Miniaturized weak affinity chromatography for ligand identification of nanodiscs-embedded G-protein coupled receptors

Abstract

Biophysical techniques that enable the screening and identification of weak affinity fragments against a target protein are at the heart of Fragment Based Drug Design approaches. In the case of membrane proteins, the crucial criteria for fragment screening are low protein consumption, unbiased conformational states and rapidity because of the difficulties in obtaining sufficient amounts of stable and functionally folded proteins. Here we show for the first time that lipidnanodisc systems (membrane-mimicking environment) and ultra-miniaturized affinity chromatography can be combined to identify specific small molecule ligands that bind to an integral membrane protein. The approach is exemplified using the AA2AR GPCR. Home-made affinity nano-columns modified with nanodiscs-embedded AA2AR (only about 1 µg of protein per column) are fully characterized by frontal chromatographic experiments. This method allows (i) to distinguish specific and unspecific ligand/receptor interactions, (ii) to assess dissociation constants, (iii) to identify the binding pocket of uncharacterized ligands using a reference compound with competition experiments. Weak affinity ligands with Kd in the low to high micromolar range be detected. At last, the applicability of this method is demonstrated with 6 fragments recently identified as ligands or non-ligands of AA2AR.

Fragment-to-Lead Medicinal Chemistry Publications in 2020

“…Among others, two studies reported promising fragment screening using Weak Affinity Chromatography (WAC) including for a GPCR target. 69 , 70 It will be interesting to see if these exploratory studies will result in lead compounds.…”

Affinity monolith chromatography: A review of general principles and recent developments

“…The presence of the secondary binding agent may also increase the possibility for non‐specific binding and result in a lower binding capacity for the affinity ligand due to steric hindrance. Biospecific adsorption in AMC has been used to prepare monoliths for the capture of antibodies by protein A or protein G; in addition, streptavidin monoliths have been used to adsorb biotinylated HSA, aptamers, and membrane proteins [60,64,172,187,198–200].…”

“…Frontal analysis using affinity monoliths coupled with MS has been applied to examine the binding of small molecules to dihydrofolate reductase [90] and to examine small ligands that may bind to adenosine A 2A receptor and compete with theophylline for this receptor (see Fig. 9) [200]. AMC has also been used in a frontal analysis format to examine the binding of IgG to protein A, protein G, and protein L [170,178,325].…”

Affinity chromatography: A review of trends and developments over the past 50 years

“…Avidin has recently been used with biotin and affinity columns in various formats to isolate proteins under native elution conditions [171]. Streptavidin-biotin interactions have also been employed in miniaturized affinity systems to identify and characterize ligands for nanodisc-embedded G-protein coupled receptors [172].…”