| Assay Method Information | |
| | HTS identification of compounds inhibiting phosphomannose isomerase (PMI) via a fluorescence intensity assay using a high concentration of mannose 6-phosphate |
| Description: | The purpose of this assay is to identify non-competititve inhibitors of human PMI. This is accomplished by using a G6PD- NADPH-coupled assay. In the assay PMI activity is detected through conversion of its product, fructose-6-phosphate, to glucose-6-phosphate catalyzed by phosphoglucose isomerase (PGI) and subsequent oxidation of glucose-6-phosphate to 6-phosphogluconolactone concomitant with NADP-to-NADPH conversion catalyzed by glucose-6-phosphate dehydrogenase (G6PDH). The NADPH is then detected via a resazurin-diaphorase fluorogenic reaction. This assay is performed in the presence of 10x-Km concentrations of the PMI substrate, mannose-6-phosphate, to help to ensure the identification of non-competitive inhibitors. 1) 9 uL of Substrate working solution was added to columns 3-24 of a Greiner 384-well black plate (cat # 784076) using a WellMate bulk dispenser (Matrix) 2) 9 ul of Substrate working solution without mannose-6-p was added to columns 1 and 2 (positive control) 3) Dose-response curves contained 10 concentrations of compounds obtained using 2-fold serial dilution. Compounds were serially diluted in 100% DMSO, and then diluted with water to 10% final DMSO concentration. 4) 2 uL compounds in 10% DMSO were transferred into columns 3-22. Columns 1-2 and 23-24 contained 4 uL of 10% DMSO. 5) 9 uL of Enzyme working solution was added to the whole plate using a Thermo Multidrop Combi dispenser. 6) Plates were incubated at room temperature for 30 min. 7) The plates were read on an Analyst plate reader (Molecular Devices), Ex544, Em590. 8) Data analysis was performed using CBIS software (ChemInnovations, Inc). |
| Affinity data for this assay | |
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