Method for designing fusion protein for selective knockdown or inhibition of mutant of protein, and use thereof

Abstract

Disclosed in the present invention are a method for designing a fusion protein for selective knockdown or inhibition of a mutant of a protein, and a use thereof. The method comprises the following step: linking a ligand to a ubiquitin ligase to obtain a fusion protein, wherein the ligand has a high affinity for the activated form of a target protein and a low affinity for the inactivated form of the target protein. An artificial protein or a small molecule drug designed by said method targets only mutant-type proteins that lead to diseased tissue, and is ineffective on wild-type proteins in normal tissue, thereby achieving safe treatment.

Description

A method for designing a mutant fusion protein for selectively knocking down or inhibiting a protein and its application Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a design method for a mutant fusion protein for selectively knocking down or inhibiting a protein and its application. Background Art

[0002] Gene mutations are the root cause of most human diseases, particularly all cancers. Diseased tissue often differs from normal tissue in that the mutation in the diseased tissue causes the mutant protein to become constitutively active. This means that the concentration of the activated protein in the mutant diseased tissue is much higher than that in the wild-type normal tissue, thus distinguishing the two.

[0003] Currently, the inhibition of protein activity mainly involves the development of small molecule inhibitors targeting the active sites of target proteins. However, relevant studies have shown that 85% of proteins are "undruggable proteins". These proteins are difficult to develop inhibitors for due to their loose and variable structures, strong competitive binding ability with other small molecules inside the cell, and easy mutation of active sites. It is extremely difficult to achieve the effect of only targeting the mutant protein that causes diseased tissues while being ineffective against the wild-type protein in normal tissues. Therefore, a new drug design method is urgently needed.

[0004] Summary of the Invention

[0005] The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. To this end, the present invention provides a method for designing a mutant fusion protein that selectively knocks down or inhibits a protein.

[0006] The present invention also provides a fusion protein designed by the above method.

[0007] The present invention also provides a fusion protein of mutant Raf 1 S257L that targets and degrades or inhibits Raf 1 protein.

[0008] The present invention also provides a fusion protein of mutant PIK3CA E545K that targets and degrades or inhibits PIK3CA protein.

[0009] The present invention also provides a nucleic acid molecule encoding the above fusion protein.

[0010] The present invention also provides a biological material containing the above nucleic acid molecule.

[0011] The present invention also provides applications of the fusion protein, nucleic acid molecule and biological material.

[0012] According to one aspect of the present invention, a method for designing a mutant fusion protein that selectively knocks down or inhibits a protein is proposed, the method comprising the following steps: linking a ligand to a ubiquitin ligase to obtain a fusion protein; the ligand has a high affinity for the activated form of the target protein and a low affinity for the inactivated form of the target protein.

[0013] In some embodiments of the present invention, the target protein includes one of Raf 1 protein and PIK3CA protein.

[0014] In some embodiments of the invention, the ligand comprises one of p85 and MEK.

[0015] In some embodiments of the present invention, the binding constant of the ligand to the activated form and the inactivated form of the protein differs by a factor of 1000 or more.

[0016] In some embodiments of the present invention, the ubiquitin ligase comprises an E3 ubiquitin ligase and a midnolin protein.

[0017] According to the second aspect of the present invention, the application of the above-mentioned method for designing a mutant fusion protein that selectively knocks down or inhibits a protein is proposed, and the application is in the preparation of a mutant fusion protein or drug that selectively knocks down or inhibits a protein.

[0018] In some embodiments of the present invention, the application is application in the preparation of a drug for treating cancer.

[0019] According to a third aspect of the present invention, a fusion protein is proposed, which is designed by the above method.

[0020] In some embodiments of the present invention, the sequence of the fusion protein is shown as SEQ ID NO: 1 or SEQ ID NO: 3.

[0021] According to a fourth aspect of the present invention, the present invention further provides a fusion protein that targets degradation or inhibits a mutant type of Raf 1 protein, wherein the fusion protein comprises a ligand MEK and a ubiquitin ligase.

[0022] In some embodiments of the invention, the ubiquitin ligase comprises an E3 ubiquitin ligase.

[0023] In some embodiments of the present invention, the amino acid sequence of the ligand MEK is shown in SEQ ID NO:5.

[0024] In some embodiments of the present invention, the amino acid sequence of the E3 ubiquitin ligase is shown in SEQ ID NO:7.

[0025] In some embodiments of the present invention, the amino acid sequence of the fusion protein is: the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence modified by substitution, deletion or addition of one or more amino acids in the amino acid sequence shown in SEQ ID NO: 1, and having the same or similar function.

[0026] In some embodiments of the present invention, the mutant form of the Raf 1 protein includes the Raf 1 S257L mutation.

[0027] According to a fifth aspect of the present invention, the present invention further provides a fusion protein that targets degradation or inhibits a mutant type of PIK3CA protein, wherein the fusion protein comprises a ligand p85 and a ubiquitin ligase.

[0028] In some embodiments of the invention, the ubiquitin ligase comprises an E3 ubiquitin ligase.

[0029] In some embodiments of the present invention, the amino acid sequence of the ligand p85 is shown in SEQ ID NO:11.

[0030] In some embodiments of the present invention, the amino acid sequence of the E3 ubiquitin ligase is shown in SEQ ID NO:7.

[0031] In some embodiments of the present invention, the amino acid sequence of the fusion protein is: the amino acid sequence shown in SEQ ID NO: 3, or an amino acid sequence modified by substitution, deletion or addition of one or more amino acids in the amino acid sequence shown in SEQ ID NO: 3, and having the same or similar function.

[0032] In some embodiments of the present invention, the mutant form of the PIK3CA protein includes a PIK3CA E545K mutation.

[0033] According to the sixth aspect of the present invention, a nucleic acid molecule is provided, comprising DNA or mRNA encoding a mutant fusion protein that targets degradation or inhibition of PIK3CA protein or a mutant fusion protein that targets degradation or inhibition of Raf 1 protein.

[0034] In some embodiments of the present invention, the fusion protein encoding the mutant type that targets and degrades or inhibits the PIK3CA protein is represented by the nucleotide sequence of SEQ ID NO: 4.

[0035] In some embodiments of the present invention, the fusion protein encoding the mutant type that targets and degrades or inhibits Raf 1 protein is represented by the nucleotide sequence of SEQ ID NO: 2.

[0036] According to a seventh aspect of the present invention, a biomaterial related to the above-mentioned nucleic acid molecule is provided, wherein the biomaterial is any one of the following 1) to 3);

[0037] 1) an expression cassette containing the above-mentioned nucleic acid molecule;

[0038] 2) a recombinant vector containing the aforementioned nucleic acid molecule or the expression cassette in 1);

[0039] 3) A recombinant cell containing the aforementioned nucleic acid molecule, the expression cassette in 1) or the recombinant vector in 2).

[0040] According to an eighth aspect of the present invention, an application of the above-mentioned fusion protein, nucleic acid molecule and biomaterial is proposed, wherein the application is for preparing a mutant fusion protein or drug for selectively knocking down or inhibiting a protein.

[0041] In some embodiments of the present invention, the protein comprises one of Raf 1 protein and PIK3CA protein.

[0042] In some embodiments of the present invention, the application is application in the preparation of a drug for treating cancer.

[0043] The technical solution of the present invention has at least the following beneficial effects: The method of the present invention utilizes a ligand with extremely high affinity for the activated form of a certain protein and extremely low affinity for the inactivated form (the binding constant difference is typically more than 1000-fold) to design an artificial protein or small molecule drug. This selectively degrades or inhibits mutant proteins with extremely high concentrations of the activated form, while retaining wild-type proteins with extremely low concentrations of the activated form (the concentration difference is typically more than 10-fold). The artificial protein or small molecule drug thus designed only targets the mutant form of the protein that causes diseased tissues and is ineffective against the wild-type protein in normal tissues, thereby achieving the purpose of safe treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] The present invention will be further described below with reference to the accompanying drawings and embodiments, in which:

[0045] FIG1 is a graph showing the expression results of Raf 1 in wild-type Raf 1 cells containing MS2 in Example 2 of the present invention;

[0046] FIG2 is a graph showing the expression results of Raf 1 in Raf 1 mutant cells containing MS2 in Example 2 of the present invention;

[0047] FIG3 is a graph showing the expression results of PIK3CA in wild-type PIK3CA cells containing PS3 in Example 2 of the present invention;

[0048] FIG4 is a graph showing the expression results of PIK3CA in PIK3CA mutant cells containing PS3 in Example 2 of the present invention. DETAILED DESCRIPTION

[0049] The following will clearly and completely describe the concept and technical effects of the present invention in conjunction with the embodiments to fully understand the purpose, features and effects of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0050] Example 1 A method for designing a mutant fusion protein or drug that selectively knocks down or inhibits a protein

[0051] This example prepares a method for designing a mutant fusion protein or drug that selectively knocks down or inhibits a protein. The specific process is as follows:

[0052] Ligands with extremely high affinity for the activated form of the target protein and extremely low affinity for the inactivated form (the difference in binding constants is usually more than 1000 times) are used to design artificial proteins or small molecule drugs; the designed artificial proteins or small molecule drugs can selectively degrade or inhibit mutant proteins of the target protein with extremely high concentrations of the activated form, while retaining the wild-type protein with extremely low concentrations of the activated form (the concentration difference is usually more than 10 times).

[0053] Example 2 A fusion protein targeting the degradation or inhibition of the mutant Raf 1 S257L Raf 1 protein

[0054] Normal cells will continue to synthesize wild-type Raf1 protein, continuously increasing the level of activated phosphorylated Raf1 (p-Raf1); on the other hand, p-Raf1 will also be rapidly converted to an inactive form of Raf1, and the Raf1 protein will also be continuously ubiquitinated and degraded: this is a dynamic balance in normal cells, ensuring that the p-Raf1 concentration is not too high (<1%). Raf1 mutations, such as the S257L mutation, break this balance, causing the concentration of activated p-Raf1 to increase significantly (more than 10%). This example uses the method in Example 1 to design an artificial protein (MS2) that targets the degradation or inhibition of the mutant Raf1 S257L of Raf1 protein. The specific process is as follows:

[0055] (1) The ligand MEK was selected to construct an artificial protein for specific binding to Raf1, and its affinity for p-Raf1 was much higher than that for inactivated Raf1 (more than 1000 times).

[0056] MEK amino acid sequence:

[0057] MEK nucleotide sequence:

[0058] (2) MEK (amino acid sequence as shown in SEQ ID NO:5, nucleotide sequence as shown in SEQ ID NO:6) and E3 ubiquitin ligase SPOP BTB (amino acid sequence as shown in SEQ ID NO:7, nucleotide sequence as shown in SEQ ID NO:8) are combined into an artificial protein (MS2) (amino acid sequence as shown in SEQ ID NO:1). This protein can degrade the high concentration of p-Raf1 of mutant Raf1, but is ineffective against the low concentration of p-Raf1 of wild-type Raf1. This is because the p-Raf1 concentration of mutant Raf1 is too high and cannot be quickly replenished after degradation; while the p-Raf1 concentration of wild-type Raf1 is extremely low and can be quickly replenished even after degradation.

[0059] Amino acid sequence of E3 ubiquitin ligase SPOP BTB:

[0060] Nucleotide sequence of E3 ubiquitin ligase SPOP BTB:

[0061] MS2 amino acid sequence:

[0062] MS2 nucleotide sequence:

[0063] The validation experiment of MS2 degrading mutant Raf1 through the ubiquitin-proteasome pathway was conducted as follows:

[0064] MS2 (nucleotide sequence shown in SEQ ID NO:2) and a control artificial protein (containing the SPOP BTB artificial protein and an unmodified RNA encoding MS2 but not translating into a protein product (nMS2), amino acid sequence shown in SEQ ID NO:9, nucleotide sequence shown in SEQ ID NO:10) were encoded in mRNA. The mRNA was dissolved in 25 mM sodium acetate buffer (pH 4-5) and vortexed with the LNP lipid component (SM102, DSPC, cholesterol, and DMG-PEG2000 dissolved in ethanol at a molar ratio of 50:10:38.5:1.5) at a mass ratio of 1:10. The volume ratio of the aqueous phase to the ethanol phase was greater than 5:1. The ethanol in the mRNA-loaded LNPs was removed by dialysis against PBS.

[0065] 300,000 cells were seeded per well of a 6-well plate in DMEM supplemented with 10% fetal bovine serum and cultured at 37°C. LNPs loaded with mRNA were added to Raf 1 wild-type (HCC56) and Raf 1 S257L mutant (HCC2998) cells and incubated for 15 hours at a concentration of 1 μg mRNA / mL. The mRNAs included mRNA encoding MS2 (nucleotide sequence shown in SEQ ID NO: 2), a control artificial protein containing SPOP BTB artificial protein S1, and an unmodified RNA encoding MS2 but not translated into a protein product (nMS2). The proteasome inhibitor MG132 (final concentration of 10 μM) (to inhibit MS2 degradation of mutant Raf 1) was added to the Raf 1 mutant and wild-type cells containing MS2 mRNA. The culture medium was then removed, the cells were washed twice with pre-chilled PBS, and pre-chilled RIPA lysis buffer was added. After incubation at 4°C for 5 minutes, the cell debris suspension was collected with a cell scraper and centrifuged at 14,000 × g for 10 minutes. The supernatant was collected for Western blotting analysis to detect protein expression in the cells.

[0066] Artificial protein sequence of the control group:

[0067] Nucleotide sequence of artificial protein in control group:

[0068] The results are shown in Figures 1-2. As can be seen from the figures, compared with the control group, MS2 was unable to degrade Raf1 in wild-type Raf1 cells (Figure 1), but only degraded Raf1 in mutant Raf1 cells (Figure 2). MG132 inhibited MS2 degradation of Raf1 in mutant Raf1 cells, indicating that MS2 degrades Raf1 through the ubiquitin-proteasome pathway.

[0069] Example 3 A fusion protein targeting degradation or inhibition of PIK3CA protein with a mutant PIK3CA E545K mutation

[0070] Normal cells will continue to synthesize wild-type PIK3CA protein, continuously increasing the level of activated phosphorylated PIK3CA (p-PIK3CA); on the other hand, p-PIK3CA will also be rapidly converted into an inactive form of PIK3CA, and the PIK3CA protein will also be continuously ubiquitinated and degraded: this is a dynamic balance in normal cells to ensure that the p-PIK3CA concentration is not too high (<1%). PIK3CA mutations, such as the E545K mutation, break this balance, causing the concentration of activated p-PIK3CA to increase significantly (more than 10%). This example uses the method in Example 1 to design a mutant PIK3CAE545K mutation fusion protein (PS3) that targets degradation or inhibition of PIK3CA protein. The specific process is as follows:

[0071] (1) p85 was selected to construct an artificial protein for specific binding to PIK3CA, and its affinity for p-PIK3CA was much higher than that for inactivated PIK3CA (more than 1000 times).

[0072] (2) p85 (amino acid sequence as shown in SEQ ID NO:11) and E3 ubiquitin ligase SPOP BTB (amino acid sequence as shown in SEQ ID NO:7, nucleotide sequence as shown in SEQ ID NO:8) were combined into an artificial protein (PS3) (amino acid sequence as shown in SEQ ID NO:3). This protein can degrade high-concentration p-PIK3CA of mutant PIK3CA, but is ineffective against low-concentration p-PIK3CA of wild-type PIK3CA. This is because the p-PIK3CA concentration of mutant PIK3CA is too high and cannot be quickly replenished after degradation; while the p-PIK3CA concentration of wild-type PIK3CA is extremely low and can be quickly replenished even after degradation.

[0073] p85 amino acid sequence:

[0074] PS3 amino acid sequence:

[0075] PS3 nucleotide sequence:

[0076] The specific steps of the experiment to verify the degradation of mutant PIK3CA by PS3 through the ubiquitin proteasome pathway are as follows:

[0077] PS3 (nucleotide sequence shown in SEQ ID NO:4) and a control artificial protein (containing the SPOP BTB artificial protein (S1) and an unmodified RNA encoding PS3 but not translating into a protein product (nPS3), amino acid sequence shown in SEQ ID NO:9, nucleotide sequence shown in SEQ ID NO:10) were encoded in mRNA. The mRNA was dissolved in 25 mM sodium acetate buffer (pH 4-5) and vortexed with lipid nanoparticles (LNPs; LNPs were prepared by dissolving SM102, DSPC, cholesterol, and DMG-PEG2000 in ethanol at a molar ratio of 50:10:38.5:1.5) at a mass ratio of 1:10. The volume ratio of the aqueous phase to the ethanol phase was greater than 5:1. The ethanol in the mRNA-loaded LNPs was removed by dialysis against PBS.

[0078] 300,000 cells were seeded per well of a 6-well plate in DMEM supplemented with 10% fetal bovine serum and cultured at 37°C. mRNA-loaded LNPs were added to PIK3CA wild-type (MCF10A) and PIK3CA E545K mutant (MCF7) cells and incubated for 15 hours at a concentration of 1 μg mRNA / mL. The mRNAs included mRNA encoding PS3 (nucleotide sequence shown in SEQ ID NO:4), a control artificial protein containing SPOP BTB artificial protein S1, and an unmodified RNA encoding PS3 but unable to produce a protein product (nPS3). The proteasome inhibitor MG132 (final concentration of 10 μM) (to inhibit PS3 degradation of mutant PIK3CA) was added to the PIK3CA mutant and wild-type cells containing PS3. The culture medium was then removed, the cells were washed twice with pre-chilled PBS, and pre-chilled RIPA lysis buffer was added. After incubation at 4°C for 5 minutes, the cell debris suspension was collected with a cell scraper and centrifuged at 14,000 × g for 10 minutes. The supernatant was collected for protein immunoblotting analysis to detect protein expression in the cells.

[0079] Nucleotide sequence of artificial protein in control group:

[0080] The test results are shown in Figures 3 and 4. As can be seen from the figures, compared with the control group, PS3 was unable to degrade PIK3CA in wild-type PIK3CA cells (Figure 3), but only degraded PIK3CA in mutant PIK3CA cells (Figure 4). MG132 inhibited PS3 degradation of PIK3CA in mutant PIK3CA cells, indicating that PS3 degrades PIK3CA through the ubiquitin-proteasome pathway.

[0081] While the embodiments of the present invention have been described in detail above with reference to the accompanying drawings, the present invention is not limited to the embodiments described above. Various modifications may be made within the scope of knowledge possessed by a person skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof may be combined with one another unless there is a conflict.

Claims

1. A method for designing a mutant fusion protein for selectively knocking down or inhibiting a target protein, characterized in that, The design method includes the following steps: connecting a ligand with a ubiquitin ligase to obtain a fusion protein; the ligand has a high affinity for the activated form of the target protein and a low affinity for the unactivated form of the target protein.

2. The design method according to claim 1, characterized in that, The target protein includes one of Raf1 protein and PIK3CA protein; the ligand includes one of p85 and MEK; the ubiquitin ligase includes E3 ubiquitin ligase and midnolin protein.

3. Application of the design method according to claim 1 or 2 in any one of the following (1)-(2): (1) Preparing a fusion protein or a drug with a mutant form of a protein that selectively knockdowns or inhibits the protein; (2) Preparing a drug for treating cancer.

4. A fusion protein, characterized in that, The fusion protein is designed by the design method according to claim 1 or 2; preferably, the sequence of the fusion protein is as shown in SEQ ID NO:1 or SEQ ID NO:

3.

5. A mutant fusion protein that targets and degrades or inhibits Raf1 protein, characterized in that, The fusion protein includes the ligand MEK and a ubiquitin ligase; preferably, the ubiquitin ligase includes E3 ubiquitin ligase and midnolin protein; preferably, the amino acid sequence of the ligand MEK is as shown in SEQ ID NO:5; preferably, the amino acid sequence of the fusion protein is: the amino acid sequence as shown in SEQ ID NO:1 or the amino acid sequence as shown in SEQ ID NO:1 after being modified by substitution, deletion or addition of one or more amino acids and having the same or similar function; preferably, the mutant form of the Raf1 protein includes Raf1 S257L mutation.

6. A fusion protein for targeted degradation or inhibition of the mutant form of the PIK3CA protein, characterized in that, The fusion protein includes the ligand p85 and a ubiquitin ligase.

7. The fusion protein according to claim 6, wherein, The ubiquitin ligase includes E3 ubiquitin ligase and midnolin protein; preferably, the amino acid sequence of the ligand p85 is as shown in SEQ ID NO:11; preferably, the amino acid sequence of the fusion protein is: the amino acid sequence as shown in SEQ ID NO:3 or the amino acid sequence as shown in SEQ ID NO:3 after being modified by substitution, deletion or addition of one or more amino acids and having the same or similar function; preferably, the mutant form of the PIK3CA protein includes PIK3CA E545K mutation.

8. A nucleic acid molecule, characterized in that, The nucleic acid molecule includes DNA or mRNA encoding the fusion protein according to claim 4, encoding the fusion protein with a mutant form of the Raf1 protein for targeted degradation or inhibition according to claim 5, or encoding the fusion protein with a mutant form of the PIK3CA protein for targeted degradation or inhibition according to claim 6 or 7.

9. A biological material related to the nucleic acid molecule, and the biological material is any one of the following 1)-3): 1) An expression cassette containing the nucleic acid molecule according to claim 8; 2) A recombinant vector containing the nucleic acid molecule according to claim 8 or the expression cassette in 1); 3) A recombinant cell containing the nucleic acid molecule according to claim 8, the expression cassette in 1) or the recombinant vector in 2).

10. Use of at least one of the fusion protein according to claim 4, the mutant fusion protein targeting degradation or inhibition of Raf1 protein according to claim 5, the mutant fusion protein targeting degradation or inhibition of PIK3CA protein according to claim 6 or 7, the nucleic acid molecule according to claim 8, and the biological material according to claim 9 in any one of the following a1)-a2): a1) Preparing a mutant fusion protein or a drug for selectively knocking down or inhibiting a protein; a2) Preparing a drug for treating cancer; preferably, the protein includes one of Raf1 protein and PIK3CA protein.

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