Screening of enzymatic ligands using integrated assay platforms

• Grant number: 24/18688-3
• Support Opportunities: Regular Research Grants
• Start date: September 01, 2025
• End date: August 31, 2028
• Field of knowledge: Physical Sciences and Mathematics - Chemistry - Organic Chemistry
• Principal Investigator: Carmen Lúcia Cardoso
• Grantee: Carmen Lúcia Cardoso
• Host Institution: Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto (FFCLRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil
• Associated researchers: Ana Maria Soares Pereira ; Anelize Bauermeister ; Claudio Viegas Junior ; Dulce Helena Siqueira Silva ; Maria Fátima das Graças Fernandes da Silva ; Taisa Magnani Dinamarco

Abstract

One of the challenges in identifying new bioactive compounds is the large number of candidates, whether of natural or synthetic origin. The use of fast and efficient separation and screening techniques to identify active molecules from natural extracts and combinatorial collections is increasing, which encourages the development and application of integrated screening platforms that employ both on-line and off-line methods. These methods facilitate and reduce the time spent on identifying active molecules. In this context, affinity selection and mass spectrometry (AS-MS) have gained relevance over conventional high-throughput screening (HTS) techniques. This method is based on affinity interactions between the biological target and the ligand, allowing for the separation of binders from non-binders. Strong binders are then analyzed by mass spectrometry, thereby reducing the number of candidates to be tested in inhibition assays. This strategy is especially valuable for complex samples such as natural extracts. The proposal of this project is to combine affinity assays (AS-MS) using the ligand fishing approach with biological activity screening assays, thereby creating a comprehensive testing platform. On-flow activity screening assays can couple chromatographic separation techniques with biological assays. The immobilized enzyme reactor (IMER) can be used in different configurations: i) directly connected to the detector; ii) before the analytical column (1D), where the enzymatic reaction product is separated and quantified on the analytical column (2D); or iii) positioned after the separation performed on the analytical column (1D), thus allowing the detection of biological activity directly in the liquid chromatography (LC) eluent (2D). By using mass spectrometry, it is possible not only to gather information on catalytic activity but also to chemically characterize the biologically active compound(s) online, thereby reducing time and labor. This platform can be applied to various enzymatic targets. Among the targets of interest are cholinesterase enzymes, specifically human acetylcholinesterase (AChEhu) and brown stink bug Euschistus heros acetylcholinesterase (AChEhe), in the search for selective inhibitors. Additionally, human recombinant butyrylcholinesterase (BChEhu) and monoamine oxidases A and B (MAO-A and MAO-B) will be explored for multitarget inhibitors. Other targets may also be included during the course of the project. (AU)