Stapling peptides, uses thereof and methods of expanding stem cells in vitro

Abstract

The application provides a stapling peptide, which is shown as SEQ ID NO. 1, SEQ ID NO. 1: CDACXATLXNIHWVCQKCGFV; wherein the stapling peptide comprises an alpha-helix and two non-natural amino acids X, X represents 2-amino-2-methyl-6-heptenoic acid; and the two non-natural amino acids X are annelated. The application also provides use of the stapling peptide and a method for expanding stem cells in vitro. Through the above technical solution, the stapling peptide can significantly enhance the activity of JMJD1C in cells, thereby promoting expansion of hematopoietic stem cells.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically, to a staple peptide, the use of the staple peptide, and a method for expanding stem cells in vitro. Background Technology

[0002] Stapling peptides are developed based on the requirement that polypeptides form α-helices to cross the cell membrane and enter the cell. Many biological processes are regulated through protein-protein interactions, such as viral self-assembly, cell growth, division, and differentiation. However, the protein-protein interface is usually too large, making it difficult for small molecule drugs to target and specifically block these interactions, thus hindering therapeutic efficacy. Protein drugs, because they struggle to cross the cell membrane, also cannot directly target intracellular interactions. Therefore, researchers have sought new drug molecules that overcome these limitations, enabling them to both cross the cell membrane and specifically target protein-protein interactions.

[0003] Studies have shown that peptides with α-helical structures and high positive charges can cross cell membranes. Therefore, researchers have developed α-helical structures using disulfide bonds and intramolecular amide bonds as scaffolds; however, these scaffolds are not stable under physiological conditions. In recent years, a method has been developed to stabilize the α-helical structure of peptides using carbon-carbon bonds as a scaffold. Peptides obtained by this method are called stapled peptides. Stapled peptides have advantages such as a higher degree of α-helix structure, strong binding affinity, ability to cross cell membranes, resistance to protease hydrolysis, and a long half-life in vivo.

[0004] Histone demethylase JMJD1C is an important protein widely involved in the development of various solid tumors, leukemia, sperm and egg development, induced pluripotent stem cell formation, platelet count, inflammatory responses, serum levels of liver-specific enzymes, and lipid metabolism regulation. Therefore, small molecule inhibitors and agonists of JMJD1C may have broad clinical applications. To date, various small molecule inhibitors and modulators of JMJD1C have been developed, demonstrating their ability to selectively kill specific types of leukemia, inhibit leukemia stem cells, promote hematopoietic stem cell proliferation, and activate certain types of immune cells.

[0005] However, there is still a need to develop a staple peptide that can enhance the activity of JMJD1C. Summary of the Invention

[0006] The purpose of this invention is to provide a staple peptide that can enhance the activity of JMJD1C.

[0007] This invention provides a staple peptide, as shown in SEQ ID NO.1.

[0008] SEQ ID NO.1: CDACXATLXNIHWVCQKCGFV;

[0009] The staple peptide contains an α-helix and two non-natural amino acids X.

[0010] X represents 2-amino-2-methyl-6-heptenic acid;

[0011] The two non-natural amino acids are cyclized.

[0012] The present invention also provides the use of the staple peptide as described above in the preparation of a drug that promotes hematopoietic function.

[0013] The present invention also provides the use of the staple peptide as described above in the preparation of a medicament for treating hematopoietic disorders.

[0014] The present invention also provides the use of the staple peptide as described above in the preparation of a medicament for promoting the expansion of hematopoietic stem cells.

[0015] The present invention also provides the use of the staple peptide as described above in the preparation of a medicament for promoting the expansion of mesenchymal stem cells.

[0016] The present invention also provides a method for expanding stem cells in vitro, the method comprising: seeding stem cells in a culture medium containing the staple peptide as described above and culturing them.

[0017] Optionally, the stem cells are hematopoietic stem cells.

[0018] Optionally, the hematopoietic stem cells are umbilical cord hematopoietic stem cells, uterine blood hematopoietic stem cells, or bone marrow hematopoietic stem cells.

[0019] Optionally, the stem cells are mesenchymal stem cells, preferably umbilical cord mesenchymal stem cells.

[0020] Optionally, the concentration of the staple peptide according to claim 1 in the culture medium is 0.1-100 μM, preferably 5-20 μM.

[0021] Through the above technical solution, the staple peptide of the present invention can significantly enhance the activity of JMJD1C in cells, thereby promoting the expansion of hematopoietic stem cells.

[0022] Other features and advantages of the present invention will be described in detail in the following detailed description section. Attached Figure Description

[0023] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used together with the following detailed description to explain the invention, but do not constitute a limitation thereof. In the drawings:

[0024] Figure 1 This shows the sequence of the staple peptide derived from the JMJD1C zinc finger domain.

[0025] Figure 2-8 The results indicate that the staple peptide SAH-JZ3 can promote the proliferation of myeloid (MV4-11, HL60), lymphoid (SEM), and T cell (Jurkat) cells, but has no significant effect on natural killer cells (NK-92).

[0026] Figure 9-10 This study describes the effects of peptide SAH-JZ3 on JMJD1C protein and JMJD1C target gene SCD in myeloid cell lines MOLM-13 and THP-1.

[0027] Figure 11 This indicates that SAH-JZ3 upregulates the number of umbilical cord blood mononuclear cells.

[0028] Figure 12 This indicates that SAH-JZ3 upregulates the number of umbilical cord blood mononuclear cells.

[0029] Figure 13 This indicates that SAH-JZ3 upregulates the number of umbilical cord mesenchymal stem cells.

[0030] Figure 14 This indicates that SAH-JZ3 does not affect the differentiation of umbilical cord mesenchymal stem cells.

[0031] Figure 15 This indicates that SAH-JZ3 enhances the ability of hematopoietic stem cells to reconstitute in recipient mice. Detailed Implementation

[0032] The specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0033] This invention provides a staple peptide, as shown in SEQ ID NO.1.

[0034] SEQ ID NO.1: CDACXATLXNIHWVCQKCGFV; which is Ac-CDAC(S5)ATL(S5)NIHWVCQKCGFV.

[0035] The staple peptide contains an α-helix and two non-natural amino acids X.

[0036] X represents 2-amino-2-methylhept-6-enoic acid;

[0037] The two non-natural amino acids are cyclized.

[0038] The cyclization can be carried out using conventional cyclization methods in the preparation of stapled peptides, such as using DMF solution, Grubbs Catalyst, 2nd Generation as catalyst, at room temperature, to carry out olefin metathesis reaction for cyclization.

[0039] The specific mechanism by which this staple peptide exerts its effect may be by antagonizing the degradation of JMJD1C protein to enhance JMJD1C stability. This staple peptide can enhance the expansion of hematopoietic stem cells and mesenchymal stem cells.

[0040] The present invention also provides the use of the staple peptide as described above in the preparation of a drug that promotes hematopoietic function.

[0041] The present invention also provides the use of the staple peptide as described above in the preparation of a medicament for treating hematopoietic disorders.

[0042] The present invention also provides the use of the staple peptide as described above in the preparation of a medicament for promoting the expansion of hematopoietic stem cells.

[0043] The present invention also provides the use of the staple peptide as described above in the preparation of a medicament for promoting the expansion of mesenchymal stem cells.

[0044] The present invention also provides a method for expanding stem cells in vitro, the method comprising: seeding stem cells in a culture medium containing the staple peptide as described above and culturing them.

[0045] Optionally, the stem cells are hematopoietic stem cells.

[0046] Optionally, the hematopoietic stem cells are umbilical cord hematopoietic stem cells, uterine blood hematopoietic stem cells, or bone marrow hematopoietic stem cells.

[0047] Optionally, the stem cells are mesenchymal stem cells, preferably umbilical cord mesenchymal stem cells.

[0048] Optionally, the concentration of the staple peptide according to claim 1 in the culture medium is 0.1-100 μM, preferably 5-20 μM.

[0049] Through the above technical solution, the staple peptide of the present invention can significantly enhance the activity of JMJD1C in cells, thereby promoting the expansion of hematopoietic stem cells.

[0050] The present invention will be further illustrated by the following examples, but the present invention is not limited thereto.

[0051] Example

[0052] By inserting staple peptides at different positions into the zinc finger domain of JMJD1C, a series of candidate staple peptides were obtained, such as... Figure 1 As shown.

[0053] Figure 1 The sequence of the binding peptide derived from the zinc finger domain of JMJD1C is shown. The full-length zinc finger domain and partial sequence of the enzyme active domain of the histone demethylase JMJD1C are shown below. Figure 1 As shown, seven peptides were obtained by adding a transmembrane peptide (TAT, sequence YGRKKRRQRRR), adding a non-natural amino acid (X) at different positions, and performing side chain cross-linking and cyclization. ZFD represents the zinc finger domain; JZ represents the zinc finger domain (ZFD) of JMJD1C; Myr represents the N-terminal myristoylation modification; JJ represents the jumonji domain of JMJD1C; and X represents the non-natural amino acid 2-amino-2-methyl-6-heptenoic acid.

[0054] Figure 2 This provides a detailed description of the staple peptide's structure. 2A represents the transhelical structure of the staple peptide; 2B gives the specific structure of X; and 2C provides the specific sequence of the polypeptide inserted into the staple peptide.

[0055] The obtained peptides are then screened. For example... Figure 2-8 As shown, the staple peptide SAH-JZ3 can promote the proliferation of myeloid (MV4-11, HL60), lymphoid (SEM), and T cell (Jurkat) cells, but has no significant effect on natural killer cells (NK-92).

[0056] Effects of different peptides on the proliferation of myeloid cell lines MV4-11 and HL60, lymphoid cell line SEM, T cell line Jurkat, and natural killer cell line NK-92. MV4-11 cells were cultured in 80% 1640 medium with 20% fetal bovine serum; HL60, SEM, and Jurkat cells were cultured in 90% 1640 medium with 10% fetal bovine serum; NK-92 cells were cultured in 75% αMEM medium with 12.5% ​​fetal bovine serum, 12.5% ​​horse serum, and 30 units / mL interleukin-2. All cultures were performed at 37°C with 5% CO2. Cells were seeded at 200,000 / mL, and a 4-hour serum-free culture step was used for peptide treatment. Cell proliferation was detected using CCK8 reagent. All experiments were performed in triplicate. A p-value less than 0.05 was considered statistically significant. Figure 9-10This study describes the effects of peptide SAH-JZ3 on JMJD1C protein and its target gene SCD in myeloid cell lines MOLM-13 and THP-1. MOLM-13 and THP-1 cells were cultured in 80% 1640 medium with 20% fetal bovine serum at 37°C and 5% CO2. Cells were seeded at 200,000 / mL, and a 4-hour serum-free culture was performed before peptide treatment. Western blots were performed using JMJD1C antibody 17-10262 (Sigma, Shanghai, China) and SCD antibody (sc-58420, Santa Cruz, Shanghai, China). All experiments were performed in triplicate, and the histogram on the right represents the statistical analysis of the triplicate experiments. A p-value less than 0.05 was considered statistically significant.

[0057] To further explore the mechanism of action of this peptide, the effects of SAH-JZ3 on intracellular genes were detected by RNA sequencing. As shown in Table 1, the main genes positively regulated by SAH-JZ3 include lipid metabolism genes SCD and FADS2, which are target genes of JMJD1C and are reduced by knockout of JMJD1C or small molecule inhibition. These results indicate that SAH-JZ3 can act as an agonist of JMJD1C in terms of target gene expression, and that SAH-JZ3 may increase the expression of JMJD1C, leading to the upregulation of its downstream genes such as SCD.

[0058] Table 1

[0059]

[0060]

[0061] Table 1 shows the RNA sequencing analysis results of SAH-JZ3-treated MOLM-13 cells. List of the top 10 genes in RNA sequencing. MOLM-13 cell culture as follows: Figure 2 As shown, RNA sequencing was performed after treatment with 20 μmol SAH-JZ3 peptide for 24 hours (including 4 hours of pre-culture). The results were sorted according to the adjusted p-index, and the top 10 genes were presented in the list.

[0062] Next, the effect of SAH-JZ3 on JMJD1C protein was examined. The results showed that SAH-JZ3 could upregulate JMJD1C protein levels. Figure 9-10 ).

[0063] The results showed that SAH-JZ3 can promote cell proliferation, upregulate the expression of JMJD1C target genes, and increase the level of JMJD1C protein, thus it may act as an agonist of JMJD1C.

[0064] The JMJD1C agonist SAH-JZ3 can promote the expansion of hematopoietic stem cells. Next, the effect of SAH-JZ3 on umbilical cord blood was examined. First, the effect of SAH-JZ3 on the number of umbilical cord blood monocytes in liquid culture medium was investigated. Figure 11 This indicates that SAH_JZ3 can upregulate the number of umbilical cord blood monocytes by 1.4-1.92 times.

[0065] Figure 11 This indicates that SAH-JZ3 upregulated the number of umbilical cord blood mononuclear cells. Umbilical cord blood was isolated from the umbilical cord of healthy newborns and obtained by centrifugation, i.e., adding an equal volume of lymphocyte separation medium and centrifuging at 2000 rpm for 20 minutes. Mononuclear cells were then seeded at 1 million / ml in 24-well culture plates using a DMEM / F12 1:1 mixture medium supplemented with 10% fetal bovine serum, 100 ng / ml SCF, 100 ng / ml FLT3L, 50 ng / ml TPO, and 10 μg / ml low-density lipoprotein. All reagents were from eBioscience. The cells were treated with 20 μmol SAH-JZ3 and cultured for 4 hours in a serum-free environment. Cell counting was performed after 7 days. All experiments were repeated three times. A p-value less than 0.05 was considered statistically significant (indicated by an asterisk).

[0066] Considering that cell proliferation does not reflect the reconstitution capacity of hematopoietic stem cells, the effect of SAH-JZ3 on colony formation capacity was further investigated. Figure 12 As shown, SAH-JZ3 can greatly promote the formation of umbilical cord cell colonies.

[0067] Figure 12 This indicates that SAH-JZ3 upregulates the number of umbilical cord blood mononuclear cells. Umbilical cord blood was isolated from the umbilical cords of healthy newborns. After treatment with SAH-JZ3 (20 μmol) for 4 hours (serum-free), cells were inoculated at 500 cells per well (24-well plate) on colony-forming medium H4434 (Stem Cell, Shanghai, China). Observations and photographs were taken after 12 days. All experiments were repeated three times.

[0068] The JMJD1C agonist SAH-JZ3 promotes mesenchymal stem cell proliferation but not differentiation. Further investigation is needed to examine the effect of SAH-JZ3 on umbilical cord mesenchymal stem cell proliferation, such as... Figure 13 As shown, SAH-JZ3 can promote the proliferation of mesenchymal stem cells.

[0069] Figure 13This indicates that SAH-JZ3 upregulates the number of umbilical cord mesenchymal stem cells. Umbilical cord mesenchymal stem cells were isolated from the umbilical cords of healthy newborns. After treatment with 20 μmol of peptide for 4 hours (serum-free), they were seeded at 3000 cells per well (96-well plate) and cultured in DMEM high-glucose + 10% fetal bovine serum. CCK8 proliferation was measured after 2 days. All experiments were repeated three times. A p-value less than 0.05 was considered statistically significant (indicated by an asterisk).

[0070] The next step is to examine the effect of SAH-JZ3 on the differentiation of umbilical cord mesenchymal stem cells, such as... Figure 14 As shown, SAH-JZ3 does not affect the differentiation of mesenchymal stem cells.

[0071] Figure 14 This indicates that SAH-JZ3 does not affect the differentiation of umbilical cord mesenchymal stem cells. Umbilical cord mesenchymal stem cells were isolated from the umbilical cords of healthy newborns. After treatment with 20 μmol of peptide for 4 hours (serum-free), they were seeded at 3000 cells per well (96-well plate) and cultured in DMEM high-glucose + 10% fetal bovine serum. RNA was extracted 2 days later for quantitative PCR detection. Genes related to mesenchymal stem cell neural development (GDNF, MAPT), muscle development (MEF2C), and skeletal development (Osteocalcin, Twist) were detected. All experiments were repeated three times. A p-value less than 0.05 was considered statistically significant (indicated by an asterisk).

[0072] SAH-JZ3 enhances the reconstitution of hematopoietic stem cells in recipient mice. The reconstitution of hematopoietic stem cells by SAH-JZ3 in recipient mice was further measured.

[0073] Figure 15 This indicates that SAH-JZ3 enhances the reconstitution of hematopoietic stem cells in recipient mice. Umbilical cord mesenchymal stem cells were isolated from the umbilical cords of healthy newborns. They were then cultured with 20 μmol of peptide for 10 days. Recipient mice were inoculated 10 days later. The figure shows the number of CD34-positive cells required for in vivo reconstitution in mice. The DMSO group required 1897 cells, the DMSO-free group required approximately 1259 cells, and the SAH-JZ3 group required only 430 cells. Six mice were used in each group, and a p-value less than 0.05 was considered statistically significant.

[0074] The preferred embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the specific details of the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0075] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

[0076] Furthermore, various different embodiments of the present invention can be combined in any way, as long as they do not violate the spirit of the present invention, they should also be regarded as the content disclosed by the present invention.

Claims

1. A stapler peptide, as shown in SEQ ID NO. 1, SEQ ID NO. 1: CDACXATLXNIHWVCQKCGFV; in, The staple peptide contains an α-helix and two non-natural amino acids X. X represents 2-amino-2-methyl-6-heptenic acid; The two non-natural amino acids are cyclized.

2. A method for expanding stem cells in vitro, characterized in that, The method includes: inoculating stem cells into a culture medium containing the staple peptide of claim 1 and culturing them.

3. The method according to claim 2, wherein, The stem cells mentioned are hematopoietic stem cells.

4. The method according to claim 3, wherein, The hematopoietic stem cells are umbilical cord hematopoietic stem cells, uterine blood hematopoietic stem cells, or bone marrow hematopoietic stem cells.

5. The method according to claim 2, wherein, The stem cells mentioned are mesenchymal stem cells.

6. The method according to claim 5, wherein, The stem cells mentioned are umbilical cord mesenchymal stem cells.

7. The method according to any one of claims 2-6, wherein, In the culture medium, the concentration of the staple peptide according to claim 1 is 0.1-100 μM.

8. The method according to claim 7, wherein, In the culture medium, the concentration of the staple peptide according to claim 1 is 5-20 μM.

Images