Covalent Modifier Discovery Using Hydrogen/Deuterium Exchange-Mass Spectrometry.

Area of Science:

• Biochemistry
• Chemical Biology
• Drug Discovery

Background:

• Covalent ligands are often excluded from high-throughput screening due to concerns about pan-assay interference compounds (PAINS).
• Novel screening strategies are needed to differentiate genuine covalent binders from PAINS.
• Hydrogen/deuterium-exchange mass spectrometry (HDX-MS) is a valuable technique for assessing protein stability and ligand-protein interactions.

Purpose of the Study:

• To develop and validate a covalent modifier screening approach utilizing HDX-MS.
• To apply HDX-MS for classifying ligands targeting peroxisome proliferator-activated receptor γ (PPARγ) and vitamin D receptor.
• To identify novel covalent modifiers and understand their interaction dynamics.

Main Methods:

• Utilized Hydrogen/deuterium-exchange mass spectrometry (HDX-MS) to evaluate ligand-protein interactions.
• Applied HDX-MS to screen and classify ligands for PPARγ and vitamin D receptor.
• Characterized the binding affinity and stability effects of identified covalent modifiers.

Main Results:

• HDX-MS successfully discriminated the strength of ligand-protein interactions.
• The HDX-MS screening identified LT175 and nTZDpa as concurrent binders to the PPARγ ligand-binding domain (PPARγ-LBD), exhibiting synergistic activation.
• Iodoacetic acid was identified as a novel covalent modifier that enhances PPARγ-LBD stability.

Conclusions:

• HDX-MS provides a robust method for covalent modifier screening, effectively distinguishing true covalent ligands.
• The study identified novel compounds and a new covalent modifier for PPARγ, offering potential for therapeutic development.
• This approach advances the identification of covalent drugs by overcoming limitations of traditional screening methods.