key: cord-0839156-7ppwb48f authors: Zhou, Jie; Xu, Wei; Liu, Zezhong; Wang, Chao; Xia, Shuai; Lan, Qiaoshuai; Cai, Yanxing; Su, Shan; Pu, Jing; Xing, Lixiao; Xie, Youhua; Lu, Lu; Jiang, Shibo; Wang, Qian title: A highly potent and stable pan-coronavirus fusion inhibitor as a candidate prophylactic and therapeutic for COVID-19 and other coronavirus diseases date: 2021-08-02 journal: Acta Pharm Sin B DOI: 10.1016/j.apsb.2021.07.026 sha: 93932029da6003b48a1b56533e84bf662ffb2e9f doc_id: 839156 cord_uid: 7ppwb48f The development of broad-spectrum antivirals against human coronaviruses (HCoVs) is critical to combat the current coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its variants, as well as future outbreaks of emerging CoVs. We have previously identified a polyethylene glycol-conjugated (PEGylated) lipopeptide, EK1C4, with potent pan-CoV fusion inhibitory activity. However, PEG linkers in peptide or protein drugs may reduce stability or induce anti-PEG antibodies in vivo. Therefore, we herein report the design and synthesis of a series of dePEGylated lipopeptide-based pan-CoV fusion inhibitors featuring the replacement of the PEG linker with amino acids in the heptad repeat 2 C-terminal fragment (HR2-CF) of HCoV-OC43. Among these lipopeptides, EKL1C showed the most potent inhibitory activity against infection by SARS-CoV-2 and its spike (S) mutants, as well as other HCoVs and some bat SARS-related coronaviruses (SARSr-CoVs) tested. The dePEGylated lipopeptide EKL1C exhibited significantly stronger resistance to proteolytic enzymes, better metabolic stability in mouse serum, higher thermostability than the PEGylated lipopeptide EK1C4, suggesting that EKL1C could be further developed as a candidate prophylactic and therapeutic for COVID-19 and other coronavirus diseases. The envelope-expressing plasmids pcDNA3.1-SARS-2-S, pcDNA3.1-SARS-S, pcDNA3.1-MERS-S, pcDNA3.1-NL63-S, pcDNA3.1-OC43-S, pcDNA3.1-WIV1-S, pcDNA3.1-Rs3367-S, pNL4-3.Luc.RE (luciferase reporter vector), and pAAV-IRES-EGFP (encodes EGFP), as well as SARS-CoV-2 S mutant envelopeexpressing plasmids, were maintained in our laboratory as previously reported 9,11 . were synthesized by using a standard solid-phase fluorenylmethoxycarbonyl (Fmoc) method with a CEM Liberty Blue (CEM Co., Matthews, NC, USA) automated microwave peptide synthesizer on Rink amide resin (0.44 mmol/g, Nankai Hecheng S&T Co., Ltd., Tianjin, China). Cholesteroylated peptides were prepared by chemoselective thioether conjugation between the peptide precursor that has a C-terminal cysteine residue and the cholesterol derivative, i.e., cholest-5-en-3-yl bromoacetate, as described previously 19 . For synthesizing fatty acid conjugated peptides, the template peptides containing a N- [1-(4,4- The protein accession numbers of MERS-CoV (AID55097.1), SARS-CoV (ABD72929.1), SARS-CoV-2 (QIC53213.1), and HCoV-OC43 (CAA83661.1) were downloaded from NCBI GenBank, and sequence alignment was conducted using MEGA and WebLogo 3. S protein-mediated cell-cell fusion was performed as previously described 9, 11 . In brief, 293T effector cells were transfected with plasmid pAAV-IRES-EGFP, encoding the SARS-CoV-2 S protein (293T/SARS-CoV-2/GFP), while Huh-7 cells, expressing various HCoV receptors on the membrane surface, were used as target cells. Huh-7 cells were first seeded in a 96-well plate at 2×10 4 cells/well for 12 h. Then the 293T/SARS-CoV-2/GFP effector cells were added and cocultured with Huh-7 cells for 2 h in DMEM medium containing 10% FBS at 37 °C. After incubation, the number of fused and unfused cells were counted randomly from five selected fields in each well under an inverted fluorescence microscope (Nikon, Tokyo, Japan), and the percent inhibition of cell-cell fusion was calculated as previously reported 9,11 . Cytotoxicity of the peptide EKL1C to the cells (Huh-7, 293T/ACE2, Caco-2) was performed as previously described 11 PsVs were constructed as described previously 11, 20 . Briefly, 293T cells were cotransfected with plasmids pNL4-3.Luc.RE and plasmid pcDNA3.1-HCoV-S (encoding the corresponding HCoV S protein) at mass ratio of 2:1, using VigoFect (Vigorous Biotechnology, Beijing, China). The supernatant containing the PsV was then collected after centrifuging at 1500×g for 10 min and maintained at −80 °C until use. The HCoV PsV inhibition assay was performed as previously described 11, 21 . constructed and produced in Huh-7 cells as described previously 9,20 . An inhibition assay was performed in a manner similar to that described above. The inhibition assay for SARS-CoV-2 was performed in a biosafety level 3 (BSL-3) laboratory as previously described 22 . Before infection, Vero-E6 cells were seeded at The inhibitory activities of peptides against HoV-OC43 replication on RD cells were assessed as described 11 . The 10 2 median tissue culture infective dose (TCID50) of OC43 was mixed with the test peptide at graded concentrations and incubated at 37 °C for 30 min, and then the mixtures were added to the RD cells (1×10 4 cells/well). After culturing for 36 h, CCK-8 solution was added to determine cytopathic effect as described above to measure EKL1C protective effect. To test the protective effect of peptides against SARS-CoV-2 infection in vivo, hACE2-Tg were used 22, 23 . A total of 15 hACE2-Tg mice were randomly assigned to three groups: viral control, prophylactic, and therapeutic groups. In the viral control group, each mouse was challenged intranasally with 10 5 TCID50 SARS-CoV-2. EKL1C (1.5 mg/kg) was administered intranasally 0.5 h before challenge in the prophylactic group and administered intranasally 0.5 h after challenge in the therapeutic group. Mice were euthanized after 4 days of infection, and lungs were collected and homogenized in Trizol (Takara, Japan). Then total RNA in the lungs were extracted for RT-qPCR assay as described above. To test the protective effect of peptides against HCoV-OC43 infection in vivo, 18 newborn mice bred from pregnant mice were randomly assigned to three groups: J o u r n a l P r e -p r o o f viral control, prophylactic, and therapeutic (each group had six 3-day-old mice). A challenge experiment was performed and evaluated in a manner similar to that described above. Mice in the prevention and treatment groups were intranasally administered with EKL1C (4 mg/kg) 30 min before or after the viral challenge. Each mouse was intranasally challenged with a viral dose of 10 2 TCID50. Afterwards, the viral titer in mice lungs was collected and assessed 9 . Assays for the stability of peptides to proteolytic enzymes, proteinase K, and trypsin were performed as previously described [24] [25] [26] . First, the concentration of peptide was set according to its 90% inhibition concentration (IC90). For proteinase K tests, solvent B (methanol) were used as mobile phases. The gradient elution program was optimized as follows: 5% phase B at 0.00-1.00 min; from 5% to 70% phase B at 1.00-2.00 min; from 70% to 95% phase B at 2.00-3.00 min; 95% phase B maintaining at 3.00-3.50 minutes; from 95% to 5% phase B at 3.50-3.60 min, followed by re-equilibration at 5% phase B until 5 min and the flow rate was 0.5 mL/min. Autosampler temperature was kept at 15 ℃ and the injection volume was set at 4 μL. EKL1C and EK1C4 were dissolved in water to produce 1 mg/mL auto-sampler vials and was analyzed according to the analytical method. To detect the significance of difference in stability between EKL1C and EK1C4, the peptides were stored under the same conditions at 4 °C, room temperature (RT) and 37 °C, respectively. Samples were collected at the same time at different days (15, 30 , 60, 100, and 120 days) and stored at −20 °C before testing. The antiviral activity of each sample was detected by PsV inhibition assay as described above. Assay for stability of peptide metabolic in serum. To compare significance of difference in metabolic stability of peptides in mouse serum, we collected mouse serum from 6-week BALB/c and mixed the serum with peptides at the concentration that could achieve IC90, estimated according to the result from the previous study. After incubation at 37 °C, samples were collected at different time points (1, 3, 6, and 18 h), followed by inactivating serum at 56 °C for 30 min. Then the mixtures were collected and stored at −20 °C before testing. The residual antiviral activity of each sample was detected by PsV inhibition assay as described above. All data were presented as the mean±standard deviation (SD) from at least three experiments. Statistical differences were analyzed with GraphPad Prism software. HCoV-OC43 replication in hACE2-Tg mice. Unpaired Student's t-test was used to compare the difference in sensitivity to trypsin, protease K, and serum between EKL1C and EK1C4 and the significance of difference in stability of EKL1C and EK1C4 stored at different temperatures. In order to design effective pan-CoV dePEGylated lipopeptides, we first conducted an alignment with the amino acid sequences of the HR2-CF of several HCoVs, including SARS-CoV, MERS-CoV, SARS-CoV-2, and HCoV-OC43. We found that HR2-CF sequences are relatively conserved among these HCoVs (Fig. 1C) . Then, we replaced the PEG flexible linker in the EK1C4 lipopeptide with HR2-CF J o u r n a l P r e -p r o o f sequence of HCoV-OC43 to design a series of dePEGylated lipopeptides consisting of different length of EK1 and HR2-CF sequences, as well as cholesterol (Chol) or palmitic acid (Palm) (Fig. 1D) . The molecular weight of the synthesized peptides was confirmed by MALDI-TOF-MS (Supporting Information Fig. S1 ). We assessed the antiviral activity of lipopeptides against SARS-CoV-2 PsV infection in Huh-7, 293T/ACE2, and Caco-2 cells, respectively. Among these lipopeptides, EKL1C was found to be the most potent, with IC50s ranging from 0.037 to 0.045 μmol/L, respectively, about 20-to 62-fold more potent than that of EK1 ( Fig. 2A) , but about 1.3-to 5-fold less potent than that of EK1C4 (IC50 ranging from 0.007 to 0.027 μmol/L). Next, we tested the potential inhibitory activity of the lipopeptides against SARS-CoV-2 S protein-mediated cell-cell fusion. As shown in Fig. 2Ba We then assessed the cytotoxicity of EKL1C and found that its 50% cytotoxic concentration (CC50) was 10, 13.81, and 8.49 μmol/L on Huh-7, Caco-2, and 293T/ACE2 cells, respectively (Supporting Information Fig. S2) , and all the selectivity indexes (SI=CC50/IC50) were in a range of 222 to 345, justifying the further study of EKL1C. Mutations in the spike protein of SARS-CoV-2 are well known, e.g., D614G and 31 . We found that EKL1C could effectively inhibit infection of all 12 mutants with IC50 below 0.15 μmol/L (Fig. 3A) . We then compared the inhibitory activity of the cholesterol-conjugated EKL1 peptide, EKL1C, with that of EK1 and EKL1 peptides (Fig. 1D) (Fig. 3Bd) . EKL1C was also highly effective against infection of pseudotyped SARSr-CoVs, including WIV1 and Rs3367, with IC50s of 0.218 and 0.046 μmol/L, respectively, about 70-and 156-fold more potent than that of EK1, and 138-and 1919-fold more potent than that of EKL1 ( Fig. 3Be-f ). We further assessed the inhibitory activity of EKL1C against authentic SARS-CoV- To compare the sensitivity of the dePEGylated lipopeptide EKL1C with the PEGylated lipopeptide EK1C4 to proteolytic enzymes, both peptides were treated with trypsin or proteinase K at 37 °C, followed by collection of samples at different time points post-treatment for detection of their inhibitory activity against SARS-CoV-2 PsV infection. As shown in Fig. 5Aa and b, the inhibitory activity of EK1C4 against SARS-CoV-2 PsV infection gradually decreased more significantly than that of EKL1C under the same treatment condition (*P<0.05, **P<0.01, ***P<0.001). These samples were also analyzed by LC-MS quantitative analysis and similar trends were obtained ( Fig. 5Ac and d) . These results suggest that the dePEGylated lipopeptide EKL1C is significantly more resistant than the PEGylated lipopeptide EK1C4 to the proteolytic enzymes trypsin and proteinase K. To compare the metabolic stability of the dePEGylated lipopeptide EKL1C with that of the PEGylated lipopeptide EK1C4 in mouse serum, we added the peptides into mouse serum. After incubation at 37 °C for 18 h, the inhibitory activity of EKL1C on SARS-CoV-2 PsV infection decreased about 20%, while that of EK1C4 decreased about 40% (*P<0.05), indicating that EKL1C has better metabolic stability than EK1C4 (Fig. 5Ae) . To detect the thermostability of lipopeptides, we tested the antiviral activities of The current COVID-19 pandemic caused by SARS-CoV-2 and its variants has posed a serious threat to the global heath and economy 27, [32] [33] [34] [35] , while future outbreaks of highly pathogenic infectious diseases may be caused by emerging and reemerging coronaviruses 36 However, recent studies have shown that some PEG-modified therapeutics could induce PEG-specific antibodies in vivo, resulting in decreased therapeutic efficacy [40] [41] [42] [43] . It was also reported that PEG conjugation at the C-terminus of a lipopeptide could enhance its sensitivity to proteolytic enzymes 18, 44 Most importantly, the dePEGylated lipopeptide EKL1C exhibited significantly stronger resistance to the proteolytic enzymes, trypsin, and proteinase, better metabolic stability in mouse sera, and higher thermostability than the PEGylated lipopeptide EK1C4, suggesting that our strategy offers a possible solution to the limitations of PEG linkers in PEGylated drugs. Since the dePEGylated lipopeptide EKL1C contains no PEG, it is unlikely to cause severe allergy-like reactions in people who have high levels of the preexisting anti-PEG antibodies. We have designed and developed a highly stable dePEGylated lipopeptide, EKL1C, with highly potent pan-CoV fusion inhibitory activity. 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