Rapid screening of neuraminidase inhibitors with the benzoic acid skeleton from Paeonia suffruticosa Andrews by solid-phase extraction with an enzyme activity switch combined with mass spectrometry analysis

Highlights

• Solid-phase extraction with an enzyme activity switch was proposed.

• Quick screening of bioactive compounds with specific skeleton was realized.

• The separation, detection and collection were performed on one instrument system.

Abstract

It is meaningful for drug discovery to discover lead compounds with specific skeletons from medicinal herbs. Screening bioactive compounds with specific skeletons by a simple and rapid strategy is still a challenging task. Solid-phase extraction (SPE) is a simple and time-saving technique in the laboratory and is often used in the concentration of natural products. It is attractive to apply the SPE in the screening of bioactive compounds with specific skeletons. To achieve this goal, SPE with an enzyme activity (EA) switch combined with mass spectrometry analysis was first proposed. The screening of benzoic acid-derived neuraminidase (NA) inhibitors from the root cortex of Paeonia suffruticosa Andrews (CPSA) was used as an example. The NA and crude extract of CPSA were incubated to form a sample solution. Subsequently, the sample was separated, detected, and collected by the SPE with an EA switch. When the detected values reduced significantly, the EA switch was triggered, and the collection was stopped. The collected eluents were treated for LC-MS/MS analysis. Finally, combining diagnostic ions and mass spectrometry data, two benzoic acid NA inhibitors were successfully screened from CPSA. In this study, the separation, detection, and collection were performed on one instrument system. Compared with the traditional isolation strategy, this strategy with the simpler operation and higher experimental efficiency could be an effective tool for the rapid screening of lead compounds with specific skeletons.

Introduction

Finding active ingredients from natural herbs is a very important way of drug development [1,2]. The bioactivity and draggability of compounds are closely related to the skeleton structures [3,4]. The discovery of active compounds with specific skeletons can reduce the blindness of lead compound discovery and improve the efficiency of drug development. The traditional method for the finding of specific skeleton compounds with bioactivities was isolation followed by biological assays. This strategy has unique advantages in the identification of bioactive compounds with certain skeletons and unreported skeletons [2,[4], [5], [6]]. However, it contains many steps, including sample extraction, isolation, purification, structure identification, and biological assays. It was not suitable for rapid screening. A simpler and faster strategy is urgently needed in the screening of bioactive compounds with certain skeletons. As a common sample processing technique in the laboratory, solid-phase extraction (SPE) is often used in the accumulation of natural products [7], [8], [9], [10], because of its simple and rapid operations. Considering this, it is attractive to apply the SPE in the screening of bioactive compounds with specific skeletons from natural resources.

As an illustrative example, neuraminidase (NA) and the root cortex of Paeonia suffruticosa Andrews (CPSA) were used as the biotarget and medicinal herb to screen the NA inhibitors with the specific skeleton. NA is a tetramer glycoprotein on the envelope of the influenza virus and plays an important role in the transmission of the virus [11], [12], [13]. One of the research hotspots of NA inhibitors is benzoic acid-derived compounds because of their simple synthesis process, simple stereochemical structure, and high bioavailability [14], [15], [16], [17]. Benzoic acid-derived inhibitors (BAIs) of NA are mainly chemo-synthesized molecules based on rational drug design, and limited numbers have been discovered from natural herbs. CPSA is a well-known medicinal herb for its effects of activating blood circulation, dissipating blood stasis, curing carbuncle and sore poison [18,19].

In this study, the SPE with an EA switch combined with mass spectrometry analysis was proposed (Fig. 1). The enzyme was incubated with the crude extract solution forming enzyme-inhibitor complexes at first. The SPE was used to separate the sample solution based on polarities. The EA switch was designed to detect and collect the fractions containing enzyme-inhibitor complexes and could exclude most of the non-target compounds. And the mass spectrometry analysis was used to pick out the specific skeleton inhibitors from the collected fractions.

As a proof-of-concept, the BAIs of NA were obtained from CPSA in three steps. Firstly, NA and CPSA crude extracts were incubated to get the sample solution containing two types of enzyme-inhibitor complexes (NA-BAIs and NA-Is), NA, and small molecules with different polarities (LP1, LP2, SP). The enzyme-inhibitor complexes and enzymes are with large polarity and could dissolve in the mobile phase. Next, the sample was separated, detected, and collected by the SPE with an EA switch. Molecules with large polarity (NA-BAIs, NA-Is, NA, LP1) were eluted first, and they were detected and collected simultaneously by the EA switch. When the detected values reduced significantly, the six-way valve of the EA switch was triggered to end the collection process. The medium and small polarity compounds (LP2, SP) flowed into the waste bottle. The collected eluents were treated for liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) analysis. Finally, the fragment pathways of benzoic acid-derived standard compounds were used as the guidance for the pinpointing of BAIs from the collected eluents. Two natural benzoic acid derived NA inhibitors were successfully identified. In this study, the complicated isolation and purification were avoided, and the separation, detection, and collection were carried out on one instrument system. In addition to the traditional use of SPE, we first applied the SPE with an EA switch combined with mass spectrometry analysis in finding the bioactive compounds with specific skeletons.