Rapid ligand fishing assay for identification of enzyme ligands in natural product library

Quezia B. Cass, Chemistry, Federal University of São Carlos, Sao Carlos, Brasil (qbcass@gmail.com)

Natural product combinatorial libraries afford a diversity of molecular framework with different levels of stereochemical complexity and biological activity and are considered a promisor source of leads for the development of new drugs.¹They possess, however, high level of difficulty for disclosing active compounds which usually involves time-consuming, high complex, and ineffective procedures either to identify or to isolate bioactive compounds. In this respect, affinity-based screening assays emerged as a useful tool for monitoring binding events aiming identification, characterization and/or isolation of active small molecules.²  The use of magnetic beads coated with biological targets such as enzymes and receptors, for instance, are used to identify weak but selective binders from natural products extracts, enabling identification of ligands directly from fractions or crude extracts. There are several advantages associated with this approach, as reutilization of the immobilized proteins (which are generally expensive), rapidly chemical characterization of ligands and investigation of the specificity of the interactions.³As an alternative to classical bioassay guide fractionation of natural product crude extracts, we have been exploring ligand fishing assays by the use of magnetic beads coated with a series of important targets enzymes such as: acetylcholinesterase from E. electricus (used as a model), phosphoenolpyruvate carboxykinase (PEPCK) from T. cruzi and arginase from L. amazonenses. Classical inhibition methods for these enzymes are based in the use of either colorimetric and/or conjugated enzyme assays, which are susceptible to false negative and positive results and interferences. Thus, for all of them, we have developed direct analysis by LC-MS/MS, in which the enzyme activity is directly monitored through the quantification of the formed product. These activity-based assays allow evaluation of the immobilized enzymes kinetics and interactions of identified ligands with the enzyme catalytic site. For the fishing assay, in order to chemically characterize the identified ligands, we have developed natural product extracts fingerprint by liquid chromatography hyphenated with high-resolution mass spectrometry (LC-HRMS). For this presentation, a critical overview of these assays will be sightseen.

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Abstract Reference & Short Personal Biography of Presenting Author

1.        N. L. Batista, A., M. dos Santos, F., Batista, J. & Cass, Q. Enantiomeric Mixtures in Natural Product Chemistry: Separation and Absolute Configuration Assignment. Molecules 23, 492 (2018).

2.        Zhuo, R., Liu, H., Liu, N. & Wang, Y. Ligand fishing: A remarkable strategy for discovering bioactive compounds from complex mixture of natural products. Molecules 21, 1516 (2016).

3.        Vanzolini, K. L. et al. Acetylcholinesterase affinity-based screening assay on Lippia gracilis Schauer extracts. J. Pharm. Biomed. Anal. 153, 232–237 (2018).

Quezia B. Cass is a full professor at the Chemistry Department of Federal University of São Carlos, SP, Brazil and coordinator of Separare – Chromatography Research Nucleus. Separation of drugs plays an important role in her research projects and a number of methods were developed for studies of achiral and chiral drugs of medical and pharmaceutical interest by multidimensional mode. Zonal bioaffinity chromatography and ligand-fishing assays has been, lately, one of her main research interests. She has been working for prospecting ligands from natural or synthetic libraries associated with drug discovery programs and green chemistry procedures. In this respect, she has been recognized for her contribution to the development of cutting-edge analytical techniques. She has authored more than 160 articles that have been published in professional scientific journals; supervised more than 52 Masters and PhD candidates, 10 research fellows and 24 undergraduate research students. She has presented more than 50 seminars in national and/or internationally recognized conferences and has worked as editor of 3 books and authored 4 book chapters. She is a member of the international scientific committee of the Pharmaceutical and Biomedical Analysis Symposium series.