KRAS targeted protein degradation chimera and application thereof

Abstract

The invention discloses a KRAS targeted protein degradation chimera and application thereof.The constructed targeted protein degradation chimera can effectively target KRAS mutants which are traditionally considered to be'non-patent medicine ', and the mutants are generally lack of deep binding pockets and are difficult to be effectively recognized by small-molecule inhibitors; the chimera shows higher target affinity and wider target coverage range, and meanwhile, the off-target risk is remarkably reduced; long-acting expression can be realized in vivo based on a plasmid DNA or mRNA delivery strategy, so that the drug delivery system has the treatment potential of'one-time administration and lasting degradation ', and is expected to greatly improve the medication compliance and the treatment effect.

Description

(I) Technical Field

[0001] This invention relates to the fields of biomedicine and genetic engineering, specifically to a protein degradation chimera capable of targeting and degrading KRAS protein and its applications. (II) Background Technology

[0002] Members of the RAS protein family (KRAS, NRAS, HRAS) are frequently mutated in human malignant tumors, with KRAS mutations being the most prominent, accounting for approximately 86% of all RAS mutations. [1] KRAS mutants are a major driver of cancers such as colorectal cancer, lung cancer, and pancreatic cancer. The core function of the KRAS protein is to act as a molecular switch regulating cellular signaling. Under normal physiological conditions, it cycles between an inactive GDP-binding state and an active GTP-binding state, transducing extracellular signals into the cell and precisely controlling key processes such as cell proliferation, survival, differentiation, and migration. When KRAS binds to GDP, the protein is inactive and is activated through nucleotide exchange from GDP to GTP. Normally, the activation / inactivation cycle is catalyzed by guanine nucleotide exchange factors and GTPase activator proteins (GAPs). Mutant KRAS proteins remain active, hydrolyzing GTP much more slowly than wild-type KRAS. The mutation reduces GAP activity, leading to constitutive activation of the KRAS effector pathway. [2] RAS continuously generates signaling cascades that activate cellular functions such as division, survival, and invasion. Therefore, developing selective RAS inhibitors that can specifically recognize and block its function has become an important direction in current targeted cancer therapy research.

[0003] KRAS plays a crucial role in mediating extracellular growth factor signaling into the nucleus, participating in the regulation of various biological processes such as cell proliferation, growth, and survival. Due to its smooth surface structure and lack of binding sites for traditional small molecule drugs, KRAS has long been considered an "untreatable" target. Recent studies have discovered a specific target pocket within its structure, driving the development of a series of inhibitors targeting the KRAS G12C mutant. Currently, sotorasib and adagrasib are approved for the treatment of advanced non-small cell lung cancer. [3, 4] Several other drug candidates are currently in clinical trials. [5]Furthermore, small molecule inhibitors specifically and directly target mutated KRAS. This strategy targets the G12C mutation in the KRAS gene, which accounts for approximately 12% of all KRAS oncogenic mutations and is only present in certain cancer subtypes, such as non-small cell lung cancer. Therefore, for the remaining approximately 88% of KRAS-mutant cancers, there is an urgent need to develop new targeted therapies to inhibit other common KRAS mutation subtypes. In addition, cancer cells can evade the therapeutic effects of KRAS G12C inhibitors through mechanisms such as activating alternative signaling pathways or acquiring novel drug-resistant mutations, further highlighting the necessity of developing broad-spectrum and combination strategies.

[0004] The core advantage of proteolytic targeting strategies lies in their ability to bind only to the target protein without inhibiting its function. Unlike traditional occupation-driven mechanisms (such as protein-protein interaction inhibitors), degradative agents employ an event-driven mechanism, achieving pharmacological effects by inducing the degradation of the target protein, thus typically exhibiting stronger efficacy than traditional inhibitors. Currently, developed degradative agents primarily target bromine domains or kinase family proteins, with only a few studies successfully applying them to "undruggable" targets (such as transcription factors).

[0005] Macromolecular degraders induce the clearance of target proteins through the ubiquitin-proteasome system. Their structure typically comprises three functional units: a binding element for recognizing the target protein (such as an intracellular single-domain antibody), a linker, and a ligand domain responsible for recruiting E3 ligases. Similarly, small-molecule degradation strategies have been developed. These strategies utilize small-molecule compounds that can bind to target proteins to couple with E3 ligase ligands, forming proteolytic-targeting chimeras (PROTACs), thereby achieving targeted protein degradation.

[0006] Previous studies have reported that DARPin K19 (hereinafter referred to as DARPin), a KRAS-specific degrader, can effectively induce the degradation of endogenous KRAS in both in vitro and in vivo experiments. Therefore, using PROTAC technology to regulate KRAS protein levels in cancer cells is a promising intervention method. (III) Summary of the Invention

[0007] The purpose of this invention is to provide a KRAS-targeting protein degradation chimera based on the ubiquitin-proteasome pathway and its applications. This chimera can effectively bind to and degrade exogenous and endogenous KRAS proteins and mutants, thereby disrupting the interaction between KRAS proteins and mutant KRAS and downstream effectors, and inhibiting the activation of downstream signaling pathways. Targeting KRAS degradation enables a broad-spectrum targeted therapeutic strategy against all tumors expressing mutant KRAS. This method relies on the proteolytic mechanism of the target protein, exhibiting excellent degradation capabilities and achieving efficient degradation of KRAS proteins, providing a novel tool for KRAS-related pathway regulation and disease intervention.

[0008] The technical solution adopted in this invention is:

[0009] This invention provides a KRAS-targeting protein degradation chimera based on the ubiquitin-proteasome pathway. The KRAS-targeting protein degradation chimera is composed of a target protein ligand and an E3 ubiquitin ligase ligand connected by a linker. The target protein ligand is Raf protein, and the E3 ubiquitin ligase ligand is VHL protein. The amino acid sequence of the Raf protein is shown as amino acids 224-393 in SEQ ID NO. 6.

[0010] Furthermore, the amino acid sequence of the VHL protein is shown as amino acids 1-213 in SEQ ID NO.6.

[0011] Furthermore, the target protein is a wild-type KRAS or a KRAS mutant. The amino acid sequence of the wild-type KRAS is shown as amino acids 269-456 in SEQ ID NO.4. The KRAS mutant is formed by mutating glycine at position 12 of the wild-type amino acid sequence to cysteine, and is denoted as KRAS mutant (G12C). The amino acid sequence is shown as amino acids 269-456 in SEQ ID NO.2.

[0012] Furthermore, the amino acid sequence of the linker chain is shown as 214-223 in SEQ ID NO.6.

[0013] The present invention also provides an application of the KRAS-targeting protein degradation chimera in the degradation of target proteins.

[0014] Furthermore, the target proteins include endogenous or exogenous KRAS proteins and the KRAS mutant G12C.

[0015] The mechanism by which the KRAS-targeting protein degradation chimera of this invention degrades the target protein is as follows: Figure 1As shown, specifically: one end is the target protein KRAS binding ligand Raf, and the other end is the substrate recognition receptor VHL of the E3 ubiquitin ligase complex. They are linked by a linker to form a transient and stable "KRAS-PROTAC-E3 Ligase" ternary complex. Once the ternary complex is formed, the recruited E3 ligase will work with the E2 ubiquitin conjugator to transfer multiple ubiquitin molecules and covalently label them to lysine residues on the KRAS protein. PROTAC acts as a molecular bridge, enabling KRAS to be polyubiquitinated under the synergistic effect of E1 / E2 / E3. The KRAS protein labeled with polyubiquitin chains will be recognized by the proteasome and degraded into short peptide fragments. The PROTAC molecules themselves are not consumed during the degradation process. After dissociation, they can enter the next catalytic cycle and be cleared in a "catalytic" manner.

[0016] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0017] First, the targeted protein degradation chimera constructed in this invention can effectively target KRAS mutants, which are traditionally considered "undruggable." These mutant proteins typically lack deep binding pockets and are difficult for small molecule inhibitors to effectively recognize. Second, the chimera exhibits higher target affinity and broader target coverage while significantly reducing off-target risks. Furthermore, the plasmid DNA or mRNA-based delivery strategy can achieve long-term expression in vivo, thus possessing the therapeutic potential of "single-dose, sustained degradation," which is expected to significantly improve medication adherence and treatment efficacy. (iv) Description of the attached drawings

[0018] Figure 1 A schematic diagram illustrating the principle of the KRAS-targeted protein degradation chimera (PROTAC).

[0019] Figure 2 The spectrum of plasmid pLV2-EF1α-VHL-Raf(51-220)-3×FLAG.

[0020] Figure 3 Example 2: Laser confocal microscope image of the chimera after co-transfection with KRAS protein targeting degradation.

[0021] Figure 4 Example 3: Western blot and bar chart of relative KRAS expression levels after co-transfection of the chimeric target exogenous KRAS protein degradation.

[0022] Figure 5 Example 4: Western blot and bar chart of relative KRAS expression level after transfection of chimera targeting endogenous KRAS protein degradation.

[0023] Figure 6Example 5: Plate clone diagram of the chimera after transfection with the target endogenous KRAS protein degradation. (V) Detailed Implementation Methods

[0024] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto:

[0025] Unless otherwise specified, the experimental methods in the following embodiments are conventional methods. Unless otherwise specified, the molecular cloning method used in this invention is mainly seamless cloning.

[0026] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0027] Sequence Description of the Invention

[0028] SEQ ID NO.1 represents the nucleotide sequence of mEmerald-3×FLAG-KRAS (G12C), where 1-717bp represents mEmerald, 718-807bp represents 3×FLAG, 808-813bp represents the linker, and 814-1377bp represents KRAS G12C.

[0029] SEQ ID NO.2 represents the amino acid sequence of mEmerald-3×FLAG-KRAS (G12C), where amino acids 1-239 represent mEmerald, 240-245 represent the linker, 246-269 represent 3×FLAG, 270-271 represent the linker, and 272-459 represent KRAS G12C.

[0030] SEQ ID NO.3 represents the nucleotide sequence of mCh-3×FLAG-KRAS (WT), where 1-708bp represents mCherry, 709-714bp represents linker, 715-798bp represents 3×FLAG, 799-804bp represents linker, and 805-1368bp represents KRAS (WT).

[0031] SEQ ID NO.4 represents the amino acid sequence of mCh-3×FLAG-KRAS (WT), where 1-236 represent mCherry, 237-242 represent linker, 243-266 represent 3×FLAG, 267-268 represent linker, and 269-456 represent KRAS (WT).

[0032] SEQ ID NO.5 represents the nucleotide sequence of VHL-Raf(51-220)-3×FLAG, where 1-639bp represents VHL, 640-669bp represents the linker, 670-1179bp represents Raf(51-220), 1180-1194bp represents the linker, and 1195-1260bp represents 3×FLAG.

[0033] SEQ ID NO.6 represents the amino acid sequence of VHL-Raf(51-220)-3×FLAG, where 1-213 represent VHL, 214-223 represent the linker, 224-393 represent Raf(51-220), 394-399 represent the linker, and 400-421 represent 3×FLAG.

[0034] SEQ ID NO.7 represents the nucleotide sequence of VHL-Raf(51-220)-miRFP670nano3, where 1-639bp represents VHL, 640-669bp represents the linker, 670-1179bp represents Raf(51-220), 1180-1191bp represents the linker, and 1192-1632bp represents miRFP670nano3.

[0035] SEQ ID NO.8 represents the amino acid sequence of VHL-Raf(51-220)-miRFP670nano3, where 1-213 represent VHL, 214-223 represent the linker, 224-393 represent Raf(51-220), 394-397 represent the linker, and 398-567 represent miRFP670nano3.

[0036] SEQ ID NO.9 represents the nucleotide sequence of VHL-DARPin-3×flag, where 1-639bp represents VHL, 640-669bp represents the linker, 670-1140bp represents DARPin, 1141-1155bp represents the linker, and 1156-1224bp represents 3×flag.

[0037] SEQ ID NO.10 represents the amino acid sequence of VHL-DARPin-3×flag, where 1-213 represent VHL, 214-223 represent the linker, 224-380 represent DARPin, 381-386 represent the linker, and 387-408 represent 3×flag.

[0038] Example 1: Design and construction of a KRAS protein-targeted degradation chimera (PROTAC):

[0039] I. Constructing target protein plasmids: Fusion expression of red fluorescent protein mCherry or green fluorescent protein mEmerald with wild-type or mutant KRAS target protein to construct plasmids mCh-KRAS (WT) and mEmerald-KRAS (G12C), specifically following these steps:

[0040] 1. Construction of pcDNA-CMV-mEmerald-3×FLAG-KRAS (G12C)

[0041] (1) Enzyme digestion of empty vector:

[0042] The empty vector pcDNA-CMV-eGFP (purchased from Miaoling Biotechnology, P64303, also known as pcDNA3.1(+) eGFP) was digested with XbaⅠ and SpeⅠ to remove the eGFP fragment, and the digested vector backbone pcDNA-CMV was recovered by gel excision.

[0043] (2) Sequence template

[0044] KRAS wild-type (WT) and KRAS mutant (G12C) were purchased from Miaoling Biotechnology (lot numbers P88851 and P31033, respectively). The plasmid PUC57-mEmerald expressing green fluorescent protein mEmerald (amino acid sequence as shown in amino acids 1-239 of SEQ ID NO.2) and the plasmid PUC57-mCh expressing red fluorescent protein mCherry (amino acid sequence as shown in amino acids 1-236 of SEQ ID NO.4) were purchased from Sangon Biotech. They were respectively constructed by Sangon Biotech synthesizing the target fragment and inserting it into the ApaI and BamHI double restriction sites of the PUC57 vector.

[0045] (3) Amplification of the target fragment

[0046] The primer sequences are designed as shown in Table 1. Using plasmid PUC57-mEmerald as a template, the first round of PCR amplification was performed using primers mEmerald-F and mEmerald-R1 in Table 1. Using the first round of PCR products as a template, the second round of PCR amplification was performed using primers mEmerald-F and mEmerald-R2 in Table 1. Using the second round of PCR products as a template, the third round of PCR amplification was performed using primers mEmerald-F and mEmerald-R3 in Table 1. The obtained products were separated into target fragments by 1% agarose gel electrophoresis, and the 807 bp mEmerald-3×FLAG fragment (FLAG fragment synthesized from primers) was recovered using the Keco gel recovery kit. Using the KRAS mutant G12C as a template, PCR amplification was performed using primers KRAS(G12C)-F and KRAS-R in Table 1. The target fragment was separated by 1% agarose gel electrophoresis, and the 564 bp KRAS(G12C) fragment was recovered using the Keco gel recovery kit.

[0047] Table 1. Primer sequences

[0048]

[0049] PCR system: 1 μL upstream primer, 1 μL downstream primer, 12.5 μL 2×Phanta MaX master MiX (Vazyme), 1 μL template, 10 μL purified water.

[0050] PCR program: 95 ℃ for 3 minutes; 95 ℃ for 15 seconds, 55 ℃ for 15 seconds, 72 ℃ for 2 minutes, 35 cycles; 72 ℃ for 5 minutes; 4 ℃.

[0051] (4) Seamless cloning of the target fragment

[0052] Two μL each of the two fragments from step (3) and the pcDNA-CMV from step (1) were ligated with 3 μL of 2×Basic Assembly Mix. The ligation product was transformed into DH5α Escherichia coli and cultured on an LB medium without ampicillin (Amp) at 37°C on a shaker for 30 minutes. After 30 minutes, the ligation product was evenly spread on an LB plate containing Amp (100 μg / mL) and cultured at 37°C for 16 hours. Clones were then picked and sent to Qingke Company for sequencing verification. Clones with correct sequencing results were amplified by placing the corresponding clones in 25 mL of LB medium and culturing at 37°C for 16 hours. Plasmids were extracted using a plasmid miniprep kit (Kangwei Century) to obtain pcDNA-CMV-mEmerald-3×FLAG-KRAS (G12C), abbreviated as mEmerald-KRAS (G12C).

[0053] 2. Construct pcDNA-CMV-mCh-3×FLAG-KRAS (WT)

[0054] Using the primers in Table 2, pcDNA-CMV-mCh-3×FLAG-KRAS (WT) was constructed in the same manner as in step 1, abbreviated as mCh-KRAS (WT).

[0055] Table 2. Primer sequences

[0056]

[0057] II. Constructing a protein-targeted imaging system: Proteins Raf and Raf(R89L) were fused with the purple fluorescent protein miRFP670nano3 for expression, and plasmids Raf-miRFP670nano3 and Raf(R89L)-miRFP670nano3 were constructed, including the following steps:

[0058] 1. Plasmid pcDNA-CMV-Raf(51-220)-miRFP670nano3

[0059] (1) Acquisition of empty vector fragments:

[0060] Using pcDNA-CMV-eGFP as a template, the empty vector fragment pcDNA-CMV was obtained through upstream and downstream primers of CMV.

[0061] Table 3. Primer sequences

[0062]

[0063] (2) Obtaining the target fragment and constructing the plasmid

[0064] Using pCMV-RAF1(human)-3×Myc-Neo (Miaoling Biotechnology, P51575) as a template, fragment 1 was obtained using the Raf(51-220) upstream primer and the Raf(51-220) downstream primer. Using PUC57-miRFP670nano3 as a template, fragment 2 was obtained using the miRFP670nano3 upstream primer and the miRFP670nano3 downstream primer. Fragment 1 (2 μL), fragment 2 (2 μL) and the empty vector fragment pcDNA-CMV (2 μL) were ligated using seamless cloning to obtain the plasmid pcDNA-CMV-Raf(51-220)-miRFP670nano3, wherein the amino acid sequence of miRFP670nano3 is shown as 398-567 in SEQ ID NO.8, and is abbreviated as Raf(51-220)-miRFP670nano3.

[0065] Table 4. Primer Sequences

[0066]

[0067] 2. Plasmid pcDNA-CMV-Raf(R89L)-miRFP670nano3

[0068] Using pcDNA-CMV-Raf(51-220)-miRFP670nano3 as a template, fragment 1 was obtained using the upstream primer of Raf(51-220) and the downstream primer of Raf(R89L); using pcDNA-CMV-Raf(51-220)-miRFP670nano3 as a template, fragment 2 was obtained using the upstream primer of Raf(R89L) and the downstream primer of miRFP670nano3; fragment 1 (2 μL), fragment 2 (2 μL), and the vector pcDNA-CMV (2 μL) were ligated using seamless cloning to obtain the plasmid pcDNA-CMV-Raf(R89L)-miRFP670nano3, abbreviated as Raf(R89L)-miRFP670nano3.

[0069] Table 5. Primer sequences are as follows:

[0070]

[0071] 3. Plasmid pcDNA-CMV-DARPin-miRFP670nano3

[0072] Using pcDNA-CMV-Raf(51-220)-miRFP670nano3 as a template, fragment 1 was obtained using the miRFP670nano3 upstream primer and the CMV downstream primer; DARPin (from the literature) was used to obtain fragment 1. [6] Using G5161 (synthesized by Miaoling Biotechnology) as a template, fragment 2 was obtained through upstream and downstream primers of DARPin. Fragment 1 (2 μL) and fragment 2 (2 μL) were ligated using seamless cloning to obtain plasmid pcDNA-CMV-DARPin-miRFP670nano3, wherein the amino acid sequence of DARPin is shown as 224-380 in SEQ ID NO. 10, abbreviated as DARPin-miRFP670nano3.

[0073] Table 6. Primer sequences are as follows:

[0074]

[0075] 4. Plasmid pcDNA-CMV-VHL-Raf(51-220)-miRFP670nano3

[0076] Using pCDH-CMV-VHL(human)-3×FLAG-EF1α-turboRFP-T2A-Puro (purchased from Miaoling Biotechnology, P27212) as a template, fragment 1 was obtained using the VHL upstream primer and VHL downstream primer; using pcDNA-CMV-Raf(51-220)-miRFP670nano3 as a template, fragment 2 was obtained using the Raf(51-220) upstream primer and miRFP670nano3 downstream primer; fragment 1 (2 μL), fragment 2 (2 μL), and pcDNA-CMV vector (2 μL) were ligated using seamless cloning to obtain plasmid pcDNA-CMV-VHL-Raf(51-220)-miRFP670nano3.

[0077] Table 7. Primer Sequences

[0078]

[0079] 5. Plasmid pcDNA-CMV-VHL-Raf(51-220)-miRFP670nano3

[0080] Using pcDNA-CMV-VHL-Raf(51-220)-miRFP670nano3 as a template, fragment 1 was obtained using the VHL upstream primer and the RAF(R89L) downstream primer; using pcDNA-CMV-VHL-Raf(51-220)-miRFP670nano3 as a template, fragment 2 was obtained using the Raf(R89L) upstream primer and the miRFP670nano3 downstream primer; fragment 1 (2 μL), fragment 2 (2 μL), and pcDNA-CMV vector (2 μL) were ligated using seamless cloning to obtain plasmid pcDNA-CMV-VHL-Raf(R89L)-miRFP670nano3, abbreviated as Raf(R89L)-miRFP670nano3.

[0081] Table 8. Primer Sequences

[0082]

[0083] III. Construct a targeted endogenous KRAS degradation system by fusing the E3 ubiquitin ligase VHL and Raf, constructing the plasmid VHL-Raf, and subcloning the sequence into an empty vector. The vector construction method includes the following steps:

[0084] 1. Obtaining empty vector fragments:

[0085] Using pLV2-EF1α-6×His-GLP1R(human)-3×FLAG-IRES-Puro (purchased from Miaoling Biotechnology, P46284) as a template, vector fragment 1 was obtained through the FLAG upstream primer and the AMP downstream primer; vector fragment 2 was obtained through the AMP upstream primer and the EF-1α downstream primer.

[0086] Table 9. Primer Sequences

[0087]

[0088] 2. Plasmid pLV2-EF1α-VHL-Raf(51-220)-3×FLAG

[0089] Using pcDNA-CMV-VHL-Raf(51-220)-miRFP670nano3 as a template, the VHL-Raf fragment was obtained through the upstream primer of VHL and the downstream primer of Raf(51-220). Using seamless cloning, vector fragment 1 (2 μL), vector fragment 2 (2 μL), and fragment VHL-Raf (2 μL) were ligated to obtain the plasmid pLV2-EF1α-VHL-Raf(51-220)-3×FLAG. The plasmid map is shown below. Figure 2 .

[0090] Table 10. Primer Sequences

[0091]

[0092] 3. Plasmid pLV2-EF1α-VHL-Raf(R89L)-3×FLAG

[0093] Using pLV2-EF1α-VHL-Raf(51-220)-3×FLAG as a template, fragment 1 was obtained using the VHL upstream primer and the RAF(R89L) downstream primer; fragment 2 was obtained using the Raf(R89L) upstream primer and the Raf(R89L) downstream primer; fragment 1 (2 μL), fragment 2 (2 μL), and vector fragment 1 (2 μL) and vector fragment 2 (2 μL) were ligated using seamless cloning to obtain plasmid pLV2-EF1α-VHL-Raf(R89L)-3×FLAG.

[0094] Table 11. Primer sequences

[0095]

[0096] 4. Plasmid pLV2-EF1α-VHL-DARPin-3×FLAG

[0097] Using pLV2-EF1α-VHL-Raf(51-220)-3×FLAG as a template, fragment 1 was obtained using VHL upstream and downstream primers; fragment 2 was obtained using DARPin upstream and downstream primers; fragment 1 (2 μL), fragment 2 (2 μL), and vector fragment 1 (2 μL) and vector fragment 2 (2 μL) were ligated using seamless cloning to obtain plasmid pLV2-EF1α-VHL-DARPin-3×FLAG.

[0098] Table 12. Primer sequences

[0099]

[0100] Example 2: Subcellular localization characterization of Raf (51-220) and KRAS

[0101] 1. Cell Culture

[0102] Human embryonic kidney cells 293T at 1×10 6 Cells were seeded at a rate of 100 cells / mL into 5 mL of DMEM medium and cultured at 37°C with 5% CO2 for 18 h. 1 mL of trypsin digest was added, and the cells were digested at 37°C for 30 s until completely digested. 1 mL of DMEM medium was added, and the cells were repeatedly pipetted to mix. After centrifugation at 1000 rpm for 5 min, the cell pellet was seeded into a 35 mm glass-bottom confocal culture dish and cultured at 37°C until the cell density reached 80% confluence, ready for plasmid transfection.

[0103] 2. Co-transfect plasmids into 293T cells

[0104] All transfection experiments were performed using Lipo8000™ transfection reagent (Beyotime) according to the manufacturer's instructions, with 2 μg of each plasmid used for transfection. The experiments were divided into the following groups:

[0105] Single expression control group: transfected separately with mEmerald-KRAS(G12C) or mCh-KRAS(WT) plasmids ( Figure 3 (a)

[0106] Mutant KRAS(G12C) co-expression group: Plasmids expressing the following three purple fluorescent proteins (miRFP670nano3) fusion proteins respectively: Raf(51-220)-miRFP670nano3 (wild-type RBD), Raf(R89L)-miRFP670nano3 (point mutation inactivated RBD), and DARPin-miRFP670nano3 (alternative binding control), were co-transfected with the mEmerald-KRAS(G12C) plasmid expressing green fluorescent protein (mEmerald) labeling. Figure 3 (b)

[0107] Wild-type KRAS(WT) co-expression group: The above three purple fluorescent fusion protein expression plasmids were co-transfected with the mCh-KRAS(WT) plasmid expressing mCh-labeled protein. Figure 3 (c)

[0108] 3. Confocal microscopy analysis confirmed that the Raf(51-220) domain specifically binds to KRAS.

[0109] After transfection in step 2, the cells were seeded into DMEM medium and cultured at 37°C with 5% CO2 for 16 h. Cells were then harvested and observed using a laser confocal microscope. The results are as follows: Figure 3 As shown.

[0110] like Figure 3 As shown in Figure a, both KRAS(G12C) and KRAS(WT) expressed individually exhibit typical cell membrane localization.

[0111] like Figure 3 As shown in Figures b and c: when Raf(51-220)-miRFP670nano3 is co-expressed with KRAS(G12C) or KRAS(WT), the two exhibit highly significant co-localization signals on the cell membrane. This directly proves that the Raf(51-220) domain can effectively and specifically bind to exogenous KRAS protein, and this binding is independent of the G12C mutation status of KRAS.

[0112] To highlight the specificity and advantages of the above combination, a strict control group was set up:

[0113] (1) Negative control (Raf mutant): When Raf(R89L)-miRFP670nano3 carrying the R89L inactivation mutation was co-expressed with KRAS, the mutant was diffusely distributed in the cytoplasm and did not co-localize with membrane-localized KRAS, indicating that the point mutation completely destroyed its ability to interact with KRAS.

[0114] (2) Alternative binder control (DARPin): Although DARPin-miRFP670nano3 can bind to KRAS, its binding leads to abnormal changes in the subcellular localization of KRAS protein, causing KRAS, which is originally located in the cell membrane, to diffuse into the cytoplasm. This suggests that the binding of DARPin may interfere with the normal membrane localization and function of KRAS.

[0115] Conclusion: This embodiment, through systematic confocal colocalization analysis, demonstrates that the chimera exhibits significant and complete colocalization on the cell membrane with various KRAS mutants (such as G12C and G12D) (PANC-1 cells are a G12D mutant cell line). This directly proves its excellent binding ability and targeting specificity with different KRAS mutants, providing a structural basis for achieving a broader target coverage. It confirms that the Raf(51-220) domain selected in this invention can efficiently and specifically bind to wild-type and G12C mutant KRAS without altering the normal membrane localization of KRAS. This characteristic is a key advantage for its use as a binding element in targeted degradation chimeras, laying the molecular foundation for subsequent efficient and physiologically relevant KRAS targeted degradation.

[0116] Example 3: Evaluation of the efficiency of targeted degradation of exogenous KRAS by Western Blot

[0117] 1. Cell Culture

[0118] Human embryonic kidney cells 293T at 1×10 6 Cells were seeded at a rate of 100 cells / mL into 5 mL of DMEM medium and cultured at 37°C with 5% CO2 for 18 h. 1 mL of trypsin digestion solution was added, and the cells were digested at 37°C for 30 s until completely digested. 1 mL of DMEM medium was added, and the cells were repeatedly pipetted to mix. After centrifugation at 1000 rpm for 5 min, the cell pellet was seeded into 12-well plates and cultured at 37°C until the cell density reached 80% confluence for plasmid transfection.

[0119] 2. Co-transfect plasmids into 293T cells

[0120] Using the method of Example 2, pcDNA-CMV-Raf(51-220)-miRFP670nano3 and pcDNA-CMV-mEmerald-3×FLAG-KRAS(G12C) were mixed. Figure 4 KRAS (G12C) Raf group), pcDNA-CMV-Raf (51-220)-miRFP670nano3 and pcDNA-CMV-mCh-3×FLAG-KRAS (WT) Figure 4The KRAS (WT) Raf group was co-transfected into 293T cells. Twenty-four hours after transfection (three replicates per group), cells were collected, lysed, and analyzed by Western blotting using GFP or Flag antibodies. In this experiment, groups transfected with pcDNA-CMV-Raf(R89L)-miRFP670nano3 instead of the aforementioned pcDNA-CMV-Raf(51-220)-miRFP670nano3 served as negative controls (i.e.,...). Figure 4 The R89L group was included, and a blank control group (corresponding to) was set up without any plasmid transfection. Figure 4 (WT), experimental results are as follows Figure 4 As shown.

[0121] Western blot analysis confirmed that the targeted degradation system effectively reduced the levels of KRAS (G12C) and KRAS (WT) proteins. Quantitative results showed that, compared with the R89L group, the targeted system had a degradation efficiency of approximately 40% for KRAS (G12C) (n=3, p < 0.001) and approximately 65% ​​for KRAS (WT) (n=3, p < 0.001).

[0122] Example 4: Evaluation of the efficiency of targeted degradation of endogenous KRAS by Western Blot

[0123] 1. Cell Culture

[0124] Human embryonic kidney cells 293T, human cervical cancer cell line (HeLa), and human pancreatic cancer cell line (PANC-1) were all processed using the methods and conditions described in Example 3.

[0125] 2. Lentiviral packaging

[0126] Using the Lipo8000™ Transfection Reagent (Beyotime) three-plasmid transfection system, pLVX (target plasmid), pSPAX2 (Miaoling Bio, P0261), and PMD2.G (Miaoling Bio, P0262) were transfected into 293T cells at a ratio of 4:3:1. The medium was changed 8 hours after transfection, and lentiviral supernatant was collected at 24 and 48 hours after the medium change. The target plasmid included pLV2-EF1α-VHL-Raf(51-220)-3×FLAG( Figure 5 pLV2-EF1α-VHL-Raf(R89L)-3×FLAG( Figure 5 R89L), pLV2-EF1α-VHL-DARPin-3×FLAG ( Figure 5 DARPin), using untransfected 293T cells as WT.

[0127] 3. Lentiviral infection

[0128] Human embryonic kidney cells 293T, human cervical cancer cells (HeLa), and human pancreatic cancer cells (PANC-1) were pre-resuscitated and cultured in good condition. Cells were evenly seeded into six-well plates and cultured overnight until cell confluence reached approximately 70%-80%. After 18-24 hours of culture, the medium was replaced with 1 mL of DMEM complete medium containing 8 μg / mL polybrene. Subsequently, 1 mL of the corresponding viral supernatant filtered through a 0.45 μm filter was added to each well, and the plates were incubated at 37°C and 5% C. Incubate for 24-48 hours under suitable conditions. After infection, replace with fresh complete culture medium and continue culturing for another 24 hours.

[0129] 4. Screening of stable cell lines

[0130] Forty-eight hours after infection, the culture medium was replaced with a selection medium containing an appropriate concentration of puromycin for selective culture. The selection concentration for 293T cells was 1 μg / mL, while the selection concentration for HeLa and PANC-1 cells was 2 μg / mL. The selection medium was changed every 2-3 days, and selection was continued for 5-7 days until all untransduced control cells died, thereby obtaining a cell line that stably expresses the targeted degradation chimera.

[0131] 5. Verification of protein degradation effect

[0132] The stable expression cell lines obtained above were seeded in 12-well plates. When the cells reached approximately 90% confluence, total protein was extracted using Beyotime RIPA lysis buffer. Protein concentration was determined and quantified using the BCA method. 30 μg of protein sample was subjected to SDS-PAGE electrophoresis and then transferred to a PVDF membrane. Western blotting was performed using KRAS antibody, Flag-tagged antibody (for detecting chimeric expression), and GAPDH antibody (as an internal control).

[0133] 4. Results Analysis

[0134] In three different cell lines, compared with the control group transduced with empty virus, the experimental groups transduced with the targeted degradation chimera all showed detectable specific bands of the Flag tag, confirming successful and stable expression of the chimera. More importantly, KRAS protein signaling was significantly reduced in all experimental groups, while GAPDH expression levels remained unchanged. This result directly demonstrates that the degradation chimera constructed in this invention can achieve effective and specific degradation of KRAS protein in various cellular backgrounds.

[0135] In this embodiment, quantitative analysis of the Western blot results shows that... Figure 5 The data show that approximately 40% of the endogenous KRAS protein was degraded in the Raf group at 293T, while approximately 70% and 80% of the endogenous KRAS degradation were observed in HeLa and PANC-1, respectively. All three groups exhibited degradation effects similar to those of DARPin. Stable integration and sustained expression of the chimera within cells were achieved through a viral delivery system. The resulting stable cell line continuously and stably degrades KRAS protein, demonstrating in principle the potential for long-term protein degradation through a single delivery.

[0136] Example 5: Assessment of cell proliferation expressing targeted degradation of endogenous KRAS using plate clones

[0137] Using the method in Example 4, HeLa transfected pLV2-EF1α-VHL-Raf(51-220)-3×FLAG( Figure 6 pLV2-EF1α-VHL-Raf(R89L)-3×FLAG( Figure 6 R89L), pLV2-EF1α-VHL-DARPin-3×FLAG ( Figure 6 (DARPin), and a blank control group without plasmid conversion (corresponding to) Figure 6 (WT); PANC-1 transfected pLV2-EF1α-VHL-Raf (51-220)-3×FLAG ( Figure 6 pLV2-EF1α-VHL-Raf(R89L)-3×FLAG( Figure 6 (R89L), and a blank control group without plasmid transformation was set up (corresponding to R89L). Figure 6 (WT). Using the methods and conditions described in Example 3, different cells (HeLa and PANC-1) in the logarithmic growth phase were digested into single cells and prepared into cell suspensions. After cell dilution, 200 cells were added to each well of a 6-well plate, and the plates were placed in a cell culture incubator. After 48 h, DMEM medium was added. During subsequent culture, the medium was changed every 48 h, and colony formation was observed. After 14 days of culture, the medium was discarded, the cells were gently washed twice with PBS, fixed with paraformaldehyde at room temperature for 15 min, then washed three times with PBS, stained with 0.1% crystal violet, gently washed three times with PBS, dried, and photographed. The results are shown below. Figure 6 As shown. Figure 6 The data shown indicate that targeted degradation of endogenous KRAS treatment exhibited significant inhibitory effects on cell proliferation in both HeLa and PANC-1 cell models.

[0138] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the technical principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

[0139] [1] COX AD, FESIK SW, KIMMELMAN AC, et al. Drugging theundruggable RAS: Mission Possible? [J]. Nature Reviews Drug Discovery, 2014,13(11): 828-851.

[0140] [2] CRUZ-MIGONI A, CANNING P, QUEVEDO CE, et al. Structure-baseddevelopment of new RAS-effector inhibitors from a combination of active and inactive RAS-binding compounds [J]. Proceedings of the National Academy ofSciences, 2019, 116(7): 2545-2550.

[0141] [3] STRATMANN JA, ALTHOFF FC, DOEBEL P, et al. Sotorasib in KRASG12C-mutated non-small cell lung cancer: A multicenter real-world experience from the compassionate use program in Germany [J]. European Journal ofCancer, 2024, 201: 113911.

[0142] [4] SURYA K D. First Approval of Adagrasib for the Treatment of Non-Small Cell Lung Cancer Harboring a KRAS G12CMutation [J]. Current MedicinalChemistry, 2024, 31(3): 266-272.

[0143] [5] MCCORMICK F. Sticking it to KRAS: Covalent Inhibitors Enter theClinic [J]. Cancer Cell, 2020, 37(1): 3-4.

[0144] [6] BERY N, MILLER A, RABBITTS T. A potent KRAS macromoleculedegrader specifically targeting tumours with mutant KRAS [J]. NatureCommunications, 2020, 11(1): 3233.

Claims

1. A ubiquitin proteasome pathway-based KRAS targeted protein degradation chimera, characterized in that, The KRAS-targeting protein degradation chimera is composed of a target protein ligand and an E3 ubiquitin ligase ligand through a linker chain; the target protein ligand is a Raf protein, the E3 ubiquitin ligase ligand is a VHL protein, and the amino acid sequence of the Raf protein is shown as amino acids 224-393 in SEQ ID NO.

6.

2. The KRAS-targeting protein degradation chimera of claim 1, wherein, The amino acid sequence of the VHL protein is shown as amino acids 1-213 in SEQ ID NO.

6.

3. The KRAS-targeting protein degradation chimera of claim 1, wherein, The target protein is KRAS wild type or KRAS mutant, the amino acid sequence of the KRAS wild type is shown as amino acids 269-456 in SEQ ID NO. 4; the KRAS mutant is a wild type amino acid sequence with the 12th glycine mutated to cysteine, and the amino acid sequence is shown as amino acids 269-456 in SEQ ID NO.

2.

4. The KRAS-targeting protein degradation chimera of claim 1, wherein, The amino acid sequence of the linker chain is shown as SEQ ID NO. 6 214-223.

5. Use of the KRAS-targeting protein degradation chimera of claim 1 in degrading target proteins.

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