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Doctoral Thesis
DOI
10.11606/T.75.2018.tde-25042018-142801
Document
Author
Full name
Buana Carvalho de Almeida
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
São Carlos, 2018
Supervisor
Committee
Canduri, Fernanda (President)
Azevedo Junior, Walter Filgueira de
Leitão, Andrei
Lago, João Henrique Ghilardi
Silva, Dulce Helena Siqueira
Title in Portuguese
Metabólitos secundários como potenciais inibidores de CDK8 (proteína quinase humana)
Keywords in Portuguese
CDK
clonagem
expressão
inibidores
metabólitos
quinase
Abstract in Portuguese
Quinases Dependentes de Ciclinas (CDKs) são holoenzimas que possuem uma subunidade catalítica, a CDK, e uma subunidade regulatória, a ciclina. CDKs são Ser/Thr quinases, ou seja, fosforilam resíduos de aminoácidos específicos. A CDK8 fosforila o CTD da RNA Pol II, sendo considerada como regulador transcricional positivo, implicando em efeitos oncogênicos. Em suma, essas descobertas a colocam como alvo de drogas em pesquisas relacionadas ao câncer. Técnicas de biologia molecular foram realizadas a fim de obter o recombinate CDK8::pET28a. A CDK8-HisTag foi expressa em Rosetta(DE3), com cauda de histidina no N-terminal (45 kDa), no entanto a proteína foi expressa como corpos de inclusão. Desta forma, a CDK8-HisTag foi solubilizada com SDS 2%, o detergente foi completamente removido e o refolding da mesma foi obtido com cromatografia líquida de alta eficiência utilizando coluna de Ni2+. A proteína reenovelada e purificada foi analisada por DC e revelou a existência de 13, 6% de hélices α, 34,5 %d e folhas β e 33,1 % de coil. O espectro de emissão de fluorescência da CDK8-HisTag mostrou comprimento de onda máximo em torno de 360 nm. A análise de docking com potenciais inibidores obtidos da espécie W. brachypetala revelou o alcaloide O-metil-hyeronimona como potencial inibidor de CDK8. Este é o primeiro relato de expressão e purificação da proteína CDK8 em E. coli, assim como do screening virtual desta proteína na presença de inibidores naturais obtidos de W. brachypetala.
Title in English
Secondary metabolites as potential inhibitors of CDK8 (human kinase protein)
Keywords in English
CDK
cloning
expression
inhibitors
kinase
metabolites
Abstract in English
Cyclin Dependent Kinases (CDK) are holoenzyme having a catalytic subunit, CDK, and a regulatory subunit, cyclin. CDKs are Ser / Thr kinases, which phosphorylate specific amino acid residues. The CDK8 phosphorylates the CTD of RNA Pol II. It is considered a positive transcriptional regulator, resulting in oncogenic effects. In short, these findings pose as a drug target in research related to cancer. Molecular biology assays were performed in order to obtain the recombinant CDK8::pET28a. CDK8-HisTag was expressed in Rosetta (DE3), with a N-terminal tail histidine (45 kDa), however, the protein was expressed as inclusion bodies. In this way, a CDK8-HisTag was solubilized with 2% SDS, then the detergent was completely removed and the refolding of the same was obtained with High Performance Liquid Chromatography using Ni2 + column. A refolded and purified protein for DC analysis revealed the existence of 13.6% helix, 34.5% beta and 33.1% coils. The fluorescence emission spectrum of CDK8-HisTag showed the maximum wavelength around 360 nm. An analysis with potential inhibitors obtained from the W. brachypetala species revealed the alkaloid O-metil-hyeronimona as a potential inhibitor of CDK8. This is the first report of expression and purification of the CDK8 protein in E. coli, as well as the virtual screening of this protein in the presence of natural inhibitors obtained from W. brachypetala.
 
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Publishing Date
2018-05-14
 
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