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LC-MS / 2D methods for screening ligands for evaluating enzyme inhibitory activity and identification of active compounds in crude extracts

Grant number: 14/11640-3
Support type:Scholarships in Brazil - Post-Doctorate
Effective date (Start): November 01, 2014
Effective date (End): December 31, 2017
Field of knowledge:Physical Sciences and Mathematics - Chemistry - Organic Chemistry
Principal researcher:Carmen Lúcia Cardoso
Grantee:Cláudia Seidl
Home Institution: Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto (FFCLRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil
Associated research grant:13/01710-1 - Enzyme ligand: new models of screening, AP.TEM
Associated scholarship(s):16/02941-5 - Development and optimization of two-dimensional liquid chromatography methodologies coupled with bioassays for discovery of enzyme inhibitors in natural product extracts, BE.EP.PD


The identification of substances with biological activity in natural extracts or synthetic collections is a crucial step and requires the use of fast and efficient techniques for separation and screening. This paradigm stimulates the development of new screening methods to facilitate and reduce the time taken to identify such substances. By employing chromatographic techniques, isolation of active principles derived from a natural extract can exceptionally be achieved in a single chromatographic step. The use of chromatographic methods 2D is a valuable strategy to obtain greater separation efficiency with reduced analysis time. 2D separations by liquid chromatography are well established and can be classified into broad, comprehensive and pseudo - "heart- cutting". In comprehensive (LCxLC ) all effluent from the first dimension, or the representative of it, is introduced into the second dimension. This is the main difference compared to the heart- cutting liquid chromatography (LC - LC) in which only the selected fractions are transferred to the second column. Classical separation methods of natural extracts involve high consumption of time, while screening offline does not result in information on individual compounds and often have false positive or false negative results. A promising alternative is the coupling of chromatographic separation techniques, particularly with multidimensional biological assays, called high throughput screening. Thus, it is possible to detect biological activity directly in the LC eluent and chemically characterize the compounds biologically active online, by MS, resulting in reduced time and labor spent in collecting the fractions for subsequent activity detection. For conducting online biological assays, the use of immobilized enzymes is a great alternative. The immobilized enzymes involve advantages such as: it enhanced stability in the presence of organic solvents and temperature variations; use of small amounts of enzyme and they can be reused. In this context, the aim of this project is to develop a rapid method for online identification of bioactive constituents of plant extracts without the need of prior purification. The extract is separated in the first dimension and the chromatographic bands are transferred to the bioreactor, where the inhibitory activity is evaluated. ICERs (capillary Immobilized enzyme reactors) containing the cholinesterase enzyme acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), were developed in our research group and were selected as model enzyme targets for this project. These enzymes are serine hydrolase responsible for the catalysis of the hydrolysis of the neurotransmitter acetylcholine at the nerve endings and synapses are targets of interest in studies of the development of drugs for Alzheimer's disease. Moreover, by acting on signal transduction in animals, there is considerable interest in the discovery of inhibitors of these enzymes to be used in agriculture as pesticides and against social insects. (AU)

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Scientific publications (6)
(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)
RAPHAEL A. DE JESUS; VILMA M. J. PRADO; VANDERSON S. PINTO; VALDENIZIA R. SILVA; LUCIANO S. SANTOS; PAULO C. L. NOGUEIRA; SANDRO NAVICKIENE; EDENIR R. PEREIRA-FILHO; ARIE F. BLANK; DANIEL P. BEZERRA; et al. Application of LC-DAD Metabolic Fingerprinting in Combination with PCA for Evaluation of Seasonality and Extraction Method on the Chemical Composition of Accessions from Lippia alba (Mill) N. E. Brown and Biological Activities. Journal of the Brazilian Chemical Society, v. 30, n. 5, p. 978-987, . (13/01710-1, 14/11640-3)
DE JESUS, RAPHAEL A.; PRADO, VILMA M. J.; PINTO, VANDERSON S.; SILVA, VALDENIZIA R.; SANTOS, LUCIANO S.; NOGUEIRA, PAULO C. L.; NAVICKIENE, SANDRO; PEREIRA-FILHO, EDENIR R.; BLANK, ARIE E.; BEZERRA, DANIEL P.; et al. Application of LC-DAD Metabolic Fingerprinting in Combination with PCA for Evaluation of Seasonality and Extraction Method on the Chemical Composition of Accessions from Lippia alba (Mill.) N. E. Brown and Biological Activities. Journal of the Brazilian Chemical Society, v. 30, n. 5, p. 978-987, . (13/01710-1, 14/11640-3)
MONTEIRO, AFIF F.; SEIDL, CLAUDIA; SEVERINO, VANESSA G. P.; CARDOSO, CARMEN LUIA; CASTRO-GAMBOA, IAN. Biotransformation of labdane and halimane diterpenoids by two filamentous fungi strains. ROYAL SOCIETY OPEN SCIENCE, v. 4, n. 11, . (14/11640-3, 13/07600-3)
VILMA M. J. PRADO; RAPHAEL A. DE JESUS; JULIO M. A. OLIVEIRA; CAMILA S. A. PEREIRA; ARIE F. BLANK; EDENIR R. PEREIRA-FILHO; QUEZIA B. CASS; JULIANA M. DE LIMA; ANTONIO G. FERREIRA; PAULO C. L. NOGUEIRA; et al. Evaluation of the Seasonality and Extraction Method on the Polar Extracts of Croton grewioides Baill. by Chromatogram Fingerprinting and Isolation of a New Triglycosylated Flavonoid. Journal of the Brazilian Chemical Society, v. 32, n. 2, p. 385-394, . (14/11640-3, 13/01710-1)
LOPES VILELA, ADRIANA FERREIRA; SEIDL, CLAUDIA; LIMA, JULIANA MARIA; CARDOSO, CARMEN LUCIA. An improved immobilized enzyme reactor-mass spectrometry-based label free assay for butyrylcholinesterase ligand screening. Analytical Biochemistry, v. 549, p. 53-57, . (14/11640-3, 14/06907-0, 16/02873-0)
LIMA, JULIANA M.; SEIDL, CLAUDIA; CUNHA, ELISE M. F.; OLIVEIRA, ARTHUR H. C.; CARDOSO, CARMEN L.. On-Flow Ligand Screening Assay Based on Immobilized Nucleoside Diphosphate Kinase B from Homo sapiens. Journal of the Brazilian Chemical Society, v. 30, n. 11, SI, p. 2308-2317, . (14/50249-8, 13/01710-1, 14/06907-0, 14/11640-3)

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