key: cord-1014945-x4592fkc
authors: Tran, Hoai Thi Thu; Le, Nguyen Phan Khoi; Gigl, Michael; Dawid, Corinna; Lamy, Evelyn
title: Common dandelion (Taraxacum officinale) efficiently blocks the interaction between ACE2 cell surface receptor and SARS-CoV-2 spike protein D614, mutants D614G, N501Y, K417N and E484K in vitro
date: 2021-03-19
journal: bioRxiv
DOI: 10.1101/2021.03.19.435959
sha: 2ebb99482368e2a0b37b33411db946df426c45ae
doc_id: 1014945
cord_uid: x4592fkc

On 11th March 2020, coronavirus disease 2019 (COVID-19), caused by the SARS-CoV-2 virus, was declared as a global pandemic by the World Health Organization (WHO). To date, there are rapidly spreading new “variants of concern” of SARS-CoV-2, the United Kingdom (B.1.1.7), the South African (B.1.351) or Brasilian (P.1) variant. All of them contain multiple mutations in the ACE2 receptor recognition site of the spike protein, compared to the original Wuhan sequence, which is of great concern, because of their potential for immune escape. Here we report on the efficacy of common dandelion (Taraxacum officinale) to block protein-protein interaction of spike S1 to the human ACE2 cell surface receptor. This could be shown for the original spike D614, but also for its mutant forms (D614G, N501Y, and mix of K417N, E484K, N501Y) in human HEK293-hACE2 kidney and A549-hACE2-TMPRSS2 lung cells. High molecular weight compounds in the water-based extract account for this effect. Infection of the lung cells using SARS-CoV-2 spike pseudotyped lentivirus particles was efficiently prevented by the extract and so was virus-triggered pro-inflammatory interleukin 6 secretion. Modern herbal monographs consider the usage of this medicinal plant as safe. Thus, the in vitro results reported here should encourage further research on the clinical relevance and applicability of the extract as prevention strategy for SARS-CoV-2 infection. Significance statement SARS-CoV-2 is steadily mutating during continuous transmission among humans. This might eventually lead the virus into evading existing therapeutic and prophylactic approaches aimed at the viral spike. We found effective inhibition of protein-protein interaction between the human virus cell entry receptor ACE2 and SARS-CoV-2 spike, including five relevant mutations, by water-based common dandelion (Taraxacum officinale) extracts. This was shown in vitro using human kidney (HEK293) and lung (A549) cells, overexpressing the ACE2 and ACE2/TMPRSS2 protein, respectively. Infection of the lung cells using SARS-CoV-2 pseudotyped lentivirus was efficiently prevented by the extract. The results deserve more in-depth analysis of dandelions’ effectiveness in SARS-CoV-2 prevention and now require confirmatory clinical evidence.

depth analysis of dandelions´ effectiveness in SARS-CoV-2 prevention and now require confirmatory clinical evidence.

In late 2019, the disease known as Corona Virus Disease 2019 or COVID-19 was first reported (1, 2). It is induced by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

Dry cough, fever, fatigue, headache, myalgias, and diarrhea are common symptoms of the disease. In severe cases people may become critically ill with acute respiratory distress syndrome (3) . The SARS-CoV-2 virus surface is covered by a large number of glycosylated S proteins, which consist of two subunits, S1 and S2. The S1 subunit recognizes and attaches to the membrane-anchored carboxypeptidase angiotensin-converting enzyme 2 (ACE2) receptor on the host cell surface through its receptor binding domain (RBD). The S2 subunit plays a key role in mediating virus-cell fusion and in concert with the host transmembrane protease serine subtype 2 (TMPRSS2), promotes cellular entry (4) . This interaction between the virus and host cell at entry site is crucial for disease onset and progression. in the RBD (6) . Preliminary data suggest a possible association between the observed increased fatality rate with the mutation D614G and it is hypothesized that a conformational change in the spike protein results in increased infectivity (7) . Free energy perturbation calculations for interactions of the N501Y and K417N mutations with both the ACE2 receptor and an antibody derived from COVID-19 patients raise important questions about the possible human immune response and the success of already available vaccines (8) . Further, increased resistance of the variants B.1.351 and B.1.1.7 to antibody neutralization has been reported; for B.1.351 this was largely due to the E484K mutation in the spike protein (9) .

Interference with the interaction site between the spike S1 subunit and ACE2 has the potential to be a major target for therapy or prevention (10) . Compounds from natural origin may offer here some protection against viral cell entry while have no or few side effects. Here we report on the inhibitory potential of dandelion on the binding of the spike S1 protein RBD to the hACE2 cell surface receptor and compared the effect of the original D614 spike protein to its D614G, N501Y, and mix (K417N, E484K, N501Y) mutations.

The common dandelion (Taraxacum officinale) belongs to the plant family Asteraceae, subfamily Cichorioideae with many varieties and microspecies. It is a perennial herb, native distributed in the warmer temperate zones of the Northern Hemisphere inhabiting fields, roadsides and ruderal sites. T. officinale is consumed as vegetable food, but also employed in European phytotherapy to treat disorders from the liver, gallbladder, digestive tract or rheumatic diseases. Modern herbal monographs consider the plant usage as safe and have evaluated the empiric use of T. officinale with a positive outcome. Therapeutic indications for the use of T. officinale are listed in the German Commission E, the European Scientific Cooperative for Phytotherapy (ESCOP) monographs (11, 12) as well as in the British Herbal Medicine Association (13). The plant contains a wide array of phytochemicals including terpenes (sesquiterpene lactones such as taraxinic acid and triterpenes), phenolic compounds (phenolic acids, flavonoids, and coumarins) and also polysaccharides (14) . The predominant phenolic compound was found to be chicoric acid (dicaffeoyltartaric acid). The other were mono-and dicaffeoylquinic acids, tartaric acid derivatives, flavone and flavonol glycosides.

The roots, in addition to these compound classes, contain high amounts of inulin (15) . Dosage forms include aqueous decoction and infusion, expressed juice of fresh plant, hydroalcoholic tincture as well as coated tablets from dried extracts applied as monopreparations (16) but also integral components of pharmaceutical remedies. Our research was conducted using water-based extracts from plant leaves. We found that leaf extracts efficiently blocked spike protein or its mutant forms to the ACE2 receptor, used in either pre-or post-incubation, and that high molecular weight compounds account for this effect. A plant from the same tribe (Cichorium intybus) could exert similar effects but with less potency. Infection of A549-hACE2-TMPRSS2 human lung cells using SARS-CoV-2 pseudotyped lentivirus was efficiently prevented by the extract.

T. officinale inhibits spike S1 -ACE2 binding

We first investigated the inhibition of interaction between SARS-CoV-2 spike protein RBD and ACE2 using extracts from T. officinale leaves. In figure 1A , the concentration dependent inhibition of Spike S1 -ACE2 binding upon treatment with T. officinale extract is given (EC50=12 mg/ml). Extracts from C. intybus, also showed a concentration dependent binding inhibition, but with less potency than T. officinale (EC50=30 mg/ml) (figure 1B). We then prepared two fractions of the dried T. officinale as well as chicory leaves, separating the extracts into a high molecular (>5kDa) and low molecular weight (<5kDa) fraction. As can be seen from figure 1C , the bioactive compounds were mostly present in the HMW fraction. Only minor activity was seen in the LMW fraction. Using hACE2 overexpressing HEK293 cells, the potential of T. officinale and C. intybus extracts to block spike binding to cells was further investigated. As can be seen from figure 2, pre-incubation of cells with T. officinale for one min. efficiently blocked cell binding of spike by 76.67% ± 2.9, and its HMW fraction by 62.5 ± 13.4% as compared to water control. After 3h, inhibition was still at 50 ± 13.6% for the extract, and 35.0 ± 20% for the HMW fraction of T.

officinale. The chicory extract was less potent in this test system; binding inhibition was seen at 37 ± 20% after 1min. and 5.6 ± 9.9%. To see whether T. officinale extract interferes with the catalytic activity of the ACE2 receptor or affects ACE2 protein expression, we treated A549-hACE2-TMPRSS2 cells with the extract for 1-24h before cell lysis and detection. No loss in cell viability was seen after extract exposure to the cells for 84h (figure 4A). No enzyme activity impairment could be detected after 1 or 24h (4B). Spike significantly downregulated ACE2 protein after 6h (4C, black bars), and this was also true for the extract, either alone (4C, white bars) or in combination with spike (black bars).

After 24h, this effect was abolished (4D). A) Viability of A549-hACE2-TMPRSS2 cells was determined using trypan blue cell staining after 84h exposure to the extract. B) Cells were incubated with TO extract or 500 ng/ml S1 protein and analysed for enzyme activity using a fluorescence kit. C-D) Cells were exposed for 6h or 24h to extract without (white bars) or with (black bars) 500 ng/ml S1 protein and analysed for ACE2 protein expression using a human ACE2 ELISA kit; a. d. 

The development of effective prevention and treatment strategies for SARS-CoV-2 infection is still at the early stages. Although the first vaccines received now marketing authorization, challenges in distribution concerns or about durable effectiveness, and risk of re-infection remain (17, 18) . Subsequent infections may possibly be milder than the first one, though.

Besides vaccination against COVID-19, blocking the accessibility of the virus to membranebound ACE2 as the primary receptor for SARS-CoV-2 target cell entry, represents an alternative strategy to prevent COVID-19. Here, different approaches exist (19) , but of course each of these treatment strategies also has its fundamental as well as translational challenges which need to be overcome for clinical utility. Technical hurdles include off-target potential, ACE2-independent effects, stability or toxicity (19) . Compounds from natural origin could be an important resource here as they are described long term and many of them have been established as safe. While in silico docking experiments suggested different common natural compounds as ACE2 inhibitors, spike binding inhibition to ACE2 has not been shown for most of them so far, which might be explained by a lack of complete coverage of ACE2 binding residues by the compounds (20) . However, for glycyrrhizin, nobiletin, and neohesperidin, ACE2 binding falls partially within the RBD contact region and thus, these have been proposed to additionally block spike binding to ACE2 (20) . The same accounts for synthetic ACE2 inhibitors, such as N-(2-aminoethyl)-1 aziridine-ethanamine (NAAE) (21) . In contrast, the lipoglycopeptide antibiotic dalbavancin has now been identified as both, ACE2 binder and SARS-CoV-2 spike-ACE2 inhibitor (22); SARS-CoV-2 infection was effectively inhibited in both mouse and rhesus macaque models by this compound. Also, for a hydroalcoholic pomegranate peel extract, blocking of spike-ACE2 interaction was shown at 74%, for its main constituents punicalagin at 64%, and ellagic acid at 36%. Using SARS-CoV-2 spike pseudotyped lentivirus infection of human kidney-2 (HK-2) cells, virus entry was then efficiently blocked by the peel extract (23) . In the present study, we could show potent ACE2-spike S1 RBD protein inhibition by T. officinale extracts using a cell-free assay and confirmed this finding by demonstrating efficient ACE2 cell surface binding inhibition in two human cell lines. We observed stronger binding of the variants D614G and N501Y to the ACE2 surface receptor of human cells, but all tested variants were sensitive to binding inhibition by T. officinale, either used before spike protein exposure or thereafter. To date, several studies indicate that the D614G viral lineage is more infectious than the D614 virus (24) . Also, the presence of characteristic mutations such as N501Y of, e. g. the so-called UK variant B.1.1.7, result in higher infectivity than the parent strain which might be due to a higher binding affinity between the spike protein and ACE2 (25) . So our findings on T. officinale extracts could here be important, as with progression of the pandemic, new virus variants of potential concern will emerge which may also reduce the efficacy of some vaccines or cause increased rates of (16) . The plant is a significant source of potassium (26, 27) and thus a warning is given because of the possible risk for hyperkalaemia. The use in children under 12 years of age, during pregnancy and lactation has not been established due to lack of adequate or sufficient data.

While ACE2 enzyme activity was not affected by T. officinale extract in the present study, ACE2

protein was transiently downregulated in the ACE2 overexpressing lung cell line, which requires more attention in ongoing studies. ACE2 is an important zinc-dependent monocarboxypeptidase in the renin-angiotensin pathway, critical in impacting cardiovascular and immune system. Disruption of the Angiotensin II/Angiotensin-(1-7) balance by ACE2 enzyme activity inhibition or protein decrease and more circulating Angiotensin II in the system, is e. g.

recognised to promote lung injury in the context of COVID-19 disease (28, 29) .

The lung would be assumed to be the primary target of interest but, ACE2 mRNA and protein expression have been found in epithelial cells of all oral tissues, especially in the buccal mucosa, lip and tongue (30) . These data concur with the observation of very high salivary viral load in SARS-CoV-2 infected patients (31, 32) . As an essential part of the upper aerodigestive tract, the oral cavity is thus believed to play a key role in the transmission and pathogenicity of SARS-CoV-2. There is high potential that prevention of viral colonization at the oral and The study was carried out using dried leaves from T. officinale (vom Achterhof, Uplengen, Germany; batch no. 37259, B370244, and P351756). Plant leaf samples were also collected at three different places in the region of Freiburg i. Br. (Germany), on 12.7.2020, and tested positive in the cell-free spike S1-ACE2 binding assay (data not shown). C. intybus was purchased from Naturideen (Germany). To subculture, all cells were first rinsed with phosphate buffered saline (PBS) then incubated with 0.25% trypsin-EDTA until detachment. All cells were cultured at 37 °C in a humidified incubator with 5% CO2/95% air atmosphere.

Dried plant material was weighted in an amber glass vial (Carl Roth GmbH, Germany) and 

A549-hACE2-TMPRSS2 (2x10 5 ) cells were seeded in a 24-well plate in high glucose DMEM medium, containing 10% heat inactivated FCS, at 37 o C, 5 % CO2. Cells were then treated with T. officinale extract with/without 500 ng/ml SARS-CoV-2 S1 Spike RBD protein for 1-24h.

Afterwards, cells were washed with PBS and lysed. 25µg protein were used for quantification of ACE2 protein (ACE2 ELISA kit), 5 µg for ACE2 enzyme activity (ACE2 activity assay kit, Abcam, Cambridge, UK) according to the manufacturer's instructions. 

After 24h SARS-CoV-2 spike pseudotyped lentivirus transduction and 60h post-infection of A549-hACE2-TMPRSS2 cells, supernatants were collected and stored at -80 °C until analysis for cytokine secretion using the human MACSplex cytokine 12-kit (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) according to manufacturer's protocol.

Extracts from dried plant leaves were prepared by adding bidistilled water (5 ml) to plant material (500 mg each). The samples were incubated in the dark at room temperature (RT) for 60 min, followed by centrifugation at 16.000 g for 3 min. The supernatants were collected and membrane filtrated (0.45 µm), resulting in the extracts. Aliquots were freeze dried for 48h to determine their yield by weight. The extracts were then further separated in a high molecular weight (HMW) and low molecular weight (LMW) fraction, using a centrifugation tube with an insert containing a molecular weight cut-off filter (5 kDa, Sartorius Stedim Biotech, Goettingen, Germany). Each HMW fraction was purified by flushing with 20 ml of water, yielding the HMW fractions, as well as LMW. The fractions were freeze dried, their yield determined by weight and stored at -20°C until use.

Cell viability was assessed using the trypan blue dye exclusion test as described before (Odongo et al., 2017). Briefly, A549-hACE2-TMPRSS2 cells were cultured for 24h, and then exposed to extracts or the solvent control (a. d.) for 84 h.

Results were analysed using the GraphPad Prism 6.0 software (La Jolla, California, USA).

Data were presented as means + SD. Statistical significance was determined by the one-way ANOVA test followed by Bonferroni correction. P values <0.05 (*) were considered statistically significant and <0.01 (**) were considered highly statistically significant.

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The authors are grateful to Prof. Dr. Stefan Pöhlmann (German Primate Center, Göttingen, Germany) for providing the human embryonic kidney 293 (HEK293) cells, stably expressing hACE2.