CN114146186B - Polypeptide drug conjugate based on sulfonium salt stabilized target HDAC and application thereof - Google Patents

Abstract

The invention relates to a sulfonium salt-stabilized target HDAC polypeptide drug conjugate and application thereof, wherein a sulfonium salt-stabilized pro-apoptosis polypeptide is utilized to be coupled with an HDAC inhibitor to form a stable polypeptide HDAC inhibitor with tumor selective toxicity, the apoptosis polypeptide chemically stabilized by the sulfonium salt has remarkably reduced nonspecific toxicity and stronger protease stability, and when the HDAC inhibitor is coupled to form the polypeptide drug conjugate, the polypeptide drug can remarkably improve the selective toxicity to tumor cells and has weaker toxicity to normal cells. The cell proliferation, apoptosis and cell cycle blocking experiments prove that the polypeptide can effectively inhibit the proliferation of malignant liver cancer, has relatively better biological safety and has potential anti-tumor clinical application prospect.

Description

Polypeptide drug conjugate based on sulfonium salt stabilized target HDAC and application thereof

Technical Field

The invention belongs to the field of bioengineering, and relates to a polypeptide drug conjugate, in particular to a polypeptide drug conjugate based on sulfonium salt stable targeting HDAC and application thereof.

Background

Cancer is a major public health problem worldwide. World health organization publishes global cancer data in 2020 that 457 thousands of new cancer cases exist in China and 300 or more thousands of death cases exist in 2020, and the number of new cancer cases in China is the first place worldwide. Lung cancer, colorectal cancer, liver cancer, gastric cancer, breast cancer and the like are the top ten cancers among the deaths. The types of tumors and the causative agents of the tumors vary in different sexes and ages, and the complexity of the tumor disease presents challenges for humans to combat the disease. Traditional methods of tumor treatment include surgery, radiation therapy and chemotherapy, but recurrence, metastasis and resistance of tumors present significant challenges for tumor treatment. Along with the rapid development of biotechnology, the understanding of human beings on the pathogenesis of tumors is more and more in depth, so that the developed anti-tumor therapeutic drugs are layered endlessly, and the research results bring great confidence to the human beings for thoroughly curing tumors. Comprehensive analysis of the human cancer genome has shown over the last decade that mutations in epigenetic regulatory proteins involved in gene expression, DNA repair and DNA replication have occurred in many cancers. Further research shows that epigenetic abnormality is involved in tumor metastasis, drug resistance and other mechanisms, so that the combination of epigenetic therapy becomes one of the important means for anti-tumor treatment. Histone Deacetylase (HDAC) plays an important role in the development and resistance of many malignant tumors (such as lymphoma, breast cancer, liver cancer, colon cancer, etc.) as an epigenetic important regulatory factor. Therefore, drug development targeting HDAC has been favored by pharmaceutical chemists. Before the day of expiration, there are 5 HDAC drugs on the global market, vorinostat, romidepsin, belinostat, panobinostat and cibutamine, respectively, mainly for peripheral T cell lymphoma and peripheral T cell lymphoma, with cibutamine being the only HDAC inhibitor approved for the treatment of solid tumor breast cancer. Currently, there are still many HDAC drugs in preclinical or clinical studies for the treatment of solid tumors or hematological cancers. Therefore, drugs targeting HDAC have a better prospect for treating tumors.

Due to the heterogeneity and complexity of cancers, treatment of cancer employs combination therapy regimens, such as targeting agents in combination with chemotherapeutic agents, dual-target inhibitors, multi-functional inhibitors, and the like. The apoptosis polypeptide can inhibit proliferation of tumor by inducing apoptosis of cells, and has broad-spectrum antitumor effect. Meanwhile, the apoptosis polypeptide has different anti-tumor mechanism with the first-line chemotherapeutic drugs, and has potential application prospect for treating chemotherapy-resistant tumor cells. Similar to biological macromolecules, polypeptide molecules have higher binding force and selectivity to targets, and have smaller off-target effect compared with small molecular drugs. The metabolic products of the polypeptide in the body are amino acids, so that the toxicity is reduced to the greatest extent. However, linear polypeptides often have difficulty maintaining the original secondary structure, and thus have poor target affinity, serum stability, and cell-penetrating ability. Chemically stable polypeptides can effectively overcome these weaknesses, and therefore, in recent decades, chemically stable polypeptides have been used for the development of various protein target inhibitors, and exhibit good tumor-inhibiting effects at cellular and animal levels.

Although HDAC inhibitors and apoptotic polypeptides are useful in the treatment of a variety of tumors, they each have some drawbacks, such as HDAC inhibitors have many toxic and side effects in clinical applications, and apoptotic polypeptides have been found in early studies to have poor biosafety due to their strong hydrophobicity, which makes them have proliferation inhibiting effects on both normal cells and tumor cells. The HDAC polypeptide inhibitor developed in the prior stage has stronger tumor stem cell selective inhibition activity, and has no toxicity to normal cells at a concentration of more than 10 times, which indicates that the introduction of the polypeptide can obviously reduce the toxicity of the HDAC small molecule inhibitor. In the early stage, it has also been reported that changing the hydrophilicity and hydrophobicity of apoptotic polypeptides can significantly affect the toxicity of polypeptides to cells. Therefore, the selective toxicity of polypeptide drugs can be expected to be improved by coupling the HDAC drugs to the polypeptides and chemically changing the structure and the hydrophobicity of the polypeptides.

Disclosure of Invention

Aiming at the problems, the invention provides a polypeptide drug conjugate targeting HDAC and application thereof, which is stable based on sulfonium salt, and the technical scheme for solving the technical problems is as follows:

the polypeptide drug conjugate body of the targeting HDAC is stabilized based on sulfonium salt, the polypeptide is coupled with the HDAC inhibitor by utilizing the sulfonium salt stabilized pro-apoptosis polypeptide to form a stable polypeptide HDAC inhibitor with tumor selective toxicity, and the polypeptide sequence is as follows: arginine-leucine-arginine-leucine-methionine-arginine-leucine-arginine-methionine-leucine-arginine-leucine, the HDAC inhibitor is a hydroxamic acid structure.

Further, the structure of the coupler is a saturated fatty chain of 5 carbons or more.

Use of a sulfonium salt-based stable HDAC-targeting polypeptide drug conjugate for the preparation of a medicament for inhibiting the enzymatic activity of the HDAC family.

Use of a sulfonium salt-based stable HDAC targeting polypeptide drug conjugate for the preparation of a medicament for inhibiting tumor cells highly expressed by HDAC.

In the invention, the sulfonium salt chemical stable cyclic peptide designed based on the reported apoptosis polypeptide 'RLL' sequence changes the hydrophobicity of the apoptosis polypeptide through the hydrophilicity of the sulfonium salt, thereby reducing the nonspecific toxicity of the apoptosis polypeptide. Meanwhile, in order to further improve the killing effect on tumors, the carbon end of the polypeptide is introduced into an HDAC inhibitor hydroxamic acid, so that the purpose of resisting tumors by the bifunctional polypeptide is achieved. The novel polypeptide drug conjugate designed by the invention has better selective toxicity at the cellular level, has better toxicity to liver cancer cells Huh7, colon cancer cells HCT116, HT29 and the like, but has weaker cytotoxicity to normal cells 293T. Fully illustrates that the designed polypeptide medicine has better biological safety while maintaining the anti-tumor effect.

Experiments such as an HDAC enzyme activity inhibition experiment, apoptosis, a cell cycle, an immunoblotting analysis and a transcriptome sequencing prove that the polypeptide drug conjugate can obviously influence the related signal path of the HDAC. Meanwhile, the hemolysis experiment is adopted to further prove that the polypeptide medicament has better biocompatibility and can not cause erythrocyte disruption in a proper concentration range. The invention is beneficial to solving the toxic and side effects of the traditional HDAC inhibitor and increasing the treatment window of clinical application.

The invention adopts a sulfonium salt method for stabilizing polypeptide to form the apoptosis cyclic peptide. The HDAC inhibitor hydroxamic acid structure is introduced into the carbon end of the polypeptide sequence by a solid phase synthesis method, and immunoblotting analysis proves that the polypeptide conjugate can effectively inhibit the intracellular biological activity of HDAC. The polypeptide can inhibit the signal path related to HDAC through apoptosis, cell cycle experiments and total RNA sequencing experiments on tumor cells.

Compared with the prior art, the invention has obvious technical progress. The modified apoptosis polypeptide is coupled with the HDAC inhibitor, so that the dual-function anti-tumor activity is achieved, the nonspecific toxicity of the polypeptide is reduced, and the polypeptide is hopeful to become an anti-tumor drug with high efficiency and low toxicity. The project firstly proves that the polypeptide conjugate can effectively inhibit the HDAC protein through a series of enzyme activity inhibition experiments, and belongs to a broad-spectrum HDAC inhibitor. Cell proliferation, apoptosis and cell cycle blocking experiments prove that the polypeptide drug conjugate can effectively inhibit proliferation of various tumor cells and induce apoptosis of the tumor cells. The polypeptides of the invention can inhibit HDAC-related signaling through transcriptome sequencing and immunoblotting experiments. The hemolysis experiment shows that the polypeptide drug conjugate has better biocompatibility and new, and the serum stability experiment proves that the cyclic peptide has better serum stability than the linear polypeptide. The research result provides a thinking for developing novel and broader and safer treatment window of multifunctional HDAC inhibitors for treating malignant solid tumors in the future.

Drawings

Fig. 1 is: a design drawing of a stable polypeptide drug conjugate targeting HDAC;

fig. 2 is: synthetic roadmaps for stable polypeptide drug conjugates targeting HDACs;

fig. 3 is: toxicity patterns of different polypeptide drugs in various tumor cells and normal cells;

fig. 4 is: haemolytic toxicity profile of different polypeptide drugs of the invention on blood cells;

fig. 5 is: the result graph of the huh7 liver cancer cell apoptosis caused by different polypeptide medicaments is shown in the invention;

fig. 6 is: different polypeptide drugs of the invention cause huh7 liver cancer cell cycle arrest result patterns;

fig. 7 is: an influence diagram of different polypeptide drugs of the invention on the acetylation level of HDAC substrates in HCT116 cells of liver cancer cells and colon cancer cells;

fig. 8 is: graph of the impact of the inventive HDAC-targeting stable polypeptide on liver cancer cell huh7 transcriptome compared to linear apoptotic peptide.

Detailed Description

The principles and features of the present invention are described below with reference to the drawings, the examples are illustrated for the purpose of illustrating the invention and are not to be construed as limiting the scope of the invention.

Example 1: design of polypeptide drug conjugates targeting HDAC

The invention adopts a sulfonium salt stabilized apoptosis polypeptide to couple with an HDAC inhibitor to design a target HDAC polypeptide drug conjugate, as shown in figure 1, we select the apoptosis polypeptide reported earlier as an entry point (the original apoptosis polypeptide sequence is RLLRLLRLRRLLRL, R is arginine, L is leucine), adopts a sulfonium salt stabilized polypeptide methodology to stabilize the polypeptide, tries to improve the stability of the polypeptide, and changes the hydrophilicity and hydrophobicity of the whole apoptosis peptide by depending on the hydrophilicity of the sulfonium salt, tries to reduce the toxicity to normal cells. To further enhance the antitumor activity of the polypeptide, we introduced the carbon-terminus of the polypeptide into the HDAC inhibitor hydroxamic acid structure, which has a broad spectrum of inhibitory activity against proteins of the HDAC family, as shown in fig. 1.

To further illustrate the selective anti-tumor effect of the stable polypeptide drug conjugate targeting HDAC, we designed three additional negative control polypeptides, linear apoptotic polypeptide conjugated HDAC inhibitor (WP 2), linear apoptotic polypeptide (WP 3) and sulfonium salt stable apoptotic cyclic peptide (WP 4), respectively, as shown in the following table. These three negative control polypeptides demonstrate that sulfonium-salt stable apoptotic cyclic peptides have lower normal cytotoxicity than linear polypeptides; the apoptosis cyclic peptide is coupled with the HDAC inhibitor to obviously improve the anti-tumor activity of the polypeptide, but the toxicity of the polypeptide to normal cells is not obviously improved, so that the hypothesis that the sulfonium salt stable apoptosis polypeptide is coupled with the HDAC inhibitor to improve the selective toxicity of tumor cells and reduce the toxicity of the polypeptide to normal cells is verified, and the concept is provided for clinically developing high-efficiency low-toxicity anti-tumor drugs.

Example 2: synthesis preparation of stable polypeptide drug conjugate targeting HDAC

(1) Resin swelling and deprotection:

the specific operation of solid-phase polypeptide synthesis is shown in the scheme of FIG. 2: solid phase synthesis of hydroxamic acid-carrying polypeptide specific 2-Chlorotityl-N-Fmoc-hydroxylamine resin (degree of loading: 0.54 mmol/g) was used. The operation is as follows: the 2-Chlorotityl-N-Fmoc-hydroxyamine resin was swelled with DCM for 15 min. Fmoc protecting groups were removed with 50% morphine (in DMF) for 30 min each time, twice in total, and then each time washed 3 times alternately with DMF/DCM, respectively.

(2) Synthesis of resin polypeptide:

the deprotected resin was prepared by mixing evenly a solution of Fmoc-6-amino-hexanoic acid (5 eq,0.4M, DMF), a solution of 1H-benzotriazole-1-yloxytripyrrolidinyl hexafluorophosphate (PyBOP) (5 eq,0.4M, DMF) and N, N-Diisopropylethylamine (DIPEA) (10 eq) and then adding the mixture to the resin and bubbling nitrogen for 1H. Removing the reaction solution, washing the resin according to the method, then removing the next amino acid, washing the resin according to the method, performing the next operation, namely after removing Fmoc protecting groups on the resin by using morpholine, adding prepared (2) Fmoc-Leu-OH or (3) Fmoc-Arg (pdf) -OH or (4) Fmoc-Leu-OH or (5) Fmoc-Leu-OH or (6) Fmoc-Met-OH or (7) Fmoc-Arg (pdf) -OH or (8) Fmoc-Leu-OH or (9) Fmoc-Arg (pdf) -OH or (10) Fmoc-Met-OH or (11) Fmoc-Leu-OH or (12) Fmoc-OH or (13) Fmoc-Leu-OH or (14) Fmoc-Leu-OH or (15) Fmoc-Leu-OH, and mixing the resin with nitrogen to the PEBOP 2; the reaction solution was removed, and the resin was washed as described above, followed by the next step. And (3) removing Fmoc at the N end of the synthesized resin peptide for acetylation, namely preparing an acetylating reagent DCM: DIEA AC2 o=8:1:0.5. The polypeptides were cleaved from the resin with trifluoroacetic acid (TFA), triisopropylsilane (TIPS) and H2O (v: v=9.5:0.25:0.25) shears, and the shears were removed. The crude polypeptide was dissolved in 70% acetonitrile/ddH 2O (v: v) as powder, formic acid was added to give a final concentration of 1%, and then 1, 3-m-phenyldibromo (2.0 eq) was dissolved in a little DMF, and then added to the polypeptide mixture to react for 24 hours. ddH20 was added to the reaction solution to reach an acetonitrile concentration of 50%, the reaction solution was filtered, and the clear filtrate was separated by high performance liquid chromatography, and the molecular weight of the polypeptide was identified by ESI mass spectrometry.

Example 3: enzyme activity inhibition effect of stable polypeptide drug conjugate of target HDAC

The identification of the inhibition effect of HDAC enzyme activity was performed using a commercially available HDAC enzyme activity detection kit, repeated at least three times for each operation, from HeLa nuclear extracts reported in various documents, the enzyme activity was performed according to the operation of the commercial kit, and finally the inhibition activity of the polypeptide drug conjugate on the HDAC protein was measured using an enzyme-labeled instrument, as shown in table 1:

TABLE 1 enzyme activity inhibition effect of different polypeptide drugs designed in the present invention on HDAC

Example 4: evaluation of killing effect of stable polypeptide drug conjugates targeting HDAC on different cells

The stable polypeptide drug conjugate targeting HDAC has obvious killing effect on tumor cells such as liver cancer cells Huh7, colon cancer cells HT29, HCT116 and the like, and has weak inhibition effect on normal cells 293T. However, the linear apoptosis polypeptide has strong inhibition effect on normal cells and tumor cells, as shown in figure 3. Cell viability was determined by CCK8 cytotoxicity assay. Cells were seeded at 4×103 in 96-well plates and treated with polypeptide drug conjugates in medium (5% serum) for 24h or 48 h and CCK8 was added to the medium and incubated for 1h. Absorbance was measured at 450nm using an enzyme-labeled instrument. Wherein the untreated cell viability was 100%.

The result shows that the stable polypeptide drug conjugate body targeting the HDAC has obvious killing effect on tumor cells such as liver cancer cells Huh7, colon cancer cells HT29, HCT116 and the like, and has weak inhibition effect on normal cells 293T.

Example 5: haemolytic toxicity study of polypeptide drug conjugate

To evaluate the nonspecific toxicity of polypeptide drug conjugates to erythrocytes, we used a mouse hemolytic toxicity assay. Fresh mouse erythrocytes were obtained by eyeball removal and placed in EP tubes containing anticoagulant (1 ml of blood plus 10. Mu.L of heparin sodium at a concentration of 10 mg/ml), rapidly centrifuged at 8600rpm/min for 10s, and clarified by washing with 0.9% physiological saline. The resulting erythrocytes were diluted to 108/mL and a series of polypeptides at different concentrations were incubated with erythrocytes for 1 hour at 37 ℃. After rapid centrifugation, the released heme in the supernatant was measured with a Nano Drop 2000c and the absorption wavelength was determined to be 570nm. As positive and negative controls, 0.1% Triton X-100 and 0.9% physiological saline were used, respectively. The percent hemolysis was calculated according to the reported formula to give = [ (sample 570nm absorbance-negative control 570nm absorbance)/(positive control 570nm absorbance-negative control 570nm absorbance) ]x100. Through hemolysis experiments, the sulfonium salt stabilized polypeptide drug conjugate has lower toxicity than the linear apoptosis polypeptide, as shown in figure 4. From this, it was confirmed that sulfonium salt-stabilized polypeptide drug conjugates can significantly reduce the nonspecific toxicity of polypeptides.

Example 6: effects of stable polypeptide conjugates targeting HDACs on apoptosis and cell cycle arrest in liver cancer cells.

In order to evaluate whether the polypeptide drug causes apoptosis of tumor cells, an Annexin V-FITC apoptosis detection kit is adopted, and the condition that the polypeptide drug causes apoptosis is detected by a flow cytometer. As shown in fig. 5, the polypeptide drug can indeed obviously induce apoptosis of tumor cells.

For cell cycle experiments, cells treated with drug for 24 hours were collected, first incubated with 70% glacial ethanol overnight at-20 ℃, PI stained, and then analyzed by flow cytometry. The results show that the polypeptide drug can obviously cause the cell cycle arrest of G1 phase of tumor cells, as shown in figure 6.

Example 7: polypeptide drug conjugates can significantly increase the substrate acetylation level of HDAC

Immunoblotting (WB) was used to study the effect of different polypeptides on the level of acetylation of HDAC substrates after treatment of tumor cells at different dosing concentrations. Through repeated WB experiments, the designed stable polypeptide medicine WP1 of the target HDAC can obviously cause the up-regulation of the acetylation level of the HDAC histone substrate, and as shown in figure 7, the medicine can obviously inhibit the activity of tumor cell HDAC.

Example 8: influence of polypeptide drug conjugate on liver cancer cell HDAC related signal channel

To further verify the effect of polypeptide drug conjugate on the whole signal pathway of liver cancer cells, we performed transcriptome sequencing, examined the effect of polypeptide drug on the whole gene expression of tumor cells, and found that the polypeptide drug has an effect on multiple signal pathways of liver cancer cells (FIG. 8A shows the difference of total gene expression of RNA microarray analysis drug-loaded groups and drug-free groups, FIG. 8B shows the total up-regulated gene and down-regulated gene numbers of the summarized drug-loaded groups and drug-free groups, and FIG. 8C shows the signal pathway of different biological functions of the whole genome analysis polypeptide drug effect in liver cancer cells.

The foregoing description of the preferred embodiments of the invention is not intended to limit the invention to the precise form disclosed, and any such modifications, equivalents, and alternatives falling within the spirit and scope of the invention are intended to be included within the scope of the invention.

Claims (2)

1. The polypeptide drug conjugate body targeting HDAC based on sulfonium salt stabilization is characterized in that a stable polypeptide HDAC inhibitor with tumor selective toxicity is formed by coupling an HDAC inhibitor with a sulfonium salt stable pro-apoptosis polypeptide, and the polypeptide sequence is as follows: arginine-leucine-arginine-leucine-methionine-arginine-leucine-arginine-methionine-leucine-arginine-leucine, the HDAC inhibitor is a hydroxamic acid structure; the structure of the sulfonium salt-based stable polypeptide drug conjugate targeting HDAC is as follows:

。

2. the use of a sulfonium salt-based stable HDAC targeting polypeptide drug conjugate according to claim 1 for the preparation of a medicament for inhibiting liver cancer or colon cancer.

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