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. 2022 Aug 5:e21800. Online ahead of print. doi: 10.1002/mas.21800

Affinity selection‐mass spectrometry in the discovery of anti‐SARS‐CoV‐2 compounds

Richard B van Breemen 1,✉, Ruth N Muchiri 1
PMCID: PMC9538385  PMID: 35929396

Abstract

Small molecule therapeutic agents are needed to treat or prevent infections by severe acute respiratory syndrome coronavirus‐2 (SARS‐CoV‐2), which is the cause of the COVID‐19 pandemic. To expedite the discovery of lead compounds for development, assays have been developed based on affinity selection‐mass spectrometry (AS‐MS), which enables the rapid screening of mixtures such as combinatorial libraries and extracts of botanicals or other sources of natural products. AS‐MS assays have been used to find ligands to the SARS‐CoV‐2 spike protein for inhibition of cell entry as well as to the 3‐chymotrypsin‐like cysteine protease (3CLpro) and the RNA‐dependent RNA polymerase complex constituent Nsp9, which are targets for inhibition of viral replication. The AS‐MS approach of magnetic microbead affinity selection screening has been used to discover high‐affinity peptide ligands to the spike protein as well as the hemp cannabinoids cannabidiolic acid and cannabigerolic acid, which can prevent cell infection by SARS‐CoV‐2. Another AS‐MS method, native mass spectrometry, has been used to discover that the flavonoids baicalein, scutellarein, and ganhuangenin, can inhibit the SARS‐CoV‐2 protease 3CLpro. Native mass spectrometry has also been used to find an ent‐kaurane natural product, oridonin, that can bind to the viral protein Nsp9 and interfere with RNA replication. These natural lead compounds are under investigation for the development of therapeutic agents to prevent or treat SARS‐CoV‐2 infection.

Keywords: affinity selection‐mass spectrometry, COVID‐19, drug discovery, natural products, SARS‐CoV‐2

1. INTRODUCTION

1.1. Affinity selection‐mass spectrometry (AS‐MS) and high‐throughput screening

The development of combinatorial chemistry in the 1980s and 1990s enabled the rapid parallel synthesis of thousands of compounds (Furka, 2018). This contributed to the assembly of combinatorial libraries of hundreds of thousands of molecules for screening as a first step in drug discovery. To keep pace with the need to identify pharmacologically active compounds within such large combinatorial libraries, new high‐throughput screening approaches were required. One approach utilized the selectivity, speed, and sensitivity of MS to screen pools of combinatorial libraries for compounds that bind to a pharmacological target. Another screening approach assayed one compound at a time for binding to a receptor or inhibition of an enzymatic reaction and usually used optical readouts such as fluorescence or absorbance or, less frequently, MS.

Each screening approach has unique advantages or limitations (Table 1). For example, assaying mixtures of compounds is faster and consumes less reagents than screening each compound separately. Furthermore, screening extracts of botanical, fungal, or microbial extracts containing complex mixtures of natural products enables the discovery of compounds with unique chemical structures that are not represented in combinatorial libraries. However, mixture screening requires an extra identification step that is not required when screening discreet compounds. Although deconvoluting the hit in a mixture of combinatorial library compounds is fast and can be automated using MS (especially high‐resolution MS), this identification step becomes more challenging when the assay hit is a new chemical entity from a natural source. Driven by the pharmaceutical industry, high‐throughput screening of discreet compounds became the most popular drug discovery approach. Meanwhile, MS‐based mixture screening continued to evolve in academic laboratories and some pharmaceutical companies and continues to grow in popularity.

Table 1.

Comparison of mass spectrometry‐based approaches and optical readout high‐throughput screening approaches for the discovery of anti‐SARS‐CoV‐2 compounds.

Feature MagMASS Native MS screening Conventional HTS
Combinatorial libraries Yes Yes Yes
Compounds per assay <1,000,000 <1000 Usually 1
Reagent consumption Low Low High
Labeled reagents (fluorophore, chromophore, radioisotope) No No Yes
Compatible with fluorescent or UV chromophores Yes Yes No
Chromatographic step Optional No No
Allosteric ligand screening Yes Yes No
Compatible with enzyme inhibition screening Yes Yes Yes
Compatible understudied targets Yes Yes No
Ranking of ligands by affinity to receptor Yes Yes No
Solution‐phase receptor screening No Yes Yes
Solid‐phase receptor screening Yes No Yes
Allows receptor reuse Yes No No
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Abbreviations: HTS, high‐throughput screening; MagMASS, magnetic microbead affinity selection screening.

MS‐based drug discovery methods that utilize an affinity separation step are suitable for screening complex mixtures of compounds including natural product extracts as well as combinatorial libraries (van Breemen, 2020). These methods involve the binding of ligands to a pharmacological receptor as the affinity interaction followed by the isolation of ligand–receptor complexes from the unbound inactive compounds in the mixture. MS is then used to characterize and identify the affinity‐extracted ligands. These MS‐based approaches are known as AS‐MS. Unlike high‐throughput screening assays based on fluorescence or absorption spectrophotometry, AS‐MS enables the detection of allosteric ligands and is compatible with natural products, which often contain strong fluorophores and chromophores (Table 1).

AS‐MS approaches include affinity column‐MS, affinity capillary electrophoresis‐MS, frontal affinity chromatography‐MS, size exclusion chromatography liquid chromatography‐MS (LC‐MS), pulsed ultrafiltration MS, native MS screening (also known as bio‐affinity characterization MS and direct affinity selection‐MS), and magnetic microbead affinity selection MS screening (MagMASS). AS‐MS uses the solution‐phase MS ionization techniques of electrospray or atmospheric pressure chemical ionization, which are compatible with on‐line LC and electrophoresis. Some AS‐MS approaches such as frontal affinity chromatography‐MS, size exclusion chromatography LC‐MS, and capillary electrophoresis‐MS require an on‐line separation step, while chromatographic separation is optional for pulsed ultrafiltration MS and MagMASS. Note that no chromatography or electrophoresis is used during native MS screening.

In the search for therapeutic agents against COVID‐19, which is caused by the severe acute respiratory syndrome‐coronavirus‐2 (SARS‐CoV‐2) (Figure 1), two AS‐MS approaches have been used to date, MagMASS (Muchiri et al., 2022; Pomplun et al., 2021) (Figure 2) and native MS screening (Littler, Liu, et al., 2021; Zhu et al., 2022) (Figure 3). Both approaches have been used in academic laboratories to find natural product inhibitors of SARS‐CoV‐2 infection and replication.

Figure 1.

Figure 1

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SARS‐CoV‐2 infection and replication cycle in the host cell. Viral cell entry and fusion begins with binding of the viral spike protein to the host cell surface protein ACE2. The release of genomic viral RNA is facilitated by the cellular enzyme TMPRSS2, and viral RNA is translated into polypeptides, pp1a and pp1ab, which are cleaved by viral proteases 3CLpro and PLpro into subunits that form the RNA‐dependent RNA polymerase (RdRp). Replication and translation of viral RNA results in the synthesis of components that are assembled into a functional virion, which exits the cell through exocytosis. Targets for pharmacological intervention include prevention of cell entry and fusion, inhibition of viral proteases, inhibition of RNA replication, and inhibition of capsid assembly and virion exocytosis.

Figure 2.

Figure 2

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Magnetic microbead affinity selection screening (MagMASS) applied to the discovery of ligands to the SARS‐CoV‐2 spike protein. After immobilization of the spike protein S1 subunit on magnetic microbeads, the immobilized receptor was incubated with a botanical extract containing potential ligands and allowed to reach equilibrium. A magnet was used to capture the magnetic microbeads containing the ligand–receptor complexes for washing with binding buffer to remove unbound compounds, and then organic solvent was used to denature the S1 protein and release the ligands for analysis using UHPLC‐MS. (Reproduced with permission from Muchiri et al., 2022.). UHPLC‐MS, ultrahigh performance liquid chromatography–tandem mass spectrometry.

Figure 3.

Figure 3

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Native MS screening applied to the discovery of inhibitors of SARS‐CoV‐2 replication. After incubation of s solution‐phase receptor such as 3CLpro or Nsp9 with a mixture of potential ligands in a low molarity volatile aqueous buffer (such as 10 mM ammonium acetate), the receptor–ligand complexes are desorbed and measured using electrospray mass spectrometry on an ultrahigh resolution mass spectrometer. During electrospray mass spectrometry, high mass receptor ions are detected as a series of multiply charged species (black), and ligand‐receptor complexes appear as a series of multiply charged peaks of slightly higher m/z values corresponding to the receptor plus the attached ligand (red). (Based on Pedro & Quinn, 2016). MS, mass spectrometry.

1.2. SARS‐CoV‐2 therapeutic targets for drug discovery

The COVID‐19 pandemic includes at least 425 million cases worldwide, 6.3 million deaths, and over 600,000 new cases daily as of June 2022 (Worldometer, 2022). Although vaccines are available, incomplete immunization and emerging variants of concern suggest that SARS‐CoV‐2 infections are likely to continue indefinitely. Therefore, new drugs are needed that may be used to treat or prevent SARS‐CoV‐2 infections.

SARS‐CoV‐2 is an RNA coronavirus with characteristic spike protein trimers on the outer surface (Figure 1) (Huang et al., 2020; Tahir ul Qamar et al., 2020). The RNA of SARS‐CoV‐2 encodes 4 main structural proteins (including the spike), 16 nonstructural proteins, and several accessory proteins (Jiang et al., 2020). Any step of the SARS‐CoV‐2 virus infection and replication cycle is a potential target for antiviral intervention including cell entry, genome replication, viral maturation, or viral release (Figure 1). AS‐MS screening assays have been reported for the discovery of potential SARS‐CoV‐2 therapeutic agents targeting cell entry (Muchiri et al., 2022), genome replication (Littler, Liu, et al., 2021), and protein processing (Zhu et al., 2022).

Cell entry of SARS‐CoV‐2 begins with the binding of the viral spike protein to the human cell surface protein angiotensin‐converting enzyme‐2 (ACE2), which is abundant on human endothelial cells in the lungs and cardiovascular system (Turner, 2015). The spike protein forms a homotrimer, and each monomer (~150 kDa) contains an S1 subunit that binds to ACE2, an S2 subunit that mediates virus fusion with the cell, and a transmembrane domain (Figure 1). After binding of the S1 subunit to ACE2, TMPRSS2 protease on the human cell membrane cleaves the spike protein at the S1/S2 and S2 sites, resulting in conformational changes that enable SARS‐CoV‐2 to enter the host cell (Hoffmann et al., 2020). AS‐MS assays have been developed to discover ligands to the S1 subunit as possible cell entry inhibitors (Muchiri et al., 2022; Pomplun et al., 2021).

Following cell entry, the viral genetic material is released and translated by the ribosomes of the host cell to produce polyproteins (pp1a and pp1ab) that require hydrolysis into structural and functional viral proteins (Figure 1). This step is catalyzed by the viral proteases 3C‐like protease (3CLpro) and the papain‐like protease (PLpro). 3CLpro is a 33.8 kDa homodimeric cysteine protease that catalyzes the hydrolysis of the viral polyprotein at Leu‐Gln* (Ser, Ala, Gly) (*marks the cleavage site) to release the nonstructural proteins Nsp5‐16 (Moeller et al., 2022). Because these proteins are important for viral replication, inhibition of 3CLpro would inhibit SARS‐CoV‐2 replication and maturation. Native MS screening has been used to discover natural ligands of 3CLpro that inhibit proteolytic activity (Zhu et al., 2022).

During replication of SARS‐CoV‐2 inside the host cell, polyprotein processing by 3CLpro releases several nonstructural proteins that facilitate RNA synthesis (Figure 1). The 12 kDa nonstructural protein 9 (Nsp9) forms a homodimer that functions as an essential RNA binding subunit of the replication complex (de et al., 2021). Because disruption of Nsp9 binding to the replication complex reduces RNA binding and SARS‐CoV proliferation, ligands to Nsp9 are potential anti‐SARS‐CoV‐2 agents (Littler, Mohanty, et al., 2021). Native MS screening has been used to discover Nsp9 ligands that can inhibit the replication of SARS‐CoV‐2 (Littler, Liu, et al., 2021).

2. DISCOVERY OF ANTI‐SARS‐COV‐2 COMPOUNDS USING MS

2.1. MagMASS

Invented in 2008 (Choi & van Breemen, 2008), MagMASS has been used to screen pools of combinatorial library compounds as well as natural product extracts (Rush et al., 2016) for ligands to a wide variety of pharmacological targets including the SARS‐CoV‐2 spike protein. MagMASS was the first AS‐MS approach to incorporate multititer well plates and automation for high‐throughput screening (Rush et al., 2017). The MagMASS process (Figure 2) begins with covalent or noncovalent immobilization of a pharmacological target (usually an enzyme or a receptor) to magnetic beads. Covalent immobilization can be accomplished using microbeads derivatized on the surface with electrophilic functionalities such as N‐hydroxysuccinimide, which reacts with nucleophiles on receptors and enzymes such as primary amino groups or thiol groups. Alternatively, water‐soluble carbodiimides may be used to form covalent crosslinks between primary amino groups on the magnetic microbeads and carboxylic acids on the target or between carboxylic acids on the surface of the microbeads and amino groups on the target molecules. Noncovalent immobilization of receptors and enzymes may be carried out using strong binding interactions between immobilized nickel cations and receptors containing His‐tags, immobilized amylose interacting with receptors containing maltose‐binding protein, and immobilized streptavidin binding to biotinylated receptors.

After immobilization of the receptor, the magnetic microbeads are incubated with mixtures containing potential ligands such as pools of combinatorial libraries or extracts containing complex natural product mixtures (Figure 2). Next, the beads are washed to remove unbound compounds while a magnetic field retains the beads containing the receptor–ligand complexes. Ligands bound to the immobilized receptor are released using a denaturing solvent or a pH change and then analyzed using MS or ultrahigh performance liquid chromatography‐MS (UHPLC‐MS). To control for nonspecific binding, beads containing denatured receptor may be incubated in parallel.

Advantages of MagMASS include the ability to screen complex mixtures, discovery of orthosteric as well as allosteric ligands, and compatibility with all types of targets, incubation buffers, cofactors, cosubstrates, and natural products (Table 1). MagMASS is ideal for insoluble or membrane‐bound receptors that cannot be assayed in solution, but soluble receptors may also be used as targets following immobilization. Disadvantages of MagMASS include the requirement of a target immobilization step, which might change the receptor binding properties, and the need for deconvolution of hits from combinatorial library mixtures or structure determination of unknown natural product hits (Table 1).

In an application of MagMASS to the discovery of natural ligands to the SARS‐CoV‐2 spike protein, Muchiri et al. (2022) immobilized the 108 kDa SARS‐CoV‐2 S1 protein containing a His‐tag on Ni2+‐nitrilotriacetic acid derivatized magnetic microbeads. As a positive control, binding was demonstrated for SBP‐1, which is a 23 amino acid peptide identical to the ACE2 α1 helix sequence recognized by the SARS‐CoV‐2 spike protein S1 subunit. Screening extracts of botanical dietary supplements resulted in the discovery of licochalcone A (K D = 6.3 ± 1.1 μM) from the licorice species Glycyrrhiza inflata (Muchiri et al., 2022), and cannabidiolic acid (5.6 ± 2.2 μM), cannabigerolic acid (19.8 ± 2.7), and tetrahydrocannabinolic acid from hemp (Cannabis sativa) (van Breemen et al., 2022). Binding of licochalcone A, cannabidiolic acid, and cannabigerolic acid to the active site of the SARS‐CoV‐2 S1 protein was confirmed by demonstrating competitive binding with SBP‐1. Cannabigerolic acid was also found to bind to an allosteric site on the spike protein S1 subunit. Functional assays using live SARS‐CoV‐2 and two early variants showed that cannabidiolic acid and cannabigerolic acid could block cell entry and infection (van Breemen et al., 2022).

Using a similar MagMASS assay with a biotinylated S1 receptor binding domain immobilized on avidin‐derivatized microbeads, Pomplun et al. (2021) screened mixtures of synthetic peptides and found a peptide (TVFGLNVWKAYSK) that bound with a Kd of 250 nM. However, this peptide did not bind to the active site where the peptide interacts with ACE2. Furthermore, the SBP‐1 peptide, which should bind to that active site, did not bind in this assay. This suggests that the biotinylated 52 kDa subunit of the S1 is a less accurate target for spike protein binding and ligand discovery than the His‐tagged 108 kDa S1 protein.

2.2. Native MS screening

Originally called bio‐affinity characterization mass spectrometry (Bruce et al., 1995), native MS screening enables the direct screening of mixtures of compounds for ligands to solution‐phase receptors (Cheng et al., 1995). Using the solution‐phase technique of electrospray ionization, native MS involves the desorption and ionization of macromolecules such as protein receptors directly from aqueous solutions while maintaining their solution‐phase (native) conformations (Figure 3). Screening using native MS requires the measurement of undissociated ligand‐receptor complexes desorbed from incubations of mixtures of potential ligands with a solution‐phase receptor. Due to the high molecular mass of receptors, the low mass of drug‐like compounds, and the need to measure small mass differences between the receptor ions and the receptor‐ligand ions, ultrahigh resolution mass spectrometers such as Fourier Transform ion cyclotron resonance instruments equipped with electrospray ionization are required.

Limitations of native MS screening include the need for an ultrahigh resolution mass spectrometer and the requirement that the receptor can be desorbed and ionized directly from the incubation solution in its native, biologically active form. Denaturing organic solvents and pH conditions must be avoided, and buffers are limited to volatile constituents that do not precipitate in the electrospray source or suppress ionization. Cofactors and co‐substrates may be used at low concentrations providing they do not interfere with ionization. Receptors must be solution‐phase, so that membrane‐bound or low solubility receptors are incompatible with native MS screening. Advantages of native MS screening include the capability of detecting allosteric as well as orthosteric ligands, compatibility with natural products, no requirement of immobilization of water‐soluble receptors, and no need for a separation step such as chromatography or electrophoresis.

Zhu et al. (2022) applied native MS screening to the discovery of natural inhibitors of the SARS‐CoV‐2 protease 3CLpro. Reversed‐phase HPLC fractions of methanolic extracts of botanicals used in traditional Chinese medicine were screened for binding to recombinant 3CLpro (1 µM) in 10 mM ammonium acetate (pH 6.9) using an FTICR mass spectrometer. By comparison with standards, the flavonoids baicalein, scutellarein, and ganhuangenin were identified as ligands of 3CLpro with K D values of 1.43, 3.85, and 1.09 μM, respectively. Subsequent enzyme assays indicated that these flavonoids were reversible inhibitors of 3CLpro with IC50 values of 0.94, 3.02, and 0.84 μM, respectively.

Littler, Liu, et al. (2021) used native MS to screen a natural product library for ligands to Nsp9, which is an essential RNA binding component of the SARS‐CoV‐2 RNA‐dependent RNA polymerase complex. The ent‐kaurane natural product, oridonin, was identified as a ligand to Nsp9 with a KD value of ~7.2 ± 1.0 μM. In enzymatic assays with Nsp9 in the SARS‐CoV‐2 RNA‐dependent RNA polymerase, oridonin reduced nucleotidyl transferase activity, and in cellular assays using live SARS‐CoV‐2, oridonin reduced viral titer following infection.

3. CONCLUSIONS AND FUTURE DIRECTIONS

Although invented to address the need for high‐throughput screening of combinatorial libraries during early drug discovery (Zhao et al., 1997), AS‐MS was quickly recognized to be suitable for the discovery of natural ligands to therapeutic targets in complex mixtures such as botanical extracts (Liu et al., 2001). Compared with conventional high‐throughput screening, AS‐MS offers advantages such as compatibility with any type of pharmacological target, ligand, matrix, or assay buffer, detection of allosteric as well as orthosteric ligands, and no requirement for labeled reagents (Table 1).

The speed and flexibility of AS‐MS have enabled the rapid development and application of assays for the discovery of anti‐SARS‐CoV‐2 compounds. These reports of early drug discovery for COVID‐19 originated in academic laboratories. As AS‐MS becomes more widely adopted by pharmaceutical and biotechnology companies, the pace of drug discovery, including the discovery of new drugs based on natural product lead compounds, should be expected to accelerate.

ACKNOWLEDGMENT

This review is dedicated to the mentorship and biomedical pioneering research of Prof. Catherine Fenselau.

Biographies

Richard B. van Breemen is a Professor of Pharmaceutical Sciences in the College of Pharmacy and serves on the faculty of the Linus Pauling Institute and the Global Hemp Innovation Center at Oregon State University. Richard received his PhD in Pharmacology and Experimental Therapeutics from the Johns Hopkins University School of Medicine in 1985 before carrying out postdoctoral research in laser desorption mass spectrometry at Johns Hopkins. After teaching chemistry at North Carolina State University and medicinal chemistry and pharmacognosy at the University of Illinois at Chicago, Richard joined the faculty of Oregon State University in 2018. Richard received the Harvey W. Wiley Award from the AOAC International in 2008, the Varro E. Tyler Prize from the American Society of Pharmacognosy in 2017, and is a Fellow of the International Carotenoid Society. He is a member of the USP Dietary Supplements Admission, Evaluation, & Labeling Expert Committee and has served on the Editorial Board of the Journal of AOAC International since 2016. Richard is also on the editorial board of Assay and Drug Development Technologies. Richard's research concerns the discovery of pharmacologically active natural products using affinity selection‐mass spectrometry, the discovery and development of natural products that prevent cancer and neurological degenerative diseases, and the investigation of the safety and efficacy of botanical dietary supplements. Richard has published nearly 400 research papers and book chapters concerning natural products, botanical dietary supplements and the use of mass spectrometry for drug discovery and development from natural product sources, and he has mentored over 80 graduate students and postdoctoral fellows.

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Ruth N. Muchiri is a researcher and lab manager in the Department of Pharmaceutical Sciences of the College of Pharmacy and Linus Pauling Institute at Oregon State University. She obtained her BS in chemistry and biochemistry from the University of Nairobi and her PhD in bioorganic chemistry from Michigan State University. During here predoctoral research, Dr. Muchiri developed a biosynthetic approach to produce the chemotherapy drug and natural product paclitaxel. She carried out postdoctoral research at the University of Illinois at Chicago College of Pharmacy with Dr. Richard van Breemen before moving to Oregon State University in 2018. Her research is at the forefront of affinity selection‐mass spectrometry assays for the discovery of pharmacologically active natural products. Dr. Muchiri's research concerns the discovery and development of safe and effective bioactive natural products, including those with anticancer or antiviral activity. She is using biomedical mass spectrometry including affinity selection‐mass spectrometry for the discovery of bioactive compounds from botanicals. Dr. Muchiri also combines LC‐MS/MS with cell‐based assays and enzyme assays to confirm the bioactivities of natural products identified through the application of affinity selection MS.

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Breemen RB, Muchiri RN. Affinity selection‐mass spectrometry in the discovery of anti‐SARS‐CoV‐2 compounds. Mass Spectrometry Reviews, 2022;e21800. 10.1002/mas.21800

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