Polypeptide TAT-FSHR-pep and application thereof
By designing the polypeptide TAT-FSHR-pep to compete to block FSHR/β-arrestins interaction, restoring the FSHR pro-apoptotic function, solving the problem of easy desensitization of drugs in the prior art, achieving efficient and low-toxic treatment for ovarian cancer, and expanding to the application of other FSHR-dependent cancers.
Patent Information
- Application Number
- CN202510545998.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-28
- Publication Date
- 2025-07-25
AI Technical Summary
There is a lack of polypeptide drugs in the prior art that competes to block FSHR/β-arrestins interactions to restore FSHR pro-apoptotic function. Traditional GPCR-targeted drugs are susceptible to desensitization or off-target effects, and ovarian cancer treatment faces problems such as high systemic toxicity and recurrence.
The peptide TAT-FSHR-pep was designed to simulate the binding epitope of FSHR and β-arrestins, and competitively inhibited their interactions. The TAT-permeable peptide sequence was fused to efficiently penetrate the cell membrane, specifically interfering with the inhibition of FSHR proapoptotic function by β-arrestins.
It significantly inhibits the proliferation of ovarian cancer cells, reduces toxicity to healthy tissues, avoids off-target risks, extends the treatment response cycle, and expands to the treatment of other FSHR-dependent cancers, with broad clinical application prospects.
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Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a polypeptide TAT-FSHR-pep and its application. Background Art
[0002] Ovarian cancer is the most lethal malignant tumor in the female reproductive system. Among them, serous ovarian cancer faces severe challenges in clinical treatment due to its strong invasiveness, easy metastasis, and chemotherapy resistance. Although traditional chemotherapy can temporarily inhibit tumor progression, it has problems such as high systemic toxicity and easy recurrence, and there is an urgent need to develop highly efficient and low-toxic targeted therapies for specific molecular mechanisms.
[0003] Membrane protein receptors are common cancer treatment targets. As G protein-coupled receptors, the expression of FSHR (follicle-stimulating hormone receptor) is found in 50% - 70% of serous ovarian cancers. It is mainly selectively expressed in ovarian granulosa cells and plays a key role in the occurrence and development of ovarian cancer. Moreover, importantly, no obvious expression of FSHR is found in healthy cells outside the ovaries in women, so its function may be organ- or cell-dependent, which provides a good opportunity for targeted treatment of ovarian cancer.
[0004] As a G protein-coupled receptor (GPCR), the role of FSHR in ovarian cancer is regulated by β-arrestins. This interaction mediates the function of FSHR in the occurrence and development of ovarian cancer. It is worth noting that the analysis of ovarian cancer data in the Kaplan-Meier Plotter database shows that the expression of β-arrestins (β-arrestin1 and β-arrestin2) is increased in ovarian cancer tissues, and their expression is often related to poor prognosis. There is a significant difference in the median survival time between high-expression patients and low-expression patients. And the median survival time of FSHR high-expression patients is significantly longer than that of low-expression patients. The high expression of FSHR in ovarian cancer may be a means for the body to clear ovarian cancer because some studies suggest that FSHR may mediate the apoptosis of ovarian cancer cells, while β-arrestins block the role of FSHR; if β-arrestins are knocked out, it can induce cell apoptosis. As a typical seven-transmembrane G protein-coupled receptor, the carboxyl-terminal region of FSHR may play a key role in the interaction with β-arrestins.
[0005] Existing tumor treatment strategies targeting GPCRs mostly focus on the development of receptor agonists or antagonists. However, such small molecule drugs are easily restricted by receptor desensitization or off-target effects. The carboxyl terminus of FSHR, as the key domain for binding to β-arrestins, the discovery of its specific interaction interface provides a new direction for precise intervention. However, there is currently no reported polypeptide drug that competitively blocks the FSHR / β-arrestins interaction to selectively restore the pro-apoptotic function of FSHR. Summary of the Invention
[0006] In view of the deficiencies of the prior art, the present invention provides a polypeptide TAT-FSHR-pep and its application. By mimicking the binding epitope of FSHR and β-arrestins, it competitively inhibits their interaction. This design is expected to relieve the negative regulation of β-arrestins on the FSHR signaling pathway, reconstruct the pro-apoptotic effect mediated by FSHR, and at the same time avoid the desensitization risk of traditional GPCR-targeted drugs, providing a candidate drug with both tissue specificity and mechanism innovation for ovarian cancer treatment.
[0007] The present invention is achieved through the following technical solutions:
[0008] A polypeptide TAT-FSHR-pep, the polypeptide TAT-FSHR-pep is selected from the polypeptides of the following (i) or (ii):
[0009] (i) A polypeptide having the amino acid sequence shown in SEQ ID NO.1;
[0010] (ii) A polypeptide having the amino acid residues at positions 12-77 of the amino acid sequence shown in SEQ ID NO.1.
[0011] A gene encoding the above polypeptide TAT-FSHR-pep, the gene is selected from the genes of the following (iii) or (iv):
[0012] (iii) A gene having the nucleotide sequence shown in SEQ ID NO.2;
[0013] (iv) A gene having the nucleotides at positions 34-231 from the 5'-end of the nucleotide sequence shown in SEQ ID NO.2.
[0014] An expression cassette, recombinant vector or recombinant bacterium containing the above gene.
[0015] Use of the above polypeptide TAT-FSHR-pep, the above gene or the above expression cassette, recombinant vector or recombinant bacterium in the preparation of a drug for preventing and / or treating FSHR-dependent cancer.
[0016] Preferably, the FSHR-dependent cancers include ovarian cancer, thyroid cancer, and prostate cancer.
[0017] Preferably, the drug is any pharmaceutically acceptable dosage form.
[0018] A pharmaceutical composition for treating ovarian cancer, wherein the active ingredient of the pharmaceutical composition is the above-mentioned polypeptide TAT-FSHR-pep or the above-mentioned gene.
[0019] Preferably, it further comprises a pharmaceutically acceptable drug delivery carrier.
[0020] The principle of the present invention is as follows:
[0021] The present invention designs a polypeptide based on the amino acid sequence of the carboxyl-terminal region of FSHR, which can competitively replace the interaction between FSHR and β-arrestins. And since this interfering polypeptide cannot transmit the information mediated by the complete FSHR protein, it will disrupt the function of β-arrestins, thereby achieving the purpose of inhibiting the proliferation of ovarian cancer cells. At the same time, when developing the polypeptide as a drug, it needs to enter ovarian cancer cells to play a role, so the TAT transmembrane peptide sequence is added to its amino terminus.
[0022] The beneficial effects of the present invention are as follows:
[0023] (1) The polypeptide TAT-FSHR-pep designed by the present invention specifically targets the interaction interface between the carboxyl terminus of FSHR and β-arrestins. Since FSHR is highly expressed in ovarian cancer but hardly expressed in normal non-ovarian tissues, this polypeptide can selectively act on tumor cells, significantly reducing the toxicity to healthy tissues and improving the treatment safety.
[0024] (2) The polypeptide TAT-FSHR-pep of the present invention competitively blocks the binding between FSHR and β-arrestins. On the one hand, it relieves the inhibition of the pro-apoptotic function of FSHR by β-arrestins and restores the apoptotic effect of cancer cells mediated by FSHR; on the other hand, it inhibits the β-arrestins-dependent pro-proliferation signaling pathway, achieving a "dual anti-cancer" effect and effectively curbing tumor growth and metastasis.
[0025] (3) Compared with small molecule agonists / antagonists that are prone to cause receptor desensitization or drug resistance problems, the polypeptide TAT-FSHR-pep of the present invention intervenes in the interaction between the receptor and regulatory proteins, directly regulates downstream signal branches, avoids interfering with the overall activity of FSHR, reduces the off-target risk from the source, and prolongs the treatment response cycle.
[0026] (4) The polypeptide TAT-FSHR-pep of the present invention fuses with the TAT cell-penetrating peptide sequence, which can efficiently penetrate cell membranes and tumor tissue barriers, ensuring the enrichment of drug molecules at the lesion site. Meanwhile, the short peptide sequence designed based on the natural interaction epitope of FSHR has both structural stability and low immunogenicity, laying a foundation for in vivo application.
[0027] (5) Clinical data analysis shows that high expression of β-arrestins is strongly correlated with poor prognosis in ovarian cancer patients. The polypeptide TAT-FSHR-pep of the present invention is expected to reverse its pro-cancer effect and extend the survival of patients by targeting and inhibiting the function of β-arrestins. In addition, for the new mechanism of the FSHR / β-arrestins axis, it can bypass the traditional chemotherapy resistance pathway and provide an alternative treatment plan for drug-resistant ovarian cancer.
[0028] (6) In addition to ovarian cancer, FSHR is also specifically expressed in some tumors such as thyroid cancer and prostate cancer. The polypeptide TAT-FSHR-pep of the present invention can be extended to the treatment of other FSHR-dependent cancers through the same mechanism of action, showing broad clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS
[0029] Figure 1 Purification of the polypeptide TAT-FSHR-pep in Example 1;
[0030] Figure 2 Effect of the polypeptide TAT-FSHR-pep on the proliferation of ovarian cancer cells SKOV3 and CAOV3 in Example 2;
[0031] Figure 3 Effect of the polypeptide TAT-FSHR-pep on the cAMP level of ovarian cancer cells SKOV3 and CAOV3 in Example 3;
[0032] Figure 4 Effect of the polypeptide TAT-FSHR-pep on the expression level of β-arrestins in Example 3: Lane 1 is the cancer cell control group; Lane 2 is the polypeptide TAT-FSHR-pep treatment group. DETAILED DESCRIPTION OF THE INVENTION
[0033] The present invention will be further described in detail below with reference to the drawings and specific examples.
[0034] Unless otherwise specified, the technical means used in the following examples are all conventional means well known to those skilled in the art. The experimental methods without specific conditions mentioned are all conventional methods in the art.
[0035] The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.
[0036] The ovarian cancer SKOV3 cells, CAOV3 cells, etc. in the following examples are all derived from ATCC (American Type Culture Collection).
[0037] Example 1 Obtaining of Polypeptide TAT-FSHR-pep
[0038] This example provides the process for obtaining the TAT-FSHR-pep polypeptide with transmembrane activity, and the specific steps are as follows:
[0039] (1) Extract the total RNA of SKOV3 cells, reverse transcribe to obtain cDNA, and use the cDNA as a template to obtain the nucleic acid sequence of the FSHR carboxyl-terminal polypeptide by PCR. The specific upstream and downstream primers are as follows:
[0040] Upstream primer (SEQ ID NO.3): 5’-CCATCTCGAGATGTACGGTCGTAAAAAACG TCGTCAGCGTCGTCGTACCAAAAACTTTCGCAGAGATTTC-3’;
[0041] Downstream primer (SEQ ID NO.4): 5’-CGTCGGATCCGTTTTGGGCTAAATGACTTA GAG-3’ (SEQ IDNO.8).
[0042] Insert the TAT coding sequence into the upstream primer, add XhoI and BamHI restriction enzyme sites to the upstream and downstream primers respectively, and construct the expression vector pWaldo-TAT-FSHR-pep by restriction enzyme digestion and ligation method, and screen the recombinants.
[0043] Among them, pWaldo is a vector obtained by inserting GFP-8His into pET28(a+). Its synthesis can refer to the literature: Drew, D.E., von Heijne, G., Nordlund, P. & de Gier, J.W. Green fluorescent protein as an indicator to monitor membrane protein overexpression in Escherichia coli. FEBS Lett 507, 220-4 (2001).
[0044] (2) Transform the recombinant with correct sequencing into the Escherichia coli BL21(DE3) expression strain. Inoculate the recombinant strain into 200 mL of LB medium and culture overnight at 37°C. Transfer 50 mL of the overnight culture to 1 L of LB medium and culture at 37°C until OD 600Between 0.5 and 0.6, IPTG with a final concentration of 0.1 mM was added to induce protein expression, and the culture was continued at 20 °C for 20 h. The cells were collected by centrifugation at 6000 g for 15 min for protein purification.
[0045] (3) The cells were suspended in 120 mL of lysis buffer (20 mM Tris-HCl, pH 7.5; 300 mM NaCl, and 5% glycerol), 60 μg / mL of lysozyme, 200 U of DnaseI, and 100 mM PMSF were added, and the cells were disrupted at 800 bar. The cell debris was removed by centrifugation at 10000 rpm at 4 °C for 30 min. The supernatant was passed through a nickel column, and the washing buffer (20 mM Tris-HCl, pH 7.5; 300 mM NaCl, 5% glycerol, and 30 mM imidazole) was used to wash 100 mL to remove the impurity proteins. The elution buffer (20 mM Tris-HCl, pH 7.5; 300 mM NaCl, 5% glycerol, and 300 mM imidazole) was used for elution, and the protein was collected and further purified by a molecular sieve column (buffer: 20 mM Tris-HCl, pH 7.5; 300 mM NaCl, and 5% glycerol) to obtain a TAT-FSHR-pep polypeptide and GFP fusion protein with a higher purity.
[0046] (4) To further obtain the TAT-FSHR-pep polypeptide, 100 μL of TEV (tobacco etch virus) protease with a concentration of 5 mg / mL was added to the protein sample, and the sample was digested overnight at 4 °C. The sample was passed through the nickel column again to remove the incompletely digested fusion protein and the digested GFP component. The finally collected sample was the purified TAT-FSHR-pep polypeptide, which was concentrated to about 3 mg / mL through a concentration tube with a molecular weight cut-off of 1000 for the functional research of the subsequent examples.
[0047] As Figure 1 shown, a TAT-FSHR-pep polypeptide with a higher purity (>92%) can be obtained according to the above protocol.
[0048] The amino acid sequence of the polypeptide TAT-FSHR-pep is shown in SEQ ID NO.1 and consists of 77 amino acid residues. Among them, the 1-11 amino acid residues form a region with the function of crossing the cell membrane, that is, the transmembrane peptide TAT. The positions of this region and the 12-77 amino acids can be interchanged, and it can be replaced by other sequences with similar functions. The 12-77 amino acid residues constitute a region that can interact with the β-arrestins protein, that is, FSHR-pep.
[0049] The nucleotide sequence of the polypeptide TAT-FSHR-pep encoding gene is shown in SEQ ID NO.2, with a length of 231bp; among them, the nucleotide sequence encoding the transmembrane peptide TAT is from the 1st to the 33rd position starting from the 5'-end, and the nucleotide sequence encoding FSHR-pep is from the 34th to the 231st position.
[0050] Example 2 Application research of polypeptide TAT-FSHR-pep
[0051] 1. Experimental steps
[0052] MTT assay: SKOV3 cells and CAOV3 cells were seeded in 96-well plates. After adherent for 12 h, 0.2 mg / mL polypeptide TAT-FSHR-pep was added. After 2 h, 0.1 mg / mL polypeptide TAT-FSHR-pep was added again, and the cells with PBS added were used as the control. After 48 h of drug treatment, 10 μL of 5 mg / mL MTT solution was added to the cell wells, and incubated on a low-speed shaker for 3 - 4 h. Subsequently, the mixture of MTT and culture medium was removed, and 100 μL of DMSO solution was added. Due to the presence of MTT, viable cells will form crystals, and the crystals will then dissolve in the DMSO solution. The absorbance at a wavelength of 490 nm was detected by an enzyme-linked immunosorbent assay detector.
[0053] 2. Experimental results
[0054] The results of the MTT assay are as Figure 2 shown. Compared with the PBS control group, the absorbance values of SKOV3 cells and CAOV3 cells treated with TAT-FSHR-pep at a wavelength of 490 nm were significantly decreased ( ** P < 0.01), indicating that this polypeptide can effectively inhibit the proliferation activities of the two ovarian cancer cell lines.
[0055] This example confirmed by the MTT method that the polypeptide TAT-FSHR-pep can significantly inhibit the proliferation of ovarian cancer cells, and the effect is statistically significant ( ** P < 0.01), providing direct evidence for its anti-cancer mechanism by interfering with the FSHR / β-arrestins interaction. This result laid an important experimental foundation for the subsequent development of new ovarian cancer treatment drugs targeting the GPCR-β-arrestins axis.
[0056] Example 3 Mechanism study of polypeptide TAT-FSHR-pep
[0057] In order to further study the mechanism of action of the polypeptide TAT-FSHR-pep in inhibiting ovarian cancer, the following experiments were designed in this example.
[0058] 1. Experimental steps
[0059] (1) The effect of treating SKOV3 and CAOV3 cells with polypeptide TAT-FSHR-pep for 48 h on the cAMP level in the cell culture medium was detected using an ELISA kit.
[0060] The cell culture medium was centrifuged at 1000 r / min for 10 min at room temperature, the supernatant was taken, 0.1 mol / L hydrochloric acid was added to the supernatant, and it was pipetted and mixed well. After standing at room temperature for 15 min, Tris solution was added to neutralize the pH, and then added to the enzyme-linked immunosorbent assay (ELISA) plate, and the cAMP concentration was detected using an enzyme-labeling instrument.
[0061] (2) The effect of treating SKOV3 cells with polypeptide TAT-FSHR-pep for 48 h on the protein expression levels of β-arrestin-1 and β-arrestin-2 was detected by Western blot.
[0062] After treating with polypeptide TAT-FSHR-pep for 48 h, the supernatant was removed, the cells were washed with PBS, and the cells were collected after trypsin digestion. Proteins were separated by SDS-PAGE, transferred to a membrane, incubated with the primary antibody overnight at 4 °C, the membrane was washed with TBST, incubated with the secondary antibody at room temperature for 1 h, and then developed.
[0063] 2. Experimental results
[0064] The ELISA results were as Figure 3 shown. The cAMP concentrations in the SKOV3 and CAOV3 cells in the TAT-FSHR-pep treatment group were significantly increased compared with the control group, indicating that polypeptide TAT-FSHR-pep releases the G protein-coupled activity of FSHR by blocking the binding of β-arrestins to FSHR, and restores the function of the Gαs / cAMP / PKA signaling pathway. As a second messenger, the increase in cAMP level may activate the downstream molecular mechanisms of apoptosis promotion or proliferation inhibition.
[0065] The WB results were as Figure 4 shown. The protein expressions of β-arrestin-1 and β-arrestin-2 in the SKOV3 cells in the TAT-FSHR-pep treatment group were almost completely inhibited (the bands in lane 2 almost disappeared), suggesting that polypeptide TAT-FSHR-pep may act through the following mechanisms: (1) directly competitively blocking the binding of β-arrestins to FSHR, resulting in the ubiquitination and degradation of free β-arrestins; (2) inhibiting the transcription or translation of β-arrestins through negative feedback regulation.
[0066] The experimental results of this example indicate that the downregulation of β-arrestins releases the G protein-coupled activity of FSHR, reactivates the cAMP pathway, and may promote cancer cell apoptosis or inhibit proliferation (consistent with the MTT results of Example 2). At the same time, β-arrestins themselves can drive tumor progression by activating pro-proliferation pathways such as MAPK / ERK, and their downregulation further weakens the survival and proliferation ability of cancer cells. It is suggested that the polypeptide TAT-FSHR-pep may play an inhibitory role in ovarian cancer by inhibiting the overexpression of β-arrestins, dissociating the interaction between β-arrestin-1 and β-arrestin-2 and FSHR, and the FSHR released by β-arrestins will restore the vitality of the G protein pathway - increasing cAMP.
[0067] The experimental results of the above examples completely reveal the action chain of the polypeptide TAT-FSHR-pep in inhibiting ovarian cancer: the polypeptide TAT-FSHR-pep blocks the interaction between FSHR / β-arrestins, thereby causing β-arrestins to be degraded or their expression to be inhibited, and then activating the FSHR-G protein pathway (the cAMP concentration increases), as well as turning off the pro-cancer signal of β-arrestins, ultimately synergistically inhibiting the proliferation of ovarian cancer.
[0068] The experimental results of the above examples are highly consistent with the previous mechanism hypothesis, that is, the polypeptide TAT-FSHR-pep competitively binds to β-arrestins, blocks their binding to FSHR, dissociates the inhibition of the pro-apoptotic function of FSHR by β-arrestins, restores the FSHR / cAMP pro-apoptotic pathway, and at the same time inhibits the expression / function of β-arrestins, ultimately resulting in a decrease in the proliferation ability of cancer cells. This result provides direct evidence for the molecular mechanism of the polypeptide TAT-FSHR-pep and supports its potential therapeutic strategy as a target for β-arrestins overexpressing ovarian cancer. Moreover, the polypeptide TAT-FSHR-pep shows significant inhibitory effects on two different ovarian cancer cell lines (SKOV3 and CAOV3), suggesting that its action may not be limited to a single cell type and has potential broad-spectrum anti-ovarian cancer activity.
[0069] The embodiments described above are only a part of the embodiments of the present invention, not all of them. The detailed description of the embodiments of the present invention is not intended to limit the scope of the present invention claimed, but merely represents the selected embodiments of the present invention. The protection scope of the present invention shall be subject to the scope claimed in the claims. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.
Claims
1. A polypeptide TAT-FSHR-pep, characterized in that, The polypeptide TAT-FSHR-pep is selected from the polypeptides of the following (i) or (ii): (i) A polypeptide having the amino acid sequence shown in SEQ ID NO.1; (ii) A polypeptide having the amino acid residues at positions 12 - 77 of the amino acid sequence shown in SEQ ID NO.
1.
2. A gene encoding the polypeptide TAT-FSHR-pep as claimed in claim 1, characterized in that, The gene is selected from the genes of the following (iii) or (iv): (iii) A gene having the nucleotide sequence shown in SEQ ID NO.2; (iv) A gene having the nucleotides at positions 34 - 231 from the 5'-end of the nucleotide sequence shown in SEQ ID NO.
2.
3. An expression cassette, recombinant vector or recombinant bacterium containing the gene according to claim 2.
4. Use of the polypeptide TAT-FSHR-pep according to claim 1, the gene according to claim 2, or the expression cassette, recombinant vector or recombinant bacterium according to claim 3 in the preparation of a drug for preventing and / or treating FSHR-dependent cancer.
5. The application according to claim 4, wherein The FSHR-dependent cancer includes ovarian cancer, thyroid cancer, and prostate cancer.
6. The application according to claim 4, characterized in that The drug is any pharmaceutically acceptable dosage form.
7. A pharmaceutical composition for anti-ovarian cancer, characterized in that, The active ingredient of the pharmaceutical composition is the polypeptide TAT-FSHR-pep according to claim 1 or the gene according to claim 2.
8. The pharmaceutical composition for anti-ovarian cancer according to claim 7, wherein It also includes a pharmaceutically acceptable drug delivery carrier.