Development, validation and quantitative assessment of an enzymatic assay suitable for small molecule screening and profiling: A case-study
| Autor: | Svetlana Mukhina, Kai Shih Er, Grant Carr, Nidhi Johal, Vicente Sancenon, Aishwarya Sundaram, Wei Hau Goh, Saravanakumar Dhakshinamoorthy |
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| Rok vydání: | 2015 |
| Předmět: |
Inhibitor
lcsh:QR1-502 Computational biology Bioinformatics Biochemistry lcsh:Microbiology DiFMUP 6 8-difluoro-4-methylumbelliferyl phosphate Structural Biology Phosphatase Quantitative assessment Molecular Biology AP - Alkaline phosphatase lcsh:QH301-705.5 Mechanism-of-action Reaction conditions Chemistry Assay development In vitro toxicology CV coefficient of variation Z′ Z prime pNPP p-nitrophenol phosphate Small molecule AP alkaline phosphatase lcsh:Biology (General) pNP p-nitrophenol Initial phase Signal variability Screening Molecular Medicine Original Article DEA diethanolamine KM Michaelis constant Vmax maximal reaction velocity SD standard deviation DiFMU 6 8-difluoro-4-methylumbelliferone |
| Zdroj: | Biomolecular Detection and Quantification Biomolecular Detection and Quantification, Vol 4, Iss C, Pp 1-9 (2015) |
| ISSN: | 2214-7535 |
| DOI: | 10.1016/j.bdq.2015.03.001 |
| Popis: | The successful discovery and subsequent development of small molecule inhibitors of drug targets relies on the establishment of robust, cost-effective, quantitative, and physiologically relevant in vitro assays that can support prolonged screening and optimization campaigns. The current study illustrates the process of developing and validating an enzymatic assay for the discovery of small molecule inhibitors using alkaline phosphatase from bovine intestine as model target. The assay development workflow includes an initial phase of optimization of assay materials, reagents, and conditions, continues with a process of miniaturization and automation, and concludes with validation by quantitative measurement of assay performance and signal variability. The assay is further evaluated for dose–response and mechanism-of-action studies required to support structure–activity-relationship studies. Emphasis is placed on the most critical aspects of assay optimization and other relevant considerations, including the technology, assay materials, buffer constituents, reaction conditions, liquid handling equipment, analytical instrumentation, and quantitative assessments. Examples of bottlenecks encountered during assay development and strategies to address them are provided. |
| Databáze: | OpenAIRE |
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