Development of a high throughput scintillation proximity assay for hepatitis C virus NS3 protease that reduces the proportion of competitive inhibitors identified
| Autor: | Marvin D. Schulman, Molly J. Price-Jones, Anne Fowler, John Anson, Russell B. Lingham, Kelvin T. Hughes |
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| Rok vydání: | 2000 |
| Předmět: |
0301 basic medicine
medicine.medical_treatment High-throughput screening Peptide Biology Viral Nonstructural Proteins 01 natural sciences Biochemistry Binding Competitive Analytical Chemistry 03 medical and health sciences Enzyme activator medicine Enzyme Inhibitors chemistry.chemical_classification NS3 Protease Molecular biology Enzyme assay 0104 chemical sciences Enzyme Activation 010404 medicinal & biomolecular chemistry 030104 developmental biology Scintillation proximity assay Enzyme chemistry biology.protein Molecular Medicine Scintillation Counting Biotechnology |
| Zdroj: | Journal of biomolecular screening. 5(3) |
| ISSN: | 1087-0571 |
| Popis: | A screening assay has been developed for hepatitis C virus (HCV) NS3 protease using the scintillation proximity assay (SPA) technology. The sequence of the peptide substrate used was taken from the site cleaved by the enzyme in the mature nonstructural protein of HCV. The peptide was biotinylated at the N-terminus and tritiated at the C-terminus so that a decrease in signal was detected as a result of enzyme activity. IC(50) values were calculated for the cleaved product, and it was shown that the value obtained was dependent on the substrate concentration used. The effect of substrate concentration on the inhibition of HCV NS3 protease was further highlighted in a mock screening assay, using colored natural product samples, in which the hit rate was altered by a change in substrate concentration. An increase in substrate concentration reduced the proportion of competitive inhibitors identified. This study highlighted the importance of optimizing the components used in SPA assays in order to obtain an assay format valid for high throughput screening. |
| Databáze: | OpenAIRE |
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