Harringtonine: A more effective antagonist for Omicron variant.

Fusion with host cell membrane is the main mechanism of infection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we propose that a new strategy to screen small-molecule antagonists blocking SARS-CoV-2 membrane fusion. Using cell membrane chromatography (CMC), we found that harringtonine (HT) simultaneously targeted SARS-CoV-2 S protein and host cell surface TMPRSS2 expressed by the host cell, and subsequently confirmed that HT can inhibit membrane fusion. HT effectively blocked SARS-CoV-2 original strain entry with the IC50 of 0.217 µM, while the IC50 in delta variant decreased to 0.101 µM, the IC50 in Omicron BA.1 variant was 0.042 µM. Due to high transmissibility and immune escape, Omicron subvariant BA.5 has become the dominant strain of the SARS-CoV-2 virus and led to escalating COVID-19 cases, however, against BA.5, HT showed a surprising effectiveness. The IC50 in Omicron BA.5 was even lower than 0.0019 µM. The above results revealed the effect of HT on Omicron is very significant. In summary, we characterize HT as a small-molecule antagonist by direct targeting on the Spike protein and TMPRSS2.

Establishment of angiotensin-converting enzyme 2 and cluster of differentiation 147 dual target cell membrane chromatography based on SNAP-tag technology for screening anti severe acute respiratory syndrome coronavirus 2 active components

Patients have different responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections and these may be life-threatening for critically ill patients. Screening components that act on host cell receptors, especially multi-receptor components, is challenging. The in-line combination of dual-targeted cell membrane chromatography and a liquid chromatography-mass spectroscopy (LC-MS) system for analyzing angiotensin-converting enzyme 2 (ACE2) and cluster of differentiation 147 (CD147) receptors based on SNAP-tag technology provides a comprehensive solution for screening multiple components in complex samples acting on the two receptors. The selectivity and applicability of the system were validated with encouraging results. Under the optimized conditions, this method was used to screen for antiviral components in Citrus aurantium extracts. The results showed that 25 μmol /L of the active ingredient could inhibit virus entry into cells. Hesperidin, neohesperidin, nobiletin, and tangeretin were identified as antiviral components. In vitro pseudovirus assays and macromolecular cell membrane chromatography further verified the interaction of these four components with host-virus receptors, showing good effects on some or all of the pseudoviruses and host receptors. In conclusion, the in-line dual-targeted cell membrane chromatography LC-MS system developed in this study can be used for the comprehensive screening of antiviral components in complex samples. It also provides new insight into small-molecule drug-receptor and macromolecular-protein-receptor interactions.

Establishment of angiotensin-converting enzyme 2 and cluster of differentiation 147 dual target cell membrane chromatography based on SNAP-tag technology for screening anti severe acute respiratory syndrome coronavirus 2 active components.

Patients have different responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections and these may be life-threatening for critically ill patients. Screening components that act on host cell receptors, especially multi-receptor components, is challenging. The in-line combination of dual-targeted cell membrane chromatography and a liquid chromatography-mass spectroscopy (LC-MS) system for analyzing angiotensin-converting enzyme 2 (ACE2) and cluster of differentiation 147 (CD147) receptors based on SNAP-tag technology provides a comprehensive solution for screening multiple components in complex samples acting on the two receptors. The selectivity and applicability of the system were validated with encouraging results. Under the optimized conditions, this method was used to screen for antiviral components in Citrus aurantium extracts. The results showed that 25 µmol /L of the active ingredient could inhibit virus entry into cells. Hesperidin, neohesperidin, nobiletin, and tangeretin were identified as antiviral components. In vitro pseudovirus assays and macromolecular cell membrane chromatography further verified the interaction of these four components with host-virus receptors, showing good effects on some or all of the pseudoviruses and host receptors. In conclusion, the in-line dual-targeted cell membrane chromatography LC-MS system developed in this study can be used for the comprehensive screening of antiviral components in complex samples. It also provides new insight into small-molecule drug-receptor and macromolecular-protein-receptor interactions.

Investigating interactions between chloroquine/hydroxychloroquine and their single enantiomers and angiotensin-converting enzyme 2 by a cell membrane chromatography method.

Chloroquine and hydroxychloroquine have been studied since the early clinical treatment of SARS-CoV-2 outbreak. Considering these two chiral drugs are currently in use as the racemate, high-expression angiotensin-converting enzyme 2 cell membrane chromatography was established for investigating the differences of two paired enantiomers binding to angiotensin-converting enzyme 2 receptor. Molecular docking assay and detection of SARS-CoV-2 spike pseudotyped virus entry into angiotensin-converting enzyme 2-HEK293T cells were also conducted for further investigation. Results showed that each single enantiomer could bind well to angiotensin-converting enzyme 2, but there were differences between the paired enantiomers and corresponding racemate in frontal analysis. R-Chloroquine showed better angiotensin-converting enzyme 2 receptor binding ability compared to S-chloroquine/chloroquine (racemate). S-Hydroxychloroquine showed better angiotensin-converting enzyme 2 receptor binding ability than R-hydroxychloroquine/hydroxychloroquine. Moreover, each single enantiomer was proved effective compared with the control group; compared with S-chloroquine or the racemate, R-chloroquine showed better inhibitory effects at the same concentration. As for hydroxychloroquine, R-hydroxychloroquine showed better inhibitory effects than S-hydroxychloroquine, but it slightly worse than the racemate. In conclusion, R-chloroquine showed better angiotensin-converting enzyme 2 receptor binding ability and inhibitory effects compared to S-chloroquine/chloroquine (racemate). S-Hydroxychloroquine showed better angiotensin-converting enzyme 2 receptor binding ability than R-hydroxychloroquine/hydroxychloroquine (racemate), while the effect of preventing SARS-CoV-2 pseudovirus from entering cells was weaker than R-hydroxychloroquine/hydroxychloroquine (racemate).

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

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 KD 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 ACE2h 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 ACE2h 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 ACE2h cells.

Cell membrane chromatography for the analysis of the interaction between chloroquine and hydroxychloroquine with ACE2 receptors.

The recent emergence of the novel pathogenic coronavirus disease 2019 (COVID-19) is responsible for a worldwide pandemic. In sight of this, there has been growing interest in the use of chloroquine (CQ) and hydroxychloroquine (HCQ) as potential treatments. In this study, we use angiotensin converting enzyme 2 (ACE2) over-expressed cell membrane chromatography (CMC) to study the interaction of CQ and HCQ with ACE2 receptor. Both CQ and HCQ were retained on the ACE2/CMC column. Then we analyzed the binding character of CQ and HCQ to ACE2 by CMC frontal analysis, ionic force investigation and competitive binding experiment. Results showed that CQ and HCQ KD values obtained from the CMC frontal analysis method were 8.22(±0.61) × 10-7 M and 11.70(±2.44) × 10-7 M. Compare to CQ, HCQ has the weaker affinity with ACE2. The action force of CQ, HCQ and ACE2 is mainly ionic force. CQ and HCQ have different degrees of competitive binding relationship with ACE2. Our study revealed the interaction of CQ and HCQ with ACE2 receptor, which provides new insights for the use of CQ and HCQ in the treatment of COVID-19. Moreover, this biomimetic drug screening method is expected to open the door for rapid targeting and separating bioactive ingredients active towards ACE2 receptor.

Chloroquine and hydroxychloroquine as ACE2 blockers to inhibit viropexis of 2019-nCoV Spike pseudotyped virus.

BACKGROUND: The novel coronavirus disease (2019-nCoV) has been affecting global health since the end of 2019 and there is no sign that the epidemic is abating . The major issue for controlling the infectious is lacking efficient prevention and therapeutic approaches. Chloroquine (CQ) and Hydroxychloroquine (HCQ) have been reported to treat the disease, but the underlying mechanism remains controversial. PURPOSE: The objective of this study is to investigate whether CQ and HCQ could be ACE2 blockers and used to inhibit 2019-nCoV virus infection. METHODS: In our study, we used CCK-8 staining, flow cytometry and immunofluorescent staining to evaluate the toxicity and autophagy of CQ and HCQ, respectively, on ACE2 high-expressing HEK293T cells (ACE2h cells). We further analyzed the binding character of CQ and HCQ to ACE2 by molecular docking and surface plasmon resonance (SPR) assays, 2019-nCoV spike pseudotyped virus was also used to observe the viropexis effect of CQ and HCQ in ACE2h cells. RESULTS: Results showed that HCQ is slightly more toxic to ACE2h cells than CQ. Both CQ and HCQ could bind to ACE2 with KD = (7.31 ± 0.62)e-7 M and (4.82 ± 0.87)e-7 M, respectively. They exhibit equivalent suppression effect for the entrance of 2019-nCoV spike pseudotyped virus into ACE2h cells. CONCLUSIONS: CQ and HCQ both inhibit the entrance 2019-nCoV into cells by blocking the binding of the virus with ACE2. Our findings provide novel insights into the molecular mechanism of CQ and HCQ treatment effect on virus infection.