Application of small molecule peptide development based on mitochondrial membrane Atad3a-VDAC1 interaction in lung cancer treatment

Abstract

The invention discloses application of small molecule peptide development based on mitochondrial membrane Atad3a-VDAC1 interaction in lung cancer treatment, and belongs to the technical field of bioactive peptides. The inhibitory peptide has a sequence as shown in any one of SEQ ID NO.1-6. The invention finds that the expression of the mitochondrial protein Atad3a is related to the anti-PD-L1 treatment effect for the first time, the Atad3a small molecule inhibitory peptide is developed by taking Atad3a as a target spot, the Atad3a small molecule inhibitory peptide has the function of inhibiting the interaction of Atad3a and VDAC1, can be used for treating tumors such as lung cancer and the like, and can be combined with anti-PD-L1 medicines to further improve the anti-PD-L1 treatment effect.

Description

Technical Field

[0001] The present invention belongs to the technical field of bioactive peptides, and specifically relates to the development and application of small molecule peptides based on the interaction between mitochondrial membrane Atad3a and VDAC1 in the treatment of lung cancer. Background Art

[0002] According to the latest assessment data from the World Health Organization's International Agency for Research on Cancer (IARC), 19.96 million new cancer cases and 9.74 million cancer deaths were reported worldwide in 2022. Lung cancer leads in both morbidity and mortality among all cancers. Therefore, strengthening lung cancer prevention and treatment is particularly urgent.

[0003] Clinical treatments for lung cancer include surgery, chemotherapy, radiotherapy, targeted therapy, and immunotherapy. In recent years, immunotherapy, as an emerging treatment approach, has achieved certain survival benefits in multiple clinical trials for non-small cell lung cancer, demonstrating positive results through immune checkpoint inhibitors such as PD-L1 monoclonal antibodies. However, immunotherapy resistance is common, with approximately 40% to 60% of patients failing to benefit from immunotherapy or combined immunotherapy. Therefore, developing combination treatment strategies to address immunotherapy resistance is crucial for the effective treatment of lung cancer. Summary of the Invention

[0004] In response to the deficiencies in the prior art, the present invention provides an application of a small molecule peptide developed based on the interaction between mitochondrial membrane Atad3a-VDAC1 in the treatment of lung cancer. The purpose of the present invention is to provide a combined immunotherapy regimen for lung cancer, which improves the response rate of immunotherapy by targeting cancer cells and igniting the "cold" tumor microenvironment.

[0005] To achieve the above-mentioned purpose, the technical solution adopted by the present invention to solve the technical problem is: The object of the present invention is to provide an Atad3a inhibitory peptide having a sequence shown in any one of SEQ ID NOs. 1 to 6, specifically as follows: SSFSPNTGKKNAKIK(SEQ ID NO.1); PNTGKKNAKIKTGYK (SEQ ID NO. 2); GYKREHINLGCDMDF(SEQ ID NO.3); EHINLGCDMDFDIAG(SEQ ID NO.4); LGCDMDFDIAGPSIR(SEQ ID NO.5); EFGGSIYQKVNKKLE (SEQ ID NO. 6).

[0006] Another object of the present invention is to provide a composition comprising the above inhibitory peptide as an active ingredient.

[0007] Furthermore, the composition also includes pharmaceutically acceptable excipients of the above inhibitory peptide.

[0008] Another object of the present invention is to provide a preparation for inhibiting the binding of Atad3a to VDAC1, which comprises the above-mentioned inhibitory peptide or composition as an active ingredient.

[0009] Another object of the present invention is to provide use of the above inhibitory peptide or composition in the preparation of a product for preventing and / or treating tumors caused by the binding of Atad3a to VDAC1.

[0010] Furthermore, the tumor is lung cancer or non-small cell lung cancer.

[0011] Another object of the present invention is to provide a drug for treating lung cancer, which contains the above inhibitory peptide, composition or preparation as an active ingredient.

[0012] Furthermore, the dosage form of the drug includes any pharmaceutically acceptable dosage form.

[0013] Another object of the present invention is to provide a drug that can enhance the efficacy of anti-PD-L1 drugs, which contains the above-mentioned inhibitory peptide, composition or preparation as an active ingredient.

[0014] Another object of the present invention is to provide a combination drug for improving the therapeutic efficacy of lung cancer, which comprises the above-mentioned inhibitory peptide, composition or preparation, and an anti-PD-L1 drug.

[0015] Beneficial effects of the present invention: The present invention discovered for the first time that the expression of the molecular marker Atad3a is correlated with the efficacy of anti-PD-L1 therapy. By targeting Atad3a, a small molecule inhibitory peptide for Atad3a was developed to inhibit the interaction between Atad3a and VDAC1. The peptide can be used to treat tumors such as lung cancer. Moreover, it can be used in combination with anti-PD-L1 drugs to further enhance the efficacy of anti-PD-L1 therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a graph showing the correlation between Atad3a expression and the efficacy of anti-PD-L1 therapy in NSCLC patients; Figure 2 To detect the interaction between Atad3a and VDAC1; Figure 3 The expression of cGAS-STING downstream cytokines IFN-β, TNF-α, and IL-6 in A549 cells after treatment with inhibitory peptides; Figure 4Western-blot was used to detect the effect of inhibitory peptide treatment on the oligomerization of VDAC1 and BAX in A549 cells; Figure 5 q-PCR was used to detect the release of cmtDNA in A549 cells after treatment with inhibitory peptides; Figure 6 The immunoprecipitation results obtained by anti-Atad3a antibody in A549 cells treated with peptides 2, 3, and 24; Figure 7 Flow cytometry was used to detect the expression of CD274 in A549 cells after treatment with inhibitory peptides; Figure 8 Flow cytometry was used to detect the expression of CD274 in BMDMs cells co-cultured with A549 cells treated with inhibitory peptides; Figure 9 To investigate the effects of combined treatment with inhibitory peptides and PD-L1 monoclonal antibodies on tumor growth and survival in mice; Figure 10 Flow cytometry was used to detect the effects of combined treatment with inhibitory peptide and PD-L1 monoclonal antibody on infiltrating CD274 + The influence of macrophages. DETAILED DESCRIPTION

[0017] The specific embodiments of the present invention are described below to facilitate understanding of the present invention by those skilled in the art. However, it should be clear that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, as long as various changes are within the spirit and scope of the present invention as defined and determined by the appended claims, these changes are obvious, and all inventions and creations utilizing the concepts of the present invention are protected.

[0018] Example 1 Development of inhibitory peptides Through extensive screening, it was first discovered that Atad3a expression is associated with the efficacy of anti-PD-L1 therapy (see Figure 1 ), then NSCLC cells were washed and collected in PBS and incubated with the cross-linking reagent EGS (200 μM) at 30°C for 40 min. The samples were subjected to IP experiments and SDS-PAGE analysis using anti-VADC1 antibodies. It was found that Atad3a can form a complex with VDAC1 on the mitochondrial outer membrane, and the two interact ( Figure 2 ).

[0019] To disrupt the interaction between Atad3a and VDAC1, 24 peptides were designed, each containing 10 amino acids. A549 cells were treated with these peptides, and the expression of cGAS-STING downstream cytokines IFN-β, TNF-α, and IL-6 was detected. The results are shown in Figure 3 .

[0020] like Figure 3 As shown, peptides 2, 3, 6, 7, 8, and 24 among the inhibitory peptides can promote the expression of cGAS-STING downstream cytokines IFN-β, TNF-α, and IL-6, promote the oligomerization of VDAC1, and downregulate the oligomerization of BAX.

[0021] Western-blot was further used to detect the oligomerization of VDAC1 and BAX in A549 cells treated with peptides 2, 3, 6, 7, 8, and 24. The specific WB process is as follows: Non-small cell lung cancer cell lysates were prepared using RIPA lysis buffer containing protease inhibitors. Proteins were loaded onto 10% sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, USA). The membranes were incubated with Tris-buffered saline (TBS) blocking buffer containing 5% milk and subsequently incubated with primary antibodies. Results are shown in Figure 4 .

[0022] like Figure 4 As shown, treatment with peptides 2, 3, 6, 7, 8, and 24 promoted the oligomerization of VDAC1 and downregulated the oligomerization of BAX.

[0023] It was determined that peptides 2, 3, 6, 7, 8, and 24 in the inhibitory peptides can be used as small molecule peptides to inhibit the binding of Atad3a and VDAC1. Their specific sequences are as follows: Peptide 2:SSFSPNTGKKNAKIK (SEQ ID NO.1) Peptide 3:PNTGKKNAKIKTGYK (SEQ ID NO.2) Peptide 6: GYKREHINLGCDMDF (SEQ ID NO.3) Peptide 7: EHINLGCDMDFDIAG (SEQ ID NO. 4) Peptide 8: LGCDMDFDIAGPSIR (SEQ ID NO. 5) Peptide 24: EFGGSIYQKVNKKLE (SEQ ID NO. 6).

[0024] Example 2 Effects of inhibitory peptides on lung cancer cells 1. Detect the expression level of cmtDNA in A549 cells treated with peptides 2, 3, and 24 by q-PCR. The specific process is as follows: A 10 μL reaction mixture containing 2× ChamQSYBR qPCR Master mix (Vazyme, Cat#Q311-02), 10 μL of forward and reverse primers, and 4.2 μL of sample DNA was loaded onto a CFX96 Real-Time PCR Detection System (Bio-Rad). The q-PCR reaction program was as follows: denaturation at 95°C for 30 seconds, followed by denaturation at 95°C for 10 seconds, denaturation at 60°C for 30 seconds, and denaturation at 72°C for 1 minute, for a total of 40 cycles. Dissociation curves were plotted. Results are shown in the table. Figure 5 .

[0025] like Figure 5 As shown in Figure 2, q-PCR assays showed that treatment with peptides 2, 3, and 24 increased cmtDNA release. Furthermore, immunoprecipitation obtained by anti-Atad3a antibody in A549 cells treated with peptides 2, 3, and 24 (10 μM, 24 h) was detected by IP assays. Figure 6 As shown, the level of co-precipitated VDAC1 was significantly reduced compared to control cells.

[0026] 2. The expression of CD274 in A549 cells treated with peptides 2, 3, and 24 (10 μM, 24 h) was detected by flow cytometry. The results are shown in Figure 7 .like Figure 7 As shown, treatment with peptides 2, 3, and 24 upregulated the expression of CD274 in A549 cells.

[0027] 3. First, A549 cells were treated with peptides 2, 3, and 24 (10 μM, 24 h), and then the treated A549 cell culture supernatant was co-cultured with BMDMs cells. The expression of CD274 in BMDMs cells was detected by flow cytometry. The results are shown in Figure 8 .

[0028] like Figure 8 As shown in the results, peptides 2, 3, and 24 could inhibit the interaction between Atad3a and VDAC1 and promote the release of cmtDNA. In addition, the culture supernatant of A549 cells treated with peptides 2, 3, and 24 also promoted the expression of CD274 in BMDMs.

[0029] 4. Tail vein injection of LLC cell suspension (1 × 10 6An orthotopic lung cancer mouse model was established using LLC cells resuspended in 200µL PBS. For treatment experiments, mice were randomly divided into four treatment groups (n = 6 each): saline, peptide (inhibitory peptide), PD-L1 monoclonal antibody (Bioxcell Catalog No. BE0101), and peptide + PD-L1 monoclonal antibody.

[0030] Mice in the saline group received intraperitoneal injections of saline every other day. Mice in the peptide group received intraperitoneal injections of 5 mg / kg peptide on days 7, 10, and 13. Mice in the PD-L1 monoclonal antibody group received intraperitoneal injections of monoclonal antibody (200 μg / mouse) on days 8, 11, and 14. For combination therapy with peptide and antibody, mice received intraperitoneal injections of peptide (5 mg / kg) on ​​days 7, 10, and 13; and monoclonal antibody (200 μg / mouse) on days 8, 11, and 14. Mice were sacrificed by cervical dislocation on day 28.

[0031] Lung tumor tissue was dissected and cut into 1 mm 3 The fragments were digested into single cell suspensions using 1 mg / mL collagenase type IV and deoxyribonuclease I. CD274 was quantified by flow cytometry. + The mean fluorescence intensity (MFI) of macrophages was shown in Figure 2. Figure 9 and Figure 10 .

[0032] like Figure 9 and Figure 10 As shown in the results, peptide alone had a certain inhibitory effect on tumor growth, while peptide combined with anti-PD-L1 treatment could significantly inhibit tumor growth and prolong the survival of mice. + The number of macrophages also increased significantly.

[0033] Finally, it should be noted that the above specific implementation methods are only used to illustrate the technical solutions of the present invention and are not limiting. Although the present invention has been described in detail with reference to examples, those skilled in the art should understand that the technical solutions of the present invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. An Atad3a inhibitory peptide, characterized in that: The inhibitory peptide has a sequence shown in any one of SEQ ID NOs. 1 to 6.

2. A composition, characterized in that The composition contains the inhibitory peptide according to claim 1 as an active ingredient.

3. The composition according to claim 2, characterized in that The composition further comprises the inhibitory peptide according to claim 1 in a pharmaceutically acceptable excipient.

4. A preparation for inhibiting the binding of Atad3a and VDAC1, characterized in that The preparation contains the inhibitory peptide according to claim 1, or the composition according to claim 2 or 3 as an active ingredient.

5. Use of the inhibitory peptide according to claim 1, the composition according to claim 2 or 3, or the preparation according to claim 4 in the preparation of a product for preventing and / or treating tumors caused by the binding of Atad3a to VDAC1.

6. The use according to claim 4, characterized in that The tumor is lung cancer or non-small cell lung cancer.

7. A drug for treating lung cancer, characterized in that: The drug contains the inhibitory peptide according to claim 1, the composition according to claim 2 or 3, or the preparation according to claim 4 as an active ingredient.

8. The drug according to claim 7, characterized in that The dosage form of the drug includes any pharmaceutically acceptable dosage form.

9. A drug capable of enhancing the efficacy of anti-PD-L1, characterized in that: The drug contains the inhibitory peptide according to claim 1, the composition according to claim 2 or 3, or the preparation according to claim 4 as an active ingredient.

10. A combined drug for improving the therapeutic effect of lung cancer, characterized in that: It comprises the inhibitory peptide according to claim 1, the composition according to claim 2 or 3 or the preparation according to claim 4, and an anti-PD-L1 drug.

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