Cyclic peptide mutant and use and product thereof for preparation of coronavirus inhibitor

Abstract

Provided in the present application are a cyclic peptide mutant and use and product thereof for the preparation of a coronavirus inhibitor, belonging to the technical field of cyclic peptide drugs. The present application is optimized on the basis of the cyclic peptide 6L3-3P11R. The obtained cyclic peptide mutant can resist the enzyme digestion effect of pancreatic enzymes and exhibits further improved antiviral activity, which is of great significance for expanding antiviral applications of cyclic peptide drugs.

Description

Cyclic peptide mutants and their applications and products in the preparation of coronavirus inhibitors

[0001] Cross-reference of related applications

[0002] This application claims priority to Chinese Patent Application No. 202411653348.0, filed on November 19, 2024, entitled "Cyclic Peptide Mutant and Its Application and Product in the Preparation of Coronavirus Inhibitors", the entire contents of which are incorporated herein by reference. Technical Field

[0003] This application belongs to the field of cyclic peptide drug technology, specifically relating to cyclic peptide mutants and their applications and products in the preparation of coronavirus inhibitors. Background Technology

[0004] Coronaviruses are the cause of many diseases, and they mutate very easily. Taking SARS-CoV-2 (hereinafter referred to as the novel coronavirus, or COVID-19) as an example, the development and promotion of broad-spectrum drugs and universal vaccines are key measures to combat SARS-CoV-2. Currently approved anti-COVID-19 drugs mainly target the main protease and RNA-dependent RNA polymerase, and their safety and efficacy still require further verification.

[0005] Cyclic peptide drugs have advantages such as structural diversity, strong biological activity, target specificity, biological stability, low drug resistance, and high customizability. Therefore, the discovery and development of drugs using the unique structural characteristics of cyclic peptides has become an important research direction in recent years.

[0006] As described in the invention patent application number 202311261559.5, 6L3-3P11R cyclic peptide is a cyclic peptide that can bind to the receptor-binding domain (RBD) on the spike protein (S) of the novel coronavirus and inhibit the infection of pseudoviruses of various novel coronavirus mutant strains, including Omeprone.

[0007] However, analysis revealed that trypsin is widely distributed in the human body, and 6L3-3P11R contains arginine, whose antiviral activity is easily affected by trypsin digestion, thus limiting its practical use. Summary of the Invention

[0008] The previously discovered cyclic peptide 6L3-3P11R can inhibit the infection of various SARS-CoV-2 pseudoviruses. However, 6L3-3P11R is sensitive to trypsin treatment, and trypsin is widely distributed in the human body. Therefore, the purpose of this application is to provide an optimized SARS-CoV-2 cyclic peptide inhibitor that can resist the enzymatic cleavage of trypsin. At the same time, the cyclic peptide is optimized to have a better inhibitory effect.

[0009] On the one hand, this application provides a cyclic peptide mutant.

[0010] The cyclic peptide mutant before mutation was:

[0011] Ac = Acetyl group;

[0012] L Y = L-type tyrosine;

[0013] The cyclic peptide mutant includes amino acid modifications, at least one of which involves adding a carbon atom; the amino acid modifications give the cyclic peptide mutant resistance to trypsin cleavage.

[0014] Specifically, the number of carbon atoms added can be n, where n is an integer; alternatively, the number of carbon atoms added is 1.

[0015] In some specific embodiments, the amino acid modification includes, but is not limited to: (1) cysteine ​​methylation; or (2) arginine replacement with high arginine.

[0016] The cysteine ​​methylation described herein involves the substitution of a methyl group with cysteine, which adds one carbon atom.

[0017] The high-arginine mentioned above is a homologous amino acid to arginine, and the high-arginine has one more carbon atom than arginine. When not part of a peptide chain, the molecular formula of high-arginine is C7H. 16 N4O2, the molecular formula of arginine is C6H 14 N4O2.

[0018] Based on the aforementioned amino acid modifications, the amino acid modifications may also selectively include: (3) replacing L-tyrosine with L-phenylalanine.

[0019] Specifically, the cyclic peptide mutant is selected from any of the following:

[0020] 1)6L3-3P11R12 Me The C sequence is:

[0021] Ac = Acetyl group;

[0022] L Y = L-type tyrosine;

[0023] Me C = methylated cysteine;

[0024] Among them, Ac and LThe amino group at the N-terminus of Y is linked to the amino group, and the hydroxyl group in the carboxyl group of the last amino acid G at the C-terminus of the polypeptide chain is replaced by amino-NH2 to form an amide.

[0025] 2) The 6L3-3P11hR sequence is:

[0026] Ac = Acetyl group;

[0027] L Y = L-type tyrosine;

[0028] hR = High arginine;

[0029] Among them, Ac and L The amino group at the N-terminus of Y is linked to the amino group, and the hydroxyl group in the carboxyl group of the last amino acid G at the C-terminus of the polypeptide chain is replaced by amino-NH2 to form an amide.

[0030] The 6L3-1F3P11hR sequence is:

[0031] Ac = Acetyl group;

[0032] L F = L-type phenylalanine;

[0033] hR = High arginine;

[0034] Among them, Ac and L The amino group at the N-terminus of F is linked to the amino group, and the hydroxyl group in the carboxyl group of the last amino acid G at the C-terminus of the polypeptide chain is replaced by amino-NH2 to form an amide.

[0035] On the other hand, this application provides the application of the aforementioned cyclic peptide mutant in the preparation of coronavirus inhibitors.

[0036] Optionally, the coronavirus mentioned is sabezia coronavirus.

[0037] The aforementioned sarbene coronavirus includes, but is not limited to, SARS-CoV-2 or coronaviruses with more than 85% homology to SARS-CoV-2, which can be more than 90% or 95%, or more than 90.1% or 96.9%.

[0038] Examples include coronaviruses with more than 85% homology to SARS-CoV-2, such as bat coronavirus RaTG13 or pangolin coronavirus GD / 1 / 2019.

[0039] Prior Art "Niu S, Wang J, Bai B, Wu L, Zheng A, Chen Q, Du P, Han P, Zhang Y, Jia Y, Qiao C, Qi J, Tian WX, Wang HW, Wang Q, Gao GF. Molecular basis of cross-species ACE2 interactions with SARS-CoV-2-like viruses of pangolin origin. EMBO J. 2021Aug 16;40(16):e107786.doi:10.15252 / embj.2021107786.Epub 2021Jun 8.Erratumin:EMBO J.2022Jan 4;41(1):e109962.doi:10.15252 / embj.2021109962.Erratumin:EMBO J.2022Jan 04;41(1):EMBJ2021109962.doi:10.15252 / embj.2021109962.PMID:34018203;PMCID:PMC8209949.” recorded that RaTG13 and the RBD amino acid sequence of the novel coronavirus have a homology of 90.1%.

[0040] Prior Art "Niu S, Wang J, Bai B, Wu L, Zheng A, Chen Q, Du P, Han P, Zhang Y, Jia Y, Qiao C, Qi J, Tian WX, Wang HW, Wang Q, Gao GF. Molecular basis of cross-species ACE2 interactions with SARS-CoV-2-like viruses of pangolin origin. EMBO J. 2021Aug 16;40(16):e107786.doi:10.15252 / embj.2021107786.Epub 2021Jun 8.Erratumin:EMBO J.2022Jan 4;41(1):e109962.doi:10.15252 / embj.2021109962.Erratumin:EMBO J.2022Jan 04;41(1):EMBJ2021109962.doi:10.15252 / embj.2021109962.PMID:34018203;PMCID:PMC8209949.” recorded that the amino acid sequence homology between GD / 1 / 2019 and the RBD of the novel coronavirus reached 96.9%.

[0041] Optionally, the coronavirus includes any one or more of the SARS-CoV-2 prototype strain or variant strain.

[0042] The mutant strains mentioned include, but are not limited to, any one or more of the following: Delta B.1.617.2, Omicron BA.1, Omicron BA.2, Omicron BA.4 / 5, Omicron BF.7, Omicron BQ.1, Omicron BQ.1.1, Omicron XBB, Omicron XBB.1.5, Omicron CH.1.1, and Omicron BA.2.86.

[0043] Furthermore, this application provides the use of the aforementioned cyclic peptide mutant in the preparation of products that alleviate symptoms of infection caused by coronaviruses.

[0044] The symptoms of infection caused by the coronavirus include, but are not limited to, any one or more of the following: respiratory infection symptoms, fever, muscle pain, diarrhea, joint pain, hypoxemia, general weakness, conjunctival congestion, tearing, dry and itchy eyes, increased eye discharge, nausea and vomiting, loss of appetite, chest tightness, and palpitations.

[0045] The respiratory infection symptoms include, but are not limited to, any one or more of the following: dry throat, sore throat, cough, nasal congestion, runny nose, pneumonia, and difficulty breathing.

[0046] On the other hand, this application provides a drug including the aforementioned cyclic peptide mutant.

[0047] The drug comprises at least the aforementioned cyclic peptide mutant as an active ingredient, and may also selectively include other active ingredients.

[0048] Optionally, the dosage form of the drug, according to the route of administration, includes, but is not limited to: gastrointestinal administration, injection administration, respiratory administration, and ocular administration.

[0049] The drug may also include pharmaceutical excipients.

[0050] The pharmaceutical excipients include, but are not limited to, any one or more of the following: excipients, buffers, emulsifiers, stabilizers, diluents, binders, preservatives, and lubricants.

[0051] Specifically, the excipient is selected from at least one of microcrystalline cellulose, lactose, pregelatinized starch, cyclodextrin, carboxymethyl cellulose, and mannitol.

[0052] Specifically, the buffer is selected from at least one of sodium dihydrogen phosphate, sodium bicarbonate, ammonium bicarbonate, sodium acetate, citrate, histidine, and succinate.

[0053] Specifically, the emulsifier is selected from at least one of magnesium stearate, zinc stearate, calcium stearate, glyceryl stearate, sorbitan isostearate, sorbitan oleate, glyceryl oleate, and polyglycerol-3 polyricinoleate.

[0054] Specifically, the stabilizer is selected from at least one of acacia gum, agar, alginic acid, cellulose ether, and carboxymethyl chitosan.

[0055] Specifically, the diluent is selected from at least one of erythritol, mannitol, sorbitol, xylitol, lactose, sucrose, corn starch, potato starch, calcium phosphate, calcium citrate, and crystalline cellulose.

[0056] Specifically, the adhesive is selected from at least one of ethanol, starch paste, syrup, hydroxypropyl methylcellulose, sodium carboxymethyl cellulose, sodium alginate, and polyvinylpyrrolidone.

[0057] Specifically, the preservative is selected from at least one of methylparaben, propylparaben, methylparaben, ethylparaben, propylparaben, chlorobutanol, thimerosal, mercuric oxycyanide, phenoxyethanol, chlorhexidine, benzoic acid, sodium benzoate, chlorocresol, benzalkonium bromide, benzalkonium chloride, and ethylparaben.

[0058] Specifically, the lubricant is selected from at least one of magnesium stearate, stearic acid, sodium chloride, sodium oleate, sodium lauryl sulfate, and poloxamer.

[0059] This application also provides genetically engineered products for expressing, producing, or delivering the aforementioned cyclic peptide mutants.

[0060] As is generally known to those skilled in the art, the genetically engineered products may include any form such as nucleic acid molecules and genetically engineered cells.

[0061] On the other hand, this application provides a method for inhibiting coronaviruses, including administering the aforementioned cyclic peptide mutant to a subject in need.

[0062] In some implementations, the coronavirus is sabezoar; the sabezoar includes SARS-CoV-2 or a virus with more than 85% homology to SARS-CoV-2.

[0063] In some implementations, the coronaviruses with more than 85% homology to SARS-CoV-2 include bat coronavirus RaTG13 or pangolin coronavirus GD / 1 / 2019.

[0064] In some implementations, the coronavirus includes any one or more of the SARS-CoV-2 prototype strain or variant strain.

[0065] In some embodiments, the variant strains include any one or more of Delta B.1.617.2, Omicron BA.1, Omicron BA.2, Omicron BA.4 / 5, Omicron BF.7, Omicron BQ.1, Omicron BQ.1.1, Omicron XBB, Omicron XBB.1.5, Omicron CH.1.1, and Omicron BA.2.86.

[0066] On the other hand, this application provides a method for alleviating symptoms of infection caused by coronavirus, including administering the aforementioned cyclic peptide mutant to subjects in need.

[0067] In some implementations, the symptoms of infection caused by the coronavirus include any one or more of the following: respiratory symptoms, fever, muscle pain, diarrhea, joint pain, hypoxemia, general weakness, conjunctival congestion, tearing, dry and itchy eyes, increased eye discharge, nausea and vomiting, loss of appetite, chest tightness, and palpitations.

[0068] In some implementations, the respiratory infection symptoms include any one or more of the following: dry throat, sore throat, cough, nasal congestion, runny nose, pneumonia, and difficulty breathing.

[0069] The beneficial effects of this application are:

[0070] Based on the previously discovered 6L3-3P11R cyclic peptide, this application further optimized it, and the resulting cyclic peptide mutant improved its resistance to common trypsin in the human body and further enhanced its antiviral activity, which is of great significance for improving the antiviral application of cyclic peptide drugs. Attached Figure Description

[0071] Figure 1 shows 6L3-3P11R12 Me Chromatographic analysis of C-cyclic peptide.

[0072] Figure 2 shows 6L3-3P11R12 Me Mass spectrum of C-cyclic peptide.

[0073] Figure 3 shows the chromatographic analysis of the 6L3-3P11hR cyclic peptide.

[0074] Figure 4 shows the mass spectrum of the 6L3-3P11hR cyclic peptide.

[0075] Figure 5 shows the chromatographic analysis of the 6L3-1F3P11hR cyclic peptide.

[0076] Figure 6 shows the mass spectrum of the 6L3-1F3P11hRc cyclic peptide.

[0077] Figure 7 shows the neutralizing effect of 6L3-3P11R on BF.7 SARS-CoV-2 pseudovirus in the presence of trypsin.

[0078] Figure 8 shows the effect of 6L3-3P11R12 in the presence of trypsin. Me Neutralizing effects of C, 6L3-3P11hR and 6L3-1F3P11hR cyclic peptides on BF.7 SARS-CoV-2 pseudovirus

[0079] Figure 9 shows the neutralizing effects of 6L3-3P11hR and 6L3-1F3P11hR on different SARS-CoV-2 pseudoviruses.

[0080] Figure 10 shows the thermal stability verification of the cyclic peptides 6L3-3P11hR and 6L3-1F3P11hR. Detailed Implementation

[0081] The present application will be further described in detail below with reference to specific embodiments. The following embodiments are not intended to limit the present application, but only to illustrate the present application. Unless otherwise specified, the experimental methods used in the following embodiments are generally performed under conventional conditions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0082] This application modifies the 6L3-3P11R cyclic peptide, which can be found in the disclosure of invention patent application number 202311261559.5. The modified peptide yields 6L3-3P11R12. Me C, 6L3-3P11hR cyclic peptide and 6L3-1F3P11hR cyclic peptide. All cyclic peptides were synthesized and purified by Anhui Guoping Pharmaceutical Co., Ltd.

[0083] 6L3-3P11R12 Me The C sequence is:

[0084] Ac = Acetyl group;

[0085] L Y = L-type tyrosine;

[0086] Me C = methylated cysteine;

[0087] Among them, Ac and L The amino group at the N-terminus of Y is linked to the amino group, and the hydroxyl group in the carboxyl group of the last amino acid G at the C-terminus of the polypeptide chain is replaced by an amino group (-NH2) to form an amide.

[0088] The 6L3-3P11hR sequence is:

[0089] Ac = Acetyl group;

[0090] L Y = L-type tyrosine;

[0091] hR = High arginine;

[0092] Among them, Ac and L The amino group at the N-terminus of Y is linked to the amino group, and the hydroxyl group in the carboxyl group of the last amino acid G at the C-terminus of the polypeptide chain is replaced by an amino group (-NH2) to form an amide.

[0093] The 6L3-1F3P11hR sequence is:

[0094] Ac = Acetyl group;

[0095] L F = L-type phenylalanine;

[0096] hR = High arginine;

[0097] Among them, Ac and L The amino group at the N-terminus of F is linked to the amino group, and the hydroxyl group in the carboxyl group of the last amino acid G at the C-terminus of the polypeptide chain is replaced by an amino group (-NH2) to form an amide.

[0098] The structural formulas of each cyclic peptide are as follows:

[0099] 6L3-3P11R12 Me The structural formula of the C-cyclic peptide is as follows:

[0100] The structural formula of the 6L3-3P11hR cyclic peptide is as follows:

[0101] The structural formula of the 6L3-1F3P11hR cyclic peptide is as follows:

[0102] Example 1: Preparation and Identification of Cyclic Peptides

[0103] Cyclic peptides were synthesized according to the standard fluorene methoxycarbonyl (Fmoc) solid-phase peptide synthesis (SPPS) protocol, with peptide synthesis proceeding sequentially from the C-terminus to the N-terminus.

[0104] (1) 0.5 g of Rink Amide MBHA resin was treated with a dimethylformamide (DMF) solution containing 20% ​​piperidine to remove the Fmoc protecting group. Subsequently, 10 mL of a DMF solution containing 0.3 mmol of Fmoc-Gly-OH (or Fmoc-Cys(Trt)-OH), 0.3 mmol of 1-hydroxybenzotriazole (HOBT), and 5% diisopropylcarbodiimide (DIC) was added to the resin, and the reaction was carried out under nitrogen bubbling conditions for 1.5 h. The resin was then thoroughly washed three times with DMF and dichloromethane (DCM), and the Fmoc protecting group was removed again with a DMF solution containing 20% ​​piperidine.

[0105] (2) To couple the subsequent amino acid, the resin was mixed with 10 mL of freshly prepared DMF solution containing 0.9 mmol Fmoc-AA-OH (the subsequent amino acid), 0.9 mmol HOBT, and 9% DIC, and the coupling reaction was carried out under nitrogen bubbling conditions for 1 hour. The above de-Fmoc and coupling steps were repeated iteratively until the desired peptide sequence and length were synthesized.

[0106] (3) The N-terminal bromoacetyl group is obtained by coupling bromoacetic acid with the free amino group (N-terminal tyrosine) at the N-terminus of the polypeptide.

[0107] (4) The peptide was then cleaved from the resin, precipitated with diethyl ether (Et2O), and redissolved in dimethyl sulfoxide (DMSO). The thioether cyclization reaction was then carried out at pH 8.0 for 1 hour. Finally, trifluoroacetic acid (TFA) was added to the acidified solution, and purification was performed using reversed-phase high-performance liquid chromatography (RP-HPLC). The mobile phase consisted of an aqueous solution containing 0.1% trifluoroacetic acid and acetonitrile containing 0.1% trifluoroacetic acid (MeCN; mobile phase B). The synthesized cyclic peptide was detected by electrospray ionization mass spectrometry (ESI-MS), and its purity was confirmed by analytical HPLC.

[0108] 6L3-3P11R12 Me The chromatograms and mass spectra of C, 6L3-3P11hR cyclic peptide, and 6L3-1F3P11hR are shown in Figures 1-6. Based on the preparation method and the chromatograms and mass spectra, the structural formulas of the above three cyclic peptides can be determined as described above.

[0109] Example 2: Inhibitory effect of cyclic peptide 6L3-3P11R on SARS-CoV-2 pseudovirus in the presence of trypsin

[0110] (1) The day before the experiment, trypsin was used to digest Vero cells in the logarithmic growth phase, counted, and re-seeded in 96-well plates. When the cell density reaches 80-100% after 18-24 hours, the cells can be used for the experiment.

[0111] (2) Take out the BF.7 pseudovirus from -80℃ on the day of the experiment (recorded in "Tong Z, Tong J, Lei W, Xie Y, Cui Y, Jia G, Li S, Zhang Z, Cheng Z, Xing X, Ma H, Deng L, Zhang R, Zhao X, Liu K, Wang Q, Qi J, Huang H, Song R, Su Z, Wu G, Lou J, Gao GF. synergistic bispecific strategy of rescuing antibodies for SARS-CoV-2 escape variants, including BA.2.86,EG.5.1,and JN.1.Cell Rep.2024 Jun 25;43(6):114338.doi:10.1016 / j.celrep.2024.114338.Epub 2024 Jun 8. The article PMID:38850530” is available to the public from the applicant and may only be used to repeat the experiments in this application and may not be used for other purposes. The pseudovirus was thawed on ice and then diluted with complete culture medium (DMEM medium, 10% FBS) to 1000TU / 50μL / well.

[0112] (3) Dilution of cyclic peptide (initial concentration of 6.25 μM, 2 replicates, 4-fold serial dilution of 6L3-3P11R cyclic peptide, 11 gradients).

[0113] (4) Add trypsin to the diluted cyclic peptide solution to make a final concentration of 4 μg / mL, mix the liquid well, and incubate at 37°C for 30 min.

[0114] (5) Diluted BF.7 pseudovirus was poured into 10cm cell culture dishes and added to 96-well plates (1:1 volume ratio of cyclic peptide to cyclic peptide, i.e., 60μL trypsin-treated cyclic peptide + 60μL pseudovirus per replicate well), and mixed once by pipetting. The 96-well plates were incubated at 37℃ for 1 hour. After 40-50 minutes of incubation, the Vero cells prepared in advance were removed from the incubator, the cell supernatant was discarded, and 100μL of the mixture of cyclic peptide and virus was added. After incubation at 37℃ for 15 hours, the green fluorescence was detected by CQ1 microscopy, and photographed and counted.

[0115] A blank control with only culture medium was set up, and a challenge control containing an equal amount of pseudovirus but no cyclic peptide was set up.

[0116] (6) Data analysis: The EC50 of the cyclic peptide was calculated using GraphPad. 50(The half-maximal effective concentration, i.e., the concentration of the test substance that can cause 50% of the maximum effect in a pharmacological experiment). The calculation method is: inhibition rate = 1 - (fluorescence count in experimental group - fluorescence count in blank control group) / (fluorescence count in challenge control group - fluorescence count in blank control group) * 100 (%).

[0117] II. Results and Analysis

[0118] The experimental results are shown in Figure 7. The cyclic peptide 6L3-3P11R has a neutralizing effect on BF.7 pseudovirus, EC 50 The value was 2731 nM, which was significantly lower than the effect in the absence of pancreatic enzymes, with a decrease of more than 500 times.

[0119] Example 3 Cyclic Peptide 6L3-3P11R12 Me Inhibitory effects of C, 6L3-3P11hR and 6L3-1F3P11hR on SARS-CoV-2 pseudovirus in the presence of trypsin

[0120] (1) The day before the experiment, trypsin was used to digest Vero cells in the logarithmic growth phase, counted, and re-seeded in 96-well plates. When the cell density reaches 80-100% after 18-24 hours, the cells can be used for the experiment.

[0121] (2) Take out the BF.7 pseudovirus from -80℃ on the day of the experiment (recorded in "Tong Z, Tong J, Lei W, Xie Y, Cui Y, Jia G, Li S, Zhang Z, Cheng Z, Xing X, Ma H, Deng L, Zhang R, Zhao X, Liu K, Wang Q, Qi J, Huang H, Song R, Su Z, Wu G, Lou J, Gao GF. synergistic bispecific strategy of rescuing antibodies for SARS-CoV-2 escape variants, including BA.2.86,EG.5.1,and JN.1.Cell Rep.2024 Jun 25;43(6):114338.doi:10.1016 / j.celrep.2024.114338.Epub 2024 Jun 8. The article PMID:38850530” is available to the public from the applicant and may only be used to repeat the experiments in this application and may not be used for other purposes. The pseudovirus was thawed on ice and then diluted with complete culture medium (DMEM medium, 10% FBS) to 1000TU / 50μL / well.

[0122] (3) Dilution of cyclic peptide (initial concentration 6.25 μM, 2 replicates, cyclic peptide 6L3-3P11R12) Me C, 6L3-3P11hR and 6L3-1F3P11hR were diluted 4-fold in 11 serial dilutions.

[0123] (4) Add trypsin to the diluted cyclic peptide solution to a final concentration of 4 μg / mL, mix the liquid well, and incubate at 37°C for 30 min.

[0124] (5) Diluted BF.7 pseudovirus was poured into 10cm cell culture dishes and added to 96-well plates (1:1 volume ratio of cyclic peptide to cyclic peptide, i.e., 60μL trypsin-treated cyclic peptide + 60μL pseudovirus per replicate well), and mixed once by pipetting. The 96-well plates were incubated at 37℃ for 1 hour. After 40-50 minutes of incubation, the Vero cells prepared in advance were removed from the incubator, the cell supernatant was discarded, and 100μL of the mixture of cyclic peptide and virus was added. After incubation at 37℃ for 15 hours, the green fluorescence was detected by CQ1 microscopy, and photographed and counted.

[0125] A blank control with only culture medium was set up, and a challenge control containing an equal amount of pseudovirus but no cyclic peptide was set up.

[0126] (6) Data analysis: EC50 of each cyclic peptide was calculated using GraphPad. 50 (The half-maximal effective concentration, i.e., the concentration of the test substance that can cause 50% of the maximum effect in a pharmacological experiment). The calculation method is: inhibition rate = 1 - (fluorescence count in experimental group - fluorescence count in blank control group) / (fluorescence count in challenge control group - fluorescence count in blank control group) * 100 (%).

[0127] II. Results and Analysis

[0128] The experimental results are shown in Figure 8. In the presence of trypsin, the cyclic peptide 6L3-3P11R12 Me C, 6L3-3P11hR, and 6L3-1F3P11hR have a neutralizing effect on BF.7 pseudovirus. EC 50 The concentrations were 56.5 nM, 2.71 nM, and 1.3 nM, respectively, all of which showed superior inhibitory effects compared to the cyclic peptide 6L3-3P11R (2731 nM).

[0129] Example 4: Neutralization experiment of cyclic peptides 6L3-3P11hR and 6L3-1F3P11hR with SARS-CoV-2 pseudovirus

[0130] I. Experimental Methods

[0131] 1. Preparation of fake viruses

[0132] This application provides the following 12 pseudoviruses: the SARS-CoV-2 prototype strain (SARS-CoV-2-PT) and 11 variant strains—Delta (B.1.617.2), Omicron (BA.1), Omicron (BA.2), Omicron (BA.4 / 5), Omicron (BF.7), Omicron (BQ.1), Omicron (BQ.1.1), Omicron (XBB), Omicron (XBB.1.5), Omicron (CH.1.1), and Omicron (BA.2.86).

[0133] Among them, pseudoviruses of the SARS-CoV-2 prototype strain (SARS-CoV-2-PT) and its variants Delta (B.1.617.2), Omicron (BA.1, BA.2, BA.4 / 5, BF.7, BQ.1.1, XBB, XBB.1.5, BA.2.86) are recorded in “Tong Z, Tong J, Lei W, Xie Y, Cui Y, Jia G, Li S, Zhang Z, Cheng Z, Xing X, Ma H, Deng L, Zhang R, Zhao X, Liu K, Wang Q, Qi J, Huang H, Song R, Su Z, Wu G, Lou J, Gao GF. Deciphering a reliable synergistic bispecific strategy of rescuing antibodies for SARS-CoV-2 escape variants, including BA.2.86, EG.5.1, and JN.1. Cell Rep.2024 Jun The article “25;43(6):114338.doi:10.1016 / j.celrep.2024.114338.Epub 2024Jun 8.PMID:38850530” is available to the public from the applicant and may only be used to repeat the experiments in this application and may not be used for other purposes.

[0134] The pseudoviruses of the SARS-CoV-2 variant strains BQ.1 and CH.1.1 are described in the article “Liu C,Xu S,Zheng Y,Xie Y,Xu K,Chai Y,Luo T,Dai L,Gao GF.Mosaic RBD nanoparticle elicits immunodominant antibody responses across sarbecoviruses.Cell Rep.2024May 28;43(5):114235.doi:10.1016 / j.celrep.2024.114235.Epub 2024May 14.PMID:38748880.”, which are available to the public from the applicant and may only be used to repeat the experiments in this application and may not be used for other purposes.

[0135] 2. Neutralization Experiment

[0136] (1) The day before the experiment, trypsin was used to digest Vero cells in the logarithmic growth phase, counted, and re-seeded in 96-well plates. When the cell density reaches 80-100% after 18-24 hours, the cells can be used for the experiment.

[0137] (2) On the day of the experiment, each fake virus packaged in step 1 was taken out from -80℃ and placed on ice to thaw. Then, the fake virus was diluted with complete culture medium (DMEM medium, 10% FBS) to 1000TU / 50μL / well.

[0138] (3) Dilution of cyclic peptides (initial concentration of 100 μM, 2 replicates, 4-fold serial dilution of two cyclic peptides, 6L3-3P11hR and 6L3-1F3P11hR, 14 gradients).

[0139] (4) Pour the diluted pseudoviruses into 10cm cell culture dishes, add them to 96-well plates (1:1 volume ratio with cyclic peptide, i.e., 60μL diluted cyclic peptide + 60μL pseudovirus per replicate well), and mix once by pipetting. Incubate the 96-well plates at 37℃ for 1 hour; after 40-50 minutes of incubation, remove the prepared Vero cells from the incubator, discard the cell supernatant, and add 100μL of the cyclic peptide and virus mixture. After incubation at 37℃ for 15 hours, detect green fluorescence using a CQ1 microscope and take pictures and count the cells.

[0140] A blank control with only culture medium was set up, and a challenge control containing an equal amount of pseudovirus but no cyclic peptide was set up.

[0141] (5) Data analysis: EC50 of each cyclic peptide was calculated using GraphPad. 50(The half-maximal effective concentration, i.e., the concentration of the test substance that can cause 50% of the maximum effect in a pharmacological experiment). The calculation method is: inhibition rate = 1 - (fluorescence count in experimental group - fluorescence count in blank control group) / (fluorescence count in challenge control group - fluorescence count in blank control group) * 100 (%).

[0142] II. Results and Analysis

[0143] The experimental results are shown in Figure 9. The cyclic peptides 6L3-3P11hR and 6L3-1F3P11hR showed good neutralizing effects against pseudoviruses of PT, Delta, BA.1, BA.2, BA.4 / 5, BF.7, BQ.1, BQ.1.1, XBB, XBB.1.5, CH.1.1, and BA.2.86 viral strains. EC 50 The values ​​are in the range of 1.1 nM to 1.1 μM.

[0144] Example 5: Affinity determination of cyclic peptides 6L3-3P11hR and 6L3-1F3P11hR with RBD

[0145] Intermolecular interactions were detected using surface plasmon resonance (SPR) on a Biacore 8K biomacromolecule interaction analysis system manufactured by GE Healthcare. The supernatant of expressed RBD (containing expressed RBD-Fc protein) was captured using a protein A chip (Cytiva, 29127555) as the stationary phase, while the mobile phase consisted of the cyclic peptide to be detected. Kinetic parameters were then analyzed and plotted using BIA evaluation software. The analysis was performed at an isothermal temperature of 25°C.

[0146] Experimental steps:

[0147] (1) Expression of viral RBD-Fc protein and microarray fixation

[0148] The viral RBD-Fc protein to be expressed in this application comes from the following sources: PT RBD, Delta RBD, Omicron BA.2 RBD, Omicron BQ.1.1 RBD, Omicron XBB.1.5 RBD, Omicron JN.1 RBD, RaTG13 RBD, and GD / 1 / 2019 RBD.

[0149] For details on the expression plasmids and their construction methods for expressing PT RBD, Omicron BA.2 RBD, RaTG13 RBD, GD / 1 / 2019 RBD and Fc fusion protein, please refer to "Liu H, Wu L, Liu B, Xu K, Lei W, Deng J, Rong X, Du P, Wang L, Wang D, Zhang X, Su C, Bi Y, Chen H, Liu WJ, Qi J, Cui Q, Qi S, Fan R, Jiang J, Wu G, Gao GF, Wang Q. Two pan-SARS-CoV-2 nanobodies and their multivalent derivatives effectively prevent Omicron infections in mice. Cell Rep Med. 2023 Feb 21;4(2):100918.doi:10.1016 / j.xcrm.2023.100918.Epub 2023 Jan 12. PMID:36702124; PMCID:PMC9834170.” Expression plasmids used to express the remaining viral RBD-Fc proteins were prepared using the same method, differing only in that the viral RBD sequences were replaced. The BQ.1.1 RBD sequence is shown in SEQ ID No.1, the XBB.1.5 RBD sequence is shown in SEQ ID No.2, and the JN.1 RBD sequence is shown in SEQ ID No.3.

[0150] SEQ ID No. 1:

[0151] SEQ ID No. 2:

[0152] SEQ ID No. 3:

[0153] After obtaining the expression plasmids for expressing the viral RBD-Fc protein, protein expression and microarray fixation were performed as follows: The expression plasmids for expressing the viral RBD-Fc protein were mixed with PEI at a ratio of 1:3 and transfected into 293T cells. After 2 days, the supernatant was collected, filtered, and fixed into a Protein A microarray (Cytiva, 29127555) according to the preset capture program. PBST solution was used as the buffer (PBS buffer contains 0.05% Tween 20, volume percentage).

[0154] (2) Dissolve the cyclic peptides 6L3-3P11hR and 6L3-1F3P11hR in DMSO and prepare a 10mM stock solution. Following the GE Healthcare Life Science Procedure 29264621AA, the peptides were serially diluted with 1.05×PBST (1.05×PBS buffer containing 0.05% Tween 20, volume percentage) and 5% DMSO PBST solution (1×PBS buffer containing 0.05% Tween 20, 5% DMSO, volume percentage), with each concentration being 100μL.

[0155] (3) Using the Single-cycle kinetics method, the sample position was displayed according to the pre-programmed instructions, and the sample was added to a 96-well plate. The test solution flowed sequentially through the chip, and the real-time response value was recorded. The data were processed using BIAevaluation Version 4.1 (GE Healthcare) software to calculate the affinity of cyclic peptides 6L3-3P11hR and 6L3-1F3P11hR for RBD-Fc protein.

[0156] The results of surface plasmon resonance experiments to detect its binding affinity (nM) are as follows:

[0157] The affinity constants for cyclic peptide 6L3-3P11hR and SARS-CoV-2 PT RBD protein were 45500 nM; for cyclic peptide 6L3-3P11hR and SARS-CoV-2 Delta RBD protein, the affinity constant was 47800 nM; for cyclic peptide 6L3-3P11hR and SARS-CoV-2 BA.2 RBD protein, the affinity constant was 4.07 nM; for cyclic peptide 6L3-3P11hR and SARS-CoV-2 BQ.1.1 RBD protein, the affinity constant was 3.17 nM; for cyclic peptide 6L3-3P11hR and SARS-CoV-2 XBB.1.5 RBD protein, the affinity constant was 4.69 nM; and for cyclic peptide 6L3-3P11hR and SARS-CoV-2 JN.1... The affinity constant of the RBD protein is 4.73 nM; the affinity constants of the cyclic peptide 6L3-3P11hR and bat coronavirus RaTG13 RBD protein are 65200 nM, and the affinity constants of the cyclic peptide 6L3-3P11hR and pangolin coronavirus GD / 1 / 2019 RBD protein are 35100 nM.

[0158] The affinity constants for cyclic peptide 6L3-1F3P11hR and SARS-CoV-2 PT RBD protein are 30500 nM; for cyclic peptide 6L3-1F3P11hR and SARS-CoV-2 Delta RBD protein, the affinity constant is 31900 nM; for cyclic peptide 6L3-1F3P11hR and SARS-CoV-2 BA.2 RBD protein, the affinity constant is 3.55 nM; for cyclic peptide 6L3-1F3P11hR and SARS-CoV-2 BQ.1.1 RBD protein, the affinity constant is 1.81 nM; for cyclic peptide 6L3-1F3P11hR and SARS-CoV-2 XBB.1.5 RBD protein, the affinity constant is 3.05 nM; and for cyclic peptide 6L3-1F3P11hR and SARS-CoV-2 JN.1... The affinity constant of the RBD protein is 3.15 nM; the affinity constants of the cyclic peptide 6L3-1F3P11hR and bat coronavirus RaTG13 RBD protein are 45900 nM, and the affinity constants of the cyclic peptide 6L3-1F3P11hR and pangolin coronavirus GD / 1 / 2019 RBD protein are 31500 nM.

[0159] The above data indicate that the cyclic peptides 6L3-3P11hR and 6L3-1F3P11hR have a strong affinity for the RBD of various SARS-CoV-2 variants. Simultaneously, the cyclic peptides also exhibit binding ability to bat coronaviruses and pangolin coronaviruses.

[0160] Example 6: Evaluation Experiment of the High Temperature Tolerance of Cyclic Peptides

[0161] (1) The day before the experiment, trypsin was used to digest Vero cells in the logarithmic growth phase, counted, and re-seeded in 96-well plates. When the cell density reaches 80-100% after 18-24 hours, the cells can be used for the experiment.

[0162] (2) On the day of the experiment, the Omicron (BF.7) pseudovirus was taken out from -80℃ and placed on ice to thaw. Then, the pseudovirus was diluted with complete culture medium (DMEM medium, 10% FBS) to 1000TU / 50μL / well.

[0163] (3) After heating the cyclic peptide at 160℃ for 10 min, remove it and dilute the cyclic peptide (initial concentration of 6.25 μM, 2 replicates, 4-fold serial dilution of the two cyclic peptides 6L3-3P11hR and 6L3-1F3P11hR, 11 gradients).

[0164] (4) Pour the diluted pseudovirus into 10cm cell culture dishes and add it to 96-well plates (1:1 volume ratio with cyclic peptide, i.e., 60μL of diluted cyclic peptide + 60μL of pseudovirus per replicate well), and mix once by pipetting. Incubate the 96-well plates at 37℃ for 1 hour; after 40-50 minutes of incubation, remove the prepared Vero cells from the incubator, discard the cell supernatant, and add 100μL of the mixture of cyclic peptide and virus. After incubation at 37℃ for 15 hours, detect green fluorescence using a CQ1 microscope and take pictures and count the cells.

[0165] A blank control with only culture medium was set up, and a challenge control containing an equal amount of pseudovirus but no cyclic peptide was set up.

[0166] (5) Data analysis: EC50 of each cyclic peptide was calculated using GraphPad. 50 (The half-maximal effective concentration, i.e., the concentration of the test substance that can cause 50% of the maximum effect in a pharmacological experiment). The calculation method is: inhibition rate = 1 - (fluorescence count in experimental group - fluorescence count in blank control group) / (fluorescence count in challenge control group - fluorescence count in blank control group) * 100 (%).

[0167] The experimental results are shown in Figure 10. After heating at 160℃ for 10 min, the cyclic peptides 6L3-3P11hR and 6L3-1F3P11hR both showed good neutralizing activity against the pseudovirus of the BF.7 virus strain. 50 The values ​​were 4.1 nM and 2.9 nM, indicating that the cyclic peptides 6L3-3P11hR and 6L3-1F3P11hR have good thermal stability.

Claims

1. A cyclic peptide mutant, characterized in that, The cyclic peptide mutant before mutation was: Ac = Acetyl group; L Y = L-type tyrosine; The cyclic peptide mutant includes amino acid modifications, which occur at any one or more amino acid sites, and these modifications endow the cyclic peptide mutant with resistance to trypsin cleavage.

2. The cyclic peptide mutant according to claim 1, characterized in that, The amino acid modification includes at least one type of modification that increases carbon atoms, and the number of carbon atoms increased is preferably 1.

3. The cyclic peptide mutant according to claim 2, characterized in that, The amino acid modifications include: (1) cysteine ​​methylation; or (2) arginine replacement with high arginine.

4. The cyclic peptide mutant according to claim 3, characterized in that, The amino acid modifications also include: (3) replacing L-tyrosine with L-phenylalanine.

5. The cyclic peptide mutant according to claim 4, characterized in that, The cyclic peptide mutant is selected from any of the following: 1)6L3-3P11R12 Me The C sequence is: Ac = Acetyl group; L Y = L-type tyrosine; Me C = methylated cysteine; Among them, Ac and L The amino group at the N-terminus of Y is linked to the amino group, and the hydroxyl group in the carboxyl group of the last amino acid G at the C-terminus of the polypeptide chain is replaced by amino-NH2 to form an amide. 2) The 6L3-3P11hR sequence is: Ac = Acetyl group; L Y = L-type tyrosine; hR = High arginine; Among them, Ac and L The amino group at the N-terminus of Y is linked to the amino group, and the hydroxyl group in the carboxyl group of the last amino acid G at the C-terminus of the polypeptide chain is replaced by amino-NH2 to form an amide. The 6L3-1F3P11hR sequence is: Ac = Acetyl group; L F = L-type phenylalanine; hR = High arginine; Among them, Ac and L The amino group at the N-terminus of F is linked to the amino group, and the hydroxyl group in the carboxyl group of the last amino acid G at the C-terminus of the polypeptide chain is replaced by amino-NH2 to form an amide.

6. The use of the cyclic peptide mutant according to any one of claims 1-5 in the preparation of coronavirus inhibitors.

7. The application according to claim 6, characterized in that, The coronavirus is sabeclovirus; the sabeclovirus includes SARS-CoV-2 or viruses with more than 85% homology to SARS-CoV-2.

8. The application according to claim 7, characterized in that, The coronaviruses mentioned that share more than 85% homology with SARS-CoV-2 include bat coronavirus RaTG13 or pangolin coronavirus GD / 1 / 2019.

9. The application according to claim 7, characterized in that, The coronaviruses mentioned include any one or more of the SARS-CoV-2 prototype strain or variant strains.

10. The application according to claim 9, characterized in that, The mutant strains include any one or more of Delta B.1.617.2, Omicron BA.1, Omicron BA.2, Omicron BA.4 / 5, Omicron BF.7, Omicron BQ.1, Omicron BQ.1.1, Omicron XBB, Omicron XBB.1.5, Omicron CH.1.1, and Omicron BA.2.

86.

11. The use of the cyclic peptide mutant according to any one of claims 1-5 in the preparation of products that alleviate symptoms of infection caused by coronavirus.

12. The application according to claim 11, characterized in that, The symptoms of infection caused by the coronavirus include any one or more of the following: respiratory symptoms, fever, muscle pain, diarrhea, joint pain, hypoxemia, general weakness, conjunctival congestion, tearing, dry and itchy eyes, increased eye discharge, nausea and vomiting, loss of appetite, chest tightness, and palpitations.

13. The application according to claim 12, characterized in that, The respiratory infection symptoms include any one or more of the following: dry throat, sore throat, cough, nasal congestion, runny nose, pneumonia, and difficulty breathing.

14. A drug comprising the cyclic peptide mutant according to any one of claims 1-5.

15. The medicament according to claim 14, characterized in that, The dosage forms of the drugs, according to the route of administration, include gastrointestinal administration, injection administration, respiratory administration, and ocular administration.

16. The medicament according to claim 15, characterized in that, The drug also includes pharmaceutical excipients.

17. The medicament according to claim 16, characterized in that, The pharmaceutical excipients include any one or more of the following: excipients, buffers, emulsifiers, stabilizers, diluents, binders, preservatives, and lubricants.

18. Genetically engineered products for expressing, producing, or delivering the cyclic peptide mutant of any one of claims 1-5.

19. The genetically engineered product according to claim 18, characterized in that, This includes nucleic acid molecules and genetically engineered cells.

20. A method for inhibiting coronaviruses, characterized in that, This includes administering the cyclic peptide mutant of any one of claims 1-5 to a subject in need.

21. The method according to claim 20, characterized in that, The coronavirus is sabeclovirus; the sabeclovirus includes SARS-CoV-2 or viruses with more than 85% homology to SARS-CoV-2.

22. The method according to claim 21, characterized in that, The coronaviruses mentioned that share more than 85% homology with SARS-CoV-2 include bat coronavirus RaTG13 or pangolin coronavirus GD / 1 / 2019.

23. The method according to claim 21, characterized in that, The coronaviruses mentioned include any one or more of the SARS-CoV-2 prototype strain or variant strains.

24. The method according to claim 23, characterized in that, The mutant strains include any one or more of Delta B.1.617.2, Omicron BA.1, Omicron BA.2, Omicron BA.4 / 5, Omicron BF.7, Omicron BQ.1, Omicron BQ.1.1, Omicron XBB, Omicron XBB.1.5, Omicron CH.1.1, and Omicron BA.2.

86.

25. A method for alleviating symptoms of infection caused by coronavirus, characterized in that, This includes administering the cyclic peptide mutant of any one of claims 1-5 to a subject in need.

26. The method according to claim 25, characterized in that, The symptoms of infection caused by the coronavirus include any one or more of the following: respiratory symptoms, fever, muscle pain, diarrhea, joint pain, hypoxemia, general weakness, conjunctival congestion, tearing, dry and itchy eyes, increased eye discharge, nausea and vomiting, loss of appetite, chest tightness, and palpitations.

27. The method according to claim 26, characterized in that, The respiratory infection symptoms include any one or more of the following: dry throat, sore throat, cough, nasal congestion, runny nose, pneumonia, and difficulty breathing.

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