Abstract
Structural studies of integral membrane proteins have been limited by the intrinsic conformational flexibility and the need to stabilize the proteins in solution. Stabilization by mutagenesis was very successful for structural biology of G protein-coupled receptors (GPCRs). However, it requires heavy protein engineering and may introduce structural deviations. Here we describe the use of specific calixarenes-based detergents for native GPCR stabilization. Wild type, full length human adenosine A2A receptor was used to exemplify the approach. We could stabilize native, glycosylated, non-aggregated and homogenous A2AR that maintained its ligand binding capacity. The benefit of the preparation for fragment screening, using the Saturation-Transfer Difference nuclear magnetic resonance (STD-NMR) experiment is reported. The binding of the agonist adenosine and the antagonist caffeine were observed and competition experiments with CGS-21680 and ZM241385 were performed, demonstrating the feasibility of the STD-based fragment screening on the native A2A receptor. Interestingly, adenosine was shown to bind a second binding site in the presence of the agonist CGS-21680 which corroborates published results obtained with molecular dynamics simulation. Fragment-like compounds identified using STD-NMR showed antagonistic effects on A2AR in the cAMP cellular assay. Taken together, our study shows that stabilization of native GPCRs represents an attractive approach for STD-based fragment screening and drug design.
Highlights
G protein-coupled receptors (GPCR) represent one of the largest family of integral membrane proteins and constitute highly druggable targets[1,2,3,4,5]
Fragment screening could be valuable in the case of GPCRs, for the development of allosteric modulators that can overcome the selectivity issue of orthosteric ligands[17,37]
By comparison to the Carr-Purcell-Meiboom-Gill sequence (CPMG) experiment used in the TINS technology, the so-called Saturation-Transfer Difference (STD) NMR experiment provides structural information through the discrimination of solvent exposed and buried hydrogens of the ligand bound to the receptor[43,44]
Summary
G protein-coupled receptors (GPCR) represent one of the largest family of integral membrane proteins and constitute highly druggable targets[1,2,3,4,5]. Fragment screening has been performed using biological and biophysical assays, including SPR38–41 and NMR-based TINS technology[41,42] These techniques have proved to be valuable, it is of high importance to develop orthogonal methods that enable robust identification and validation of fragment hits. To allow drug design and fragment screening on wild-type GPCRs using STD experiments, we have developed a strategy using calixarene-based detergent to solubilize and stabilize native, full length and functional GPCRs. We have lately reported on a systematic solubilization method for membrane proteins that allows screening for suitable detergents[45,46,47] and have described the use of novel calixarene-based detergents[46,48,49,50]. The STD-NMR screening results illustrate the advantage of the experiment to obtain rapid structural information and gain additional insight into the ligand interaction
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
