Reliable Analysis of the Interaction between Specific Ligands and Immobilized Beta-2-Adrenoceptor by Adsorption Energy Distribution

Abstract

Although a comparatively robust method, immobilized protein-based techniques have displayed limited precision and inconsistent results due to a lack of strategy for the accurate selection of drug adsorption models on the protein surface. We generated the adsorption data of three drugs on immobilized beta-2-adrenoceptor (β-AR) by frontal affinity chromatography-mass spectrometry (FAC-MS) and site-specific competitive FAC-MS. Using adsorption energy distribution (AED) calculations, we achieved the best adsorption models for the binding of salbutamol, terbutaline, and pseudoephedrine to immobilized β-AR. The Langmuir model proved to be desirable for describing the adsorptions of salbutamol and terbutaline on immobilized β-AR, while the bi-Langmuir model was favorable to characterize the adsorption of pseudoephedrine on the receptor. Relying on the accurate determination of association constants, we presented an efficient approach for β-AR ligand screening based on the loss of breakthrough time of an indicator drug caused by the inclusion of competitive drugs in the mobile phase. We concluded that the current strategy enables the reliable and accurate analysis of G protein-coupled receptor (GPCR)-drug interaction. The percentage change in the breakthrough time for drugs can provide useful information for estimating their binding affinity to the receptor. This approach builds a powerful platform for high-throughput ligand screening.

Site-Specific Immobilization of β2-AR Using O6-Benzylguanine Derivative-Functionalized Supporter for High-Throughput Receptor-Targeting Lead Discovery

“…When the incubating time reached 8 h, we achieved the highest expression of the receptor. Comparing with the culture of his-tagged β 2 -AR (154.1 ± 4.2 μg/mL) in LB media, , we concluded that the yield clearly increased. This was reasonable since SNAP fusion proteins are successfully and efficiently expressed on cell surface, in the cytoplasm, in the endoplasmic reticulum, and in the nucleus. , Taking inspiration from previous reports on maltose-binding protein and thioredoxin A, , we hold that SNAP is a very efficient C-terminal fusion partner guiding the expression into the inner membrane; introduction of SNAP into GPCRs will give functional expression of lipid embedded receptor.…”

Characterization of the adsorption site energies and heterogeneous surfaces of porous materials

“…The use of the above equation requires a set of initial estimation of F(E i ) as the method maximizes the influence of data over the final distribution. The initial guess used to determine the site energy distribution function is based on the 'total ignorance' guess: [72][73][74] (57)…”

“…Recently, the method was successfully used for protein-ligand screening. 74 With sufficient number of iterations, this method can unambiguously discriminate different types of adsorption sites having energy difference of less than 4-5 kJ/mol. 75 The main limitation of this method is that it cannot distinguish between the adsorption sites having energies less than 2 kJ/mol.…”

Solid-Supported Proteins in the Liquid Chromatography Domain to Probe Ligand-Target Interactions

“…FAC–MS has been employed to assess the adsorption data of three drugs (salbutamol, terbutaline, and pseudoephedrine) and the beta-2-adrenoreceptor (β 2 -AR) attached to polystyrene amino microspheres (Li et al, 2018). Adsorption data of the selected drugs obtained by FAC–MS and site-specific studies have helped to investigate the adsorption models for the binding of each ligand through adsorption energy distribution calculations.…”