CN116693626A - Staple peptides and uses thereof and methods for expanding stem cells in vitro - Google Patents
Staple peptides and uses thereof and methods for expanding stem cells in vitro Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/06—Antianaemics
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- C12N2501/998—Proteins not provided for elsewhere
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
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Abstract
The invention provides a staple peptide, which is shown as SEQ ID NO.1, wherein SEQ ID NO.1: cdactxatlxnihvcqkcgfv; wherein the staple peptide comprises an alpha-helix and two unnatural amino acids X, wherein X represents 2-amino-2-methyl-6-heptenoic acid; the two unnatural amino acids X are cyclized. The invention also provides the application of the staple peptide and a method for expanding stem cells in vitro. Through the technical scheme, the staple peptide can remarkably enhance the activity of JMJD1C in cells, so that the expansion of hematopoietic stem cells is promoted.
Description
Technical Field
The invention relates to the field of biotechnology, in particular to a staple peptide, application of the staple peptide and a method for expanding stem cells in vitro.
Background
Stapled peptides were developed based on the need for polypeptides to form alpha-helices to enter cells through the cell membrane. The regulation of various life processes in organisms is achieved by protein-protein interactions. Such as virus self-assembly, cell growth, division, differentiation, and the like. The interface of the protein-protein interaction is usually too large, so that the small molecular medicine is difficult to target and position, the interaction is blocked with high efficiency and specificity, and a good therapeutic effect is shown. Protein drugs cannot achieve the effect of directly targeting intracellular interactions because of difficulty in passing through cell membranes, and researchers have begun to seek a new drug molecule capable of overcoming the shortcomings of both cell membrane entry and specific targeting protein-protein interactions.
Studies have shown that polypeptides with an alpha-helical structure and a positive charge can cross cell membranes. Therefore, α -helical structures have been developed that utilize disulfide bonds and intramolecular lactam bonds as scaffolds, but none of these scaffolds exist stably under physiological conditions. In recent years, a method for stabilizing the alpha-helical structure of a polypeptide using a carbon-carbon bond as a scaffold has been developed, and the polypeptide obtained by this method becomes a staple peptide (Stapled peptides). The staple peptide has the advantages of higher alpha-helix degree, strong binding capacity, capability of passing through cell membranes, difficult hydrolysis by protease, long half-life in organisms and the like.
Histone demethylase JMJD1C is an important protein and is widely involved in the generation of various solid tumors, leukemia, sperm-egg development, induction of multipotent stem cell formation, platelet number, inflammatory reaction, liver characteristic enzyme level in serum, lipid metabolism regulation and the like. Therefore, small molecule inhibitors and agonists of JMJD1C may have wide clinical application value. Up to now, a variety of JMJD1C small molecule inhibitors and modulators have been developed that find their ability to selectively kill certain types of leukemia, inhibit leukemia stem cells, promote hematopoietic stem cell expansion, and activate certain types of immune cells.
However, there remains a need to develop a stapled peptide that can enhance the activity of JMJD 1C.
Disclosure of Invention
The invention aims to provide a stapling peptide capable of enhancing the activity of JMJD 1C.
The invention provides a staple peptide, which is shown as SEQ ID NO.1,
SEQ ID NO.1：CDACXATLXNIHWVCQKCGFV；
wherein the staple peptide comprises an alpha-helix and two unnatural amino acids X,
x represents 2-amino-2-methyl-6-heptenoic acid;
the two unnatural amino acids X are cyclized.
The invention also provides application of the staple peptide in preparing a medicament for promoting hematopoiesis.
The invention also provides the use of a staple peptide as described above in the manufacture of a medicament for the treatment of a hematopoietic disorder.
The invention also provides application of the staple peptide in preparing a medicament for promoting the expansion of hematopoietic stem cells.
The invention also provides application of the staple peptide in preparation of a medicament for promoting mesenchymal stem cell expansion.
The present invention also provides a method of expanding stem cells in vitro, the method comprising: the stem cells were inoculated into a medium containing the stapled peptide as described above for culturing.
Optionally, wherein the stem cells are hematopoietic stem cells.
Optionally, the hematopoietic stem cells are umbilical cord hematopoietic stem cells, uterine blood hematopoietic stem cells or bone marrow hematopoietic stem cells.
Alternatively, wherein the stem cells are mesenchymal stem cells, preferably umbilical cord mesenchymal stem cells.
Alternatively, wherein the concentration of the stapling peptides according to claim 1 in the culture medium is 0.1-100. Mu.M, preferably 5-20. Mu.M.
Through the technical scheme, the staple peptide can remarkably enhance the activity of JMJD1C in cells, so that the expansion of hematopoietic stem cells is promoted.
Additional features and advantages of the invention will be set forth in the detailed description which follows.
Drawings
The accompanying drawings are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate the invention and together with the description serve to explain, without limitation, the invention. In the drawings:
FIG. 1 is a sequence showing the JMJD1C zinc finger domain derived staple peptide.
FIGS. 2-8 show that the stapled peptide SAH-JZ3 promotes expansion of myeloid (MV 4-11, HL 60), lymphoid (SEM), T-cell (Jurkat) cells, but has no significant effect on natural killer cells (NK-92).
FIGS. 9-10 show the effect of the polypeptide SAH-JZ3 on the JMJD1C protein and JMJD1C target gene SCD in myeloid cell lines MOLM-13 and THP-1.
FIG. 11 shows SAH-JZ3 up-regulates cord blood mononuclear cell numbers.
FIG. 12 shows SAH-JZ3 up-regulates cord blood mononuclear cell numbers.
FIG. 13 shows that SAH-JZ3 up regulates umbilical cord mesenchymal stem cell numbers.
FIG. 14 shows that SAH-JZ3 does not affect umbilical cord mesenchymal stem cell differentiation.
FIG. 15 shows that SAH-JZ3 enhances hematopoietic stem cell reconstitution in recipient mice.
Detailed Description
The following describes specific embodiments of the present invention in detail with reference to the drawings. It should be understood that the detailed description and specific examples, while indicating and illustrating the invention, are not intended to limit the invention.
The invention provides a staple peptide, which is shown as SEQ ID NO.1,
SEQ ID NO.1: cdactxatlxnihvcqkcgfv; i.e., ac-CDAC (S5) ATL (S5) NIHWVCQKCGFV.
Wherein the staple peptide comprises an alpha-helix and two unnatural amino acids X,
x represents 2-amino-2-methyl-6-heptenoic acid (2-amino-2-methylhept-6-enoic acid);
the two unnatural amino acids X are cyclized.
Wherein the cyclization can be carried out by adopting a conventional cyclization method in the preparation of a staple peptide, for example, adopting DMF solution, using Grubbs Catalyst,2nd Generation as a Catalyst, reacting at room temperature, and carrying out olefin metathesis reaction.
The specific mechanism by which the staple peptide acts may be by antagonizing JMJD1C protein degradation to improve JMJD1C stability. The staple peptide can enhance the expansion of hematopoietic stem cells and mesenchymal stem cells.
The invention also provides application of the staple peptide in preparing a medicament for promoting hematopoiesis.
The invention also provides the use of a staple peptide as described above in the manufacture of a medicament for the treatment of a hematopoietic disorder.
The invention also provides application of the staple peptide in preparing a medicament for promoting the expansion of hematopoietic stem cells.
The invention also provides application of the staple peptide in preparation of a medicament for promoting mesenchymal stem cell expansion.
The present invention also provides a method of expanding stem cells in vitro, the method comprising: the stem cells were inoculated into a medium containing the stapled peptide as described above for culturing.
Optionally, wherein the stem cells are hematopoietic stem cells.
Optionally, the hematopoietic stem cells are umbilical cord hematopoietic stem cells, uterine blood hematopoietic stem cells or bone marrow hematopoietic stem cells.
Alternatively, wherein the stem cells are mesenchymal stem cells, preferably umbilical cord mesenchymal stem cells.
Alternatively, wherein the concentration of the stapling peptides according to claim 1 in the culture medium is 0.1-100. Mu.M, preferably 5-20. Mu.M.
Through the technical scheme, the staple peptide can remarkably enhance the activity of JMJD1C in cells, so that the expansion of hematopoietic stem cells is promoted.
The invention is further illustrated by the following examples, which are not intended to be limiting in any way.
Examples
The binding peptide modification was performed by inserting different positions in the JMJD1C zinc finger domain, resulting in a series of candidate binding peptides, as shown in figure 1.
FIG. 1 shows the sequence of JMJD1C zinc finger domain derived staple peptide. The full length of zinc finger domain and partial sequence of enzyme activity domain of histone demethylase JMJD1C are shown in figure 1, and 7 polypeptides are obtained by adding membrane penetrating peptide (TAT, sequence YGRKRRQRRRR), adding unnatural amino acid (X) at different positions and performing side chain crosslinking cyclization. ZFD represents zinc finger domain; JZ represents JMJD1C zinc finger domain (zinc finger domain, ZFD); myr represents a myristoylation modification of the N-terminus; JJ represents the enzyme catalytic domain (jumonji domain) of JMJD 1C; x represents the unnatural amino acid 2-amino-2-methyl-6-heptenoic acid.
Fig. 2 is a detailed staple peptide construction. 2A represents a staple peptide trans-helical structure; 2B gives the specific structure of X; 2C gives the specific sequence of the polypeptide inserted into the staple peptide.
The resulting polypeptides are then screened. As shown in FIGS. 2-8, the staple peptide SAH-JZ3 promotes expansion of myeloid (MV 4-11, HL 60), lymphoid (SEM), T-cell (Jurkat) cells, but has no significant effect on natural killer cells (NK-92).
Effects of different polypeptides on proliferation of myeloid cell lines MV4-11, HL60, lymphoid cell line SEM, T cell line Jurkat, natural killer cell line NK-92. MV4-11 is cultured by adopting 80 percent 1640 culture medium and 20 percent fetal bovine serum; HL60, SEM, jurkat were cultured with 90%1640 medium+10% foetal calf serum; NK-92 using 75% alpha MEM medium +12.5% fetal bovine serum +12.5% horse serum +30 units/mL interleukin 2 culture. All cultures were performed at 5% carbon dioxide, 37 ℃. Cells were seeded at 20 ten thousand/mL and polypeptide treatment was performed using a 4 hour serum-free culture step. Cell proliferation assays were performed using CCK8 reagent. All experiments were performed in 3 complete independent replicates. P means less than 0.05 is considered statistically significant. FIGS. 9-10 show the effect of the polypeptide SAH-JZ3 on the JMJD1C protein and JMJD1C target gene SCD in myeloid cell lines MOLM-13 and THP-1. MOLM-13 and THP-1 were cultured with 80%1640 medium and 20% fetal bovine serum at 37℃under 5% carbon dioxide. Cells were seeded at 20 ten thousand/mL and polypeptide treatment was performed using a 4 hour serum-free culture step. The antibody used in Western blot was JMJD1C antibody 17-10262 (Sigma, shanghai, china), SCD antibody (sc-58420, santa Cruz, shanghai, china). All experiments were subjected to 3 complete independent replicates and the right histogram was statistical analysis of the 3 replicates. P means less than 0.05 is considered statistically significant.
To further explore the mechanism of action of this polypeptide, the effect of SAH-JZ3 on intracellular genes was examined by RNA sequencing. As shown in Table 1, major genes positively regulated by SAH-JZ3 include lipid metabolism genes SCD, FADS2, etc., which are target genes for JMJD1C and are reduced by the knockout or small molecule inhibition of JMJD 1C. These results indicate that SAH-JZ3 can act as an agonist of JMJD1C on the one hand on target gene expression, and that SAH-JZ3 can increase JMJD1C expression so that its downstream genes such as SCD and the like are up-regulated on the other hand.
TABLE 1
Table 1 shows the results of SAH-JZ3 treated MOLM-13 cell RNA sequencing analysis. List of 10 genes before RNA sequencing. MOLM-13 cell culture As shown in FIG. 2, RNA sequencing was performed after 24 hours of treatment with 20. Mu. Mol SAH-JZ3 polypeptide (including 4 hours of preculture), the results were ordered according to the regulatory p finger, and the first 10 genes were selected and presented in the list.
Next, the effect of SAH-JZ3 on JMJD1C protein was examined, and the results showed that SAH-JZ3 was able to up-regulate JMJD1C protein levels (FIGS. 9-10).
The results show that SAH-JZ3 can promote cell proliferation, up-regulate JMJD1C target gene expression, and increase JMJD1C protein level, thus acting as an agonist of JMJD 1C.
The JMJD1C agonist SAH-JZ3 promotes hematopoietic stem cell expansion. The effect of SAH-JZ3 on cord blood was next examined. The effect of SAH-JZ3 on cord blood mononuclear cell number in liquid medium was first examined. FIG. 11 shows that SAH_JZ3 can up-regulate cord blood mononuclear cell count 1.4-1.92-fold.
FIG. 11 shows SAH-JZ3 up-regulates cord blood mononuclear cell numbers. The umbilical cord blood is separated from the umbilical cord of a healthy neonate, and umbilical cord blood mononuclear cells are obtained by adopting a centrifugal separation method, namely adding an equal volume of lymphocyte separation liquid, and centrifuging at 2000rpm for 20 minutes, and 100 ten thousand/ml of umbilical cord blood mononuclear cells are inoculated on a 24-hole culture plate, wherein the culture medium is DMEM/F12 1:1, 10% fetal bovine serum and 100ng/ml SCF,100ng/ml FLT3L,50ng/ml TPO, 10. Mu.g/ml low density lipoprotein, all reagents from eBioscience. A4 hour serum free incubation step was used for the 20. Mu. Mole SAH-JZ3 treatment. Cell counts were performed 7 days later. All experiments were repeated 3 times. P means less than 0.05 was considered statistically significant (indicated by asterisks).
The effect of SAH-JZ3 on colony forming ability was further examined considering that cell proliferation did not reflect the reconstitution ability of hematopoietic stem cells. As shown in FIG. 12, SAH-JZ3 greatly promotes umbilical cord cell colony formation.
FIG. 12 shows SAH-JZ3 up-regulates cord blood mononuclear cell numbers. Cord blood was isolated from healthy neonatal umbilical cord. After that, SAH-JZ3 (20. Mu. Mol) treatment was performed for 4 hours (serum-free), and 500 colony forming medium H4434 (Stem Cell, shanghai, china) was added to each well (24-well plate) for inoculation culture. Observation photographing was performed after 12 days. All experiments were repeated 3 times.
The JMJD1C agonist SAH-JZ3 promotes mesenchymal stem cell proliferation but does not promote differentiation. Further, the effect of SAH-JZ3 on proliferation of umbilical cord mesenchymal stem cells was examined, and as shown in FIG. 13, SAH-JZ3 promoted proliferation of mesenchymal stem cells.
FIG. 13 shows that SAH-JZ3 up regulates umbilical cord mesenchymal stem cell numbers. Umbilical cord mesenchymal stem cells were isolated from healthy neonatal umbilical cord. After that, the cells were subjected to 20. Mu. Mol polypeptide treatment for 4 hours (serum-free), and inoculated and cultured in 3000 cells per well (96-well plate) in DMEM high-sugar +10% fetal bovine serum. CCK8 proliferation assay was performed 2 days later. All experiments were repeated 3 times. P means less than 0.05 was considered statistically significant (indicated by asterisks).
Next, the effect of SAH-JZ3 on the differentiation of umbilical cord mesenchymal stem cells was examined, and as shown in FIG. 14, SAH-JZ3 did not affect the differentiation of mesenchymal stem cells.
FIG. 14 shows that SAH-JZ3 does not affect umbilical cord mesenchymal stem cell differentiation. Umbilical cord mesenchymal stem cells were isolated from healthy neonatal umbilical cord. After that, the cells were subjected to 20. Mu. Mol polypeptide treatment for 4 hours (serum-free), and inoculated and cultured in 3000 cells per well (96-well plate) in DMEM high-sugar +10% fetal bovine serum. RNA was extracted after 2 days for quantitative PCR detection. The mesenchymal stem cell neural development genes (GDNF, MAPT), the muscle development gene (MEF 2C) and the skeletal development gene (Osteocalcin, twist) are detected. All experiments were repeated 3 times. P means less than 0.05 was considered statistically significant (indicated by asterisks).
SAH-JZ3 enhances the ability of hematopoietic stem cells to reconstitute in recipient mice. The ability of SAH-JZ3 to reconstitute hematopoietic stem cells in recipient mice was further measured.
FIG. 15 shows that SAH-JZ3 enhances hematopoietic stem cell reconstitution in recipient mice. Umbilical cord mesenchymal stem cells were isolated from healthy neonatal umbilical cord. Followed by 20 micromolar polypeptide culture for 10 days. Recipient mice were vaccinated 10 days later. The figure shows how many CD34 positive cells are needed to achieve in vivo reconstitution in mice. The DMSO group required 1897, the group without DMSO required approximately 1259, and the SAH-JZ3 group required only 430. 6 mice in each group, P values less than 0.05 were considered to be significantly different.
The preferred embodiments of the present invention have been described in detail above with reference to the accompanying drawings, but the present invention is not limited to the specific details of the above embodiments, and various simple modifications can be made to the technical solution of the present invention within the scope of the technical concept of the present invention, and all the simple modifications belong to the protection scope of the present invention.
In addition, the specific features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various possible combinations are not described further.
Moreover, any combination of the various embodiments of the invention can be made without departing from the spirit of the invention, which should also be considered as disclosed herein.
Claims (10)
1. A staple peptide is shown in SEQ ID NO.1,
SEQ ID NO.1：CDACXATLXNIHWVCQKCGFV；
wherein the staple peptide comprises an alpha-helix and two unnatural amino acids X,
x represents 2-amino-2-methyl-6-heptenoic acid;
the two unnatural amino acids X are cyclized.
2. Use of a staple peptide according to claim 1 for the manufacture of a medicament for promoting hematopoiesis.
3. Use of a staple peptide according to claim 1 for the manufacture of a medicament for the treatment of a hematopoietic disorder.
4. Use of a staple peptide according to claim 1 for the manufacture of a medicament for promoting expansion of hematopoietic stem cells.
5. Use of a staple peptide according to claim 1 for the manufacture of a medicament for promoting mesenchymal stem cell expansion.
6. A method of expanding stem cells in vitro, the method comprising: the stem cells are inoculated into a medium containing the staple peptide of claim 1 for culturing.
7. The method of claim 6, wherein the stem cells are hematopoietic stem cells.
8. The method of claim 6, wherein the hematopoietic stem cells are umbilical cord hematopoietic stem cells, uterine blood hematopoietic stem cells, or bone marrow hematopoietic stem cells.
9. The method according to claim 6, wherein the stem cells are mesenchymal stem cells, preferably umbilical cord mesenchymal stem cells.
10. The method according to any one of claims 6-9, wherein the concentration of the stapling peptides according to claim 1 in the medium is 0.1-100 μm, preferably 5-20 μm.
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| CN117756905A (en) * | 2023-12-22 | 2024-03-26 | 潍坊医学院 | Staple peptide and pharmaceutical application thereof |
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