Chimeric ubiquitin ligases targeting KRAS degradation, their preparation methods and applications

Abstract

This invention belongs to the field of biomedicine and discloses a chimeric ubiquitin ligase for targeted degradation of KRAS, its preparation method, and its applications. The chimeric ubiquitin ligase comprises a KRAS-binding domain and an E3 ubiquitin ligase-binding protein; the KRAS-binding domain includes at least one of RAF1(52-188), ARAF, PIP5K1A, RASSF2, APP, KRAS, and RAF1. This invention also discloses a nucleic acid molecule encoding the above-mentioned chimeric ubiquitin ligase; a vector comprising the above-mentioned nucleic acid molecule; a host cell comprising the above-mentioned nucleic acid molecule or vector; a method for preparing the chimeric ubiquitin ligase; and the application of this enzyme in the preparation of formulations that degrade KRAS protein or drugs for treating KRAS-related cancers. The chimeric ubiquitin ligase of this invention can degrade different mutant KRAS.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a chimeric ubiquitin ligase that targets the degradation of KRAS, its preparation method, and its application. Background Technology

[0002] RAS is a common mutated gene in tumors. In normal cells, RAS activation occurs in the downstream pathway of the cell membrane growth factor receptor family. The RAS protein is a membrane-bound GTP / GDP-binding protein (GTPase), activated when bound to GTP and inactivated when bound to GDP. RAS isoforms are mainly encoded by three genes: KRAS, NRAS, and HRAS. KRAS is the most prevalent mutant in the RAS family and is associated with 22% of human tumors, making it a frequently mutated gene in the three leading causes of cancer death in humans: lung cancer (17%), colorectal cancer (33%), and pancreatic cancer (61%). Common KRAS mutations mainly occur at codons 12, 13, 61, and 146 in exon 2, with mutations at positions 12 and 13 accounting for 90% of all mutations.

[0003] Currently, the inhibition of protein activity mainly involves the development of small molecule inhibitors targeting the active sites of the target protein. However, research shows that, excluding KRAS proteins, 85% of proteins are "undruggable proteins." These proteins are difficult to inhibit due to their loose and variable structures, strong competitive binding ability with other small molecules within the cell, and the susceptibility of mutations at their active sites. Even though some small molecule inhibitors targeting KRAS have been developed, the binding area between these inhibitors and KRAS is too small. Even slight mutations or conformational changes in KRAS can affect the binding of small molecule inhibitors.

[0004] Furthermore, degrading protein at the protein level to reduce its accumulation in cells is also an important strategy for attenuating protein expression, with protein degradation being a crucial method for reducing its accumulation in cells. Mainstream protein-targeted degradation technologies involve constructing chimeric molecules to link the target protein to an E3 ligase, thereby promoting the degradation of the target protein; a typical example is PROTAC (Protein Degradation Targeted Chimera). It has been reported that many proteins can be rapidly degraded using PROTAC technology. However, this technology only allows one type of PROTAC to degrade one mutant KRAS, and cannot degrade multiple mutant KRAS, thus lacking universal applicability. Summary of the Invention

[0005] The present invention aims to at least solve one of the technical problems existing in the prior art. To this end, the present invention proposes a chimeric ubiquitin ligase that targets and degrades KRAS, capable of degrading different mutant KRAS.

[0006] This invention also proposes a nucleic acid molecule.

[0007] The present invention also proposes a carrier.

[0008] The present invention also proposes a host cell.

[0009] The present invention also proposes a method for preparing the chimeric ubiquitin ligase that targets and degrades KRAS.

[0010] This invention also proposes the application of the chimeric ubiquitin ligase that targets and degrades KRAS in the preparation of products.

[0011] According to one aspect of the present invention, a chimeric ubiquitin ligase for targeted degradation of KRAS is provided, comprising a KRAS-binding domain and an E3 ubiquitin ligase-binding protein; wherein the KRAS-binding domain comprises at least one of RAF1(52-188), ARAF, PIP5K1A, RASSF2, APP, KRAS, and RAF1.

[0012] The amino acid sequence of RAF1(52-188) is as follows:

[0013] or

[0014] The amino acid sequence shown in SEQ ID NO:1 is modified by substitution, deletion or addition of one or more amino acids, and the amino acid sequence has the same or similar function.

[0015] The amino acid sequence of the ARAF is as follows:

[0016] SEQ ID NO:2: ASEPPRGPPANGAEPSRAVGTVKVYLPNKQRTVVTVRDGMSVY; or

[0017] The amino acid sequence shown in SEQ ID NO:2 is modified by substitution, deletion or addition of one or more amino acids, and the amino acid sequence has the same or similar function;

[0018] The amino acid sequence of PIP5K1A is as follows:

[0019] SEQ ID NO:3: ASASASSGPSSSVGFSSFDPAVPSCTLSSAASGIKRPMASEVLEAR QDSYISLVPYASGMPIKKIGHRSVDSSGETTYKKTTSSALKGAIQLGITHTVGSLSTKPERDVLMQDFYVVESIFFPSEGSNLTPAHHYNDFRFKTYAPVAFRYFRELFGIRPDDYLYSLCSEPLIELCS SGASGSLFYVSSDDEFIIKTVQHKEAEFLQKLLPGYYMNLNQNPRTLLPKFYGLYCVQAGGKNIRIVVMNNLLPRSVKMHIKYDLKGSTYKRRASQKEREKPLPTFKDLDFLQDIPDGLFLDADMYNALC KTLQRDCLVLQSFKIMDYSLLMSIHNIDHAQREPLSSETQYSVDTRRPAPQKALYSTAMESIQGEARRGGTMETDDHMGGIPARNSKGERLLLYIGIIDILQSYRFVKKLEHSWKALVHDGDTVSVHRPG OR

[0020] The amino acid sequence shown in SEQ ID NO:3 is modified by substitution, deletion or addition of one or more amino acids, and has the same or similar function.

[0021] The amino acid sequence of RASSF2 is as follows:

[0022] SEQ ID NO:4:ASDYSHQTSLVPCGQDKYISKNELLLHLKTYNLYYEGQNLQLRH REEEDEFIVEGLLNISWGLRRPIRLQMQDDNERIRPPPSSSSWHSGCNLGAQGTTLKPLTVPKVQISEVDAPPEGDQMPSSTDSRGLKPLQEDTPQLMRTRSDVGVRRRGNVRTPSDQRRIRRHRFSINGHFYNHKTSVFTPA OR

[0023] The amino acid sequence shown in SEQ ID NO:4 is modified by substitution, deletion or addition of one or more amino acids, and the amino acid sequence has the same or similar function.

[0024] The amino acid sequence of the APP is as follows:

[0025] SEQ ID NO:5: ASLPGLALLLLAAWTARALEVPTDGNAGLLAEPQIAMFCGRLN; or

[0026] The amino acid sequence shown in SEQ ID NO:5 is modified by substitution, deletion or addition of one or more amino acids, and has the same or similar function.

[0027] The amino acid sequence of the KRAS is as follows:

[0028] SEQ ID NO:6:ASTEYKLVVVGAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQ VVIDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHHYREQIK RVKDSEDVPMVLVGNKCDLPSRTVDTKQAQDLARSYGIPFIETSAKTRQGVDDAFYT LVREIRKHKEKMSKDGKKKKKKSKTKCVIMG; or

[0029] The amino acid sequence shown in SEQ ID NO:6 is modified by substitution, deletion or addition of one or more amino acids, and the amino acid sequence has the same or similar function.

[0030] The amino acid sequence of RAF1 is as follows:

[0031] SEQ ID NO:7: ASEHIQGAWKTISNGFGFKDAVFDGSSCISPTIVQQFGYQRRASD; or

[0032] The amino acid sequence shown in SEQ ID NO:7 is a functionally identical or similar amino acid sequence after one or more amino acids have been substituted, deleted, or added.

[0033] According to a preferred embodiment of the present invention, at least the following beneficial effects are achieved:

[0034] In the chimeric ubiquitin ligase proposed in this invention, all or part of the RAF1 protein, ARAF, PIP5K1A, RASSF2, APP, and KRAS proteins can serve as natural binding proteins that specifically bind to and activate KRAS. Specifically, RAF1 protein is a downstream protein of KRAS and only binds to activated KRAS, while mutant KRAS is almost always in an activated state. This means that most mutant KRAS can bind to the RAF1 domain, i.e., the chimeric ubiquitin ligase including the RAF1 domain can effectively degrade KRAS, solving the problem of reduced binding affinity of existing inhibitors (PROTACs) to KRAS due to KRAS mutations. Furthermore, because the KRAS-binding domain in this invention has a strong binding affinity to various mutant KRAS, the chimeric ubiquitin ligase including the KRAS-binding domain can effectively degrade different mutant KRAS, thus making it applicable to the treatment of various cancers and broad in scope.

[0035] In some embodiments of the present invention, the E3 ubiquitin ligase is selected from any one of the HECT domain family, RING domain family, and U.box domain family E3 ubiquitin ligases.

[0036] In some preferred embodiments of the present invention, the E3 ubiquitin ligase is selected from the RING domain family of E3 ubiquitin ligases. Specifically, the E3 ubiquitin ligase is an adaptor protein / Cullin protein / E2 ubiquitin conjugase recruitment domain.

[0037] Specifically, the E3 ubiquitin ligase-binding protein is part or all of the adaptor protein.

[0038] More specifically, the E3 ubiquitin ligase-binding protein is part of the adaptor protein, and its amino acid sequence is as follows:

[0039] a)SEQ ID NO:8:MGLDYKDDDDKASGSVNISGQNTMNMVKVPECRLADELG OR

[0040] b) The amino acid sequence shown in SEQ ID NO:8, after being modified by substitution, deletion or addition of one or more amino acids, and having the same or similar function.

[0041] In some embodiments of the present invention, the chimeric ubiquitin ligase further includes a ligase protein having an amino acid sequence as shown in SEQ ID NO:9:SGSGSG or SEQ ID NO:10:SGSG.

[0042] In some embodiments of the present invention, the amino acid sequence of the chimeric ubiquitin ligase is as follows:

[0043] a)SEQ ID NO:11:MGDYKDDDDKASSKTSNTIRVFLPNKQRTVVNVRNGMSLH DCLMKALKVRGLQPECCAVFRLLHEHKGKKARLDWNTDAASLIGEELQVDFLDHVPLTTHNFARKTFLKLAFCDICQKFLLNGFRCQTCGYKFHEHCSTKVPTMCVDWSGSGSGSVNISGQNTMNMVKVPECRLADELGGLWENSRFTDCCLCVAGQEFQAHK OR

[0044] b) The amino acid sequence shown in SEQ ID NO:11, after being modified by substitution, deletion or addition of one or more amino acids, and having the same or similar function.

[0045] In some embodiments of the present invention, the amino acid sequence of the chimeric ubiquitin ligase formed by the ARAF and the E3 ubiquitin ligase-binding protein is as follows:

[0046] SEQ ID NO:12: MGDYKDDDDKASEPPRGPPANGAEPSRAVGTVKVYLPNKQRT VVTVRDGMSVYDSLDKALKVRGLNQDCCVVYRLIKGRKTVTAWDTAIAPLDGEELIVEVLEDVPLTMHNFVRKTFFSLAFCDFCLKFLFHGFRCQTCGYKFHQHCSSKVPTVCVDMSTNRQQPSRFYHSVQDLSGGSRQHEAPSNRPLNELLTPQGPSPRTQHCDPEHFPFPAPANAPLQRIRSTSTPNVHMVSTTAPMDSNLIQLTGQSFSTDAAGSRGGSDGTPRGSPSPASVSSGRKSPHSKSPAEQRERKSLADDKKKVKNLGYRDSGYYWEVPPSEVQLLKRIGTGSFGTVFRGRWHGDVAVKVLKVSQPTAEQAQAFKNEMQVLRKTRHVNILLFMGFMTRPGFAIITQWCEGSSLYHHLHVADTRFDMVQLIDVARQTAQGMDYLHAKNIIHRDLKSNNIFLHEGLTVKIGDFGLATVKTRWSGAQPLEQPSGSVLWMAAEVIRMQDPNPYSFQSDVYAYGVVLYELMTGSLPYSHIGCRDQIIFMVGRGYLSPDLSKISSNCPKAMRRLLSDCLKFQREERPLFPQILATIELLQRSLPKIERSASEPSLHRTQADELPACLLSAARLVPGSGSGSVNISGQNTMNMVKVPECRLADELGGLWENSRFTDCCLCVAGQEFQAHKAILAARSPVFSAMFEHEMEESKKNRVEINDVEPEVFKEMMCFIYTGKAPNLDKMADDLLAAADKYALERLKVMCEDALCSNLSVENAAEILILADLHSADQLKTQAVDFINYHASDVLETSGWKSMVVSHPHLVAEAYRSLASAQCPFLGPPRKRLKQSGS; or

[0047] An amino acid sequence that is the same as or similar in function to the amino acid shown in SEQ ID NO:12 after substitution, deletion or addition of one or more amino acids.

[0048] In some embodiments of the present invention, the amino acid sequence of the chimeric ubiquitin ligase formed by PIP5K1A and the E3 ubiquitin ligase-binding protein is as follows:

[0049] SEQ ID NO:13: MGDYKDDDDKASASASSGPSSSVGFSSFDPAVPSCTLSSAASGI; or

[0050] The amino acid sequence shown in SEQ ID NO:13 is a functionally identical or similar amino acid sequence after substitution, deletion or addition of one or more amino acids.

[0051] In some embodiments of the present invention, the amino acid sequence of the chimeric ubiquitin ligase formed by RASSF2 and the E3 ubiquitin ligase-binding protein is as follows:

[0052] SEQ ID NO:14:MGDYKDDDDKASDYSHQTSLVPCGQDKYISKNELLLHLKTYN LYYEGQNLQLRHREEEDEFIVEGLLNISWGLRRPIRLQMQDDNERIRPPPSSSSWHSGCNLGAQGTTLKPLTVPKVQISEVDAPPEGDQMPSSTDSRGLKPLQEDTPQLMRTRSDVGVRRRGNVRTPS DQRRIRRHRFSINGHFYNHKTSVFTPAYGSVTNVRINSTMTTPQVLKLLLNKFKIENSAEEFALYVVHTSGEKQKLKATDYPLIARILQGPCEQISKVFLMEKDQVEEVTYDVAQYIKFEMPVLKSFI QKLQEEEDREVKKLMRKYTVLRLMIRQRLEEIAETPATIGSGSGSVNISGQNTMNMVKVPECRLADELGGLWENSRFTDCCLCVAGQEFQAHKAILAARSPVFSAMFEHEMEESKKNRVEINDVEPEV or

[0053] The amino acid sequence shown in SEQ ID NO:14 is a functionally identical or similar amino acid sequence after substitution, deletion or addition of one or more amino acids.

[0054] In some embodiments of the present invention, the amino acid sequence of the chimeric ubiquitin ligase formed by the APP and the E3 ubiquitin ligase binding protein is as follows:

[0055] SEQ ID NO:15:MGDYKDDDDKASLPGLALLLLAAWTARALEVPTDGNAGLLAEPQIAMFCGRLNMHMNVQNGKWDSDPSGTKTCIDTKEGILQYCQEVYPELQITNVVEANQPVTIQNWCKRGRKQCKTHPHFVIPYRCLVGEFVSDALLVPDKCKFLHQERMDVCETHLHWHTVAKETCSEKSTNLHDYGMLLPCGIDKFRGVEFVCCPLAEESDNVDSADAEEDDSDVWWGGADTDYADGSEDKVVEVAEEEEVAEVEEEEADDDEDDEDGDEVEEEAEEPYEEATERTTSIATTTTTTTESVEEVVREVCSEQAETGPCRAMISRWYFDVTEGKCAPFFYGGCGGNRNNFDTEEYCMAVCGSAMSQSLLKTTQEPLARDPVKLPTTAASTPDAVDKYLETPGDENEHAHFQKAKERLEAKHRERMSQVMREWEEAERQAKNLPKADKKAVIQHFQEKVESLEQEAANERQQLVETHMARVEAMLNDRRRLALENYITALQAVPPRPRHVFNMLKKYVRAEQKDRQHTLKHFEHVRMVDPKKAAQIRSQVMTHLRVIYERMNQSLSLLYNVPAVAEEIQDEVDELLQKEQNYSDDVLANMISEPRISYGNDALMPSLTETKTTVELLPVNGEFSLDDLQPWHSFGADSVPANTENEVEPVDARPAADRGLTTRPGSGLTNIKTEEISEVKMDAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIATVIVITLVMLKKKQYTSIHHGVVEVDAAVTPEERHLSKMQQNGYENPTYKFFEQMQNGSGSGSVNISGQNTMNMVKVPECRLADELGGLWENSRFTDCCLCVAGQEFQAHKAILAARSPVFSAMFEHEMEESKKNRVEINDVEPEVFKEMMCFIYTGKAPNLDKMADDLLAAADKYALERLKVMCEDALCSNLSVENAAEILILADLHSADQLKTQAVDFINYHASDVLETSGWKSMVVSHPHLVAEAYRSLASAQCPFLGPPRKRLKQSGS; or

[0056] The amino acid sequence shown in SEQ ID NO:15 is a functionally identical or similar amino acid sequence after substitution, deletion or addition of one or more amino acids.

[0057] In some embodiments of the present invention, the amino acid sequence of the chimeric ubiquitin ligase formed by the KRAS and the E3 ubiquitin ligase-binding protein is as follows:

[0058] SEQ ID NO:16:MGDYKDDDDKASTEYKLVVVGAGGVGKSALTIQLIQNHFVDE YDPTIEDSYRKQVVIDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHHYREQIKRVKDSEDVPMVLVGNKCDLPSRTVDTKQAQDLARSYGIPFIETSAKTRQGVDDAFYTLVREIRKHKEKMSKDGKKKKKKSKTKCVIMGSGSGSVNISGQNTMNMVKVPECRLADELG OR

[0059] The amino acid sequence shown in SEQ ID NO:16 is a functionally identical or similar amino acid sequence after substitution, deletion or addition of one or more amino acids.

[0060] In some embodiments of the present invention, the amino acid sequence of the chimeric ubiquitin ligase formed by RAF1 and the E3 ubiquitin ligase-binding protein is as follows:

[0061] SEQ ID NO:17: MGDYKDDDDKASEHIQGAWKTISNGFGFKDAVFDGSSCISPTI VQQFGYQRRASDDGKLTDPSKTSNTIRVFLPNKQRTVVNVRNGMSLHDCLMKALKVRGLQPECCAVFRLLHEHKGKKARLDWNTDAASLIGEELQVDFLDHVPLTTHNFARKTFLKLAFCDICQKFLLNGFRCQTCGYKFHEHCSTKVPTMCVDWSNIRQLLLFPNSTIGDSGVPALPSLTMRRMRESVSRMPVSSQHRYSTPHAFTFNTSSPSSEGSLSQRQRSTSTPNVHMVSTTLPVDSRMIEDAIRSHSESASPSALSSSPNNLSPTGWSQPKTPVPAQRERAPVSGTQEKNKIRPRGQRDSSYYWEIEASEVMLSTRIGSGSFGTVYKGKWHGDVAVKILKVVDPTPEQFQAFRNEVAVLRKTRHVNILLFMGYMTKDNLAIVTQWCEGSSLYKHLHVQETKFQMFQLIDIARQTAQGMDYLHAKNIIHRDMKSNNIFLHEGLTVKIGDFGLATVKSRWSGSQQVEQPTGSVLWMAPEVIRMQDNNPFSFQSDVYSYGIVLYELMTGELPYSHINNRDQIIFMVGRGYASPDLSKLYKNCPKAMKRLVADCVKKVKEERPLFPQILSSIELLQHSLPKINRSASEPSLHRAAHTEDINACTLTTSPRLPVFGSGSGSVNISGQNTMNMVKVPECRLADELGGLWENSRFTDCCLCVAGQEFQAHKAILAARSPVFSAMFEHEMEESKKNRVEINDVEPEVFKEMMCFIYTGKAPNLDKMADDLLAAADKYALERLKVMCEDALCSNLSVENAAEILILADLHSADQLKTQAVDFINYHASDVLETSGWKSMVVSHPHLVAEAYRSLASAQCPFLGPPRKRLKQSGS; or

[0062] An amino acid sequence which is the same as or similar to the amino acid shown in SEQ ID NO:17 after substitution, deletion or addition of one or more amino acids and has the same or similar function.

[0063] According to a second aspect of the invention, a nucleic acid molecule is proposed comprising DNA or mRNA encoding a chimeric ubiquitin ligase that targets and degrades KRAS.

[0064] According to a third aspect of the invention, a carrier comprising the nucleic acid molecule is provided.

[0065] In some embodiments of the present invention, the carrier is an expression carrier.

[0066] Specifically, the vector comprising the nucleic acid molecule can be used for cloning or for producing expression vectors, which can be plasmids, viruses, bacteriophages, or any other vectors commonly used in genetic engineering.

[0067] Specifically, in addition to the nucleic acid molecule, the vector may also include eukaryotic or prokaryotic elements for controlling expression, such as regulatory sequences, enhancers, promoters, signal sequences, etc., for initiating and terminating transcription and / or translation.

[0068] In some embodiments of the present invention, the vector includes bacterial, fungal, insect, viral, or mammalian vectors.

[0069] According to a fourth aspect of the invention, a host cell is provided, comprising the nucleic acid molecule or the vector, wherein the host cell is a prokaryotic cell or a eukaryotic cell.

[0070] Specifically, the nucleic acid molecule or the vector can be introduced into a suitable host cell. The nucleic acid molecule can be introduced into the genome of the host cell; the vector can exist in the cytoplasm in an extrachromosomal form or be incorporated into the chromosome of the host cell.

[0071] In some embodiments of the present invention, the prokaryotic cell is a bacterial cell.

[0072] In some embodiments of the present invention, the eukaryotic cells include yeast cells, filamentous fungal cells, mammalian cells, or insect cells.

[0073] According to a fifth aspect of the present invention, a method for preparing the chimeric ubiquitin ligase that targets KRAS degradation is provided, comprising the following steps: culturing the host cells of the fourth aspect to obtain the chimeric ubiquitin ligase.

[0074] According to a sixth aspect of the present invention, the application of the chimeric ubiquitin ligase targeting KRAS degradation in the preparation of a product, said product being any of the following:

[0075] a) Preparations that degrade KRAS protein;

[0076] b) Medications for treating KRAS-related cancers.

[0077] In some embodiments of the present invention, the KRAS protein includes at least one of wild-type KRAS protein and mutant KRAS protein.

[0078] In some embodiments of the present invention, the mutant KRAS protein includes a KRAS protein in which a mutation has occurred at at least one amino acid position of G12, G13, S17, P34, A59, Q61, or A146.

[0079] In some embodiments of the present invention, the KRAS-related cancers include, but are not limited to, at least one of colorectal cancer, lung cancer, pancreatic cancer, liver cancer, and breast cancer. Attached Figure Description

[0080] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0081] Figure 1 This is a Western blotting result diagram of Example 2 of the present invention; where C - blank control group, no transfection performed; S1 - control group, transfected with SPOP (167-374); DS13(-) - transfected with DS13 expression vector, without MG132 (proteasome inhibitor); DS13(MG132) - transfected with DS13 expression vector, with MG132 added; RS14(-) - transfected with RS14 expression vector, without MG132 added; RS14(MG132) - transfected with RS14 expression vector, with MG132 added; Flag-tag indicates protein expression of S1, DS13, and RS14; HSP90 - internal reference protein;

[0082] Figure 2 This is a Western blotting result diagram of Example 3 of the present invention; where C - blank control group, no transfection performed; S1 - control group, transfected with SPOP (167-374); DS13(-) - transfected with DS13 expression vector, without MG132; DS13(MG132) - transfected with DS13 expression vector, with MG132 added; RS14(-) - transfected with RS14 expression vector, without MG132 added; RS14(MG132) - transfected with RS14 expression vector, with MG132 added; Flag-tag indicates the protein expression of S1, DS13, and RS14; HSP90 - internal reference protein;

[0083] Figure 3This is a Western blotting result diagram of Example 4 of the present invention; where C - blank control group, no transfection performed; S1 - control group, transfected with SPOP (167-374); DS13(-) - transfected with DS13 expression vector, without MG132; DS13(MG132) - transfected with DS13 expression vector, with MG132 added; RS14(-) - transfected with RS14 expression vector, without MG132 added; RS14(MG132) - transfected with RS14 expression vector, with MG132 added; Flag-tag indicates the protein expression of S1, DS13 and RS14; HSP90 - internal reference protein;

[0084] Figure 4 This is a Western blotting result diagram of Example 5 of the present invention; where C - blank control group, no transfection performed; S1 - control group, transfected with SPOP (167-374); DS13(-) - transfected with DS13 expression vector, without MG132; DS13(MG132) - transfected with DS13 expression vector, with MG132 added; RS14(-) - transfected with RS14 expression vector, without MG132 added; RS14(MG132) - transfected with RS14 expression vector, with MG132 added; Flag-tag indicates the protein expression of S1, DS13 and RS14; HSP90 - internal reference protein. Detailed Implementation

[0085] The embodiments of the present invention are described in detail below. These embodiments are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0086] In the description of this invention, unless otherwise explicitly defined, terms such as transfection and culture should be interpreted broadly, and those skilled in the art can reasonably determine the specific meaning of the above terms in this invention in conjunction with the specific content of the technical solution.

[0087] In the description of this invention, references to terms such as "one embodiment," "some embodiments," etc., indicate that a specific feature, material, or characteristic described in connection with that embodiment is included in at least one embodiment of the invention. In this specification, illustrative expressions of the above terms do not necessarily refer to the same embodiment. Furthermore, the specific features, materials, or characteristics described may be combined in any suitable manner in one or more embodiments.

[0088] Unless otherwise specified, the experimental methods used in the examples are conventional methods; unless otherwise specified, the materials and reagents used are commercially available.

[0089] Example 1

[0090] This embodiment prepared a chimeric ubiquitin ligase RS14 that targets the degradation of KRAS. The specific process is as follows:

[0091] (1) Search the PDB database for the protein RAF1 that can bind to KRAS, and extract the protein fragment RAF1 (52-188) that binds to KRAS (amino acid sequence as shown in SEQ ID NO:1: ASSKTSNTIRVFLPNKQRTV VNVRNGMSLHDCLMKALKVRGLQPECCAVFRLLHEHKGKKARLDWNTDAASLIGE ELQVDFLDHVPLTTHNFARKTFLKLAFCDICQKFLLNGFRCQTCGYKFHEHCSTKVPT MCVDW); then bind RAF1 (52-188) to the adaptor protein SPOP (167-374) (abbreviated as S1, which is an E3 ubiquitin ligase binding protein used to bind to Cullin protein in the E3 ubiquitin ligase (adaptor protein / Cullin protein / E2 ubiquitin conjugation enzyme recruitment domain);Its amino acid sequence is shown in SEQ ID NO:8: MGLDYK DDDDKASGSVNISGQNTMNMVKVPECRLADELGGLWENSRFTDCCLCVAGQEFQAHKAILAARSPVFSAMFEHEMEESKKNRVEINDVEPEVFKEMMCFIYTGKAPNLDKMADDLLAAADKYALERLKVMCEDALCSNLSVENAAEILILADLHSADQLKTQAVDFINYHASDVLETSGWKSMVVSHPHLVAEAYRSLASAQCPFLGPPRKRLKQSGS. These are linked by a connecting protein (amino acid sequence shown in SEQ ID NO:9: SGSGSG) to form the chimeric ubiquitin ligase RS14 (its amino acid sequence is shown in SEQ ID NO:11: MGDYKDDDDKASSKTSNTIRVFLPN). KQRTVVNVRNGMSLHDCLMKALKVRGLQPECCAVFRLLHEHKGKKARLDWNTDAASLIGEELQVDFLDHVPLTTHNFARKTFLKLAFCCDICQKFLLNGFRCQTCGYKFHEHCSTKVPTMCVDWSGSGSGSVNISGQNTMNMVKVPECRLADELGGLWENSRFTDCCLCVAG QEFQAHKAILAARSPVFSAMFEHEMEESKKNRVEINDVEPEVFKEMMCFIYTGKAPNLDKMADDLLAAADKYALERLKVMCEDALCSNLSVENAAEILILADLHSADQLKTQAVDFINYHASDVLETSGWKSMVVSHPHLVAEAYRSLASAQCPFLGPPRKRLKQSGS). ;

[0092]

[0093] Example 2

[0094] This embodiment tested whether RS14 could knock down wild-type KRAS in HT29 cells (colon cancer cells). The specific process is as follows:

[0095]

[0096] 1×10 seedlings were planted on a six-hole plate. 6 HT29 cells were cultured in McCoy's 5A medium supplemented with 10% fetal bovine serum (FBS) and 1× penicillin / streptomycin, and incubated overnight at 37°C with 5% CO2. Afterward, the McCoy's 5A medium was replaced with 1 mL of OPTI medium, and 2 μg of the S1, DS13, and RS14 expression vectors were transfected into the HT29 cells using the commercial transfection reagent Lipofectamine Messenger MAX. Six hours after transfection, total cellular protein was extracted for Western blotting analysis. The results are shown below. Figure 1 As shown, C is the blank control group, without transfection; S1 is the control group, transfected with SPOP (167-374); DS13(-) is transfected with the DS13 expression vector without MG132 (proteasome inhibitor); DS13(MG132) is transfected with the DS13 expression vector with MG132 added; RS14(-) is transfected with the RS14 expression vector without MG132 added; RS14(MG132) is transfected with the RS14 expression vector with MG132 added; Flag-tags indicate the protein expression of S1, DS13, and RS14 (because S1, DS13, and RS14 all have Flag-tag tags); HSP90 is used as an internal control protein. It can be seen that compared with the control group, DS13 significantly reduces the grayscale of the KRAS band, while the reduction effect of RS14 is not significant. This indicates that DS13 can knock down wild-type KRAS, while RS14 has no significant knockdown effect on wild-type KRAS.

[0097] Example 3

[0098] This embodiment tested whether RS14 could knock down the G12V mutant KRAS in SW480 cells (human colon cancer cells). The specific process is as follows:

[0099] 1×10 seedlings were planted on a six-hole plate. 6 SW480 cells were cultured in Leibovitz's L-15 medium (containing 10% FBS and 1× penicillin / streptomycin) at 37°C with 5% CO2 overnight. Afterward, the Leibovitz's L-15 medium was replaced with 1 mL of OPTI medium, and 2 μg of the S1, DS13, and RS14 expression vectors were transfected into the SW480 cells using the commercial transfection reagent Lipofectamine MessengerMAX. Six hours after transfection, total cellular protein was extracted for Western blotting analysis. The results are shown below. Figure 2As shown, the specific labeling is the same as in Example 2. It can be seen that, compared to the control group, RS14 significantly reduced the grayscale of the KRAS band, while the reduction effect of DS13 was not significant. This indicates that RS14 can knock down the G12V mutant KRAS, while DS13 has no significant knockdown effect on the G12V mutant KRAS.

[0100] Example 4

[0101] This embodiment tested whether RS14 could knock down the G12S mutant KRAS in A549 cells (human non-small cell lung cancer cells). The specific process is as follows:

[0102] 1×10 seedlings were planted on a six-hole plate. 6 A549 cells were cultured in a medium (Ham's F-12K containing 10% FBS and 1× penicillin / streptomycin) at 37°C with 5% CO2 overnight. Afterward, the Ham's F-12K medium was replaced with 1 mL of OPTI medium, and 2 μg of the S1, DS13, and RS14 expression vectors were transfected into A549 cells using the commercial transfection reagent Lipofectamine MessengerMAX. Six hours after transfection, total cellular protein was extracted for Western blotting analysis. The results are shown below. Figure 3 As shown, the specific labeling is the same as in Example 2. It can be seen that, compared with the control group, RS14 significantly reduced the grayscale of the KRAS band, while the reduction effect of DS13 was not significant. This indicates that RS14 can knock down the G12S mutant KRAS, while the knockdown effect of DS13 on the G12S mutant KRAS is not significant.

[0103] Example 5

[0104] This embodiment tested whether RS14 could knock down the G12D mutant KRAS in PANC1 cells (human pancreatic cancer cells). The specific process is as follows:

[0105] 1×10 seedlings were planted on a six-hole plate. 6 PANC1 cells were cultured overnight in DMEM medium (containing 10% FBS and 1× penicillin / streptomycin) at 37°C with 5% CO2. Afterward, the DMEM medium was replaced with 1 mL of OPTI medium, and 2 μg of the S1, DS13, and RS14 expression vectors were transfected into PANC1 cells using the commercial transfection reagent Lipofectamine Messenger MAX. Six hours after transfection, total cellular protein was extracted for Western blotting analysis. The results are shown below. Figure 4As shown, the specific labeling is the same as in Example 2. It can be seen that, compared to the control group, RS14 significantly reduces the grayscale of the KRAS band more effectively than DS13. This indicates that RS14 can knock down the G12D mutant KRAS.

[0106] As can be seen from the above examples, RS14 can degrade a variety of mutant KRAS but not wild-type KRAS. This may be because the RAF1 protein is more likely to bind to activated KRAS (i.e. mutant KRAS).

[0107] The embodiments of the present invention have been described in detail above. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. Use of a chimeric ubiquitin ligase for the manufacture of a product, characterized in that, The product is a drug for treating a KRAS-related cancer; The KRAS-related cancer includes at least one of colorectal cancer, lung cancer, pancreatic cancer, liver cancer, breast cancer; The chimeric ubiquitin ligase comprises a KRAS binding domain and an E3 ubiquitin ligase binding protein; The KRAS binding domain is RAF1 (52-188); The amino acid sequence of the RAF1 (52-188) is SEQ ID NO: 1; The amino acid sequence of the E3 ubiquitin ligase binding protein is SEQ ID NO: 8; The amino acid sequence of the chimeric ubiquitin ligase is SEQ ID NO:

11.

2. Use according to claim 1, characterized in that, The KRAS protein is a mutant KRAS protein.

3. Use according to claim 2, characterized in that, The mutant KRAS protein is a KRAS protein mutated at a G12, G13, or Q61 amino acid position.

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