EstablishingTrypanosoma cruzifarnesyl pyrophosphate synthase as a viable target for biosensor driven fragment‐based lead discovery
| Autor: | U. Helena Danielson, Helena Nordström, Tom Dzus, Valeria Napolitano, Giulia Opassi, Gerhard Klebe, Arne Lundin, Francesca Magari |
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| Rok vydání: | 2020 |
| Předmět: |
Chagas disease
Thermal shift assay Trypanosoma cruzi Fragment-based lead discovery Farnesyl pyrophosphate SPR Biochemistry Pyrophosphate Catalysis Article Cofactor 03 medical and health sciences chemistry.chemical_compound Magnesium Luciferase Molecular Biology 030304 developmental biology 0303 health sciences biology 030302 biochemistry & molecular biology Biochemistry and Molecular Biology Geranyltranstransferase fragment screening Articles Surface Plasmon Resonance binding efficiency biology.organism_classification farnesyl pyrophosphate synthase chemistry biology.protein Target protein Biokemi och molekylärbiologi |
| Zdroj: | Protein Science Protein Science : A Publication of the Protein Society |
| ISSN: | 1469-896X 0961-8368 |
| DOI: | 10.1002/pro.3834 |
| Popis: | Procedures for producing and exploring Trypanosoma cruzi farnesyl pyrophosphate synthase (tcFPPS) for surface plasmon resonance (SPR) biosensor‐driven fragment‐based discovery have been established. The method requires functional sensor surfaces with high sensitivity for extended times and appropriate controls. Initial problems with protein stability and lack of useful reference compounds motivated optimization of experimental procedures and conditions. The improved methods enabled the production of pure, folded and dimeric protein, and identified procedures for storage and handling. A new coupled enzymatic assay, using luciferase for detection of pyrophosphate, was developed and used to confirm that the purified enzyme was active after purification and storage. It also confirmed that sensor surfaces prepared with structurally intact protein was active. An SPR‐biosensor assay for fragment library screening and hit confirmation was developed. A thermal shift assay was used in parallel. A library of 90 fragments was efficiently screened by both assays at a single concentration in the presence and absence of the catalytic cofactor Mg2+. Hits were selected on the basis of response levels or ΔT m > 1°C and selectivity for tcFPPS in the presence of Mg2+. Characterization of hits by SPR showed that all had low affinities and the relationships between steady‐state responses and concentrations were not sufficiently hyperbolic for determination of KD‐values. Instead, ranking could be performed from the slope of the linear relationship at low concentrations. This pilot screen confirms that the procedures developed herein enables SPR‐biosensor driven fragment‐based discovery of leads targeting tcFPPS, despite the lack of a reference compound. Significance Statement To enable the discovery of drugs, it is essential to have access to relevant forms of the target protein and valid biochemical methods for studying the protein and effects of compounds that may be evolved into drugs. We have established methods for the discovery of drugs for treatment of American Trypanosomiasis (Chagas disease), using farnesyl pyrophosphate synthase from Trypanosoma cruzi as a target. |
| Databáze: | OpenAIRE |
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