Objective

HTS remains the key methodology for finding hit and lead compounds within the pharmaceutical industry. HTS utilises automated robotic platforms to screen large compound libraries against biological targets at a single concentration to identify novel chemical hits for project teams. Hit compounds are confirmed by follow up assays that yield pIC₅₀ values. Biophysical methods are rapidly gaining traction in post HTS cascades to confirm target engagement prior to commencement of expensive chemistry. Differential scanning fluorimetry (DSF) is one such biophysical technique that can provide an indication of a hit compound binding to a target protein. Melting temperatures (Tm) of the target protein can be determined and compared to the Tm measured in the presence of a potential hit compound. The difference in melting temperature (ΔTm) measured is concommitant with target compound binding. This study evaluates the benefits of using DSF in HTS as a triage assay with the aim of validating hit compounds post HTS campaigns. DSF was performed on three target proteins from recent HTS campaigns where biophysical data was of interest; a kinase, a GTPase and a lipase using compounds identified as active by their pIC₅₀ values. Microscale Thermophoresis (MST) and Surface Plasmon Resonance (SPR) were performed and compared to the DSF data, to observe if there was agreement between these biophysical techniques.