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 (ACE2
h
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 ACE2
h
cells.
Results
Results showed that HCQ is slightly more toxic to ACE2
h
cells than CQ. Both CQ and HCQ could bind to ACE2 with
K
D
= (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 ACE2
h
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.
The current worldwide outbreak of the coronavirus disease 2019 (COVID‐19) has been declared a public health emergency. The angiotensin‐converting enzyme II (ACE2) has been reported as the primary host‐cell receptor for severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2), the causative virus of COVID‐19. In this study, we screened ACE2 ligands from Radix Scutellariae and investigated its suppressive effect on SARS‐CoV‐2 spiked pseudotyped virus in vitro. HEK293T cells stably expressing ACE2 receptors (ACE2 cells) were used to provide the receptor for the ACE2/cell membrane chromatography (CMC) method used for analysis. The SARS‐CoV‐2‐spiked pseudotyped virus was used to examine the anti‐viropexis effect of the screened compounds in ACE2 cells. Molecular docking and the surface plasmon resonance (SPR) assay were used to determine the binding properties. Oroxylin A exhibited an appreciable suppressive effect against the entrance of the SARS‐CoV‐2‐spiked pseudotyped virus into ACE2 cells, which showed good binding to ACE2 as determined using SPR and CMC. Oroxylin A was shown to be a potential candidate in the treatment for COVID‐19 by virtue of its blocking the entrance of SARS‐CoV‐2 into ACE2 cells by specifically binding to the ACE2 receptor.
Mast cells play an important role in inflammatory and allergic diseases. MAS-related G protein-coupled receptor X2 (MRGPRX2) is a novel G protein-coupled receptor in mast cells that mediates drug-induced anaphylactoid reactions. Piperine has been reported to have anti-inflammatory and anti-allergic pharmacological activities. However, whether the pharmacological effects are regulated by MRGPRX2 has not yet been reported. The purpose of this study was to assess the anti-anaphylactoid effect of Piperine and to explore its potential mechanism. The anti-anaphylactoid effect of Piperine was assessed by an in vivo mouse hindpaw extravasation model. Mast cell intracellular calcium mobilization was measured by a calcium imaging assay. An enzyme immunoassay was used to evaluate the release of pro-inflammatory factors from stimulated mast cells. Activated mast cell related signals were assessed by western blot. A cell membrane chromatography assay was used to determine the binding characteristics of Piperine and MRGPRX2. The results showed that Piperine suppressed mast cell intracellular Ca 2+ mobilization, inhibited cytokines and chemokines release, and down-regulated the phosphorylation level of phospholipase Cγ1, protein kinase C, inositol 1,4,5-triphate receptor, P38, protein kinase B, and ERK. Meanwhile, Piperine can bind to MRGPRX2 as a specific antagonist. Hence, Piperine can be served as a novel therapeutic drug candidate for MRGPRX2-mediated anaphylactoid reactions.
K E Y W O R D Santianaphylactoid effects, Ca 2+ influx, mast cells, MRGPRX2, Piperine
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.