At-Line Cellular Screening Methodology for Bioactives in Mixtures Targeting the α7-Nicotinic Acetylcholine Receptor

Abstract

The α7-nicotinic acetylcholine receptor (α7-nAChR) is a ligand-gated ion channel expressed in different regions of the central nervous system (CNS). The α7-nAChR has been associated with Alzheimer's disease, epilepsy, and schizophrenia, and therefore is extensively studied as a drug target for the treatment of these diseases. Important sources for new compounds in drug discovery are natural extracts. Since natural extracts are complex mixtures, identification of the bioactives demands the use of analytical techniques to separate a bioactive from inactive compounds. This study describes screening methodology for identifying bioactive compounds in mixtures acting on the α7-nAChR. The methodology developed combines liquid chromatography (LC) coupled via a split with both an at-line calcium (Ca 2+ )-flux assay and highresolution mass spectrometry (MS). This allows evaluation of α7-nAChR responses after LC separation, while parallel MS enables compound identification. The methodology was optimized for analysis of agonists and positive allosteric modulators, and was successfully applied to screening of the hallucinogen mushroom Psilocybe Mckennaii. The crude mushroom extract was analyzed using both reversed-phase and hydrophilic interaction liquid chromatography. Matching retention times and peak shapes of bioactives found with data from the parallel MS measurements allowed rapid pinpointing of accurate masses corresponding to the bioactives.

Liquid chromatographic nanofractionation with parallel mass spectrometric detection for the screening of plasmin inhibitors and (metallo)proteinases in snake venoms

“…As chromatographic resolution is essentially maintained, the results of the bioassay readout can be constructed into a bioactivity chromatogram, which can be accurately correlated with the parallel obtained MS chromatogram. Besides venom research, the nanofractionation technique also has shown to be useful for drug-drug interaction profiling [ 23 ], environmental analysis [ 28 ], and profiling natural extracts [ 29 ].…”

Bioactivity Profiling of Small-Volume Samples by Nano Liquid Chromatography Coupled to Microarray Bioassaying Using High-Resolution Fractionation

“…In order to address this limitation, an orthogonal separation using two different separation mechanisms (e.g., RP and HILIC) can be applied and was in fact demonstrated by us and others. 6,7,38 Similar approaches are anticipated to be applied to the picofractionation platform, however, at later stages of its development.…”

Antivenom Neutralization of Coagulopathic Snake Venom Toxins Assessed by Bioactivity Profiling Using Nanofractionation Analytics

“…The nanofractionation analytics described herein, therefore, serves as a valuable complementary antivenomics tool in antivenom research. Furthermore, such analytical approaches are also useful for studying environmental pollutants, plant extracts, and metabolic mixtures [38][39][40]. Ultimately, our data on antivenom neutralization can guide future research on the development of next generation antivenoms for treating snakebite.…”