CN112830975A - Pro-apoptotic bicyclic polypeptide with stable α-helical conformation and preparation method and application - Google Patents
Pro-apoptotic bicyclic polypeptide with stable α-helical conformation and preparation method and application Download PDFInfo
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- CN112830975A CN112830975A CN202110140359.9A CN202110140359A CN112830975A CN 112830975 A CN112830975 A CN 112830975A CN 202110140359 A CN202110140359 A CN 202110140359A CN 112830975 A CN112830975 A CN 112830975A
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Abstract
本发明公开了一种α‑螺旋构象稳定的促凋亡双环多肽及其制备方法与应用，其包含促凋亡功能肽段、肿瘤靶向功能肽段、转角氨基酸残基及含硫醚分子骨架，促凋亡肽段为富含赖氨酸和亮氨酸的两亲性序列，转角氨基酸为脯氨酸，所述含硫醚分子骨架通过巯基‑炔基点击化学将两分子巯基丙酸加成到含炔基非天然氨基酸侧链，并通过大环内酰胺化获得。该多肽的制备方法简单有效，且通过在促凋亡肽功能肽段的第i个和i+7个氨基酸残基位置进行双环化，可以显著提高环化效率，与线性多肽相比，双环多肽α‑螺旋程度更高，稳定性更强，血液稳定性良好，此外，该双环多肽可分别靶向细胞表面整合素受体以及细胞内线粒体，选择性扰乱整合素高表达肿瘤细胞线粒体膜电位，该促凋亡双环多肽具有良好的肿瘤治疗应用前景。
The invention discloses a pro-apoptosis bicyclic polypeptide with stable α-helical conformation, a preparation method and application thereof, and comprises a pro-apoptosis functional peptide segment, a tumor targeting functional peptide segment, a turn amino acid residue and a thioether-containing molecular skeleton , the pro-apoptotic peptide segment is an amphiphilic sequence rich in lysine and leucine, the turn amino acid is proline, and the thioether-containing molecular skeleton combines two molecules of mercaptopropionic acid by mercapto-alkynyl click chemistry. into alkynyl-containing non-natural amino acid side chains and obtained by macrolactamation. The preparation method of the polypeptide is simple and effective, and by bicyclizing the i-th and i+7 amino acid residue positions of the pro-apoptotic peptide functional peptide segment, the cyclization efficiency can be significantly improved. The α-helix degree is higher, the stability is stronger, and the blood stability is good. In addition, the bicyclic polypeptide can target integrin receptors on the cell surface and mitochondria in cells respectively, and selectively disturb the mitochondrial membrane potential of tumor cells with high integrin expression. The pro-apoptotic bicyclic polypeptide has a good application prospect in tumor therapy.
Description
Technical Field
The invention belongs to the field of biological medicines, and particularly relates to a pro-apoptotic bicyclic polypeptide with stable alpha-helix conformation, and a preparation method and application thereof.
Background
The apoptosis-promoting peptide KLA (KLAKLAKKLAKLAK) is a section of alpha-helix amphiphilic polypeptide with antibacterial activity, can target mitochondria and destroy mitochondrial membranes, leads to the release of cytochrome C and induces apoptosis, and is widely reported to be used for treating malignant tumors. However, linear KLA polypeptides have problems of poor stability, poor cell penetration, poor cell activity, and the like. The literature reports that the pro-apoptotic peptide KLA is delivered intracellularly, mainly by coupling ligands, using receptor-mediated endocytosis. For example by further modifying a cyclic NGR polypeptide ligand, an AHNP polypeptide ligand or by folate in the pro-apoptotic peptide KLA sequence. Therefore, the apoptosis-promoting peptide KLA can effectively target cell mitochondria and destroy mitochondrial membranes, so that cytochrome C is released, an apoptosis pathway of cells is activated, and the apoptosis-promoting peptide KLA is finally used for treating malignant tumors, so that the apoptosis-promoting peptide drug gradually becomes a research hotspot.
Under the existence of the advantages of polypeptide drugs relative to small molecule drugs, the traditional polypeptide drugs still have various problems, such as poor stability of linear polypeptides, poor cell penetration ability, no targeting of functional polypeptides, no functionality of targeted polypeptides, and single targeting of receptors in cells or on cell surfaces. Therefore, modification and modification aiming at the defects of the polypeptide drugs still remain great challenges.
Disclosure of Invention
The invention solves the technical problems of poor metabolic stability, low cell penetration capability and weak targeting property of the traditional polypeptide.
The applicant develops a plurality of novel polypeptide stabilizing systems in earlier researches, finds that the stability of the polypeptide can be effectively improved through chemical cyclization, the cell penetrating capacity of the polypeptide is enhanced, the activity of the polypeptide is improved, meanwhile, different side chain chemical modifications can also obviously influence the conformation, the amphipathy and other biophysical properties of the polypeptide, but the monocyclic polypeptide lacks tumor targeting, and based on the research results, the applicant further constructs the alpha-helix conformation stable bicyclic polypeptide with the thioether side chain and the double targeting capacity through photoinitiation thiol-alkyne click chemistry.
According to the first aspect of the invention, the apoptosis-promoting bicyclic polypeptide with stable alpha-helix conformation is provided, the apoptosis-promoting bicyclic polypeptide has a stable alpha-helix secondary structure and comprises an apoptosis-promoting functional peptide segment, a tumor-targeting functional peptide segment, a corner amino acid residue and a thioether-containing molecular skeleton, wherein the apoptosis-promoting functional peptide segment and the tumor-targeting functional peptide segment are connected through the corner amino acid residue and form the bicyclic structure of the thioether-containing molecular skeleton by utilizing connecting molecules.
Preferably, the linker molecule is any one of mercaptopropionic acid, thioglycolic acid or mercaptobutyric acid.
Preferably, the turn amino acid residue is any one of proline or glycine, proline and glycine being the majority of protein turn amino acids, and this turn function may provide flexibility between the pro-apoptotic and targeting peptide segments.
Preferably, the tumor targeting functional peptide segment is targeting alphavβ3Any one of an RGD sequence of integrin, an octreotide fCYwKTCT sequence of a targeted somatostatin receptor, a D-2-Nal-CYwKVCT sequence of a lanreotide sequence and a polypeptide sequence YCDGFYACYMDV sequence of a targeted Her2 receptor.
Preferably, the thioether-containing molecular skeleton is located at the ith amino acid position in the sequence of the apoptosis-promoting functional peptide segment, and the cyclization position of the bicyclic structure is initiated by the amino acid residue of the thioether-containing side chain, cyclized with the side chain of the amino acid residue at the i +4 position, the i +7 position or the i +11 position in the direction of the C-terminal end of the polypeptide, and cyclized with the amino acid residue at the N-terminal end of the polypeptide to form the bicyclic molecular skeleton.
Cyclization using positions i and i +4, i and i +7, i and i +11 can significantly improve cyclization efficiency because the amino acid side chains at positions i and i +4, i +7, i +11 are on the same side of the helix in the polypeptide sequence of the alpha-helix structure.
Preferably, the structure of the pro-apoptotic bicyclic polypeptide is represented by formula 1:
or, the structure of the pro-apoptotic bicyclic polypeptide is shown as formula 2:
according to another aspect of the present invention, there is provided a method for preparing a pro-apoptotic bicyclic polypeptide with stable α -helix conformation, comprising the steps of:
s1, synthesizing a linear polypeptide sequence Fmoc-WRGDfVPKLAKLKKLAKLK (alloc) K-Resin by adopting an Fmoc solid-phase polypeptide synthesis technology, wherein X is an unnatural amino acid with an alkynyl side chain;
s2, removing an Fmoc group from tryptophan at the tail end of a linear peptide sequence Fmoc-WRGDfVPKLAKLXKKLAKLK (alloc) K-Resin to obtain linear polypeptide;
s3, forming a precursor of a molecular skeleton containing a thioether side chain by utilizing a photo-initiated thiol-alkyne click chemical reaction: adding 2 equivalents of photoinitiator 2, 2-dimethylolpropionic acid DMPA and 4 equivalents of mercaptopropionic acid into 1 equivalent of linear polypeptide product obtained in the step S2, and carrying out irradiation reaction for 2 hours under a 365nm ultraviolet lamp;
s4, carrying out macrocyclic lactamization by using the reaction product obtained in the step S3 to construct a bicyclic framework containing a thioether structure;
and S5, cutting the cyclized polypeptide from the resin, purifying by using a high performance liquid chromatography, and freeze-drying to obtain the target bicyclic polypeptide.
Preferably, in step S5, 2.5% H is used2The polypeptide was cleaved from the resin by a cleavage solution consisting of O, 2.5% TIS and 95% TFA.
According to another aspect of the present invention, there is provided the use of a conformationally stabilized pro-apoptotic bicyclic polypeptide as described above in the treatment of tumors.
Generally, compared with the prior art, the technical scheme of the invention mainly has the following beneficial effects:
(1) creatively constructs a bicyclic polypeptide skeleton with dual effects of therapeutic property and targeting property and containing a thioether structure, and the bicyclic polypeptide has stable alpha-helix secondary conformation; the double targeting function is realized in the same polypeptide framework by combining a pro-apoptosis peptide segment which can change the membrane permeability of mitochondria to break mitochondria so as to promote the apoptosis and an RGD peptide segment which has alpha v beta 3 integrin overexpressed by targeting tumor cells; meanwhile, the thioether-containing bicyclic molecular skeleton formed by utilizing mercaptoalkyne click chemistry has low non-specific toxicity and can further increase the stability and cell penetration capacity of the polypeptide.
(2) The bicyclic polypeptide containing thioether bond molecular skeleton is creatively synthesized by using a light reaction, and cyclization efficiency can be obviously improved by carrying out double cyclization at the i and i +4 or i and i +7 or i and i +11 amino acid residue positions of the apoptosis-promoting peptide segment, wherein the cyclization efficiency can reach 80%.
(3) Compared with linear polypeptide, the constructed bicyclic polypeptide has higher alpha-helix degree, good alpha-helix stability, lower hemolysis rate, good blood stability and low non-specific cytotoxicity, and has more obvious mitochondrial killing capability on integrin high-expression tumor cells, smaller killing capability on normal cells and good tumor treatment application prospect.
Drawings
Figure 1 is a synthetic scheme of a pro-apoptotic bicyclic polypeptide with alpha-helical conformational stability.
FIG. 2 is a liquid phase diagram of a purified linear polypeptide.
FIG. 3 is a liquid phase diagram of the purified bicyclic polypeptide, Cyclic.
FIG. 4 is a liquid phase diagram of Cyclic-1 after separation from the two isomers.
FIG. 5 is a liquid phase diagram of Cyclic-2 after separation from the two isomers.
FIG. 6 is a mass spectrum of a purified linear polypeptide.
FIG. 7 shows the mass spectrum of Cyclic-1 after separation from the two isomers.
FIG. 8 shows the mass spectrum of Cyclic-2 after separation from the two isomers.
FIG. 9 is a circular dichroism chart of linear polypeptide and bicyclic polypeptides, Cyclic-1 and Cyclic-2.
FIG. 10 is a graph showing a statistical hemolysis ratio of linear polypeptide and bicyclic polypeptide Cyclic at different concentrations.
FIG. 11 is a graph of cytotoxicity statistics for linear and bicyclic polypeptides with different cell cultures.
Figure 12 is a fluorescence micrograph of linear and bicyclic polypeptides incubated with 4T 1.
FIG. 13 is a fluorescence micrograph of linear and bicyclic polypeptides incubated with MCF-7.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.
Example 1
This example provides an alpha-helix conformationally stabilized pro-apoptotic bicyclic polypeptide comprising a pro-apoptotic peptide functional peptide fragment, targeting alphavβ3The sequence of the apoptosis-promoting peptide functional peptide segment is KLAKLXXKLLAKLKK, the sequence is a linear sequence rich in lysine, X is an unnatural amino acid with an alkynyl side chain, the apoptosis-promoting peptide functional peptide segment is connected with RGD through proline to reduce mutual interference between the apoptosis-promoting peptide and RGD, meanwhile, the turning cyclization is facilitated, the flexibility of a connection part is improved, and meanwhile, a connecting molecule mercaptopropionic acid is utilized to construct a double-ring structure of the thioether-containing molecular skeleton on the linear polypeptide through a mercapto-alkyne click chemical reaction.
The amino acids in the sequence of the apoptosis-promoting peptide functional peptide segment are all L-type amino acids, have alpha-helix conformation and can exist more stably in vivo.
The unnatural amino acid X with alkynyl side chain is used for replacing the position of the sixth amino acid alanine A on the apoptosis-promoting peptide KLAKLAKKLAKLAK, because in the sequence of the apoptosis-promoting peptide KLA, the sequence mainly comprises three amino acids of lysine K, leucine L and alanine A, wherein lysine is a positively charged amino acid, leucine is taken as a hydrophobic amino acid and has a significant influence on the amphiphilicity of the polypeptide sequence, and the amphiphilic structure has a significant influence on the mitochondrion targeting capability of the polypeptide, so that the unnatural amino acid X with alkynyl side chain is used for replacing alanine as a ring forming site, and correspondingly the position of the 13 th amino acid alanine A in the apoptosis-promoting peptide KLAKLAKKLAKLAK is replaced by lysine.
The thioether-containing molecular skeleton is located at the ith amino acid position in the peptide segment sequence with the apoptosis promoting function, and the cyclization position of the double-ring structure is initiated by taking the amino acid residue of the thioether-containing side chain as the initial, cyclizes with the i +7 th amino acid residue side chain in the direction of the C end of the polypeptide and cyclizes with the amino acid residue at the N end of the polypeptide to form the double-ring molecular skeleton.
The structure of the apoptosis-promoting bicyclic polypeptide is shown as formula 1:
or, the structure of the pro-apoptotic bicyclic polypeptide is shown as formula 2:
in another alternative embodiment, the linker molecule used for the ring formation may also be any of thioglycolic acid or thioglycolic acid.
In an alternative embodiment, the corner amino acid may also be glycine.
In another alternative embodiment, the tumor targeting peptide segment may also be any one of the polypeptide sequences ycdgfyacyldv targeting the somatostatin receptor octreotide fCYwKTCT, the lanreotide sequence D-2-Nal-CYwKVCT, and the Her2 receptor.
In another alternative embodiment, the cyclization position of the bicyclic structure is initiated by the amino acid residue of the thioether-containing side chain, cyclizes with the side chain of the i +4 position or i +11 position amino acid residue in the C-terminal direction of the polypeptide and cyclizes with the N-terminal amino acid residue of the polypeptide to form a bicyclic molecular skeleton.
Example 2
The present embodiment provides a method for preparing a pro-apoptotic bicyclic polypeptide with stable α -helix conformation, as shown in fig. 1 of the specification, the specific preparation process is as follows:
s1, synthesizing a linear peptide sequence Fmoc-WRGDfVPKLLXXKKLAKLK (Alloc) K-Resin containing an allyloxycarbonyl Alloc side chain protecting group by adopting an Fmoc solid-phase synthesis method, wherein the Alloc side chain protecting group is positioned on lysine on the right side of the sequence, X is an unnatural amino acid with alkynyl, the terminal of RGD is connected with tryptophan, the tryptophan has an absorption peak at 280nm, and the concentration of the polypeptide can be determined by a microplate reader, and the linear peptide sequence Fmoc-WRGDfVPKLLXXKKLAKLK (Alloc) K-Resin has the following specific structure:
the specific synthetic process is as follows:
400mg Rink resin was weighed, swollen with N, N-dimethylformamide DMF for 20min, deprotected with a deprotection solution (50% morpholine, 50% DMF, (V: V)) to remove the Fmoc protecting group from the resin, washed three times with DMF, then washed three times with dichloromethane DCM, washed three times with DMF and then sequentially added DMF-solubilized amino acid starting material and HATU mixture, each amino acid was linked for two hours and each amino acid was reacted twice in the same procedure to increase the conversion efficiency.
The Fmoc solid-phase synthesis method is adopted to synthesize the polypeptide, Fmoc is used as a protecting group of alpha-amino acid, is stable under acidic conditions and is not influenced by reagents such as TFA and the like, and deprotection can be realized by applying mild alkali treatment, so that a side chain can be protected by a Boc protecting group which is easy to remove by acid.
S2, removing an Fmoc group from tryptophan at the tail end of a linear peptide sequence Fmoc-WRGDfVPKLAKLXXKLLAKLK (alloc) K-Resin;
the specific process is as follows:
adding 0.1 equivalent of tetrakis (triphenylphosphine) palladium and 4 equivalents of 1, 3-dimethyl barbituric acid into 1 equivalent of a linear peptide sequence containing an allyloxycarbonyl Alloc side chain protecting group, and reacting twice for two hours each time by using dichloromethane as a solvent. After the reaction, excess tetrakis (triphenylphosphine) palladium (Pd (PPh3)4) attached to the resin was washed away using sodium diethyldithiocarbamate (copper reagent) dissolved in DMF to obtain NH2-WRGDfVPKLAKLXKKLAKLKK-Resin；
S3, utilizing light to initiate a thiol-alkynyl click chemical reaction to form a thioether-containing side chain molecular skeleton precursor;
the specific process is as follows:
at 1 equivalent of resin NH with polypeptide2-WRGDfVPKLAKLKKLAKLKK-Resin is dissolved in N-methylpyrrolidone NMP, 2 equivalents of photoinitiator 2, 2-dimethylolpropionic acid DMPA and 4 equivalents of mercaptopropionic acid are added, and the mercaptopropionic acid and alkyne are subjected to addition reaction by irradiating under 365nm ultraviolet light for 2 hours.
S4, carrying out macrocyclic lactamization by using the reaction product of the previous step to construct a bicyclic framework containing a thioether structure;
the specific process is as follows:
adding 2 equivalents of benzotriazole-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate PyBop, 2 equivalents of 1-hydroxybenzotriazole HOBT and 2 equivalents of N-methylmorpholine NMM into 1 equivalent of the reactant in the previous step, and placing the mixture in a shaking table for reaction for 12 hours.
S5, utilizing 2.5% H to cyclize the polypeptide2And cutting off a shearing solution prepared by mixing O, 2.5% TIS and 95% TFA, purifying by using High Performance Liquid Chromatography (HPLC), and performing freeze-drying to obtain the target bicyclic polypeptide.
Wherein, the preparation parameters of the high performance liquid chromatography are as follows:
the undegraded polypeptides were analyzed by reverse phase HPLC (Agilent ZORBAX SB-Aq: 4.6X 250mm, 5 μm, flow rate 1.0mL/min, 220 nm). The analytical conditions were a gradient elution of solvent B from 10% to 90% in 40 minutes (solvent A: water with 1% o TFA (v/v), solvent B: acetonitrile).
The liquid phase chromatogram combining the linear polypeptide and the cyclic peptide is shown in the attached figures 2-3 in the specification, and the retention time of the purified linear polypeptide is 15.067min, while the retention time of the double-cyclized polypeptide and the linear polypeptide is increased, so that the hydrophobicity of the double-cyclized polypeptide is improved, and the retention time of the double-cyclized polypeptide is concentrated in two similar double peaks which are 18.373min and 18.767min respectively according to the attached figure 3, so that the two isomers can be obtained by separation and purification.
Further separating and purifying by high performance liquid chromatography, and obtaining two bicyclic isomers, namely Cyclic-1 and Cyclic-2 according to the order of peak-appearing time, wherein the obtained liquid chromatogram is shown as 4-5 and respectively corresponds to the following structural formula:
in order to further verify that the expected product is obtained by synthesis, mass spectrum detection is respectively carried out on two different bicyclic isomers, a mass spectrum manufacturer adopts a Waters model Q-TOF, a mobile phase adopts methanol and water, and the analysis condition is that the methanol is 10-90% within 5 minutes;
FIGS. 6-8 are mass spectra of purified linear and Cyclic peptides, Cyclic-1 and Cyclic-2, respectively, wherein [ M +5 ]]+Represents a molecular weight of five positive charges, [ M +4 ]]+Represents a molecular weight of four positive charges, [ M +3 ]]+Represents the molecular weight with three positive charges, and the analysis of mass spectrum shows that the bicyclic polypeptide with correct molecular weight is successfully obtained.
Cyclization can effectively restrict the conformation of the polypeptide, the conformation of the polypeptide is considered to have important influence on the penetration capacity of the polypeptide, the polypeptide can form a plurality of secondary structures such as alpha-helix, beta-fold and the like, the secondary structures of linear polypeptide and bicyclic polypeptide are inspected by circular dichroism, and the linear polypeptide and the bicyclic polypeptide are respectively dissolved in 30mM Sodium Dodecyl Sulfate (SDS). Circular dichroism data were collected by Jasco J-810 at room temperature under the following detection conditions: step resolution was 0.5nm, velocity was 20nm/s, 10 accumulations, response time 1s, bandwidth 1nm, path length 10mm, and all circular dichroism spectra were converted to average residue molar ovality.
The obtained circular dichroism chromatogram is shown in figure 9, and it can be seen that a positive peak appears at about 190 of the bicyclic polypeptide, and two negative peaks appear at 208nm and 222nm, which are characteristic peaks of a very obvious alpha-helix structure, and compared with the peak width of the linear polypeptide at 222nm, the peak width is obviously enhanced and broadened, which shows that the helix degree of the constructed bicyclic polypeptide is improved relative to the linear polypeptide, and further proves that the bicyclic polypeptide is successfully obtained, because the alpha-helix content of the cyclized polypeptide is obviously improved relative to the linear polypeptide, and two alpha-helix characteristic negative peaks appear at 208nm and 222 nm.
The hemolysis of the fresh rabbit blood was investigated by incubating the linear polypeptide and the bicyclic polypeptide with different concentrations with the fresh rabbit blood for 1h, and as shown in fig. 10, it can be found that both the bicyclic polypeptide and the linear polypeptide have low hemolysis and good biosafety.
To investigate the tumor cell selective inhibitory effect of the bicyclic polypeptides, 0. mu.M, 5. mu.M, 10. mu.M, 20. mu.M, 40. mu.M of the linear polypeptide and the bicyclic polypeptide and alpha. were used, respectivelyvβ3Integrin high expression cell mouse melanoma cell B16, mouse breast cancer cell 4T1 and alphavβ3Integrin low expression cell human breast cancer cell MCF-7, alphavβ3And incubating the normal liver cells L02 with low integrin expression for 24h, dyeing by using AB dye, and detecting the OD value by using a microplate reader so as to detect the cell survival rate. As shown in fig. 11, it can be seen that both the bicyclic peptide and the linear peptide exhibit lower non-specific cytotoxicity for normal cell lines, while the bicyclic peptide has better effect on cell killing than the linear peptide and has better effect on cells with high integrin expression for tumor cell lines, thereby verifying the selective pro-apoptotic effect of the bicyclic polypeptide on tumor cells.
To verify the effect of the bicyclic polypeptides on targeting mitochondria, mitochondrial membrane potential of cells was characterized by mitochondrial staining with JC-1 dye. 4T1 cells and MCF-7 cells were plated at 1.2X 105Density of seeds/well in the confocal dishAdding 10% fetal calf serum culture medium at 37 deg.C and 5% CO2Culturing for 24h under constant temperature and atmosphere conditions. Polypeptide with a final concentration of 15 μ M is then added and incubated 24, then the newly configured JC-1 stain is added for staining for 10min, followed by 3 washes with PBS buffer. The sample is placed under an inverted fluorescence microscope to be observed and pictures are taken as shown in figures 12-13, and linear polypeptide is selected as a contrast, so that the bicyclic polypeptide has obvious capacity of influencing mitochondrial membrane potential compared with the linear polypeptide, and has more obvious effect compared with an integrin high-expression cell line, so that the bicyclic polypeptide can be proved to be capable of targeting an integrin receptor on the cell surface and mitochondria in the cell, selectively disturbing the integrin high-expression tumor cell mitochondrial membrane potential, and has good application prospect in tumor treatment.
It will be understood by those skilled in the art that the foregoing is only a preferred embodiment of the present invention, and is not intended to limit the invention, and that any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the scope of the present invention.
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