Publication date: Jun 17, 2025

Despite progress made in addressing the COVID-19 pandemic, numerous challenges persist, including breakthrough infections (BTI) and reinfections with SARS-CoV-2. There is an urgent need for broad-spectrum antiviral agents with multifaceted functionalities. Dihydrotanshinone I (DHT), a lipophilic monomer component of Salvia miltiorrhiza, exhibits diverse pharmacological activities, including antioxidant, anti-inflammatory, antibacterial and immunomodulatory effects. The efficacy and mechanisms underlying its “multi-target” advantages in treating COVID-19 warrant further investigation. To investigate the inhibitory activity and mechanisms of DHT against pan-SARS-CoV-2 invasion. We examined the activity of DHT against SARS-CoV-2 Variants of Concern (VOCs) using the viral replicon system, pseudo virus-based entry assays, and a human ACE2 transgenic mouse model. The enzymatic activity and time-of-drug-addition assays were conducted to elucidate the viral life cycle targeted by DHT. Drug resistance induction assay, next-generation sequencing (NGS) analysis and molecular docking were employed to confirm DHT’s target sites. Further ELISA, RT-qPCR, and western blot assays revealed the mechanism and anti-inflammatory activity of DHT. As a potent antiviral agent, DHT showed activity against SARS-CoV-2 VOCs with EC values ranging from 0. 3-4. 0 μM. DHT binds to two conserved sites on the spike N-terminal domain (NTD), inhibiting viral entry for both original and variant strains. Furthermore, DHT modulates the interaction between the spike and Toll-like receptor 4 (TLR4), stimulating nuclear factor-erythroid 2-related factor 2 (NRF2)-dependent immune response to attenuate cytokine production. Moreover, multiple administrations of DHT provide therapeutic benefits in mouse model of COVID-19. The dual functionality of DHT is accomplished through binding to the spike NTD and inhibiting spike-activated inflammation, making it effective in preventing SARS-CoV-2 invasion. Our findings offer a conceptual framework for the rational design of prospective multitargeted inhibitors against SARS-CoV-2 spike NTD.

Semantics

Original Article