Polypeptide for inhibiting SAE1 / SAE2 interaction and application thereof

Abstract

The invention provides an active polypeptide for inhibiting SAE1 / SAE2 interaction. The active polypeptide comprises an amino acid sequence as shown in at least one of a formula (1), a formula (2) and a formula (3) and salt of the amino acid sequence, wherein the formula (1) is Ac-X1X2X3YDX4QIX5LWG-NH2; the formula (2) is as follows: Ac-X3YDX4QIX5-NH2; and a formula (3): Ac-YDX4QIX5L-NH2. The series of novel polypeptides provided by the invention can play a role in resisting tumors (lymphoma, colorectal cancer, head and neck cancer and the like) by inhibiting the interaction of SAE1 / SAE2 proteins in cancer cells, and have the advantages of high anti-tumor activity, stability, difficulty in inactivation and the like.

Description

Technical Field

[0001] The present invention belongs to the field of polypeptides, and more particularly, relates to polypeptides and their anti-tumor applications. Background Art

[0002] SUMOylation is an important post-translational modification of proteins, regulating their localization, activation, and transport, thereby influencing various cellular physiological activities, including signal transduction, cell cycle regulation, proliferation, and apoptosis. SUMOylation has been shown to be closely associated with the development and progression of various cancers, manifesting as abnormal upregulation of protein expression in these pathways. Therefore, targeted SUMOylation inhibition holds great promise for cancer therapy.

[0003] SUMO activating enzyme E1 (SAE) is the only activating enzyme in SUMOylation. It consists of two subunits, SAE1 and SAE2. SAE activates the C-terminus of the SUMO protein through a two-step hydrolysis reaction that consumes ATP and transfers it to the conjugating enzyme E2. Studies have shown that SAE is highly correlated with disease phenotypes such as progression, metastasis, and prognosis in various cancers, including hepatocellular carcinoma, colorectal cancer, and gastric cancer, making SAE a promising target for oncology drug development. In recent years, a number of SAE inhibitors have been reported, including small molecules such as TAK-981 and COH000 and natural products such as ginkgo bilobalic acid. However, these inhibitors have various limitations that limit their potential as pharmaceuticals. For example, ginkgo bilobalic acid can induce neuronal cell death. In a Phase 1b clinical study of TAK-981 combined with pembrolizumab for the treatment of non-small cell lung cancer and MSS colorectal cancer, 88% of patients experienced treatment-emergent adverse events. Summary of the Invention

[0004] Polypeptides are a type of organic compound formed by amino acids connected by peptide bonds. They have advantages such as high activity and low toxicity and are widely used in drug research. Compared with small chemical molecules, peptide inhibitors have significant advantages such as better target specificity, strong affinity, and less accumulation in the body to cause toxicity with protein targets. Therefore, in response to the problems or potential safety risks of current SAE inhibitors, the inventors of the present invention have developed a new type of polypeptide that inhibits the SAE1 / SAE2 interaction by targeting SAE1, thereby destroying the normal function of the SUMO E1 enzyme and inhibiting the SUMOylation pathway. In addition, because the SAE1 / SAE2 interaction interface is less conserved than the active site of SAE, polypeptides that inhibit the SAE1 / SAE2 interaction are more easily selectively inhibited, thereby reducing the possibility of adverse reactions and drug resistance.

[0005] The first object of the present invention is to provide an active polypeptide.

[0006] The second object of the present invention is to provide the use of an active polypeptide in preparing an inhibitor for inhibiting the SAE1 / SAE2 protein interaction.

[0007] The third object of the present invention is to provide polypeptide inhibitors for preventing or treating tumors such as lymphoma, colorectal cancer, head and neck cancer, etc.

[0008] The fourth object of the present invention is to provide a pharmaceutical composition for preventing or treating tumors such as lymphoma, colorectal cancer, head and neck cancer, etc.

[0009] To achieve the above objectives, the present invention provides an active polypeptide that inhibits SAE1 / SAE2 interaction, wherein the active polypeptide comprises an amino acid sequence as represented by at least one of formula (1), formula (2), and formula (3), and a salt thereof:

[0010] Formula (1): Ac-X1X2X3YDX4QIX5LWG-NH2

[0011] Formula (2): Ac-X3YDX4QIX5-NH2

[0012] Formula (3): Ac-YDX4QIX5L-NH2

[0013] X1 is Ala or Arg;

[0014] X2 is Ala or Tyr;

[0015] X3 is Gln or Tyr;

[0016] X4 is Arg or Asn or Ser or Asp or Thr;

[0017] X5 is Arg or His;

[0018] According to a preferred embodiment, the active polypeptide comprises a sequence represented by formula (1), formula (2), formula (3) or a salt thereof.

[0019] In a preferred technical solution, the sequence of the active polypeptide is:

[0020] SEQ1: Ac-QYDRQIR-NH2

[0021] SEQ2: Ac-YDRQIRL-NH2

[0022] SEQ3: Ac-AAQYDRQIRLWG-NH2

[0023] The inventors have discovered a series of active polypeptides through research, the general structural formula of which is shown in Formula I. The above active polypeptides can significantly inhibit the SAE1 / SAE2 protein interaction.

[0024] The present invention provides a pharmaceutical composition for preventing or treating tumors such as lymphoma, colorectal cancer, and head and neck cancer. The pharmaceutical composition contains the above-mentioned active polypeptide or its salt, or a mixture of the above-mentioned polypeptide or its salt, and a pharmaceutically acceptable carrier.

[0025] The pharmaceutical composition of the present invention can be prepared into various conventional dosage forms known in the art, including but not limited to tablets suitable for oral administration (including various coated tablets, sustained-release or controlled-release tablets), lozenges, capsules (including soft capsules and hard capsules), granules, dispersible powders, aqueous or oily suspensions, emulsions, elixirs or syrups, etc.; creams, ointments, gels, aqueous or oily solutions or suspensions, etc. suitable for topical use; powders or liquid aerosols suitable for inhalation, sterile aqueous or oily intravenous, subcutaneous or intramuscular injections, suppositories, etc. suitable for parenteral administration.

[0026] Those skilled in the art will appreciate that the appropriate amounts of the anti-tumor peptide as the active ingredient and the pharmaceutically acceptable carrier in the pharmaceutical composition of the present invention can be determined according to conventional methods in the art. Those skilled in the art will also appreciate how to prepare pharmaceutical compositions containing the anti-tumor peptide of the present invention.

[0027] The novel polypeptides of the present invention can be synthesized by methods known to those skilled in the art, such as solid phase synthesis, and purified by methods known to those skilled in the art, such as high performance liquid chromatography.

[0028] The advantages of the present invention include providing a novel polypeptide that inhibits the SAE1 / SAE2 protein interaction in cancer cells, exerting anti-tumor effects against lymphomas, colorectal cancer, head and neck cancer, and other tumors. The polypeptide's steric configuration fits snugly into the SAE2 protein pocket and tightly interacts with the protein through a network of hydrophobic and hydrogen-bonding interactions, thereby strongly inhibiting the SAE1 / SAE2 interaction and exerting an anti-tumor effect. The novel polypeptide of the present invention exhibits high anti-tumor activity and is stable and resistant to inactivation. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The graph shows the inhibitory effect of SEQ1-SEQ3 on SAE1 / SAE2 interaction determined by homogeneous time-resolved fluorescence (HTRF) experiment. DETAILED DESCRIPTION

[0030] The present invention will be further described below in conjunction with specific examples. The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources. It should be understood that these examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention.

[0031] Example 1. Solid-phase synthesis, cleavage, purification and identification of SEQ1-SEQ 3 and SEQ NC.

[0032] The present invention utilizes conventional polypeptide solid phase synthesis method to synthesize polypeptides, and obtains polypeptide products through the steps of resin activation, polypeptide condensation, cleavage, purification and identification.

[0033] Preparation of peptide SEQ1: Ac-QYDRQIR-NH2, solid purity greater than 95%, ESI: 1017.5043 [MH]-;

[0034] Preparation of peptide SEQ2: Ac-YDRQIRL-NH2, solid purity greater than 95%, ESI: 1002.5298 [MH]-;

[0035] Preparation of peptide SEQ3: Ac-AAQYDRQIRLWG-NH2, solid purity greater than 95%, ESI: 1517.69 [MH]-;

[0036] The peptide SEQ NC: Ac-RQIY-NH2 was prepared as a control group, and the solid purity was greater than 95%.

[0037] Example 2: Experiment on the inhibition of SAE1 / SAE2 interaction at the polypeptide molecular level.

[0038] Homogeneous time-resolved fluorescence (HTRF) was used to establish an in vitro screening system for SAE1 / SAE2 interaction inhibitors to evaluate the ability of peptides to inhibit SAE1 / SAE2 interaction at the molecular level.

[0039] (1) Expression and purification of N-terminal GST-tagged SAE1 and N-terminal His-tagged SAE2. Prokaryotic expression vectors pGEX-4T-1-SAE1 and pET28a-SAE2 were constructed and SAE1 and SAE2 were expressed in large quantities in the strain Rosetta (DE3). Purification methods such as affinity chromatography, ion exchange chromatography, and GST-tagged magnetic beads were then used to obtain purified SAE1 and SAE2 proteins expressed by the recombinant plasmids.

[0040] (2) Preparation of stock solution: The synthesized peptides are numbered one by one, such as SEQ1-SEQ3, SEQ NC. Then 1 mg of each is weighed and dissolved in DMSO to an initial concentration of 10 mM.

[0041] (3) Reaction conditions: SAE1 and SAE2 were diluted to their respective concentrations in reaction buffer (20 mM Hepes 7.5, 50 mM NaCl, 1 mM DTT, 0.1% Tween 20, 5 mM MgCl2). Tb cryptate Gold-labeled His-tag antibody (Cat. No. 61HI2TLA, Univ) and d2-labeled GST-tag antibody (Cat. No. 61GSTDLA, Univ) were diluted to their respective concentrations in Terbium detection buffer (Cat. No. 61DB10RDF, Univ) and mixed. The reaction was performed at room temperature in a 384-well plate. Three replicates were performed for each experimental group.

[0042] (4) Serial dilution: Add 4 μL of peptide to tube 1 of the eight-tube PCR strip, and add 2 μL of DMSO to tubes 2-8. Take 2 μL of peptide from tube 1 and dilute it in tube 2, then take out 2 μL and add it to tube 3, and so on until the end of tube 8. After dilution in tube 8, take out 2 μL of peptide and discard it. Add 18 μL of reaction buffer to each tube, vortex to mix thoroughly, and then add 2 μL to each well of the 384-well plate.

[0043] (5) Add 4 μL of SAE2 protein diluent and incubate at room temperature for 0.5 h. Add 4 μL of SAE1 protein diluent and incubate at room temperature for another 1 h.

[0044] (6) Add 10 μL of label antibody dilution solution and continue incubation at room temperature for 1 h.

[0045] (7) Signal value detection: A multifunctional microplate reader (Synergy Reader) was used to detect the fluorescence signal. The excitation light was 340 nm, the emission light was 620 and 665 nm, and the final reading was the 665 nm / 620 nm ratio.

[0046] (8) Data processing: (i) Calculate the mean fluorescence value of the sample, including "SAE1+SAE2" as the positive control and "SAE1" as the negative control; (ii) Then calculate the inhibition (%) according to the following formula: Inhibition (%) = 100*(positive control - sample) / (positive control - negative control). Graphpad Prism 10 software was used to fit the inhibition curve and calculate the IC, with the log value of the final concentration of the small molecule as the horizontal axis and the inhibition rate (%) as the vertical axis. 50 The results are shown in Table 1 below. The corresponding inhibition curve is drawn. Figure 1 shown.

[0047] Table 1. IC of peptides inhibiting SAE1 / SAE2 interaction 50 (Unit: μM)

[0048] Peptide name <![CDATA[IC 50 (μM)]]> Peptide SEQ1 23.08 Peptide SEQ2 43.93 Peptide SEQ3 60.79

[0049] Figure 1 The inhibition rates of SEQ1-SEQ3 on SAE1 / SAE2 interaction at different concentrations are shown. Compared with the peptide SEQ NC as the control group, SEQ1-SEQ3 all showed significantly higher inhibition rates. The maximum inhibition rates of SEQ1-SEQ3 were all higher than 50%, and the maximum inhibition rates of SEQ1 and SEQ3 were higher than 60%. Therefore, Figure 1 The results demonstrated that SEQ1-SEQ3 had a significant inhibitory effect on SAE1 / SAE2 interaction.

[0050] The above description is only a preferred embodiment of the present invention. It should be pointed out that ordinary technicians in this technical field can make several improvements and modifications without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An active polypeptide, characterized in that The active polypeptide is used to inhibit the SAE1 / SAE2 interaction and comprises an amino acid sequence represented by at least one of the following formulas (1), (2), and (3), and a salt thereof: Formula (1): Ac-X1X2X3YDX4QIX5LWG-NH2; Formula (2): Ac-X3YDX4QIX5-NH2; Formula (3): Ac-YDX4QIX5L-NH2; X1 is Ala or Arg; X2 is Ala or Tyr; X3 is Gln or Tyr; X4 is Arg or Asn or Ser or Asp or Thr; X5 is Arg or His.

2. The active polypeptide according to claim 1, characterized in that The active polypeptide is Ac-QYDRQIR-NH2.

3. The active polypeptide according to claim 1, characterized in that The active polypeptide is Ac-YDRQIRL-NH2.

4. The active polypeptide according to claim 1, characterized in that The active polypeptide is Ac-AAQYDRQIRLWG-NH2. 5 . A polypeptide inhibitor for inhibiting SAE1 / SAE2 interaction, comprising the active polypeptide according to any one of claims 1 to 4 .

6. Use of the active polypeptide according to any one of claims 1 to 4 in the preparation of an inhibitor for inhibiting SAE1 / SAE2 interaction.

7. Use of the polypeptide inhibitor according to claim 5 in the preparation of a drug for treating cancer.

8. The use according to claim 7, characterized in that The cancer is lymphoma, colorectal cancer, or head and neck cancer.

9. A pharmaceutical composition for preventing or treating cancer, characterized in that: The pharmaceutical composition contains the active polypeptide according to any one of claims 1 to 4, or the polypeptide inhibitor according to claim 5, and a pharmaceutically acceptable carrier.

10. The use according to claim 9, characterized in that The cancer is lymphoma, colorectal cancer, or head and neck cancer.

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