CN114380889B - Humanized ACE2 polypeptide blocker and design and synthesis method and application thereof - Google Patents
Humanized ACE2 polypeptide blocker and design and synthesis method and application thereof Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- G—PHYSICS
- G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
- G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
- G16B15/00—ICT specially adapted for analysing two-dimensional or three-dimensional molecular structures, e.g. structural or functional relations or structure alignment
- G16B15/30—Drug targeting using structural data; Docking or binding prediction
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Abstract
The application discloses a humanized ACE2 polypeptide blocker, a design and synthesis method and application thereof, wherein the amino acid sequence of the humanized ACE2 polypeptide blocker comprises proline and tryptophan. The humanized ACE2 polypeptide blocker can competitively bind with ACE2, so that the specific binding between coronavirus S protein and ACE2 is reduced, and the humanized ACE2 polypeptide blocker has good application prospect as a coronavirus inhibiting drug.
Description
Technical Field
The application relates to a humanized ACE2 polypeptide blocker, a design and synthesis method and application thereof, belonging to the technical field of virus-inhibiting drug polypeptides.
Background
The novel coronavirus SARS-COV-2 Spike glycoprotein is identified by combining human angiotensin converting enzyme 2 (ACE 2) receptor in human body and adsorbed on the surface of host cell, then penetrates into susceptible cell through host cell membrane, and replicates and recombines in cell to generate progeny virus, and the susceptible cell releases the progeny virus, thus completing rapid propagation of virus in human body and further causing various infections.
In general, anti-neocoronavirus drugs can be categorized into two broad categories, one that prevents binding of the virus to the host cell and the other that prevents production of the new virus within the host cell. If the virus is to be prevented from binding to the host cell, the target of action is the S protein (spike glycoprotein) or ACE2. The S protein antibody is difficult to obtain in a short time and is more complex, and the competitive combination of the small molecular polypeptide with a simpler design structure and the S protein is a simple and easy method. Binding of the polypeptide inhibitor to the key site of ACE2 blocks further invasion of the S protein and thus alleviates further infection by the virus.
Disclosure of Invention
According to one aspect of the application, a humanized ACE2 polypeptide blocker is provided, and the humanized ACE2 polypeptide blocker can competitively bind to ACE2, so that the specific binding between coronavirus S protein and ACE2 is reduced, and the humanized ACE2 polypeptide blocker has good application prospect as a coronavirus inhibiting drug.
A human ACE2 polypeptide blocker, the amino acid sequence of which comprises proline and tryptophan.
Optionally, the amino acid sequence of the humanized ACE2 polypeptide blocker is selected from at least one of TrpTrp Pro ProTrpTrp, trpTrp Pro ProTrpTrp, trpTrp Pro ProProTrpTrp, trp Pro Ala ProProProTrpTrp and Trp ProTrp Pro ProProTrpTrp,
or at least one amino acid sequence selected from TrpTrp Pro ProTrpTrp, trpTrp Pro ProTrpTrp, trpTrp Pro ProProTrpTrp, trp Pro Ala ProProProTrpTrp and Trp ProTrp Pro ProProTrpTrp by substitution, deletion and/or addition of one or more amino acids without affecting its biological activity.
Optionally, the amino acid in the humanized ACE2 polypeptide blocker is a D-amino acid and/or an L-amino acid.
Optionally, the amino acid sequence of the polypeptide is selected from at least one of the following:
TrpTrp Pro ProTrpTrp wherein Trp at position 2, pro at position 4, trp at position 5 and Trp at position 6 are D-amino acids, trp at position 1, pro at position 3 are L-amino acids;
TrpTrp Pro ProTrpTrp wherein Trp at position 2, pro at position 3, pro at position 4, trp at position 5, trp at position 6 is a D-amino acid and Trp at position 1 is an L-amino acid;
TrpTrp Pro ProProTrpTrp wherein Trp at position 2, pro at position 3, pro at position 5, trp at position 6, trp at position 7 are D-amino acids, trp at position 1, pro at position 4 are L-amino acids;
trp Pro Ala ProProProTrpTrp wherein Trp at position 1, pro at position 2, ala at position third, pro at position 4, pro at position 6, trp at position 7, trp at position 8 are D-amino acids and Pro at position 5 is L-amino acid;
trp ProTrp Pro ProProTrpTrp wherein Trp at position 1, pro at position 2, trp at position 3, pro at position 4, pro at position 6, trp at position 7, trp at position 8 are D-amino acids and Pro at position 5 is L-amino acid.
Optionally, the humanized ACE2 polypeptide blocker has a stable spatial structure.
The conventional polypeptide has a flexible linear structure and can freely rotate, and proline is added into the sequence for changing the bond angle, so that the polypeptide exists in a folded or rigid structure, and finally the humanized ACE2 polypeptide blocker has good rigidity, stable structure and difficult rotation.
Alternatively, the humanized ACE2 polypeptide blocker may specifically bind to ACE2.
Optionally, the degradation rate of the humanized ACE2 polypeptide blocker is less than 60% when incubated in plasma at 37 ℃ for 8 hours.
Optionally, the degradation rate of the humanized ACE2 polypeptide blocker is 40% -50% after incubation for 8h at 37 ℃ in plasma.
According to another aspect of the present application there is provided a method of synthesizing a human ACE2 polypeptide blocker, the method comprising synthesizing a human ACE2 polypeptide blocker having a sequence as described in any one of the preceding claims.
According to another aspect of the present application, there is provided a method of designing a humanized ACE2 polypeptide blocker, the method comprising the steps of: and (3) virtually docking and analyzing the designed humanized ACE2 polypeptide blocker with ACE2 through the sequence of the humanized ACE2 polypeptide blocker with the aid of a computer, optimizing the sequence structure of the humanized ACE2 polypeptide blocker, and scoring to determine a final sequence to obtain the humanized ACE2 polypeptide blocker.
Optionally, the sequence of the humanized ACE2 polypeptide blocker through computer aided design is specifically as follows: the lead polypeptide is designed based on the lead compound.
Alternatively, the lead compound is (Z) -3,3' - (2- (((3, 5-bis (1H-indol-3-yl) -1H-pyrrol-2-yl) methylene) -2H-pyrrol-3, 5-diyl) bis (1H-indole).
Optionally, the virtual docking analysis of the designed humanized ACE2 polypeptide blocker and ACE2 specifically includes: the type of interaction between the amino acid residues between the designed humanized ACE2 polypeptide blocker and ACE2 was determined and the high affinity region of ACE2 was determined.
Optionally, the high affinity region of ACE2 is located between amino acids 348 to 401 of the ACE2 sequence.
Optionally, the optimized sequence structure of the humanized ACE2 polypeptide blocker is specifically as follows: according to the docking analysis result, the sequence structure of the humanized ACE2 polypeptide blocker is adjusted to enable amino acid residues on the humanized ACE2 polypeptide blocker to extend to the high affinity region.
Optionally, the scoring determines the final sequence specifically as: and scoring the optimized sequence to obtain the humanized ACE2 polypeptide blocker sequence.
Optionally, the scoring determines the final sequence specifically as: and scoring the optimized sequence, and selecting a humanized ACE2 polypeptide blocker with the binding force smaller than-7.
According to another aspect of the application, there is provided an application of at least one of the human ACE2 polypeptide blocker according to any one of the above, the human ACE2 polypeptide blocker synthesized by any one of the above synthesis methods, and the human ACE2 polypeptide blocker designed by any one of the above design methods as a coronavirus-inhibiting drug.
Alternatively, the coronavirus comprises a novel coronavirus.
The novel coronavirus is SARS-CoV-2.
According to another aspect of the present application, there is provided a coronavirus inhibitory drug comprising at least one of the human ACE2 polypeptide blocker as defined in any one of the above, a human ACE2 polypeptide blocker synthesized according to any one of the above synthesis methods, and a human ACE2 polypeptide blocker designed by any one of the above design methods.
Alternatively, the coronavirus comprises a novel coronavirus.
The novel coronavirus is SARS-CoV-2.
As one implementation mode, the application designs a kind of polypeptide compounds with different sequence compositions, then uses molecular docking software to carry out molecular docking with specific sites on ACE2, gradually improves the sequence and the space structure of the compounds by evaluating the molecular docking result, and finally obtains the molecular structure with bioactivity. The application synthesizes relevant corresponding polypeptides based on molecular docking results, discovers that the polypeptide to be detected has a long decay period in a blood stability experiment, further utilizes a kit to test the binding capacity of a designed compound to ACE2, proves that the designed compound has the capacity of competing with a novel coronavirus S protein for binding to ACE2, and lays a foundation for the research and development of a novel coronavirus polypeptide antagonist.
In order to achieve the above purpose, the technical scheme adopted by the application is as follows:
design of human ACE2 polypeptide blocker based on ACE2 locus and application thereof in novel coronavirus, wherein the human ACE2 polypeptide blocker is composed of the following five sequences WwPpww, wwppww, wwpPpww, wpapPpww and wppPpww, wherein lowercase represents D-amino acid uppercase represents L-amino acid
The humanized ACE2 polypeptide blocker has specific spatial structure and sequence information, and is characterized in that the polypeptide core structure consists of proline and tryptophan, the L/D-configuration of amino acid can be replaced, and amino acid residues can be increased or decreased based on the existing sequence
The humanized ACE2 polypeptide blocker can exist stably in normal human blood for a long time and is not easy to degrade, and specifically, the incubation time is 0h, 0.083h, 0.5h, 1h, 2h, 4h, 8h, 24h and 48h respectively.
The humanized ACE2 polypeptide blocker can competitively bind to ACE2 to reduce specific binding of novel coronavirus S protein and ACE2, and specifically, the concentration of the test polypeptide is 50 mu M and 100 mu M respectively.
As one embodiment, the present application provides a humanized ACE2 polypeptide blocker that specifically binds ACE2 to inhibit the S protein of novel coronavirus SARS-CoV-2;
alternatively, the amino acid of the humanized ACE2 polypeptide blocker may be in the L/D configuration.
Optionally, the humanized ACE2 polypeptide blocker has a specific spatial structure and sequence, and amino acid residues can be added/deleted on the premise of ensuring a core structure;
optionally, the sequence core of the humanized ACE2 polypeptide blocker consists of proline and tryptophan interleaved.
Optionally, the humanized ACE2 polypeptide blocker is stable in normal human blood for a long period of time and is not easily degraded;
specifically, the incubation time is 0h, 0.083h, 0.5h, 1h, 2h, 4h, 8h, 24h, 48h, respectively.
Optionally, the humanized ACE2 polypeptide blocker is capable of competitively binding ACE2 to inhibit specific binding of novel coronavirus S protein to ACE 2;
specifically, the concentrations of the tested humanized ACE2 polypeptide blockers were 50 μm and 100 μm, respectively.
As one embodiment, the application provides a method for designing an ACE 2-based polypeptide antagonist and application thereof in inhibiting novel coronavirus SARS-CoV-2. And (3) carrying out molecular docking on the designed polypeptide and angiotensin converting enzyme 2 (ACE 2) at a specific inhibitory site by using molecular docking software, comprehensively analyzing the result, and screening the polypeptide with ACE2 inhibitory activity. The half-life period of the screened polypeptide is obviously improved through detection, the usability of the designed polypeptide is enhanced, and the binding capacity of the designed compound ACE2 is tested by using a kit, so that the polypeptide has the capacity of competing with the novel coronavirus S protein for binding ACE2, lays a foundation for the research and development of novel coronavirus polypeptide antagonists, and can be used as a potential medicament for inhibiting the novel coronavirus.
The application realizes the de novo design of the polypeptide/peptoid by the aid of a computer, so that the interaction between proteins and polypeptides is deeply known, the development and design of new drugs are guided, the labor and development period is reduced, and the economic cost is greatly saved.
The humanized ACE2 polypeptide blocker designed by the application can be combined with a targeting protein, and the compound synthesis step is simplified by optimizing the structure of the humanized ACE2 polypeptide blocker, so that the low-cost high-flux output of active ingredients is facilitated.
The human ACE2 polypeptide blocker has a stable structure, and plasma stability experiments prove that the human ACE2 polypeptide blocker can exist in body fluid of a complex circulatory system stably for a long time, which provides more possibility for intake and preparation of the human ACE2 polypeptide blocker.
The humanized ACE2 polypeptide blocker designed by means of a computer can be combined with S protein in a competitive mode to form ACE2, so that the humanized ACE2 polypeptide blocker has a prospect of developing medicines.
The application has the beneficial effects that:
(1) The humanized ACE2 polypeptide blocker provided by the application can competitively bind with ACE2, thereby reducing the specific binding of coronavirus S protein and ACE2, laying a foundation for the research and development of coronavirus polypeptide antagonists, and having good application prospect as a coronavirus inhibiting drug.
(2) The humanized ACE2 polypeptide blocker provided by the application has a stable structure, has a longer half-life, can stably exist in normal human blood for a long time, is not easy to degrade, and enhances the usability.
(3) The design method of the humanized ACE2 polypeptide blocker provided by the application realizes the de novo design of polypeptides/peptoids through the assistance of a computer, optimizes the structure of the polypeptides, simplifies the synthesis steps of the compounds, and is beneficial to the low-cost and high-flux output of active ingredients. Not only reduces the manpower and research and development period, but also greatly saves the economic cost.
Drawings
FIG. 1 is a spatial interaction analysis of a humanized ACE2 polypeptide blocker P1 with ACE 2;
FIG. 2 is an analysis of amino acid residue interactions of human ACE2 polypeptide blocker P1 with ACE 2;
FIG. 3 is an ACE2 key spatial structure analysis;
FIG. 4 shows the results of plasma stability analysis of humanized ACE2 polypeptide blockers P1-P5;
fig. 5 and 6 show the results of competitive inhibition assays of the S protein by the humanized ACE2 polypeptide blockers P1-P5.
FIG. 7 is a structural formula of the lead compound (Z) -3,3' - (2- (((3, 5-bis (1H-indol-3-yl) -1H-pyrrol-2-yl) methylene) -2H-pyrrol-3, 5-diyl) bis (1H-indole).
Detailed Description
The present application is described in detail below with reference to examples, but the present application is not limited to these examples.
In the present application, pro and P represent proline; trp and W represent tryptophan, ala and a represent alanine.
Example 1: virtual docking analysis of humanized ACE2 polypeptide blocker and ACE2
Computer-aided de novo design of polypeptides comprises the steps of:
the sequence of leader polypeptide P1 was designed based on the leader compound (Z) -3,3' - (2- (((3, 5-bis (1H-indol-3-yl) -1H-pyrrol-2-yl) methylene) -2H-pyrrol-3, 5-diyl) bis (1H-indole) (structure shown in FIG. 7), and was analyzed and optimized for structure.
The interaction force between polypeptide and protein mainly comprises Van der Waals force, hydrophobic action, hydrogen bonding action, electrostatic interaction and other forms. Thus, to understand the results obtained by virtual docking, to evaluate the interaction between polypeptide and protein, we selected leader polypeptide P1 and target protein (ACE 2) as reference, the interaction force was analyzed as follows (see fig. 1):
from the composition of the lead compound P1, we analyzed the interaction forces between amino acid residues between the polypeptides P1 and ACE2 in detail, determined that the hydrophobic effect is the most dominant force between the two, followed by the hydrogen bonding effect, and we determined the highly affinity regions of P1 for ACE2, where both are more likely to interact and the forces are stronger (see fig. 2). The analysis results show that ARG-514, GLU-398, GLU-402 and the like have hydroxyl, carbonyl, amino and the like which are easy to form hydrogen bond structures, the structures are easy to interact with peptide bonds and R groups in polypeptides to form stronger binding force, and the structures with high hydrophobicity such as PHE-40, PHE-390 and TYR-385 are also present, so that the structures are easy to react with alkyl and phenyl.
According to the butt joint result of the polypeptide P1, the sequence structure is modified to extend the amino acid residues on the polypeptide to the parts which are easier to generate interaction, the interaction among the residues is integrated into a space structure (see figure 3), the cavity of the most-affine polypeptide in the whole ACE2 is determined, and a plurality of polypeptide segments with similar capability are finally obtained by adjusting the sequence in the cavity. The final polypeptide sequences and scoring results are shown in Table 1 below, and the polypeptides mainly comprise an overall structure skeleton consisting of proline and tryptophan, and the molecular docking structure also has better score, and accords with the optimization of the action of local amino acid residues and the optimization of cavities.
Table 1 design polypeptide sequences and molecular docking scores
Example 2: plasma stability analysis of polypeptides
(1) Co-incubation of polypeptides with plasma
5 polypeptides (P1-P5) are respectively prepared into mixed solutions with the concentration of 100 mu M, 40 mu L of the mixed solutions are added into a 1.5mL EP tube, 360 mu L of human plasma is added and uniformly mixed, the final concentration of the polypeptides in the plasma is 10 mu M, and the mixed solutions are respectively incubated for 0h, 0.083h, 0.5h, 1h, 2h, 4h, 8h, 24h and 48h at 37 ℃. And (3) pretreating the plasma sample by adopting a salting-out assisted all-direction liquid-liquid extraction method to obtain a sample to be analyzed.
(2) HPLC-MS analysis
The used instrument is a UPLC-Q-TOF liquid chromatography-mass spectrometer (Agilent);
high performance liquid chromatography conditions: mobile phase A is formic acid solution with volume fraction of 0.1%; mobile phase B was acetonitrile (containing 0.1% formic acid by volume); the chromatographic column is as follows: infinityLabPorosill 120EC-C18 (50 mm. Times.2.1 mm,2.7 μm); the flow rate is 0.35mL/min, the column temperature is 50 ℃, and the sample injection amount is 10 mu L; the elution gradient is 0-2 min:5% -10% B; 2-7 min:10% -98% of B; 7-9.5 min:98% -100% of B;9.6 to 12 minutes: 5% B.
The mass spectrum conditions are as follows: the electrospray ionization source is in a positive ion mode, the acquisition range is 100-1200m/z, and the capillary voltage is 3500V; the dryer temperature was 350 ℃; the fragmentation voltage was 150V.
(3) Experimental results
The time is taken as the abscissa, the 0h concentration is taken as 100%, the time point concentration and the 0h concentration percentage are taken as the ordinate, as shown in fig. 4, 5 polypeptides can exist stably in plasma (the curves of P1 and P2 overlap), and the degradation is only 40% -50% after incubation at 37 ℃ for 8h.
Example 3: analysis of competitive binding of polypeptide to ACE2 by S protein (polypeptide co-incubated with ACE2 protein)
By usingThe detection is carried out by using a COVID-19Spike-ACE2binding detection kit:
(1) Preparation of mixed solution of polypeptide and ACE2 protein and co-incubation
Respectively preparing polypeptides P1-P5 into 50 mu M solution and 100 mu M solution by using a diluent, respectively taking 100 mu L of the solutions, adding 10 mu L of 10 XACE 2 working solution into the solutions, vortexing the solutions, and incubating the solutions at 37 ℃ for 1 hour for the next operation;
as a negative control, 100uL of diluent plus 10 uL of 10 XACE 2 working solution was used.
(2) Detection of binding rate of ACE2 protein and S protein by enzyme-linked immunosorbent assay
The 96-well plate is coated with SARS-CoV-2S1 protein binding domain, 100 mu L of the mixed solution of the polypeptide and ACE2 in the step (1) is added, the mixture is incubated for 2.5 hours, and unbound ACE2 protein is washed out by washing liquid; adding 100 mu L of primary antibody, namely goat-derived anti-ACE 2 antibody combined with the S1 protein-ACE 2 protein complex, incubating for 1 hour, and washing with a washing solution; adding 100 mu L of secondary antibody, namely HRP marked anti-goat-lgG, and washing with washing liquid; adding 100 mu L of a color reagent TBM, and incubating for 30 minutes; the reaction was stopped by adding a stop solution, and the absorbance was measured at 450nm, and the absorbance was proportional to the amount of the S1 protein-ACE 2 protein complex.
(3) Experimental results
As shown in fig. 5, the polypeptides were incubated with ACE2 protein for 1 hour, and the 100 μm group of P1, P2, P3 was able to inhibit the binding of S1 protein to ACE2 protein at 30.3%, 11.1% and 9.5%, respectively. The P1-P5 can inhibit the combination of the novel coronavirus S protein and ACE2 by competitive combination with ACE2, and has application prospect as a competitive inhibitor of the novel coronavirus S protein.
Example 4: analysis of competitive binding of polypeptide to ACE2 by S protein (polypeptide and ACE2 protein are not co-incubated)
By usingThe detection is carried out by using a COVID-19Spike-ACE2binding detection kit:
(1) Preparation of mixed solution of polypeptide and ACE2 protein
Respectively preparing polypeptides P1-P5 into 50 mu M solution and 100 mu M solution by using a diluent, adding 10 mu L10 XACE 2 working solution into 100 mu L of the solution, and directly performing the next operation after vortex;
as a negative control, 100uL of diluent plus 10 uL of 10 XACE 2 working solution was used.
(2) Detection of binding rate of ACE2 protein and S protein by enzyme-linked immunosorbent assay
The 96-well plate is coated with SARS-CoV-2S1 protein binding domain, 100 mu L of the mixed solution of the polypeptide and ACE2 in the step (1) is added, the mixture is incubated for 2.5 hours, and unbound ACE2 protein is washed out by washing liquid; adding 100 mu L of primary antibody, namely goat-derived anti-ACE 2 antibody combined with the S1 protein-ACE 2 protein complex, incubating for 1 hour, and washing with a washing solution; adding 100 mu L of secondary antibody, namely HRP marked anti-goat-lgG, and washing with washing liquid; adding 100 mu L of a color reagent TBM, and incubating for 30 minutes; the reaction was stopped by adding a stop solution, and the absorbance was measured at 450nm, and the absorbance was proportional to the amount of the S1 protein-ACE 2 protein complex.
(3) Experimental results
As shown in fig. 6, the 100 μm group of P1 showed inhibition with 20.8% inhibition when not incubated. The P1 has strong capability of inhibiting the combination of the novel coronavirus S protein and ACE2 by combining with the ACE2 in a competitive way, and has good application prospect as a competitive inhibitor of the novel coronavirus S protein.
While the application has been described in terms of preferred embodiments, it will be understood by those skilled in the art that various changes and modifications can be made without departing from the scope of the application, and it is intended that the application is not limited to the specific embodiments disclosed.
Claims (3)
1. A human ACE2 polypeptide blocker, which is characterized in that the amino acid sequence of the human ACE2 polypeptide blocker is Trp Trp Pro Pro Trp Trp, wherein Trp at position 2, pro at position 4, trp at position 5 and Trp at position 6 are D-amino acids, trp at position 1 and Pro at position 3 are L-amino acids.
2. Use of the humanized ACE2 polypeptide blocker of claim 1 in the preparation of a medicament for inhibiting a novel coronavirus.
3. A novel coronavirus inhibitor comprising the humanized ACE2 polypeptide blocker of claim 1.
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