Drug oligopeptide for assisting targeted therapy, composition and application

Abstract

The invention relates to a drug oligopeptide assisting in targeted therapy, a composition and application, and relates to the technical field of biological medicine, and the amino acid sequence of the drug oligopeptide is shown as SEQ ID NO: 1 or SEQ ID NO: 2. According to the oligopeptide disclosed by the invention, the S1070 site on the EGFR is subjected to binding, blocking and inhibiting, the binding of the tail part of the C tail end of the EGFR with NEK7, P85 and GRB2 is inhibited, and the activation of a downstream signal channel is blocked, so that the sensitivity degree of liver cancer cells to the lenvatinib is promoted; aiming at a liver cancer patient with drug resistance of the lenvatinib, the sensitive degree of liver cancer cells to the lenvatinib is effectively promoted through combined use of the lenvatinib and the oligopeptide disclosed by the invention, and the curative effect of the lenvatinib is improved.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly relates to a drug short peptide, a composition and an application for assisting targeted therapy. Background Art

[0002] In liver cancer, inhibiting tyrosine kinase (TK) through targeting is a very important first-line treatment method. Among them, lenvatinib (Lenvatinib Mesilate), as a first-line drug, is widely used clinically. However, some patients will show characteristics of resistance to lenvatinib, and the response rate of lenvatinib in clinical treatment experiments is 20%. Lenvatinib resistance is a pain point in the treatment of liver cancer.

[0003] NIMA (never in mitosis A)-related kinase 7 (NEK7), as a drug resistance-related gene, epidermal growth factor receptor (EGFR) signaling plays a crucial role in regulating the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase / protein kinase B (PI3K / AKT) signaling pathways [Lemmon, M. A., Schlessinger, J. & Ferguson, K. M. The EGFR family: not so prototypical receptor tyrosine kinases. Cold Spring Harb Perspect Biol 6, a020768 (2014). Endres, N. F., Engel, K., Das, R., Kovacs, E. & Kuriyan, J. Regulation of the catalytic activity of the EGF receptor. Curr Opin Struct Biol 21, 777-784 (2011). Avraham, R. & Yarden, Y. Feedback regulation of EGFR signalling: decision making by early and delayed loops. Nature Reviews Molecular Cell Biology 12, 104-117 (2011). Cantor, A. J., Shah, N. H. & Kuriyan, J. Deep mutational analysis reveals functional trade-offs in the sequences of EGFR autophosphorylation sites. Proc Natl Acad Sci U S A 115, E7303-E7312 (2018).].After phosphorylation occurs at the C-terminal tail of EGFR, growth factor receptor-bound protein 2 (GRB2) can bind to it through its SH2 domain, subsequently recruiting SOS protein and activating the MAPK signaling pathway [Boykevisch, S. et al. Regulation of rassignaling dynamics by Sos-mediated positive feedback. Curr Biol 16, 2173-2179 (2006). Lin, C. W. et al. A two-component protein condensate of the EGFR cytoplasmic tail and Grb2 regulates Ras activation by SOS at the membrane. Proc Natl Acad Sci U S A 119, e2122531119 (2022).]. It has been reported that the SH2 domain of P85 (the regulatory subunit of PI3K) can bind to the EGFR complex and relieve the inhibitory contact on P110 (the catalytic subunit of PI3K), enabling the catalytic site of P110 to access the substrate and receive further activation signals, thereby activating the PI3K / AKT signaling pathway [Suenaga, A. et al. Novel mechanism of interaction of p85 subunit of phosphatidylinositol 3-kinase and ErbB3 receptor-derived phosphotyrosyl peptides. J Biol Chem 280, 1321-1326 (2005). Hu, P. et al. Interaction of phosphatidylinositol 3-kinase-associated p85 with epidermal growth factor and platelet-derived growth factor receptors. Molecular and Cellular Biology 12, 981-990 (1992). Hennessy, B. T., Smith, D. L., Ram, P. T., Lu, Y. & Mills, G. B. Exploiting the PI3K / AKT pathway for cancer drug discovery. Nat Rev Drug Discov 4, 988-1004 (2005).].

[0004] At present, many short peptides have been found to bind to important proteins, thereby inhibiting the activation of downstream proteins. However, there are no drugs or blocking peptides targeting the C-terminal tail of EGFR on the market. In view of this, the present invention provides a drug short peptide, a composition and an application for adjuvant targeted therapy. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a drug short peptide, a composition and an application for adjuvant targeted therapy. The purpose is to bind and block the S1070 site on EGFR, inhibit the binding of the C-terminal tail of EGFR (the fragment after 979aa) to NEK7, P85, and GRB2, block the activation of downstream signal pathways, thereby promoting the sensitivity of liver cancer cells to lenvatinib and increasing the efficacy of lenvatinib.

[0006] The technical solution of the present invention to solve the above technical problems is as follows:

[0007] In the first aspect, a drug short peptide for adjuvant targeted therapy, the amino acid sequence of the drug short peptide is as shown in SEQ ID NO: 1 or SEQ ID: 2.

[0008] On the basis of the above technical solution, the present invention can also be improved as follows.

[0009] In the second aspect, a nucleic acid encodes the drug short peptide for adjuvant targeted therapy.

[0010] In the third aspect, a drug composition includes the drug short peptide for adjuvant targeted therapy.

[0011] Further, the drug composition further includes lenvatinib.

[0012] In the fourth aspect, an application of a drug composition is to use the drug composition to prepare a drug for treating liver cancer.

[0013] Further, the drug short peptide in the drug composition blocks the binding of the S1070 site on EGFR, inhibits the binding of the C-terminal tail of EGFR to NEK7, and / or P85, and / or GRB2, so as to block the activation of downstream signal pathways, thereby promoting the sensitivity of liver cancer cells to lenvatinib.

[0014] In the fifth aspect, a drug for treating liver cancer includes the drug short peptide for adjuvant targeted therapy and lenvatinib.

[0015] Further, the drug for treating liver cancer further includes pharmaceutically acceptable excipients.

[0016] Furthermore, the adjuvant includes DMSO.

[0017] Furthermore, the dosage form of the anti-hepatocellular carcinoma drug includes an injection.

[0018] The beneficial effects of the present invention are as follows: The short peptide of the present invention inhibits the binding of the C-terminal tail (the fragment after 979aa) of EGFR to NEK7, P85, and GRB2 by binding and blocking the S1070 site on EGFR, blocking the activation of the downstream signaling pathway, thereby promoting the sensitivity of hepatocellular carcinoma cells to lenvatinib; for hepatocellular carcinoma patients resistant to lenvatinib, by jointly using lenvatinib and the short peptide of the present invention, it effectively promotes the sensitivity of hepatocellular carcinoma cells to lenvatinib and increases the efficacy of lenvatinib. Description of the Drawings

[0019] Figure 1 It is a schematic diagram of the screening process of the short peptide of the drug for adjuvant targeted therapy of the present invention;

[0020] Figure 2 It is a verification diagram of the present invention that can be verified at the tumor cell level experiment;

[0021] Figure 3 It is a verification diagram of the present invention that can inhibit the proliferation, invasion and metastasis ability of tumor cells; among them, A is cell proliferation and cytotoxicity (CCK-8 method) to verify the ability of the short peptide to inhibit tumor proliferation; B is Transwell migration or invasion assay to verify the ability of the short peptide to inhibit tumor invasion and metastasis; C is the statistical chart of the results of the Transwell migration or invasion assay experiment;

[0022] Figure 4 It is a verification diagram of the present invention in the hepatocellular carcinoma organoid model; among them, A is the representative diagram before and after using the drug; B is the statistical chart of cell viability in the organoid with short peptide 10 alone; C is the statistical chart of cell viability in the organoid with the combination of short peptide 10 and lenvatinib;

[0023] Figure 5 It is a verification diagram of the present invention by Surface Plasmon Resonance (SPR);

[0024] Figure 6 It is an experimental diagram of the present invention verifying its ability to inhibit the proliferation of drug-resistant hepatocellular carcinoma in a mouse in vivo model; among them, A is the representative picture of all tumor masses; B is the statistical chart of tumor mass volume; C is the statistical chart of tumor mass weight. Detailed Embodiments

[0025] The principles and features of the present invention are described below. The examples given are only used to explain the present invention and are not intended to limit the scope of the present invention. For those without specific technologies or conditions indicated in the examples, they shall be in accordance with the technologies or conditions described in the literature in this field or in accordance with the product specifications. For the reagents or instruments without the manufacturer indicated, they are all conventional products that can be purchased through regular channels.

[0026] Example

[0027] 1. Screening of drug peptides for adjuvant targeted therapy.

[0028] In this example, the protein structure of amino acid residues 979 - 1099 in the epidermal growth factor receptor (EGFR) protein was obtained from the AlphaFold2 database. At the same time, the structure of the NIMA (never in mitosis A)-related kinase 7 (NEK7) protein was also obtained from this database. Through the analysis of the NEK7 protein structure, polypeptide sequences were extracted based on the characteristics of the protein secondary structure, and polypeptides of 10 - 15 amino acids were constructed.

[0029] The binding situation between the constructed amino acid polypeptides and the 979 - 1099 sequence structure of the EGFR protein was analyzed. The above - constructed polypeptides were docked through the HPEPDOCK 2.0 Server, and the top 4 polypeptide structures before scoring were obtained (Table 1), and the possible binding sites of the EGFR protein were obtained.

[0030] Table 1 Polypeptide sequences

[0031]

[0032] Furthermore, the FlexPepDock and InterfaceAnalyzer modules of the Rosetta 3.12 software were used to perform more sampling, scoring, and analysis on the 5 obtained polypeptides. Finally, 2 polypeptides (Pep_1 and Pep_10) with reasonable structures were obtained ( Figure 1 )

[0033] Among them, Pep_1: KRMHACTASS;

[0034] Pep_10: NFRIEKKIGR.

[0035] Since the binding site of the constructed polypeptide to the protein structure of amino acid residues 979 - 1099 in the EGFR protein is inside the cell membrane, in order to allow the polypeptide to better enter the membrane, in this embodiment, the TAT sequence is added to these two polypeptides to promote the entry of the short peptide into the cell membrane, namely Pep_1 (SEQ ID NO: 1): YGRKKRRQRRRAKRMHACTASS; Pep_10 (SEQ ID NO: 2): YGRKKRRQRRRANFRIEKKIGR. The two fragments are synthesized by amino acid short peptide synthesis, and the synthesis is outsourced to a third-party company. The steps of solid-phase peptide synthesis (SPPS) are as follows:

[0036] (1) Preparation of the solid-phase carrier:

[0037] The carboxyl group of the first amino acid of the target peptide is covalently bound to the solid-phase carrier (usually resin). Commonly used solid-phase carriers include Rink Amide MBHA resin, etc.

[0038] (2) Deprotection:

[0039] An alkaline solvent (such as piperidine) is used to remove the amino protecting group (such as Fmoc) of the first amino acid. This step ensures that the amino group can participate in subsequent condensation reactions.

[0040] (3) Activation and crosslinking:

[0041] The carboxyl group of the next amino acid is activated with an activator (such as DCC, HATU, etc.) to make it reactive. The activated amino acid reacts with the deprotected amino group in the previous step to form a peptide bond.

[0042] (4) Washing and neutralization:

[0043] After the reaction is completed, the reaction system is washed with an appropriate solvent (such as DMF) to remove unreacted activators and by-products. Then, the reaction system is neutralized with an alkaline solution (such as piperidine) to prepare for the next round of reaction.

[0044] (5) Repeat the operation:

[0045] Repeat the above deprotection, activation and crosslinking steps to gradually increase the length of the polypeptide chain until the desired polypeptide sequence is synthesized.

[0046] (6) Cleavage and purification:

[0047] After the synthesis is completed, the polypeptide is cleaved from the solid-phase carrier with a cleavage agent (such as TFA), and all protecting groups are removed. Finally, the polypeptide is purified by methods such as high performance liquid chromatography (HPLC).

[0048] 2. Conduct experimental tests on lenvatinib-resistant hepatocellular carcinoma cell lines and human organoids.

[0049] 2.1 Experimental methods.

[0050] In the lenvatinib-resistant Hep3B cell line: The Hep3B cell line (China Center for Type Culture Collection (CCTCC)) was cultured for 6 months with increasing doses of lenvatinib (Selleck) at different concentrations ranging from 3 μM to 30 μM.

[0051] Human organoids of hepatocellular carcinoma patients (Collector: Huang Qibo, Time: 2021 - 2022, Location: Surgical Building of Tongji Hospital): After tumor resection, the hepatocellular carcinoma patients' samples were cultured (Ethical number: TJ - IRB20211163). Peptide 10 was added according to the concentration gradient (10 μM, 20 μM, 40 μM), and after treatment with combined use of peptide 10 (3 μM) and lenvatinib (10 μM), experiments were carried out according to different experimental procedures.

[0052] 2.2 Experimental results.

[0053] Experiments were conducted on lenvatinib-resistant hepatocellular carcinoma cell lines and human organoids of patients. The results showed that Pep_10 most significantly inhibited the activation of downstream MAPK and PI3K / AKT signaling pathways. After combination with lenvatinib, it increased the drug sensitivity of hepatocellular carcinoma cells and produced a significant hepatocellular carcinoma cell killing effect (10 μM) ( Figures 2 to 4 )

[0054] Among them, Figure 2 This is the experimental verification figure for the present invention to verify that it can be experimentally verified at the tumor cell level; inhibiting the binding of the C-terminal tail of EGFR (the fragment after 979aa) to NEK7, P85, and GRB2, thereby inhibiting the activation of the EGFR downstream pathway, and the experimental figure showing that the effect of peptide 10 is better than that of peptide 1.

[0055] Figure 3 This is the experimental verification figure for the present invention to verify its ability to inhibit tumor cell proliferation and invasion and metastasis; among them, A is cell proliferation and cytotoxicity (CCK - 8 method) to verify the ability of the peptide to inhibit tumor proliferation; B is Transwell migration or invasion assay to verify the ability of the peptide to inhibit tumor invasion and metastasis; C is the statistical chart of the results of the Transwell migration or invasion assay experiment;

[0056] Figure 4Verification diagram of the present invention in a hepatocellular carcinoma organoid model; wherein, A is a representative diagram before and after using the drug; B is a statistical chart of cell viability in organoids with only peptide 10; C is a statistical chart of cell viability in organoids with combined use of peptide 10 and lenvatinib.

[0057] 3. SPR experiment.

[0058] 3.1 SPR experimental method.

[0059] (1) Amine coupling of M40623:

[0060] The experiment used 1×HBS-EP+ (pH 7.4) buffer as the running buffer. Referring to the amine coupling kit, M40623 was coupled to channel 3 of the CM5 chip at a flow rate of 10 μL / min. First, the chip surface was activated with freshly prepared 1:1 50 mM N-Hydroxysuccinimide (NHS) and 200 mM 1-Ethyl-3(3-Dimethylaminopropy)CarbodiimidHydrochloride (EDC) for 420 s; then M40623 was diluted to 50 μg / mL with 10 mM sodium acetate at pH 4.0 and flowed through channel 3 of the chip for 900 s; finally, it was blocked with Ethanolamine-HCl for 420 s. The coupling level of M40623 on channel 3 was approximately 2600 RU.

[0061] (2) Solvent calibration:

[0062] Solutions containing 4% DMSO, 5% DMSO, and 6% DMSO were prepared respectively using PBST (0.05% P20) solution (Table 2), and then the 4% and 6% DMSO solutions were used to prepare a solvent calibration curve solution according to the ratio (unit: μL).

[0063] Table 2 Preparation of solvent calibration curve

[0064]

[0065] (3) Binding experiment:

[0066] When analyzing the interaction between proteins and small molecules, PBST (0.05% P20, 5% DMSO) solution was used as the running and dilution buffer. First, the small molecule was dissolved in DMSO to prepare a small molecule solution with a concentration of 10 mM, and then the solution was diluted 20-fold with PBST (0.05% P20) so that the content of DMSO in the solution was 5%. After that, gradient dilution was carried out using the same dilution buffer containing 5% DMSO. During the experiment run, channel 1 was used as the reference channel and channel 3 was used as the active channel. The diluted small molecules were flowed through channels 1, 2, 3, and 4 in sequence according to the concentration gradient, and the multi-cycle kinetic mode was adopted, as shown in Tables 3 and 4.

[0067] Table 3 Detection settings for the binding kinetics of protein and small molecule

[0068]

[0069] Table 4 Detection parameters for the binding kinetics of protein and small molecule

[0070]

[0071] (4) Data analysis:

[0072] The data was analyzed using the Biacore T200 analysis software (version 3.1), the reference channel and zero-concentration background signals were subtracted, and the 1:1 binding analysis model was selected.

[0073] 3.2 Experimental results.

[0074] Through the Surface Plasmon Resonance (SPR) experiment, it was found that there was a direct binding relationship between Pep_10 and the intracellular segment (669 - 1210 aa) of EGFR, further verifying the feasibility that Pep_10 blocks the S1070 site on EGFR, inhibits the binding of EGFR to NEK7, P85, and GRB2, and blocks the activation of downstream signaling pathways ( Figure 5 ).

[0075] 4. Tumor-bearing experiment.

[0076] 4.1 Test method.

[0077] The Hep3B lenvatinib-resistant line in the logarithmic growth phase was digested with trypsin, the supernatant was removed by centrifugation, and the cells were resuspended with PBS and the cell concentration was adjusted to 2×10 6 cells / ml. Injection was carried out under the armpits of BALB / c mice (Beijing Vital River Laboratory Animal Technology Co., Ltd.). Starting from the 8th day after inoculation, the longest diameter (a) and the shortest diameter (b) of the tumor were measured every 1 - 3 days using a vernier caliper, and the tumor volume was calculated (V = a × b × 0.5 × b mm3 )。

[0078] On the 11th day, the mice were grouped and given different drugs, including a placebo group, a lenvatinib group (30 mg / kg / d), a short peptide group (3 mg / mL, 100 μL), and a combination drug group (lenvatinib, 10 mg / kg, short peptide 10, 1 mg / mL, 100 μL). At the end of the experiment, the mice were sacrificed, and the tumor tissues were removed for volume measurement and weight detection.

[0079] 4.2 Experimental results.

[0080] Hep3B lenvatinib-resistant subcutaneous tumors were implanted in nude mice and treated with Pep_10 (1 mg / mL, 100 μL) and lenvatinib (10 mg / kg). It was found that after using Pep_10, the drug sensitivity of the lenvatinib-resistant line of hepatocellular carcinoma cells to lenvatinib was increased ( Figure 6 from A to C in).

[0081] In summary, the present invention first discovered the EGFR S1070 site, the activation of the downstream MAPK and PI3K / AKT signaling pathways, which promoted the resistance of hepatocellular carcinoma cells to lenvatinib. At the same time, through molecular simulation, it was found that Pep_10 (the sequence is shown in SEQ ID NO: 2) binds to and blocks the S1070 site on EGFR to inhibit the binding of the C-terminal tail (979-1210 aa) of EGFR to NEK7, P85, and GRB2, thereby blocking the activation of the downstream signaling pathway.

[0082] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention.

Claims

1. A short peptide drug for auxiliary targeted therapy, characterized in that: The amino acid sequence of the drug short peptide is shown as SEQ ID NO: 1 or SEQ ID:

2.

2. A nucleic acid, characterized in that The nucleic acid encodes a short peptide drug for auxiliary targeted therapy as described in claim 1.

3. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the short peptide drug for auxiliary targeted therapy as claimed in claim 1.

4. A pharmaceutical composition according to claim 3, characterized in that: The pharmaceutical composition also includes lenvatinib.

5. Use of a pharmaceutical composition, characterized in that: The pharmaceutical composition according to claim 3 or 4 is used to prepare an anti-liver cancer drug.

6. The use of a pharmaceutical composition according to claim 5, characterized in that: The drug short peptide in the pharmaceutical composition blocks the binding to the S1070 site on EGFR, inhibits the binding of the C-terminal tail of EGFR with NEK7, or / and P85, or / and GRB2, thereby blocking the activation of downstream signaling pathways, thereby promoting the sensitivity of liver cancer cells to lenvatinib.

7. An anti-liver cancer drug, characterized in that: The anti-liver cancer drug includes the short peptide drug for auxiliary targeted therapy as described in claim 1 and lenvatinib.

8. The anti-liver cancer drug according to claim 7, characterized in that: The anti-liver cancer drug also includes pharmaceutically acceptable excipients.

9. The anti-liver cancer drug according to claim 8, characterized in that: The auxiliary material includes DMSO.

10. The anti-liver cancer drug according to claim 7, characterized in that: The dosage form of the anti-liver cancer drug includes injection.

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