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Study of new methodologies for cathepsin D assays in the search for inhibitors

Grant number: 09/17538-8
Support Opportunities:Scholarships in Brazil - Doctorate (Direct)
Effective date (Start): March 01, 2010
Effective date (End): January 31, 2015
Field of knowledge:Physical Sciences and Mathematics - Chemistry - Organic Chemistry
Principal Investigator:Paulo Cézar Vieira
Grantee:Vivian Estevam Cornélio
Host Institution: Centro de Ciências Exatas e de Tecnologia (CCET). Universidade Federal de São Carlos (UFSCAR). São Carlos , SP, Brazil


Cathepsin D is an aspartyl endopeptidase involved in many pathological processes when overexpressed. In addition, it is responsible for degradation of hemoglobin ingested by parasites of schistosomiasis. For these reasons cathepsin D is considered an important target for chemotherapeutic intervention. The assays commonly performed to evaluate the inhibition of this enzyme monitor reaction products generated from the cleavage of fluorogenic peptide substrates or human hemoglobin in the presence of inhibitors that may exhibit autofluorescence/absorbance at the same wavelength used. Considering this problem, this paper presents the development and application of four methodologies for cathepsin D assays. In two methodologies the assays were performed in solution, and in one of them was used isolated bovine cathepsin D, and the other employed an extract of proteins taken from Schistosoma mansoni adult worms. A third assay was developed to evaluate inhibitors by means of a CatD-IMER bioreactor coupled to a multidimensional system in which it is possible to monitor the immobilized enzyme activity directly by quantifying the product formed. Finally, the last methodology was carried out by immobilizing the substrate human hemoglobin in the quartz crystal. The hydrolysis of hemoglobin by cathepsin D was evaluated by using a quartz crystal microbalance technique, a method that monitor the enzyme activity by mass variations. For the enzymatic assays, twenty plant extracts were evaluated, which led to the isolation and identification of the alkaloid evolitrine, the compound 4-hydroxy-3-methoxycinnamaldehyde and the flavonoids orientin and isovitexina. It were also tested 31 pure compounds, among them, the substance (z)-2-(pentadec-5-enyl)benzene-1,4-diol, which showed significant activity both against the isolated enzyme as the protein extract of S. mansoni. Finally, recombinant cathepsin D from S. mansoni was cloned and expressed using E. coli system, and in vitro studies of isolated natural product compounds showed excellent results regarding the limonoid cedrelona against somules and adult worms of S. mansoni. (AU)

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Scientific publications
(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)
CORNELIO, VIVIAN E.; PEDROSO, MARIELE M.; AFONSO, ANDRE S.; FERNANDES, JOAO B.; DA SILVA, M. FATIMA G. F.; FARIA, RONALDO C.; VIEIRA, PAULO C.. New approach for natural products screening by real-time monitoring of hemoglobin hydrolysis using quartz crystal microbalance. Analytica Chimica Acta, v. 862, p. 86-93, . (09/17538-8)
CORNELIO, VIVIAN E.; MALUF, FERNANDO V.; FERNANDES, JOAO B.; DA SILVA, MARIA FATIMA G. F.; OLIVA, GLAUCIUS; GUIDO, RAFAEL V. C.; VIEIRA, PAULO C.. Isolation of Tiliroside from Spiranthera odoratissima as Inhibitor of Trypanosoma cruzi Glyceraldehyde-3-phosphate Dehydrogenase by Using Bioactivity-Guided Fractionation. Journal of the Brazilian Chemical Society, v. 28, n. 3, SI, p. 512-519, . (09/17538-8, 13/07600-3)
CORNELIO, VIVIAN ESTEVAM; DE MORAES, MARCELA CRISTINA; DOMINGUES, VANESSA DE CASSIA; FERNANDES, JOAO BATISTA; DAS GRACAS FERNANDES DA SILVA, MARIA FATIMA; CASS, QUEZIA BEZERRA; VIEIRA, PAULO CEZAR. Cathepsin D immobilized capillary reactors for on-flow screening assays. Journal of Pharmaceutical and Biomedical Analysis, v. 151, p. 252-259, . (13/01710-1, 09/17538-8)

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