CN117567561A - Surface-loaded hypoxia activatable covalent inhibitor for polypeptide assembly and preparation method thereof - Google Patents
Surface-loaded hypoxia activatable covalent inhibitor for polypeptide assembly and preparation method thereof Download PDFInfo
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- CN117567561A CN117567561A CN202311545531.4A CN202311545531A CN117567561A CN 117567561 A CN117567561 A CN 117567561A CN 202311545531 A CN202311545531 A CN 202311545531A CN 117567561 A CN117567561 A CN 117567561A
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- polypeptide
- assembly
- covalent
- hypoxia
- inhibitor
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- 229920001184 polypeptide Polymers 0.000 title claims abstract description 163
- 102000004196 processed proteins & peptides Human genes 0.000 title claims abstract description 163
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 163
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- 229940125808 covalent inhibitor Drugs 0.000 title claims abstract description 104
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- 238000000034 method Methods 0.000 claims description 16
- XJLXINKUBYWONI-DQQFMEOOSA-N [[(2r,3r,4r,5r)-5-(6-aminopurin-9-yl)-3-hydroxy-4-phosphonooxyoxolan-2-yl]methoxy-hydroxyphosphoryl] [(2s,3r,4s,5s)-5-(3-carbamoylpyridin-1-ium-1-yl)-3,4-dihydroxyoxolan-2-yl]methyl phosphate Chemical compound NC(=O)C1=CC=C[N+]([C@@H]2[C@H]([C@@H](O)[C@H](COP([O-])(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](OP(O)(O)=O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 XJLXINKUBYWONI-DQQFMEOOSA-N 0.000 claims description 15
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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
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Organic Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Genetics & Genomics (AREA)
- Biophysics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Immunology (AREA)
- Gastroenterology & Hepatology (AREA)
- Epidemiology (AREA)
- Molecular Biology (AREA)
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Abstract
A hypoxia activatable covalent inhibitor loaded on the surface of a polypeptide assembly and a preparation method thereof relate to the technical field of polypeptide nano-drugs. The covalent inhibitor comprises: the polypeptide self-assembly motif heptapeptide EIISIIE sequence comprises a ligand assembly motif-amq-EIS with a non-covalent targeting protein function of an inhibitor and a warhead assembly motif bipmNI-EIS with an hypoxia response group with an activatable covalent linking function, and the hypoxia activatable covalent inhibitor loaded on the surface of a polypeptide assembly is prepared by a ternary co-grouping mode. The invention has the advantages that: the hypoxia activatable covalent inhibitor loaded on the surface of the polypeptide assembly promotes the proximity effect of a ligand-warhead, and the hypoxia activated covalent linkage target protein enhances the inhibition effect, reduces the toxic and side effects of off-target, and has good tumor enrichment, long retention capacity and excellent tumor inhibition effect. The preparation method is simple, easy for industrial production and wide in application field range.
Description
Technical Field
The invention relates to the technical field of polypeptide nano-drugs, in particular to a preparation and performance characterization method of a hypoxia activatable covalent inhibitor loaded on the surface of a polypeptide assembly.
Technical Field
Proteins are essential to almost all intrinsic physiological processes. However, the pathological mechanisms of many diseases are closely related to abnormal expression of proteins, and thus it is necessary to develop inhibitors that effectively modulate the biological activity of target proteins. Although non-covalent inhibitors of proteins have been widely developed, the complex microenvironment places greater demands on the efficacy of protein inhibitors. Conventional targeted covalent inhibitors are constructed by linking electrophilic warheads, which can be covalently coupled to proteins, to reversible ligands, to effect covalent binding to adjacent nucleophilic residues, thereby enhancing the affinity and potency of the ligand inhibitor. However, this approach requires proper structural matching between the covalent inhibitor and the binding site of the target protein, and strict structural matching and the necessity of adjacent nucleophilic residues limit its design development. Loading of the surface of the assembly with different ligands has proven to be an effective method of establishing multivalent effects between the ligands and their corresponding receptors. Thus, we hypothesize that the simultaneous introduction of reversible ligands and electrophilic warheads on the surface of the assembly has great potential to facilitate the proximity-induced covalent binding process, thereby inspiring the design of targeted covalent inhibitors based on the assembly.
Considering the potential risk of non-targeted covalent binding of non-activated electrophilic warheads to normal protein dysfunction, covalent inhibitors are susceptible to off-target side effects. Electrophilic warheads with controlled reactivity have been designed to increase the selective binding capacity of covalent inhibitors. The introduction of a stimulus responsive group into a covalent inhibitor enables the control of covalent binding of a target protein using external or internal stimuli. Although stimulus-responsive groups have great potential in controlling covalent binding, there are still few covalent inhibitors containing stimulus-responsive warheads. In the hypoxia responsive group, 2-nitroimidazole is capable of generating a azophilic intermediate that is covalently reactive with cysteine sulfhydryl groups during the reduction of NTR that is overexpressed by the hypoxia environment. Therefore, the 2-nitroimidazole group is reasonably introduced, so that the method has important significance and wide application prospect in constructing the stimulus response covalent inhibitor and improving the targeted covalent inhibition.
Disclosure of Invention
The object of the present invention is to solve the above-mentioned drawbacks of the prior art and to provide a hypoxia activatable covalent inhibitor containing responsive 2-nitroimidazole based on polypeptide assemblies by rational design. The reversible ligand and the hypoxia response electrophilic warhead are loaded on the surface of the polypeptide assembly body, so that the proximity-induced covalent bonding can be promoted, the hypoxia activatable polypeptide nano covalent inhibitor is reduced under the action of NTR (non-covalent bonding) to generate a azonia intermediate, and the azonia intermediate can be covalently connected with cysteine residues of non-covalent bonding proteins, thereby improving the selectivity of covalent bonding, enhancing the effect of inhibiting abnormal proteins, promoting the retention of the activatable nano covalent inhibitor at tumor sites and improving the tumor inhibition effect. The preparation method of the polypeptide nano-drug is simple, the reaction condition is mild, and the operation is simple.
In order to achieve the above purpose, the technical scheme provided by the invention is as follows:
a surface-loaded hypoxia activatable covalent inhibitor of a polypeptide assembly, the covalent inhibitor comprising: polypeptide self-assembly motif heptapeptide EIISIIE sequence, ligand assembly motif one (amq-EIS) containing inhibitor with noncovalent targeting protein function and warhead assembly motif two (pmNI-EIS) containing hypoxia response group with activatable covalent linking function; the hypoxia activatable covalent inhibitor loaded on the surface of the polypeptide assembly is prepared by the following method:
s1: designing and synthesizing a polypeptide sequence EIISIIE with a dumbbell structure and strong self-assembly capability, wherein the sequence can be self-assembled to form an assembly body with a beta-sheet secondary structure, the polypeptide sequence is synthesized by a standard Fmoc solid-phase polypeptide synthesis (SPPS) method, piperidine is used as a deprotection agent, benzotriazole-N, N, N ', N' -tetramethylurea Hexafluorophosphate (HBTU) is used as a condensing agent, and N, N-Diisopropylethylamine (DIEA) is used as catalytic alkali;
s2: on the basis of a polypeptide self-assembly sequence EIISIIE, covalently connecting a ligand inhibitor to the polypeptide sequence by an amide condensation method to obtain a polypeptide assembly motif I containing the ligand inhibitor with a noncovalent targeting protein function;
s3: is provided withSynthesizing 1-methyl-4-phenylacetic acid-2-nitroimidazole warhead group with Nitroreductase (NTR) activating reactivity; based on Suzuki coupling reaction, 1-bis (diphenylphosphine) dicyclopentadienyl iron palladium (II) dichloride (Pd (dppf) Cl) in 1, 4-dioxane solvent 2 ) And potassium carbonate (K) 2 CO 3 ) Reacting 4-bromo-2-nitroimidazole with 4-borate-methyl phenylacetate to produce 1-methyl-4-methyl phenylacetate-2-nitroimidazole; then, hydrolyzing methyl ester groups by sodium hydroxide (NaOH) treatment to generate NTR (n-methyl-4-phenylacetic acid-2-nitroimidazole) (pmNI) groups, and covalently connecting the hypoxia response groups to an EIISIIE polypeptide sequence by an amide condensation method to obtain a polypeptide assembly motif II containing the hypoxia response groups and having an activatable covalent connection function;
s4: and (3) co-assembling the three polypeptides and the polypeptide assembly motifs of the derivatives thereof in the steps S1, S2 and S3 in an aqueous solution according to a certain component proportion to obtain a polypeptide co-assembly system solution, and annealing to obtain the hypoxia activatable covalent inhibitor loaded on the surface of the polypeptide assembly.
Characterization of the Performance of surface-loaded hypoxia activatable covalent inhibitors of polypeptide assemblies
S5: and (2) placing the hypoxia activatable covalent inhibitor loaded on the surface of the polypeptide assembly body obtained in the step (S4) under an NTR reduction condition, and testing the covalent connection performance, wherein the covalent connection performance test proves that the polypeptide nano covalent inhibitor can be in covalent connection with cysteine and cysteine-containing proteins in the reduction process.
S6: further cell experiments and animal experiments are carried out on the hypoxia activatable covalent inhibitor loaded on the surface of the polypeptide assembly body obtained in the step S4, and the polypeptide nano covalent inhibitor is proved to improve the killing power of cancer cells under hypoxia, and has excellent tumor retention and tumor growth inhibition performances.
In a further embodiment of the invention, the polypeptide self-assembly motif is a dumbbell-type amphiphilic polypeptide sequence, such as glutamate-isoleucine-serine-isoleucine-glutamate.
In a further embodiment of the invention, the polypeptide assembly motif comprising a ligand inhibitor having a non-covalent binding protein function is an inhibitor functionalized polypeptide comprising a 4-aminoquinazoline.
In a further embodiment of the invention, the polypeptide assembly motif comprising a hypoxia responsive group having an activatable covalent attachment function is a functionalized polypeptide comprising a hypoxia responsive 1-methyl-4-phenylacetate-2-nitroimidazole.
In a further embodiment of the present invention, in step S4: the ratio of polypeptide assembly motifs containing ligand inhibitors with non-covalent binding protein functions to the total number of moles of the polypeptide co-assembly system is between 0.1% and 50%; the proportion of the polypeptide assembly motif II containing the hypoxia response group with the activatable covalent connection function to the total mole number of the polypeptide co-assembly system is between 0.1 and 50 percent; the mass concentration of the substances of the polypeptide co-assembly system solution is between 0.1 micromoles per liter and 10 millimoles per liter; the annealing temperature of the polypeptide co-assembly system solution is between 10 and 100 ℃, the annealing time is between 0.1 and 100 hours, preferably between 1 and 48 hours, the solvent is buffer solution or water, and the water is ultrapure water, deionized water or Milli-Q water.
In a further embodiment of the present invention, in step S5: the reductive response covalent linking conditions of the surface-loaded hypoxia activatable covalent inhibitor of the polypeptide assembly are nitroreductase NTR, reduced coenzyme nicotinamide adenine dinucleotide phosphate NADPH, and cysteine Cys; the NTR concentration ranges from 0.1 microgram per milliliter to 100 microgram per milliliter, the NADPH concentration ranges from 0.1 millimole per liter to 1 millimole per liter, and the Cys concentration ranges from 0.01 millimole per liter to 1 millimole per liter; the temperature is 37 ℃ and the reduction time is 0.1-1 hour.
In a further embodiment of the present invention, in step S6: the cancer cells used in the cell experiments were human epidermal cancer cells (a 431).
The hypoxia activatable covalent inhibitor loaded on the surface of the polypeptide assembly provided by the invention can efficiently respond to the NTR zymogen site to generate a azonia electrophilic intermediate in a hypoxia environment, and can be covalently connected with nucleophilic cysteine residues, so that the affinity and effectiveness of a non-covalent ligand and the selectivity of covalent connection are improved.
The polypeptide assembly surface-loaded hypoxia activatable covalent inhibitor provided by the invention has good tumor enrichment capability, and covalent connection of solid tumor hypoxia activation and target protein endows the nano covalent inhibitor with excellent tumor retention capability. The hypoxia activatable covalent inhibitor loaded on the surface of the polypeptide assembly can be combined with ligand distribution on the assembly, and the advantage of stimulus response can strongly inhibit the function of abnormal proteins. The hypoxia activatable covalent inhibitor loaded on the surface of the polypeptide assembly has application value in efficiently inhibiting tumor growth.
The invention has the advantages and beneficial effects that:
(1) The polypeptide sequence adopted by the invention has good biocompatibility and bioactivity, the amino acid is used as a structural unit of protein in organisms, the polypeptide sequence has good biocompatibility, and thermodynamic and kinetic control in the assembly process can be realized by reasonably designing and regulating and controlling the molecular structure in the assembly sequence. (2) According to the invention, the polypeptide sequence of the dumbbell-shaped amphiphilic beta-sheet assembled secondary structure is reasonably designed, so that the assembled polypeptide assembly is obtained. (3) According to the invention, a ligand inhibitor with a non-covalent binding protein function and a warhead group with an activatable covalent connection function are covalently connected into a dumbbell-type self-assembled polypeptide, and a hypoxia activatable covalent inhibitor loaded on the surface of a polypeptide assembly is obtained in a co-assembly mode. The polypeptide nanometer covalent inhibitor can promote adjacent induced covalent bonding by simultaneously introducing reversible ligand and hypoxia response electrophilic warhead on a polypeptide assembly bracket. (4) The hypoxia activatable covalent inhibitor loaded on the surface of the polypeptide assembly provided by the invention can efficiently respond to the NTR zymogen site to generate a azonia electrophilic intermediate in a hypoxia environment, and can be covalently connected with nucleophilic cysteine residues, so that the affinity and effectiveness of a non-covalent ligand and the selectivity of covalent connection are improved. (5) The hypoxia activatable covalent inhibitor loaded on the surface of the polypeptide assembly provided by the invention can be effectively and covalently connected with target protein in a solid tumor hypoxia environment, and is beneficial to prolonging tumor retention and enhancing tumor growth inhibition effect. (6) The preparation method of the hypoxia activatable covalent inhibitor loaded on the surface of the polypeptide assembly is simple, the reaction condition is mild, the operation is simple and convenient, the industrialization is easy, and the polypeptide assembly has great application potential in the aspects of anti-inflammation, anti-tumor, antibacterial and the like.
Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.
Drawings
FIG. 1 is a diagram showing the molecular structural formulae of hypoxia activatable warhead, dumbbell-type polypeptide self-assembly sequence and functionalized polypeptide according to the embodiment of the present invention (1:1-methyl-4-phenylacetic acid-2-nitroimidazole synthesis route, 2:glutamate-isoleucine-serine-isoleucine-glutamate, 3:4-aminoquinazoline-glutamate-isoleucine-serine-isoleucine-glutamate, 4:1-methyl-4-phenyl-2-nitroimidazole-glutamate-isoleucine-methionine-glutamate);
FIG. 2 is a diagram of ultra high performance liquid chromatography (UPLC) and inductively coupled plasma mass spectrometry (ESI-MS) of a hypoxia activatable warhead in an embodiment of the present invention;
FIG. 3 is a UPLC and ESI-MS plot of an embodiment of the invention before and after NTR/NADPH+Cys action in a hypoxia activatable warhead;
FIG. 4 is a graph of circular dichroism spectrum (CD) obtained by assembling a surface-loaded hypoxia activatable covalent inhibitor of a polypeptide assembly in an embodiment of the present invention;
FIG. 5 is an atomic force microscope and transmission electron microscope of an assembled hypoxia activatable covalent inhibitor of a polypeptide assembly surface load according to an embodiment of the present invention;
FIG. 6 is a graph of a micro-thermophoresis (MST) assay for binding of a surface-loaded hypoxia activatable covalent inhibitor to a target protein kinase of a polypeptide assembly in an embodiment of the present invention;
FIG. 7 is a UPLC and ESI-MS diagram of a surface-loaded hypoxia activatable covalent inhibitor of a polypeptide assembly of an embodiment of the present invention before and after NTR/NADPH+Cys action under hypoxic conditions;
FIG. 8 is a mass spectrum of the interaction of a surface-loaded hypoxia activatable covalent inhibitor of a polypeptide assembly of an embodiment of the present invention with NTR/NADPH+ reduced insulin B-chain under hypoxic conditions;
FIG. 9 is a graph showing inhibition of target protein kinase activity by a surface-loaded hypoxia activatable covalent inhibitor of a polypeptide assembly in accordance with an embodiment of the present invention under hypoxic conditions;
FIG. 10 is a flow cytometer analysis of cellular uptake of surface-loaded hypoxia activatable covalent inhibitors of polypeptide assemblies in embodiments of the present invention;
FIG. 11 is a laser confocal image of the cellular uptake pathway of a surface-loaded hypoxia activatable covalent inhibitor of a polypeptide assembly in an embodiment of the present invention;
FIG. 12 is a cytotoxicity assay of a surface-loaded hypoxia activatable covalent inhibitor of a polypeptide assembly of an embodiment of the present invention under normoxic and hypoxic conditions;
FIG. 13 is an assay for the intracellular phosphorylation inhibition of a target protein by a surface-loaded hypoxia activatable covalent inhibitor of a polypeptide assembly in an embodiment of the present invention;
FIG. 14 is an in vivo biodistribution imaging of a surface-loaded hypoxia activatable covalent inhibitor of a polypeptide assembly in an embodiment of the present invention;
FIG. 15 is a graph showing the distribution of the surface-loaded hypoxia activatable covalent inhibitor of the polypeptide assemblies according to the present invention;
FIG. 16 is an in vivo anti-tumor study of a surface-loaded hypoxia activatable covalent inhibitor of a polypeptide assembly in an embodiment of the present invention;
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. The experimental methods in the following examples are conventional methods unless otherwise specified.
The test materials used in the examples described below, unless otherwise specified, were purchased from conventional biochemical reagent stores.
The quantitative tests in the following examples were all set up with three replicates, and the data are the mean or mean ± standard deviation of the three replicates.
The method for preparing the surface-supported hypoxia activatable covalent inhibitor of the polypeptide assembly provided by the invention is further described below with reference to specific examples.
Example 1
The present embodiment provides a method for preparing a surface-loaded hypoxia activatable covalent inhibitor of a polypeptide assembly, the hypoxia activatable covalent inhibitor comprising: a polypeptide self-assembly motif, a ligand assembly motif containing an inhibitor with a non-covalent targeting protein function and a warhead assembly motif containing a hypoxia response group with an activatable covalent linking function; the preparation process comprises the following steps:
s1: synthesizing 1-methyl-4-phenylacetic acid-2-nitroimidazole through suzuki coupling reaction;
based on Suzuki coupling reaction, 1-bis (diphenylphosphine) dicyclopentadienyl iron palladium (II) dichloride (Pd (dppf) Cl) in 1, 4-dioxane solvent 2 ) And potassium carbonate (K) 2 CO 3 ) Reacting 4-bromo-2-nitroimidazole with 4-borate-methyl phenylacetate to produce 1-methyl-4-methyl phenylacetate-2-nitroimidazole; then, the methyl ester group is hydrolyzed by sodium hydroxide (NaOH) treatment to generate NTR responding to 1-methyl-4-phenylacetic acid-2-nitroimidazole (pmNI) group for solid phase amide condensation to synthesize an assembly motif II as shown in figure 1;
s2: designing a synthetic polypeptide sequence by a solid-phase polypeptide synthesis method:
polypeptide self-assembly motifs: the solid phase resin is added with 0.25 millimole, the polypeptide sequence is synthesized by a standard Fmoc Solid Phase Polypeptide Synthesis (SPPS) method, piperidine is taken as a deprotection agent, benzotriazole-N, N, N ', N' -tetramethylurea Hexafluorophosphate (HBTU) is taken as a condensing agent, and the product purity is greater than or equal to 99 percent through high performance liquid chromatography separation and purification.
S3: three polypeptides designed and synthesized in step S2 are as follows: the polypeptide self-assembly motif glutamic acid-isoleucine-serine-isoleucine-glutamic acid, the ligand assembly motif 4-aminoquinazoline-glutamic acid-isoleucine-serine-isoleucine-glutamic acid containing noncovalently targeting protein functions, and the warhead assembly motif di-1-methyl-4-phenyl-2-nitroimidazole-glutamic acid-isoleucine-methionine-glutamic acid containing a hypoxia responsive group with activatable covalent linking function. Annealing and assembling the three polypeptide assembly motifs in an aqueous solution at the annealing temperature of 80 ℃ for 24 hours according to a formula with a molar ratio of 90:5:5 to obtain the hypoxia activatable covalent inhibitor loaded on the surface of the polypeptide assembly, and observing the obtained nano assembly structure under an atomic force microscope and a field emission transmission electron microscope to obtain the nano belt structure, as shown in figure 5.
Example 2
1-methyl-4-phenylacetic acid-2-nitroimidazole (pmNI) prepared in the embodiment 1 of the invention is dissolved in PBS solution, and is subjected to ultra performance liquid chromatography mass spectrometry UPLC-MS characterization. As shown in fig. 2.
Experimental results: from the characterization of 2UPLC and ESI-MS in the figure, it can be observed that the 1-methyl-4-phenylacetic acid-2-nitroimidazole target molecule with the purity of more than or equal to 99% is successfully prepared.
Example 3
1-methyl-4-phenylacetic acid-2-nitroimidazole (pmNI) prepared in example 1 of the present invention was dissolved in PBS solution (100. Mu.M), followed by addition of 10. Mu.g/mL NTR reductase, 0.5mM coenzyme NADPH and 100. Mu.M Cys, respectively. The mixed solution is N 2 The reaction is carried out for 15min under the atmosphere, and the characterization of ultra-high performance liquid chromatography mass spectrometry (UPLC-MS) is carried out. As shown in fig. 3.
Experimental results: from the UPLC characterization of FIG. 3, it can be observed that after the action of NTR/NADPH+Cys under the hypoxia condition, the absorption peak corresponding to pmNI disappears, a new peak with more polarity appears, and the combination of mass spectrum data results in the formation of the corresponding covalent connection product with cysteine.
Example 4
The surface-supported hypoxia activatable covalent inhibitor of the polypeptide assembly prepared in example 1 of the present invention was subjected to Circular Dichroism (CD) characterization, as shown in fig. 4.
Experimental results: from the circular dichroism spectrum, the polypeptide nanometer covalent inhibitor has a positive peak at 200nm and a negative peak at 222nm, and is a typical beta-sheet secondary structure, which shows strong self-assembly trend.
Example 5
The assembled structure obtained by annealing in example 1 of the present invention was observed under an Atomic Force Microscope (AFM) and a field emission Transmission Electron Microscope (TEM), as shown in fig. 5.
Experimental results: obvious nano-belt structure can be observed from atomic force microscope and field emission transmission electron microscope pictures, which shows that the hypoxia activatable covalent inhibitor loaded on the surface of the polypeptide assembly can be successfully assembled into ordered nano-structure.
Example 6
The surface-loaded hypoxia activatable covalent inhibitor of the polypeptide assembly prepared in example 1 of the present invention was evaluated for affinity to protein EGFR by MST assay. The labeled EGFR solution was diluted with PBS to a concentration of 160nM and various concentrations of polypeptide nanocovalent inhibitors were added, and the dissociation constant (Kd) values between the protein EGFR and ligand-containing polypeptide nanocovalent inhibitors were obtained using NT analysis software, as shown in fig. 6.
Experimental results: the Kd value of the hypoxia activatable covalent inhibitor loaded on the surface of the polypeptide assembly obtained from MST test and EGFR target protein shows that the polypeptide nano covalent inhibitor has good affinity with the target protein.
Example 7
To the surface-supported hypoxia activatable covalent inhibitor solution of the polypeptide assembly prepared in example 1 of the present invention, 1. Mu.g/mL NTR reductase, 0.05mM coenzyme NADPH and 10. Mu.M Cys were added, respectively. The mixed solution is N 2 The reaction is carried out for 15min under the atmosphere, and the characterization of ultra-high performance liquid chromatography mass spectrometry (UPLC-MS) is carried out. As shown in fig. 7.
Experimental results: from the UPLC characterization of FIG. 7, it can be observed that after the NTR/NADPH+Cys is acted under the anaerobic condition, the absorption peak corresponding to the warhead pmNI-EIS in the polypeptide nanometer covalent inhibitor disappears, a new peak appears, and the combination of mass spectrum data results in the generation of the corresponding covalent connection product with cysteine.
Example 8
5. Mu.g/mL of NTR reductase, 0.25mM of coenzyme NADPH and 20. Mu.M of TCEP-reduced insulin were added to the surface-supported hypoxia activatable covalent inhibitor solution of the polypeptide assembly prepared in example 1 of the present invention, respectively. The mixed solution is N 2 And (3) reacting for 15min under the atmosphere, desalting, and performing mass spectrum characterization. As shown in fig. 8.
Experimental results: as can be seen from the mass spectrum characterization of FIG. 8, after NTR/NADPH+ insulin is subjected to hypoxia, the warhead pmNI-EI in the polypeptide nano covalent inhibitor is covalently connected with the B chain of the reduced insulin.
Example 9
The enzyme activity of the polypeptide assembly prepared in example 1 of the present invention was analyzed after the surface-supported hypoxia activatable covalent inhibitor acted on EGFR kinase under hypoxia conditions, as shown in FIG. 9.
Experimental results: the half inhibitory concentration values of the polypeptide nano covalent inhibitor and EGFR target protein obtained from the enzyme activity inhibition test of fig. 9 show that the polypeptide nano covalent inhibitor can effectively inhibit the activity of the target protein.
Example 10
The surface-supported hypoxia activatable covalent inhibitor of the polypeptide assembly prepared in example 1 of the present invention was tested for cellular uptake by flow cytometry.
A431 cells were grown at 2×10 5 Cell density of/well was plated in 6-well plates, cultured in an incubator for 12 hours, and then cultured in a hypoxic environment for another 12 hours. Fresh medium containing polypeptide nano-covalent inhibitor is added and culture is continued for 2, 4, 8 or 12 hours. After incubation to a predetermined time, the medium was discarded, after washing the cells three times with PBS, the cells were digested with trypsin, collected and washed twice with PBS, and the cells were resuspended in PBS for flow cytometry analysis as shown in fig. 10.
Experimental results: the results of flow cytometry show that the fluorescence intensity of the polypeptide nano covalent inhibitor in the A431 cells is gradually increased along with the extension of the incubation time, which indicates that the polypeptide nano covalent inhibitor is successfully absorbed by the A431 cells, and the absorption amount is continuously increased along with the extension of the absorption time.
Example 11
The surface of the polypeptide assembly prepared in the embodiment 1 of the invention is loaded with the hypoxia activatable covalent inhibitor, and the cell uptake route of the nano covalent inhibitor is monitored by a confocal laser scanning microscope.
4T1 cells were grown at 2X 10 5 Is spread on a confocal glass dish and cultured in an incubator for 12 hours. And after further incubation for 12 hours under hypoxic conditions, cells were washed with PBS and pretreated with endocytic pathway inhibitor 50 μm Chlorpromazine (CPZ), 1mM methyl- β -cyclodextrin (mβ -CD) or 50 μm Amiloride (AMI) for 1 hour, fresh medium containing polypeptide nano-covalent inhibitor was added and incubation was continued for 12 hours. After washing the cells three times with PBS, the cells were fixed with 4% paraformaldehyde for 20 minutes and stained with nuclear dye DAPI for 20 minutes. Observations were made by Confocal Laser Scanning Microscopy (CLSM), as shown in fig. 11.
The results of the experiments show that CLSM images resulted in a significant decrease in fluorescence signal of the polypeptide nano-covalent inhibitor when a431 cells were pretreated with CPZ compared to a431 cells pretreated with PBS. In contrast, cancer cells pretreated with AMI and mβ -CD showed only a partial decrease or comparable fluorescence intensity, respectively. These results indicate that clathrin-mediated endocytosis is the primary internalization pathway for polypeptide nanocovalent inhibitors into a431 cells.
Example 12
The polypeptide nanometer covalent inhibitor has the advantage that the surface loaded hypoxia activatable covalent inhibitor of the polypeptide assembly prepared in the embodiment 1 of the invention can evaluate the hypoxia enhanced tumor cell killing capability through normoxicity and cytotoxicity under hypoxia.
Cytotoxicity of the polypeptide nanocovalent inhibitor was detected by MTT method: a431 cells were grown at 6×10 3 Cell density of/well cells were seeded in 96-well plates and incubated in an incubator under normal or hypoxic conditions for 24 hours. Fresh medium containing different concentrations of polypeptide nano-covalent inhibitor was added to 96-well plates and incubation was continued for 12 hours under normoxic or hypoxic conditions, A431 cellsThe culture was washed twice with fresh medium and further incubated under normoxic or anoxic conditions for 36 hours. Subsequently, 10. Mu.l of MTT solution was added to each well and incubation was continued in the incubator for 4 hours. After 4 hours, the culture broth was discarded, 100. Mu.l of dimethyl sulfoxide was added to each well, and the absorbance at 490nm was measured with a microplate reader.
Experimental results: as shown in fig. 12MTT results, polypeptide nano-covalent inhibitors showed more effective killing effect on a431 cells under hypoxic conditions. Enhanced cytotoxicity under hypoxic conditions suggests that hypoxia-activated covalent binding of warhead to cysteine residues occurs, thereby increasing the inhibitory potency of the covalent inhibitor.
Example 13
The hypoxia activatable covalent inhibitor loaded on the surface of the polypeptide assembly in the embodiment of the invention evaluates the mechanism of killing tumor cells by the nano covalent inhibitor by detecting the change of the phosphorylation content of the target protein in cells.
The level of phosphorylated EGFR was tested by enzyme-linked immunosorbent assay (ELISA). Proteins were collected from a431 cells treated with PBS and nano-covalent inhibitors under hypoxic conditions, with or without washing. The collected proteins were added appropriately to wells containing the antibody mixture and incubated for 1 hour at room temperature. After washing the wells with ELISA wash buffer, 3', 5' -Tetramethylbenzidine (TMB) substrate was added to each well and incubated for 15 minutes in the dark at room temperature. After adding the stop solution to each well, the absorbance at 450nm was measured with a microplate reader.
Experimental results: as shown in the ELISA results of fig. 13, the surface-loaded hypoxia activatable covalent inhibitor of the polypeptide assemblies induced significantly reduced expression of phosphorylated EGFR without washing, and maintained significant inhibition of its phosphorylation after washing treatment, as compared to PBS group. This result suggests that hypoxia-activated covalent binding can strongly inhibit EGFR phosphorylation.
Example 14
The surface-supported hypoxia activatable covalent inhibitor of the polypeptide assembly prepared in example 1 of the present invention was subjected to in vivo biodistribution and various tissue distribution studies.
A431 cells (5×10) 6 ) Subcutaneously injecting the mixture into the back and right lower limbs of female BALB/c nude mice, and establishing a tumor-bearing mouse model. Mice were randomly grouped after 14 days of tumor growth, subcutaneously injected with a surface-loaded hypoxia activatable covalent inhibitor, and then imaged using a living imaging system to monitor the living distribution of polypeptide nanocovalent inhibitors after 2, 4, 8, 12, 24 hours of injection, respectively. After 24 hours, mice were sacrificed, and major organs (heart, liver, spleen, lung, kidney) and tumor tissues were collected and ex vivo tissue imaging was performed to observe the distribution of the polypeptide nano-covalent inhibitor in each tissue, as shown in fig. 14 and 15.
Experimental results: the results of in vivo imaging in fig. 14 show that a distinct fluorescent signal of the polypeptide nano-covalent inhibitor can be observed near the tumor tissue, indicating that the polypeptide nano-covalent inhibitor can be successfully enriched at the tumor site. In addition, a distinct fluorescent signal of the polypeptide nano-covalent inhibitor was still observed at the tumor site 24 hours after administration of the mice, indicating that the nano-covalent inhibitor has a longer residence time at the tumor site. The fluorescence imaging results of the isolated major organs (heart, liver, spleen, lung, kidney) and tumor tissues of fig. 15 show that the fluorescence intensity of the polypeptide nano-covalent inhibitor in tumor tissues is significantly higher than that of the normal organs, and these results demonstrate that the polypeptide nano-covalent inhibitor can be enriched in tumor sites and has a longer residence time. This phenomenon demonstrates the long-term retention of the covalent inhibitor in tumor tissue, indicating proximity-induced covalent binding to the target protein.
Example 15
The surface-loaded hypoxia activatable covalent inhibitor of the polypeptide assembly prepared in the embodiment 1 of the invention is subjected to in vivo anti-tumor treatment effect research.
A431 cells (5×10) 6 ) Subcutaneously injected into the back and right lower limbs of female BALB/c mice to establish a tumor-bearing mouse model. When the tumor volume reaches 100mm 3 At this time, mice were randomly grouped and subjected to subcutaneous injectionDifferent treatments, the surface-loaded hypoxia activatable covalent inhibitor of the polypeptide assembly served as the experimental group and PBS served as the control group. Once every two days, four times in total. In addition, tumor volumes of each mouse were monitored and recorded every other day, as shown in fig. 16.
Experimental results: as shown in FIG. 16, compared with the free ligand inhibitors of the amo-EIS and the amo-EIS & EIS binary co-assembly non-covalent inhibitor, the tumor volume of mice in the treatment group of the hypoxia activatable covalent inhibitor is obviously reduced, which indicates that the hypoxia activatable covalent inhibitor loaded on the surface of the polypeptide assembly is activated to form covalent connection in a hypoxia environment, has stronger treatment effect and can obviously inhibit tumor growth.
It should be noted that other raw material ratios and preparation method parameters are also possible in addition to those exemplified in examples 1 to 15. In addition, the present invention does not relate to diagnosis and treatment of diseases, and the provided inhibitor can be used for preparing medicines for treating cancers, and although the obtained products are acted on living mice in the above embodiments, the results are not directly obtained for diagnosing and treating diseases, but only for verifying the relevant effects of the inhibitor obtained by the present invention, and providing basis for preparing medicines for treating relevant diseases.
Claims (9)
1. A surface-supported hypoxia activatable covalent inhibitor of a polypeptide assembly, the covalent inhibitor comprising: a polypeptide self-assembly motif heptapeptide EIISIIE sequence, which comprises a ligand assembly motif I with an inhibitor having a non-covalent targeting protein function and a warhead assembly motif II with a hypoxia response group having an activatable covalent linking function; the hypoxia activatable covalent inhibitor loaded on the surface of the polypeptide assembly is prepared by the following method:
s1: designing and synthesizing 1-methyl-4-phenylacetic acid-2-nitroimidazole warhead group with nitroreductase NTR activating reaction activity; based on Suzuki coupling reaction, 1-bis (diphenylphosphine) dicyclopentadienyl iron palladium (II) dichloride Pd (dppf) Cl in 1, 4-dioxane solvent 2 And potassium carbonate K 2 CO 3 4-bromo-2-nitro when presentReacting the methylimidazole with methyl 4-borate-phenylacetate to produce 1-methyl-4-methyl phenylacetate-2-nitroimidazole; then, hydrolyzing the methyl ester group by sodium hydroxide NaOH to generate NTR response 1-methyl-4-phenylacetic acid-2-nitroimidazole pmNI group, which is used for constructing an activatable covalently linked warhead motif for SPPS (solid phase polypeptide synthesis) of standard Fmoc;
s2: designing and synthesizing a polypeptide sequence EIISIIE with a dumbbell structure and strong self-assembly capability, wherein the sequence can be self-assembled to form an assembly body with a beta-sheet secondary structure, the polypeptide sequence is synthesized by a SPPS (specific surface plasmon resonance) method through a standard Fmoc solid-phase polypeptide synthesis method, piperidine is used as a deprotection agent, benzotriazole-N, N, N ', N' -tetramethylurea hexafluorophosphate HBTU is used as a condensing agent, and N, N-diisopropylethylamine DIEA is used as catalytic alkali;
s3: on the basis of polypeptide self-assembly motifs EIISIIE, a ligand inhibitor and a hypoxia response group are covalently connected to the polypeptide sequence by an amide condensation method, so that a first polypeptide assembly motif containing the ligand inhibitor and having a non-covalent targeting protein function and a second polypeptide assembly motif containing the hypoxia response group and having an activatable covalent connection function are obtained; and (3) co-assembling the three polypeptide assembly motifs to obtain a polypeptide co-assembly system solution, and annealing to obtain the hypoxia activatable covalent inhibitor loaded on the surface of the polypeptide assembly.
2. The surface-supported hypoxia activatable covalent inhibitor of a polypeptide assembly according to claim 1, wherein:
the polypeptide self-assembly motifs EIISIIE include, but are not limited to, glutamic acid-isoleucine-serine-isoleucine-glutamic acid.
3. The surface-supported hypoxia activatable covalent inhibitor of a polypeptide assembly according to claim 1, wherein:
the polypeptide assembly motif containing ligand inhibitors having non-covalent binding protein function is an inhibitor functionalized polypeptide including, but not limited to, 4-aminoquinazoline.
4. The surface-supported hypoxia activatable covalent inhibitor of a polypeptide assembly according to claim 1, wherein:
the polypeptide assembly motif containing the hypoxia response group and having the function of activating covalent connection is a functionalized polypeptide containing the hypoxia response 1-methyl-4-phenylacetic acid-2-nitroimidazole.
5. The surface-supported hypoxia activatable covalent inhibitor of a polypeptide assembly according to claim 1, wherein:
the ratio of polypeptide assembly motifs containing ligand inhibitors with non-covalent binding protein functions to the total number of moles of the polypeptide co-assembly system is between 0.1% and 50%; the proportion of the polypeptide assembly motif II containing the hypoxia response group with the activatable covalent connection function to the total mole number of the polypeptide co-assembly system is between 0.1 and 50 percent; the mass concentration of the substances of the polypeptide co-assembly system solution is between 0.1 micromoles per liter and 10 millimoles per liter; the annealing temperature of the polypeptide co-assembly system solution is between 10 and 100 ℃, the annealing time is between 0.1 and 100 hours, the solvent is buffer solution or water, and the water is ultrapure water, deionized water or Milli-Q water.
6. The surface-supported hypoxia activatable covalent inhibitor of a polypeptide assembly according to claim 1, wherein:
the reduction response covalent linking conditions of the hypoxia activatable covalent inhibitor are nitroreductase NTR, reduced coenzyme nicotinamide adenine dinucleotide phosphate NADPH, and cysteine Cys; the NTR concentration ranges from 0.1 microgram per milliliter to 100 microgram per milliliter, the NADPH concentration ranges from 0.1 millimole per liter to 1 millimole per liter, and the Cys concentration ranges from 0.01 millimole per liter to 1 millimole per liter; the temperature is 37 ℃ and the time for reducing covalent connection is 0.1-1 hour.
7. A polypeptide assembly surface-supported hypoxia activatable covalent inhibitor prepared by the method of any one of claims 1-6.
8. Use of a surface-supported hypoxia activatable covalent inhibitor of a polypeptide assembly as claimed in claim 7, wherein: can be used for preparing antitumor drugs to improve cancer cell killing power under hypoxia.
9. The use of a surface-supported hypoxia activatable covalent inhibitor of a polypeptide assembly according to claim 8, wherein: the cancer cells are human epidermal cancer cells a431.
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