ThermoFMN - A Thermofluor Assay Developed for Ligand-Screening as an Alternative Strategy for Drug Discovery

Padua, R.A.P.; Tomaleri, Giovani Pinton; Reis, Renata A.G.; David, Juliana S.; Silva, Valeria C.; Pinheiro, Matheus Pinto; Nonato, Maria Cristina

doi:10.5935/0103-5053.20140157

Cited by 10 publications

(10 citation statements)

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“…We also investigate the effect of the removal of this protuberant domain in inhibitor binding. Since Sm DHODHΔloop was found inactive, we applied differential scanning fluorimetry (ThermoFMN, [35]) on both constructs to compare the thermal stability effects with IC 50 against our library of atovaquone analogues [30] (Figs 2 and 7).…”

Section: Resultsmentioning

confidence: 99%

Structural basis for the function and inhibition of dihydroorotate dehydrogenase from Schistosoma mansoni

et al. 2020

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Schistosomiasis is a serious public health problem, prevalent in tropical and subtropical areas, especially in poor communities without access to safe drinking water and adequate sanitation. Transmission has been reported in 78 countries, and its control depends on a single drug, praziquantel, which has been used over the past 30 years. Our work is focused on exploiting target‐based drug discovery strategies to develop new therapeutics to treat schistosomiasis. In particular, we are interested in evaluating the enzyme dihydroorotate dehydrogenase (DHODH) as a drug target. DHODH is a flavoenzyme that catalyzes the stereospecific oxidation of (S)‐dihydroorotate (DHO) to orotate during the fourth and only redox step of the de novo pyrimidine nucleotide biosynthetic pathway. Previously, we identified atovaquone, used in the treatment of malaria, and its analogues, as potent and selective inhibitors against Schistosoma mansoni DHODH (SmDHODH). In the present article, we report the first crystal structure of SmDHODH in complex with the atovaquone analogue inhibitor 2‐((4‐fluorophenyl)amino)‐3‐hydroxynaphthalene‐1,4‐dione (QLA). We discuss three major findings: (a) the open conformation of the active site loop and the unveiling of a novel transient druggable pocket for class 2 DHODHs; (b) the presence of a protuberant domain, only present in Schistosoma spp DHODHs, that was found to control and modulate the dynamics of the inhibitor binding site; (c) a detailed description of an unexpected binding mode for the atovaquone analogue to SmDHODH. Our findings contribute to the understanding of the catalytic mechanism performed by class 2 DHODHs and provide the molecular basis for structure‐guided design of SmDHODH inhibitors. Database The structural data are available in Protein Data Bank (PDB) database under the accession code number https://www.rcsb.org/structure/6UY4.

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Section: Resultsmentioning

confidence: 99%

Structural basis for the function and inhibition of dihydroorotate dehydrogenase from Schistosoma mansoni

et al. 2020

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“…In order to better understand some physical–chemical properties of Moojase, we have performed the thermofluor assay of this toxin in the presence of different conditions of pH, salt concentrations and chemical additives. This technique assesses the effect of solvent conditions and ligands on the protein thermostability, based on temperature-dependent protein unfolding [44,45]. Thermofluor assay is composed by two high-throughput screening kits in which an initial screening for global parameters is performed, followed by a search for protein-specific additives.…”

Section: Discussionmentioning

confidence: 99%

New Insights on Moojase, a Thrombin-Like Serine Protease from Bothrops moojeni Snake Venom

Amorim

Menaldo

Carone

et al. 2018

Toxins

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Snake venom serine proteases (SVSPs) are enzymes that are capable of interfering in various parts of the blood coagulation cascade, which makes them interesting candidates for the development of new therapeutic drugs. Herein, we isolated and characterized Moojase, a potent coagulant enzyme from Bothrops moojeni snake venom. The toxin was isolated from the crude venom using a two-step chromatographic procedure. Moojase is a glycoprotein with N-linked glycans, molecular mass of 30.3 kDa and acidic character (pI 5.80–6.88). Sequencing of Moojase indicated that it is an isoform of Batroxobin. Moojase was able to clot platelet-poor plasma and fibrinogen solutions in a dose-dependent manner, indicating thrombin-like properties. Moojase also rapidly induced the proteolysis of the Aα chains of human fibrinogen, followed by the degradation of the Bβ chains after extended periods of incubation, and these effects were inhibited by PMSF, SDS and DTT, but not by benzamidine or EDTA. RP-HPLC analysis of its fibrinogenolysis confirmed the main generation of fibrinopeptide A. Moojase also induced the fibrinolysis of fibrin clots formed in vitro, and the aggregation of washed platelets, as well as significant amidolytic activity on substrates for thrombin, plasma kallikrein, factor Xia, and factor XIIa. Furthermore, thermofluor analyses and the esterase activity of Moojase demonstrated its very high stability at different pH buffers and temperatures. Thus, studies such as this for Moojase should increase knowledge on SVSPs, allowing their bioprospection as valuable prototypes in the development of new drugs, or as biotechnological tools.

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“…Outra preocupação para a otimização dos parâmetros utilizados durante os experimentos de expressão se baseou na tentativa de maximizar a produção de FMN em E.coli. O FMN, utilizado como cofator em DHODH, se encontra no interior da estrutura da proteína [64] e a sua ligação é parte integrante do processo de enovelamento da SmDHODH. Uma dificuldade na biossíntese de FMN em E.coli poderia levar à produção de proteína deficiente em FMN, o que poderia não somente ser responsável por uma população de SmDHODH inativa, assim como estruturalmente instável.…”

Section: Teste De Atividade Das Proteínas Na Presença De Inibidoresunclassified

Caracterização bioquímica, biofísica e estudos inibitórios da enzima diidroorotato desidrogenase de Schistosoma mansoni

Costacurta¹

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AGRADECIMENTOSAgradeço primeiramente à minha orientadora, pelas inúmeras oportunidades que me concedeu, pela confiança e pelo exemplo de profissional que é.Ao meu time, LCP, pelas idéias e incansáveis discussões. Sem o apoio de vocês este projeto não se realizaria! Joane, Paty, Valéria, Diego, Ricardo Soares, Guilherme e Vinícius, obrigada! Em especial quero agradecer à Renata, Ricardo de Pádua, Iara e Giovani. Muito obrigado pela força! Aos Profs. Dr. Antonio Jose da Costa Filho, Dr. Flávio da Silva Emery, Dra. Eliane Candiani Arantes Braga e Dra. Renata Fonseca Vianna Lopez, por disponibilizarem seus laboratórios e equipamentos para a execução deste projeto. À Faculdade de Ciências Farmacêuticas de Ribeirão Preto, aonde tive a oportunidade de me formar como farmacêutica com excelência, de trabalhar como técnica de laboratório e de realizar meu mestrado. Aos meus pais, que tanto me apoiaram para que eu pudesse sempre seguir com meus sonhos e objetivos. Aos meus irmãos, parentes e amigos, que sempre estiveram ao meu lado: Marina, muitos outros! Ao meu marido e filha, meus amores e minha inspiração. "O sucesso nasce do querer, da determinação e persistência em se chegar a um objetivo. Mesmo não atingindo o alvo, quem busca e vence obstáculos, no mínimo fará coisas admiráveis." José de Alencar i RESUMO COSTACURTA, J. S. D. Caracterização bioquímica, biofísica e estudos inibitórios da enzima diidroorotato desidrogenase de Schistosoma mansoni. 2014. 79f. Dissertação (Mestrado). Faculdade de Ciências Farmacêuticas de Ribeirão Preto -Universidade de São Paulo, Ribeirão Preto, 2014.Ping-Pong, de acordo com o que já foi descrito para as outras DHODHs, com os seguintes valores de KM e kcat: KDHO= 174 ± 18 µM; KQo= 159 ± 18 µM; e kcat= 27 ± 1 s -1 para a SmDHODH e KDHO= 286 ± 31 µM; KQo= 354 ± 38 µM; e kcat= 78 ± 4 s -1 para a HsDHODH. Foram identificados compostos químicos com potencial inibitório na faixa de 794  3 M a 19,1  0,1 nM para a SmDHODH e de 33,9  0,1 M a 37,2  0,1 nM para a HsDHODH. Os resultados deste trabalho aliado aos estudos estruturais em desenvolvimento pelo nosso laboratório serão utilizados não só para a completa caracterização da enzima, mas também para o futuro planejamento de ligantes específicos baseados na estrutura e função protéica, como uma importante ferramenta no combate à esquistossomose.Palavras-chave: esquistossomose; diidroorotato desidrogenase; caracterização bioquímica e biofísica; estudos inibitórios.iii ABSTRACT COSTACURTA, J. S. D. Biochemical, biophysical and inhibitory studies of dihydroorotate dehydrogenase from Schistosoma mansoni. 2014. 79f. Dissertation (Master). Faculdade de Ciências Farmacêuticas de Ribeirão Preto -Universidade de São Paulo, Ribeirão Preto, 2014.iv enzyme, as well as for the development of specific inhibitors of SmDHODH, as an important tool in the fight against schistosomiasis.

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ThermoFMN - A Thermofluor Assay Developed for Ligand-Screening as an Alternative Strategy for Drug Discovery

Cited by 10 publications

References 0 publications

Structural basis for the function and inhibition of dihydroorotate dehydrogenase from Schistosoma mansoni

Structural basis for the function and inhibition of dihydroorotate dehydrogenase from Schistosoma mansoni

New Insights on Moojase, a Thrombin-Like Serine Protease from Bothrops moojeni Snake Venom

Caracterização bioquímica, biofísica e estudos inibitórios da enzima diidroorotato desidrogenase de Schistosoma mansoni

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