Tulobuterol

Protein functionalized surface has the potential to develop new assays for determining the drug-like properties of potential compounds and discovering specific partners of G protein-coupled receptors (GPCRs). However, a universal method for purifying and immobilizing functional GPCRs has remained elusive. To this end, we developed a general and rapid way to purify and immobilize β₂-adrenergic receptor (β₂AR) by silicon-specific peptide. We screened CotB1p as a tag from six silica-binding peptides (minTBP-1, CotB1p, SB₇, Car₉, and Si_4-1) by examining their affinity to macroporous silica gel. We investigated the adsorption and desorption of CotB1p-tagged β₂-adrenoceptor (β₂AR-CotB1p) under diverse conditions to propose a protocol for receptor purification and immobilization. Under optimized conditions, β₂AR immobilization were achieved by directly immersing cell lysates harboring the receptor with silica gel, and the elution of the receptor without demonstratable contaminants was realized by including l-arginine/L-lysine in the elutes. This allows purification of the receptor from Escherichia coli (E.coli) lysates with a purity of 95 %. The immobilized receptor was utilized as a stationary phase to reveal the tag impact on ligand-binding outputs by comparing the CotB1p-strategy with a typical covalent method. The K_As of salbutamol, chlorprenaline, tulobuterol, and terbutaline on β₂AR-CotB1p column were 1.26 × 10⁶, 6.59 × 10⁶, 7.90 × 10⁶, and 8.97 × 10⁵ M^-1 respectively, which were two orders of magnitude higher than those on the Halo-β₂AR column. The whole immobilization was accomplished within 30 min without the requirement of any special treatment, resulting in enhanced accuracy for determining receptor-ligand binding parameters. Taken together, CotB1p-mediated strategy is simple, rapid, and universal for purification or immobilization of unstable biomolecules like GPCRs for analytical and biological applications.

Cortex Morin Radicis (CMR) is the dried root bark of Morus alba. L. It has a variety of effects such as antibacterial, anti-tumour, treatment of cardiovascular diseases or upper respiratory tract disease and so on. The pursuit for drugs selected from Cortex Mori Radicis having improved therapeutic efficacy necessitates increasing research on new assays for screening bioactive compounds with multi-targets. In this work, we applied immobilized β₁-AR and β₂-AR as the stationary phase in chromatographic column to screen bioactive compounds from Cortex Morin Radicis. Specific ligands of the two receptors (e.g. esmolol, metoprolol, atenolol, salbutamol, methoxyphenamine, tulobuterol and clorprenaline) were utilized to characterize the specificity and bioactivity of the columns. We used high performance affinity chromatography coupled with ESI-MS to screen targeted compounds of Cortex Morin Radicis. By zonal elution, we identified morin as a bioactive compound simultaneously binding to β₁-AR and β₂-AR. The compound exhibited the association constants of 3.10 × 10⁴ and 2.60 × 10⁴ M^-1 on the β₁-AR and β₂-AR column. On these sites, the dissociation rate constants were calculated to be 0.131 and 0.097 s^-1. Molecular docking indicated that the binding of morin to the two receptors occurred on Asp200, Asp121, and Val122 of β₁-AR, Asn312, Thr110, Asp113, Tyr316, Gly90, Phe193, Ile309, and Trp109 of β₂-AR. Likewise, mulberroside C was identified as the bioactive compound binding to β₂-AR. The association constants and dissociation rate constants were calculated to be 1.08 × 10⁴ M^-1 and 0.900 s^-1. Molecular docking also indicated that mulberroside C could bind to β₂-AR receptor on its agonist site. Taking together, we demonstrated that the chromatographic strategy to identify bioactive natural products based on the β₁-AR and β₂-AR immobilization, has potential for screening bioactive compounds with multi-targets from complex matrices including traditional Chinese medicines.