key: cord-0708117-b0fig8oc
authors: Gu, Chenjian; Wu, Yang; Guo, Huimin; Zhu, Yuanfei; Xu, Wei; Wang, Yuyan; Zhou, Yu; Sun, Zhiping; Cai, Xia; Li, Yutang; Liu, Jing; Huang, Zhong; Yuan, Zhenghong; Zhang, Rong; Deng, Qiang; Qu, Di; Xie, Youhua
title: Protoporphyrin IX and verteporfin potently inhibit SARS-CoV-2 infection in vitro and in a mouse model expressing human ACE2
date: 2020-12-09
journal: Sci Bull (Beijing)
DOI: 10.1016/j.scib.2020.12.005
sha: 31ba1498eab7e63397b7e142f1ce392f420c595c
doc_id: 708117
cord_uid: b0fig8oc

The SARS-CoV-2 infection is spreading rapidly worldwide. Efficacious antiviral therapeutics against SARS-CoV-2 is urgently needed. Here, we discovered that protoporphyrin IX (PpIX) and verteporfin, two FDA-approved drugs, completely inhibited the cytopathic effect produced by SARS-CoV-2 infection at 1.25 µmol/Land 0.31 µmol/L respectively, and their EC50 values of reduction of viral RNA were at nanomolar concentrations. The selectivity indices of PpIX and verteporfin were 952.74 and 368.93, respectively, suggesting broad margin of safety. Importantly, PpIX and verteporfin prevented SARS-CoV-2 infection in mice adenovirally transduced with human ACE2. The compounds, sharing a porphyrin ring structure, were shown to bind viral receptor ACE2 and interfere with the interaction between ACE2 and the receptor-binding domain of viral S protein. Our study suggests that PpIX and verteporfin are potent antiviral agents against SARS-CoV-2 infection and sheds new light on developing novel chemoprophylaxis and chemotherapy against SARS-CoV-2.

The infection of SARS-CoV-2 has spread around the world since December 2019. As of July 6, 2020, there are nearly 11 million confirmed cases globally, of which more than five hundred thousand died (https://www.who.int/emergencies/diseases/novel-coronavirus-2019). Although the pandemic has been contained in some countries, the numbers of confirmed cases and deaths worldwide are expected to continue to rise. is mostly supportive, including non-specific antivirals and symptom-alleviating therapies [1] . Ventilations and intensive care are required for severe cases, calling for early intervention to prevent symptoms from deteriorating [1, 2] .

In vitro experiment showed that remdesivir targeting viral RNA-dependent RNA polymerase (RdRp) effectively inhibited SARS-CoV-2 replication [3, 4] . The compassionate use of remdesivir for patients with severe COVID-19 indicated that clinical improvement was observed in 36 of 53 patients (68%) [5] . Remdesivir was reported to shorten the time to recovery in adults hospitalized with COVID-19 and evidence of lower respiratory tract infection in a double-blind, randomized, placebo-controlled trial, though conflicting trial results have also been reported [6, 7] .

Several repurposed drugs have been tested in vitro for inhibition of SARS-CoV-2 infection and some of them were tested in clinical trial [8] [9] [10] [11] . Among them, chloroquine and hydroxychloroquine have been shown to inhibit SARS-CoV-2 infection in vitro, while the clinical trials of hydroxychloroquine reported 4 controversial results [4, [12] [13] [14] . The effective concentrations (presented as the concentration for 50% of maximal effect (EC50) on the reduction of viral RNA) of most previously selected drugs are in the micromolar (µmol/L) concentration range.

On the other hand, neutralizing antibodies against SARS-CoV-2 are also being intensively studied [15] [16] [17] . In general, more efficacious antiviral therapeutic agents against SARS-CoV-2 with good safety profile are urgently needed.

In this work, in search of novel antivirals that can effectively inhibit SARS-CoV-2

infection, we set out to screen an FDA-approved drug library of 3200 small molecules via observation of viral CPE (Cytopathic effect) in Vero E6 cells, followed by evaluation of the antiviral effect of candidate compounds in vitro and in mice transduced intranasally with the recombinant adenovirus 5 expressing human ACE2

(Ad5-hACE2). We discovered that protoporphyrin IX (PpIX) and verteporfin displayed a potent antiviral activity and prevent SARS-CoV-2 infection.

African green monkey kidney Vero E6 cells and human embryonic kidney HEK293T cells were cultured at 37 ºC with 5% CO 2 in Dulbecco's modified Eagle medium (DMEM) (Gibco, Carlsbad, USA) containing 2 mmol/L L-glutamine, 50 U/mL penicillin, 100 mg/mL streptomycin, and 10% (vol/vol) fetal bovine serum (Gibco).

Vero E6 cells after SARS-CoV-2 infection were maintained in DMEM containing 2 mmol/L L-glutamine, 50 U/mL penicillin, 100 mg/mL streptomycin, and 2% (vol/vol) fetal bovine serum.

A clinical isolate of SARS-CoV-2, nCoV-SH01 (GenBank: MT121215.1) [18] , was propagated in Vero E6 cells and the viral titer was determined as plaque forming units 

To detect the viral nucleocapsid protein (N protein), anti-N polyclonal antibodies were generated using standard immunization of BALB/c mice with recombinant N 

Cryo-electron microscopy structures of the full-length human ACE2 and a neutral amino acid transporter B 0 AT1 complex with an overall resolution of 2.9 Å have been reported [22] . The structure files were downloaded from Protein Data Bank (PDB ID: 

Cell-cell fusion was performed as described previously [23] . The inhibitory value of protoporphyrin IX or verteporfin-treated group was presented relative to that of the DMSO-treated group which was set as 100%, respectively. 10 

Pseudovirions were produced by co-transfection HEK293T cells with psPAX2, pLenti-NanoLuc, and plasmid encoding either C-terminally 19 amino acids truncated SARS-CoV-2 S or VSV-G by using polyetherimide (PEI). The supernatants were 

In the binding assay of viral S protein receptor binding domain (RBD), the was used for final signal detection. followed by HRP-conjugated goat anti-rabbit IgG secondary antibody (1:5000 dilution, Invitrogen). Immobilon Western Chemiluminescent HRP Substrate (Thermo Fisher Scientific) was used for signal development. 

Mouse lung tissues were fixed in 4% paraformaldehyde solution. Tissue homogenates (1 g/mL) were prepared by homogenizing perfused lung tissues using an automatic sample grinding instrument (Jingxin, Shanghai, China) for 1 minute in TRIzol reagent. The homogenates were centrifuged at 12,000 rpm for 10 minutes at 4 °C. The supernatant was collected for viral RNA extraction.

Mouse lungs were fixed in 4% paraformaldehyde solution. Tissue paraffin sections 

BLI assays were carried out in 96-well black plates using an OctetRED96 device (Pall 

Data were analyzed using GraphPad Prism 7 and were presented as mean ± SEM. The dose response curves of viral RNA levels or cell viability versus the drug concentrations were plotted and evaluated by Prism 7. Statistical significance was determined using unpaired two-tailed Student's t test for single variables and two-way ANOVA followed by Bonferroni posttests for multiple variables.

Vero E6 cells (Fig. 1b) , respectively. The EC50 of remdesivir was comparable to the previous report [4] . Cell viability assay was performed, resulting in a viability-compound concentration curve (Fig. 1b) , from which the CC50 

We next analyzed the relationship between the antiviral effect and treatment timing of protoporphyrin IX and verteporfin. As shown in Fig. 2a Fig. 2b , c) were 15 significantly lower than that of the DMSO-treated group (group VIII in Fig. 2b, c) .

Importantly, pre-treatment alone resulted in the complete inhibition of SARS-CoV-2 infection (group IV in Fig. 2b, c) . In addition, treatment of cells with protoporphyrin IX or verteporfin after viral infection showed a 64.6% or 95.4% reduction of viral RNA production, respectively (group VII in Fig. 2b, c) . The results of immunofluorescence analysis on intracellular viral N protein were consistent with those of viral RNA measurement (Fig. 2d) . Collectively, the results indicate that protoporphyrin IX and verteporfin can prevent SARS-CoV-2 infection and also suppress established SARS-CoV-2 infection to some degree.

The preventive effect was further tested by the pre-treatment of cells with either compound at a constant concentration and later infection with an increasing virus titer (Fig. 3a) . As shown in Fig. 3b , c, no CPE or viral N protein expression was detected in protoporphyrin IX or verteporfin pre-treated cells even if the inoculated viral titer was raised by 16 folds (200 PFU to 3200 PFU). Pre-treatment of the virus with protoporphyrin IX (100 µmol/L) or verteporfin (20 µmol/L) had no effect on viral infectivity (Fig. S1 ).

Protoporphyrin IX and verteporfin share a structure formed by four pyrrole rings (Fig.   4a ) and thus likely act through a common antiviral mechanism. The above results suggest that both drugs act by inhibiting an early step in viral infection. One possible antiviral mechanism was that the drugs bind or modify an essential cellular factor(s) required for viral infection and inhibit its/their functions. We thus investigated firstly by molecular docking analysis whether human ACE2, the viral receptor, might be the target of the compounds. The ACE2 peptidase domain (PD) from the human ACE2-B 0 AT1 complex (PDB ID: 6m18) [22] was used for docking with protoporphyrin IX and verteporfin (Fig. 4a ). The result with the highest ranking is exhibited in Fig. 4b , which represents the molecular model of protoporphyrin IX or 16 verteporfin binding to PD. Protoporphyrin IX is located in the shallow-pocket-like space in the PD, with a binding energy of -5.60 kcal/mol. Similar result was obtained from the docking of verteporfin with PD (with a binding energy of -5.35 kcal/mol). We next used BLI (Biolayer Interferometry) assay to evaluate the binding between ACE2 and these two compounds. As shown in Fig. 4d , protoporphyrin IX and verteporfin indeed bind to ACE2-Fc. The K D of protoporphyrin IX and verteporfin binding to ACE2-Fc were calculated to be 3.897 × 10 -5 mol/L and 1.15 × 10 -4 mol/L, respectively. Therefore, structural simulation by molecular docking and direct drug-protein binding assay support the binding of both drugs to viral receptor ACE2.

Based on the molecular docking and the experimental data, both drugs likely interfere 17 with the interaction between ACE2 and RBD via binding ACE2, which would impair viral entry. We first tested this possibility using a cell-cell fusion assay. HEK293T cells that express SARS-CoV-2 S protein served as the effector cells and those co-expressing human ACE2 and GFP as the target cells (Fig. 5a) . (Fig. 5b) .

To more directly demonstrate the interference of the compounds with the interaction of ACE2 to RBD, we designed an ELISA assay, in which protoporphyrin IX or verteporfin was added to 96-well plate pre-coated with ACE2-Fc or His-RBD. After incubation, unbound drugs were washed away. His-RBD or ACE2-Fc was added to the drug-treated wells pre-coated with ACE2-Fc or His-RBD. The results showed that both drugs could prevent the binding of His-RBD to pre-coated ACE2-Fc, while they had no effect on the binding of ACE2-Fc to pre-coated His-RBD (Fig. 5c) . The data suggest that protoporphyrin IX and verteporfin most likely bind to ACE2 and interfere 18 with the binding of RBD to ACE2, which is consistent with the results of the cell-cell fusion and molecular docking abovementioned. Western blot and RT-qPCR respectively (Fig. S2a, b) . (Fig. 6c) . Much fewer cells expressed viral N protein in the protoporphyrin IX and verteporfin groups compared to the DMSO group (Fig. 6c ). Viral RNA levels in the lung samples taken from the protoporphyrin IX and verteporfin groups were significantly lower than that from the DMSO group (4 log reduction), and were close to that from the negative control (Fig. 6b) . The sections of lung tissues from the DMSO group displayed a variety of lesions including perivascular to interstitial inflammatory cell infiltrates, and necrotic cell debris. In contrast, the sections of lung 19 tissues from the protoporphyrin IX and verteporfin groups showed no obvious histopathological change, so did those from the non-infected mice (the NC group) (Fig. 6d) . These results indicate that protoporphyrin IX and verteporfin also effectively inhibit SARS-CoV-2 infection in mouse model.

Protoporphyrin IX and verteporfin have been approved and used in the treatment of human diseases. Protoporphyrin IX is the final intermediate in the protoporphyrin IX iron complex (heme) biosynthetic pathway [24] . Heme is an important cofactor for oxygen transfer and oxygen storage [25] and is a constituent of hemoproteins which play a variety of roles in cellular metabolism [26] . The light-activable photodynamic effect of protoporphyrin IX was used for cancer diagnosis [27] and approved by FDA for treatment of bronchial and esophageal cancers and early malignant lesions of the skin, bladder, breast, stomach, and oral cavity [28, 29] . Verteporfin was approved for the treatment of age-related macular degeneration [30] . The potential of verteporfin for the treatment of cancers, such as prostatic cancer, breast cancer, and pancreatic ductal adenocarcinoma has been investigated [31] . Verteporfin also has been reported to inhibit autophagy at an early stage by suppressing autophagosome formation [32] .

A study of clinical pharmacokinetics of verteporfin showed that in healthy volunteers who were infused with verteporfin 6 to 14 mg/m 2 of body surface area over 1.5 to 45 minutes, Cmax (peak concentration) of verteporfin was 1.24-2.74 μg/mL [33] . The Cmax value is approximately 2.4 to 5.2-fold higher than the EC90 value that was obtained in this study (0.73 μmol/L, i.e. 0.52 μg/mL). Protoporphyrin IX is the metabolite of 5-aminolevulinic acid (5-ALA) in human body. After administration of 5-ALA 2 mg/kg p.o., the average Cmax of protoporphyrin IX was 27.44 μg/mL [34] , which is about 20-fold higher than the EC90 value in this study (2.45 μmol/L, i.e.

1.38 μg/mL). These data indicate that the two drugs can reach a plasma concentration that is much higher than the in vitro effective antiviral concentration. In the mouse 20 model in this study, protoporphyrin IX and verteporfin exhibited effective inhibition of SARS-CoV-2 infection without notable toxicity.

Both protoporphyrin IX and verteporfin have a porphyrin ring structure formed by four pyrrole rings. It is most likely that they share a similar mechanism of antiviral action. In the experiment when either drug was added prior to viral infection, viral RNA production was inhibited even if the relevant drug was not added in the later virus infection and post-infection stages (group IV in Fig. 2b, c) . Furthermore, increasing viral titer did not relieve the inhibition of the drugs added before viral infection (Fig. 3b, c) . A logical hypothesis is that both drugs act by inhibiting an early On the other hand, protoporphyrin IX and verteporfin were able to inhibit viral RNA production to some degree when they were added after viral infection (group VII in 

The authors declare that they have no conflict of interest. Nuclei were stained with DAPI. ELISA. The binding of His-RBD or ACE2-Fc to drug-treated pre-coated ACE-Fc or

His-RBD was measured by absorbance at 450 nm. Statistical significance was determined using the unpaired two-tailed Student's t test. *** P < 0.001. Data from triplicate wells were analyzed. 

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This study was supported by the National Science and Technology Major Project