Immobilization of M3 Muscarinic Receptor to Rapidly Analyze Drug-Protein Interactions and Bioactive Components in a Natural Plant.

Despite its increasing application in pursing potential ligands, the capacity of receptor affinity chromatography is greatly challenged as most current research studies lack a comprehensive characterization of the ligand-receptor interaction, particularly when simultaneously determining their binding thermodynamics and kinetics. This work developed an immobilized M3 muscarinic receptor (M3R) affinity column by fixing M3R on amino polystyrene microspheres via the interaction of a 6-chlorohexanoic acid linker with haloalkane dehalogenase. The efficiency of the immobilized M3R was tested by characterizing the binding thermodynamics and kinetics of three known drugs to immobilized M3R using a frontal analysis and the peak profiling method, as well as by analyzing the bioactive compounds in Daturae Flos (DF) extract. The data showed that the immobilized M3R demonstrated good specificity, stability, and competence for analyzing drug-protein interactions. The association constants of (-)-scopolamine hydrochloride, atropine sulfate, and pilocarpine to M3R were determined to be (2.39 ± 0.03) × 104, (3.71 ± 0.03) × 104, and (2.73 ± 0.04) × 104 M-1, respectively, with dissociation rate constants of 27.47 ± 0.65, 14.28 ± 0.17, and 10.70 ± 0.35 min-1, respectively. Hyoscyamine and scopolamine were verified as the bioactive compounds that bind to M3R in the DF extract. Our results suggest that the immobilized M3R method was capable of determining drug-protein binding parameters and probing specific ligands in a natural plant, thus enhancing the effectiveness of receptor affinity chromatography in diverse stages of drug discovery.

Immobilization of peroxisome proliferator-activated receptor gamma and the application in screening modulators of the receptor from herbal medicine.

Screening and identification of potential compounds from herbal medicine is a prevailing way to find a lead for the development of innovative drugs. This promotes the development of new methods that are feasible in complex matrices. Here, we described a one-step reversible methodology to immobilize nuclear peroxisome proliferator-activated receptor gamma (PPARγ) onto amino microsphere coated with a DNA strand specifically binding to the receptor. The specific interaction allowed us to achieve the immobilization of PPARγ by mixing the DNA modified microspheres with E. coli lysates expressing the receptor. Characterization of the immobilized receptor was carried out by morphology and binding specificity analysis. Feasibility of immobilized PPARγ in the drug-receptor interaction analysis was performed by an injection amount-dependent method. Besides, immobilized PPARγ was also applied in screening modulators of the receptor from Coptidis Rhizoma extract. The binding of the screened compounds to PPARγ was examined by time-resolved fluorescence resonance energy transfer assay. The results showed that immobilized PPARγ was stable for thirty days with a high-specificity of ligand recognition at the subtype receptor level. Berberine and palmatine were the bioactive compounds of Coptidis Rhizoma specifically binding to PPARγ. The two compounds exhibited half maximal inhibitory concentrations of 4.11 and 2.98 µM during their binding to the receptor. We concluded that the current method is possible to become a common strategy for the immobilization of nuclear receptors, and the immobilized receptor is a high throughput method for recognizing and separating the receptor modulators from complex matrices including herbal medicine.

Preparation and characterization of immobilized 5-HT1A receptor stationary phase for high throughput screening of the receptor-binding ligands from complex systems like Curcuma wenyujin Y. H. Chen et C. Ling extract.

The incidence of depression has increased significantly during the COVID-19 pandemic. This disease is closely associated with serotonin 1A (5-HT1A) receptor and often treated by complex prescription containing Curcuma wenyujin Y. H. Chen et C. Ling. Therefore, we hypothesized that this herb contains bioactive compounds specially binding to the receptor. However, the rapid discovery of new ligands of 5-HT1A receptor is still challenging due to the lack of efficient screening methods. To address this problem, we developed and characterized a novel approach for the rapid screening of ligands by using immobilized 5-HT1A receptor as the chromatographic stationary phase. Briefly, haloalkane dehalogenase was fused at the C-terminal of 5-HT1A receptor, and the modified 5-HT1A receptor was immobilized on amino-microspheres by the reaction between haloalkane dehalogenase and 6-chlorohexanoic acid linker. Scanning electron microscope and X-ray photo-electron were used to characterize the morphology and element of the immobilized receptor. The binding of three specific ligands to 5-HT1A receptor was investigated by two different methods. Moreover, we examined the feasibility of 5-HT1A receptor colume in high throughput screening of new ligands from complex systems as exemplified by Curcuma wenyujin Y. H. Chen et C. Ling. Gweicurculactone, 2-hydroxy-1-(3,4-dihydroxybenzene)-7-(4'-hydroxybezene)-heptane and curcuminol F were identified as the ligands of 5-HT1A receptor with the binding energies of -7.06 kcal/mol, -7.77 kcal/mol and -5.26 kcal/mol, respectively. Collectively, these results indicated that the immobilized 5-HT1A receptor was capable of screening bioactive compound from complex system, providing an effective methodology for high throughput screening.

Immobilized beta2-adrenergic receptor: A powerful chromatographic platform for drug discovery and evaluation of drug-like property for natural products.

Drug discovery based on natural products like medicinal herbs remains challenging due to the technique limitations for rapidly screening and validating leads. To address the challenges, we employ the immobilized ß2- adrenergic recepotor (ß2-AR), an identified target of asthma, as the stationary phase in chromatographic column to screen compounds extracted from Stemonae Radix, Playtycodonis Radix, and Glycyrrhizae Radix et Rhizoma. To analyze binding properties of the extracted compounds to the immobilized receptors, we measured their retention behavior in the receptor chromatography and compared with six clinical asthma drugs. We identified tuberostemonine, platycodin D, and glycyrrhizic acid as the potential leads against asthma by our ß2-AR chromatography coupled with mass spectrum (MS). The association constants of the three compounds to ß2-AR were 2.85 × 10-5, 2.55 × 10-4, and 4.07 × 10-6 M with the dissociation rate constants of 6.91 ± 0.35, 11.88 ± 0.60, and 9.49 ± 0.64 min-1, respectively. Tuberostemonine, a pentacyclic Stemona alkaloids, presented the most optimum values of binding efficiency index (BEI) and surface efficiency index (SEI) as close to the diagonal of SEI-BEI optimization plane when it is compared with platycodin D, glycyrrhizic and the six clinical drugs. Our results suggest that tuberostemonine is a promising natural product to be developed for treating asthma because it exhibits better drug-like binding properties to ß2-AR than the clinical drugs. As such, we demonstrate a chromatographic strategy to identify bioactive natural products based on the ß2-AR immobilization, which can be widely adopted to screen natural products from mixture of herbal extracts.