
Tipo
Tese de doutorado
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Introdução: No Brasil, ocorrem, de forma endêmica, arboviroses como Dengue, Zika, Chikungunya e Febre amarela, que possuem um vetor comum, o mosquito Aedes aegypti. As enzimas NS2B-NS3 dos flavivírus e as tripsinas nos Aedes aegypti desempenham funções essenciais no ciclo viral e na digestão de Aedes spp, respectivamente, o que justifica a busca por inibidores dessas enzimas como opções para romper esses ciclos. Nesse contexto, a técnica phage display tem sido uma aliada. Os inibidores Boophilina domínio 1 (D1) e Sunflower trypsin inhibitor (SFTI), são representantes das famílias de inibidores Kunitz-BPTI (I2) e sunflower cyclic trypsin inhibitor (família I12), respectivamente, e inibem serinoproteases na ordem de nano molar. Objetivo: Selecionar através da técnica de phage display, inibidores das bibliotecas BoophilinaD1 e SFTI, com a utilização de 3 alvos (proteases ZIKV, DENV2 e tripsina5607). Seguido pela caracterização cinética e in silico dos mutantes mais frequentes. Métodos: As proteases DENV2Pro e tripsina5607, foram expressas em bactéria E coli SHuffleT7 e purificadas por afinidade. As bibliotecas de inibidores foram sintetizadas utilizando técnicas moleculares de PCR, utilizando-se o plasmídeo pCANTAB5E e bactérias E. coli TG1. As seleções foram realizadas para enzimas do vírus ZIKVPro, DENV2Pro e do mosquito vetor tripsina5607. Os mutantes de BoophilinaD1 mais frequentes, selecionados para a enzima do ZIKVPro foram produzidos e utilizados nos ensaios cinéticos para determinação do perfil inibitório para as enzimas (ZIKVPro e DENV2Pro), utilizados também na modelagem, docking molecular e refinamento para as enzimas de flavivírus, via MODELLER, ClusPro e RosettaDock, respectivamente. Resultados: As enzimas alvo purificadas apresentaram bandas predominantes e ativas em torno de 28 e 26 kDa, para DENV2Pro e tripsina5607, respectivamente. A ZIKVPro usada nas seleções foi gentilmente cedida por Dr. Martin Wurtele. Os títulos das bibliotecas utilizadas nos processos de seleção foram de 2,9 x 106 CFU e 2,1 x 105 CFU para BoophilinaD1 e SFTI, respectivamente. Dentre os inibidores BophD1 mut selecionados, 76,5% apresentam aminoácido básico Arg ou Lys na posição P1 e 70,6% e 58,8% Ala na posição P1’e P4’, respectivamente. O inibidor BoophD1 WT e os BoophD1 mut12 e mut14 apresentam Ki entre 1 e 2 nM para tripsina bovina e aproximadamente 100 e 300 nM para as enzimas ZIKVPro e DENV2Pro, respectivamente. As análises in silico de docking molecular indicam diferenças de interação dos mutantes com as enzimas. Os mutantes da biblioteca SFTI selecionados para os três alvos são, em sua maioria, compartilhados e sem consenso. Subtraindo-se os ligantes inespecíficos, destaca-se as sequências (P1-P4’) ARQLL, KSQWD e LSRHP por serem representativas para ZIKVPro, DENV2Pro e tripsina5607, respectivamente. Esses SFTIs mutantes foram sintetizados e serão utilizados futuramente nos ensaios cinéticos para caracterização inibitória com diferentes alvos. Discussão: As enzimas proteolíticas desempenham papéis importantes em diversas funções, no caso das DENV2Pro e ZIKVPro, estão relacionadas com a clivagem da poliproteína e no caso da tripsina5607, relacionada a digestão de proteínas ingeridas pela larva de Ae. aegypti. Obtê-las de forma recombinante, solúvel e ativa é importante para aprofundar nossos conhecimentos sobre elas e fornecem informações para o desenvolvimento de inibidores com alta afinidade, podendo ser um inibidor específico ou pan-inibidores (para diferentes flavivírus). Conclusão: Este trabalho traz dados importantes sobre a busca de inibidores para as proteases de flavivírus e de mosquito utilizando bibliotecas de inibidores em sistema phage display. Inibidores para ZIKVPro foram selecionados por phage display e apresentaram constantes de inibição em 10-7 M assim como para a DENV2Pro, estes resultados sugerem que os inibidores selecionados são candidatos a pan-inibidores para proteases de flavivírus.
Introduction: In Brazil, there are endemic arboviruses such as Dengue, Zika, Chikungunya and yellow fever, which have a common vector, the Aedes aegypti mosquito. The NS2B-NS3 enzymes of flaviviruses and trypsins in Aedes aegypti play essential roles in the viral cycle and digestion of Aedes spp., which justifies the search for inhibitors of these enzymes as options to break these cycles. In this context, the phage display technique has been an ally. The Boophilin domain 1 (D1) and Sunflower trypsin inhibitor (SFTI) inhibitors are representatives of the Kunitz-BPTI (I2) and sunflower cyclic trypsin inhibitor (I12 family) families, respectively, and inhibit serine proteases in nanomolar order. Objective: To select, through the phage display technique, inhibitors from BoophilinaD1 and SFTI libraries, using 3 proteases as targets (ZIKV, DENV2 and trypsin5607). Followed by kinetic and in silico characterization of the most frequent mutants. Methods: DENV2Pro and trypsin5607 proteases were expressed in E coli SHuffleT7 bacteria and affinity purified. Inhibitor libraries were synthesized using molecular PCR techniques, using the pCANTAB5E plasmid and E coli TG1 bacteria. Selections were performed for enzymes from the ZIKVPro, DENV2Pro and mosquito vector trypsin5607. The most frequent BoophilinaD1 mutants, selected for ZIKVPro, were produced and used in kinetic assays to determine the inhibitory profile for enzymes (ZIKVPro and DENV2Pro ), also used in modeling, molecular docking and refinement for flavivirus enzymes, via MODELLER, ClusPro and RosettaDock, respectively. Results: The target purified showed predominant and active bands around 28 and 26 kDa, for DENV2Pro and trypsin5607, respectively. The ZIKVPro used in the selections was kindly provided by Dr. Martin Wurtele. The titles of the libraries used in the selection processes were 2.9 x 106 CFU and 2.1 x 105 CFU for BoophilinaD1 and SFTI, respectively. Among the BophD1 mut inhibitors selected, 76.5% present basic amino acid Arg or Lys in position P1 and 70.6% and 58.8% Ala in position P1' and P4', respectively. The BoophD1 wt inhibitor and the BoophD1 mut12 and mut14 present Ki between 1 and 2 nM for bovine trypsin and approximately 100 and 300 nM for the ZIKVPro and DENV2Pro enzymes, respectively. Molecular docking in silico analyzes indicates differences in the interaction of the mutants with the enzymes. The SFTI library mutants selected for the three targets are, for the most part, shared and without consensus. By subtracting the nonspecific ligands, the sequences (P1-P4') ARQLL, KSQWD and LSRHP stand out because they are representative for ZIKVPro, DENV2Pro and trypsin5607, respectively. These SFTIs mutants were synthesized and will be used in the future in kinetic assays for inhibitory characterization with different targets. Discussion: The proteolytic enzymes play important roles in several functions, in the case of DENV2Pro and ZIKVPro, are related to polyprotein cleavage and in the case of trypsin5607, related to protein digestion ingested by the larva of Ae. aegypti. Obtaining them recombinantly, soluble, and active is important to deepen our knowledge about them and provide information for the development of inhibitors with high affinity, which may be a specific or polyspecific inhibitor (for different flaviviruses). Conclusion: This work brings important data about search for inhibitors for flavivirus and mosquito proteases, and the use of inhibitor libraries in a phage display system to obtain inhibitors. Inhibitors selected for ZIKVPro were selected by phage display and showed inhibitory constants of 10-7 M as well as for DENV2Pro. These results suggest that selected inhibitors are candidates for pan-inhibitors for flavivirus proteases.
Introduction: In Brazil, there are endemic arboviruses such as Dengue, Zika, Chikungunya and yellow fever, which have a common vector, the Aedes aegypti mosquito. The NS2B-NS3 enzymes of flaviviruses and trypsins in Aedes aegypti play essential roles in the viral cycle and digestion of Aedes spp., which justifies the search for inhibitors of these enzymes as options to break these cycles. In this context, the phage display technique has been an ally. The Boophilin domain 1 (D1) and Sunflower trypsin inhibitor (SFTI) inhibitors are representatives of the Kunitz-BPTI (I2) and sunflower cyclic trypsin inhibitor (I12 family) families, respectively, and inhibit serine proteases in nanomolar order. Objective: To select, through the phage display technique, inhibitors from BoophilinaD1 and SFTI libraries, using 3 proteases as targets (ZIKV, DENV2 and trypsin5607). Followed by kinetic and in silico characterization of the most frequent mutants. Methods: DENV2Pro and trypsin5607 proteases were expressed in E coli SHuffleT7 bacteria and affinity purified. Inhibitor libraries were synthesized using molecular PCR techniques, using the pCANTAB5E plasmid and E coli TG1 bacteria. Selections were performed for enzymes from the ZIKVPro, DENV2Pro and mosquito vector trypsin5607. The most frequent BoophilinaD1 mutants, selected for ZIKVPro, were produced and used in kinetic assays to determine the inhibitory profile for enzymes (ZIKVPro and DENV2Pro ), also used in modeling, molecular docking and refinement for flavivirus enzymes, via MODELLER, ClusPro and RosettaDock, respectively. Results: The target purified showed predominant and active bands around 28 and 26 kDa, for DENV2Pro and trypsin5607, respectively. The ZIKVPro used in the selections was kindly provided by Dr. Martin Wurtele. The titles of the libraries used in the selection processes were 2.9 x 106 CFU and 2.1 x 105 CFU for BoophilinaD1 and SFTI, respectively. Among the BophD1 mut inhibitors selected, 76.5% present basic amino acid Arg or Lys in position P1 and 70.6% and 58.8% Ala in position P1' and P4', respectively. The BoophD1 wt inhibitor and the BoophD1 mut12 and mut14 present Ki between 1 and 2 nM for bovine trypsin and approximately 100 and 300 nM for the ZIKVPro and DENV2Pro enzymes, respectively. Molecular docking in silico analyzes indicates differences in the interaction of the mutants with the enzymes. The SFTI library mutants selected for the three targets are, for the most part, shared and without consensus. By subtracting the nonspecific ligands, the sequences (P1-P4') ARQLL, KSQWD and LSRHP stand out because they are representative for ZIKVPro, DENV2Pro and trypsin5607, respectively. These SFTIs mutants were synthesized and will be used in the future in kinetic assays for inhibitory characterization with different targets. Discussion: The proteolytic enzymes play important roles in several functions, in the case of DENV2Pro and ZIKVPro, are related to polyprotein cleavage and in the case of trypsin5607, related to protein digestion ingested by the larva of Ae. aegypti. Obtaining them recombinantly, soluble, and active is important to deepen our knowledge about them and provide information for the development of inhibitors with high affinity, which may be a specific or polyspecific inhibitor (for different flaviviruses). Conclusion: This work brings important data about search for inhibitors for flavivirus and mosquito proteases, and the use of inhibitor libraries in a phage display system to obtain inhibitors. Inhibitors selected for ZIKVPro were selected by phage display and showed inhibitory constants of 10-7 M as well as for DENV2Pro. These results suggest that selected inhibitors are candidates for pan-inhibitors for flavivirus proteases.
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Citação
MANZATO, Verônica de Moraes. Construção de bibliotecas de inibidores de serinoproteases em sistema phage display: seleção de inibidores para enzimas de Flavivírus e do mosquito Aedes Aegypti. 2023. 114 f. Tese (Doutorado em Biologia Molecular) - Escola Paulista de Medicina, Universidade Federal de São Paulo (UNIFESP). São Paulo, 2023.