A strategy for screening trypsin inhibitors from traditional Chinese medicine based on a monolithic capillary immobilized enzyme reactor coupled with offline liquid chromatography and mass spectrometry

Abstract

† These authors contributed equally to this work.A novel strategy was successfully developed for screening trypsin inhibitors in traditional Chinese medicines based on monolithic capillary immobilized enzyme reactors combined with liquid chromatography-tandem mass spectrometry. Organic polymer based monolithic enzyme reactors were firstly prepared by covalently bonding trypsin to a poly(glycidyl methacrylate-co-poly (ethylene glycol) diacrylate) monolith by the ring-opening reaction of epoxy groups. The activity and kinetic parameters of the obtained monolithic trypsin reactors were systematically evaluated using microliquid chromatography. Fourier transform infrared spectroscopy and scanning electron microscopy were also used to characterize the monolithic trypsin reactors. The resulting functional and denatured monolithic trypsin reactors were applied as affinity solidphase extraction columns, and offline coupled with a liquid chromatography-tandem mass spectrometry system to construct a binding affinity screening platform. Subsequently, the proposed platform was applied for screening trypsin binders in a Scutellaria baicalensis Georgi extract. Three compounds, namely scutellarin, baicalin, and wogonoside were identified, and their inhibitory activities were further confirmed via an in vitro enzymatic inhibition assay. Additionally, molecular docking was also performed to study the interactions between trypsin and these three compounds.

Deviation of Trypsin Activity Using Peptide Conformational Imprints

“…As shown in Table 2, among all the PPT/PCIMPs, PPT/PCIMPs 233-245+G had the best kinetic parameters. The K m value of PPT (0.36 mM) was almost similar to that of the PPT/PCIMPs 233-245+G (0.42 mM), which could be due to the high feasibility of forming an enzyme-substrate complex, and also a lower diffusion restraint imposed on the flow of the substrate and product molecules from the grafted polymer matrix of the MPs [43,44]. The V max values were found to be 3.2 × 10 -3 mMs −1 and 1.47 × 10 -3 mMs −1 for PPT and PPT/PCIMPs 233-245+G , in which the V max was decreased for PPT/PCIMPs when compared to the free enzyme.…”

Recent advances on discovery of enzyme inhibitors from natural products using bioactivity screening

“…This approach has been widely used in active compounds screening fields. Lin et al [68] developed a new affinity monolith chromatography method to screen trypsin inhibitors from the extract of Scutellaria baicalensis Georgi. In their work, an immobilized enzyme reactor combining SPE was proposed for the first time.…”

Affinity monolith chromatography: A review of general principles and recent developments

“…The most frequently used organic monoliths in AMC are based on copolymers of glycidyl methacrylate (GMA) and ethylene glycol dimethacrylate (EDMA) [19,44,50,61–65]. GMA/EDMA monoliths can be readily synthesized and are commercially available under the trade name “convective interaction media” (CIM) [15,20,44].…”

“…AMC can be utilized to capture solutes and screen for targets that bind to enzymes that have been placed in monoliths. For example, dihydrofolate reductase in a silica monolith has been used to detect inhibitors for this enzyme [90], and screening of trypsin inhibitors has been achieved by integrating a GMA/EDMA monolithic trypsin reactor offline with LC‐MS [65]. However, it is more common for an enzyme to be used in a monolith to create an IMER for biocatalysis [100,228–231].…”