Thermodynamic characterisation of covalent ligand binding

Area of Science:

• Biochemistry
• Chemical Biology
• Drug Discovery

Background:

• Differential scanning fluorimetry (DSF) is a standard technique for protein thermal stability assessment and ligand binding studies.
• Covalent binders are less commonly evaluated using DSF.
• This study investigates the role of covalent bonds in protein thermal stabilization.

Purpose of the Study:

• To evaluate the contribution of covalent bonds to protein thermal stabilization using DSF.
• To assess the utility of DSF for screening covalent binders, particularly electrophilic fragments.
• To compare DSF measurements with covalent labeling data.

Main Methods:

• Proteins were incubated with selective covalent binders and non-selective reactive electrophiles.
• Differential scanning fluorimetry (DSF) was employed to measure thermal stabilization.
• DSF results were compared with covalent labeling data for electrophilic fragments.

Main Results:

• Selective covalent bond formation generally increased protein thermal stabilization.
• Non-selective irreversible binding was neutral or destabilizing.
• In fragment screening, increased thermal shift correlated with irreversible labeling, while high reactivity often led to destabilization.

Conclusions:

• DSF is a valuable complementary method for triaging covalent fragment hits.
• Fragments exhibiting both labeling and stabilization are promising starting points for covalent ligand development.
• This approach aids in identifying covalent ligands with molecular recognition-driven binding.