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

Abstract

The present invention relates to a HDAC-targeted HDAC-stabilized polypeptide drug conjugate based on sulfonium salt stabilization and its application. The HDAC inhibitor is coupled to a pro-apoptosis polypeptide stabilized by sulfonium salt to form a stable polypeptide HDAC inhibitor with tumor selective toxicity. , the apoptotic polypeptide chemically stabilized by sulfonium salts, the non-specific toxicity is significantly reduced, and the stability of the protease is stronger. When the polypeptide drug conjugate is formed by coupling HDAC inhibitors, the polypeptide drug can significantly improve the selection of tumor cells Sexual toxicity, but less toxic to normal cells. Cell proliferation, cell apoptosis, and cell cycle arrest experiments prove that the polypeptide of the present invention can effectively inhibit the proliferation of malignant liver cancer, has relatively better biological safety, and has potential anti-tumor clinical application prospects.

Description

HDAC-targeting HDAC-stabilized peptide drug conjugates based on sulfonium salts and their applications

technical field

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

Background technique

Cancer is a major public health problem worldwide. According to the 2020 global cancer data published by the World Health Organization, there will be 4.57 million new cancer cases and more than 3 million deaths in China in 2020. The number of new cancer cases in China ranks first in the world. Lung cancer, colorectal cancer, liver cancer, gastric cancer, and breast cancer are among the top ten cancers in terms of death toll. The types of tumors and the causes of tumors are different in different genders and ages. The complexity of tumor diseases has brought challenges to human beings to overcome this disease. Traditional tumor treatment methods include surgery, radiotherapy and chemotherapy, but tumor recurrence, metastasis and drug resistance pose great challenges to tumor treatment. With the rapid development of biotechnology, human beings have a deeper and deeper understanding of the pathogenesis of tumors. Therefore, anti-tumor therapeutic drugs have been developed in an endless stream. These research results have brought great confidence to humans in completely curing tumors. Comprehensive analyzes of human cancer genomes over the past decade have revealed that epigenetic regulatory proteins involved in gene expression, DNA repair, and DNA replication are mutated in many cancers. Further studies have shown that epigenetic abnormalities are involved in tumor metastasis, drug resistance and other mechanisms, so combined epigenetic therapy has become one of the important means of anti-tumor therapy. Histone deacetylase (HDAC), as an important epigenetic regulator, plays an important role in the development and drug resistance of many malignant tumors (such as lymphoma, breast cancer, liver cancer, colon cancer, etc.). Therefore, the development of drugs targeting HDACs has been favored by medicinal chemists. As of the deadline, there are 5 HDAC drugs on the global market, namely vorinostat, romidepsin, belistat, panobinostat and chidamide, which are mainly used for peripheral T-cell lymphoma and peripheral T-cell lymphoma. cell lymphoma, among which chidamide is the only HDAC inhibitor approved for the treatment of solid tumor breast cancer. At present, there are still many HDAC drugs in preclinical or clinical research for the treatment of solid tumors or blood cancers. Therefore, drugs targeting HDACs have good prospects for the treatment of tumors.

Due to the heterogeneity and complexity of cancer, cancer treatment adopts combination therapy, such as the combination of targeted drugs and chemotherapy drugs, dual-target inhibitors, multifunctional inhibitors, etc. The apoptotic polypeptide can inhibit tumor proliferation by inducing cell apoptosis, and has a broad-spectrum anti-tumor effect. At the same time, the anti-tumor mechanism of apoptotic polypeptide is different from that of first-line chemotherapy drugs, and it also has potential application prospects in the treatment of chemotherapy-resistant tumor cells. Similar to biomacromolecules, polypeptide molecules also have higher binding force and selectivity for targets, and have smaller off-target effects than small molecule drugs. The metabolites of polypeptides in the body are amino acids, which minimizes toxicity. However, linear peptides are often difficult to maintain the original secondary structure, so they have poor target affinity, serum stability and cell membrane penetration ability. Chemically stable peptides can effectively overcome these weaknesses. Therefore, in the past ten years, chemically stable peptides have been used in the research and development of various protein target inhibitors, and have shown good tumor inhibitory effects at the cell and animal levels.

Although HDAC inhibitors and apoptotic polypeptides can be used in the treatment of various tumors, they each have some shortcomings. It has strong hydrophobicity, which makes it inhibit the proliferation of both normal cells and tumor cells, so its biological safety is poor. The previously developed HDAC peptide inhibitors have strong selective inhibitory activity on tumor stem cells, but are not toxic to normal cells at a concentration greater than 10 times, indicating that the introduction of peptides can significantly reduce the toxicity of HDAC small molecule inhibitors. It was also reported earlier that changing the hydrophilicity and hydrophobicity of the apoptotic polypeptide can significantly affect the toxicity of the polypeptide to cells. Therefore, coupling HDAC drugs to peptides and changing the structure and hydrophobicity of peptides by chemical means is expected to improve the selective toxicity of peptide drugs.

Contents of the invention

The technical problem to be solved in the present invention is to provide a HDAC-targeted polypeptide drug conjugate based on sulfonium salt stabilization and its application. The technical solution for solving the above technical problem in the present invention is as follows:

Based on sulfonium salt-stabilized peptide-drug conjugates targeting HDAC, sulfonium salt-stabilized pro-apoptotic peptides are coupled to HDAC inhibitors to form stable peptide HDAC inhibitors with tumor-selective toxicity. The peptide sequence is: arginine -leucine-leucine-arginine-leucine-methionine-arginine-leucine-arginine-leucine-arginine-methionine-leucine -Leucine-Arginine-Leucine, HDAC inhibitors have a hydroxamic acid structure.

Further, the structure of the coupler is a saturated aliphatic chain with more than 5 carbons.

Application of HDAC-targeting HDAC-stabilized polypeptide drug conjugates based on sulfonium salts in the preparation of drugs for inhibiting HDAC family enzyme activity.

Application of HDAC-targeting HDAC-stabilized polypeptide drug conjugates based on sulfonium salts in the preparation of drugs for inhibiting tumor cells with high HDAC expression.

In the present invention, we designed a sulfonium salt chemically stable cyclic peptide based on the reported sequence of the apoptotic polypeptide "RLL", and changed the hydrophobicity of the apoptotic polypeptide through the hydrophilicity of the sulfonium salt, thereby reducing its non-specific toxicity. At the same time, in order to further improve its killing effect on tumors, we introduced the HDAC inhibitor hydroxamic acid into the carbon-terminus of the polypeptide, so as to realize the anti-tumor purpose of the bifunctional polypeptide. The novel polypeptide drug conjugate designed by the present invention has better selective toxicity at the cellular level, and has better toxicity to liver cancer cells Huh7 and colon cancer cells HCT116 and HT29, but has weaker cytotoxicity to normal cells 293T . It fully demonstrates that the peptide drug we designed can maintain the anti-tumor effect while having good biological safety.

Through HDAC enzyme activity inhibition experiments, cell apoptosis, cell cycle, western blot analysis, transcriptome sequencing and other experiments, the present invention proves that the polypeptide drug conjugate can significantly affect HDAC-related signaling pathways. At the same time, we used hemolysis experiments to further verify that the peptide drug has good biocompatibility and will not cause red blood cell fragmentation in an appropriate concentration range. The invention is beneficial to solving the toxic and side effects of traditional HDAC inhibitors and increasing the therapeutic window of its clinical use.

The invention adopts the method of stabilizing the polypeptide with sulfonium salt to form the apoptosis cyclic peptide. The HDAC inhibitor hydroxamic acid structure was introduced into the carbon-terminus of the polypeptide sequence by solid-phase synthesis, and Western blot analysis proved that the polypeptide conjugate could effectively inhibit the biological activity of HDAC in cells. The polypeptide can inhibit HDAC-related signaling pathways through cell apoptosis, cell cycle experiments and total RNA sequencing experiments on tumor cells.

Compared with the prior art, the technical progress of the present invention is remarkable. The invention couples the modified apoptotic polypeptide with HDAC inhibitor, which not only achieves bifunctional anti-tumor activity, but also reduces its non-specific toxicity. This project is expected to become an anti-tumor drug with high efficiency and low toxicity. The project firstly proved through a series of enzyme activity inhibition experiments that our polypeptide conjugate can effectively inhibit HDAC protein, which belongs to broad-spectrum HDAC inhibitor. Experiments on cell proliferation, apoptosis, and cell cycle arrest prove that our peptide drug conjugates can effectively inhibit the proliferation and induce apoptosis of various tumor cells. Transcriptome sequencing and immunoblotting experiments prove that the polypeptide of the present invention can inhibit HDAC-related signaling pathways. The hemolysis test shows that the peptide-drug conjugate has better biocompatibility, and the serum stability test proves that the cyclic peptide has better serum stability than the linear peptide. The research results provide an idea for the future development of novel, multifunctional HDAC inhibitors with a wider and safer therapeutic window for the treatment of malignant solid tumors.

Description of drawings

Figure 1 is a design diagram of a stable peptide-drug conjugate targeting HDAC;

Figure 2 is a synthetic route diagram of a stable peptide-drug conjugate targeting HDAC;

Fig. 3 is: the toxicity profile of different polypeptide drugs of the present invention in various tumor cells and normal cells;

Fig. 4 is: the hemolytic toxicity diagram of different polypeptide drugs of the present invention to blood cells;

Fig. 5 is a graph showing the results of apoptosis of huh7 liver cancer cells caused by different polypeptide drugs of the present invention;

Fig. 6 is a graph showing the results of cycle arrest of huh7 liver cancer cells caused by different polypeptide drugs of the present invention;

Fig. 7 is a graph showing the influence of different polypeptide drugs of the present invention on the acetylation level of HDAC substrates in liver cancer cells and colon cancer cells HCT116 cells;

Fig. 8 is a graph showing the influence of the HDAC-targeting stable polypeptide of the present invention on the transcriptome of liver cancer cell huh7 compared with the linear apoptotic peptide.

Detailed ways

The principles and features of the present invention are described below in conjunction with the accompanying drawings, and the examples given are only used to explain the present invention, and are not intended to limit the scope of the present invention.

Example 1: Design of HDAC-targeted polypeptide drug conjugates

The present invention adopts the mode of sulfonium salt-stabilized apoptotic polypeptide coupled with HDAC inhibitors to design HDAC-targeting polypeptide drug conjugates, as shown in Figure 1, we choose the apoptotic polypeptide reported earlier as the entry point (the original apoptotic polypeptide sequence : RLLRLRLLRLRLLRL, R is arginine, L is leucine), the polypeptide is stabilized by using the sulfonium salt-stabilized polypeptide methodology, trying to improve the stability of the polypeptide, and relying on the hydrophilicity of the sulfonium salt to change the overall apoptosis Hydrophilic and hydrophobic properties of the peptide in an attempt to reduce toxicity to normal cells. In order to further improve the anti-tumor activity of the polypeptide, we introduced the carbon-terminal of the polypeptide into the HDAC inhibitor hydroxamic acid structure, which has a broad-spectrum inhibitory activity on HDAC family proteins, as shown in Figure 1.

In order to further illustrate the selective anti-tumor effect of HDAC-targeting stable peptide-drug conjugates, we designed three other negative control peptides, as shown in the table below, which are linear apoptosis peptide-coupled HDAC inhibitor (WP2), linear Apoptotic polypeptide (WP3) and sulfonium salt stabilized apoptotic cyclic peptide (WP4). These three negative control peptides show that the apoptotic cyclic peptide stabilized by sulfonium salts has lower normal cytotoxicity than the linear peptide; the apoptotic cyclic peptide coupled with HDAC inhibitors significantly improves the anti-tumor activity of the peptide, but its effect on normal The toxicity of the cells was not significantly improved, thus verifying our conjecture that the sulfonium salt-stabilized apoptotic peptide-coupled HDAC inhibitor can increase the selective toxicity of tumor cells and reduce its toxicity to normal cells. Anticancer drugs provide ideas.

Example 2: Synthesis and preparation of stable peptide drug conjugates targeting HDAC

(1) Resin swelling and deprotection:

The specific operation of solid-phase peptide synthesis is shown in the roadmap in Figure 2: the solid-phase synthesis of peptides with hydroxamic acid uses a special 2-Chlorotityl-N-Fmoc-hydroxylamine resin (loading degree: 0.54mmol/g). The procedure was as follows: 2-Chlorotityl-N-Fmoc-hydroxylamine resin was swollen with DCM for 15 minutes. Use 50% morphine (dissolved in DMF) to remove the Fmoc protecting group for 30 minutes each time, twice in total, and then wash with DMF/DCM alternately for 3 times.

(2) Synthesis of resin polypeptide:

The above-mentioned deprotected resin, the prepared (1) Fmoc-6-amino-caproic acid (5eq, 0.4M, DMF) solution, 1H-benzotriazol-1-yloxytripyrrolidinyl hexafluorophosphate ( PyBOP) (5eq, 0.4M, DMF) solution and N,N-diisopropylethylamine (DIPEA) (10eq) were mixed well, then added to the resin and blown with nitrogen for 1h. Take out the reaction solution, wash the resin according to the above method and then connect the next amino acid, that is, take off the reaction solution, wash the resin according to the above method, and then proceed to the next step, that is, after removing the Fmoc protecting group on the resin with morpholine, the prepared Well (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-Arg(pdf)-OH or (13) Fmoc-Leu-OH or (14) Fmoc-Leu-OH or (15) Fmoc-Arg(pdf)-OH, mix PyBOP and DIPEA solution and add Nitrogen gas was blown in the resin for 2 hours; the reaction solution was removed, and the resin was washed by the above method before proceeding to the next step. Remove the N-terminal Fmoc of the synthesized resin peptide for acetylation, that is, prepare the acetylation reagent DCM: DIEA:AC2O=8:1:0.5. Cut the polypeptide from the resin with trifluoroacetic acid (TFA), triisopropylsilane (TIPS) and H2O (v:v:v=9.5:0.25:0.25) shear fluid, and remove the shear fluid. Dissolve the powdered crude peptide in 70% acetonitrile/ddH2O (v:v), add formic acid to make the final concentration 1%, then dissolve 1,3-m-phenylenedibromide (2.0eq) in a little DMF, Add to the peptide mixture and react for 24 hours. Add ddH20 to the reaction solution to make the acetonitrile concentration reach 50%, filter the reaction solution, separate the clarified filtrate by high performance liquid chromatography, and identify the molecular weight of the polypeptide by ESI mass spectrometry.

Example 3: Enzyme Activity Inhibition Effect of Stable Polypeptide Drug Conjugate Targeting HDAC

The identification of the inhibitory effect of HDAC enzyme activity was carried out with a commercially purchased HDAC enzyme activity detection kit, and each operation was repeated at least three times. HDAC enzymes came from HeLa nuclear extracts reported in many literatures. The enzyme activity results were based on commercial reagents. The operation of the box is carried out, and finally the inhibitory activity of the peptide drug conjugate on the HDAC protein is measured with a microplate reader, as shown in Table 1:

Table 1 Different polypeptide drugs designed by the present invention are to HDAC enzyme activity inhibitory effect figure

Example 4: Evaluation of the Killing Effects of Stable Polypeptide Drug Conjugates Targeting HDAC on Different Cells

The stable peptide drug conjugate targeting HDAC has obvious killing effect on tumor cells such as liver cancer cells Huh7, colon cancer cells HT29, HCT116, etc., but has a weak inhibitory effect on normal cells 293T. However, the linear apoptotic polypeptide has a strong inhibitory effect on both normal cells and tumor cells, as shown in FIG. 3 . Cell viability was determined by CCK8 cytotoxicity assay. The cells were inoculated at 4×10 3 in a 96-well plate, treated with the peptide drug conjugate dissolved in the culture medium (5% serum) for 24 hours or 48 hours, and CCK8 was added to the culture medium and incubated for 1 hour. Absorbance was measured at 450 nm using a microplate reader. The survival rate of untreated cells was 100%.

The results showed that the stable peptide drug conjugate targeting HDAC had obvious killing effect on tumor cells such as liver cancer cells Huh7, colon cancer cells HT29, HCT116, etc., but had a weak inhibitory effect on normal cells 293T.

Example 5: Study on hemolytic toxicity of polypeptide drug conjugates

In order to evaluate the non-specific toxicity of the peptide drug conjugate to red blood cells, we used the mouse hemolytic toxicity experiment. Fresh mouse erythrocytes were obtained by enucleation and placed in EP tubes containing anticoagulants (1ml of blood plus 10μL of heparin sodium with a concentration of 10mg/ml), centrifuged at 8600rpm/min for 10s, and washed with 0.9% saline Supernatant clarified. The obtained erythrocytes were diluted to 108/mL, and a series of polypeptides with different concentrations were incubated with erythrocytes at 37°C for 1 hour. After rapid centrifugation, Nano Drop 2000c was used to measure the hemoglobin released in the supernatant, and the measured absorption wavelength was 570nm. 0.1% Triton X-100 and 0.9% normal saline were used as positive and negative controls respectively. The percentage of hemolysis was calculated according to the reported formula: % hemolysis=[(absorption at 570 nm of the sample-absorption at 570 nm of the negative control)/(absorption at 570 nm of the positive control-absorption at 570 nm of the negative control)]×100. Through hemolysis experiments, we found that the peptide-drug conjugates stabilized by sulfonium salts have lower toxicity than linear apoptotic peptides, as shown in Figure 4. Thus, it can be confirmed that the polypeptide-drug conjugate stabilized by sulfonium salt can significantly reduce the non-specific toxicity of the polypeptide.

Example 6: Effects of HDAC-targeted stable polypeptide conjugates on apoptosis and cell cycle arrest of liver cancer cells.

In order to evaluate whether the peptide drug induces tumor cell apoptosis, we use the Annexin V-FITC cell apoptosis detection kit to detect the apoptosis caused by the peptide drug by flow cytometry. As shown in Figure 5, the peptide drug can indeed significantly induce tumor cell apoptosis.

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

Example 7: The polypeptide drug conjugate can significantly increase the acetylation level of HDAC substrates

Western blotting (WB) was used to study the effect of different polypeptides on the acetylation level of HDAC substrates after treating tumor cells with different concentrations. After repeated WB experiments, we found that our designed stable peptide drug WP1 targeting HDAC can significantly increase the acetylation level of HDAC histone substrates, as shown in Figure 7, revealing that our drug can significantly inhibit tumor cell HDAC activity.

Example 8: Effects of polypeptide drug conjugates on HDAC-related signaling pathways in liver cancer cells

In order to further verify the effect of the peptide drug conjugate on the entire signaling pathway of liver cancer cells, we performed transcriptome sequencing to detect the effect of the peptide drug on the entire gene expression of tumor cells, and found that the peptide drug had significant effects on multiple signaling pathways of liver cancer cells. Influenced (Fig. 8A is the difference of the total gene expression of the RNA microarray analysis drug-dosing group and no drug-dosing group, Fig. 8B is the number of total up-regulated genes and down-regulated genes of the drug-dosed group and no drug-dosed group, Fig. 8C is a genome-wide analysis of the signaling pathways of different biological functions affected by polypeptide drugs in liver cancer cells.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the protection of the present invention. within range.

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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