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.

A comprehensive guide to the pharmacologic regulation of angiotensin converting enzyme 2 (ACE2), the SARS-CoV-2 entry receptor

“…In an earlier study, a compound coined SSAA09E2 has been shown to block the binding of the SARS-CoV spike protein to ACE2 and to inhibit SARS-CoV infection in ACE2 expressing HEK-293T cells with an EC 50 of 3.1 μM ( Adedeji et al, 2013 ). Chloroquine and hydroxychloroquine, in addition to their pH elevating effects in endosomes, bind to ACE2 ( N. Wang et al, 2020 ), impair the terminal glycosylation of ACE2 ( Vincent et al, 2005 ) and inhibit SARS-CoV replication ( Al-Bari, 2017 ; Keyaerts, Vijgen, Maes, Neyts, & Van Ranst, 2004 ). They also prevent the entrance of SARS CoV-2 spike protein into ACE2 expressing cell lines (N. Wang et al, 2020).…”

Chloroquine and Hydroxychloroquine Interact Differently with ACE2 Domains Reported to Bind with the Coronavirus Spike Protein: Mediation by ACE2 Polymorphism

“…In fact, both inhibit the SARS-CoV-2 viral replication [ 8 ], decrease antigen processing and its presentation [ 9 , 10 ], and decrease the cellular activity via low secretion of inflammatory cytokines and type 1 interferon [ 5 ]. CQ and HCQ might also interfere with angiotensin-converting enzyme 2 (ACE2) receptor which is involved in COVID-19 and its symptoms [ 11 ]. Strong interactions have been reported between the SARS-CoV-2 RBD domain of the S protein and ACE2 [ 12 ].…”

Repositioning of histamine H1 receptor antagonist: Doxepin inhibits viropexis of SARS-CoV-2 Spike pseudovirus by blocking ACE2

“…ACE2-overexpressing HEK293T cell (ACE2-HEK293T) line was constructed by Genomeditech (Shanghai, China) and its over-expression of ACE2 has been verified in our previous research ( Wang et al, 2020 ). ACE2-HEK293T was cultured at 37 °C and 5% CO 2 in Dulbecco's modification of eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, 100 μg/ml streptomycin, and 4 μg/ml puromycin.…”