HPLC–MS/MS For Screening Neuraminidase Inhibitors

In a recent study published in the Journal of Chromatography B, a research team from Fusan University in Shanghai, China explored the potential antiviral components in Baphicacanthus cusia, a herb that is widely used for the treatment of colds and the flu in traditional Chinese medicine (1). This study, led by Zhihong Cheng, introduces an affinity ultrafiltration (AUF) coupled with high performance liquid chromatography–tandem mass spectrometry (HPLC-MS/MS) method for screening NA inhibitors from natural products, particularly focusing on stems and leaves of Baphicacanthus cusia.

Neuraminidase inhibitors are a key class of drugs that are known for their ability to help treat and prevent influenza. Baphicacanthus cusia is known to contain NA inhibitors, which makes the study of this plant useful in the context of improving treatment for influenza.

Influenza is a highly contagious viral infection that affects the respiratory system. It is estimated that there will be 29-54 million flu illnesses in the United States, during the 2023-2024 season, according to the Centers for Disease Control and Prevention (CDC) (3). Although most healthy Americans recover from influenza and its symptoms relatively quickly, those with compromised immune systems can suffer serious complications. A 2018 study conducted by the CDC found that approximately 8% of Americans contract the flu every year, although this number ranges from 3–11% depending on vaccination rates and external factors (2,3).

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AUF, the method used by the research team, was first developed in 1997 (1). It involves rapidly screening active compounds targeting enzymes (1). AUF is increasingly used for enzyme inhibitor screening because of its effectiveness, coupled with the capability of LC–MS to identify structures (1). As a result, it was a major contributing factor why the research team deduced AUF could be used to explore the antiviral components in Baphicacanthus cusia.

Most of the team’s investigation focused on the factors affecting the enzyme–ligand interaction. These factors include enzyme concentration, sample concentration, temperature and incubation time, and pH of the buffer, to name a few. By optimizing these factors, the research team identified five active alkaloids, which include 2,4(1H,3H)-quinazolinedione, 4(3H)-quinazolinone, 2(3H)-benzoxazolone, tryptanthrin, and indirubin (1). Then, the research team analyzed their NA inhibitory activity. Molecular docking analysis further elucidated the binding mechanisms, emphasizing hydrogen bond and π-cation interactions within the binding sites (1).

The developed AUF method proved effective in identifying potential NA inhibitors from B. cusia, offering a straightforward and efficient screening approach.

References

(1) Fan, X.; Li, Y.; Wu, T; Cheng, Z. Screening and Identification of Neuraminidase Inhibitors from Baphicacanthus cusia by a Combination of Affinity Ultrafiltration, HPLC-MS/MS, Molecular Docking, and Fluorescent Techniques. J. Chromatogr. B. 2023, 1231, 123924. DOI: 10.1016/j.jchromb.2023.123924

(2) Takers, J. I.; Olsen, S. J.; Reed, C. Seasonal Incidence of Symptomatic Influenza in the United States. Clin. Inf. Diseases 2018, 66 (10), 1511–1518. DOI: 10.1093/cid/cix1060

(3) CDC, Key Facts About Influenza (Flu). Available at: https://www.cdc.gov/flu/about/keyfacts.htm (accessed 03-20-2024).