Magnetic Microbead Affinity Selection Screening (MagMASS) of Botanical Extracts for Inhibitors of 15-Lipoxygenase

Abstract

To expedite the identification of active natural products in complex mixtures such as botanical extracts, a Magnetic Microbead Affinity Selection Screening (MagMASS) procedure was developed. This technique utilizes target proteins immobilized on magnetic beads for rapid bioaffinity isolation of ligands from complex mixtures. A MagMASS method was developed and validated for 15-lipoxygenase. As a proof of concept, several North American prairie plants used medicinally by Native Americans were extracted with MeOH and screened. A hit from an extract of Proserpinaca palustris, also known as mermaid weed, was flagged for further characterization using high-resolution tandem mass spectrometry, dereplication, and identification using XCMS online. Through the application of high-resolution product ion tandem mass spectrometry, comparison with natural product databases and confirmation using standards, the hit was identified as quercitrin, which is a known inhibitor of 15-lipoxygenase. The overall workflow of MagMASS is faster and more amendable to automation than alternative methods designed for screening botanical extracts or complex mixtures of combinatorial libraries.

Affinity selection–mass spectrometry for the discovery of pharmacologically active compounds from combinatorial libraries and natural products

“…Rush et al 56 used MagMASS to screen a series of North American prairie plants for inhibitors of the anti‐inflammation target 15‐lipoxygenase 45 . This work was significant in that it described the application of mass spectrometry metabolomics software (in this case XCMS online, Scripps Institute) 57 to facilitate the automation of AS‐MS data processing.…”

“…54 In a variation of MagMASS, solution-phase receptors were incubated with a mixture of ligands and then immobilized on magnetic microbeads for separation from the unbound compounds. 55 Rush et al 56 used MagMASS to screen a series of North American prairie plants for inhibitors of the anti-inflammation target 15-lipoxygenase. 45 This work was significant in that it described the application of mass spectrometry metabolomics software (in this case XCMS online, Scripps Institute) 57 to facilitate the automation of AS-MS data processing.…”

An Integrated Strategy for the Detection, Dereplication, and Identification of DNA-Binding Biomolecules from Complex Natural Product Mixtures

“…Whereas a detailed review of drug discovery applications using ligand fishing is beyond the scope of this discussion, , it is worth noting that this conceptual approach has been interpreted and applied in diverse ways, including within the field of natural products. ,− Notwithstanding these accounts, we noted the relative dearth of reports pertaining to the utilization of DNA as a biological target in ligand-fishing-based studies despite its favorable history as a focal point for biomedical interventions. − Considering our group’s collaborative interests in the discovery of natural products that selectively inhibit different types of pediatric and triple-negative breast cancers, − we found this point intriguing given the expansive role that DNA-targeting agents have played in the field of cancer chemotherapy. − For these reasons, our goal was to develop a ligand-fishing system that could be used for the detection and identification of DNA-binding molecules from natural product mixtures. In this report, we describe our efforts to implement a natural product drug discovery pipeline using an ultrafiltration-based assay system linked with hyphenated mass spectrometry to seamlessly detect, dereplicate, and, in some cases, identify compounds from complex mixtures of natural products.…”

Solving the Competitive Binding Equilibria between Many Ligands: Application to High-Throughput Screening and Affinity Optimization

“…Indirect AS–MS methods involve a four-step process (Figure ): (1) incubation of a target biomolecule, such as a protein, with a pool of small molecules, often hundreds or thousands; (2) separation of nonbinding compounds from the protein–ligand complexes; (3) dissociation of the protein–ligand complexes, and (4) identification of the ligands by MS. The different AS–MS methods mainly differ in how the protein–ligand complexes and the small molecules in solution are separated, some approaches include ultrafiltration, − immobilized protein AS, , magnetic beads, , and size-exclusion chromatography. − …”

“…(3) dissociation of the protein−ligand complexes, and (4) identification of the ligands by MS. The different AS−MS methods mainly differ in how the protein−ligand complexes and the small molecules in solution are separated, 15 some approaches include ultrafiltration, 16−18 immobilized protein AS, 19,20 magnetic beads, 21,22 and size-exclusion chromatography. 23−31 Optimizing experimental conditions to distinguish highaffinity ligands from low-affinity ones is an essential step in AS−MS screening.…”