Development, validation and quantitative assessment of an enzymatic assay suitable for small molecule screening and profiling: A case-study

Publication date: June 2015 Source:Biomolecular Detection and Quantification, Volume 4 Author(s): Vicente Sancenon , Wei Hau Goh , Aishwarya Sundaram , Kai Shih Er , Nidhi Johal , Svetlana Mukhina , Grant Carr , Saravanakumar Dhakshinamoorthy 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 an...
Source: Biomolecular Detection and Quantification - Category: Molecular Biology Source Type: research