Furin, a potential therapeutic target for COVID-19
Canrong WU,a,1Yueying YANG,b,1Yang LIU,bPeng ZHANG,bYaliWANG,bQiqi WANG, b Yang XU,bMingxue LI,bMengzhu ZHENG,a,* Lixia CHEN,b,* &Hua LIa,b,*
aHubei Key Laboratory of Natural Medicinal Chemistry and Resource Evaluation, School of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China
bWuya College of Innovation, Key Laboratory of Structure-Based Drug Design & Discovery, Ministry of Education, Shenyang Pharmaceutical University, Shenyang 110016, China
A novel coronavirus (SARS-CoV-2) infectious disease has broken out in Wuhan, Hubei Province since December 2019, and spread rapidly from Wuhan to other areas, which has been listed as an international concerning public health emergency. We compared the Spike proteins from four sources, SARS-CoV-2, SARS-CoV, MERS-CoV and Bat-CoVRaTG13, and found that the SARS-CoV-2 virus sequence had redundant PRRA sequences. Through a series of analyses, we propose the reason why SARS-CoV-2is more infectious than other coronaviruses. And through structure-based virtual ligand screening, we foundpotentialfurin inhibitors, which might be used in the treatment of new coronary pneumonia.
Keywords: SARS-CoV-2；Spike proteins；Furin；Inhibitors；Virtual screening
In December 2019, a series of acute respiratory diseases occurred in Wuhan, Hubei Province, China and then spread rapidly from Wuhan to other areas. As of February 17, 2020, a total of 71,444 patients have been diagnosed and 1,775 have died worldwide. This is caused by a novel coronavirus, which was named as "2019-nCoV" by the World Health Organization, and diseases caused by 2019-nCoV was COVID-19. 2019-nCoV, as a close relative of SARS-CoV, was classified as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by the International Committee on Taxonomy of Viruses (ICTV) on February 11, 2020.
Coronaviruses (CoVs) are mainly composed of four structural proteins, including Spike (S), membrane (M), envelope (E) and nucleocapsid (N) . Spike, a trimeric glycoprotein of CoVs, determines diversity of CoVs and host tropism, and mediates CoVs binding to host cells surface-specific receptors and virus-cell membrane fusion . Current research found that SARS-CoV-2 belongs to the beta coronavirus genus, and speculated that it may interact with angiotensin-converting enzyme 2 (ACE2) on the surface of human cells through Spike protein, thereby infecting human respiratory epithelium cell . Letko M and Munster Vthen identified the receptor for SARS-CoV-2 entry into human cells to be ACE2 .
Coronavirus Spike protein plays a key role in the early stages of viral infection, with the S1 domain responsible for receptor binding and the S2 domain mediating membrane fusion . The process of SARS-CoV infecting the host involves two indispensable cleaving processes which affect the infectious capacity of SARS-CoV. First, Spike was cleaved into receptor-bound N-terminal S1 subunit and membrane-fusion C-terminal S2 subunit by host proteases at S1/S2 cleavage site (such as type II transmembrane serine protease (TMPRSS2), cathepsins B and L) [6,7]. Second, after CoVs are endocytosed by the host, the lysosomal protease mediates cleavage of S2 subunit (S2’ cleavage site) and releases the hydrophobic fusion peptide to fuse with the host cell membrane .
Furin, a kind of proprotein convertases (PCs), is located in the trans-Golgi network (TGN) and activated by acid pH . Furin can cleave precursor proteins with specific motifs to produce mature proteins with biological activity. The first (P1) and fourth (P4) amino acids at the
amino acid, ↓:cleavage site). If the P2 position is basic lysine or arginine, the cleavage efficiency
can be improved by about 10 times . Kibler KV et al. demonstrated that the Spike protein S1/S2 and S2′ cleavage sites of the infectious bronchitis virus (IBVs) Beaudette strain can be recognized by fruin, which is a distinctive feature of IBV-Beaudette with other IBVs and has stronger infection ability [11,12]. Based on the characteristics of furin's recognition substrate sequence, some short peptide inhibitors have been developed, such as Decanoyl-Arg-Val-Lys-Arg-chloromethylketone (Dec-RVKR-CMK) and modified α1-antitrypsin Portland (α1-PDX). However, the non-specific and irreversible inhibitory effects on all members of the PC family limit their application [10, 13]. No small molecule inhibitor of furin with good effect and high specificity has been found so far.
The epidemiological observations showed the infectious capacity of SARS-CoV-2 is stronger than SARS-CoV, so there are likely to be other mechanisms to make the infection of SARS-CoV-2 easier. We suppose the main possibilities as follows, first, SARS-CoV-2 RBD combining with ACE2 may have other conformations; second, the SARS-CoV-2 Spike protein can also bind to other receptors besides ACE2; third, Spike is more easily cleaved by host enzymes and easily fuses with host cell membrane. We compared the Spike proteins from four sources, SARS-CoV-2, SARS-CoV, MERS-CoV and Bat-CoVRaTG13, and found that the SARS-CoV-2 virus sequence had redundant PRRA sequences. Through a series of analyses, this study propose that one of the important reasons for the high infectivity of SARS-CoV-2 is a redundant furin cut site in its Spike protein.And through structure based virtual ligand screening, we proposed possible furin inhibitors, which might be potentially used in the treatment of COVID-19.
2.1 Homology Spike protein blast and sequence alignment.
The Spike protein of(GB:QHR63250.1) was downloaded from NCBI nucleotide database. The protein sequence were aligned with whole database using BLASTp to search for homology viral Spike protein (Alogorithm parameters, Max target sequences: 1000, Expect threshold: 10). Multiple-sequence alignment was conducted in BLASTp online and analysis with DNAMAN and Jalview. The evolutionary history was inferred using the Neighbor-Joining method in MEGA 7 software package. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test wasdetermined by 500 replicates. The Spike protein sequence analyses were conducted in snapgene view.
2.2 Furin cleavage site prediction
The prediction of furin cleavage sites were carried out in ProP 1.0 Server (http://www.cbs.dtu.dk/services/ProP/).
2.3 Compounds database
Approved drug database was from the subset of ZINC database, ZDD (ZINC drug database) containing 2924 compounds . Natural products database was constructed by ourselves, containing 1066 chemicals separated from traditional Chinese herbals in own lab and natural-occurring potential antiviral components and derivatives. Antiviral compounds library contains 78 known antiviral drugs and reported antiviral compounds through literature search.
2.4 Homology modeling and molecular docking
3.1 Bioinformatics analysis reveals furin cut site in Spike protein of SARS-CoV-2
Table 1.Furin cleavage probability of Spike sequence homology
aScores are predicted by ProP 1.0 Server. Scores above 0.5 mean furin cleavable.
bIdentities compared with SARS-CoV-2 Spike protein.
3.2 Homology modeling and protein-protein docking calculation
3.3. Virtual ligand screening of furin protein
The structure-based virtual ligand screening method was used to screen potential furin protein inhibitors through ICM 3.7.3 modeling software (MolSoft LLC, San Diego, CA) from a ZINC Drug Database (2924 compounds), a small in-house database of natural products (including reported common antiviral components from traditional Chinese medicine) and derivatives (1066 compounds), and an antiviral compounds library contains 78 known antiviral drugs and reported antiviral compounds. Compounds with lower calculated binding energies (being expressed with scores and mfscores) are considered to have higher binding affinities with the target protein.
Table 2. Potential furin inhibitors from the ZINC drug database
Vitamin B9, necessary material for the growth and reproduction of body cells
Antineoplastic, antirheumatic effects
Treatment of Parkinson's
Folic acid supplement
Intermediate for serine
Treatment of nausea and vomiting induced by chemotherapy
Treatment of gastrohelcosis
Table 3.Potential furin inhibitors from in-house natural product database
Antioxidation, anti-tumor, treatment
Antioxidant effect, anti-tumor, anti-virus
formamido-1,4a-dimethyl-6- methylene-5-((E)-2-(2-oxo-2,5- dihydrofuran- 3-yl)ethenyl) decahydronaphthalen-2-yl
formamido-1,4a-dimethyl-6- methylene-5-((E)-2-(2-oxo-2,5- dihydrofuran-3-yl)ethenyl) decahydronaphthalen-2-yl
Table 4. Potential furin inhibitors from the common antiviral drugs database
DNA topoisomerase II inhibitor
Human immunodeficiency virus Protease (HIV PR)
Hepatitis C virus Serine protease NS3/4A (HCV NS3/4A) Modulator
HIV-1 nucleotide reverse transcriptase inhibitor
HIV, HBV nucleotide reverse transcriptase inhibitor
Thymidine kinase of herpesvirus
Hepatitis C virus Serine protease NS3/4A (HCV NS3/4A) Modulator
Human immunodeficiency virus Integrase (HIV IN)
1.C-C chemokine receptor type 5 (CCR5)
2.CCR5 messenger RNA(CCR5 mRNA)
Inhibitor of cytochrome P450 3A (CYP3A) enzymes
Nucleoside analogue reverse transcriptase inhibitor used in the treatment of HIV