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

Abstract

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.

Keywords: 2019-nCoV; ACE2; Chloroquine; Hydroxychloroquine.

Fig. 1

Effect of CQ and HCQ on the viability of ACE2^h cells. A. Western blotting analysis of the expression levels of ACE2 protein in EOL-1 cells, AT2 cells, HSAEpC cells, and ACE2^h cells. B. Viability of ACE2^h cells treated with CQ or HCQ for 24 h. C. The toxicity of HCQ and CQ on ACE2^h cells at different time points. D. Calcium (Ca²⁺) flux change in ACE2^h cells. E. The apoptosis of ACE2^h cells treated with CQ or HCQ for 24 h. The experiments were repeat three times. Data are presented as mean ± S.D. (* p < 0.05, ** p < 0.01, ***p < 0.001, compared with HEK293T, or concentration was 0, or HCQ 20 μM, ^#p < 0.05 compared with HCQ 10 μM at corresponding time points).

Fig. 2

Effects of CQ and HCQ on the LC3 levels of ACE2^h cells. ACE^h cells were treated with different doses of CQ or HCQ for 24 h. (A) Effects of CQ and HCQ on the fluorescent staining of FITC-LC3 and TRITC-Phalloidin in ACE^h cells. (B) The representative blots of autophagy proteins and changes of LC3-II/LC3-I ratio. The experiments were repeat three times. Data are presented as mean ± S.D. * p < 0.05, ** p < 0.01, *** p < 0.001 compared with control.

Fig. 3

Binding character of CQ and HCQ with ACE2. A. Structural formulas of CQ and HCQ. B. SPR analysis of CQ or HCQ and ACE2. C. Molecular docking of CQ and HCQ with ACE2.

Fig. 4

Effect of CQ and HCQ on the entrance of 2019-nCoV spike pseudotyped virus into ACE2^h cells. The experiments were repeat three times. Data are presented as mean ± S.D. * p < 0.05, ** p < 0.01, *** p < 0.001 compared with group 0.