Sygnature Discovery\u2019s EPSA assay is an SFC-based measurement that estimates a compound\u2019s exposed polar surface area (EPSA). This can be used to assess a compound\u2019s ability to shield polarity and for it to form intramolecular hydrogen bonds.1<\/sup> By comparing the EPSA to the topological polar surface area (TPSA), we can assess how much of the polarity has been hidden. This method provides a rapid assessment that can be useful in drug discovery particularly for beyond rule of five (bRo5) molecules, including bifunctional degraders, as compounds with a lower EPSA have a greater chance of being cell permeable.2<\/sup><\/p>\n\n\n\n The EPSA assay workflow begins with preparation of test compound solutions from 10 mM DMSO stocks. These are transferred to vials or plates for automated analysis by Supercritical Fluid Chromatography (SFC) coupled with mass spectrometry (MS). The test compounds are analysed using a gradient of supercritical CO2<\/sub> and organic modifier against calibration standards of known EPSA. The EPSA value is calculated from the retention time of the test compound against the calibration line.\u00a0 Compound identity is confirmed by MS. The calibration standard mixture comprises analytes with an established EPSA1<\/sup>, and the chromatographic parameters have been optimised to minimise overall analytical runtime and provide increased capacity.<\/p>\n\n\n\n Representative retention time and EPSA are shown below<\/p>\n\n\n Antipyrine<\/p>\n\n <\/div>\n <\/div>\n <\/td>\n \n \n \n \n \n 0.61<\/p>\n\n <\/div>\n <\/div>\n <\/td>\n \n \n \n \n \n 61<\/p>\n\n <\/div>\n <\/div>\n <\/td>\n <\/tr>\n Desipramine<\/p>\n\n <\/div>\n <\/div>\n <\/td>\n \n \n \n \n \n 0.95<\/p>\n\n <\/div>\n <\/div>\n <\/td>\n \n \n \n \n \n 87<\/p>\n\n <\/div>\n <\/div>\n <\/td>\n <\/tr>\n Pindolol<\/p>\n\n <\/div>\n <\/div>\n <\/td>\n \n \n \n \n \n 1.17<\/p>\n\n <\/div>\n <\/div>\n <\/td>\n \n \n \n \n \n 103<\/p>\n\n <\/div>\n <\/div>\n <\/td>\n <\/tr>\n Diclofenac Sodium<\/p>\n\n <\/div>\n <\/div>\n <\/td>\n \n \n \n \n \n 1.79<\/p>\n\n <\/div>\n <\/div>\n <\/td>\n \n \n \n \n \n 135<\/p>\n\n <\/div>\n <\/div>\n <\/td>\n <\/tr>\n m-Nitrobenzoic Acid<\/p>\n\n <\/div>\n <\/div>\n <\/td>\n \n \n \n \n \n 2.24<\/p>\n\n <\/div>\n <\/div>\n <\/td>\n \n \n \n \n \n 157<\/p>\n\n <\/div>\n <\/div>\n <\/td>\n <\/tr>\n <\/tbody>\n\n <\/table>\n <\/div>\n<\/div>\n\n<\/div>\n\t\t\n\t <\/section>\n\n<\/div>\n\t\t\n\t <\/section>\n\n\n\n Standard reproducibility was assessed by analyzing 50 injections of calibration mixture across inter-assay experiments as well as following a solvent exchange and cylinder replacement. Excellent reproducibility with consistent retention times was observed.<\/p>\n\n\n\n This assay provides a rapid and robust assessment of the exposed polar surface area of compounds that can aid in the identification of compounds with the best chance of displaying acceptable cell permeability. This technique can be particularly useful for drug discovery projects involving bRo5 compounds.<\/p>\n\n\n\n Sygnature Discovery\u2019s EPSA validation demonstrates a fast and reliable SFC\u2011MS method for estimating exposed polar surface area and understanding how effectively compounds can shield polarity. With optimised chromatography, consistent retention times and strong reproducibility, the assay supports rapid permeability assessment\u2014especially valuable for bRo5 molecules and bifunctional degraders.<\/p>\n","protected":false},"featured_media":0,"template":"","category":[683,719],"resource_tag":[],"class_list":["post-13511","technical-note","type-technical-note","status-publish","hentry","category-dmpk","category-physicochemical-profiling"],"acf":[],"yoast_head":"\nProtocol Summary<\/h2>\n\n\n\n
<\/p>\n\n\n\nValidation Results<\/h2>\n\n\n\n
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References<\/h3>\n\n\n\n
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