Polypeptide inhibitor of targeted orphan nuclear receptor and application of polypeptide inhibitor

Abstract

The invention belongs to the field of biological medicine, and relates to a polypeptide inhibitor of a targeted nuclear receptor. The invention particularly relates to a polypeptide inhibitor for inhibiting diseases caused by orphan nuclear receptors such as TR4, TR2, COUP-TFI, COUP-TFII and TLX. Experiments show that high binding affinity between the polypeptide and various orphan nuclear receptors such as TR4, TR2, COUP-TF I, COUP-TF II and TLX can be detected, and stable and efficient peptide drug choices are provided for treating cancers and metabolic diseases caused by the typical orphan nuclear receptors and other various related physiological diseases.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to polypeptide inhibitors targeting nuclear receptors, and specifically relates to polypeptide inhibitors that inhibit diseases caused by orphan nuclear receptors such as TR4, TR2, COUP-TFⅠ, COUP-TFⅡ and TLX. Background Art

[0002] The nuclear receptors (NRs) family is the largest family of transcription factors in metazoans. NRs are widely present in higher organisms, especially farmed animals and humans. NRs widely regulate the expression of genes related to reproduction, development, and metabolism. Abnormalities will lead to cancer, immune abnormalities, and various metabolic diseases such as diabetes. As of April 2020, 13% of the drugs approved by the U.S. Food and Drug Administration (FDA) target nuclear receptors. However, the drugs currently developed have poor specificity and obvious side effects, and some can even cause severe heart failure. Therefore, the development of specific inhibitors for nuclear receptors has become a key scientific issue that needs to be urgently addressed in the field of biomedicine.

[0003] NRs are divided into steroid hormone receptors such as thyroid hormone receptor THR, retinoic acid receptor RAR, glucocorticoid receptor GR and vitamin D receptor VDR, as well as orphan nuclear receptors whose ligands have not yet been determined, such as peroxisome proliferator-activated receptor PPAR, liver X receptor LXR, testis receptor TR, hepatocyte nuclear factor HNF-4, photoreceptor cell-specific nuclear receptor PNR. NRs have received extensive attention since the 1980s. A large number of studies in the past 40 years have found that NRs are involved in the regulation of many physiological processes, including insulin sensitivity, glucose and lipid metabolism, gluconeogenesis, lipid synthesis, atherosclerosis, bone formation, inflammatory response and reproductive development.

[0004] Orphan nuclear receptors are typical members of NRs, and their ligands have not been identified yet. In recent years, orphan nuclear receptors have been found to be involved in the regulation of many physiological processes. For example, the COUP-TFⅠ protein regulates the important nervous system in the human body and plays an important regulatory role in epilepsy, autism, and motor performance disorders; abnormal expression of COUP-TFⅡ can cause congenital heart defects, accompanied by growth retardation and mental defects; testicular orphan nuclear receptor proteins TR2 and TR4 form a large complex to affect hepatosplenomegaly and inflammatory responses by regulating the expression of fetal γ-globin. TR4 induces the expression of stearoyl-CoA desaturase 1 gene SCD1 to increase fat mass and reduce insulin sensitivity; TR4 activates proopiomelanocortin POMC, thereby triggering Cushing's disease and promoting the growth of adrenocorticotropic hormone tumors; TR4 also promotes the formation of myocardial foam cells by activating the expression of CD36, leading to atherosclerosis; at the same time, TR4 becomes an inhibitor of liver cancer occurrence and a stimulator of liver cancer metastasis by regulating the expression of genes such as CDK2 and p21. Similarly, TR4 also plays a dual role in the occurrence and metastasis of prostate cancer; in addition, TR4 also affects HBV-induced liver cancer by regulating the expression of hepatitis B virus HBV, and affects the occurrence of breast cancer by regulating downstream key pathogenic genes. Against the background that NRs such as TR4 cause so many physiological diseases, researchers found that the zinc finger juxtaposed gene 1 (JAZF1) can inhibit the roles played by orphan nuclear receptors such as TR4 in various diseases, which makes JAZF1 have a very bright prospect in the treatment of diseases caused by TR4 and other orphan nuclear receptors. Therefore, it is of great importance to develop polypeptide inhibitors targeting orphan nuclear receptors such as TR4, TR2, COUP-TFⅠ, COUP-TFⅡ, and TLX using JAZF1 as a template. Summary of the Invention

[0005] The object of the present invention is to provide a series of polypeptide inhibitors targeting orphan nuclear receptors, specifically referring to polypeptide drugs that inhibit diseases caused by orphan nuclear receptors such as TR4, TR2, COUP-TFⅠ, COUP-TFⅡ, and TLX.

[0006] The present invention provides a series of polypeptide inhibitors, which are characterized in that the polypeptide inhibitors comprise polypeptides with any of the following amino acid sequences:

[0007] (1) QQPTYVALSYINRFMTDAA (SEQ ID No.1), (2) RRPTYVALSYINRFMTDYR (SEQ ID No.3), (3)RRPTYLALSYIRRFLTDYR(SEQ ID No.5), (4)KRPTYLALSYIRRFLTDYR(SEQ ID No.7), (5)HRPTYLALSYIRRFLTDYR(SEQ ID No.9), (6)DRPTYLALSYIRRFLTDYR(SEQ ID No.11), (7)ERPTYLALSYIRRFLTDYR(SEQ ID No.13), (8)RKPTYLALSYIRRFLTDYR(SEQ ID No.15), (9)RHPTYLALSYIRRFLTDYR(SEQ ID No.17), (10)RDPTYLALSYIRRFLTDYR(SEQ ID No.19), (11)REPTYLALSYIRRFLTDYR(SEQ ID No.21), (12)RRPTYYALSYIRRFLTDYR(SEQ ID No.23), (13)RRPTYMALSYIRRFLTDYR(SEQ ID No.25), (14)RRPTYPALSYIRRFLTDYR(SEQ ID No.27), (15)RRPTYIALSYIRRFLTDYR(SEQ ID No.29), (16)RRPTYVALSYIRRFLTDYR(SEQ ID No.31), (17)RRPTYFALSYIRRFLTDYR(SEQ ID No.33), (18)RRPTYWALSYIRRFLTDYR(SEQ ID No.35), (19)RRPTYLALSYIKRFLTDYR(SEQ ID No.37), (20)RRPTYLALSYIHRFLTDYR(SEQ ID No.39), (21)RRPTYLALSYIDRFLTDYR(SEQ ID No.41), (22)RRPTYLALSYIERFLTDYR(SEQ ID No.43), (23) RRPTYLALSYIRRFYTDYR (SEQ ID No. 45), (24) RRPTYLALSYIRRFMTDYR (SEQ ID No. 47), (25) RRPTYLALSYIRRFPTDYR (SEQ ID No. 49), (26) RRPTYLALSYIRRFITDYR (SEQ ID No. 51), (27) RRPTYLALSYIRRFVTDYR (SEQ ID No. 53), (28) RRPTYLALSYIRRFFTDYR (SEQ ID No. 55), (29) RRPTYLALSYIRRFWTDYR (SEQ ID No. 57), (30) RRPTYLALSYIRRFLTDMR (SEQ ID No. 59), (31) RRPTYLALSYIRRFLTDPR (SEQ ID No. 61), (32) RRPTYLALSYIRRFLTDIR (SEQ ID No. 63), (33) RRPTYLALSYIRRFLTDLR (SEQ ID No. 65), (34) RRPTYLALSYIRRFLTDVR (SEQ ID No. 67), (35) RRPTYLALSYIRRFLTDFR (SEQ ID No. 69), (36) RRPTYLALSYIRRFLTDWR (SEQ ID No. 71), (37) RRPTYLALSYIRRFLTDYK (SEQ ID No. 73), (38) RRPTYLALSYIRRFLTDYH (SEQ ID No. 75), (39) RRPTYLALSYIRRFLTDYD (SEQ ID No. 77), (40) RRPTYLALSYIRRFLTDYE (SEQ ID No. 79).

[0008] The polypeptide inhibitor is characterized in that for the above amino acid sequence, multiple amino acid residues can be added separately or simultaneously at both ends, the group at the C-terminus can also be OH, NH2, etc., the group at the N-terminus can also be a modified group such as H atom, acetyl group, formyl group, etc., and pegylation and other modifications conducive to entering the cell membrane can also be carried out in the middle, or it is a synthetic polypeptide. Experiments have been carried out on these polypeptides, and the results are similar.

[0009] The polypeptide inhibitor of the present invention can be used alone or in combination for the treatment of diseases caused by orphan nuclear receptors such as TR4, TR2, COUP-TFⅠ, COUP-TFⅡ, and TLX.

[0010] The present invention provides a series of polypeptide inhibitors targeting orphan nuclear receptors such as TR4, TR2, COUP-TFⅠ, COUP-TFⅡ, and TLX. Experiments have proved that there is a high binding affinity between the polypeptides and the above-mentioned multiple orphan nuclear receptors. The binding force of the polypeptides of the present invention to orphan nuclear receptors such as TR4, TR2, COUP-TFⅠ, COUP-TFⅡ, and TLX is more than 100 times higher than that of the natural wild-type inhibitor, and can effectively inhibit the functions of the above-mentioned multiple orphan nuclear receptors in related diseases.

[0011] According to one aspect of the present invention, the present invention provides a series of polypeptide inhibitors for use in the preparation of therapeutic drugs for diseases caused by orphan nuclear receptors such as TR4, TR2, COUP-TFⅠ, COUP-TFⅡ, and TLX.

[0012] The diseases include prostate cancer, liver cancer, breast cancer, bladder cancer, endometrial cancer, melanoma, Cushing's disease, diabetes, obesity, atherosclerosis, reproductive dysfunction, abnormal neurodevelopment, skeletal dysplasia, intellectual disability, inflammatory response, leukemia, retinopathy, immune abnormality, and aging, etc., but the scope is not limited.

[0013] According to another aspect of the present invention, the present invention provides a series of pharmaceutical compositions, which comprise the active polypeptide and a pharmaceutically acceptable carrier.

[0014] The pharmaceutical compositions include external creams, injections, nasal medications, pulmonary inhalations, tablets, capsules, pills, granules, syrups, other oral liquids, suppositories, etc.

[0015] The pharmaceutically acceptable carriers include conventional diluents, wetting agents, excipients, absorption promoters, surfactants, disintegrants, fillers, binders, lubricants, adsorption carriers, etc. in the pharmaceutical field. When necessary, flavoring agents, sweetening agents, etc. can also be added. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1The structural details and mechanism of action for the inhibitory effect of the natural wild-type JAZF1 polypeptide; among them Figure 1 In A, it is the structural details of the interaction between the natural wild-type JAZF1 polypeptide and TR4; Figure 1 In B, it is the principle of the JAZF1 polypeptide (polypeptide (1)) inhibiting the function of TR4: hindering the binding of TR4 to the co-activator; Figure 1 In C, it is that the JAZF1 polypeptide (polypeptide (1)) significantly inhibits the transcriptional activation of target genes by TR4 in cells.

[0017] Figure 2 For JAZF1 51-69 Measurement results of the binding constants of the wild-type polypeptide (polypeptide (1)) and mutant polypeptides Mut1 and Mut2 to TR4; JAZF1 51-69 The wild-type polypeptide of JAZF1 is polypeptide (1) QQPTYVALSYINRFMTDAA; the mutant polypeptide Mut1 is polypeptide (2) RRPTYVALSYINRFMTDYR with four-site mutations; the mutant polypeptide Mut2 (polypeptide (3-40)) is a mutant modification based on the mutant polypeptide Mut1 (note: the mutated amino acids are in bold). Mut2 in the figure is polypeptide (3) RRPTYLALSYIRRFLTDYR.

[0018] Figure 3 For JAZF1 51-69 Measurement results of the binding constants of the natural wild-type polypeptide (polypeptide (1)) and the mutant polypeptide Mut2 (polypeptide (3)) to TR2.

[0019] Figure 4 For JAZF1 51-69 Measurement results of the binding constants of the natural wild-type polypeptide (polypeptide (1)) and the mutant polypeptide Mut2 (polypeptide (3)) to COUP-TFⅠ.

[0020] Figure 5 For JAZF1 51-69 Measurement results of the binding constants of the natural wild-type polypeptide (polypeptide (1)) and the mutant polypeptide Mut2 (polypeptide (3)) to COUP-TFⅡ.

[0021] Figure 6 For JAZF1 51-69 Measurement results of the binding constants of the natural wild-type polypeptide (polypeptide (1)) and the mutant polypeptide Mut2 (polypeptide (3)) to TLX.

[0022] Figure 7 For JAZF1 and the mutant Mut2 (corresponding to polypeptide (3)), they significantly inhibit the transcriptional activation of target genes by TR4 in cells. Specific implementation mode

[0023] The present invention will be further described below in conjunction with specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts belong to the scope of protection of the present invention.

[0024] Based on the sequence of JAZF1 protein, the present invention determines the minimum fragment length of JAZF1 protein participating in the binding of TR4 protein through experiments: JAZF1 51-69 , and uses this fragment polypeptide (polypeptide (1) QQPTYVALSYINRFMTDAA) as a TR4 inhibitory polypeptide for research. A mutant polypeptide Mut1 (polypeptide (2) RRPTYVALSYINRFMTDYR) with better inhibitory effect is screened. Based on Mut1, more high-binding polypeptides Mut2 (3-40) are further screened, which can be used as highly efficient inhibitors of nuclear orphan receptors TR4, TR2, COUP-TFⅠ, COUP-TFⅡ, and TLX.

[0025] Example 1. Analysis of the structure of the TR4-JAZF1 complex

[0026] (1) Select the fragment TR4 341-596 and JAZF1 39-79 of the binding of TR4 and JAZF1 reported in the previous study (Nakajima, T., Fujino, S., Nakanishi, G., Kim, Y.S. and Jetten, A.M. (2004) TIP27: a novel repressor of the nuclear orphan receptor TAK1 / TR4. Nucleic Acids Res., 32, 4194-4204. DOI: 10.1093 / nar / gkh741) for complex crystal screening. After certain preliminary studies, it is determined to connect JAZF1 51-75 to the end of TR4 341-596 to construct TR4 341-596 -JAZF1 51-75Chimeras. In the present invention, pET28a-TEV-MBP and BL21(DE3) are disclosed in the literature "Yunlong Liu, Lulu Ma, Min Li, Zizi Tian, Meiting Yang, Xi Wu, Xue Wang, Guohui Shang, Mengjia Xie, Yiyun Chen, Xin Liu, Lun Jiang, Wei Wu, Chaoqun Xu, Liqun Xia, Gonghui Li, Shaodong Dai, Zhongzhou Chen. Structures of human TR4 LBD -JAZF1 and TR4 DBD -DNA complexes reveal the molecular basis of transcriptional regulation”, Nucleic Acids Research, 51, 3, 2023, 1443-1457, https: / / doi.org / 10.1093 / nar / gkac1259” (described in the literature as “modified pET28a vector encoding a 6×His-maltose-binding protein (MBP) fusion tag and a tobacco etch virus (TEV) protease cleavage site”). The public can obtain the above biological materials from the applicant. The obtained above biological materials are only used for repeating the experiments of the present invention and cannot be used for other purposes. The chimeric fusion MBP protein is expressed by fusion. The MBP-TR4 341-596 -JAZF1 51-75 fusion protein is subjected to affinity chromatography using the Amylose Resin chromatography medium purchased from NEB. After the fusion protein is cleaved by TEV protease, the chimeric TR4 341-596 -JAZF1 51-75 is separated from the MBP protein. The chimeric protein TR4 341-596 -JAZF1 51-75 is further separated and purified by affinity chromatography. The protein buffer used in the purification process has the following composition: 20 mM Bis-Tris propane pH 8.5, 1 M NaCl. The expression processes of MBP-JAZF1 51-69 and TR4 341-596 -JAZF1 51-69 and the mutant proteins are similar.

[0027] (2)For the TR4 separated and purified in step (1) 341-596 -JAZF1 39-79 Perform protein crystal screening on the chimeric protein. The vapor diffusion method is used to screen for crystals, including the sitting-drop method and the hanging-drop method. The sitting-drop method is used for the initial screening of crystals, and the sitting-drop method and the hanging-drop method are used for crystal optimization. The sitting-drop method for screening crystals mainly uses a 48-well crystal plate for the initial screening of crystals, providing a closed environment. The crystal plate is placed in the dark at 4°C and 16°C for crystal cultivation. After obtaining protein crystals by using the sitting-drop method, in order to obtain larger and easier-to-pick crystals, the hanging-drop method is usually used for crystal optimization. In this study, a 24-well hanging-drop plate and a silanized coverslip are mainly used to optimize the initially screened crystals. The hanging-drop plate is placed in a suitable temperature environment and stored in the dark. After a certain cultivation time, the cultivation situation is observed. The crystal cultivation conditions suitable for X-ray diffraction are: 1.6 M (NH4)2SO4, 0.1 M HEPES pH 7.5, 0.2 M NaAc, 5% MPD.

[0028] (3)Pick up the protein crystals suitable for diffraction and transport them to the Shanghai Synchrotron Radiation Source for diffraction data collection. Set the wavelength to The parameter of the distance to the CCD is set to 550 mm, and a diffraction pattern is collected every 1°, obtaining a set of data. Using the TR4 348-582 protein structure (PDB ID: 3P0U) as a model for molecular replacement, and then alternately optimize the resulting PDB file and MTZ file with the Refmac5 program and manually build the model with COOT, and finally resolve the complex structure (PDB ID: 7XVA).

[0029] (4) Figure 1 In A, TR4 341-596 -JAZF1 51-75 For the chimeric complex structure, display the details of the interaction between TR4 341-596 and JAZF1 51-75 in the structure. The side chains of the amino acid residues participating in the interaction are shown. The formed hydrophobic interaction regions are represented by translucent ellipses, the hydrogen bond interactions are represented by dotted lines, and the water molecules are represented by red spheres.

[0030] Example 2. JAZF1 inhibits TR4 from binding to the transcriptional co-activator

[0031] (1) Nuclear receptor members such as TR4 form a transcriptional initiation complex by binding to transcriptional co-activators, thereby completing the transcriptional activation of target genes. Select the TR4 341-596 protein and TR4 341-596 -JAZF1 51-69The chimeric protein was expressed as MBP-TR4 by fusing with the MBP-tagged protein respectively. 341-596 With MBP-TR4 341-596 -JAZF1 51-69 fusion protein.

[0032] (2) Take 100 μL of the MBP protein affinity chromatography medium Amylose Resin, centrifuge to remove alcohol, add 1 mL of ddH2O, mix well, and remove the supernatant. Repeat this three times to remove residual alcohol as much as possible.

[0033] (3) Prepare the binding buffer: 20 mM HEPES pH 7.0, 1 M NaCl, 2 mM β-Me, 0.1% (v / v) Triton X-100. Wash the chromatography medium three times with 1 mL of the binding buffer, and centrifuge to remove the supernatant for the last time at 4°C.

[0034] (4) Add 800 μL of the binding buffer to the equilibrated affinity resin, and add 0.1 mg of the MBP fusion protein from step (1) respectively. Mix well and incubate on a rotator at 4°C for 10 min. After centrifuging to remove the supernatant at 4°C, add 1 mL of the binding buffer and wash once. Centrifuge to remove the supernatant at 4°C to remove the MBP fusion protein that has not bound to the chromatography medium.

[0035] (5) Add 800 μL of the binding buffer, and add the transcriptional coactivator interacting proteins SRC-1 and CBP fused with 6×His tag respectively at a certain molar ratio. Mix well and incubate on a rotator at 4°C for 2 h.

[0036] (6) After centrifuging to remove the supernatant at 4°C, add 1 mL of the binding buffer for washing. Each time, add the binding buffer, mix well, and centrifuge at 4°C. Repeat the washing 5 times, and try to remove the supernatant as completely as possible after the last washing.

[0037] (7) Add the binding buffer containing 20 mM maltose to elute the sample, and take the eluate for SDS-PAGE detection.

[0038] (8) Figure 1 In B, JAZF1 blocks the binding of TR4 to the transcriptional coactivator. The results of the Pull-down experiment show that the transcriptional coactivators SRC-1 and CBP can bind to TR4, but the interaction between the two is inhibited by JAZF1, indicating that JAZF1 blocks the binding of TR4 to the transcriptional coactivator.

[0039] Example 3. JAZF1 inhibits the transcriptional activation of target genes by TR4

[0040] (1) The COS-7 cells (purchased from the ATCC cell bank) that have been stably passaged three times after freeze-thawing were evenly seeded in a 24-well plate at a passage ratio of 1:3. The culture medium conditions were: 10% FBS (purchased from Cellmax), 90% DMEM medium (purchased from Gibco). The cells were cultured under the conditions of 5% CO2 and 37°C.

[0041] (2) After the cells were cultured to a density of 60 - 70%, the FBS concentration in the cell culture medium was reduced to 2%, and the cells were starved for 2 h. After the treatment, plasmid transfection was performed on the cells using Lipofectamine 3000 transfection reagent (purchased from Invitrogen). First, prepare Solution A (for each well of the 24-well plate: 25 μL of Opti-MEM medium + 1.25 μL of Lipofectamine 3000 transfection reagent), and Solution B (for each well of the 24-well plate: 25 μL of Opti-MEM medium + 1 μL of P3000 Reagent + 250 ng of pGL3-POMC plasmid + 250 ng of pCDNA3.1-TR4 plasmid (for JAZF1 treatment, an additional 250 ng of pCDNA3.1-JAZF1 plasmid needs to be added) + 25 ng of pRL-TK plasmid). After the prepared Solution A and Solution B were allowed to stand for 5 min, Solution A and Solution B were mixed evenly and allowed to stand for 15 min;

[0042] (3) Add 50 μL of the mixed solution to each well of the 24-well plate, and shake while adding to make it evenly distributed. After 6 h of transfection, increase the FBS concentration in the medium to 10%, and continue to culture for 18 h, then detect the dual luciferase activity.

[0043] (4) 24 h after transfection, take out the 24-well plate, discard the medium, add PBS (purchased from Gibco) along the wall to wash twice and aspirate the washing solution. Add 150 μL of Luciferase buffer (purchased from Promega) to each well for digestion for 40 min, and place it in an incubator at 25°C. After digestion, divide each well into two 70 μL and transfer them to two wells of a 96-well plate, and store in the dark. Use a TECAN microplate reader to detect the relative light unit (RLU) at a wavelength of 560 nm.

[0044] (5) After detecting the RLU at least three times, immediately add 70 μL of Dual-Glo Stop&Glo Regent solution (purchased from Promega) to each well, blow it evenly, and place it on a horizontal shaker and shake slowly at room temperature for 20 min, paying attention to avoiding light. After the incubation, use a TECAN microplate reader to detect the RLU at a wavelength of 465 nm.

[0045] (6) Using Renilla luciferase (RLU at 465 nm wavelength) as an internal reference, the ratio of firefly luciferase (RLU at 560 nm wavelength) to Renilla luciferase RLU represents the transcriptional activation level of the target gene. At least three replicate experiments were performed for each treatment.

[0046] (7) Figure 1 In C, JAZF1 inhibited the transcriptional activation of the target gene by TR4. The experimental data was normalized using the CK (TR4 overexpression treatment) result as a control. The results showed that overexpression of TR4 significantly activated the expression of the target gene, while overexpression of JAZF1 on this basis significantly inhibited the transcriptional activation of the target gene by TR4.

[0047] Example 4. Modification and screening of polypeptide inhibitors

[0048] (1) Based on the resolved TR4 341-596 -JAZF1 51-75 complex structure (PDB ID: 7XVA), the JAZF1 51-69 polypeptide (polypeptide (1) QQPTYVALSYINRFMTDAA) was subjected to sequence mutation modification. First, some single-point mutants with higher binding affinity for TR4 were screened according to the binding experiment. Subsequently, multiple mutation sites were integrated to screen a four-amino acid site mutant polypeptide Mut1 (polypeptide (2) RRPTYVALSYINRFMTDYR) with significantly improved binding affinity. Further increasing the number of mutation sites resulted in a seven-amino acid site mutant polypeptide Mut2 (polypeptide (3) RRPTYLALSYIRRFLTDYR) with higher binding affinity.

[0049] (2) Figure 2 are the measurement results of the binding constants of different inhibitory polypeptides to TR4. The binding affinity of the JAZF1 polypeptide fused with MBP protein to the dye-labeled TR4 protein was detected. The binding constant was detected by microscale thermophoresis (MST) experiment. The binding buffer used was: 20 mM HEPES pH 7.0, 300 mM KCl. The experimental results were as follows: the binding constant of the JAZF1 wild-type WT polypeptide (polypeptide (1) QQPTYVALSYINRFMTDAA) to TR4 was 2081.0 nM; the binding constant of the Mut1 polypeptide (polypeptide (2) RRPTYVALSYINRFMTDYR) to TR4 was 40.1 nM; the binding constant of the Mut2 polypeptide (polypeptide (3) RRPTYLALSYIRRFLTDYR) to TR4 was 8.0 nM. The binding ability of the Mut2 polypeptide to the TR4 protein was about 260 times higher than that of the WT polypeptide.

[0050] (3) Mutate and modify the Mut2 polypeptide sequence. Select the 7 mutation sites in the Mut2 polypeptide that are modified compared to the wild-type polypeptide, and perform single amino acid site substitution one by one. The types of mutant polypeptides constructed are shown in Table 1.

[0051] Table 1. Detection of the binding constants of Mut2 polypeptide single amino acid site mutants to TR4 (unit: nM)

[0052] Note: X: is the abbreviation of amino acid residue, representing Y, M, P, I, L, V, F, W, R, K, H, D, E.

[0053] (4) Table 1 shows the single amino acid site mutations carried out on the Mut2 polypeptide and the measurement results of the binding constants of each mutant polypeptide to TR4. Another 37 polypeptides were screened based on the Mut2 polypeptide, and they also had a high binding affinity for the TR4 protein, with binding constants ranging from 4.4 nM to 16.5 nM.The specifically selected high-binding-force polypeptide sequences are: polypeptide (4) KRPTYLALSYIRRFLTDYR, polypeptide (5) HRPTYLALSYIRRFLTDYR, polypeptide (6) DRPTYLALSYIRRFLTDYR, polypeptide (7) ERPTYLALSYIRRFLTDYR, polypeptide (8) RKPTYLALSYIRRFLTDYR, polypeptide (9) RHPTYLALSYIRRFLTDYR, polypeptide (10) RDPTYLALSYIRRFLTDYR, polypeptide (11) REPTYLALSYIRRFLTDYR, polypeptide (12) RRPTYYALSYIRRFLTDYR, polypeptide (13) RRPTYMALSYIRRFLTDYR, polypeptide (14) RRPTYPALSYIRRFLTDYR, polypeptide (15) RRPTYIALSYIRRFLTDYR, polypeptide (16) RRPTYVALSYIRRFLTDYR, polypeptide (17) RRPTYFALSYIRRFLTDYR, polypeptide (18) RRPTYWALSYIRRFLTDYR, polypeptide (19) RRPTYLALSYIKRFLTDYR, polypeptide (20) RRPTYLALSYIHRFLTDYR, polypeptide (21) RRPTYLALSYIDRFLTDYR, polypeptide (22) RRPTYLALSYIERFLTDYR, polypeptide (23) RRPTYLALSYIRRFYTDYR, polypeptide (24) RRPTYLALSYIRRFMTDYR, polypeptide (25) RRPTYLALSYIRRFPTDYR, polypeptide (26) RRPTYLALSYIRRFITDYR, polypeptide (27) RRPTYLALSYIRRFVTDYR, polypeptide (28) RRPTYLALSYIRRFFTDYR, polypeptide (29) RRPTYLALSYIRRFWTDYR, polypeptide (30) RRPTYLALSYIRRFLTDMR, polypeptide (31) RRPTYLALSYIRRFLTDPR, polypeptide (32) RRPTYLALSYIRRFLTDIR, polypeptide (33) RRPTYLALSYIRRFLTDLR, polypeptide (34) RRPTYLALSYIRRFLTDVR, polypeptide (35) RRPTYLALSYIRRFLTDFR, polypeptide (36) RRPTYLALSYIRRFLTDWR, polypeptide (37) RRPTYLALSYIRRFLTDYK, polypeptide (38) RRPTYLALSYIRRFLTDYH, polypeptide (39) RRPTYLALSYIRRFLTDYD, polypeptide (40) RRPTYLALSYIRRFLTDYE.Only the protected amino acid sequences are written here. For the polypeptide inhibitors, multiple amino acid residues can be added separately or simultaneously at both ends. The group at the C-terminus can also be OH or NH2, etc., and the group at the N-terminus can also be H or modified with Ac, formyl, etc. PEGylation and other modifications that help enter the cell membrane can also be carried out in the middle, or they can be synthesized. These polypeptides have been experimented with, and the results are similar to Table 2.

[0054] Table 2. Detection of the binding constants of Mut2 polypeptides with modified or added amino acids at both ends to TR4, TR2, COUP-TFⅠ, COUP-TFⅡ, and TLX (unit: nM)

[0055] Note: 3N2, 3N4, 3N6, 3C2, 3C4, 3C6 represent adding 2, 4, and 6 amino acid residues to the N-terminus or C-terminus of the polypeptide sequence of SEQ ID No.5 respectively; 3-NH2 and 3-OCH3 represent that the protecting groups at the C-terminus of the polypeptide of SEQ ID No.5 are NH2 and OCH3; Acetyl-3, Formyl-3, PEG-3 represent that the protecting groups at the N-terminus of the polypeptide of SEQ ID No.5 are acetyl, formyl, and PEG. TAT and Penetratin represent the cell-penetrating peptide CPP.

[0056] Example 5. Detection of the binding constants of polypeptide inhibitors with multiple orphan nuclear receptors

[0057] According to sequence similarity, potential nuclear receptors among all nuclear receptors were selected for experimental testing:

[0058] (1) Detection of the binding constant of the polypeptide inhibitor with the orphan nuclear receptor TR2

[0059] Select JAZF1 51-69 The WT polypeptide (polypeptide (1) QQPTYVALSYINRFMTDAA) and the Mut2 polypeptide (polypeptide (3) RRPTYLALSYIRRFLTDYR) were subjected to a binding experiment with TR2. As Figure 3 , the binding experiment results showed that the binding constant of the WT polypeptide with TR2 was 4100.5 nM; the binding constant of the Mut2 polypeptide with TR2 was 15.6 nM. The binding ability of the Mut2 polypeptide to the TR2 protein was more than 260 times higher than that of the WT polypeptide.

[0060] Select the 37 high-binding-force polypeptides screened in step (4) of Example 4 for a binding experiment with TR2. As shown in the results of Table 3, the 37 high-binding-force polypeptides screened in step (4) of Example 4 also had a high binding force level with TR2, and the binding constant was from 8.6 nM to 28.3 nM.

[0061] Table 3. Detection of the binding constant between Mut2 polypeptide single amino acid site mutants and TR2 (unit: nM)

[0062] Note: X: is the abbreviation of amino acid residue, representing Y, M, P, I, L, V, F, W, R, K, H, D, E.

[0063] (2) Detection of the binding constant between polypeptide inhibitors and orphan nuclear receptor COUP-TFⅠ

[0064] Select JAZF1 51-69 WT polypeptide (polypeptide (1) QQPTYVALSYINRFMTDAA) and Mut2 polypeptide (polypeptide (3) RRPTYLALSYIRRFLTDYR) were subjected to a binding experiment with COUP-TFⅠ. As Figure 4 , the binding experiment results showed that the binding constant between the WT polypeptide and COUP-TFⅠ was 9989.2 nM; the binding constant between the Mut2 polypeptide and COUP-TFⅠ was 66.0 nM. The binding ability of the Mut2 polypeptide to the COUP-TFⅠ protein was more than 150 times higher than that of the WT polypeptide.

[0065] Select 37 high-binding-force polypeptides screened in step (4) of Example 4 for a binding experiment with COUP-TFⅠ. As shown in the results of Table 4, the 37 high-binding-force polypeptides screened in step (4) of Example 4 also had a relatively high binding force level with COUP-TFⅠ, and the binding constant was from 42.1 nM to 104.0 nM.

[0066] Table 4. Detection of the binding constant between Mut2 polypeptide single amino acid site mutants and COUP-TFⅠ (unit: nM)

[0067] Note: X: is the abbreviation of amino acid residue, representing Y, M, P, I, L, V, F, W, R, K, H, D, E.

[0068] (3) Detection of the binding constant between polypeptide inhibitors and orphan nuclear receptor COUP-TFⅡ

[0069] Select JAZF1 51-69 WT polypeptide (polypeptide (1) QQPTYVALSYINRFMTDAA) and Mut2 polypeptide (polypeptide (3) RRPTYLALSYIRRFLTDYR) were subjected to a binding experiment with COUP-TFⅡ. As Figure 5, The combined experimental results show that the binding constant of the WT polypeptide to COUP-TFⅡ is 9531.4 nM; the binding constant of the Mut2 polypeptide to COUP-TFⅡ is 35.7 nM. The binding ability of the Mut2 polypeptide to the COUP-TFⅡ protein is more than 260 times higher than that of the WT polypeptide.

[0070] Thirty-seven high-binding polypeptides screened in step (4) of Example 4 were selected for binding experiments with COUP-TFⅡ. As shown in the results of Table 5, the 37 high-binding polypeptides screened in step (4) of Example 4 also have a high binding level with COUP-TFⅡ, and the binding constant ranges from 19.6 nM to 76.8 nM.

[0071] Table 5. Detection of the binding constants of single amino acid site mutants of the Mut2 polypeptide to COUP-TFⅡ (unit: nM)

[0072] Note: X: is the abbreviation of amino acid residue, representing Y, M, P, I, L, V, F, W, R, K, H, D, E.

[0073] (4) Detection of the binding constant of the polypeptide inhibitor to the orphan nuclear receptor TLX

[0074] Select JAZF1 51-69 The WT polypeptide (polypeptide (1) QQPTYVALSYINRFMTDAA) and the Mut2 polypeptide (polypeptide (3) RRPTYLALSYIRRFLTDYR) were used for binding experiments with TLX. As Figure 6 , the combined experimental results show that the binding constant of the WT polypeptide to TLX is 38303.5 nM; the binding constant of the Mut2 polypeptide to TLX is 90.2 nM. The binding ability of the Mut2 polypeptide to the TLX protein is more than 420 times higher than that of the WT polypeptide.

[0075] Thirty-seven high-binding polypeptides screened in step (4) of Example 4 were selected for binding experiments with TLX. As shown in the results of Table 6, the 37 high-binding polypeptides screened in step (4) of Example 4 also have a high binding level with TLX, and the binding constant ranges from 69.4 nM to 205.5 nM.

[0076] Table 6. Detection of the binding constants of single amino acid site mutants of the Mut2 polypeptide to TLX (unit: nM)

[0077] Note: X: is the abbreviation of amino acid residue, representing Y, M, P, I, L, V, F, W, R, K, H, D, E.

[0078] Example 6. JAZF1 mutants inhibit the transcriptional activation of target genes by TR4

[0079] Cell experiments were carried out according to the method of Example 3. Renilla luciferase was used as an internal reference, and the ratio of firefly luciferase to Renilla luciferase RLU represented the transcriptional activation level of the target gene. Each treatment was repeated at least three times. JAZF1 wild type and Mut2 (corresponding polypeptide (3) RRPTYLALSYIRRFLTDYR) were selected for experiments with TR4. Figure 7 JAZF1 and its mutants inhibit the transcriptional activation of target genes by TR4. The experimental data were normalized with the results of CK (TR4 overexpression treatment) as a control. The results showed that overexpression of TR4 significantly activated the expression of the target gene. On this basis, simultaneous overexpression of JAZF1 significantly inhibited the transcriptional activation of the target gene by TR4, and the inhibitory ability of the mutant Mut2 was significantly higher, and the result was even better than that of the blank control. The experimental results of other Mut2 inhibiting the transcriptional activation of target genes by TR4, TR2, COUP-TFⅠ, COUP-TFⅡ, and TLX were similar to this result, and the effects were all significantly better than those of the wild type.

[0080] The present invention has been described in detail above. Only the protected amino acid sequences are written here. Multiple amino acid residues can be added separately or simultaneously at both ends of the polypeptide inhibitor. The group at the C-terminus can also be OH or NH2, etc., and the group at the N-terminus can also be modified with an H atom or an acetyl group, a formyl group, etc. PEGylation and other modifications that help enter the cell membrane can also be carried out in the middle, or it can be synthesized. These polypeptides have been experimented, and the results are similar to Table 2. For those skilled in the art, without departing from the purpose and scope of the present invention and without unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations, and conditions. Although specific examples of the present invention are given, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to include any changes, uses, or improvements to the present invention, including those that depart from the scope disclosed in this application and are made with conventional techniques known in the art. Some basic features can be applied according to the scope of the following appended claims.

Claims

1. A series of polypeptide inhibitors, characterized in that, The polypeptide-related material contains a polypeptide with any of the following amino acid sequences: (1) QQPTYVALSYINRFMTDAA (SEQ ID No. 1), (2) RRPTYVALSYINRFMTDYR (SEQ ID No. 3), (3) RRPTYLALSYIRRFLTDYR (SEQ ID No. 5), (4) KRPTYLALSYIRRFLTDYR (SEQ ID No. 7), (5) HRPTYLALSYIRRFLTDYR (SEQ ID No. 9), (6) DRPTYLALSYIRRFLTDYR (SEQ ID No. 11), (7) ERPTYLALSYIRRFLTDYR (SEQ ID No. 13), (8) RKPTYLALSYIRRFLTDYR (SEQ ID No. 15), (9) RHPTYLALSYIRRFLTDYR (SEQ ID No. 17), (10) RDPTYLALSYIRRFLTDYR (SEQ ID No. 19), (11) REPTYLALSYIRRFLTDYR (SEQ ID No. 21), (12) RRPTYYALSYIRRFLTDYR (SEQ ID No. 23), (13) RRPTYMALSYIRRFLTDYR (SEQ ID No. 25), (14) RRPTYPALSYIRRFLTDYR (SEQ ID No. 27), (15) RRPTYIALSYIRRFLTDYR (SEQ ID No. 29), (16) RRPTYVALSYIRRFLTDYR (SEQ ID No. 31), (17) RRPTYFALSYIRRFLTDYR (SEQ ID No. 33), (18) RRPTYWALSYIRRFLTDYR (SEQ ID No. 35), (19) RRPTYLALSYIKRFLTDYR (SEQ ID No. 37), (20) RRPTYLALSYIHRFLTDYR (SEQ ID No. 39), (21) RRPTYLALSYIDRFLTDYR (SEQ ID No. 41), (22) RRPTYLALSYIERFLTDYR (SEQ ID No. 43), (23) RRPTYLALSYIRRFYTDYR (SEQ ID No. 45), (24) RRPTYLALSYIRRFMTDYR (SEQ ID No. 47), (25) RRPTYLALSYIRRFPTDYR (SEQ ID No. 49), (26) RRPTYLALSYIRRFITDYR (SEQ ID No. 51), (27) RRPTYLALSYIRRFVTDYR (SEQ ID No.53), (28) RRPTYLALSYIRRFFTDYR (SEQ ID No.55), (29) RRPTYLALSYIRRFWTDYR (SEQ ID No.57), (30) RRPTYLALSYIRRFLTDMR (SEQ ID No.59), (31) RRPTYLALSYIRRFLTDPR (SEQ ID No.61), (32) RRPTYLALSYIRRFLTDIR (SEQ ID No.63), (33) RRPTYLALSYIRRFLTDLR (SEQ ID No.65), (34) RRPTYLALSYIRRFLTDVR (SEQ ID No.67), (35) RRPTYLALSYIRRFLTDFR (SEQ ID No.69), (36) RRPTYLALSYIRRFLTDWR (SEQ ID No.71), (37) RRPTYLALSYIRRFLTDYK (SEQ ID No.73), (38) RRPTYLALSYIRRFLTDYH (SEQ ID No.75), (39) RRPTYLALSYIRRFLTDYD (SEQ ID No.77), (40) RRPTYLALSYIRRFLTDYE (SEQ ID No.79).

2. The polypeptide-related biological material according to claim 1, wherein The above sequences can have multiple amino acid residues added simultaneously or separately at both ends of the above amino acid sequences. The group at the C-terminus can also be OH or NH2, etc., and the group at the N-terminus can also be a modified group such as an H atom, an acetyl group, or a formyl group. Pegylation and other modifications that help enter the cell membrane can also be carried out in the middle, or it is a synthetic polypeptide.

3. A biological material related to the polypeptide according to any one of claims 1-2, characterized in that: The biological material is any one of the following: A1) A nucleic acid molecule encoding the polypeptide according to any one of claims 1-2; A2) An expression cassette containing the nucleic acid molecule described in A1); A3) A recombinant vector containing the nucleic acid molecule described in A1) or containing the expression cassette described in A2); A4) A recombinant microorganism containing the nucleic acid molecule described in A1) or containing the expression cassette described in A2) or containing the recombinant vector described in A3); A5) An animal cell line containing the nucleic acid molecule described in A1) or containing the expression cassette described in A2) or containing the recombinant vector described in A3); A6) A plant cell line containing the nucleic acid molecule described in A1) or containing the expression cassette described in A2) or containing the recombinant vector described in A3); A7) A recombinant cell that produces the polypeptide according to any one of claims 1-2.

4. The biomaterial according to claim 3, characterized in that, The nucleic acid molecule described in A1), the coding sequence of its coding strand is shown in any one of B1)-B40): B1) The coding sequence of the coding strand is shown in SEQ ID No.2; B2) The coding sequence of the coding strand is shown in SEQ ID No.4; B3) The coding sequence of the coding strand is as shown in SEQ ID No. 6; B4) The coding sequence of the coding strand is as shown in SEQ ID No. 8; B5) The coding sequence of the coding strand is as shown in SEQ ID No. 10; B6) The coding sequence of the coding strand is as shown in SEQ ID No. 12; B7) The coding sequence of the coding strand is as shown in SEQ ID No. 14; B8) The coding sequence of the coding strand is as shown in SEQ ID No. 16; B9) The coding sequence of the coding strand is as shown in SEQ ID No. 18; B10) The coding sequence of the coding strand is as shown in SEQ ID No. 20; B11) The coding sequence of the coding strand is as shown in SEQ ID No. 22; B12) The coding sequence of the coding strand is as shown in SEQ ID No. 24; B13) The coding sequence of the coding strand is as shown in SEQ ID No. 26; B14) The coding sequence of the coding strand is as shown in SEQ ID No. 28; B15) The coding sequence of the coding strand is as shown in SEQ ID No. 30; B16) The coding sequence of the coding strand is as shown in SEQ ID No. 32; B17) The coding sequence of the coding strand is as shown in SEQ ID No. 34; B18) The coding sequence of the coding strand is as shown in SEQ ID No. 36; B19) The coding sequence of the coding strand is as shown in SEQ ID No. 38; B20) The coding sequence of the coding strand is as shown in SEQ ID No. 40; B21) The coding sequence of the coding strand is as shown in SEQ ID No. 42; B22) The coding sequence of the coding strand is as shown in SEQ ID No. 44; B23) The coding sequence of the coding strand is as shown in SEQ ID No. 46; B24) The coding sequence of the coding strand is as shown in SEQ ID No. 48; B25) The coding sequence of the coding strand is as shown in SEQ ID No. 50; B26) The coding sequence of the coding strand is as shown in SEQ ID No. 52; B27) The coding sequence of the coding strand is as shown in SEQ ID No. 54; B28) The coding sequence of the coding strand is as shown in SEQ ID No. 56; B29) The coding sequence of the coding strand is as shown in SEQ ID No. 58; B30) The coding sequence of the coding strand is as shown in SEQ ID No. 60; B31) The coding sequence of the coding strand is as shown in SEQ ID No. 62; B32) The coding sequence of the coding strand is as shown in SEQ ID No. 64; B33) The coding sequence of the coding strand is as shown in SEQ ID No. 66; B34) The coding sequence of the coding strand is as shown in SEQ ID No. 68; B35) The coding sequence of the coding strand is as shown in SEQ ID No. 70; B36) The coding sequence of the coding strand is as shown in SEQ ID No. 72; B37) The coding sequence of the coding strand is as shown in SEQ ID No. 74; B38) The coding sequence of the coding strand is as shown in SEQ ID No. 76; B39) The coding sequence of the coding strand is as shown in SEQ ID No. 78; B40) The coding sequence of the coding strand is as shown in SEQ ID No.

80.

5. Use of one or more of the polypeptide inhibitors according to any one of claims 1-2, alone or in combination, in the preparation of inhibitors of orphan nuclear receptors such as TR4, TR2, COUP-TFⅠ, COUP-TFⅡ, and TLX.

6. Use of one or more of the polypeptide inhibitors according to any one of claims 1-2, alone or in combination, in the preparation of drugs for diseases caused by orphan nuclear receptors such as TR4, TR2, COUP-TFⅠ, COUP-TFⅡ, and TLX.

7. The polypeptide inhibitor according to any one of claims 1-2, characterized in that, The one or more polypeptide inhibitors, alone or in combination, can be made into various pharmaceutically acceptable dosage forms, including topical creams, injections, nasal medications, pulmonary inhalations, tablets, capsules, pills, granules, syrups, and other oral liquids and suppositories.

8. A drug or pharmaceutical composition, characterized in that: The drug or drug composition contains the polypeptide according to any one of claims 1-2.

9. Use of the polypeptide according to any one of claims 1-2 and / or the biological material according to claim 3 or 4 in the preparation of drugs for diseases caused by orphan nuclear receptors such as TR4, TR2, COUP-TFⅠ, COUP-TFⅡ, and TLX.

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