Since the outbreak of the 2019 coronavirus disease (COVID-19) in Wuhan, China, it has spread to almost every country, causing more than 6.28 million deaths and widespread economic problems. Although many developed countries have implemented mass vaccination programs that have helped stem the spread of the disease, developing countries that have struggled to obtain doses have attempted to reduce their case numbers. In addition, studies have shown reduced vaccine effectiveness in certain immunocompromised individuals. There is still an urgent need for effective treatments against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections. Naval Medical University researchers have studied anisodamine, used in traditional Chinese medicine, to inhibit SARS-CoV-2 infection in cell models. The study is published in the journal Communications on biochemical and biophysical research. Anisodamine is a naturally occurring tropane alkaloid found in plants of the nightshade family, including Datura.
Study: Anisodamine Effectively Inhibits SARS-CoV-2 Infection In Vitro, Targeting Its Main Protease. Photo Credit: SKK Pix/Shutterstock
The SARS-CoV-2 strain was isolated from a COVID-19 patient at Changhai Hospital and inoculated into Vero-E6 cells. Initial cell viability and lactate dehydrogenase assays were performed in 96-well plates at concentrations of 4×10^3 cells/well. HEK293 cells overexpressing human angiotensin converting enzyme 2 (hACE2) were obtained commercially for pseudovirus entry assays, as was the spike pseudovirus. Molecular Operating Environment software was used for molecular dynamics simulations and statistical analysis consisted of a two-tailed unpaired Student’s t-test.
Anisodamine was tested for cytotoxic effects in Vero E6 cells using CCK-8 and lactate dehydrogenase (LDH) release assays and showed no reduction in cell viability or production of LDH – even at concentrations up to 800 mg/L. At 1600 mg/l a slight decrease in cell survival was seen, but below this threshold the drug is probably safe to use. Subsequently, Vero E6 cells were infected with SARS-CoV-2 and treated with different concentrations of anisodamine. Inhibitory activity was assessed 48 hours post-infection by quantifying viral copy number using immunofluorescence. As expected, the controls showed significantly increased copies of SARS-CoV-2, but the anisodamine successfully reduced SARS-CoV-2 replication in a dose-dependent manner. At the most successful concentrations – 100 mg/L and 200 mg/L – a ~70% and 90% reduction in replication was noted.
Treatment was then further analyzed using pseudovirus neutralization assays, measuring inhibition of SARS-CoV-2 spike pseudovirus entry into hACE2-transfected HEK-293 cells. Once again, both 100 mg/L and 200 mg/L were found to be effective in reducing viral entry, with ~60% reduced transduction efficiency. To investigate the mechanisms behind this, researchers examined the binding affinities of anisodamine to different active sites of primary proteins for SARS-CoV-2 infection, including the spike receptor binding domain (RBD), Mpro, PLpro, RdRp, ACE2 and GRP78 . Docking simulation studies showed that anisodamine had the highest binding affinity to the Mpro active site with a value of -6.63 kcal/mol and formed three hydrogen bonds with residues at this active site. Similar binding affinities were predicted for the active sites of PLpro (-5.52 kcal/mol) forming hydrogen bonding and π-π interaction. Lower binding affinities were demonstrated for the active sites of all other targets.
China has traditionally used anisodamine to improve circulation, but it’s gained popularity outside the country to treat a range of ailments. Researchers have speculated that anisodamine might activate the nicotinic acetylcholine receptor alpha7 subunit and the cholinergic anti-inflammatory pathway, which might prevent the cytokine storms that are so often fatal in patients with severe COVID-19. Whether the virus can affect the disease in humans remains to be seen. However, the authors have successfully demonstrated that anisodamine inhibits viral entry into cells in both Vero E6 models and cells expressing hACE2. They also identified which proteins it binds to. More research is needed, but anisodamine appears to be an interesting candidate for further study and may even head towards clinical trials.
- Wei Wei, Ni Kong, Ming-Zhen Liu, Ting Han, Jun-Feng Xu, Chong Liu, Anisodamine potently inhibits SARS-CoV-2 infection in vitro and targets its main protease, Biochemical and Biophysical Research Communications, 2022, ISSN 0006-291X, https://doi.org/10.1016/j.bbrc.2022.05.024, https://www.sciencedirect.com/science/article/pii/S0006291X22007203