Enabling STD-NMR fragment screening using stabilized native GPCR: A case study of adenosine receptor

Area of Science:

• Biochemistry
• Structural Biology
• Pharmacology

Background:

• Integral membrane protein structural studies are challenging due to flexibility and the need for stabilization.
• Mutagenesis-based stabilization of G protein-coupled receptors (GPCRs) is effective but can alter protein structure.

Purpose of the Study:

• To develop a method for stabilizing native GPCRs without extensive protein engineering.
• To demonstrate the utility of stabilized native GPCRs for fragment-based drug discovery using Saturation-Transfer Difference nuclear magnetic resonance (STD-NMR).

Main Methods:

• Stabilization of the wild-type human adenosine A2A receptor using specific calixarene-based detergents.
• Utilizing Saturation-Transfer Difference nuclear magnetic resonance (STD-NMR) for fragment screening.
• Conducting competition binding experiments and cellular assays (cAMP) to validate findings.

Main Results:

• Native, homogenous, and functional adenosine A2A receptor was stabilized, retaining ligand binding capacity.
• STD-NMR successfully identified binding interactions of known ligands (adenosine, caffeine) and fragments.
• Fragment-like compounds identified via STD-NMR exhibited antagonistic effects on A2A receptor activity.

Conclusions:

• Calixarene-based detergents provide an effective means to stabilize native GPCRs for structural and functional studies.
• This stabilization approach facilitates fragment-based screening using STD-NMR, offering a viable strategy for drug design.
• The study validates the use of native GPCR stabilization for identifying novel drug leads.