FTSA and SPR for identifying small molecule ligands and PAINs

The power of biophysical methods in drug discovery, utilising FTSA and SPR to validate kinase p38Ξ± ligands and screen PAINs compounds.

FTSA and SPR for identifying small molecule ligands and PAINs

Abstract

Biophysical methods are attractive techniques in drug discovery, for both primary screening and orthogonal validation of hits. Here we describe aΒ dual method, employing Fluorescent Thermal Shift assay (FTSA) andΒ surface plasmon resonance (SPR), to interrogate ligands of the kinase p38Ξ± as well as several known pan-assay interference compounds (PAINs).

This combinatorial approach allows for independent verification ofΒ biophysical parameters such as KD, kon, koff, Ξ”G, Ξ”S, and Ξ”H, which may further guide chemical development of a ligand series. Affinity valuesΒ obtained from FTSA curves allow for insight into compound binding.Β Ligand–p38 interaction data were in good agreement with previousΒ literature.Β Aggregators and Fluorescence quenchers appeared to reduce Fluorescence signal in the FTSAs, causing artificially high shifts in Tm values,Β whereas redox compounds caused either a depression of FTSA signal or
shifts in affinity that did not agree between FTSA and SPR.

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