Polypeptide for male contraception and preparation method and application thereof
By designing the peptide TAT-AT2R-C, which contains a membrane-penetrating peptide and an AT2R functional region, the downstream AT2R signaling pathway is blocked, overcoming the shortcomings of existing male contraceptive methods and achieving a safe and reversible contraceptive effect, making it suitable for industrial production.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-01-16
- Publication Date
- 2026-04-10
AI Technical Summary
Existing male contraceptive methods have issues with permanence, inconvenience, or reliability. There is a lack of safe, effective, and reversible non-hormonal contraceptive drugs, and the AT2R peptide sequence design makes it difficult to maintain stability in the intracellular environment and specifically interfere with signaling pathways.
A polypeptide containing a membrane-penetrating peptide and an AT2R functional region was designed. By blocking the downstream signaling pathway of AT2R, the preparation method includes genetic engineering and protein purification. The purified polypeptide TAT-AT2R-C can specifically interfere with the binding of AT2R to β-arrestin, thereby achieving a contraceptive effect.
The peptide TAT-AT2R-C significantly reduces sperm count and motility, inhibits fertility, and has good reversibility. Reproductive function is restored after drug discontinuation, avoiding endocrine disorders. It has high safety and reversibility, making it suitable for industrial production.
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Figure CN121824784A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a polypeptide for male contraception, and a preparation method and application thereof. BACKGROUND
[0002] At present, the male contraception methods available worldwide are extremely limited, mainly limited to vasoligation, condom and external sperm collection. Vasoligation, as a permanent sterilization operation, has the risk of difficulty in recanalization and low success rate; and condom and external sperm collection are difficult to meet the needs of couples for long-term and reliable contraception due to inconvenience or reliability problems. Therefore, developing a safe, effective and reversible non-hormonal male contraceptive drug has become an urgent and promising task in the field of reproductive health.
[0003] In the process of exploring new targets for male contraception, angiotensin II type 2 receptor (AT2R) has attracted the attention of researchers. AT2R belongs to the G protein-coupled receptor (GPCR) family and plays a crucial role in the male reproductive system. Existing studies have shown that AT2R is highly expressed in human sperm, and its expression level is positively correlated with sperm motility and forward movement ability, and negatively correlated with the proportion of immotile sperm. In addition, clinical data further confirm that the expression of AT2R is abnormal in patients with idiopathic oligoasthenospermia, and the expression level of AT2R in sperm is significantly reduced in patients with azoospermia. These findings collectively reveal that AT2R is a key factor for maintaining the normal physiological function of sperm, suggesting that human intervention in the function of this receptor may be an effective strategy to regulate male fertility.
[0004] However, how to translate the findings of this basic research into a feasible contraceptive drug still faces huge technical bottlenecks. As a typical GPCR, AT2R is located on the cell membrane, and its function depends on the interaction with intracellular downstream signal proteins (such as G protein and β-arrestin), thereby activating a complex signal pathway network to precisely regulate the functions of sperm. The interaction of GPCR with downstream effectors is mainly mediated by its intracellular domain, among which the carboxy (C) terminal, the largest domain of the intracellular region, plays a core role in this process.
[0005] Polypeptide drugs are very suitable for such reversible contraception strategy due to their high specificity, low side effects and rapid metabolism in vivo. Once the administration is stopped, the inhibited sperm function is expected to recover quickly, thus achieving the "on-demand control" of fertility. However, the conversion of this theory into a feasible drug faces a core technical bottleneck: how to rationally design the polypeptide sequence itself. Although the intracellular carboxy-terminal of AT2R is an ideal target for intervention of its function, it is a daunting challenge to directly screen from its lengthy natural sequence a key functional fragment capable of efficiently and specifically competing with the downstream signaling pathway. The designed polypeptide must remain stable in the complex intracellular environment and accurately block specific protein-protein interactions with its unique spatial conformation, rather than non-specifically interfering with other cellular processes. There is a lack of specific polypeptide sequences derived from AT2R in the prior art that can effectively perform this function.
[0006] In addition, although the technology of delivering polypeptides into cells using a cell-penetrating peptide is relatively mature, how to effectively integrate this delivery capability with a precisely designed core functional polypeptide with biological activity to create a new male contraceptive drug is still an unsolved comprehensive problem. In summary, there is an urgent need in the art for a new technical solution to overcome the drawbacks of existing male contraception methods and to solve the technical obstacles of converting intracellular targets such as AT2R into effective drugs. SUMMARY
[0007] In view of the deficiencies of the prior art, the present application provides a polypeptide for male contraception, as well as a preparation method and application thereof. The polypeptide has the effect of inhibiting male reproductive ability in the short term and can competitively disrupt the downstream signaling pathway of AT2R, thereby affecting sperm motility and concentration, etc., to achieve the purpose of contraception.
[0008] The present application is realized by the following technical solutions:
[0009] A polypeptide, wherein the amino acid sequence of the polypeptide is shown as SEQ ID NO. 1, or shown as the 12th-60th amino acid residues of SEQ ID NO. 1.
[0010] A polypeptide, wherein the polypeptide comprises a first functional region capable of penetrating the cell membrane and a second functional region capable of interfering with the function of angiotensin II type 2 receptor; wherein the amino acid sequence of the second functional region has at least 90% homology with the 12th-60th amino acid residues of SEQ ID NO. 1, or the second functional region is a functionally equivalent variant obtained by adding, deleting, replacing or modifying one or more amino acids to the 12th-60th amino acid residues of SEQ ID NO. 1.
[0011] Preferably, the first functional region and the second functional region can be interchanged in their preceding and following positions in the polypeptide, and the first functional region can be replaced by other sequences with the same function.
[0012] A method for preparing the above-mentioned polypeptide includes the following steps:
[0013] Step 1) Extract total RNA from AC16 cardiomyocytes, clone the DNA fragment encoding the C-terminal polypeptide sequence of angiotensin II type 2 receptor by RT-PCR, construct the expression vector pWaldo-TAT-AT2R-C by enzyme digestion and ligation, transform it into E. coli expression strain BL21(DE3) after correct sequencing, and optimize the expression system.
[0014] Step 2) Inoculate the recombinant strain into the culture medium, induce protein expression with IPTG, and continue culturing for 20 h. After centrifugation, collect the cells for protein purification. Obtain the TAT-AT2R-C and GFP fusion protein with high purity by passing through a nickel column and a molecular sieve chromatography column.
[0015] Step 3) Add TEV protease to the fusion protein sample obtained in Step 2), digest overnight at 4°C, remove GFP component by passing through a nickel column, and finally collect the purified TAT-AT2R-C polypeptide sample.
[0016] An isolated nucleic acid molecule that encodes the aforementioned polypeptide.
[0017] Preferably, the nucleotide sequence of the nucleic acid molecule is as shown in SEQ ID NO.2, or as shown in SEQ ID NO.2 from position 34 to 180 from the 5' end.
[0018] A recombinant expression vector comprising the aforementioned nucleic acid molecules.
[0019] A host cell comprising the aforementioned nucleic acid molecule or the aforementioned recombinant expression vector.
[0020] The above-mentioned peptides are used in the preparation of male contraceptive drugs.
[0021] Preferably, the polypeptide achieves the purpose of inhibiting male reproductive capacity in the short term by blocking the downstream signaling pathway of angiotensin II type 2 receptor.
[0022] The beneficial effects of this invention are as follows:
[0023] (1) The peptide contraceptive effect of the present invention is clear, reliable, and reversible. Key in vivo animal experiments have confirmed that the peptide can significantly reduce the sperm count and inhibit sperm motility in male mice, thereby directly leading to a decline in their fertility, demonstrating clear contraceptive efficacy. More importantly, this inhibitory effect is completely reversible. After stopping the administration, the sperm parameters and fertility of the mice can be restored, perfectly meeting the core requirements of an ideal male contraceptive: "high efficiency and reversibility".
[0024] (2) The peptide of this invention has a specific mechanism of action, and in vitro molecular experiments have revealed its unique mechanism of action. This peptide does not exert its effect by affecting the expression level of the AT2R receptor itself, but rather by specifically interfering with its downstream signaling pathways, as confirmed by the significant reduction in the expression levels of β-arrestin-1 and β-arrestin-2. This hormone-free pathway of action avoids the systemic side effects such as endocrine disorders that may be caused by traditional hormonal contraceptives, thus offering greater safety.
[0025] (3) The core functional region of the polypeptide of the present invention is directly derived from the carboxyl terminus sequence of AT2R itself, which enables it to act as a highly specific “competitive inhibitor” to precisely block the binding of AT2R to downstream signaling proteins (such as β-arrestin). This design based on the natural structure ensures the precision of its action and good biocompatibility.
[0026] (4) Based on the combined in vitro and in vivo experiments, the polypeptide of the present invention, while effectively exerting its contraceptive effect, did not show significant toxic effects on target cells (such as HEK293 and GC-1 spg cells), and its effect is reversible and non-hormonal. These results collectively indicate that the polypeptide, as a novel contraceptive drug, has a high expected safety profile.
[0027] (5) The polypeptides of the present invention can be produced by mature gene engineering recombinant expression technology. The production method is relatively mature, easy to control, has high purity, and is easy to scale up, laying a solid technical foundation for subsequent industrial production and drug development, and has good clinical translation potential and market application prospects. Attached Figure Description
[0028] Figure 1 The effect of peptide TAT-AT2R-C on mouse fertility in Example 2 is shown in Figure A: a statistical graph of sperm count in each group of mice; a statistical graph of sperm motility in each group of mice; and a statistical graph of fertility rate in each group of mice.
[0029] Figure 2The effects of peptide TAT-AT2R-C on intracellular protein expression in Example 3 are shown in Figure A: the effect of TAT-AT2R-C on the expression of β-arrestin-1 and AT2R; and the effect of TAT-AT2R-C on the expression of β-arrestin-2 and AT2R. Detailed Implementation
[0030] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.
[0031] Unless otherwise specified, the technical means used in the following embodiments are all conventional means well known to those skilled in the art, and the experimental methods without specific conditions are all conventional methods in the art.
[0032] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.
[0033] The human cardiomyocytes AC16, human embryonic kidney HEK293, and mouse spermatogonia GC-1spg used in the following examples were all derived from ATCC (American Type Culture Collection).
[0034] The mice used in the following examples were all C57BL / 6 black mice, purchased from the Experimental Animal Center of Xuzhou Medical University.
[0035] Example 1: Preparation of peptide TAT-AT2R-C
[0036] The specific steps for preparing the TAT-AT2R-C polypeptide with penetrating activity are as follows:
[0037] 1. Construct expression vectors and screen recombinants
[0038] Total RNA was extracted from AC16 cardiomyocytes, and cDNA was obtained by reverse transcription. The nucleic acid sequence of the AT2R C-terminal polypeptide was obtained by PCR using the cDNA as a template. The upstream and downstream primers are as follows:
[0039] Upstream primer (SEQ ID NO.3): 5'-CCATCTCGAGATGTACGGTCGTAAAAAACGTCGTCAGCGTCGTCGTCCGTTTCTGTATTGTTTTGTTG-3';
[0040] Downstream primer (SEQ ID NO.4): 5'-CGTCGGATCCAGACACAAAGGTCTCCATTTCTC-3'.
[0041] The TAT coding sequence was inserted into the upstream primer, and XhoI and BamHI restriction sites were added to the upstream and downstream primers, respectively. The expression vector pWaldo-TAT-AT2R-C was constructed by restriction enzyme ligation method, transformed into Escherichia coli BL21(DE3) expression strain, and recombinants were screened.
[0042] 2. Obtain high-purity TAT-AT2R-C and GFP fusion protein
[0043] The recombinant strain was inoculated into 100 mL LB medium and cultured overnight at 37°C. It was then transferred to 2 L LB medium and cultured at 37°C until OD (October Expiratory Count) was reached. 600 Add approximately 0.5-0.6 g of IPTG to a final concentration of 0.3 mM to induce protein expression. Continue culturing at 20°C for 20 h. Collect cells by centrifugation at 6000 g for 15 min for protein purification.
[0044] Cells were suspended in 100 mL of lysis buffer (20 mM Tris-HCl, pH=7.5, 300 mM NaCl, 5% glycerol), and 50 μg / mL lysozyme, 200 U Dnase I, and 100 mM PMSF were added. Cells were lysed at 800 bar and centrifuged at 10,000 rpm for 30 min at 4°C to remove cell debris. The supernatant was passed through a nickel column, washed with 80 mL of wash buffer (2 mM Tris-HCl, pH=7.5, 300 mM NaCl, 5% glycerol, 30 mM imidazole) to remove contaminating proteins, and eluted with elution buffer (20 mM Tris-HCl, pH=7.5, 300 mM NaCl, 5% glycerol, 30 mM imidazole) to collect proteins. The proteins were then passed through a molecular sieve column (buffer: 20 mM Tris-HCl, pH=7.5, 300 mM NaCl, 5% glycerol, 30 mM imidazole). Further purification with NaCl (5% glycerol) yields high-purity TAT-AT2R-C and GFP fusion proteins.
[0045] 3. The TAT-AT2R-C polypeptide was purified and obtained.
[0046] 100 μL of 5 mg / mL TEV (tobacco mosaic virus) protease was added to the fusion protein sample, and the sample was digested overnight at 4°C. The sample was then passed through a nickel column to remove any incompletely digested fusion protein and the digested GFP fraction. The final collected TAT-AT2R-C peptide sample was concentrated to approximately 6 mg / mL using a 1,000 molecular weight cutoff concentration tube for functional studies.
[0047] The purified polypeptide TAT-AT2R-C has an amino acid sequence as shown in SEQ ID NO.1, consisting of 60 amino acid residues, including a first functional region and a second functional region. Residues 1-11 constitute the first functional region, the membrane-penetrating peptide TAT, which has the function of penetrating the cell membrane. Residues 12-60 constitute the second functional region, AT2R-C, which interferes with the function of AT2R. The positions of the first and second functional regions can be interchanged, and the first functional region can be replaced by other sequences with similar functions.
[0048] The nucleotide sequence of the gene encoding the polypeptide TAT-AT2R-C is shown in SEQ ID NO.2, with a length of 180 bp; wherein, positions 1-33 from the 5' end are the nucleotide sequence encoding the transmembrane peptide TAT, and positions 34-180 are the nucleotide sequence encoding AT2R-C.
[0049] Example 2: In vivo experiments of peptide TAT-AT2R-C
[0050] 1. Experimental Procedure
[0051] Blood was drawn from the heart of mice after anesthesia, followed by rinsing with physiological saline. The epididymis and testis were then quickly removed. Sperm from the epididymal tail were collected using the diffusion method. The removed mouse epididymal tail was placed in 1 mL of 37℃ physiological saline, chopped, and allowed to stand for 1 min. The mixture was then filtered through a 400-mesh filter to prepare a sperm suspension. 50 μL of the sperm suspension was placed in 950 μL of sperm culture medium and incubated at 37℃ for 5 min. After mixing, 20 μL was added to a cell counting chamber, photographed under an optical microscope, and used for sperm counting.
[0052] 10 μL of the filtered sperm suspension was added to a sperm testing plate, and sperm motility was tested using the SAS sperm analysis system (Cys Medical, Beijing).
[0053] Male C57BL / 6J mice aged 8-12 weeks were administered the peptide TAT-AT2R-C (10 mg / kg) intravenously, while the control group received an equal volume of physiological saline. The administration was once daily for 21 days. Afterward, two mature female mice were introduced into each male's cage and kept together for 10 days before being separated (with continued daily administration during this period). Pregnancy and farrowing in the females were monitored. After separation from the females, the male mice were discharged for 30 days and then re-enclosed with two mature female mice for 10 days, with continued observation of pregnancy and farrowing in the females.
[0054] 2. Experimental Results
[0055] like Figure 1 As shown, the peptide TAT-AT2R-C can significantly inhibit sperm motility in male mice. Figure 1(A) Reduce sperm count ( Figure 1 (B), thereby inhibiting the fertility of mice ( Figure 1 (C), and after stopping the drug, the sperm count and motility will recover, and eventually the male mice will regain their fertility.
[0056] Example 3: In vitro experiments of peptide TAT-AT2R-C
[0057] 1. Experimental Procedure
[0058] HEK293 and GC-1 spg cells were treated with TAT-AT2R-C peptide, and total protein was extracted. Proteins of different molecular weights were separated by SDS-PAGE and then transferred to PVDF membranes via a semi-dry / wet transfer method. The membranes were then incubated with PBS containing 5% skim milk powder at room temperature for 2 h, followed by incubation with primary antibody at 4°C overnight. The membranes were then washed three times with PBST, incubated with secondary antibody at room temperature for 1 h, washed again with PBST, and then chemiluminescence buffer was added. The membranes were exposed to X-ray film, developed, and fixed. After image scanning, the bands were analyzed using ImageJ software. The effect of the peptide on the expression of proteins such as β-arrestin-1, β-arrestin-2, and AT2R was investigated.
[0059] 2. Experimental Results
[0060] like Figure 2 As shown, the peptide TAT-AT2R-C had no significant effect on the expression of AT2R protein in HEK293 and GC-1 spg cells, but it could significantly reduce β-arrestin-1 ( Figure 2 (A, P = 0.035) and β-arrestin-2 ( Figure 2 The expression of B, P = 0.041).
[0061] The experimental results of the above embodiments show that the polypeptide TAT-AT2R-C of the present invention can interfere with the downstream signaling pathway of AT2R, inhibit the expression of β-arrestin-1 and β-arrestin-2, thereby blocking the functions of sperm concentration and motility regulated by AT2R and its downstream signals, and achieving the purpose of inhibiting male reproduction in the short term. This inhibition will recover as the polypeptide degrades, and reproductive capacity can be rapidly restored after drug withdrawal.
[0062] The embodiments described above are only some, not all, of the embodiments of the present invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments. The scope of protection of the present invention is determined by the scope claimed in the claims. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.
Claims
1. A polypeptide, characterized in that, The amino acid sequence of the polypeptide is shown in SEQ ID NO.1, or in amino acid residues 12-60 of SEQ ID NO.
1.
2. A polypeptide, characterized in that, The polypeptide comprises a first functional region capable of penetrating the cell membrane and a second functional region capable of interfering with the function of angiotensin II type 2 receptor; wherein the amino acid sequence of the second functional region has at least 90% homology with amino acid residues 12-60 of SEQ ID NO.1, or the second functional region is a functionally equivalent variant obtained by adding, deleting, substituting or modifying one or more amino acids from amino acid residues 12-60 of SEQ ID NO.
1.
3. The polypeptide according to claim 2, characterized in that, The first and second functional regions can be interchanged in their preceding and following positions within the polypeptide, and the first functional region can be replaced by other sequences having the same function.
4. A method for preparing a polypeptide as described in any one of claims 1-3, characterized in that, Includes the following steps: Step 1) Extract total RNA from AC16 cardiomyocytes, clone the DNA fragment encoding the C-terminal polypeptide sequence of angiotensin II type 2 receptor by RT-PCR, construct the expression vector pWaldo-TAT-AT2R-C by enzyme digestion and ligation, transform it into E. coli expression strain BL21(DE3) after correct sequencing, and optimize the expression system. Step 2) Inoculate the recombinant strain into the culture medium, induce protein expression with IPTG, and continue culturing for 20 h. After centrifugation, collect the cells for protein purification. Obtain the TAT-AT2R-C and GFP fusion protein with high purity by passing through a nickel column and a molecular sieve chromatography column. Step 3) Add TEV protease to the fusion protein sample obtained in Step 2), digest overnight at 4°C, remove GFP component by passing through a nickel column, and finally collect the purified TAT-AT2R-C polypeptide sample.
5. An isolated nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the polypeptide as described in any one of claims 1-3.
6. The isolated nucleic acid molecule according to claim 5, characterized in that, The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO.2, or as shown in SEQ ID NO.2 from position 34 to 180 from the 5' end.
7. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the nucleic acid molecule as described in claim 6.
8. A host cell, characterized in that, The host cell contains the nucleic acid molecule as described in claim 6, or the recombinant expression vector as described in claim 7.
9. The use of the polypeptide according to any one of claims 1-3 in the preparation of male contraceptive drugs.
10. The application according to claim 9, characterized in that, The polypeptide inhibits male fertility in the short term by blocking the downstream signaling pathway of the angiotensin II type 2 receptor.