Daphnetin may protect from SARS-CoV-2 infection by reducing ACE2

Abstract

To combat the SARS-CoV-2 pandemic, innovative prevention strategies are needed, including reducing ACE2 expression on respiratory cells. This study screened approved drugs in China for their ability to downregulate ACE2. Daphnetin (DAP) was found to significantly reduce ACE2 mRNA and protein levels in PC9 cells. DAP exerts its inhibitory effects on ACE2 expression by targeting HIF-1α and JAK2, thereby impeding the transcription of the ACE2 gene. The SARS-CoV-2 pseudovirus infection assay confirmed that DAP-treated PC9 cells exhibited decreased susceptibility to viral infection. At therapeutic doses, DAP effectively lowers ACE2 expression in the respiratory systems of mice and humans. This suggests that DAP, already approved for other conditions, could be a new preventive measure against SARS-CoV-2, offering a cost-effective and accessible way to reduce SARS-CoV-2 spread.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-024-79734-z.

Introduction

The pathogenic agent known as Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) primarily infects humans through the respiratory tract, resulting in damage to the respiratory system and various organs, leading to the manifestation of Coronavirus Disease 2019 (COVID-19). Since its initial emergence in late 2019, SARS-CoV-2 has continued to spread globally, significantly impacting the world economy and society. The ongoing pandemic has also seen the emergence of new viral variants, including alpha, beta, gamma, delta, omicron, as well as the omicron subvariants JN.1, KP.3 and LB.1^1,2. The mutant strain exhibits enhanced ability to evade immunity conferred by currently available vaccines and antibodies, leading to recurrent global outbreaks of COVID-19 ^3,4. Infection with the JN.1 variant is particularly lethal for older individuals, with case fatality rates increasing with age (0.3% for 50–59 years; 1.2% for 60–69 years; 4.7% for 70–79 years; 16.3% for 80 years and older)⁵. Despite vaccination remaining the primary preventive measure against SARS-CoV-2 infection, breakthrough infections frequently occur due to the virus’s significant variability. Furthermore, previous reports indicate that the emergence of new large-scale SARS-CoV-2 mutant strains may hinder the timely production and administration of new vaccines. Individuals previously vaccinated with the original virus strain may experience enhanced susceptibility to infection by the new mutant virus. This susceptibility is attributed to prior infection with the original SARS-CoV-2 strain prior to vaccination, resulting in a significant decrease in antibody titers against the new mutant strain RBD in SARS-CoV-2 following three doses of vaccination¹. Hence, there is a pressing necessity for the development of a novel preventive approach for SARS-CoV-2. Prior research has demonstrated that SARS-CoV-2 primarily infects human cells by binding to the angiotensin-converting enzyme 2 (ACE2) receptor through its surface spike protein⁶. ACE2 is a member of the angiotensin converting enzyme family of dipeptidyl carboxypeptidase enzymes, which play a role in the regulation of vasoconstriction and blood pressure. SARS-CoV-2 infects cells expressing ACE2 located on the luminal surface of epithelial cells in the respiratory tract. Inhibition of ACE2 expression has been shown to potentially prevent the emergence of new SARS-CoV-2 variants⁷. The lower expression of ACE2 in younger children, attributed to a 20% reduction compared to adults, is believed to contribute to their decreased susceptibility to COVID-19⁸. Additionally, reduced ACE2 expression due to allergies may also lower the risk of SARS-CoV-2 infection⁹. Therefore, the identification of a long-term effective drug that can inhibit ACE2 expression in the human body could be advantageous for the prevention of novel coronavirus infections.

Research has demonstrated that ursodeoxycholic acid (UDCA), a pharmaceutical agent utilized for the treatment of liver disease, possesses the ability to suppress ACE2 expression, thereby impeding viral entry into cells. This suggests the potential utility of UDCA in the prevention of SARS-CoV-2 infection¹⁰. While Chinese medicine has been integral in the management of COVID-19 in China, the precise mechanism of action remains unclear¹¹. It is hypothesized that certain components of Chinese medicine may exhibit similar ACE2-inhibiting properties as UDCA, thus potentially serving as preventive agents against SARS-CoV-2 infection. To identify a Chinese medicine monomer suitable for clinical application in effectively inhibiting the expression of ACE2, luciferase reporter plasmids labeled with the upstream regulatory sequence of ACE2 were utilized to screen a library of natural product monomers previously used in clinical settings. The results indicated that DAP demonstrated significant efficacy in inhibiting the transcription of ACE2 mRNA in the PC9 cell line. Furthermore, it was observed that DAP and its analogs downregulated ACE2 expression in respiratory cell lines, reduced cell membrane ACE2 levels, and subsequently hindered the infection of SARS-CoV-2 pseudovirus. We repeat our experiments in mice and demonstrate that DAP administration at Low dose or conventional dose reduces ACE2 in lung. We then demonstrate a reduction of ACE2 levels in the nasal epithelium of volunteers receiving clinically approved doses of DAP. The above experimental results provide a new economically and effectively method for protecting people who are in close contact with Covid-19 patients from SARS-CoV-2 infection.

Results

Library screening

To identify compounds that can inhibit the transcription of ACE2 mRNA, we carried out a screen based on a compound library of natural product monomers that have been used in clinic (Supplementary Material 2). In order to ensure the detection efficiency of library screening, we first identified a cell line PC9 with high expression of ACE2 by qRTPCR method (Extended Fig. 1). To carry out the primary screen, PC9 cells which transfected with ACE2-URS-Luc plasmid then treated with 50 µM concentrations of compounds after plating. After 24 h, the luciferase activity was detected by ELIASA (Fig. 1a). The results suggest that some drugs can cause significant changes in luciferase expression at 50 µM concentration (Fig. 1b). Repeated verification experiments proved that Eupatilin, Resveratrol, and Hesperetin significantly increased luciferase expression (Fig. 1c), and that Mycophenolate mofetil, Daphnetin (DAP), and Acenocoumarol significantly decreased luciferase expression (Fig. 1d). Because of the post-transcriptional regulation mechanism, we then repeatedly verified the above experimental results of luciferase by RTPCR, and we found that only DAP, and Acenocoumarol significantly downregulating the ACE2 mRNA level (Fig. 1e).

Fig. 1.

High-throughput screening of small molecule compounds inhibit ACE2 expression. (a) Schematic representation of high-throughput screening to identify small-molecule compounds significantly affect ACE2 transcription. UCDA used as positive control. (Library screening concentration, 50 µM). (b) Two-dimensional analysis of components library screening.X-axis represented the percentage of renilla luciferase activity in the treated group relative to the untreated group. Y-axis represented the relative luciferase activity .Y = 0.75 indicates that relative luciferase activity decreased by 25% after drug treatment. X = 50 indicates that the internal reference gene expression level after drug treatment is 50% of that in the untreated group. The points marked in red are Eupatilin, Resveratrol, and Hesperetin that up-regulate the relative luciferase activity.The points marked in green in the figure are Mycophenolate mofetil, Daphnetin, and Acenocoumarol, respectively, three drugs that reduce the relative luciferase activity. (c, d) Luciferase reporter assay in PC9 cells showing the transcriptional activity in the ACE2 promoter upon treatment with Acenocoumarol, DAP, Mycophenolate mofetil, Eupatilin, Reseveratrol and Hesperetin, (concentration, 50 µM). n = 3 independent experiments; one-way ANOVA adjusted for multiple comparisons; bars, standard deviations. (e) qPCR showing the levels of ACE2 upon treatment with Acenocoumarol, DAP, Mycophenolate mofetil, Eupatilin, and Reseveratrol in human PC9 cells (50 µM) and the effect increases with increasing concentration. Housekeeping gene, GAPDH; n = 3 independent samples; n = 3 independent experiments; one-way ANOVA adjusted for multiple comparisons; bars, standard deviations.

DAP and its analogs regulate ACE2 expression in vivo

DAP and Acenocoumarol belongs to coumarins, and we chose its analogues such as Daphnoretin (DT), umbelliferone (UMB) and 4-methyl umbelliferone (4-MU) to test the effect on ACE2 expression (Fig. 2a). We found that the basic structure of umbelliferone is necessary to inhibit the expression of ACE2, and the modification of methyl group at position 4 decreased the activity (Fig. 2b). DT belongs to dicoumarin, but it significantly increased the level of ACE2 mRNA. We also found that DAP more than UCDA could significantly inhibit the transcription of ACE2 mRNA (Fig. 2b). The dose curve of the effect of DAP on the expression of ACE2 mRNA showed that 10–100 µM could significantly down-regulate the expression of ACE2 mRNA, and the higher the dose of DAP, the more obvious the down-regulation of ACE2 mRNA expression (Fig. 2c). We then detected the expression of ACE2 on the cell surface of cultured PC9 cells, and found that DAP and UMB significantly reduced the expression of ACE2 on the cell surface of PC9 (Fig. 2d-e). The subsequent western blot analysis of whole cell lysate suggested that the expression of ACE2 on the cell surface of PC9 was caused by the decrease of total protein expression of ACE2 (Fig. 2f). Subsequent SARS-CoV-2 pseudovirus infection experiments confirmed that the decrease of ACE2 expression on the surface of PC9 cells after DAP treatment significantly inhibited the infection of PC9 cells with the same titer of SARS-CoV-2 pseudovirus (Fig. 2g).

Fig. 2.

DAP inhibits ACE2 expressionin vitro. (a) Molecular structure of Daphnoretin (DT), Umbelliferone (UMB), Daphnetin (DAP), 4-Methyumbelliferone (4-MU). (b) qPCR showing that the effects of different drug treatments (concentration, 50µM) on ACE2 expression in human PC9 cells. Housekeeping gene, GAPDH; n = 3 independent samples; Unpaired two-tailed t-test; error bars, s.d. (c) qPCR showing that treatment with DAP reduces the levels of ACE2 in human PC9 cells and the effect increases with increasing concentration. Housekeeping gene, GAPDH; n = 3 independent samples; Unpaired two-tailed t-test; error bars, s.d. (d) Immunofluorescence staining for ACE2 in human PC9 cells after DAP, UMB treating (48 h). n = 3 independent samples. Observed by confocal microscope, 63x oil mirror. DAP concentration, 100µM; UMB concentration, 200µM. (e) Quantitative analysis of (d). (f) Immunoblot for ACE2 in PC9 cells after treatment with DAP, UMB for different days. (g) Pseudovirus infection of PC9 cells-treated with DAP for 48 h.

DAP reduces ACE2 in mice and humans

We want to further understand whether DAP can reduce the expression of ACE2 protein in the respiratory system of mice. We compared ACE2 expression in the respiratory of mice treated with DAP vs. control mice. Because the standard therapeutic dosage of DAP in clinic is 18 mg/kg/day, then we set the DAP dose as 9, 18, and 36 mg/kg/day for 3 days (Fig. 3a). Our results indicate treat mice with DAP at low or routine dose (9 and 18 mg/kg/day) can significantly decrease the ACE2 mRNA expression level in Lung (Fig. 3b). Immunofluorescence staining of ACE2 in mouse lung tissue also clearly confirmed that DAP treatment could significantly inhibit the expression of ACE2 protein (Fig. 3c-d).

Fig. 3.

DAP inhibits ACE2 expressionin vivo. (a) Schematic of the experiment performed in C57 mice. (b) qPCR showing that gavage treatment of different doses of DAP for 3 days reduces the levels of ACE2 in mice lung. Housekeeping gene, GAPDH; n = 3 independent samples; Unpaired two-tailed t-test; error bars, s.d. (c) The typical immunofluorescence staining images of mice lung tissue. Control group (saline, n = 3) vs. experimental group (DAP, n = 3). Nikon fluorescence microscope, 20x. (d) Quantitative analysis of (c). (e) Schematic representation of the study design. 4 healthy individuals received 18 mg per kg per day of DAP for 5 days. ACE2 levels were measured by qPCR in nasal epithelial cells collected with nasal swabs. Day 0 corresponds to samples collected immediately before starting DAP treatment. Samples were collected during drug administration and again at day 30–33 and 35–40 to assess the washout of DAP. (f) QPCR measurement of the levels of ACE2 in nasal epithelial cells collected with nasal swabs. Each dot represents one individual measurement; lines connect dots from the same individual. Housekeeping gene, GAPDH; n = 4 individuals.

Because of the differences in drug distribution and drug metabolism between human body, cultured cells, and mice, it is necessary to further test whether the clinical dose of DAP is enough to down-regulate the expression of ACE2 in respiratory tract of human being. This will be the key experiment whether DAP can prevent SARS-CoV-2 infection in human being. In view of the fact that DAP is a commonly used prescription drug in China and its long-term safety has been widely confirmed, we recruited six volunteers from the Wannan Medical college and treated them with DAP at the standard therapeutic dosage of 18 mg per kg per day for 5 days (Supplementary Table 4). The nasal epithelial cells of the volunteers were collected using nasopharyngeal swabs, and the levels of ACE2 were measured at multiple time points before, during and after treatment with DAP (see Methods and Fig. ​Fig. 3e). Results showed that in humans, DAP also significantly reduces the levels of ACE2 in the nasal epithelium, which is a prime site of SARS-CoV-2 infection (Fig. 3f). We had registered the clinical study in China Clinical Research Registration Center, the registration number is ChiCTR2300077554 (13/11/2023).

DAP control ACE2 levels through inhibit HIF-1α signaling pathway

Because even though DAP has been used clinically in China for more than 40 years to treat vasoocclusive vasculitis, its target protein has not been clearly proved. We want to further determine through which target protein DAP affects the expression of ACE2. Referring to the methods in our previous work¹². We performed transcriptome sequencing, and the Volcano plot showed that the up-regulated (764) differential genes (DEGs) were basically equal to those down- regulated (705) in DAP treatment vs. control. Heatmap of DEGs showed that the up-regulation and down-regulation trends of the groups treated with DAP and DMSO were completely opposite, and the differences within each three groups were small, suggesting that the sequencing results were reliable. By analyzed the down-regulated DEGs, the main non-redundant KEEG and GO terms enriched in DAP treated group were related to HIF-1 pathway, NABA CORE MATRISOME, Glycolysis, cell migration, and et, al (EX-Fig. 2.c, d). We also found that the down-regulated terms of PC9 cells treated with DAP were all can be regulated by HIF1 transcription factors (Fig. 4a, Supplementary Material 3). It has been reported that the upstream regulatory sequence of ACE2 has a binding site of HIF-1α, and the increased activity of HIF-1α transcription factor can significantly promote the expression of ACE2¹³. So, we first use small molecule docking software to test whether HIF-1α can be combined with DAP. We found that DAP can bind to HIF-1α amino acid 264–313 domain (Fig. 4b-d), which is responsible for forming dimer with HIF-1β to combine into transcription-active HIF1 complex. Molecular docking analysis indicates that the C/O atoms of the DAP compound can form 3 hydrogen bonds with the H on ARG 311 (positively charged) of HIF1a (Fig. 4d). We also measured the binding kinetics of DAP to purified HIF-1α using Surface Plasmon Resonance (SPR, Biacore) methods. As shown in Fig. 4e, DT binds to HIF-1α with a fast-on and fast-off rate and a measured KD of 38.93 µM (Fig. 4f). Above results indicated that DAP is an inhibitor of HIF-1α. The HIF-1α agonist ml228¹⁴ or HIF-1α inhibitor Bavachinin¹⁵ also correspondingly significantly promote or inhibit the expression level of ACE2 promoter reporter gene (Fig. 4g-i). Furthermore, Cocl2 and ml228, inducers of HIF1a, can significantly induce the expression of HIF1a and further promote the increase of ACE2 expression, which can be inhibited by DAP (Fig. 4j-k).

Fig. 4.

The DAP direct binding with HIF-1α and as inhibitor to downregulate ACE2 expression. (a) DAP treated PC9 cells down-regulated genes governed by TFs predict by Metascape. (b) DAP binding to HIF1α in a cartoon model and a cartoon plus surface model (c) (PDB: 4H6J). (d) The predicted position of the direct binding of DAP and HIF-1α. (e) SPR (relative units (RU)) observed when immobilized HIF1α was exposed to a series of concentrations of DAP. (f) Steady State Analysis of SPR assays shown in Fig. 4e. (g-h) Luciferase reporter assay in PC9 cells showing the transcriptional activity in the ACE2 promoter upon treatment with HIF-1α activator ml228 (1 µM). (g), or HIF-1α inhibitor Bavachinin (20 µM) (h) for 24 h. (i) Relative mRNA levels of ACE2 in PC9 cells after HIF-1α inhibitor Bavachinin (20 µM) treated for 48 h. (j-h) Relative mRNA levels of HIF-1α (j) or ACE2 (h) in PC9 cells after different treatments for 48 h. Concerntration: Cocl2 (300 µM), DAP (100 µM), ml228 (1 µM); “Cocl2 + DAP” and “ml228 + DAP” mean those two drugs are added at the same time. N = 3 independent experiments; Unpaired two-tailed t-test; bars, mean ± standard deviation; *,P < 0.05; **,P < 0.01; ***,P < 0.001; ****,P < 0.0001.

DAP as an antagonist of JAK2 to inhibit STAT3 activation induced ACE2 expression

Since DAP is more effective than UCDA in inhibiting the expression of ACE2, we speculate that it may have multiple targets. Our NGS data also suggest that DAP treatment inhibit JAK2/STAT3 signaling pathway. Inhibitors (Fedratinib (Fed) and Brevilin A (6-OAP)) of JAK2/STAT3 signaling pathway can also effectively inhibit the expression of ACE2 (Fig. 5a). Because JAK2/STAT3 signaling pathway has been reported to regulate the expression of ACE2^16,17. Therefore, we further studied the influence of DAP on JAK2/STAT3 signaling pathway. Using small molecule reverse docking virtual screening method, we use DAP to dock JAK2/STAT3 signaling pathway protein set, suggests that DAP may bind to JAK2. DAP predominantly interacts closely with JAK2 ATP binding domain and establishes 2 hydrogen bond interactions (red), 6 hydrophobic interaction (yellow) and 2 Covalent bond (green) (Fig. 5d). We found that DAP can (Fig. 5b-d) inhibit JAK2 phosphorylation, and then the activation of STAT3 is inhibited (Fig. 5e), and finally the transcription of ACE2 mRNA is inhibited. More interestingly, we reported that DT is an agonist of JAK2¹², and DT treatment can promote the transcription of ACE2 mRNA (Fig. 2b) and increase the level of ACE2 mRNA.

Fig. 5.

DAP is a new JAK2 antagonist and inhibits JAK2/STAT3 signaling pathway to down-regulate ACE2 expression. (a) Relative mRNA levels of ACE2 in PC9 cells with different treatments for 2 days. Fed, 100 nM; 6-OAP, 2.5 µM; DAP, 50 µM. (b) Molecular docking of DAP and JAK2 in a cartoon model (PDB: 2B7A). (c) DAP binding to JAK2 in a surface model. (d) Molecular docking assay predicted position of the direct binding of DAP and JAK2. (e) Western blot analyses of ACE2 in PC9 cells with different treatments for 12 h.

Discussion

Virus infection necessitates the involvement of cell surface receptors, with the level of expression and mutations of these receptors potentially influencing the ability of the virus to infect host cells. For instance, HIV relies on the CCR5 receptor for infection, and an elevated expression level of this receptor indicates heightened susceptibility to the virus¹⁸. Conversely, individuals with a 32AA deletion mutation in the CCR5 receptor are unable to bind with HIV, thereby rendering them resistant to infection¹⁹. Consequently, targeting the host ACE2 protein for direct inhibition may prove to be an effective therapeutic strategy.

Data from our library screening indicates that Resveratrol may significantly enhance the expression of ACE2, potentially increasing susceptibility to Covid-19 infection in individuals who have been taking both drugs for an extended period. Conversely, our research shows that Mycophenolate mofetil (MMF) is a potent inhibitor of ACE2 expression. MMF, commonly used as a therapeutic drug for autoimmune diseases, possesses immunosuppressive properties that can lead to serious infections as a side effect²⁰, Subsequent qRTPCR experiments revealed that MMF does not decrease the level of ACE2 mRNA, suggesting a post-transcriptional mechanism at play. Therefore, we have excluded this drug as a potential candidate for preventing SARS-CoV-2 infection. Additionally, we have identified DAP and Acenocoumarol as the next most promising drugs following MFF. These monocoumarins share similar structures and have demonstrated long-term use without significant adverse effects. DAP has shown various pharmacological properties, such as analgesic, anti-pyretic, anti-arthritic, anti-inflammatory, and antioxidant effects. However, the specific target of DAP remains unclear, leading to a lack of understanding of its detailed biological mechanism²¹. By analysis our NGS data, it was determined that DAP primarily inhibits the activation of HIF-1α transcription factors. Given that the HIF-1α signaling pathway is known to regulate the expression of ACE2 ¹³, further investigation was conducted to assess the impact of DAP on this pathway. Specifically, the translocation of HIF-1α to the nucleus and its dimerization with HIF-1β are essential for enabling the hypoxic response²². Our findings indicate that DAP binds to the amino acid domain 264–313 of HIF-1α, which is crucial for dimer formation with HIF-1β to form the transcriptionally active HIF1 complex. This binding of DAP to HIF-1α ultimately inhibits its activity. Interestingly, recent reports indicate that SARS-CoV-2 infection can enhance the expression and stability of hypoxia-inducible factor-1α (HIF-1α)^22,23. The upregulation and stabilization of HIF-1α promote glycolysis²⁴, which in turn facilitates SARS-CoV-2 infection and cytokine production^25,26. The resultant cytokine storm, triggered by the activation of the HIF-1α and subsequently glycolysis, can lead to severe respiratory distress syndrome and potentially result in patient mortality²⁷. Our transcriptome sequencing results suggest that the down-regulation of HIF-1 signaling pathway (EX-Fig. 2. c, d) caused by DAP treatment will be accompanied by the down-regulation of key genes related to glycolysis (EX-Fig. 2. e, f, d). Consequently, the inhibitory effect of DAP on the HIF signaling pathway and subsequently glycolysis presents a promising therapeutic avenue for treating patients with severe SARS-CoV-2 infection. Of course, these needs confirming by very rigorous clinical experiments in the future.

The effectiveness and safety of multi-target drugs surpass those of single-target drugs, and the design of new antiviral medications often adheres to a multi-target approach²⁸. Based on our findings, DAP appears to be a promising multi-target molecule. DAP has the capability to bind to the ATP binding domain of JAK2, thereby inhibiting JAK2 phosphorylation, leading to the inhibition of STAT3 activation and subsequently the transcription of ACE2 mRNA.

This discovery presents a potential therapeutic option for preventing SARS-CoV-2 entry into cells. Importantly, the mechanism of action of this drug on host cells renders it unaffected by viral mutations, ensuring continued efficacy even in the presence of new mutations. Additionally, the efficacy of DAP as a treatment option may surpass that of other interventions such as vaccines and monoclonal antibodies. DAP is characterized by its ease of oral administration, convenient storage, affordability, and suitability for large-scale production. Moreover, DAP exhibits good tolerability and safety profiles, allowing for prolonged use. Notably, DAP has a history of use in China among vulnerable populations, such as individuals with coronary heart disease and thromboangiitis obliterans, demonstrating its favorable tolerability and minimal adverse effects in these patients²⁹. This suggests the potential utility of DAP as a prophylactic agent against COVID-19 in vulnerable populations. We posit that DAP may emerge as a valuable tool in combating SARS-CoV-2 infection.

In conclusion, the findings indicate that DAP, an approved medication for other clinical conditions, has the potential to serve as a novel preventive agent against SARS-CoV-2 infection. By significantly reducing ACE2 expression within its therapeutic dosage range, DAP could provide a cost-effective and readily available strategy for mitigating the spread of the virus and the severity of the disease. The results support the further investigation and potential clinical application of DAP as a preventive measure in the ongoing fight against COVID-19.

Materials and methods

Materials

All antibodies used in the experiments are list in Supplementary Table 1. All chemicals used in the experiments are list in Supplementary Table 2.

Cell culture

All the cell lines used in this study were purchased from the cell bank of Institute of Biochemistry, Chinese Academy of Sciences, with STR identification certificates. The culture method and culture condition in strict accordance with the cell bank of Institute of Biochemistry’s protocol. Three days before RNA extraction to identify the expression of ACE2, all cell culture media were replaced by Dulbecco’s Modified Eagle Medium (DMEM, D0822, Sigma-Aldrich) + 10% fetal bovine serum (FBS, Biological Industries). PC9 cell line was cultured with DMEM + 10% FBS.

Luciferase reporter assay

hACE2 URS containing (-2069/+20) of ACE2 were cloned into pGL6-control vectors (Junji) which was comprised of firefly luciferase (F-luc). The details about reporter plasmids are showed in Ex-Fig. 3. PC9 cells were transfected with 5 µg reporter plasmid and 0.2ug pRL-TK plasmid (renilla luciferase reporter vector) using an electroporator (Lonza) and then inoculated into 96-well plates. After transfection for 24 h, the cells were treated with drugs for 24 h .Cells were harvested to access the luciferase activity using Dual Luciferase Reporter Gene Assay Kit (Beyotime) with the normalization to pRL-TK.

Real-time fluorescent quantitative PCR

Total RNA was extracted from cells with the TRIzol (Invitrogen), chloroform and other reagents, the concentration and purity of RNA were determined. Then 1 µg RNA was reverse transcribed into cDNA according to the steps of PrimeScript™ RT reagent Kit (TaKaRa), and qPCR was performed by SYBR Green qPCR Mix kit (biosharp) with specific primers (Supplementary Table 3) on a real-time fluorescent quantitative PCR machine (Bio-rad). The results were normalized to housekeeping genes. All qPCR reactions were performed in triplicate.

Surface labeling of membrane proteins

Surface labeling of membrane proteins was carried out as previously described^12,30. PC9 cells were initially seeded in confocal dishes and cultured to an appropriate density. Subsequently, the cells were exposed to various drugs (experimental group: DAP, DT, UMB, UDCA, 4-MU; control group: DMSO) for a duration of 48 h. Following this treatment period, the original medium was replaced with media containing anti-ACE2 antibodies (diluted 1:100 in DMEM with 1% BSA and 5% FBS), and the cells were incubated at 37 °C for 60 min. The samples were then washed three times with 1x PBS at room temperature, fixed with 4% PFA for 30 min, and washed an additional three times. Subsequently, the cells were incubated with secondary antibodies for 2 h. Imaging was performed using a confocal microscope equipped with a 63x oil immersion lens.

Western blot

RIPA Lysis Buffer (Beyotime) containing Protease inhibitor (Roche) was prepared on ice, and total cellular protein was extracted by adding suitable Lysis Buffer according to the amount of cells. Part of the protein supernatant was used to determine the protein concentration using BCA Protein Assay Kit (Biosharp). Equal amounts of protein samples (around 20 µl per lane) were separated with 4–12% SDS-PAGE (GenScript) and then transferred to 0.22 μm PVDF membrane (Millipore). After 30 min of blocking (QuickBlock™ Blocking Buffer, Beyotime), the membrane was incubated with corresponding protein antibody at 4 °C overnight. TBST was washed 3 times for 5 min each time and co-incubated with the corresponding secondary antibody for 2 h at room temperature. After washing again, the immunoblotting was detected with Enhanced ECL chemiluminescence kit (Servicebio) on an imaging system (Bio-rad). All the antibodies employed in this study were listed in Supplementary Table 1.

SARS-CoV-2 pseudovirus infection

PC9 cells were uniformly seeded in 96-well plates, and the drug was added when the cell density fused to 30–40%. The experimental group was replaced with fresh medium containing 100 µM DAP and the control group was replaced with fresh medium containing DMSO. Three wells in each group were cultured for 48 h. Then 10 µl SARS-CoV-2-Spike(B.1.1.529) (GFP-Luciferase) Pseudovirus (1 × 10⁶ TU/ml, viral titer test with HEK293T-ACE2) (Yeasen Biotechnology Co., Ltd, Shanghai) was added to each well and put back into the incubator for infection. Observe and photograph after 48 h.

Animal experiments

Animal experiments conducted in accordance with the regulations on the management of experimental animals issued by the State Commission for Science and Technology and the implementing rules on the management of medical experimental animals issued by the Ministry of Health of China, with the approval of the Laboratory Animal Welfare and Ethics Committee of Wannan Medical College (WNMC-AWE-2023459). All experimental procedures were done in compliance with the ARRIVE guidelines (PLoS Bio 8(6), e1000412,2010). C57 mice were obtained from Henan SKBEX Biotechnology Co., Ltd (Henan, China), and housed in a 12-hour-to-12-hour light-dark alternating SPF animal house with a humidity of 45–65% and a temperature of 20–24 ° C. Regular mice food and drinking water. Age-matched male mice were used. Mice were randomly divided into experimental group and control group; we used 3 mice per group. Sample sizes were determined based on previous experimental experience. No samples were excluded from the analysis. DAP (9 mg/kg/d, 18 mg/kg/d, 36 mg/kg/d) was given by gavage to mice in the experimental group and normal saline was given to mice in the control group. Three days later, Mice were euthanatized by cervical dislocation after inhaling isoflurane anesthesia and perfused with normal saline, the expression of ACE2 was detected by RNA extraction from lung tissue. At the same time, the tissue was made into frozen sections for ACE2 immunofluorescence staining, and the fluorescence pictures were taken under the condition of the same exposure time and the same emission energy. Three blind cases on the data statistical analysis.

Immunofluorescence staining of frozen sections

The tissues place in 4% paraformaldehyde, 20% sucrose and 30% sucrose solution respectively for dehydration and precipitation, after 12 h, embedded with OCT. The thickness of frozen section machine was 20 μm, and the tissues were stored at room temperature for more than 30 min to prevent from sloughing for further experiments or stored at -20 °C. Frozen sections from the refrigerator were stored at room temperature for 30 min before. Sections was washed in 1 x PBS three times for 3 ~ 5 min each time, and antigen retrieval was performed for 15 min. The whole tissue sections were circled with immune-histochemical pen, then blocked with QuickBlock™ Blocking Buffer (Beyotime) for immunol staining, 37 °C for 1 h, and incubated with primary antibody (ACE2), 4 °C overnight. The next day, sections was washed with 1 x PBS and the secondary antibody was incubated at room temperature for 2 h. washing 3 times with 1x PBS, Anti-fluorescence quenching reagent (Biosharp) was added. then seal the section.

ACE2 measurement in nasal epithelial cells of volunteers recruitment

This study was approved by the Medical Ethics Committee of Wannan Medical College to advertise for volunteers among Wannan Medical College teachers and students (Ref. No. 2023 − 205). The study was registered in China Clinical Research Registration Center, the registration number is ChiCTR2300077554. The inclusion criteria were about 160 cm for girls and 170 cm for boys, with a BMI of 18.5-24.99. The exclusion criteria were postoperative hemorrhage, hepatic and renal insufficiency, severe hypertension, pregnant and lactating women, and allergic to DAP capsules (Xidian medicine company). Six students and teachers were recruited with informed consent.

Human study design and exclusion criteria

DAP capsules were given twice daily for 5 days at a clinically approved dose of 18 mg per kilogram per day (Supplementary Table 4). Nasal epithelial samples for ACE2 measurement were collected using nasal swabs on day 0 (before the first DAP administration), day 3 and 5 (before DAP administration), respectively. After drug clearance, samples were taken again on day 33 and 40. For samples with no detectable RNA, their volunteers were excluded from the study. ACE2 mRNA level detection. RNA was extracted from samples taken from nasal swabs using the TIANamp Virus DNA/ RNA Fast Kit (TIANGEN) as described in the instructions, and ACE2 mRNA levels were measured using qPCR. The results show the relative expression of housekeeping genes. The experiments have been performed in accordance with the Declaration of Helsinki. This study was approved by the Medical Ethics Committee of Wannan Medical College to advertise for volunteers among Wannan Medical College teachers and students (Ref. No. 2023 − 205). The study was registered in China Clinical Research Registration Center, the registration number is ChiCTR2300077554. The registration date is 13/11/2023.

Surface plasmon resonance

HIF-1a protein for SPR were purchased from abcam (ab154478). BSA were immobilized on a CM5 chip using a Biacore T200 biosensor with a blank channel as a negative control. Binding assays were performed at 25 °C using Biacore buffer (10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES; pH 7.4), 150 mM NaCl, 0.05 mM ethylenediaminetetraacetic acid (EDTA)) containing the indicated small molecules injected at 30 µl per min for 1 min using a Biacore T200 Control Software. Proteins were allowed to dissociate during perfusion with Biacore buffer at 30 µl per min for 10 min and then desorbed with 2 M MgCl₂. All binding affinities were derived by Biacore T200 Evaluation Software using Steady State analysis (Biacore, Uppsala, Sweden).

Molecular docking

Haddock2.4 online software was used for protein-ligand docking^31,32. Docking was carried out according to the optimized parameters for protein ligand docking. From the top ten clusters with the best docking results, the number 1 best structure with the highest absolute value of Z-Score and the largest Cluster size was selected as the reliable docking result for further analysis.

Statistical analysis

All data were presented as mean ± SEM for at least 3 independent experiments. Statistical analysis using GraphPad Prism 7. The data were analyzed by unpaired two-tailed student’s t-test and analysis of variance. P < 0.05 was considered significant.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Abbreviations

COVID‑19

Coronavirus disease 2019

SARS‑CoV‑2

Severe acute respiratory syndrome coronavirus 2

ACE2

Angiotensin-converting enzyme 2

DAP

Daphnetin

UDCA

Ursodeoxycholic acid

URS

Upstream regulatory sequence

UMB

Umbelliferone

4-MU

4-methyl umbelliferone

Author contributions

“Xiao-hui Xu and Zhao-huan Zhang designed experiments; Qian-wen Yang, Chang-ling Yue, Lei hu, Cao Yin and Xu Zhao carried out the cell culture, cell transfection, RT-PCR and western blot experiments. Xiao-hui Xu, Yan-xia Ding, Lei Hu analyzed the NGS data. Meng Chen，Yun-yun Ling and Ying-xin Zhou conducted the docking and binding assay experiments. Lei Hu, Xu Zhao and Hai Huang cultured cells and done the IHC experiments and animal experiments. Xiao-hui Xu, Lei Hu and Zhao-huan Zhang wrote the manuscript. All authors reviewed the manuscript.”

Funding

This work was supported by the National Natural Science Foundation of China (no. 81671263), the Scientific Research and Innovation Team, Education Department of Anhui Province, China (no. 2023AH010072), the Anhui Provincial Natural Science Foundation(no. 2208085MH221), the Key Scientific Research Program of Wannan Medical College (WK2020Z16), the Key Projects for National Science Research of Education Department of Anhui Province (No.KJ2021A0851, KJ2021A0825, 2024AH051929) and the Scientific research project of brain science research institute of Wannan Medical College.

Data availability

The data within the manuscript or supplementary information files are available from the corresponding author upon reasonable request. Please contact Dr. Xu: xhxu@wnmc.edu.cn.

Declarations

Competing interests

The authors declare no competing interests.

Clinical ethics statement

Our study was conducted in accordance with the principles of the Declaration of Helsinki and was approved by the Medical Ethics Committee of Wannan Medical College to advertise for volunteers among Wannan Medical College teachers and students (Ref. No. 2023 − 205). The study was registered in China Clinical Research Registration Center, the registration number is ChiCTR2300077554. The registration date is 13/11/2023.

Animal ethics statement

Animal experiments conducted in accordance with the regulations on the management of experimental animals issued by the State Commission for Science and Technology and the implementing rules on the management of medical experimental animals issued by the Ministry of Health of China, with the approval of the Laboratory Animal Welfare and Ethics Committee of Wannan Medical College (WNMC-AWE-2023459). All experimental procedures were done in compliance with the ARRIVE guidelines (PLoS Bio 8(6), e1000412,2010).