Method for evaluating the success of a chimeric ubiquitin ligase design and applications

Abstract

The application belongs to the technical field of biology, and discloses a method for evaluating the success of chimeric ubiquitin ligase design and application. The evaluation method comprises the following steps: designing a chimeric ubiquitin ligase; constructing a ubiquitin complex; performing sequence alignment using AF2 to calculate five alignment models; if the alignment models simultaneously satisfy the conditions of RMSD < 2, the overlapping volume accounts for < 7%, and the shortest distance between the lysine residues of the protein of interest and the C-terminal glycine residues of ubiquitin is < 7 nm, the models are qualified; if the number of qualified models is greater than or equal to 2, the chimeric ubiquitin ligase design is successful. The application also proposes a chimeric ubiquitin ligase screened by the above method and application of the chimeric ubiquitin ligase in preparation of products for treating various diseases. The above evaluation method proposes an evaluation principle, can accurately judge whether the chimeric ubiquitin ligase design is successful, provides theoretical guidance for improvement of the chimeric ubiquitin ligase with failed design, and has universal applicability.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biotechnology, and particularly relates to a method for evaluating the design success of a chimeric ubiquitin ligase and application thereof. BACKGROUND

[0002] Targeted proteolysis is a new model for human disease diagnosis and treatment. At present, the methods mainly used for targeted proteolysis include the construction of protein proteolysis targeting chimeras (PROTAC) and lysosome targeting chimeras (LYTAC). However, their application is limited by the following: 1) limited to small molecules that bind to the protein of interest (POI); 2) failure in clinical translation of small molecules with different metabolisms between humans and experimental animals. Based on existing protein interactions, replacing molecular ligands with protein ligands to bind to the protein of interest is undoubtedly more universal, and also has a lower metabolic risk. With the development of structural biology and computational biology, this possibility has become a reality. Researchers have designed a series of chimeric ubiquitin ligases, including a protein of interest binding domain and a ubiquitin ligase domain, which can target any cytosolic protein of interest. Due to the multiple choices of affinity binders and thousands of ubiquitin ligases, different combinations can match multiple ubiquitin ligases for each protein of interest, that is, multiple chimeric ubiquitin ligases can be designed. However, whether the currently designed chimeric ubiquitin ligases are qualified is rarely verified, and there is also a lack of theoretical guidance for the improvement of chimeric ubiquitin ligases that fail to be designed. SUMMARY

[0003] The present application aims to solve at least one of the technical problems existing in the prior art. To this end, the present application proposes a method for evaluating the design success of a chimeric ubiquitin ligase, which proposes the principles for evaluation, can accurately determine whether the design of the chimeric ubiquitin ligase is successful, and provides theoretical guidance for the improvement of chimeric ubiquitin ligases that fail to be designed, and has universal applicability.

[0004] The present application also proposes an improvement method for chimeric ubiquitin ligases that fail to be designed.

[0005] The present application also proposes a chimeric ubiquitin ligase capable of targeting and degrading a protein of interest, which is screened by using the evaluation method.

[0006] The present application also proposes the use of the chimeric ubiquitin ligase in the preparation of a product for treating at least one of a tumor, a cardiovascular disease, an immune disease, a nutritional and metabolic disease, a nervous system disease, a respiratory system disease, a digestive system disease, and an infectious disease.

[0007] According to one aspect of the present application, a method for evaluating the design success of a chimeric ubiquitin ligase is proposed, which comprises the following steps:

[0008] S1: designing a chimeric ubiquitin ligase for targeting and degrading a protein of interest;

[0009] S2: determining the family of the natural E3 ubiquitin ligase to which the chimeric ubiquitin ligase belongs, and constructing a ubiquitin complex of the natural E3 ubiquitin ligase; the ubiquitin complex is a natural E3 ubiquitin ligase / ubiquitin, and the natural E3 ubiquitin ligase is a second linker protein / linker protein / E2 ubiquitin ligase recruiting domain;

[0010] S3: calculating the structure of the chimeric ubiquitin ligase / protein of interest complex by using AlphaFold2 (AF2) software to obtain five models; by performing sequence alignment on the chimeric ubiquitin ligase and the natural E3 ubiquitin ligase to which the chimeric ubiquitin ligase belongs, the chimeric ubiquitin ligase / protein of interest complex is positioned in the ubiquitin complex to obtain five alignment models, and the following parameters are evaluated and recorded: (1) root mean square deviation (RMSD) of sequence alignment of each model; (2) in each alignment model, the proportion of the overlapping volume of the chimeric ubiquitin ligase / protein of interest complex and the linker protein to the volume of the linker protein except the alignment part; (3) the shortest distance between the lysine residue of the protein of interest after positioning and the glycine residue at the C-terminus of the ubiquitin;

[0011] S4: evaluating whether the parameters of the five alignment models satisfy the following conditions: (1) RMSD < 2; (2) the proportion of the overlapping volume < 7%; (3) the shortest distance between the lysine residue of the protein of interest after positioning and the glycine residue at the C-terminus of the ubiquitin < 7 nm; if the above three conditions are met at the same time, the alignment model is a qualified model; if at least one of the above three conditions is not met, the alignment model is an unqualified model;

[0012] S5: counting the number of qualified models, if the number of qualified models is ≥ 2, the design of the chimeric ubiquitin ligase is successful, and the chimeric ubiquitin ligase can target the degradation of the protein of interest.

[0013] In some embodiments of the present application, the method for designing the chimeric ubiquitin ligase in step S1 comprises the following steps: determining a protein of interest binding domain according to the protein of interest to be degraded; and connecting the protein of interest binding domain with a first linker protein to form the chimeric ubiquitin ligase.

[0014] Specifically, the method for determining the protein of interest binding domain comprises: searching for proteins or polypeptides interacting with the protein of interest from a database, and selecting a fragment capable of binding to the protein of interest to obtain the protein of interest binding domain.

[0015] Specifically, the database includes any one of MCE database, PDB database, Proteinatlas database, InterPro database, Binding DB database, String database, GENATLAS database. The database is not limited to the above-mentioned databases, and a suitable database can be selected as needed.

[0016] Specifically, the first linker protein is a protein capable of binding to the linker protein in the ubiquitin complex.

[0017] Specifically, the first linker protein is the same as the second linker protein, or the first linker protein is part of the second linker protein.

[0018] In some embodiments of the present application, the chimeric ubiquitin ligase in step S2 is designed based on the natural E3 ubiquitin ligase, so the family of the natural E3 ubiquitin ligase to which it belongs should be determined first.

[0019] In some embodiments of the present application, the family of the natural E3 ubiquitin ligase includes any one of the HECT domain family, the RING domain family, and the U.box domain family.

[0020] In some preferred embodiments of the present application, the natural E3 ubiquitin ligase belongs to the RING domain family; the first linker protein is SPOP (167-374), the second linker protein is SPOP (full length), and the linker protein is Cullin protein.

[0021] In some embodiments of the present application, the ubiquitin complex constructed in step S2 is saved to Pymol software.

[0022] Specifically, AlphaFold2 in step S3 is a neural network system based on attention that processes the internal and external relationships of amino acid sequences and is trained in an end-to-end manner to understand the graph structure, and performs reasoning based on the implicit graph constructed thereby. The entire AF2 algorithm includes three parts: DataPipeline, Evoformer, and Structuremodule. When performing protein structure reasoning, AF2 inputs the amino acid sequence and outputs the three-dimensional structure coordinates to realize end-to-end structure prediction.

[0023] In some embodiments of the present application, step S3 performs sequence alignment of the chimeric ubiquitin ligase and the natural E3 ubiquitin ligase to which it belongs using Pymol software.

[0024] Specifically, since the first linker protein in the chimeric ubiquitin ligase is the same as or partially the same as the second linker protein in the native E3 ubiquitin ligase, the chimeric ubiquitin ligase / protein of interest complex can be located in the ubiquitin complex by sequence alignment of the first linker protein and the second linker protein, thereby obtaining the alignment model.

[0025] In some preferred embodiments of the present application, the native E3 ubiquitin ligase is SPOP / CUL3 / RBX1, and the ubiquitin complex is SPOP / CUL3 / RBX1 / UB.

[0026] In some preferred embodiments of the present application, the protein of interest of the chimeric ubiquitin ligase is PLK1, and the protein interacting therewith is a DARPin protein. AF2 calculates 5 SPOP(167-374) / DARPin / PLK1 models. Each model is subjected to sequence alignment with the above-mentioned native E3 ubiquitin ligase SPOP / CUL3 / RBX1 by Pymol software, and the 5 SPOP(167-374) / DARPin / PLK1 models are located in SPOP / CUL3 / RBX1 / UB, respectively, to obtain 5 alignment models. The following parameters are evaluated and recorded: (1) the RMSD of each model subjected to sequence alignment; (2) in each alignment model, the proportion of the overlapping volume of SPOP(167-374) / DARPin / PLK1 and CUL3 to the volume of CUL3, except for the alignment part; (3) the shortest distance between the lysine residue of PLK1 after location and the C-terminal glycine residue of UB.

[0027] In some embodiments of the present application, RMSD is used to evaluate the arrangement of the first linker protein in the chimeric ubiquitin ligase. The higher the RMSD, the higher the denaturation degree of the BTB domain in the first linker protein, which will lead to the inability of the chimeric ubiquitin ligase to tightly bind to the linker protein.

[0028] In some embodiments of the present application, the proportion of the overlapping volume of the chimeric ubiquitin ligase / protein of interest complex and the linker protein to the volume of the linker protein is used to evaluate the steric hindrance of the binding of the chimeric ubiquitin ligase and the linker protein. The larger the above-mentioned proportion, the higher the steric hindrance of the binding, which is not conducive to the binding of the two.

[0029] In some embodiments of the present application, the shortest distance between the lysine residue of the protein of interest after location and the C-terminal glycine residue of the ubiquitin is used to evaluate the degree of ubiquitination. The closer the distance, the more conducive to the progress of ubiquitination.

[0030] However, the ranges of the above three parameters must also be within certain ranges to accurately evaluate each alignment model, and the specific ranges are shown in step S4.

[0031] According to a second aspect of the present application, an improved method for designing a failed chimeric ubiquitin ligase is provided, comprising the following steps: replacing the binding domain of the protein of interest in the chimeric ubiquitin ligase, and / or modifying the partial amino acid sequence of the chimeric ubiquitin ligase to obtain a new chimeric ubiquitin ligase; and then evaluating the new chimeric ubiquitin ligase according to the evaluation method provided in the first aspect to determine whether the improvement is successful.

[0032] Specifically, the failed chimeric ubiquitin ligase generally has less than 2 qualified models among the 5 alignment models, so that a new binding domain of the protein of interest is selected by selecting other binding proteins of the protein of interest; and / or the partial amino acid sequence of the chimeric ubiquitin ligase is modified so that more than 2 (including 2) alignment models can simultaneously satisfy the following three conditions: (1) RMSD < 2; (2) the overlap volume ratio < 7%; (3) the shortest distance between the lysine residue of the protein of interest and the glycine residue at the C-terminus of the ubiquitin after positioning < 7 nm. Further, the new chimeric ubiquitin ligase can successfully degrade the protein of interest.

[0033] According to a third aspect of the present application, a chimeric ubiquitin ligase capable of targeted degradation of the protein of interest is provided.

[0034] According to a fourth aspect of the present application, the chimeric ubiquitin ligase is used in the preparation of a product for treating at least one of a tumor, a cardiovascular disease, an immune disease, a nutritional and metabolic disease, a nervous system disease, a respiratory system disease, a digestive system disease, and an infectious disease.

[0035] According to a preferred embodiment of the present application, at least the following beneficial effects are achieved:

[0036] The present application evaluates whether the design of the chimeric ubiquitin ligase is successful, wherein the criteria for the evaluation are given, the judgment of the method has high accuracy, and improvement of the chimeric ubiquitin ligase which fails in design is provided. The present application can help researchers to accurately design the chimeric ubiquitin ligase with function and improve research efficiency. Without knowing whether the designed chimeric ubiquitin ligase is qualified, the subsequent research is carried out, and the error is found in the subsequent research, so that the research efficiency is reduced. In addition, the evaluation method of the present application has universal applicability, and the chimeric ubiquitin ligase for various interested proteins can be designed. Finally, the chimeric ubiquitin ligase with function can be screened according to the evaluation method of the present application, and the product containing the chimeric ubiquitin ligase is prepared and used for treating the corresponding diseases, so that a better treatment effect can be achieved. BRIEF DESCRIPTION OF DRAWINGS

[0037] The present application will be further described below in combination with the drawings and examples, wherein:

[0038] Figure 1 Figure 1 is the Western-blotting detection result diagram of Example 1 of the present application; wherein, C - control group, D1 - transfer Flag-DARPin, S1 - transfer Flag-SPOP(167-374) group, DS1 - transfer Flag-DARPin / SPOP(167-374), scRNA - non-functional control RNA, siRNA - knock down PLK1 RNA; Flag-tag indicates the expression of the protein connected therewith; HSP90 as an internal reference protein;

[0039] Figure 2 Figure 2 is the Western-blotting detection result diagram of Example 2 of the present application; wherein, C - control group; S1 - transfer Flag-SPOP(167-374) group; FS2(-) - transfer Flag-F1324 / SPOP(167-374), without proteasome inhibitor MG132; FS2(MG132) - transfer Flag-F1324 / SPOP(167-374), with MG132; Flag-tag indicates the expression of the protein connected therewith; GAPDH as an internal reference protein;

[0040] Figure 3Figure for Western-blotting detection result of Example 3 of the present application; wherein, C - control group; S1 - group of introducing Flag-SPOP (167-374); YS3(-) - group of introducing Flag-YAP1 (50-105) / SPOP (167-374) without adding MG132; YS3 (MG132) - group of introducing Flag-YAP1 (50-105) / SPOP (167-374) with adding MG132; Flag-tag indicates the expression of the protein connected therewith; HSP90 as internal reference protein;

[0041] Figure 4 Figure for Western-blotting detection result of Example 4, 5, 6 of the present application; wherein, C - control group; S1 - group of introducing Flag-SPOP (167-374); DS12(-) - group of introducing Flag-DARPin.K27 / SPOP (167-374) without adding MG132; DS12 (MG132) - group of introducing Flag-DARPin.K27 / SPOP (167-374) with adding MG132; DS13(-) - group of introducing Flag-DARPin.K27.1 / SPOP.1 (167-374) without adding MG132; DS13 (MG132) - group of introducing Flag-DARPin.K27.1 / SPOP.1 (167-374) with adding MG132; RS14(-) - group of introducing Flag-Raf1 (52-188) / SPOP (167-374) without adding MG132; RS14 (MG132) - group of introducing Flag-Raf1 (52-188) / SPOP (167-374) with adding MG132; Flag-tag indicates the expression of the protein connected therewith; HSP90 as internal reference protein;

[0042] Figure 5 Figure for Western-blotting detection result of Example 7 of the present application; wherein, C - control group; E1 - group of introducing Flag-ELOB (2-118); ME15(-) - group of introducing Flag-MAX (2-83) / ELOB (2-118) without adding MG132; ME15 (MG132) - group of introducing Flag-MAX (2-83) / ELOB (2-118) with adding MG132; Flag-tag indicates the expression of the protein connected therewith; GAPDH as internal reference protein. DETAILED DESCRIPTION

[0043] The embodiments of the present application are described in detail below, and the described embodiments are exemplary only, and are not to be understood as limiting the present application.

[0044] In the description of the present application, if the first, second, only for the purpose of distinguishing technical features described to, and can not be understood as indicating or implying the relative importance or implied indicated the number of technical features or implied indicated the technical features of the order.

[0045] In the description of the present application, unless otherwise expressly limited, the words construction, positioning and the like should be broadly understood, the skilled in the art can be combined with the specific content of the technical solution to determine the specific meaning of the above words in the present application.

[0046] In the description of the present application, the description of the reference terms "one embodiment", "some embodiments" and the like means that the specific features, structures, materials or characteristics described in conjunction with the embodiment are contained in at least one embodiment of the present application. In the present specification, the illustrative expression of the above terms does not necessarily refer to the same embodiment. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments in a suitable manner.

[0047] The test method used in the examples is a conventional method unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, can be obtained from commercially available reagents and materials.

[0048] Example 1

[0049] This embodiment designs a chimeric ubiquitin ligase DS1, and whether the design is successful is evaluated, the specific process is:

[0050] (1) The protein of interest in this embodiment is PLK1, and a protein DARPin that can bind PLK1 is searched from the MCE database as the protein of interest binding domain; the DARPin is connected with the first linker protein SPOP (167-374) to form a chimeric ubiquitin ligase DS1. The amino acid sequence of DS1 is shown in SEQ ID NO: 1: MEQKLISEEDL GGLDYKDDDDKASGSDLGKKLLEAARAGQDDEVRILIANGADVNAVDNTGLTPLHLAAVSGHLEIVEVLLKHGADVDAADVYGFTPLHLAAMTGHLEIVEVLLKYGADVNAFDMTGSTPLHLAADEGHLEIVEVLLKYGSGSGSGSVNISGQNTMNMVKVPECRLADELGGLWENSRFTDCCLCVAGQEFQAHKAILAARSPVFSAMFEHEMEESKKNRVEINDVEPEVFKEMMCFIYTGKAPNLDKMADDLLAAADKYALERLKVMCEDALCSNLSVENAAEILILADLHSADQLKTQAVDFINYHASDVLETSGWKSMVVSHPHLVAEAYRSLASAQCPFLGPPRKRLKQSGS.

[0051] (2) Since the natural E3 ubiquitin ligase SPOP in the chimeric ubiquitin ligase DS1 belongs to the Cullin-RING BTB-box family, an SPOP-based ubiquitin complex is constructed, which is SPOP / CUL3 / RBX1 / UB (corresponding to the second linker protein / connector protein / E2 ubiquitin ligase recruitment domain / ubiquitin), and the ubiquitin complex is saved in the Pymol software.

[0052] (3) The structure of the DS1 / PLK1 complex is calculated by AF2 software, and 5 models are obtained; the sequence alignment of DS1 and SPOP is performed by Pymol software, so as to position the 5 DS1 / PLK1 complex models in SPOP / CUL3 / RBX1 / UB, and 5 alignment models are obtained, and the RMSD values of the 5 alignment models, the proportion of the overlapping volume of the DS1 / PLK1 complex and CUL3 to the volume of CUL3, and the shortest distance between the lysine residues of PLK1 and the C-terminal glycine residues of UB are evaluated and recorded.

[0053] (4) The RMSD values of the 5 alignment models are 0.559, 0.402, 0.445, 0.468, and 0.808 in turn, all of which are <2;

[0054] The ratio of the overlapping volume of the DS1 / PLK1 complex and CUL3 to the volume of CUL3 is 5.34%, 7.10%, 2.51%, 4.22%, 3.95%, respectively, wherein the ratio of the first, third, fourth and fifth alignment models is less than 7%, and the ratio of the second alignment model is more than 7%;

[0055] The shortest distance between the lysine residue of PLK1 and the C-terminal glycine residue of UB is 4.28 nm, 4.72 nm, 4.56 nm, 3.96 nm, and 4.87 nm, respectively, all of which are less than 7 nm;

[0056] It can be seen that only the first, third, fourth and fifth alignment models satisfy the above three conditions simultaneously, and are qualified models, and the number of qualified models is 4, which is more than 2. Therefore, it can be determined that the design of DS1 is successful, and PLK1 can be successfully knocked out.

[0057] The above results are verified in the following experiments: Hep3B cells (human liver cancer cells) are divided into 6 groups, one group is a control group (C) without treatment; the other groups are Hep3B cells into which Flag-DARPin (D1), Flag-SPOP (167-374) (S1), Flag-DARPin / SPOP (167-374) (DS1), non-functional control RNA (scRNA) and PLK1 knockdown RNA (siRNA) are introduced, respectively. After culture, proteins are collected and Western-blotting experiments are performed, and the results are shown in Figure 1 Figure 1 It is shown that the introduction of only DARPin or SPOP (167-374) does not affect the expression of PLK1 protein; and the introduction of the chimeric ubiquitin ligase DS1 significantly reduces the expression of PLK1 protein, which is comparable to the effect of introducing siRNA, indicating that the chimeric ubiquitin ligase can significantly knock out PLK1 protein.

[0058] ​The amino acid sequence of D1 is shown in SEQ ID NO: 2: MGLDYKDDDDKASGSDLGKKLLEAARAGQ DDEVRILIANGADVNAVDNTGLTPLHLAAVSGHLEIVEVLLKHGADVDAADVYGFTP LHLAAMTGHLEIVEVLLKYGADVNAFDMTGSTPLHLAADEGHLEIVEVLLKYGS. The amino acid sequence of S1 is shown in SEQ ID NO: 3: MGLDYKDDDDKASGSVNISGQNTMNMVKV PECRLADELGGLWENSRFTDCCLCVAGQEFQAHKAILAARSPVFSAMFEHEMEESKKNRVEINDVEPEVFKEMMCFIYTGKAPNLDKMADDLLAAADKYALERLKVMCEDALCSNLSVENAAEILILADLHSADQLKTQAVDFINYHASDVLETSGWKSMVVSHPHLVAEAYRSLASAQCPFLGPPRKRLKQSGS.

[0059] Example 2

[0060] In this example, a chimeric ubiquitin ligase FS2 is designed, and whether the design is successful is evaluated, and the specific process is as follows:

[0061] (1) The protein of interest in this example is BCL6, and the polypeptide F1324 that can bind to BCL6 is found in the MCE database as the protein of interest binding domain; F1324 is connected with the first linker protein SPOP (167-374) to form a chimeric ubiquitin ligase FS2. The amino acid sequence of FS2 is shown in SEQ ID NO: 4: MGLDYKDDDDKAS GLWYTDIRMSWRVPGSGSGSGSVNISGQNTMNMVKVPECRLADELGGLWENSRFTDCCLCVAGQEFQAHKAILAARSPVFSAMFEHEMEESKKNRVEINDVEPEVFKEMMCFIYTGKAPNLDKMADDLLAAADKYALERLKVMCEDALCSNLSVENAAEILILADLHSADQLKTQAVDFINYHASDVLETSGWKSMVVSHPHLVAEAYRSLASAQCPFLGPPRKRLKQSGS.

[0062] (2) The ubiquitin complex based on SPOP, which is SPOP / CUL3 / RBX1 / UB, is constructed, and the ubiquitin complex is saved in Pymol software.

[0063] (3) Calculate the structure of FS2 / BCL6 complex with AF2 software, get 5 models; through sequence alignment of FS2 and SPOP with Pymol software, 5 FS2 / BCL6 complex models are positioned to SPOP / CUL3 / RBX1 / UB, 5 alignment models are obtained, the RMSD values of 5 alignment models are evaluated and recorded, the proportion of the overlapping volume of FS2 / BCL6 complex and CUL3 to the volume of CUL3, and the shortest distance between the lysine residues of BXL6 and the C-terminal glycine residues of UB.

[0064] (4) The RMSD values of the 5 alignment models are 1.188, 0.503, 1.217, 0.501, 1.421, respectively, all <2;

[0065] The proportion of the overlapping volume of FS2 / BCL6 complex and CUL3 to the volume of CUL3 is 6.40%, 7.56%, 4.50%, 4.63%, 5.94%, respectively, the first, third, fourth and fifth alignment models are <7%, the second alignment model is >7%;

[0066] The shortest distance between the lysine residues of BXL6 and the C-terminal glycine residues of UB is 5.88 nm, 4.73 nm, 5.47 nm, 5.18 nm, 5.05 nm, respectively, all <7 nm;

[0067] It can be seen that only the first, third, fourth and fifth alignment models in the 5 alignment models meet the above three conditions at the same time, which are qualified models, the number of qualified models is 4, >2. Therefore, it can be determined that the design of FS2 is successful, and BCL6 can be successfully knocked out.

[0068] The above results are verified in the following experiments: Hep3B cells (human liver cancer cells) are divided into 4 groups, one group is the control group (C), without treatment; the other groups are respectively: transfected with Flag-SPOP (167-374) (S1); transfected with Flag-F1324 / SPOP (167-374) (FS2), without proteasome inhibitor MG132; transfected with Flag-F1324 / SPOP (167-374) (FS2), with MG132 Hep3B cells. After culture, the protein is collected, and Western-blotting experiment is carried out, and the results are shown in Figure 2 . Figure 2As shown, only SPOP (167-374) was introduced, and the expression of BCL6 protein was not affected; the expression of BCL6 protein was significantly weakened when the chimeric ubiquitin ligase FS2 was introduced without MG132; the expression of BCL6 protein was not weakened when the chimeric ubiquitin ligase FS2 was introduced with MG132, because MG132 inhibited the degradation of BCL6 protein. It is shown that the chimeric ubiquitin ligase FS2 can significantly knock out BCL6 protein.

[0069] Example 3

[0070] In this example, the chimeric ubiquitin ligase YS3 is designed, and whether the design is successful is evaluated. The specific process is as follows:

[0071] (1) The protein of interest in this example is TEAD1, and the protein YAP1 that can bind TEAD1 is found from the MCE database, and the protein fragment YAP1 (50-105) is used as the protein binding domain of interest; YAP1 (50-105) is connected with the first linker protein SPOP (167-374) to form the chimeric ubiquitin ligase YS3. The amino acid sequence of YS3 is shown in SEQ ID NO: 5: MGDYKDDDDKASSHMAGHQIVHVRGDSETDLEALFNAVMNPKTANVPQTVPMRLRKLPDSFFKPPEPKSHSGSGSGSVNISGQNTMNMVKVPECRLADELGGLWENSRFTDCCLCVAGQEFQAHKAILAARSPVFSAMFEHEMEESKKNRVEINDVEPEVFKEMMCFIYTGKAPNLDKMADDLLAAADKYALERLKVMCEDALCSNLSVENAAEILILADLHSADQLKTQAVDFINYHASDVLETSGWKSMVVSHPHLVAEAYRSLASAQCPFLGPPRKRLKQSGS.

[0072] (2) The SPOP-based ubiquitin complex, which is SPOP / CUL3 / RBX1 / UB, is constructed, and the ubiquitin complex is saved in the Pymol software.

[0073] (3) Calculate the structure of YS3 / TEAD1 complex with AF2 software, and obtain 5 models; through sequence alignment of YS3 and SPOP with Pymol software, 5 YS3 / TEAD1 complex models are positioned in SPOP / CUL3 / RBX1 / UB, 5 alignment models are obtained, the RMSD values of the 5 alignment models are evaluated and recorded, the proportion of the overlapping volume of YS3 / TEAD1 complex and CUL3 to the volume of CUL3, and the shortest distance between the lysine residues of TEAD1 and the C-terminal glycine residues of UB.

[0074] (4) The RMSD values of the 5 alignment models are 0.504, 0.550, 0.631, 0.624, and 0.822, respectively, all of which are less than 2;

[0075] The proportion of the overlapping volume of YS3 / TEAD1 complex and CUL3 to the volume of CUL3 is 8.03%, 4.63%, 8.24%, 12.32%, and 2.31%, respectively, the 2nd and 5th alignment models are less than 7%, and the 1st, 3rd, and 5th alignment models are all greater than 7%;

[0076] The shortest distance between the lysine residues of TEAD1 and the C-terminal glycine residues of UB is 4.94 nm, 6.63 nm, 6.32 nm, 6.49 nm, and 6.11 nm, respectively, all of which are less than 7 nm;

[0077] It can be seen that only the 2nd and 5th alignment models in the 5 alignment models satisfy the above three conditions at the same time, which are qualified models, and the number of qualified models is 2. Therefore, it can be determined that the design of YS3 is successful, and TEAD1 can be successfully knocked out.

[0078] The above results are verified in the following experiments: Hela cells (human cervical cancer cells) are divided into 4 groups, one group is the control group (C) without treatment; the other groups are respectively Flag-SPOP (167-374) (S1), Flag-YAP1 (50-105) / SPOP (167-374) (YS3) without MG132, and Flag-YAP1 (50-105) / SPOP (167-374) (YS3) with MG132. After culture, the protein is collected and Western-blotting experiment is carried out, and the results are shown in Figure 3 . Figure 3As shown, only SPOP (167-374) was introduced, and the expression of TEAD1 protein was not affected; the expression of TEAD1 protein was significantly weakened when the chimeric ubiquitin ligase YS3 was introduced without MG132; the expression of TEAD1 protein was not weakened when the chimeric ubiquitin ligase YS3 was introduced with MG132, because MG132 inhibited the degradation of TEAD1 protein. It is shown that the chimeric ubiquitin ligase YS3 can significantly knock out TEAD1 protein.

[0079] Example 4

[0080] In this example, a chimeric ubiquitin ligase DS12 is designed, and whether the design is successful is evaluated, and the specific process is as follows:

[0081] (1) The protein of interest in this example is KRAS, and the protein DARPin.K27 that can bind KRAS is found from the MCE database, which is used as the protein binding domain; the DARPin.K27 is connected with the first linker protein SPOP (167-374) to form the chimeric ubiquitin ligase DS12. The amino acid sequence of DS12 is shown in SEQ ID NO: 6: MEQKLISEEDLGGLDYKDDDDKASGSDLGKKLLEAARAGQDDEVRILMANGADVNAHDTFGFTPLHLAALYGHLEIVEVLLKNGADVNADDSYGRTPLHLAAMRGHLEIVEVLLKYGADVNAADEEGRTPLHLAAKRGHLEIVEVLLKNGADVNAQDKFGKTAFDISIDNGNEDLAEILQKLNGSGSGSGSVNISGQNTMNMVKVPECRLADELGGLWENSRFTDCCLCVAGQEFQAHKAILAARSPVFSAMFEHEMEESKKNRVEINDVEPEVFKEMMCFIYTGKAPNLDKMADDLLAAADKYALERLKVMCEDALCSNLSVENAAEILILADLHSADQLKTQAVDFINYHASDVLETSGWKSMVVSHPHLVAEAYRSLASAQCPFLGPPRKRLKQSGS.

[0082] (2) The SPOP-based ubiquitin complex, which is SPOP / CUL3 / RBX1 / UB, is constructed, and the ubiquitin complex is saved in the Pymol software.

[0083] (3) Calculate the structure of the DS12 / KRAS complex with AF2 software, obtain 5 models; through sequence alignment of DS12 and SPOP with Pymol software, 5 DS12 / KRAS complex models are positioned in SPOP / CUL3 / RBX1 / UB, 5 alignment models are obtained, the RMSD values of the 5 alignment models are evaluated and recorded, the proportion of the overlapping volume of DS12 / KRAS complex and CUL3 to the volume of CUL3, and the shortest distance between the lysine residues of KRAS and the C-terminal glycine residues of UB.

[0084] (4) The RMSD values of the 5 alignment models are 0.443, 0.596, 0.899, 0.578, 0.589, all <2;

[0085] The proportion of the overlapping volume of DS12 / KRAS complex and CUL3 to the volume of CUL3 is 6.60%, 1.28%, 9.90%, 11.08%, 8.64%, the 3rd, 4th and 5th alignment models are all >7%, and the 1st and 2nd alignment models are <7%;

[0086] The shortest distance between the lysine residues of KRAS and the C-terminal glycine residues of UB is 7.75 nm, 6.46 nm, 9.05 nm, 8.95 nm, 7.76 nm, the 1st, 3rd, 4th and 5th alignment models are all >7 nm, and only the 2nd alignment model is <7 nm;

[0087] It can be seen that only the 2nd alignment model in the 5 alignment models meets the above three conditions at the same time, which is a qualified model, the number of qualified models is 1, <2. Therefore, it can be determined that the design of DS12 is failed and cannot successfully knock out KRAS.

[0088] The above results are verified in the following experiments: Hela cells (human cervical cancer cells) are divided into 4 groups, one group is the control group (C), without treatment; the other groups are respectively Flag-SPOP (167-374) (S1) transfected; Flag-DARPin.K27 / SPOP (167-374) (DS12) transfected without MG132; Flag-DARPin.K27 / SPOP (167-374) (DS12) transfected with MG132 Hela cells. After culture, the protein is collected and Western-blotting experiment is carried out, and the results are shown in Figure 4 . Figure 4 It is shown that only introducing SPOP (167-374) does not affect the expression of KRAS protein; and introducing the chimeric ubiquitin ligase DS12 without and with MG132, the expression of KRAS protein is not weakened. It shows that the chimeric ubiquitin ligase DS12 cannot knock out KRAS protein.

[0089] Example 5

[0090] This example improves the chimeric ubiquitin ligase DS12 designed to fail in Example 4, i.e. makes appropriate changes to part of the sequence in DS12 to form a new chimeric ubiquitin ligase DS13 (including DARPin.K27.1 and SPOP.1 (167-374)), and evaluates whether the design of DS13 is successful. The specific process is as follows:

[0091] (1) In this example, part of the sequence in the chimeric ubiquitin ligase DS12 is changed to obtain a new chimeric ubiquitin ligase DS13. The amino acid sequence of DS13 is shown in SEQ ID NO: 7: MGASGSDLGKKLLEAARAG QDDEVRILMANGADVNAHDTFGFTPLHLAALYGHLEIVEVLLKNGADVNADDSYGRTPLHLAAMRGHLEIVEVLLKYGADVNAADEEGRTPLHLAAKRGHLEIVEVLLKNGADVNAQDKFGKTAFDISIDNGNEDLAEILQKLNGSGSGSGSVNISGQNTMNMVKVPECRLADELGGLWENSRFTDCCLCVAGQEFQAHKAILAARSPVFSAMFEHEMEESKKNRVEINDVEPEVFKEMMCFIYTGKAPNLDKMADDLLAAADKYALERLKVMCEDALCSNLSVENAAEILILADLHSADQLKTQAVDFINYHASDVLETSGWKSMVVSHPHLVAEAYRSLASAQCPFLGPPRKRLKQSGSDYKDDDDKS.

[0092] (2) The ubiquitin complex is constructed as SPOP / CUL3 / RBX1 / UB, and the ubiquitin complex is saved into Pymol software.

[0093] (3) The structure of the DS13 / KRAS complex is calculated using AF2 software, 5 models are obtained; through sequence alignment of DS13 and SPOP by Pymol software, 5 DS13 / KRAS complex models are positioned in SPOP / CUL3 / RBX1 / UB, 5 alignment models are obtained, the RMSD value of the 5 alignment models is evaluated and recorded, the proportion of the overlapping volume of the DS13 / KRAS complex and CUL3 to the volume of CUL3, and the shortest distance between the lysine residue of KRAS and the C-terminal glycine residue of UB.

[0094] (4) The RMSD values of the 5 alignment models are 0.441, 0.424, 0.383, 0.409, 0.667, all < 2;

[0095] The proportion of the overlapping volume of the DS13 / KRAS complex with CUL3 to the volume of CUL3 is 1.65%, 2.54%, 6.62%, 12.42%, 11.60%, the 4th and 5th alignment models are > 7%, and the 1st, 2nd and 3rd alignment models are < 7%;

[0096] The shortest distance between the lysine residue of KRAS and the C-terminal glycine residue of UB is 5.59 nm, 6.12 nm, 6.47 nm, 7.82 nm, 8.80 nm, the 4th and 5th alignment models are > 7 nm, and the 1st, 2nd and 3rd alignment models are < 7 nm;

[0097] It can be seen that the 1st, 2nd and 3rd alignment models in the 5 alignment models simultaneously satisfy the above three conditions, which are qualified models, the number of qualified models is 3, > 2. Therefore, it can be determined that the design of DS13 is successful, and can successfully knock out KRAS.

[0098] The above results are verified in the following experiment: plasmids of Flag-DARPin.K27.1 / SPOP.1(167-374)(DS13) without MG132 and Flag-DARPin.K27.1 / SPOP.1(167-374)(DS13) with MG132 are respectively introduced into Hela cells. After culture, the protein is collected and Western-blotting experiment is performed, and the results are shown in Figure 4 Figure 4 It is shown that after introducing the chimeric ubiquitin ligase DS13 without MG132, the expression of KRAS protein is significantly weakened; and after adding MG132, the expression of KRAS protein is not weakened. It is shown that the chimeric ubiquitin ligase DS13 can knock out KRAS protein.

[0099] Example 6

[0100] In this embodiment, the chimeric ubiquitin ligase DS12 which failed in the design in Example 4 is improved to form the chimeric ubiquitin ligase RS14, and whether the design of the chimeric ubiquitin ligase RS14 is successful is evaluated, and the specific process is as follows:

[0101] ​(1) This embodiment finds a new protein Raf1 that can bind KRAS from the PDB database, and takes the protein fragment Raf1(52-188) as the binding domain of the protein of interest; connects Raf1(52-188) with the first linker protein SPOP(167-374), and makes appropriate changes to part of the sequence to form the chimeric ubiquitin ligase RS14. The amino acid sequence of RS14 is shown in SEQ ID NO: 8: MGDYKDDDDKASSKTSNTIRVFLPNKQRTVVNVRNGMSLHDCLMKALKVRGLQPECCAVFRLLHEHKGKKARLDWNTDAASLIGEELQVDFLDHVPLTTHNFARKTFLKLAFCDICQKFLLNGFRCQTCGYKFHEHCSTKVPTMCVDWSGSGSGSVNISGQNTMNMVKVPECRLADELGGLWENSRFTDCCLCVAGQEFQAHKAILAARSPVFSAMFEHEMEESKKNRVEINDVEPEVFKEMMCFIYTGKAPNLDKMADDLLAAADKYALERLKVMCEDALCSNLSVENAAEILILADLHSADQLKTQAVDFINYHASDVLETSGWKSMVVSHPHLVAEAYRSLASAQCPFLGPPRKRLKQSGS.

[0102] (2) Construct the ubiquitin complex as SPOP / CUL3 / RBX1 / UB, and save the ubiquitin complex to the Pymol software.

[0103] (3) Calculate the structure of the RS14 / KRAS complex using the AF2 software, obtain 5 models; sequence alignment of RS14 and SPOP is performed by Pymol software, so as to position the 5 RS14 / KRAS complex models into SPOP / CUL3 / RBX1 / UB, obtain 5 alignment models, evaluate and record the RMSD values of the 5 alignment models, the proportion of the overlapping volume of RS14 / KRAS complex and CUL3 to the volume of CUL3, and the shortest distance between the lysine residues of KRAS and the C-terminal glycine residues of UB.

[0104] (4) The RMSD values of the 5 alignment models are 0.370, 0.334, 0.397, 0.402, and 0.576, all <2;

[0105] The proportion of the overlapping volume of RS14 / KRAS complex and CUL3 to the volume of CUL3 is -1.34%, 1.67%, -0.53%, -0.61%, and -2.27%, all <7%;

[0106] The shortest distance between the lysine residues of KRAS and the C-terminal glycine residues of UB is 5.07 nm, 6.11 nm, 5.29 nm, 4.72 nm, 6.44 nm, all < 7 nm;

[0107] It can be seen that the five alignment models all meet the above three conditions at the same time, and are qualified models, and the number of qualified models is 5, > 2. Therefore, it can be determined that the design of RS14 is successful, and can successfully knock out KRAS.

[0108] The above results are verified in the following experiment: respectively, Flag-Raf1(52-188) / SPOP(167-374)(RS14) without MG132; and Flag-Raf1(52-188) / SPOP(167-374)(RS14) with MG132 plasmid is introduced into Hela cells. After culture, the protein is collected, and Western-blotting experiment is carried out, and the results are shown in Figure 4 Figure 4 It is shown that the introduction of the chimeric ubiquitin ligase RS14 without MG132 significantly reduces the expression of KRAS protein; and with MG132, the expression of KRAS protein is not reduced. It is shown that the chimeric ubiquitin ligase RS14 can knock out KRAS protein.

[0109] Example 7

[0110] In this embodiment, the chimeric ubiquitin ligase ME15 is designed, and whether the design is successful is evaluated, and the specific process is as follows:

[0111] (1) The protein of interest in this embodiment is c-MYC, the protein MAX which can bind c-MYC is found from the PDB database, and the protein fragment MAX(2-83) is used as the protein binding domain of interest; MAX(2-83) is connected with the first linker protein ELOB(2-118) to form the chimeric ubiquitin ligase ME15. The amino acid sequence of ME15 is shown in SEQ ID NO: 9: MADKRAHHNALERKRRDHIKDSFHSLRDSVPSLQGEKASRAQILDKATEYIQYMRRKNHTHQQDIDDLKRQNALLEQQVRALEGSDVFLMIRRHKTTIFTDAKESSTVFELKRIVEGILKRPPDEQRLYKDDQLLDDGKTLGECGFTSQTARPQAPATVGLAFRADDTFEALCIEPFSSPPELPDVMKPQDSGSSANEQAVQGLDYKDDDDKGS.

[0112] ​(2) Since the natural E3 ubiquitin ligase ELOB in the chimeric ubiquitin ligase ME15 belongs to the Cullin-RING BC-box family, an ELOB-based ubiquitin complex, ELOB / ELOC / CUL2 / RBX1 / UB, is constructed and saved in Pymol software.

[0113] (3) The structure of ME15 / c-MYC complex is calculated by AF2 software to obtain 5 models; sequence alignment of ME15 and ELOB is performed by Pymol software, so that the 5 ME15 / c-MYC complex models are positioned in ELOB / ELOC / CUL2 / RBX1 / UB to obtain 5 alignment models, and the RMSD values of the 5 alignment models, the proportion of the overlapping volume of ME15 / c-MYC complex and CUL2 to the volume of CUL2, and the shortest distance between the lysine residues of c-MYC and the C-terminal glycine residues of UB are evaluated and recorded.

[0114] (4) The RMSD values of the 5 alignment models are 1.247, 1.490, 1.373, 1.332, and 1.335, all < 2;

[0115] The proportion of the overlapping volume of ME15 / c-MYC complex and CUL2 to the volume of CUL2 is 5.21%, 9.69%, 4.49%, 9.85%, and 8.02%, the first and third alignment models are < 7%, and the second, fourth, and fifth alignment models are > 7%;

[0116] The shortest distance between the lysine residues of c-MYC and the C-terminal glycine residues of UB is 3.68 nm, 5.05 nm, 4.01 nm, 3.09 nm, and 6.01 nm, all < 7 nm;

[0117] It can be seen that only the first and third alignment models in the 5 alignment models simultaneously satisfy the above three conditions, which are qualified models, and the number of qualified models is 2. Therefore, it can be determined that the design of ME15 is successful, and c-MYC can be successfully knocked out.

[0118] The above results are verified in the following experiments: Hela cells are divided into 4 groups, one group is a control group (C) without treatment; the other groups are respectively Flag-ELOB (2-118) (E1) transfected, Flag-MAX (2-83) / ELOB (2-118) (ME15) transfected without MG132, and Flag-MAX (2-83) / ELOB (2-118) (ME15) transfected with MG132. After culture, the proteins are collected and Western-blotting experiment is performed, and the results are shown in Figure 5 . Figure 5As shown, only ELOB (2-118) was introduced, and the expression of c-MYC protein was not affected; the expression of c-MYC protein was significantly weakened when the chimeric ubiquitin ligase ME15 was introduced without MG132; the expression of c-MYC protein was not weakened when the chimeric ubiquitin ligase ME15 was introduced with MG132, because MG132 inhibited the degradation of c-MYC protein. It is shown that the chimeric ubiquitin ligase ME15 can significantly knock out c-MYC protein.

[0119] The above has described the embodiments of the present application in detail, but the present application is not limited to the above-described embodiments, and various changes can be made within the knowledge of those skilled in the art without departing from the spirit of the present application. In addition, the embodiments of the present application and the features in the embodiments can be combined with each other without conflict.

Claims

1. A method for evaluating the successful design of a chimeric ubiquitin ligase, characterized in that, Includes the following steps: S1: A chimeric ubiquitin ligase designed for targeted degradation of proteins of interest; S2: Determine the family of natural E3 ubiquitin ligase to which the chimeric ubiquitin ligase belongs, and construct the ubiquitin complex of the natural E3 ubiquitin ligase; the ubiquitin complex is a natural E3 ubiquitin ligase / ubiquitin, and the natural E3 ubiquitin ligase is a second adaptor protein / connector protein / E2 ubiquitin conjugation enzyme recruitment domain. S3: The structure of the chimeric ubiquitin ligase / interest protein complex was calculated using AlphaFold2 software, resulting in 5 models. The chimeric ubiquitin ligase / interest protein complex was located in the ubiquitin complex by sequence alignment between the chimeric ubiquitin ligase and its native E3 ubiquitin ligase, resulting in 5 alignment models. The following parameters were evaluated and recorded: (1) the root mean square deviation (RMSD) of the sequence alignment for each model; (2) the proportion of the overlap volume between the chimeric ubiquitin ligase / interest protein complex and the ligand protein in each alignment model, excluding the alignment portion, to the volume of the ligand protein; (3) the shortest distance between the lysine residue of the interest protein and the glycine residue at the C-terminus of the ubiquitin after localization. S4: Evaluate whether the parameters of the five alignment models meet the following conditions: (1) RMSD < 2; (2) the overlap volume ratio < 7%; (3) the shortest distance between the lysine residue of the protein of interest and the glycine residue of the C-terminus of the ubiquitin after localization < 7 nm; if all three conditions are met, the alignment model is a qualified model; if at least one of the three conditions is not met, the alignment model is an unqualified model. S5: Count the number of qualified models. If the number of qualified models is ≥2, then the design of the chimeric ubiquitin ligase is successful and can target and degrade the protein of interest.

2. The evaluation method according to claim 1, characterized in that, Step S1, the method for designing the chimeric ubiquitin ligase, includes the following steps: determining the protein-binding domain of interest based on the protein to be degraded; and linking the protein-binding domain of interest to a first adaptor protein to form the chimeric ubiquitin ligase.

3. The evaluation method according to claim 2, characterized in that, The method for determining the protein-binding domain is as follows: search a database for proteins or polypeptides that interact with the protein of interest, and select fragments that can bind to the protein of interest to obtain the protein-binding domain.

4. The evaluation method according to claim 2, characterized in that, The first adaptor protein is a protein capable of binding to the linker protein in the ubiquitin complex.

5. The evaluation method according to claim 2, characterized in that, The first adaptor protein is the same as the second adaptor protein, or the first adaptor protein is a part of the second adaptor protein.

6. The evaluation method according to claim 1, characterized in that, The family of natural E3 ubiquitin ligases includes any one of the HECT domain family, RING domain family, and U.box domain family.

7. The evaluation method according to claim 1, characterized in that, In step S3, the sequence of the chimeric ubiquitin ligase was compared with that of its natural E3 ubiquitin ligase using Pymol software.

8. An improved method for designing a failed chimeric ubiquitin ligase, characterized in that, Includes the following steps: Replace the protein-of-interest binding domain in the chimeric ubiquitin ligase and / or change a portion of the amino acid sequence of the chimeric ubiquitin ligase to obtain a new chimeric ubiquitin ligase; then evaluate the new chimeric ubiquitin ligase according to the evaluation method described in any one of claims 1 to 7 to determine whether the improvement is successful.

9. A chimeric ubiquitin ligase capable of targeting and degrading proteins of interest, screened using the evaluation method described in any one of claims 1 to 7.

10. The use of the chimeric ubiquitin ligase of claim 9 in the preparation of a product for treating at least one of tumors, cardiovascular diseases, immune diseases, nutritional and metabolic diseases, nervous system diseases, respiratory system diseases, digestive system diseases, and infectious diseases.

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