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Dihydrotanshinone I targets the spike N-terminal domain to inhibit SARS-CoV-2 entry and spike-induced inflammation
Chan Yang1, Kunyu Lu2, Jinshen Wang2
1Department of Respiratory and Critical Care Medicine, Institute of Respiratory and Critical Care Medicine, Dongguan People's Hospital Biobank, the Tenth Affiliated Hospital of Southern Medical University, Southern Medical University, Guangdong 523059, PR China; NMPA Key Laboratory for Research and Evaluation of Drug Metabolism & Guangdong Provincial Key Laboratory of New Drug Screening, School of Pharmaceutical Sciences, Southern Medical University, Guangzhou 510515, PR China.
Background:
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.
Purpose:
To investigate the inhibitory activity and mechanisms of DHT against pan-SARS-CoV-2 invasion.
Methods:
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.
Results:
As a potent antiviral agent, DHT showed activity against SARS-CoV-2 VOCs with EC50 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.
Conclusions:
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.
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