EPSA

About the Assay

Sygnature Discovery’s EPSA assay is an SFC-based measurement that estimates a compound’s exposed polar surface area (EPSA). This can be used to assess a compound’s ability to shield polarity and for it to form intramolecular hydrogen bonds.¹ 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

Protocol Summary

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 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.  Compound identity is confirmed by MS.

Validation Results

The calibration standard mixture comprises analytes with an established EPSA¹, and the chromatographic parameters have been optimised to minimise overall analytical runtime and provide increased capacity.

Representative retention time and EPSA are shown below

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.

Summary

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.

References

1. Goetz et al, J. Med. Chem., 2014, 57, 2920−2929.
2. Caron et al, ACS Med. Chem. Lett., 2021, 12, 13-23.