key: cord-0951546-8jejswuk
authors: Wang, Nan; Han, Shengli; Liu, Rui; Meng, Liesu; He, Huaizhen; Zhang, Yongjing; Wang, Cheng; Lv, Yanni; Wang, Jue; Li, Xiaowei; Ding, Yuanyuan; Fu, Jia; Hou, Yajing; Lu, Wen; Ma, Weina; Zhan, Yingzhuan; Dai, Bingling; Zhang, Jie; Pan, Xiaoyan; Hu, Shiling; Gao, Jiapan; Jia, Qianqian; Zhang, Liyang; Ge, Shuai; Wang, Saisai; Liang, Peida; Hu, Tian; Lu, Jiayu; Wang, Xiangjun; Zhou, Huaxin; Ta, Wenjing; Wang, Yuejin; Lu, Shemin; He, Langchong
title: Chloroquine and hydroxychloroquine as ACE2 blockers to inhibit viropexis of 2019-nCoV Spike pseudotyped virus
date: 2020-08-14
journal: bioRxiv
DOI: 10.1101/2020.06.22.164665
sha: c16223043e2ec68377ffb31e8f9c887c95bcae74
doc_id: 951546
cord_uid: 8jejswuk

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.

. At the same time, in a clinical trial of 197 2019-nCoV positive patients in 98

China, CQ showed a significant therapeutic effect without severe adverse reactions (Mingxing et 99 al., 2020) . The above evidence suggests that the adverse effects of CQ treatment in 2019-nCoV 100 posotive patients may be lower than that of HCQ. The curative effect and mechanism of the anti-101 2019-nCoV of CQ and HCQ are still controversial. 102

In this study, we found that CQ and HCQ can antagonize ACE2 and inhibit the entry of 2019-nCoV 103 spike pseudotyped virus into ACE2 expressed HEK293T cells (ACE2 h cells). Carlsbad, CA, USA). The survival rate of ACE2 h cells was calculated using the following formula: Immunofluorescence assays 164 ACE2 h cells (2×10 3 ) were seeded on 24 mm×24 mm coverslips. and incubated overnight at 37 °C 165 with 5 % CO2 .10 μM, 20 μM or 40 μM CQ and HCQ were added to the slides and treated for 24 h. 166

The slides were then fixed with 4 % paraformaldehyde, followed with 0.5% Triton X-100 for 5 min 167 and 5% BSA solution for 1 h at 26°C after washing three times with PBS. The cells were then 168 continuously incubated with LC3 primary antibody at 37°C for 3 h, and the fluorescent secondary 169 antibody at 26°C for 2 h followed with TRITC-Phalloidin stain for 30 min at 26°C . Finally, the 170 cells were mounted with 50 μL of DAPI-containing anti-fluorescence quenching reagent. All the 171 cells were observed using a laser confocal fluorescence microscope. Data are presented as the mean ± standard error of the mean (SD) and were statistically analyzed 202 using analysis of variance (ANOVA). Two-tailed tests were used for comparisons between two 203 groups, and differences were considered statistically significant at p <0.05. 204

The expression of ACE2 protein in human lung and bronchial-related cells was higher than that in 207 HEK293T cells. The expression of ACE2 protein in ACE2 h cells was significantly higher than that 208 in other cells, indicating that ACE2 h cells were successfully constructed. It has been reported that 209 AT2 cells express the highest ACE2 receptors in lung and bronchial cells (Zou et al., 2020) . We 210 confirmed that the highest expression of the ACE2 protein occurred in AT2 cells. In addition, this is 211 the first report that EOL-1 cells also express the ACE2 protein ( Figure 1A) . 212

As shown in Figure 1B , CQ and HCQ had no significant effect on the activity of ACE2 h cells when 213 the concentration was less than 50 μM, and the survival rate of ACE2 h cells could be reduced in a dose-dependent manner when the concentration was above 50 μM. The inhibition of HCQ on the 215 activity of ACE2 h cells was more significant than that of CQ. It can be concluded that the toxicity 216 of HCQ was higher than that of CQ on ACE2 h cells at different time points at the same 217 concentrations (Figure 1C) . At a concentration of 20 μM, the statistical difference appeared at 6 h. 218

Ca 2+ is an essential second messenger in several cell pathways, as shown in Figure 1D , and CQ or 219 HCQ rarely affects Ca 2+ influx change in ACE2 h cells. Figure 1E shows that within 24 h, the 220 concentrations of both drugs had no significant effect on apoptosis. 221

Autophagosome is a spherical structure and as an essential marker for autophagy, and LC3 is known 223 to be stably associated with the autophagosome membranes. LC3 includes two forms LC3-I and 224

LC3-II, LC3-I is found in the cytoplasm, whereas LC3-II is membrane-bound and converted from 225 LC3-I to initiate formation and lengthening of the autophagosome. Therefore, to investigate the 226

DAPI staining were used. Activating lysosomal (green) and filamentous actin (F-actin, red) was 228 detected after stimulation with 10, 20 and 40 μM of CQ and HCQ in ACE2 cells (Figure 2A) . 229

autophagy proteins LC3-I and LC3-II by Western blotting. We found that the expression level of 231 LC3 and LC3-II increased in CQ and HCQ-treated ACE2 h cells ( Figure 2B ). The protein level of 232 the LC3-II/LC3-I ratio was significantly increased compared to the control group ( Figure 2B ). All 233 of these results suggested that CQ and HCQ could induce LC3-mediated autophagy in ACE2 h cells. 234

The SARS-CoV-2 virus infects its host cells through binding to the ACE2 protein 236 followed by cleavage of the spike protein by human TMPRSS2, we focused on whether 237 CQ or HCQ could bind with ACE2. A virtual molecular docking test was performed to investigate 238 the binding character of CQ and HCQ with ACE2. The chemical structure of both drugs are showed 239 in Figure 3A . Figure 3B shows that both CQ and HCQ can bind to R393 and D350 (both in green) 240 of ACE2 with their quinoline and imino groups. In addition, due to the replacement of a methyl 241 group by a hydroxymethyl group, HCQ can form two additional hydrogen bonds with D350 and 242 S44 (in red).We further used SPR to confirm the binding between CQ or HCQ and ACE2. The 243 binding constant KD of these two compounds and ACE2 protein were (7.31±0.62)e-7 and 244 Data are presented as mean ± S.D. (*p < 0.05, **p < 0.01, ***p < 0.001, compared with HEK293T, 319 or concentration was 0, or HCQ 20 μM, # p < 0.05 compared with HCQ 10 μM at corresponding 320 time points). 321 

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