A drug control system based on KFERQ sequences, its construction method and application

Abstract

This invention belongs to the field of biomedical technology, specifically relating to a drug control system based on the KFERQ sequence, its construction method, and its application. This drug control system is composed of a KFERQ sequence, an HCV-NS3 sequence, and a protein-targeting sequence recombined together. During recombination, the protein-targeting sequence is located at the N-terminus or C-terminus of the recombinant sequence. This invention provides a novel strategy for endogenous protein degradation by recombinating the KFERQ sequence with the HCV-NS3 sequence and the endogenous protein-targeting sequence, and utilizing the self-cleavage property of the HCV-NS3 protease to regulate the degradation of endogenous proteins.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a drug control system based on KFERQ sequences, its construction method, and its application. Background Technology

[0002] DNA knockout and RNA interference, two techniques for regulating protein expression at the genomic and mRNA levels, are now widely used and are undoubtedly powerful tools for altering protein expression levels and functions. However, both methods regulate proteins indirectly, and are limited in terms of specificity, speed, and reversibility. For example, off-target effects can occur with CRISPR / Cas9 (Clustered Regulatory Interspaced Short Palindromic Repeats / CRISPR-Associated Protein 9) technology; long-lived proteins require more time to be consumed and may even be completely resistant to DNA and RNA-targeted consumption methods; and damaged genomes may be difficult to recover without reversing target protein expression after external influences are removed. These limitations restrict these methods as research tools and clinical treatments.

[0003] Currently, the main intracellular protein degradation pathways identified include the lysosomal pathway, the ubiquitin-proteasome pathway, and the autophagy pathway. The ubiquitin-proteasome system primarily degrades soluble, short-lived, and misfolded monoliths of proteins, while the lysosomal system primarily degrades long-lived proteins, protein aggregates, and damaged organelles. Both autophagy and the ubiquitin-proteasome system can degrade substrates through ubiquitination. To overcome the limitations imposed by DNA knockout and RNA interference, several methods based on cellular self-degradation targeting specific protein degradation have been developed, including PROTAC (proteolysis-targeting chimeras), LYTAC (lysosome-targeting chimeras), and AUTAC (autophagy-targeting chimeras). Although these new technologies for rapid targeted protein degradation offer many advantages, these degradation systems lack certain regulatory switching capabilities. Therefore, developing a widely applicable drug control system that enables the switching of endogenous protein degradation is of great significance. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the present invention aims to provide a drug control system based on the KFERQ sequence, its construction method, and its application. This drug control system recombines the KFERQ sequence with the HCV-NS3 sequence and the endogenous protein targeting sequence, and utilizes the self-cleavage property of the HCV-NS3 protease to regulate the degradation of endogenous proteins, thereby providing a novel strategy for endogenous protein degradation.

[0005] Chaperon-mediated autophagy (CMA) is a type of autophagy targeting proteins containing a pentapeptide motif biochemically associated with KFERQ. This CMA-targeting motif is present in all known proteins that serve as CMA substrates. Studies have shown that non-CMA substrates to which the KFERQ sequence attaches (fused) can also comply with CMA, provided the KFERQ sequence is exposed. Therefore, the inventors utilize CMA as our degradation pathway for endogenous proteins.

[0006] Hepatitis C virus (HCV) is a member of the Flaviviridae family of viruses. It has a 9.6 kb positive-sense RNA genome encoding a long, multi-protein precursor of approximately 3000 amino acids. This precursor is proteased after being translated into at least 10 individual proteins by cellular and viral proteases, including four structural proteins (C, E1, E2, and p7) and six non-structural (NS) proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B). The NS3 protein is a multifunctional protein with a serine protease domain in the N-terminal third and a helicase domain in the C-terminal third. The NS3-4A serine protease is a non-covalent heterodimeric complex formed by two HCV-encoded proteins, the N-terminal serine protease domain of NS3 (catalytic subunit), and the NS4A cofactor (activating subunit). The NS3-4A serine proteases are responsible for proteolytic cleavage at four junctions in the HCV polyprotein precursor: NS3 / NS4A (autocleavage), NS4A / NS4B, NS4B / NS5A, and NS5A / NS5B. Without special treatment, the proteases will cleave themselves from the protein, but the synthesis of the protein after a specified time can be prevented by applying cell-permeable HCV-NS3 protease inhibitors, such as simeprevir, danoprevir, asunaprevir (ASV), and ciluprevir.

[0007] Meanwhile, the hepatitis C virus NS3 protease, due to its small protease domain (19 kDa) and monomeric nature, exhibits unusual but well-defined substrate specificity and can be effectively inhibited by non-toxic cell permeability inhibitors. Furthermore, non-CMA substrates attached (fused) to the KFERQ sequence can obey CMA upon KFERQ exposure, thereby rapidly targeting and degrading proteins.

[0008] Therefore, to achieve the above objectives, the present invention provides a drug control system based on the KFERQ sequence, wherein the drug control system is composed of the KFERQ sequence, the HCV-NS3 sequence (the cleavage site is adjacent to HCV-NS3 and forms a single element) and a protein targeting sequence, wherein, during the recombination of the three, the protein targeting sequence is located at the N-terminus or C-terminus of the recombinant sequence.

[0009] Furthermore, in the above technical solution, the HCV-NS3 sequence is an element composed of a cleavage site and an adjacent HCV-NS3.

[0010] HCV-NS3 is an enzyme that cleaves cleavage sites. Its active site changes depending on the sequence order. Under normal conditions, the cleavage site is located at the N-terminus of HCV-NS3 to achieve stable cleavage efficiency. However, when the KFERQ sequence is located at the N-terminus of the recombinant sequence, HCV-NS3 is reordered (i.e., cyclically arranged) and can also achieve stable cleavage efficiency at the N-terminus of the cleavage site. In this case, we call it mHCV-NS3 (abbreviated as H, just like HCV-NS3).

[0011] Furthermore, in the above technical solution, the drug control system is a recombination of the KFERQ-mHCV-NS3(KH) sequence and the protein targeting sequence.

[0012] Furthermore, in the above technical solution, the protein targeting sequence is an endogenous protein sequence, which is any one of the following: an intracellularly overexpressed EGFP sequence, an endogenous AKT sequence, or an endogenous pAKT sequence.

[0013] Furthermore, in the above technical solution, the drug is ASV (anaspirin). Anaspirin is a hepatitis C virus NS3 / NS4A protease inhibitor and can be used to treat hepatitis C.

[0014] This invention also provides a method for constructing a drug control system based on the KFERQ sequence, comprising the following steps: using FV185 as a plasmid vector, firstly recombining the HCV-NS3 sequence with the KEFRQ sequence to obtain a KFERQ-mHCV-NS3(KH) or HCV-NS3-KFERQ(HK) recombinant sequence, and then inserting a protein targeting sequence at the N-terminus or C-terminus of the obtained recombinant sequence to obtain a recombinant plasmid.

[0015] Furthermore, in the above technical solution, when the protein targeting sequence is endogenous AKT, a GFP nanobody sequence or a 2xAKTin sequence is inserted at the C-terminus.

[0016] Preferably, the recombinant plasmid includes a KH-protein targeting sequence.

[0017] The present invention also provides an application of the above-mentioned drug control system in the degradation of exogenously overexpressed EGFP and the degradation of endogenous oncogenic proteins.

[0018] Furthermore, in the above technical solution, when applied to degrading EGFP overexpressed in cells, the plasmid containing the drug control system with the protein targeting sequence of EGFP is transfected into the recipient cells for 6-8 hours, then ASV drug is added for treatment, and the cells are cultured for another 24 hours before the EGFP degradation is detected.

[0019] Furthermore, in the above technical solution, when applied to the degradation of endogenous oncogenic proteins, after transfecting recipient cells with a plasmid containing a drug control system with a protein targeting sequence of 2xAKTin for 6-8 hours, ASV drug is added for treatment, and the cells are cultured for another 24 hours to detect the degradation of AKT and pAKT.

[0020] The beneficial effects of this invention are:

[0021] This invention provides a novel strategy for endogenous protein degradation by recombinating the KFERQ sequence with the HCV-NS3 sequence and the endogenous protein targeting sequence, and by utilizing the self-cleavage property of the HCV-NS3 protease to regulate the degradation of endogenous proteins.

[0022] This invention experimentally determined that placing the KFERQ sequence at the N-terminus of a recombinant polypeptide results in higher recognition and binding efficiency of the chaperone protein Hsc70, providing a good template for the insertion and replacement of other regulatory elements.

[0023] The drug-controlled degradation system containing KEFRQ-mHCV-NS3(KH) designed in this invention can target endogenous and exogenous proteins and significantly reduce the levels of overexpressed EGFP and endogenous AKT and pAKT in cells, achieving significant degradation of intracellular EGFP and endogenous oncogenic proteins, while effectively inhibiting cell proliferation activity, providing a basis for cancer treatment. Attached Figure Description

[0024] Figure 1 This is a schematic diagram of the composition of the recombinant plasmid of the present invention;

[0025] Figure 2 This is a schematic diagram illustrating the mechanism of action of KH-E in cells according to the present invention;

[0026] Figure 3 This is a diagram showing the changes in EGFP protein after ASV-regulated KH-E degradation according to the present invention.

[0027] Figure 4 This is a graph showing the green fluorescence level after ASV-regulated KH-E degradation according to the present invention.

[0028] Figure 5 This is a graph showing the changes in green fluorescence content and intensity after ASV-regulated KH-E degradation according to the present invention.

[0029] Figure 6 This is a schematic diagram illustrating the mechanism of action of ASV in regulating KH-G to target and degrade EGFP in this invention.

[0030] Figure 7 This is a fluorescence microscopy diagram showing the level and intensity of EGFP overexpression regulated by ASV in KH-G degradation of intracellularly overexpressed EGFP, as detected by the present invention.

[0031] Figure 8 The flow cytometry diagram shows the level and intensity of EGFP overexpressed in cells, which is regulated by ASV to degrade KH-G.

[0032] Figure 9 This invention provides a protein imprinting assay to detect the level and intensity of EGFP overexpression regulated by ASV in KH-G degradation of intracellularly overexpressed EGFP.

[0033] Figure 10 This is a diagram showing the changes in intracellular AKT and pAKT levels after KH-A was regulated by ASV according to the present invention.

[0034] Figure 11 This is a diagram showing the proliferation changes of 293T and MDA-MB-231 cells after KH-A knockdown of AKT and pAKT, as detected by the CCK8 experiment of this invention.

[0035] Figure 12 This is a comparison of the fluorescence intensity of KH-A and mKH-A in nude mice according to the present invention;

[0036] Figure 13 This is a comparison chart showing the differences in tumor mass and volume between KH-A and mKH-A in nude mice according to the present invention. Detailed Implementation

[0037] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the raw materials used in the following embodiments are all commercially available products and can be purchased from the market. Methods not specifically described are performed according to conventional methods in the art.

[0038] Lentiviral plasmid vector FV185 was purchased from Tianjin Sheweis Biotechnology Co., Ltd.

[0039] The present invention will be further described in detail below with reference to embodiments:

[0040] Example 1: Construction of recombinant plasmids

[0041] Using the lentiviral plasmid vector FV185 as the vector, a backbone was obtained using inverse amplification PCR with appropriate primers. Based on homologous sequences, the validated Cleavage site-HCV-NS3 or mHCV-NS3-Cleavage site sequences were synthesized by Sangon Biotech. The nucleotide sequences of the Cleavage site-HCV-NS3 and mHCV-NS3-Cleavage site sequences are shown in Seq_1 and Seq_2, respectively. These sequences were then recombined with a KEFRQ sequence (nucleotide sequence shown in Seq_3) to obtain KH (KEFRQ-mHCV-NS3) or HK (HCV-NS3-KEFRQ) recombinant sequences. An EGFP sequence (derived from LentiCRISPR v2-EGFP, addgene#167188, nucleotide sequence shown in Seq_4) was then inserted at the N-terminus or C-terminus of the obtained KH or HK recombinant sequence to obtain a recombinant plasmid containing the above sequences. The recombinant plasmid can be a KH-EGFP (KH-E), HK-EGFP (HK-E), EGFP-KH (E-KH), or EGFP-HK (E-HK) sequence, where the amino acid sequences of KH-E and E-HK are shown in Seq_7 and Seq_8, respectively.

[0042] The Q in the KFERQ sequence of the recombinant plasmid was mutated to A to obtain the mK(KFERA) sequence, resulting in recombinant plasmids of the types mKH-E (KEFRA-mHCV-NS3-EGFP) or E-HmK (EGFP-HCV-NS3-KEFRA). The amino acid sequences of mKH-E and E-HmK are shown in Seq_9 and Seq_10, respectively.

[0043] Inserting either a GFP nanobody (nucleotide sequence shown in Seq_5) or a 2xAKTin (nucleotide sequence shown in Seq_6) sequence at the C-terminus of the KH recombinant sequence yields plasmids containing the KH-GFP nanobody (KH-G) and KH-2xAKTin (KH-A) sequences, respectively, with amino acid sequences shown in Seq_11 and Seq_12. Mutating Q to A in the above recombinant sequence KFERQ yields the mKH-GFP nanobody (mKH-G) and mKH-2xAKTin (mKH-A) sequences, with amino acid sequences shown in Seq_13 and Seq_14, respectively. Schematic diagrams of some recombinant plasmids are shown below. Figure 1 As shown.

[0044] Example 2: ASV regulates KH-E to target and degrade its own EGFP

[0045] Taking human embryonic kidney cells HEK-293T as an example, the plasmids constructed in Example 1 were divided into 6 groups. Group 1 was transfected with the EGFP-HCV-NS3-KFERA (E-HmK) plasmid; groups 2 and 3 were transfected with the EGFP-HCV-NS3-KFERQ (E-HK) plasmid; group 4 was transfected with the KFERA-mHCV-NS3-EGFP (mKH-E) plasmid; and groups 5 and 6 were transfected with the KFERQ-mHCV-NS3-EGFP (KH-E) plasmid. The medium was changed 6 hours after plasmid transfection. DMSO was added to the culture medium of 293T cells in groups 2 and 5, while 10 μM ASV was added to the culture medium of the remaining 4 groups. After culturing for another 24 hours, the degradation mechanism of EGFP in cells regulated by ASV is shown in the diagram below. Figure 2 As shown; after extracting total protein from these 6 groups of 293T cells, the EGFP protein level was detected, and the results are as follows. Figure 3 As shown. From Figure 3 It can be seen that the KH-E recombinant protein can significantly degrade EGFP in the presence of ASV. The EGFP degradation was observed using fluorescence microscopy, and the results are as follows: Figure 4 As shown; simultaneously, flow cytometry was used to determine the content and intensity changes of green fluorescence, and the results are as follows. Figure 5 As shown. From Figure 4 and Figure 5 It can be seen that the protein degradation system composed of HCV-NS3, KFERQ and EGFP can significantly degrade EGFP recombinant protein after the addition of ASV, and the degradation efficiency of KH-EGFP is even higher.

[0046] Example 3: ASV regulates KH-G to target and degrade EGFP

[0047] To further demonstrate the usability of this drug-controlled degradation system, the KH system was compared with EGFP nanobody (a single-chain V... H The H antibody domain (possessing specific binding activity against GFP) was recombined, and based on the grouping experience in Example 2, 293T cells were directly divided into three groups, each transfected with the KH-G or mKH-G plasmid obtained in Example 1: Group 1 was transfected with the mKH-G plasmid, and Groups 2 and 3 were transfected with the KH-G plasmid. Groups 1 and 3 were treated with ASV, and Group 2 was treated with DMSO; the degradation mechanism is as follows. Figure 6 As shown, the degradation of EGFP was detected using fluorescence microscopy, flow cytometry, and protein blotting, and the results are as follows: Figure 7-9 As shown. From Figure 7-9 It can be seen that the drug-controlled degradation system does not use the previously mentioned CMA pathway, but rather the endosome microautophagy-lysosome degradation pathway that we discovered. The molecules used in this degradation system are also called endosome microautophagy-targeting chimeras (eMIATACs).

[0048] Example 4: ASV regulates KH-A to target the degradation of endogenous AKT1 and pAKT

[0049] AKTin is a short peptide that can specifically bind to AKT, and 2xAKTin has been shown to have higher AKT binding efficiency.

[0050] To verify that this system can be used to degrade endogenous oncogenic proteins, the C-terminus of the KH recombinant sequence was ligated to a 2xAKTin sequence to obtain a recombinant plasmid. The recombinant KH-2xAKTin (KH-A) or mKH-2xAKTin (mKH-A) plasmid, psPAX2, and pMD2.G were transfected into 293T cells using a 2:1.5:1 transient co-transfection method to package recombinant lentivirus. The recombinant lentivirus secreted by 293T cells was collected, filtered through a 0.22 μm filter membrane, and then used to infect 293T cells. After selection with high concentrations of puromycin (5 μg / mL) for 2-7 days, the concentration was replaced with a maintenance concentration (1 μg / mL) of puromycin for continued culture. Then, after treatment with DMSO and ASV for 24 h, total protein was extracted. Changes in AKT1 and pAKT protein levels were detected by proteoblotting. The results are shown below. Figure 10 As shown.

[0051] MDA-MB-231 cells stably overexpressing KH-A or mKH-A were constructed using the same method as the previously described construction of the 293T stable cell line. Stable 293T cells and MDA-MB-231 cells overexpressing KH-A or mKH-A were seeded at 2000 cells per well, with three replicates per group, in 96-well plates. After 24 hours, cell adhesion was achieved. The culture medium was then replaced with DMEM containing 10 μM ASV. CCK8 reagent was added at 0, 1, 2, 3, 4, and 5 days to detect cell viability and cell count. The results are as follows: Figure 11 As shown.

[0052] from Figure 10 , Figure 11 It can be seen that Western blotting analysis revealed that AKT and pAKT could be targeted and degraded to a certain extent after ASV treatment, and cell proliferation experiments also confirmed that the system could effectively inhibit the proliferation activity of 293T cells and human breast cancer cells MDA-MB-231 after degrading endogenous AKT and pAKT.

[0053] Example 5: Establishment of an orthotopic breast cancer model in nude mice

[0054] Recombinant KH-A or mKH-A plasmid, psPAX2, and pMD2.G (psPAX2 and pMD2.G are lentiviral packaging plasmids) were transfected into 293T cells using a 2:1.5:1 transient co-transfection method to obtain recombinant lentivirus. The recombinant lentivirus secreted by 293T cells was collected, filtered through a 0.22 μm filter membrane, and then used to infect MDA-MB-231 (MB-231) cells. After selection with Puromycin, stable MB-231-KH-A or MB-231-mKH-A cell lines were obtained and used to construct a nude mouse breast cancer model. The specific construction method is as follows:

[0055] (1) Take MB-231-KH-A or MB-231-mKH-A cells, digest them with 0.25% trypsin, centrifuge at 1000 rpm for 5 min at room temperature, remove the supernatant, resuspend in sterile PBS, centrifuge and wash twice.

[0056] (2) Calculate the cell count and adjust the cell concentration to 1×10⁻⁶. 7 / mL, resuspend the cells in sterile PBS to prepare a cell suspension for injection;

[0057] (3) The mice to be tested were anesthetized with isoflurane, and the inguinal regions on both sides were disinfected with povidone-iodine. 100 μL of resuspended MB-231-mKH-A cells were subcutaneously injected into the mammary fat pad under the fourth pair of nipples on the left side of the nude mouse.

[0058] (4) Inject slowly for 30 seconds. After the injection is complete, carefully rotate the syringe half a turn, withdraw the needle, and press the injection site with a cotton swab for 20-30 seconds.

[0059] (5) After nude mice rest for half an hour after injection of mKH-A cells, 100 μL of resuspended MB-231-KH-A cells were injected subcutaneously into the mammary fat pad under the fourth pair of nipples on the right side. After the injection was completed, the needle was carefully withdrawn, the injection site was gently disinfected, and the mice were put back into the cage for observation.

[0060] (6) Four days after injection of MB-231 breast cancer cells, nude mice were given oral administration at a dose of 5 mg / kg once a day.

[0061] Result determination:

[0062] ① In vivo imaging technique: Four days after tumor formation in a subcutaneous breast cancer model, all nude mice were anesthetized with isoflurane every four days. A 1× luciferase substrate was then injected intraperitoneally at a dose equal to 1 / 100 of the mouse's body weight (200 μL for 20g mice). After 10-20 minutes of reaction, the bioluminescence intensity at the tumor site was detected using a bioluminescence imaging device. The results are as follows: Figure 12 As shown in the bioluminescence imaging intensity, under the same experimental conditions and culture environment, both breast cancers increased, but the growth rate of the left tumor (MB-231-mKH-A) was significantly higher than that of the right, indicating that the KH-A system can significantly degrade endogenous AKT1 levels and inhibit tumor growth compared to mKH-A.

[0063] ② Tumor mass and volume difference analysis: After the mammary glands of nude mice grew to 16 days, the tumors from both sides of the euthanized nude mice were removed, and the size and mass of the tumors were recorded. ImageJ was used to analyze the differences, and the results are as follows: Figure 13 As shown, from Figure 13 The results showed that the tumor mass and volume in nude mice injected with MB-231-KH-A cells were significantly smaller than those in the contralateral side, further verifying that the KH system has significant degradation efficacy in a breast cancer model in female nude mice.

[0064] In summary, the drug-controlled degradation system of this invention recombines the KFERQ sequence with the HCV-NS3 sequence and endogenous protein targeting sequences, and utilizes the self-cleavage property of the HCV-NS3 protease to regulate the degradation of endogenous proteins. This significantly reduces the levels of overexpressed EGFP and endogenous AKT and pAKT in cells, especially with KFERQ exhibiting stronger degradation efficacy at the N-terminus. This research provides a drug-controlled protein degradation system that significantly degrades intracellular EGFP and endogenous oncogenic proteins while effectively inhibiting cell proliferation, providing a basis for cancer treatment.

[0065] Finally, it should be emphasized that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A drug control system based on KFERQ sequences, characterized in that, The drug control system is composed of a KFERQ sequence, an HCV-NS3 sequence, and a protein targeting sequence, wherein the protein targeting sequence is located at the N-terminus or C-terminus of the recombinant sequence; the drug is ASV.

2. The drug control system based on KFERQ sequence according to claim 1, characterized in that, The HCV-NS3 sequence is an element composed of a cleavage site and an adjacent HCV-NS3.

3. The drug control system based on KFERQ sequence according to claim 1, characterized in that, The drug control system is a recombinant of the KFERQ-mHCV-NS3 sequence and a protein targeting sequence.

4. The drug control system based on KFERQ sequence according to claim 1, characterized in that, The protein targeting sequence is an endogenous protein sequence, which is any one of the following: an overexpressed EGFP sequence, an endogenous AKT sequence, or an endogenous pAKT sequence.

5. The method for constructing a drug control system based on KFERQ sequences as described in any one of claims 1-4, characterized in that, Includes the following steps: Using FV185 as the plasmid vector, the HCV-NS3 sequence is first recombined with the KEFRQ sequence to obtain the KFERQ-mHCV-NS3 or HCV-NS3-KFERQ recombinant sequence. Then, a protein targeting sequence is inserted into the N-terminus or C-terminus of the obtained recombinant sequence to obtain the recombinant plasmid.

6. The method for constructing a drug control system based on KFERQ sequences according to claim 5, characterized in that, When the protein targeting sequence is endogenous AKT, a GFP nanobody sequence or a 2xAKTin sequence is inserted at the C-terminus; the 2xAKTin sequence is a nucleotide sequence as shown in Seq_6.

7. The method for constructing a drug control system based on KFERQ sequences according to claim 5, characterized in that, The recombinant plasmid includes the KFERQ-mHCV-NS3-protein targeting sequence.

8. The use of a drug control system according to any one of claims 1-4 in the preparation of products that degrade exogenously overexpressed EGFP or degrade endogenous oncogenic proteins.

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