Screening of inhibitors on successful covalent tyrosinase coupling with help from SpyBank.

Microbial metabolites are an important source of tyrosinase (TYR) inhibitors because of their rich chemical diversity. However, because of the complex metabolic environment of microbial products, it is difficult to rapidly locate and identify natural TYR inhibitors. Affinity-based ligand screening is an important method for capturing active ingredients in complex samples, but ligand immobilization is an important factor affecting the screening process. In this paper, TYR was used as ligand, and the SpyTag/SpyCatcher coupling system was used to rapidly construct affinity chromatography vectors for screening TYR inhibitors and separating active components from complex samples. We successfully expressed SpyTag-TYR fusion protein and SpyCatcher protein, and incubated SpyCatcher protein with epoxy-activated agarose. The SpyTag-TYR protein was spontaneously coupled with SpyCatcher to obtain an affinity chromatography filler for immobilization of TYR, and the performance of the packaging material was characterized. Finally, compound 1 with enzyme inhibitory activity was successfully obtained from the fermentation product of marine microorganism C. Through HPLC, MS, 1H NMR and 13C NMR analyses, its structure was deduced as azelaic acid, and its activity was analyzed. The results showed that this is a feasible method for screening TYR inhibitors in complex systems.

Fast screening of α-glucosidase inhibitors from Ginkgo biloba leaf by using α-glucosidase immobilized on magnetic metal-organic framework.

In this study, a ligand fishing method for the screening of α-glucosidase inhibitors from Ginkgo biloba leaf was established for the first time using α-glucosidase immobilized on the magnetic metal-organic framework. The immobilized α-glucosidase exhibited enhanced resistance to temperature and pH, as well as good thermal stability and reusability. Two ligands, namely quercitrin and quercetin, were screened from Ginkgo biloba leaf and identified by ultra-high performance liquid chromatography-tandem mass spectrometry. The half-maximal inhibitory concentration values for quercitrin and quercetin were determined to be 105.69 ± 0.39 and 83.49 ± 0.79 µM, respectively. Molecular docking further confirmed the strong inhibitory effect of these two ligands. The proposed approach in this study demonstrates exceptional efficiency in the screening of α-glucosidase inhibitors from complex natural medicinal plants, thus exhibiting significant potential for the discovery of antidiabetic compounds.

Immobilized PAD4 enzyme on magnetic nanoparticles for screening natural inhibitors from traditional Chinese medicines.

Dysregulation of peptidyl arginine deiminase 4 (PAD4) is involved in a variety of diseases including rheumatoid arthritis (RA) and Alzheimer's disease (AD), and it has emerged as potential and promising therapeutic target. However, no PAD4 inhibitor is ready for clinical use. Immobilized enzyme screening technology has gained increasing attention due to its low cost, reusability, easy separation from the reaction mixture, and resistance to changes in environmental conditions. In this study, PAD4 was immobilized on the magnetic nanoparticles (MNP) to prolong its activity stability, and a simple and rapid screening strategy of traditional Chinese medicine inhibitors based on immobilized PAD4 was established. The PAD4 enzyme was immobilized on magnetic nanoparticles (MNP) via Schiff base reaction using glutaraldehyde (GA) as crosslinking agent. Compared with free PAD4, the resulting MNP@GA@PAD4 exhibited an enhanced tolerance to temperature and storage stability, and its reusability was greatly improved with 66 % of initial enzyme activity after being recycled 10 times. The inhibitory activity of the immobilized PAD4 was assessed using two known PAD4 inhibitors GSK484 and BB-Cl-amidine. The semi-maximum inhibitory concentrations (IC50) of GSK484 and BB-Cl-amidine for MNP@GA@PAD4 were 1.00 and 0.97 µM, respectively, for free PAD4 were 0.64 and 0.85 µM, respectively. Finally, the MNP@GA@PAD4 was employed to rapid screen of natural PAD4 inhibitors from forty traditional Chinese medicines (TCMs). Under the same conditions, the controlled experiment was conducted with free PAD4. The screening results of TCMs inhibitors on MNP@GA@PAD4 and free PAD4 were similar, the alcohol extracts of Cinnamomi Cortex and Caryophylli Flos had significant inhibitory effects on PAD4 enzyme activity. The IC50 values of Cinnamomi Cortex extract for MNP@GA@PAD4 and free PAD4 were determined as 27 and 48 µg/mL, respectively. The IC50 values of Caryophylli Flos extracts for MNP@GA@PAD4 and free PAD4 were determined as 48 and 32 µg/mL, respectively. For the first time, this study proposed a method to immobilize PAD4 on magnetic materials, and developed a rapid, reusable and feasible strategy to screening natural PAD4 inhibitors from TCMs.

Xanthine oxidase immobilized cellulose membrane-based colorimetric biosensor for screening and detecting the bioactivity of xanthine oxidase inhibitors.

Xanthine oxidase (XO) is a typical target for hyperuricemia and gout, for which there are only three commercial xanthine oxidase inhibitors (XOIs): febuxostat, topiroxostat and allopurinol. However, these inhibitors have problems such as low bioactivity and several side effects. Therefore, the development of novel XOIs with high bioactivity for the treatment of hyperuricemia and gout is urgently needed. In this work we constructed a XO immobilized cellulose membrane colorimetric biosensor (XNCM) by the TEMPO oxidation, amide bond coupling and nitro blue tetrazolium chloride (NBT) loading method. As expected, the XNCM was able to detect xanthine, with high selectivity and sensitivity by colorimetric method with a distinctive color change from yellow to purple, which can be easily observed by the naked-eye in just 8 min without any complex instrumentation. In addition, the XNCM sensor performed screening of 21 different compounds and have been successfully pre-screened out XOIs with biological activity. Most importantly, the XNCM was able to quantitatively detect the IC50 values of two commercial inhibitors (febuxostat and allopurinol). All the results confirmed that the XNCM is a simple and effective tool which can be used for the accelerated screening of XOIs and has the potential to uncover additional XOIs.

A magnetic beads-based ligand fishing method for rapid discovery of monoterpene indoles as monoamine oxidase A inhibitors from Hunteria zeylanica.

In this study, a novel magnetic bead-based ligand fishing method was developed for rapid discovery of monoterpene indoles as monoamine oxidase A inhibitors from natural products. In order to improve the screening efficiency, two different magnetic beads, i.e. amine and carboxyl terminated magnetic beads, were comprehensively compared in terms of their ability to immobilize monoamine oxidase A (MAOA), biocatalytic activity and specific adsorption rates for affinity ligands. Carboxyl terminated magnetic beads performed better for MAOA immobilization and demonstrated superior performance in ligand fishing. The MAOA immobilized magnetic beads were applied to screen novel monoamine oxidase inhibitors in an alkaloid-rich plant, Hunteria zeylanica. Twelve MAOA affinity ligands were screened out, and ten of them were identified as monoterpene indole alkaloids by HPLC-Obitrap-MS/MS. Among them, six ligands, namely geissoschizol, vobasinol, yohimbol, dihydrocorynanthenol, eburnamine and (+)-isoeburnamine which exhibited inhibitory activity against MAOA with low IC50 values. To further explore their inhibitory mechanism, enzyme kinetic analysis and molecular docking studies were conducted.