Thermodynamic characterisation of covalent ligand binding

BACKGROUND AND PURPOSE: Differential scanning fluorimetry (DSF) is a common and straightforward method to evaluate the thermal stability of proteins and has been heavily used for ligand binding characterisation as well as for screening. One class of compounds that is less typically evaluated by DSF are covalent binders. Here, we assessed the contribution of the covalent bond to protein thermal stabilisation. EXPERIMENTAL APPROACH: We evaluated selective covalent binders, as well as non-selective reactive electrophiles, against five model protein targets. To assess DSF in the context of fragment-based electrophile screening, we compared DSF measurements to covalent labelling over a subset of electrophilic fragments. KEY RESULTS: We show that, in the context of selective binders, the formation of the covalent bond increases thermal stabilisation. However, it is not the covalent bond itself that stabilises the protein, because non-selective irreversible binding was typically neutral or, more often, destabilised the protein. In the context of fragment screening, the magnitude of the thermal shift tended to increase with irreversible labelling, whereas the more reactive fragments tended to destabilise the protein. CONCLUSION AND IMPLICATIONS: Taken together, we suggest DSF as a complementary approach to triage covalent fragment hits, in which fragments that show both labelling and protein stabilisation are predicted to display molecular recognition driven binding and serve as more productive starting points for covalent ligand development.