CN113755475A - ACE2 cyclopeptide mimic and application thereof in inhibiting new coronavirus - Google Patents
ACE2 cyclopeptide mimic and application thereof in inhibiting new coronavirus Download PDFInfo
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- CN113755475A CN113755475A CN202111186430.3A CN202111186430A CN113755475A CN 113755475 A CN113755475 A CN 113755475A CN 202111186430 A CN202111186430 A CN 202111186430A CN 113755475 A CN113755475 A CN 113755475A
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- 230000003278 mimic effect Effects 0.000 title description 5
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Abstract
The invention discloses an ACE2 cyclopeptide mimetic and application thereof in inhibiting new crown pseudoviruses, and particularly relates to a cyclic polypeptide molecule obtained by combining three chemical modification strategies of side chain cyclization, point mutation and dimerization of amino acids on the basis of a section of spiral polypeptide sequence of angiotensin converting enzyme 2(ACE2), which can effectively inhibit new crown pseudoviruses. The invention also encompasses different derivatives of the cyclic peptide. The ACE2 cyclopeptide mimetic provided by the invention can be further used for developing polypeptide drugs for resisting new coronavirus, and provides a valuable lead molecule for developing a rapid detection kit for the new coronavirus.
Description
Technical Field
The invention relates to an ACE2 cyclic peptide mimic capable of effectively inhibiting new coronavirus at micromolar level and application thereof.
Background
One of the most effective approaches to combat the new coronavirus is to block the protein-protein interaction between the surface spike protein RBD of the new coronavirus and the ACE2 receptor. Most antibody drugs that inhibit the new coronavirus exert biological activity by blocking the interaction of the virus RBD with ACE 2. It is noted that antibody drugs have inherent drawbacks including poor tissue penetration, low temperatures for storage and transportation, and high cost. In contrast, polypeptide drugs have several significant advantages, such as good tissue penetration, ability to be transported and stored at ambient temperature, and low production cost. ACE2 interacts mainly with the RBD of the new coronavirus through a peptide with a segment of its helical structure. Based on this, it has been proposed to use the helical polypeptide of ACE2 for the purpose of inhibiting new coronavirus. Unfortunately, the helical peptide mimetic of ACE2, which has been reported by the former, hardly inhibits the new coronavirus, and is not effective in preventing the new coronavirus from infecting human cells.
Disclosure of Invention
The invention aims at the problems in the prior art and provides an ACE2 cyclopeptide mimetic and application thereof in inhibiting novel coronavirus. Based on the polypeptide sequence of a spiral region of ACE2, the invention combines optimization of amino acid, covalent cross-linking of side chain-side chain, and reductive amination reaction of polypeptide acyl hydrazine and benzene dicarboxaldehyde to obtain an ACE2 cyclic peptide mimic capable of inhibiting new corona pseudoviruses at micromolar level. Compared with the natural helical peptide of ACE2, the polypeptide of the present invention can inhibit new coronavirus obviously and is expected to provide promising candidate molecule for the treatment and fast in vitro detection of new coronavirus.
The ACE2 cyclopeptide mimetic is a dimerization structure of cyclopeptide; the cyclopeptide is polypeptide with cyclized side chain-side chain, the C end of the cyclopeptide contains acyl hydrazide structure, and two thioether bonds are formed after the reaction of the side chain sulfydryl of two Cys in the polypeptide sequence and 4, 4' -dibromomethylbiphenyl; the first amino acid at the C-terminal of the cyclic peptide is 6-aminocaproic acid (Ahx);the dimerized structure is defined in NaCNBH3Under the action of the two polypeptide hydrazides, the two polypeptide hydrazides and benzene dicarbaldehyde (including ortho-position, meta-position and para-position benzene dicarbaldehyde) are subjected to reductive amination to generate covalent dimers.
The structure of the ACE2 cyclopeptide mimetic is shown as follows:
the ACE2 cyclopeptide mimetic is a cyclopeptide molecule obtained by using linear hydrazide peptide as a raw material and sequentially performing side chain-side chain crosslinking and reductive amination transformation on the hydrazide peptide and benzene dicarbaldehyde. The preparation process comprises the following steps:
step 1: preparation of hydrazine-2-Cl-Trt resin
The 2-CI-Trt-Cl resin was swollen with DMF and the repeated treatment of the 2-Cl-Trt-Cl resin with 5% hydrazine hydrate in DMF followed by DCM and MeOH treatment of the resin and drying gave the target hydrazine-2-Cl-Trt resin.
Step 2: solid phase assembly of hydrazide peptides
The freshly prepared hydrazine-2-Cl-Trt resin was transferred to a clean solid phase synthesis tube, the resin was washed 3 times with DMF, and the target polypeptide sequence was assembled in solid phase using the classical Fmoc method. After completion of sequence alignment, TFA cleavage reagent cleaved the resin for 2 hours and the linear hydrazide peptide was precipitated by glacial ethyl ether. The sequence of the hydrazide peptide is:
AcNH-Pro-Thr-Ile-Glu-Glu-Gln-Val-Lys-Tyr-Phe-Cys-Glu-Trp-Phe-Asp-Ala-Glu-Cys-Glu-Asp-Lys-Phe-Tyr-Leu-Ser-Ahx-CONHNH2(Ahx ═ 6-aminocaproic acid).
And step 3: side chain-side chain cyclization modification
Purifying the crude product of hydrazide peptide by high performance liquid chromatography to obtain a powder purified substance; in the presence of newly complexed DMF/H2O/NH4HCO3In the method, pure hydrazide peptide reacts with 4, 4-dibromo methyl biphenyl to form side chain-side chain cyclized hydrazide peptide.
And 4, step 4: reductive amination of hydrazide peptides with phthalaldehyde
In AcOH/MeOH, under the action of sodium cyanoborohydride, hydrazide peptide with cyclized side chain-side chain is subjected to reductive amination with 1, 3-benzene dicarbaldehyde, 1, 2-benzene dicarbaldehyde or 1, 4-benzene dicarbaldehyde to form the target product cyclopeptide dimer.
The specific process is as follows:
(1) preparation of hydrazine-2-Cl-Trt resin
Weighing 134mg of 2-CI-Trt-Cl resin (the loading capacity is 0.56mmol/g and 75 mu mol), and transferring the resin to a solid phase synthesis tube; after swelling with 4mL of DMF for 30min, the resin was washed three times with DCM and DMF, respectively; adding newly prepared 1.75mL of 5% hydrazine hydrate, shaking the resin for 30min at normal temperature, repeating the operation, adding 2.5mL of 20% MeOH/DMF, shaking the resin for 20min at normal temperature, and washing the resin with DMF for 5 times to obtain the hydrazine-2-Cl-Trt resin.
(2) Fmoc method for splicing hydrazide peptide
The hydrazine-2-Cl-Trt resin was transferred to a solid phase synthesis tube, the freshly prepared DIC/Oxyma/6-aminocaproic acid mixture (10eq DIC:10eq Oxyma: 10eq 6-aminocaproic acid, 2mL DMF) was added, and after shaking at 55 ℃ for 40min, the resin was washed thoroughly with DCM; adding 1mL of acetic anhydride reagent (acetic anhydride: 2, 6-lutidine: DMF: 5:6:89), shaking for 2min, and washing the resin with DMF for three times; adding 20% piperidine solution, shaking the resin for 8min, discarding 20% piperidine, and repeating the piperidine treatment step once; the resin was washed thoroughly three times with DMF and freshly prepared DIC/Oxyma/Fmoc-Ser (tBu) -OH mixture (10eq DIC:10eq Oxyma: 10eq Fmoc-Ser (tBu) -OH, 2mL DMF) was added and shaken at 55 ℃ for 40 min. The above procedure was repeated according to the sequence information of the polypeptide until all peptide condensation was completed. 2mL of blocking reagent (acetic anhydride: 2, 6-lutidine: DMF: 5:6:89) was used to acetylate the N-terminal amine group.
The resin was washed thoroughly with DMF and DCM in that order; after the resin was dried in the air, 6mL of TFA was added (TFA: m-cresol: water: TIPS: 88:5: 2, v/v), and after shaking at room temperature for 2 hours, the cleavage solutions were pooled, and then, ice-cold diethyl ether was added thereto, and the mixture was centrifuged to obtain a crude peptide in the form of powder; and purifying by semi-preparative HPLC, and performing vacuum freeze drying to obtain powdered purified hydrazide peptide.
(3) Side chain-side chain cyclization modification
10mg of linear hydrazide peptide in 2.0mL of DMF: h2The oxygen content of the O mixture (2:1,v is v); weighing 1.4mg of 4, 4-dibromomethylbiphenyl, and dissolving in 0.1mL of DMF; mixing the two liquids, and adding 1.0M NH4HCO3Adjusting the pH value to 8.0, reacting for 2H at normal temperature, and adding 3mL acetonitrile H2O (1:1, v: v, 0.1% TFA), purified by preparative HPLC and lyophilized to give a white powder (. about.3 mg, 30%). The side chain cyclized hydrazide polypeptide sequence is as follows:
(4) reductive amination of hydrazide peptides with phthalaldehyde
3mg of cyclized hydrazide polypeptide (. about.0.9. mu. mol) was dissolved in 600. mu.L of acetic acid: methanol (1:1, v: v); dissolving 4.1mg of 1, 3-phthalaldehyde or 1, 2-phthalaldehyde or 1, 4-phthalaldehyde in 1.0mL of acetic acid: methanol (1: 1); adding 10 μ L of newly prepared benzene dicarboxaldehyde into cyclized hydrazide polypeptide, reacting at room temperature for 1h, and reacting at 300 μ L H2O (containing 3.0mg NaBH)3CN) is added into the reaction system, vibrated for 1H at normal temperature and added with 4mL acetonitrile H2O (1:1, v: v, 0.1% TFA); after purification by preparative HPLC and freeze-drying, the desired ACE2 transpeptidomimetics were obtained as white powders (0.6mg, 20%).
The ACE2 cyclopeptide mimetic is used for preparing polypeptide medicines, and the polypeptide medicines have a remarkable inhibiting effect on new coronavirus.
The ACE2 cyclopeptide mimic disclosed by the invention is used for preparing a novel coronavirus detection kit.
The ACE2 polypeptide mimetic has been reported to be hardly effective in inhibiting new coronavirus. Compared with antibody drugs, the ACE2 cyclic peptide mimics involved in the invention have higher thermal stability, lower immunogenicity and are easy to produce at low cost.
Drawings
FIG. 1 is a schematic structural diagram of the ACE2 cyclic peptide mimetic which inhibits the novel coronavirus.
FIG. 2 is a scheme of synthesis of a target polypeptide according to the present invention.
FIG. 3 is a MS plot and high performance liquid chromatogram of side chain-side chain cyclized hydrazide peptides.
Figure 4 is an MS map and high performance liquid chromatogram of an ACE2 cyclopeptide mimetic.
FIG. 5 is the activity of ACE2 cyclopeptide mimetic to inhibit new corona pseudovirus.
Detailed description of the invention
The invention is further illustrated by the following examples:
the abbreviations referred to in the present invention have the following meanings:
| english abbreviation | Meaning of Chinese |
| Fmoc | 9-fluorenylmethoxycarbonyl group |
| tBu | Tert-butyl radical |
| Trt | Trityl radical |
| DCM | Methylene dichloride |
| DMF | N, -N dimethylformamide |
| AcOH | Acetic acid |
| TFA | Trifluoroacetic acid |
| DIC | N, N-diisopropylcarbodiimide |
| Oxyma | 2-Oxime cyanoacetic acid ethyl ester |
| TIPS | Tri-isopropyl silane |
| Tris | Tris (hydroxymethyl) aminomethane |
| NaCNBH3 | Nitrile sodium borohydride |
| HPLC | High performance liquid chromatography |
| ESI-MS | Electrospray ion source liquid phase mass spectrum |
Example 1: preparation of hydrazine-2-Cl-Trt resin
134mg of 2-CI-Trt-Cl resin (loading 0.56mmol/g, 75. mu. mol) was weighed into a solid phase synthesis tube. After swelling with 3mL of DMF for 20min, the resin was washed three times with DCM and DMF. Fresh 3.5mL of 5% hydrazine hydrate (125. mu.L hydrazine hydrate +3.33mL of DMMF) was added. Shaking at normal temperature for 30min, discarding the reaction solution, and repeating the process once. Fresh 2.5mL of 20% MeOH/DMF (2mL of DMF +0.5mL of MeOH) was added. After shaking for 20min at normal temperature, the resin was washed fully with DMF for 5 times to obtain the target hydrazine-2-Cl-Trt resin.
Example 2: fmoc solid-phase splicing hydrazide peptide
Putting hydrazine-2-Cl-Trt resin into a solid-phase synthesis tube. A DMF mixture of DIC/Oxyma/6-aminocaproic acid (10eq DIC:10eq Oxyma: 10eq 6-aminocaproic acid, 2mL DMF) was added. After shaking at 55 ℃ for 40min, the resin was washed well with DMF. 2mL of blocking reagent (acetic anhydride: 2, 6-lutidine: DMF: 5:6:89) was added. After shaking for 2min at room temperature, the resin was washed thoroughly with DMF. Adding 20% piperidine in DMF, and standing at room temperature for 8 min. The 20% piperidine solution was discarded and the piperidine treatment was repeated once. The resin was washed thoroughly three times with DMF. DMF mixture of DIC/Oxyma/Fmoc-Ser (tBu) -OH formulation (10eq DIC:10eq Oxyma: 10eq Fmoc-Ser (tBu) -OH, 2mL DMF) was added. Shaking for 40 minutes at 55 ℃ to complete the solid-phase splicing of the amino acid. The solid phase assembly of the remaining amino acids was completed as described above. The N-terminal amine group of the polypeptide was acetylated by the addition of 2mL of blocking reagent (acetic anhydride: 2, 6-lutidine: DMF ═ 5:6: 89). After fully washing with DMF and DCM, the solution was naturally dried for 10 min. 6mL of TFA cleavage solution (TFA: m-cresol: water: TIPS: 88:5:5:2, v/v) was added to the resin. After shaking at room temperature for 2 hours, the TFA cleavage liquid was pooled and a volume of ether more than 10 times that of pre-ice-cooled ether was added. The hydrazide polypeptide was obtained in a white powder state by centrifugation. Separating and purifying by semi-preparative HPLC, and freeze-drying in vacuum to obtain pure hydrazide peptide.
Example 3: side chain cyclization of hydrazide peptides
10mg of hydrazide peptide was dissolved in 2.0mL of DMF: h2O mixed solvent (2:1, v: v). 1.4mg of 4, 4-dibromomethylbiphenyl were dissolved in 0.1mL of DMF. The two liquids are mixed. Using 1.0M NH4HCO3The pH was adjusted to 8.0 (precision pH paper determination). After reacting for 2H at normal temperature, 3mL acetonitrile H is added2O mixed solvent (1:1, v: v, 0.1% TFA). HPLC purification to isolate the side chain cyclized hydrazide peptide. After lyophilization, the side chain cyclized polypeptide hydrazide (. about.3 mg, 30%) was obtained as shown in FIG. 3. Side chain cyclized hydrazide peptides schematic as follows:
example 4: dimer preparation of side chain cyclized hydrazide peptides
3mg of side chain cyclized hydrazide peptide (. about.0.9. mu. mol) was taken, dissolved in 600. mu.L of acetic acid: methanol (1:1, v: v). 4.1mg of 1, 3-benzenedicarboxaldehyde was dissolved in 1.0mLAcetic acid: in methanol. 10 mu L of 1, 3-benzene dicarbaldehyde liquid is added into the side chain cyclized hydrazide peptide solution. After reacting for 1h at normal temperature, 3.0mg NaCNBH is added3(dissolved at 300. mu.L). Shaking for 1H at normal temperature, adding 4mL acetonitrile H2O (1:1, v: v, 0.1% TFA). After HPLC isolation purification and freeze drying, ACE2 cyclopeptide mimetic was obtained as powder (0.6mg, 20%) as shown in fig. 4.
Example 5: pseudovirus neutralization assay
The plasmid pCDNA3.1(+) -HnCoV-S or pCDNA3.1(+) -SARS-S was co-transfected with HIV backbone plasmid PNL 4-3R-E-into HEK 293T cells (Invitrogen) using Lipofectamine 3000 transfection reagent to prepare SARS-CoV-2 and SARS-CoV pseudoviruses. After 48 hours, the supernatant of the cell debris was removed as a virus stock. pCDNA3.1(+) -3xFlag-hACE2 was transfected with liposome 6000 transfection reagent (Byunnan biosome) to establish 239T-ACE2 or Huh7-ACE2 cells expressing recombinant human ACE 2. 239T-hACE2 or Huh7-hACE2 cells were plated at 2X10 per well4The density of individual cells was plated in 96-well plates overnight at 37 ℃. Diluted ACE2 cyclopeptide mimetics were incubated with a titer of pseudovirus (relative luminescence units (RLU) per well in a 96 well plate ranging from 20,000 to 40,000) for 1 hour before addition to 239T-hACE2 or Huh7-hACE2 cells. After 2 days of incubation at 37 ℃, cells were harvested in cell lysis buffer containing luciferase assay reagents and assayed according to the manufacturer's protocol (Bright-GloTM luciferase assay System, Promega). The read time for luciferase assay in the experiment was 1 s.
Finally, the IC of ACE2 cyclopeptide mimetic for inhibiting new coronavirus was determined50This was 850nM, as shown in FIG. 5.
Claims (4)
1. An ACE2 cyclopeptide mimetic characterized by:
the ACE2 cyclopeptide mimetic is a dimerized structure of a cyclic peptide;
the cyclopeptide is polypeptide with cyclized side chain-side chain, the C end of the cyclopeptide contains acyl hydrazide structure, and two thioether bonds are formed after the reaction of the side chain sulfydryl of two Cys in the polypeptide sequence and 4, 4' -dibromomethylbiphenyl; the first amino acid at the C-terminal of the cyclic peptide is 6-aminocaproic acid;
the dimerized structure is defined in NaCNBH3Under the action of the two polypeptide hydrazides, the two polypeptide hydrazides and the benzene dicarboxaldehyde are subjected to reductive amination to generate covalent dimers.
2. The ACE2 cyclopeptide mimetic of claim 1, characterized by the structure:
3. use of the ACE2 cyclopeptide mimetic of claim 1, wherein:
the ACE2 cyclopeptide mimetic is used for preparing polypeptide drugs, and the polypeptide drugs have a remarkable inhibitory effect on new coronavirus.
4. Use of the ACE2 cyclopeptide mimetic of claim 1, wherein:
the ACE2 cyclopeptide mimetic is used for preparing a novel coronavirus detection kit.
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| CN112430588A (en) * | 2020-11-27 | 2021-03-02 | 中国人民解放军陆军军医大学 | Short peptide for resisting novel coronavirus infection and preparation method and application thereof |
| CN113278054A (en) * | 2021-06-24 | 2021-08-20 | 安徽大学 | Cyclic peptide combined with new coronavirus RBD site as well as preparation method and application thereof |
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| WO2017114382A1 (en) * | 2015-12-31 | 2017-07-06 | 深圳翰宇药业股份有限公司 | Method for synthesizing c-terminal modified peptide |
| CN112430588A (en) * | 2020-11-27 | 2021-03-02 | 中国人民解放军陆军军医大学 | Short peptide for resisting novel coronavirus infection and preparation method and application thereof |
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