Screening and evaluation of anti-SARS-CoV-2 components from Ephedra sinica by ACE2/CMC-HPLC-IT-TOF-MS approach

Abstract

Traditional Chinese medicines played an important role in the treatment of COVID-19 in 2020. Ephedra sinica, one of the major constituent herbs of multi-component herbal formula, has been widely used to treat COVID-19 in China. However, its active components are still unclear. The objectives of this study are to screen and evaluate active components from the traditional Chinese medicine Ephedra sinica for the treatment of COVID-19. In our study, we established an ACE2/CMC bioaffinity chromatography model, and then developed an ACE2/CMC-HPLC-IT-TOF-MS system for the active compounds screening and identification from Ephedra sinica extract. We performed molecular docking and surface plasmon resonance (SPR) assays to assess the binding characteristics (binding mode and K_D value). We used CCK-8 staining to assess the toxicity of screened compounds, and also used SARS-CoV-2 pseudovirus to observe the viropexis effect of screened compounds in ACE2^h cells. In this current work, one fraction was fished out, separated and identified as ephedrine (EP), pseudoephedrine (PEP), and methylephedrine (MEP). Binding assays showed that the three compounds could bind with ACE2 in a special way to some amino acid residues, similar to the way SARS-CoV-2 bound with ACE2. Additionally, the three compounds, especially EP, can inhibit the entrance of SARS-CoV-2 spike pseudovirus into ACE2^h cells because they can reduce the entrance ratio of pseudovirus in the pseudovirus model. Overall, the ACE2/CMC-HPLC-IT-TOF-MS system was established and verified to be suitable for ACE2-targeted bioactive compound screening. EP, PEP, and MEP with ACE2-binding features were screened out from Ephedra sinica, and acted as blockers inhibiting SARS-CoV-2 spike pseudovirus entering ACE2^h cells.

Keywords: ACE2 receptor; COVID-19; Cell membrane chromatography; Ephedra sinica.

Fig. 1

Validation of the ACE2/CMC-HPLC-IT-TOF-MS system by hydroxychloroquine. (a) Chromatogram of hydroxychloroquine retained on the ACE2/CMC column; (b) HPLC-IT-TOF-MS chromatogram of the retained fraction (R₁); (c) Direct HPLC-IT-TOF-MS chromatogram of hydroxychloroquine

Fig. 2

Application of the coupled two-dimensional system. (a) Chromatogram of EME retained on the CMC column. (b) Total ion current chromatogram of the retained fraction (R₁) on the HPLC-IT-TOF-MS system. (c) Direct total ion current chromatogram of EME on the HPLC-IT-TOF-MS system

Fig. 3

Chromatograms of the standard solutions of EP, PEP and MEP analyzed by ACE2/CMC coupled with HPLC-IT-TOF-MS system. (a) Chromatogram of standard solutions retained on the ACE2/CMC column. (b) HPLC-IT-TOF-MS chromatogram of the retained fraction (R₁). (c) Direct HPLC-IT-TOF-MS chromatogram of the standard solutions

Fig. 4

Measurement of binding affinities between the three compounds (EP, PEP and MEP) and human ACE2 (left column) or SARS-CoV-2 RBD (right column) by surface plasmon resonance assay. Purified recombinant SARS-CoV-2 RBD or recombinant human ACE2 were covalently immobilized to the sensor chip via their amine groups, and EPs flowed by. Here EPs was diluted to different concentrations (from 12.5 to 100 μM) before being injected. Data are shown as different colored lines

Fig. 5

Structural details at the interface between the three compounds (EP, PEP, and MEP) and human ACE2 (PDB ID: 6M0J). Amino acid residues in green present the same ACE2-binding sites with SARS-CoV-2 RBD reported. Amino acid residues in purple present the different ACE2-binding sites with SARS-CoV-2 RBD

Fig. 6

Effect of EP, PEP, or MEP on the viability of ACE2^h cells. (a) Viability of ACE2^h cells treated with EPs for 24 h. (b) Calcium flux change in ACE2^h cells treated with EPs

Fig. 7

Effect of EP, PEP and MEP on the entrance of SARS-CoV-2 spike pseudovirus into ACE2^h cells. Inset is the graph of EP. Data are presented as mean ± SD. *P ˂ 0.05, **P ˂ 0.01, ***P ˂ 0.001 compared with group Control