CN113150076B

CN113150076B - Synthesis method of cyclic pentapeptide and application of cyclic pentapeptide in anti-hepatitis C drugs

Info

Publication number: CN113150076B
Application number: CN202110233888.3A
Authority: CN
Inventors: 王树青; 董卫莉; 魏树琨; 欧阳波; 周界文
Original assignee: Tianjin Medical University
Current assignee: Tianjin Medical University
Priority date: 2021-03-03
Filing date: 2021-03-03
Publication date: 2022-05-31
Anticipated expiration: 2041-03-03
Also published as: CN113150076A

Abstract

The invention provides a cyclic pentapeptide, the amino acid sequence of which is as follows: cyclo-5-aspartic acid-glutamic acid-arginine-tyrosine-arginine. The cyclic pentapeptide inhibitor provided by the invention has good structural stability, can be efficiently combined with p7 channel protein, and effectively exerts the effect of resisting hepatitis C virus.

Description

Synthesis method of cyclic pentapeptide and application of cyclic pentapeptide in anti-hepatitis C drugs

Technical Field

The invention belongs to the technical field of biology, and relates to a novel cyclic pentapeptide and application thereof in anti-hepatitis C drugs.

Background

P7 has been shown to be an important protein affecting the activity of HCV infection (hepatitis C virus). p7 exists on endoplasmic reticulum, has cation transport activity and higher selectivity to calcium ion, and is the only ion channel protein in HCV. In a liposome ion flow experiment, the inhibition of the p7 protein can obviously shield ion flow on both sides of a membrane, and the titer of hepatitis C virus in extracellular supernatant is obviously eliminated by knocking out the p7 protein or carrying out mutation on the p7 protein R33Q or R35Q on a virus genome^[1,2]. It is concluded that the p7 protein functions as an ion channel and is involved in the release of hepatitis C virus. Therefore, the p7 ion channel can be used as a good drug target to carry out new drug development of polypeptides.

Small molecule drugs have been developed greatly in the application of anti-hepatitis C drugs, but because of the obvious toxic and side effects of internal organs, polypeptide drugs have great advantages. The polypeptide has good biocompatibility and low toxicity, and lays a foundation for the pharmaceutical application of the polypeptide molecules. Since the channel radius of p7 protein is in the range of 3.9-6 angstroms (7.8-12 angstroms in diameter), a 7.8 angstrom diameter tapered channel region is easier to design for a related loop polypeptide of appropriate volume to directly block the p7 channel for antiviral efficacy, compared to a 12 angstrom diameter cylindrical channel region.

Pang, S, etc^[3]Through computer simulation, the binding free energy calculation and kinetic simulation of 13 cyclic tetrapeptides (cyclic-4-proline, cyclic-4-aspartic acid, cyclic-4-arginine, cyclic-4-serine, cyclic-4-leucine, cyclic-4-glutamine, cyclic-4-asparagine, cyclic-4-threonine, cyclic-4-histidine, cyclic-4-cysteine, cyclic-4-valine, cyclic-4-methionine, and cyclic-4-isoleucine) show that the cyclic peptides may have the ability to stably bind inside hydrophobic channels, thereby exerting antiviral efficacy of inhibiting ion flow. However, practical synthetic studies have shown that cyclic tetrapeptide structure is difficult to form due to excessive tension inside the structure, and even after cyclization, the binding ability inside the hydrophobic channel may be lost due to instability of the cyclic structure, resulting in reduced or even no antiviral efficacy.

In view of the above, this patent designed and synthesized novel cyclic pentapeptide inhibitors of p7 channel protein to improve binding affinity and antiviral efficacy.

Disclosure of Invention

In our design, aspartic acid and arginine are respectively negatively charged amino acids, which are potentially hydrogen bonding forces, so that in designing novel cyclic peptides, aspartic acid and arginine are introduced to increase the binding affinity with the p7 tapered channel region. In the design, arginine is introduced to provide more hydrogen bonding force sites, and a negative charge glutamic acid is introduced while balancing the charge. In the technical aspect, in order to enable the cyclic peptide to more closely fill the conical region of the channel and ensure the structural stability of the cyclic peptide, tyrosine is added on the basis of the four former amino acid residues, so that the design of the novel cyclic pentapeptide is completed: cyclo-5-aspartic acid-glutamic acid-arginine-tyrosine-arginine.

Thus, the present invention provides, in a first aspect, a cyclic pentapeptide, wherein the configuration of each amino acid may be independently selected from D-form or L-formAnd (4) molding. Preferably, the configuration of the cyclic pentapeptide is c-DERyR, c-DERYR, c-derYr, or c-DERyR. In the cyclic pentapeptide, the alpha-amino (-NH) group of each amino acid may be methylated (-NCH) independently of each other₃)。

The cyclic peptides of the invention can be used for:

(1) anti-hepatitis C virus;

(2) inhibition of p7 protein;

(3) binding to p7 protein;

(4) reducing the radius of a p7 protein channel;

(5) repressing ion flux on p7 protein channels;

(6) and p7 protein at the amide sites of residues 15-21, 31-36 and 60-63; contracting the corresponding site conformation;

(7) binds to asparagine N9 and N16 in the p7 protein channel.

The cyclic peptide of the present invention can be prepared as a pharmaceutical composition or a kit, and thus is used for the needs of the above-mentioned various uses. The preparation method can adopt various known methods commonly used in the field.

The cyclic pentapeptide of the present invention may be applied at a final concentration of (8. + -.3) mM in the above-mentioned use.

The cyclic pentapeptide inhibitor provided by the invention has good structural stability, can be efficiently combined with p7 channel protein, and can be used for shrinking the interior of a channel by directly shielding and changing channel conformation, so that channel ion flow is more strongly inhibited, the assembly and release of hepatitis C virus are inhibited, and the drug effect of resisting the hepatitis C virus is further exerted.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate an embodiment of the invention and, together with the description, serve to explain the invention and not to limit the invention. In the drawings:

FIG. 1: molecular docking results of Cyclo-DERyR (which in this case may be abbreviated as c-DERyR).

FIG. 2: molecular docking results of Cyclo-DERYR (which may be abbreviated as c-DERYR in this case).

FIG. 3: the result of molecular docking in which the alpha-amino group of each amino acid in Cyclo-DERYR is methylated.

FIG. 4: (A) chemical structure and (B) relative nuclear magnetic spectrum of cyclic pentapeptide c-DERyR.

FIG. 5: the effect of the cyclic pentapeptide on p7 channel protein and the effect of inhibiting virus. Wherein (A) is a graphical representation of the signal shift changes of the relevant amino acid residues of the p7 channel protein before and after addition of 8mM cyclic pentapeptide at the final concentration; (B) statistical information of displacement change of corresponding residue signals of the p7 channel protein before and after 8mM cyclic pentapeptide with final concentration is dripped; (C) the method comprises the steps of adding related medicaments into Huh7.5.1 cells infected with hepatitis C virus strain JFH-1, and detecting the intracellular virus amount by utilizing RT-qPCR technology.

FIG. 6: the binding pattern of the cyclic pentapeptide on the p7 channel. Wherein (A) the binding site of the cyclic pentapeptide and the p7 channel protein is determined by a nuclear magnetic NOE experiment; (B) the cyclic pentapeptide is simulated and butted into a p7 channel by utilizing a molecular butting technology, and the site information is consistent with that determined by an experiment; (C) the illustration of the cyclic pentapeptide in the p7 channel protein shows that the cyclic peptide can form a stable complex with the p7 channel protein.

Detailed Description

For a better understanding of the present invention, reference is made to the following detailed description taken in conjunction with the accompanying drawings.

Preparation of mono-and cyclic pentapeptides

1.1 raw materials and reagents:

(1) protected amino acids and starting materials

Fmoc-Arg (Pbf) -OH, Fmoc-D-Tyr (tBu) -OH, Fmoc-Glu (OtBu) -OH, Fmoc-Asp (OtBu) -OH and the like

(2) Condensation reagent

HBTU，DIEA，

(3) Solvent(s)

DMF, DCM, acetonitrile,

(4) resin composition

2-chlorotrityl chloride resin

(5) Deprotection reagents

Piperidine derivatives

(6) Cleavage reagent

Trifluoroethanol, DCM, TFA, TIS, EDT, H₂O

(7) Nitrogen gas

(8) Anhydrous diethyl ether

(9) Precision electronic balance

1.2 synthetic process:

(1) swelling of resin

The 2-Chlorotrityl Chloride Resin was placed in a reaction tube, DCM (15ml/g) was added, and shaking was carried out for 30min.

(2) To the first amino acid

The solvent was filtered off by suction through a sand core, 3 times molar excess of Fmoc-Arg (Pbf) -OH was added, 10 times molar excess of DIEA was added, and finally DMF was added for dissolution, followed by shaking for 30min. Methanol was capped for 30min.

(3) Deprotection of the amino acid

The solvent was removed, 20% piperidine/DMF solution (15ml/g) was added for 5min, and 20% piperidine/DMF solution (15ml/g) was added for 15 min.

(4) Detection of

The piperidine solution is pumped out, dozens of particles of resin are taken, washed with ethanol for three times, added with ninhydrin, KCN and phenol solution, and heated for 5min at 105-110 ℃, and the positive reaction is obtained when the color turns dark blue.

(5) Washing machine

DMF (10ml/g) twice, methanol (10ml/g) twice, DMF (10ml/g) twice

(6) Condensation of

Protecting amino acid Fmoc-D-Tyr (tBu) -OH triple excess and HBTU triple excess, dissolving with DMF as little as possible, adding into a reaction tube, immediately adding DIEA ten-fold excess, and reacting for 30min.

(7) Detection of

And (3) pumping out the solution, taking dozens of resins, washing the resins with ethanol for three times, adding ninhydrin, KCN and phenol solutions one drop at a time, heating the mixture at 105-110 ℃ for 5min, and indicating that the reaction is complete, wherein the colorless reaction is a negative reaction.

(8) Washing machine

DMF (10ml/g) once, methanol (10ml/g) twice, DMF (10ml/g) twice

(9) Repeating the three to eight steps, connecting the rest amino acids in sequence, and removing the Fmoc protecting group of the last amino acid.

(10) DMF (10ml/g) twice, methanol (10ml/g) three times, DCM (10ml/g) three times. The resin was drained.

(11) And (3) cutting the polypeptide from the resin, and removing the solvent by a rotary evaporator to obtain the peptide fragment with the protection.

Preparing cutting fluid (10/g) 30% of trifluoroethanol, DCM 70%,

cutting time: and (4) 120 min.

(12) The protected peptide fragment DCM was dissolved, PyBop was twice excess, DIEA was ten times excess, refluxed at 45 ℃ overnight, and the solvent was removed by rotary evaporator.

(13) Removal of polypeptide protecting groups

Preparation of cleavage solution (10/g) TFA 95%, EDT 2%, TIS 2%, H₂O 1％，

Cutting time: and (4) 120 min.

(14) The lysate is blown dry as much as possible with nitrogen, washed six times with ether and then evaporated to dryness at normal temperature.

(15) Purifying the polypeptide by HPLC, and lyophilizing the purified solution to obtain the final product.

(16) Identification

And respectively taking a small amount of finished polypeptide, and performing MS molecular weight identification and HPLC analysis purity identification.

(17) Sealing and packaging white powdery polypeptide, and storing at-20 deg.C.

Structural analysis of di-and cyclic pentapeptides

The ability of cyclic pentapeptide with different chiral difference structures to inhibit p7 channel protein is calculated by simulation of drug design software. Since the structure of the protein target p7 is a C6 symmetric structure (i.e. the protein structures overlap each other every 60 degrees of rotation around the channel axis), the binding sites of the cyclic pentapeptide molecule on the p7 channel protein are asparagine N9 and N16, so that the cyclic pentapeptide molecule with chiral difference is very close to the p7 protein in the binding sites, binding conformations and interaction range, and the effect is equivalent.

Taking c-DERYR and c-DERYR as examples (in the present invention, the capital letters of amino acids represent L-type amino acids or natural amino acids, and the lowercase letters of amino acids represent D-type amino acids or unnatural amino acids), the molecular docking results are shown in FIG. 1-2, respectively, the docking score of Cyclo-DERYR is-9.10 Kcal/mol, and the docking score of Cyclo-DERYR is-9.06 Kca/lmol.

It should be noted that, since the product has a cyclic structure, when describing the cyclic pentapeptide, the cyclic pentapeptide can be initiated with any one of the amino acids (including clockwise or counterclockwise ordering), for example, c-DERyR can also be described or understood as c-ERyRD, c-RyRDE, c-yRDER, c-EDRyR, etc. In addition, as can be seen from the symmetry of the spatial structure, c-DERyR and c-derYr, c-DERYR and c-DERyR, and the like have substantially the same configuration and performance, respectively.

The alpha-amino groups (-NH) of the five amino acids constituting the cyclic peptide may each independently be methylated (-NCH)₃) We simulated the structure where all of the five amino acid alpha-amino groups were methylated, as in fig. 3, with a docking score of-9.27 Kcal/mol, indicating that it is relatively more advantageous in binding capacity and score after methylation.

We adopt the above method to select c-DERyR (ring-5-aspartic acid-glutamic acid-arginine-d tyrosine-arginine) for synthesis and identification. FIG. 4A is a chemical structural diagram of c-DERyR in which tyrosine is a dextrorotatory unnatural amino acid and the remaining four amino acid residues are levorotatory natural amino acid residues. Dissolving the cyclic peptide in DMSO to prepare a 400mM stock solution for later use; the cyclic peptide stock solution was added to 25mM MES at a final concentration of 5mM in a 6.5 pH nuclear magnetic buffer system, and subjected to TROSY-HSQC mapping at 303K (30 degrees Celsius) and NS of 4 (number of nuclear magnetic scans), and it was found that the cyclic pentapeptide could indeed detect the chemical shift signals of 5 amides, as shown in FIG. 4B.

Analysis of antiviral Effect of the three, Cyclic pentapeptide

3.1 Nuclear magnetic titration results of Cyclopentapeptide

In order to preliminarily verify that the cyclic pentapeptide can generate a certain inhibition effect on the p7 channel protein, a nuclear magnetic titration experiment is adopted to detect the conformational influence of the cyclic pentapeptide c-DERyR on the p7 protein. If cyclic peptide can be combined with p7 protein, p7 protein has obvious chemical shift change on nuclear magnetic spectrum, and thus, the potential hepatitis C resistant inhibition effect is presumed. Histidine carried by pMM-LR6 vectorAcid-and trpLE-tagged p7 plasmid was transformed into E.coli strain BL21(DE3) and expression-cultured in N15-isotope-labeled restriction M9 medium to obtain inclusion body p7 protein. Further, affinity chromatography purification of nickel ion was carried out using a buffer solution of pH6.5 prepared from 6M guanidine hydrochloride, 50mM Tris and 200mM sodium chloride to obtain fusion p7 protein with high purity, and after dialysis, this product was dissolved in 80% formic acid to cleave with hydrogen bromide, thereby finally obtaining unlabeled p7 protein dry powder. Finally, the p7 protein dry powder is recombined into the micelle of DPC (dodecyl phosphatidyl choline) to form hexamer p7 ion channel protein for subsequent nuclear magnetic titration experiment^[4]. When 8mM of cyclic pentapeptide is added into a nuclear magnetic system containing a 0.1mM p7 protein sample, the amino acid residue signal of the p7 protein has obvious chemical shift change, which proves that the cyclic pentapeptide is combined with the p7 channel protein, and the amide signals of residue numbers 15-21, 31-36 and 60-63 have obvious conformational change (figures 5A and 5B), the corresponding sites are contracted in conformation, the channel is directly shielded, the radius of the channel is further reduced, the possibility of blocking the passage of ion current is better achieved, the function of inhibiting the assembly and release of hepatitis C virus is fully exerted, and a theoretical basis is provided for the application of anti-hepatitis C drugs.

3.2 hepatitis C Virus infection cell assay

Huh7.5.1 cells were used as experimental models, and appropriate amount of cells were added to each stock solution of small molecules at a final concentration of 5uM, and after incubation for 30 minutes, the small molecules were allowed to fully enter the interior of the cells. Then, a drug-added Huh7.5.1 cell sample is infected by a JFH-1 strain of the hepatitis C virus 2a genotype, and the sample is collected for analysis of the inhibition condition of related small molecules after being cultured for 72 hours at 37 ℃ and 5% carbon dioxide. The cells adhere to the wall during the culture process, so when the samples are collected, the supernatant of each well is aspirated, the infected cells are lysed with Trizol lysate, and the total RNA is extracted using chloroform.

In the experiment, after RNA is extracted, each sample is quantified, the template amount of each sample is fixed to be 1 microgram, and cDNA is quickly obtained by reverse transcription by RT-PCR and is used for a subsequent qPCR experiment.

After collecting the data, the RNA relative expression level of HCV was calculated using the formula of Comparative ratio ^ 2 ([ delta ] CT). The difference between the CT value of HCV and the CT value of GAPDH was used to determine the Δ CT values of all samples. The Δ CT value of the NC negative control group was used as a control Δ CT value. And (3) subtracting the delta CT values of the experimental group and the NC negative control group to obtain the delta CT, and further using a 2^ (-delta CT) formula to obtain the relative expression quantity of the RNA of each sample as a representation to see the inhibition condition of each small molecule on the HCV. That is, when the ratio is low, it indicates that the RNA expression level of the HCV group is high, and the corresponding small molecule inhibition effect is good; when the ratio is high, it indicates that the RNA of the HCV group has high relative expression amount and the corresponding small molecule inhibition effect is poor. Data analysis shows that (fig. 5C), compared with a blank control without drugs, the positive control rimantadine rim has a certain hepatitis C virus inhibition effect, and after the cyclopentapeptide DERyR in the patent is used, the hepatitis C virus amount is further reduced, so that a better anti-hepatitis C virus effect is shown.

3.3 binding site of Cyclopentapeptide on p7 channel

Also using methods already available in the article^[4]The p7 channel protein²H，¹⁵The expression and purification of the N marker and the renaturation of the N marker are structures of hexamer channels, and the N marker is used for detecting the binding sites of the cyclic pentapeptide and the p7 channel protein, proving that the cyclic pentapeptide is combined with the p7 channel protein, and further playing the role of resisting the hepatitis C virus. The technique utilizes p7 channel protein as²H and¹⁵n double labelling, so that the methyl CH in the protein backbone₃The signal can be shielded, and the methyl CH of the cyclic pentapeptide under the natural abundance condition can be conveniently observed₃Or amide NH₂Of the signal of (1). When the distance of the cyclic pentapeptide is within 5 angstroms of the p7 channel protein, the related signal of the cyclic pentapeptide appears near the residue signal of the corresponding p7 channel protein, so as to presume the related binding site.

Using 0.5mM²H，¹⁵NOE mapping of N-labeled p7 channel protein without addition of Cyclopentapeptide and NMR signal acquisition with addition of 5mM final concentration of Cyclopentapeptide, as shown in FIG. 6A, by comparison between N9 andthe new signal appears in the two bands of N16, which can be identified as the related signal of the cyclic pentapeptide, thereby deducing that the binding sites of the cyclic pentapeptide on the p7 channel protein are asparagine N9 and N16.

In combination with the experiment of nuclear magnetic titration, it can be seen that the two binding sites of N9 and N16 resolved by NOE do not show obvious chemical shift change in the nuclear magnetic titration spectrum. The docking mode and structure of the cyclic pentapeptide in the p7 channel are shown in fig. 6B and 6C, and it can be speculated that when the cyclic pentapeptide is combined with two sites of N9 and N16 on the p7 channel protein, on one hand, the ion flow inside the channel is directly shielded; on the other hand, the p7 channel protein undergoes huge conformational changes in the regions of residues 15-21, 31-36 and 60-63, which further contracts and assists in the internal contraction of the channel, so that the ion flow of the channel is more strongly suppressed, the assembly and release of hepatitis C virus is inhibited, and the drug effect against hepatitis C virus is further exerted.

Reference to the literature

1.Breitinger,U.,et al.,Patch-Clamp Study of Hepatitis C p7 Channels Reveals Genotype-Specific Sensitivity to Inhibitors.Biophys J,2016.110(11):p.2419-2429.

2.Steinmann,E.,et al.,Hepatitis C virus p7 protein is crucial for assembly and release of infectious virions.PLoS Pathog,2007.3(7):p.e103.

3.Pang,S.,et al.,Cyclopeptides design as blockers against HCV p7 channel in silico.Molecular Simulation,2019.45(17):p.1419-1425.

4.OuYang,B.,et al.,Unusual architecture of the p7 channel from hepatitis C virus.Nature,2013.498(7455):p.521-5.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and should not be taken as limiting the scope of the present invention, which is intended to cover any modifications, equivalents, improvements, etc. within the spirit and scope of the present invention.

Claims

1. A cyclic pentapeptide having the amino acid sequence: cyclo-5-aspartic acid-glutamic acid-arginine-tyrosine-arginine.

2. The cyclic pentapeptide of claim 1, having the configuration c-DERyR, or c-DERyR; wherein c represents a ring, D or D represents aspartic acid, E or E represents glutamic acid, R or R represents arginine, Y or Y represents tyrosine, the capital letter of each amino acid represents an L-type amino acid, and the lowercase letter of the amino acid represents a D-type amino acid.

3. The cyclic pentapeptide of claim 1, wherein the α -amino group of each amino acid is independently methylated.

4. A pharmaceutical composition or kit comprising a cyclic pentapeptide according to any one of claims 1-3.

5. Use of a cyclic pentapeptide according to any one of claims 1 to 3 in the manufacture of a medicament for the treatment of hepatitis c.

6. Use of a cyclic pentapeptide according to any one of claims 1-3 in the preparation of an inhibitor of hepatitis c virus p7 protein.