As a complement to vaccines, small-molecule therapeutic agents are needed to treat or prevent infections by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and its variants, which cause COVID-19. Affinity selection–mass spectrometry was used for the discovery of botanical ligands to the SARS-CoV-2 spike protein. Cannabinoid acids from hemp ( Cannabis sativa ) were found to be allosteric as well as orthosteric ligands with micromolar affinity for the spike protein. In follow-up virus neutralization assays, cannabigerolic acid and cannabidiolic acid prevented infection of human epithelial cells by a pseudovirus expressing the SARS-CoV-2 spike protein and prevented entry of live SARS-CoV-2 into cells. Importantly, cannabigerolic acid and cannabidiolic acid were equally effective against the SARS-CoV-2 alpha variant B.1.1.7 and the beta variant B.1.351. Orally bioavailable and with a long history of safe human use, these cannabinoids, isolated or in hemp extracts, have the potential to prevent as well as treat infection by SARS-CoV-2.

Invented to address the high‐throughput screening (HTS) demands of combinatorial chemistry, affinity selection–mass spectrometry (AS‐MS) utilizes binding interactions between ligands and receptors to isolate pharmacologically active compounds from mixtures of small molecules and then relies on the selectivity, sensitivity, and speed of mass spectrometry to identify them. No radiolabels, fluorophores, or chromophores are required. Although many variations of AS‐MS have been devised, three approaches have emerged as the most flexible, productive, and popular, and they differ primarily in how ligand–receptor complexes are separated from nonbinding compounds in the mixture. These are pulsed ultrafiltration (PUF) AS‐MS, size exclusion chromatography (SEC) AS‐MS, and magnetic microbead affinity selection screening (MagMASS). PUF and SEC AS‐MS are solution‐phase screening approaches, and MagMASS uses receptors immobilized on magnetic microbeads. Because pools of compounds are screened using AS‐MS, each containing hundreds to thousands of potential ligands, hundreds of thousands of compounds can be screened per day. AS‐MS is also compatible with complex mixtures of chemically diverse natural products in extracts of botanicals and fungi and microbial cultures, which often contain fluorophores and chromophores that can interfere with convention HTS. Unlike conventional HTS, AS‐MS may be used to discover ligands binding to allosteric as well as orthosteric receptor sites, and AS‐MS has been useful for discovering ligands to targets that are not easily incorporated into conventional HTS such as membrane‐bound receptors.

Affinity selection-mass spectrometry, which includes magnetic microbead affinity selection-screening (MagMASS), is ideal for the discovery of ligands in complex mixtures that bind to pharmacological targets. Therapeutic agents are needed to prevent or treat COVID-19, which is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Infection of human cells by SARS-CoV-2 involves binding of the virus spike protein subunit 1 (S1) to the human cell receptor angiotensin converting enzyme-2 (ACE2). Like antibodies, small molecules have the potential to block the interaction of the viral S1 protein with human ACE2 and prevent SARS-CoV-2 infection. Therefore, a MagMASS assay was developed for the discovery of ligands to the S1 protein. Unlike previous MagMASS approaches, this new assay used robotics for 5-fold enhancement of throughput and sensitivity. The assay was validated using the SBP-1 peptide, which is identical to the ACE2 amino acid sequence recognized by the S1 protein, and then applied to the discovery of natural ligands from botanical extracts. Small molecule ligands to the S1 protein were discovered in extracts of the licorice species, Glycyrrhiza inflata. In particular, the licorice ligand licochalcone A was identified through dereplication and comparison with standards using HPLC with high-resolution tandem mass spectrometry.

The Special Feature this month is jointly authored by Drs. Ruth Muchiri and Richard van Breemen, both of the Linus Pauling Institute, Oregon State University. Their article describes affinity selection–mass spectrometry (AS‐MS) and illustrates how this general approach can be used to assess the binding of candidate molecules to immobilized or soluble receptors. AS‐MS is a family of MS‐based affinity methods that allow the screening of complex natural product extracts or combinatorial libraries without radiolabels or chromophores. The approach offers substantial improvements in speed and convenience over more conventional screening approaches and because pools of compounds can be screened, each containing hundreds to thousands of potential ligands, hundreds of thousands of compounds can be screened per day. The combination of affinity selection and mass spectrometry is a powerful tool for the isolation, characterization and identification of the active constituents of complex mixtures and this clearly written and well‐illustrated Special Feature is an excellent introduction to AS‐MS and its potential applications.

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