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

Abstract

The past decade has witnessed the great promise of strategies for ligand discovery based on surface-immobilized GPCRs. We present here a method for preparation of immobilized GPCRs. Key features include covalent immobilization with high specificity and robust application in drugreceptor interaction analysis and ligand screening. In our example assay using beta 2 -adrenergic receptor (β 2 -AR), the human DNA repair protein O 6alkylguanine-DNA alkyltransferase (hAGT) fusion receptor expressed in Escherichia coli was directly captured onto polyethylene glycol polyacrylamide (PEGA) resin. We observed even distribution and physiological functions of β 2 -AR on the resin. The immobilized β 2 -AR as a stationary phase enabled us to rapidly determine the binding of four drugs to β 2 -AR. By coupling this assay to mass spectrometry, we screened rosmarinic acid as a bioactive compound targeting β 2 -AR in Fructus Perillae. We concluded that O 6 -benzylguanine derivative-functionalized supporter is promising for specific immobilization of hAGT-tagged proteins; immobilized receptor chromatography has great potential in screening receptor-binding leads from herbal plants or traditional medicine recipes.

Covalent Inhibitor-Based One-Step Method for Endothelin Receptor A Immobilization: from Ligand Recognition to Lead Identification

“…The pioneering work by Moaddel and Wainer has established a series of membrane protein chromatographic methods for drug−protein interaction analysis. A systematic study by their group 45,46 and our work 21,22 have shown that K A values by the chromatographic methods are generally lower than those calculated by K d s by radioligand binding assay. In spite of the difference between the two sets of data, the two methods have proved to generate same affinity rank order for diverse ligands.…”

“…Bosentan showed three kinds of sites with various binding affinities. We also determined the association constants of ETA and the four ligands by the other three methods, including traditional frontal analysis, 22 self-competition method, 39 and injection amount-dependent analysis. 39 The binding affinities of these drugs and ETA are within the range of 10 3 to 10 6 M −1 , which are comparable to the previous publications.…”

“…A C-terminal 6× His-tagged version of ETA was also cloned into a pET15b vector for expressing control protein. Both EGFR-ETA and His-ETA were expressed in BL21­(DE3) cells with ampicillin resistance using an autoinduction medium . Incubating for 12 h at 37 °C, we harvested the cells by centrifugation and tested the receptor expression by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).…”

“…The tailored fusion tag selectively and covalently reacts with an immobilized substrate. Many of the engineered tags are commercially available, such as the haloalkane dehalogenase (HALO), O 6 -alkylguanine-DNA-alkyltransferase (SNAP), and a mutated carrier protein (MCP). , It is still challenging that the fusion tags are bulky and may affect the biological properties of the protein of interest, especially for G protein-coupled receptors (GPCRs) . An efficient, ideally one-step protocol remains highly desirable for CHH immobilization.…”

Development and evaluation of poly adenosine 5′‐diphosphate‐ribose polymerase 1 immobilization‐based receptor chromatography

“…In previous work, Zhao et al. immobilized β 2 ‐adrenoceptor ( β 2 ‐AR), an important drug target for respiratory diseases, onto macroporous silica gel by various immobilization methods [15–18]. The affinity columns were used to screen the bioactive compounds from TCMs, such as Siraitia grosvenorii [19] and Rhodiola crenulate [20].…”