CN112239487A - Stable polypeptide protein covalent inhibitor of targeted anti-apoptosis protein BFL-1 - Google Patents

Abstract

The invention provides a stable polypeptide protein covalent inhibitor of a target anti-apoptosis protein BFL-1, which has an amino acid sequence as follows: ac-cyclic (MIAQC) LR (Aib) IGD (Aib) FNAYYARR. The invention also provides application of the stable polypeptide protein covalent inhibitor in preparation of a medicine for targeting the anti-apoptosis protein BFL-1. Also provides application of the stable polypeptide protein covalent inhibitor in preparing a medicament for treating BFL-1 high-expression melanoma or pancreatic cancer. The invention adopts a method that methionine-cysteine on polypeptide reacts with double alkylating agent to form single sulfonium salt to stabilize BFL-1 targeted sulfonium salt cyclic peptide. Cell proliferation experiments and apoptosis experiments also prove that the polypeptide kills tumor cells with high BFL-1 expression, such as melanoma cell A375 and pancreatic cancer cell Miapaca-2, through an apoptosis way.

Description

Stable polypeptide protein covalent inhibitor of targeted anti-apoptosis protein BFL-1

Technical Field

The invention belongs to the field of bioengineering, relates to a polypeptide, and particularly relates to a stable polypeptide protein covalent inhibitor of a targeted anti-apoptosis protein BFL-1.

Background

China is a big cancer country in the world, 380 million new cases of malignant cancer in China in 2017 and 26 million cancer deaths. The data of the national Bureau of statistics show that malignant tumors have become one of the main causes of death in the Chinese population. Currently, lymphoma is one of the most rapidly growing malignancies worldwide with an average increase in annual incidence of 4%. In recent years, the incidence rate of malignant lymphoma in China is increasing rapidly, and a list of ten major malignant tumors in China is ascending. The drug resistance and the recurrence of the malignant lymphoma bring huge economic and psychological burdens to the society and families, and also bring huge challenges to the cure of the tumor. Apoptosis, also known as programmed cell death, is an extremely important biological process that plays a critical role in cell fate and cell homeostasis. The treatment of many tumors, such as chemotherapy, radiotherapy and even biological therapy, is primarily to induce apoptosis and thus achieve the goal of treating tumors. Research shows that the anti-apoptosis protein BFL-1 is a driver of oncogenes such as lymph cancer, leukemia and malignant melanoma, and is closely related to drug resistance of the cancers, for example, a lymph cancer cell with high BFL-1 expression generates drug resistance to inhibitors of BCL2 and BCL-XL, while in melanoma, the high expression of BFL-1 causes the drug resistance and metastasis of a tumor cell. Therefore, the development of selective inhibitors targeting BFL-1 brings new hope for solving the drug resistance of tumors such as malignant lymphoma and the like. The BFL-1 protein belongs to a member of the BCL-2 family, and due to the high homology of the BCL2 family, the currently developed BCL2 inhibitors are broad-spectrum inhibitors and lack selectivity for BFL-1. The mutual binding site of BFL-1 and the BH3 structural domain of ligand protein has cysteine, while other homologous proteins such as BCL2 or BCL-XL and other proteins do not have the structure, thereby providing a structural basis for designing a selective BFL-1 covalent inhibitor.

Therapeutic drugs of long interest have mainly been focused on two categories: small molecule drugs (small molecules), protein drugs (biologics). The targeted chemical space of small molecule drugs has certain limitations, protein drugs have poor stability and cannot penetrate cell membranes, and the two therapeutic drugs cannot effectively cover all confirmed important molecular targets due to the limitations of the biophysical properties of the two therapeutic drugs. Polypeptide drugs are another class of targeting molecules that have attracted much attention and interest. Similar to biological macromolecules, the polypeptide molecules also have higher binding force and selectivity for targets, and have smaller off-target effect compared with small molecule drugs. And the metabolite of the polypeptide in vivo is amino acid, thus reducing the toxicity to the utmost extent. The traditional polypeptide medicine cannot effectively form a complex secondary structure due to limited number of amino acid residues, has high degree of freedom and a random linear state in a physiological solution, reduces the specificity and is easily degraded by protease. And the cell membrane penetration ability of polypeptide drugs is not very good. The polypeptide is modified by chemical means to be stabilized into conformation with secondary structure, so that the stability of the polypeptide to protease can be increased, the cell membrane penetration capacity of the polypeptide can be enhanced, and the entropy change during the combination of the polypeptide and a target point can be reduced, thereby improving the combination capacity of the polypeptide and the target point. Through various chemical modification means, secondary structure units participating in various protein-protein interactions are extracted and modified, the interaction of original proteins is simulated by stabilizing the secondary conformation of the secondary structure units, and more importantly, the secondary structure units of the proteins can have the capacity of penetrating cell membranes through modification, so that the protein-protein interactions in cells are targeted.

Similar to other anti-apoptotic proteins of the BCL2 family, BFL-1 has a conserved hydrophobic 'notch' pocket combined with a pro-apoptotic protein BH3 structural domain, and the structural basis provides basis for developing targeted BCL2 anti-apoptotic proteins. The BH3 structural domain is a hydrophobic alpha-helical polypeptide, and the early developed polypeptide inhibitor mainly simulates pro-apoptosisThe BH3 structure of the protein is designed, for example, the full-carbon side chain stable polypeptide inhibitor SAHA 'SAHB' designed by the early United states Harvard university Walensky professor based on apoptosis protein BIM and BH3 spiral polypeptide of BID can effectively activate apoptosis channels in leukemia cells and inhibit the proliferation of the leukemia cells in vitro and in vivo. Structural biology studies have found that when BFL1 binds to pro-apoptotic protein NOXA, the cysteine at position 25, C25, of NOXA is spaced approximately the same distance from the cysteine at position 55 of BFL-1

Which facilitates the formation of disulfide bonds. Since other anti-apoptotic proteins do not have a cysteine at the position where BH3 binds, the amino acid at position C55 of BFL-1 provides a way to target the design of selective covalent inhibitors of BFL 1.

In 2016, the Walensky teaching of Harvard university in USA and the Fairlie teaching of Kunstland university in Australia successively report a selective covalent inhibitor targeting BFL-1, the polypeptide inhibitor stably improves the stability and the membrane penetrating capability of a polypeptide by a full-carbon side chain method, simultaneously, C25 of the polypeptide introduces an acrylamide group, when the polypeptide inhibitor interacts with BFL-1, the acrylamide group on a polypeptide side chain is in close space and covalently reacts with cysteine of BFL-1 to form a protein-polypeptide covalent conjugate, and the selective inhibition effect of the polypeptide inhibitor on BFL-1 is obviously improved. The polypeptide ligand stabilized based on the sulfonium salt can be used for efficiently and selectively carrying out covalent modification on protein cysteine, and methionine and cysteine on the polypeptide are subjected to double alkylation reaction with a double-halogen reagent to construct a stabilized polypeptide methodology with a single sulfonium salt on a side chain.

Disclosure of Invention

Aiming at the technical problems in the prior art, the invention provides a stable polypeptide protein covalent inhibitor of a targeted anti-apoptosis protein BFL-1, and the stable polypeptide protein covalent inhibitor of the targeted anti-apoptosis protein BFL-1 aims to solve the technical problem that the effect of a medicament in the prior art on treating melanoma or pancreatic cancer is poor.

The invention provides a stable polypeptide protein covalent inhibitor of a target anti-apoptosis protein BFL-1, the structural formula of which is shown as follows,

further, the amino acid sequence is: ac-cyclic (MIAQC) LR (Aib) IGD (Aib) FNAYYARR (shown in SEQ ID NO. 1).

The invention also provides application of the stable polypeptide protein covalent inhibitor in preparation of a medicine for targeting the anti-apoptosis protein BFL-1.

The invention also provides application of the stable polypeptide protein covalent inhibitor in preparing a medicament for treating BFL-1 high-expression melanoma or pancreatic cancer.

The invention adopts a method that methionine-cysteine on polypeptide reacts with double alkylating agent to form single sulfonium salt to stabilize BFL-1 targeted sulfonium salt cyclic peptide. By adopting the method for selective covalent modification of protein cysteine based on sulfonium salt stabilized polypeptide ligand reported in the literature, when the cyclic peptide ligand and target protein BFL-1 are mutually identified, sulfonium salt on cyclic peptide and protein BFL-1 cysteine are subjected to nucleophilic reaction under the condition of close space to realize covalent modification of protein. SDS-PAGE analysis and mass spectrometry analysis prove that the polypeptide has covalent reaction with C55 of the target protein BFL-1 after mutual recognition with the target protein. The covalent reaction of the polypeptide and the high-expression BFL-1 protein in cells is proved by immunoblotting and co-immunoprecipitation analysis. Cell proliferation experiments and apoptosis experiments also prove that the polypeptide kills tumor cells with high BFL-1 expression, such as melanoma cell A375 and pancreatic cancer cell Miapaca-2, through an apoptosis way.

Experiments such as fluorescence polarization detection, immunoblot analysis, flow cytometry analysis and cell survival prove that the polypeptide can be well combined with BFL-1 protein, and sulfonium salt on the polypeptide and cysteine on the protein BFL-1 are subjected to covalent reaction to block the combination of anti-apoptosis protein and apoptosis promoting protein, so that the apoptosis of tumor cells is promoted by the release of the apoptosis promoting protein.

Compared with the prior art, the invention has remarkable technical progress. The methionine and cysteine on the polypeptide react with a double-alkylation reagent to form sulfonium salt cyclic peptide, so that the polypeptide can be stabilized, the covalent modification can be carried out on the cysteine on the interaction site of the target protein, and the sulfonium salt cyclic peptide has an inhibiting effect on a BFL-1 high-expression melanoma cell line A375 and a pancreatic cancer cell line Miapaca-2. The polypeptide molecule of the invention blocks the combination of the anti-apoptosis protein BFL-1 and the apoptosis-promoting protein through the covalent reaction of sulfonium salt and BFL-1. The invention is beneficial to solving the problem of drug resistance of high-expression BFL-1 tumor cells, and simultaneously widens the application range of stable polypeptide.

Drawings

FIG. 1 is a diagram of the synthesis of sulfonium salt covalent inhibitor polypeptide molecules.

FIG. 2 is a graph of the covalent binding of a polypeptide to a BFL-1 protein.

FIG. 3 is a primary mass spectrum of covalent binding of the polypeptide and BFL-1 protein.

FIG. 4 shows the secondary mass spectrum of the covalent binding of the polypeptide and BFL-1 protein.

FIG. 5 is a flow cytometric analysis of the membrane penetration ability of polypeptides.

FIG. 6 shows a human melanoma cell line A375^FAMB4-MC and BFL-1 co-localization map.

FIG. 7 shows a human melanoma cell line A375^FAMB4-Co-localization of MC with mitochondria.

FIG. 8 shows that the polypeptide covalently reacts with BFL-1 in cells.

FIG. 9 shows the ability of different polypeptides to inhibit proliferation of human melanoma cells A375.

FIG. 10 is a graph showing the effect of different concentrations of polypeptide B4-MC on the level of apoptosis in human melanoma cells A375.

FIG. 11 is a schematic representation of the promotion of apoptosis of an anti-tumor covalent polypeptide inhibitor by covalent reaction with BFL-1.

FIG. 12 is a structural formula of a stable polypeptide protein covalent inhibitor targeting anti-apoptotic protein BFL-1 of the present invention.

Detailed Description

The invention is further described below with reference to the accompanying drawings.

Example 1

The invention adopts the sulfonium salt stabilized polypeptide technology reported by the prior literature (D.Wang, M.Yu, et al.chem.Sci.10,4966-4972), and through the reaction of methionine and cysteine on the polypeptide and a double-alkylation reagent, sulfonium salt cyclic peptide is formed, so that the polypeptide can be stabilized, and the sulfonium salt cyclic peptide can be subjected to covalent modification with cysteine on the interaction site of a target protein, the combination of an anti-apoptotic protein and a pro-apoptotic protein is blocked, and the release of the pro-apoptotic protein promotes the apoptosis of tumor cells.

The inventors synthesized a number of different polypeptides and focused studies on seven of them, as shown in table 1. Then, polypeptide molecules with good covalent effect are screened through covalent reaction to BFL-1. FIG. 11 is a schematic representation of the promotion of apoptosis of an anti-tumor covalent polypeptide inhibitor by covalent reaction with BFL-1.

Table one: molecular sequence of stable polypeptide protein covalent inhibitor of different target anti-apoptosis protein BFL-1

Wherein B4, B6 and B7 have the same sequence but different ring closing modes, B4 is 1,3, 5-tribromomethylbenzene, B6 is 2, 6-di (bromomethyl) pyridine, and B7 is E-1, 4-di (bromomethyl) -2-butene.

Example 2 preparation and isolation and purification of the polypeptide:

solid phase synthesis of polypeptides according to the amino acid sequence, the core steps for the preparation of the above-described stabilized polypeptides are as follows (B4-MC for example):

the specific operation steps (figure 1) are as follows:

(1) polypeptide solid phase synthesis: rink amide MBHA resin was weighed into a peptide grafting tube, Dichloromethane (DCM) was added, and nitrogen was bubbled for 30min for swelling. Adding a 50% (v/v) morpholine solution in N, N-Dimethylformamide (DMF), bubbling nitrogen for 30min, and removing the Fmoc protective group. After the resin was washed alternately with DMF and DCM, the prepared Fmoc-AA-OH (5eq,0.4M, DMF) solution, 6-chlorobenzotriazole-1, 1,3, 3-tetramethyluronium Hexafluorophosphate (HCTU) (5eq, 0.38M, DMF) solution, N, N-Diisopropylethylamine (DIPEA) (10eq) were mixed well and added to the resin under nitrogen bubbling for 1 h. The reaction solution was withdrawn, and the resin was washed as described above and then subjected to the next step. The following amino acids were the same as described above. The N-terminus of the polypeptide was acetylated with acetic anhydride and DIPEA (1:1.8 in DCM) for 30min (twice).

(2) Intramolecular cyclization: the Trt group of the side chain protecting group of cysteine (Cys) was deprotected on the resin (deprotection solution: TFA/TIS/DCM ═ 3/5/92) in portions until the solution did not yellow any more, after which the solution was cross-washed five times with DMF, DCM, respectively, and 1, 8-bis (bromomethyl) biphenyl halo reagent (2 eq) and DIPEA (4 eq) were dissolved in DMF and added to the resin for 2 hours.

(3) Polypeptide purification: with trifluoroacetic acid (TFA), Triisopropylsilane (TIPS) and H₂O (v: v: v ═ 9.5:0.25:0.25) shear cuts the polypeptide from the resin, and the shear is removed. Purifying and separating with high performance liquid chromatography, and finally determining molecular weight with Mass Spectrum (MS) to obtain pure polypeptide molecule, with specific structural formula shown in figure 12.

EXAMPLE 3 in vitro covalent attachment of polypeptide molecules to protein BFL-1

Methionine and cysteine on different polypeptides react with a double-alkylation reagent to form sulfonium salt cyclic peptide which is respectively incubated with protein, and the protein and the sulfonium salt polypeptide undergo covalent reaction, so that an obvious upward moving strip can be seen. The covalent reaction of proteins with polypeptides exhibits a dose-dependent profile. The polypeptide has better specificity, and mainly performs covalent reaction with cysteine at C55 position of BFL-1, and mutates cysteine at 55 position of BFL-1 into serine, and basically does not perform covalent reaction with BFL-1. In addition, some cysteine-containing proteins were selected to which the sulfonium salt polypeptides did not covalently react (FIG. 2).

The polypeptide B4-MC and the protein BFL-1 are selected for co-incubation, and the first-order mass spectrum verifies that the polypeptide and the BFL-1 have covalent reaction (figure 3). The polypeptide protein co-incubated sample was then subjected to enzymatic hydrolysis, and secondary mass spectrometry identified that the polypeptide was mainly chemically modified at the cysteine 55 position of BFL-1 (fig. 4).

Example 4 characterization of the Capacity of the polypeptide molecules to penetrate membranes

A375 cells were cultured in a 24-well plate, and a polypeptide compound containing a fluorescent label was added thereto, and after 4 hours, the membrane-penetrating ability of the polypeptide was analyzed by a flow cytometer. TAT is a membrane penetrating peptide, as a positive control, B5-MC is a linear polypeptide and has slightly stronger membrane penetrating capability than TAT, and B4-MC is a sulfonium salt cyclic peptide and has obviously stronger membrane penetrating capability than other polypeptides (figure 5).

The membrane penetration ability of the polypeptide molecules and the positioning of the target protein are researched by adopting a confocal microscope. After green fluorescence labeling polypeptide compounds are respectively added into cells for 4 hours, BFL-1 protein in the cells is labeled through immunofluorescence, and then cell microscopic imaging is carried out. Intracellular BFL-1 was labeled red with an antibody and the fluorescently labeled polypeptide compound emitted green, and the image after stacking the two channels appeared yellow if they were co-localized, thus demonstrating that the polypeptide inhibitor was indeed able to target intracellular BFL-1 protein (fig. 6).

Polypeptide molecule positioning is researched by adopting a confocal microscope, a fluorescence labeling polypeptide compound is added for 4 hours, mitochondria are labeled by a mitochondrial probe, and then cell microscopic imaging is carried out. Mitochondria in cells are labeled with red light by the antibody, the fluorescent-labeled polypeptide compound emits green light, and if the two are co-localized, the picture after the superposition of the two channels appears yellow, thereby indicating that the polypeptide inhibitor is mainly localized to mitochondria (fig. 7).

Example 5 polypeptide B4 covalently reacts with target protein BFL-1 in cells

In order to evaluate the in vivo covalent ability of the polypeptide with BFL-1, the most intuitive Western Blot is adopted to verify the covalent ability of the polypeptide to BFL-1 protein in BFL-1 overexpression cells. Firstly, over-expression of BFL-1 protein by 293T cells, collection of cell lysate supernatant, reaction with polypeptide, and verification of chemical covalent modification of polypeptide B4-MC and BFL-1 protein by Western Blot. Furthermore, after overexpression of the BFL-1 protein by 293T cells, the culture medium containing the B4-MC polypeptide was added and the culture was continued for 24 hours, and it was confirmed by Western Blot that the polypeptide B4-MC was chemically covalently modified with the BFL-1 protein (FIG. 8).

Example 6 polypeptide B4-MC specifically killed BFL-1 high expressing melanoma A375 cells and pancreatic cancer Miapaca-2 cells.

To evaluate the killing ability of the polypeptide on different cancer cells, BFL-1 high expression cell lines A375 and Miapaca-2 were selected as representatives. And simultaneously, BFL-1 is selected not to express a cell line Hela cell, an A549 cell and a normal cell HEK293T to eliminate the non-specific toxicity of the polypeptide B4-MC.

Cell viability was determined by MTT assay (3- (4, 5-dimethylthiozol-2-yl) -2,5-diphenylt-etrazolium bromide)). Cells were plated at 4X 10 in 96-well plates³Inoculation, treatment with polypeptide in medium (5% serum) for 24h, and addition of MTT to the medium for 4h incubation. DMSO was then added to dissolve the precipitate and absorbance was measured at 490nm using a microplate reader. Wherein the untreated cell viability was 100%.

The results show that the polypeptide B4-MC only has obvious cell proliferation inhibition and concentration dependence on A375 and Miapaca-2 cells. Has no toxic and side effects on Hela, A549 and HEK293T cells basically. These results indicate the specificity of the polypeptide. (FIG. 9)

Example 7 Effect of the polypeptide B4-MC on the apoptotic Effect

Next, whether polypeptide B4-MC killed cancer cells by the apoptotic pathway was investigated by apoptosis experiments. Collecting the cells treated by the polypeptide by pancreatin, detecting by using an FITC Annexin V apoptosis kit, and analyzing by using a flow cytometer. The results indicate that B4-MC killed a375 via the apoptotic pathway (fig. 10).

Sequence listing

<110> Shenzhen institute of university of Beijing

Shenzhen bay laboratory lawn mountain biomedical research and development transformation center

<120> stable polypeptide protein covalent inhibitor targeting anti-apoptosis protein BFL-1

<130> JSP12006102

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 18

<212> PRT

<213> 2 Ambystoma laterale x Ambystoma jeffersonianum

<400> 1

Met Ile Ala Gln Cys Leu Arg Ile Gly Asp Phe Asn Ala Tyr Tyr Ala

1 5 10 15

Arg Arg

Claims (4)

1. A stable polypeptide protein covalent inhibitor of a target anti-apoptosis protein BFL-1 is characterized in that the structural formula is shown as follows,

2. the stable polypeptide protein covalent inhibitor targeting anti-apoptotic protein BFL-1, according to claim 1, wherein its amino acid sequence is as shown in SED ID No. 1.

3. Use of a covalent inhibitor of a stable polypeptide protein as defined in claim 1, for the manufacture of a medicament for targeting the anti-apoptotic protein BFL-1.

4. Use of a covalent inhibitor of a stable polypeptide protein of claim 1 in the manufacture of a medicament for the treatment of BFL-1 high expressing melanoma or pancreatic cancer.

Images