Development of a virus-based affinity ultrafiltration method for screening virus-surface-protein-targeted compounds from complex matrixes: Herbal medicines as a case study.

Herbal medicines (HMs) are one of the main sources for the development of lead antiviral compounds. However, due to the complex composition of HMs, the screening of active compounds within these is inefficient and requires a significant time investment. We report a novel and efficient virus-based screening method for antiviral active compounds in HMs. This method involves the centrifugal ultrafiltration of viruses, known as the virus-based affinity ultrafiltration method (VAUM). This method is suitable to identify virus specific active compounds from complex matrices such as HMs. The effectiveness of the VAUM was evaluated using influenza A virus (IAV) H1N1. Using this method, four compounds that bind to the surface protein of H1N1 were identified from dried fruits of Terminalia chebula (TC). Through competitive inhibition assays, the influenza surface protein, neuraminidase (NA), was identified as the target protein of these four TC-derived compounds. Three compounds were identified by high performance liquid chromatography (HPLC) and liquid chromatography/mass spectrometry (LC/MS), and their anti-H1N1 activities were verified by examining the cytopathic effect (CPE) and by performing a virus yield reduction assay. Further mechanistic studies demonstrated that these three compounds directly bind to NA and inhibit its activity. In summary, we describe here a VAUM that we designed, one that can be used to accurately screen antiviral active compounds in HMs and also help improve the efficiency of screening antiviral drugs found in natural products.

Paving new roads toward the advancement of broad-spectrum antiviral agents.

Broad-spectrum antivirals (BSAs) have the advantageous property of being effective against a wide range of viruses with a single drug, offering a promising therapeutic solution for the largely unmet need in treating both existing and emerging viral infections. In this review, we summarize the current strategies for the development of novel BSAs, focusing on either targeting the commonalities during the replication of multiple viruses or the systemic immunity of humans. In comparison to BSAs that target viral replication, these immuno-modulatory agents possess an expanded spectrum of antiviral activity. However, antiviral immunity is a double-edged sword, and maintaining immune homeostasis ultimately dictates the health status of hosts during viral infections. Therefore, establishing an ideal goal for immuno-modulation in antiviral interventions is crucial. Herein we propose a bionic approach for immuno-modulation inspired by mimicking bats, which possess a more robust immune system for combating viral invasions, compared to humans. In addition, we discuss an empirical approach to treat diverse viral infections using traditional Chinese medicines (TCMs), mainly through bidirectional immuno-modulation to restore the disrupted homeostasis. Advancing our understanding of both the immune system of bats and the mechanisms underlying antiviral TCMs will significantly contribute to the future development of novel BSAs.

Sulforaphane is a reversible covalent inhibitor of 3-chymotrypsin-like protease of SARS-CoV-2.

The ongoing pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has posed a major public health threat worldwide and emphasizes an urgent need for effective therapeutics. Recently, Ordonez et al. identified sulforaphane (SFN) as a novel coronavirus inhibitor both in vitro and in mice, but the mechanism of action remains elusive. In this study, we independently discovered SFN for its inhibitory effect against SARS-CoV-2 using a target-based screening approach, identifying the viral 3-chymotrypsin-like protease (3CLpro ) as a target of SFN. Mechanistically, SFN inhibits 3CLpro in a reversible, mixed-type manner. Moreover, enzymatic kinetics studies reveal that SFN is a slow-binding inhibitor, following a two-step interaction. Initially, an encounter complex forms by specific binding of SFN to the active pocket of 3CLpro ; subsequently, the isothiocyanate group of SFN as "warhead" reacts covalently to the catalytic cysteine in a slower velocity, stabilizing the SFN-3CLpro complex. Our study has identified a new lead of the covalent 3CLpro inhibitors which has potential to be developed as a therapeutic agent to treat SARS-CoV-2 infection.