An eif4e / eif4g interaction inhibitor and uses thereof
By designing sulfonyl-γ-AA peptides to mimic the binding sequences of eIF4G and 4E-BP1, the problem of low affinity of existing inhibitors was solved, achieving a potent inhibition of tumor cell translation and proliferation, providing a new approach for anti-tumor drug development.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- ZHEJIANG UNIV
- Filing Date
- 2026-03-12
- Publication Date
- 2026-06-12
AI Technical Summary
Developing existing eIF4E/eIF4G interaction inhibitors is difficult, especially since small molecule eIF4E inhibitors have low affinity and may produce off-target effects, resulting in poor peptide cellular activity and difficulty in effectively inhibiting cap-dependent translation.
The sulfonyl-γ-AA peptide was designed to mimic the conserved binding sequences of eIF4G and 4E-BP1. By linking the cell-penetrating peptide and the lysosomal sorting sequence, it enters the cell to inhibit the eIF4E/eIF4G interaction, thereby inhibiting tumor cell proliferation and mediating programmed cell death.
It exhibits strong translational inhibition ability in vitro, effectively enters cells to inhibit tumor cell proliferation and induce programmed cell death, providing a new approach for anti-tumor drug development.
Smart Images
Figure FT_1 
Figure FT_2 
Figure FT_3
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biomedical technology, specifically relating to an eIF4E / eIF4G interaction inhibitor and its application. Background Technology
[0002] The initiation phase of protein translation is the most crucial, serving as both a major target for intracellular regulation and the rate-limiting step in the entire translation cascade. Most eukaryotes employ a cap-dependent translation initiation mechanism, which begins with the binding of eIF4E to the 7-methylguanine nucleotide (m2) at the 5' end of mRNA. 7 The eIF4E protein binds to the cap structure of GpppN, subsequently assembling with eIF4G (a scaffold protein) and eIF4A (an ATP-dependent helicase) to form the eIF4F complex, thereby recruiting ribosomes and initiating the translation process. The interaction between eIF4E and eIF4G bridges the gap between mRNA and ribosomes, becoming a key step in cap-dependent translation initiation in eukaryotes. Increased eIF4E expression enhances the translation of mRNAs associated with malignant tumors, including proto-oncogenes (c-myc, cyclin D1, ODC, survivin), angiogenic factors (such as FGF2 and VEGF), and degradative enzymes (such as MMP9). Therefore, the expression level of eIF4E affects the transformation of normal cells into cancer cells and participates in tumorigenesis, metastasis, and drug resistance formation. Studies have shown that eIF4E is highly expressed in prostate cancer, breast cancer, head and neck tumors, gastric cancer, colon cancer, lung cancer, skin cancer, esophageal cancer, bladder cancer, cervical cancer, and hematopoietic malignancies, making it an important target for anti-tumor drug research.
[0003] Blocking the eIF4E / eIF4G interaction is an effective strategy for inhibiting cap-dependent translation, but the development of eIF4E / eIF4G protein interaction inhibitors is currently particularly difficult. Most reported attempts focus on small molecule inhibitors of eIF4E. However, X-ray diffraction analysis of the binding between eIF4E and 4E-BP1 shows a relatively flat binding interface with no favorable sites for small molecule binding. This results in low affinity for eIF4E small molecule inhibitors and potential off-target effects. While peptides are ideal molecules for regulating protein-protein interactions (PPIs), their cellular activity is generally poor. Summary of the Invention
[0004] The purpose of this invention is to provide an eIF4E / eIF4G interaction inhibitor that mimics the conserved binding sequence of eIF4G and 4E-BP1, exhibits strong translational inhibition ability in vitro, and can effectively enter cells and inhibit intracellular translation by linking cell-penetrating peptides and lysosomal sorting sequences, thereby inhibiting tumor cell proliferation and mediating programmed cell death.
[0005] This invention provides a sulfonyl-γ-AA peptide having the following general formula; ; Where n = 0 or 1, and R1 represents a hydrogen atom, an acetyl group, or one of the following structural formulas: R2 represents one of ethyl, 2-aminoethyl, or carboxymethyl; R3 represents one of 4-aminobutyl, 4-guanidinobutyl, and 3-guanidinopropyl; R4 represents either 2-aminoethyl or ethyl; R5 represents one of benzyl or phenethyl; R6 represents one of isobutyl or 2-methylthioethyl; R7 represents one of 2-aminoethyl or ethyl; R8 represents one of (R)-benzyl or (L)-benzyl; R9 represents either ethyl or isobutyl; R10 represents a hydrogen atom or one of the following structural formulas: .
[0006] Preferably, R1 represents a hydrogen atom or the following structural formula; .
[0007] R2 represents ethyl or 2-aminoethyl; R3 represents 4-aminobutyl, 4-guanidinobutyl or 3-guanidinopropyl; R4 represents 2-aminoethyl; R5 stands for benzyl; R6 represents isobutyl or 2-methylthioethyl; R7 represents 2-aminoethyl; R8 represents (R)-benzyl; R9 represents isobutyl; R10 represents a hydrogen atom or one of the following structural formulas; , .
[0008] Preferably, it includes the following structural formula; , , , , , , .
[0009] The present invention provides an eIF4E / eIF4G interaction inhibitor comprising the sulfonyl-γ-AA peptide.
[0010] The present invention provides an anticancer drug comprising the sulfonyl-γ-AA peptide or the eIF4E / eIF4G interaction inhibitor.
[0011] Preferably, pharmaceutically acceptable excipients are also included.
[0012] This invention provides the use of the sulfonyl-γ-AA peptide or the eIF4E / eIF4G interaction inhibitor in the preparation of antitumor drugs for prevention and / or treatment.
[0013] Preferably, the antitumor effect includes inhibiting the translation level and proliferation of tumor cells and promoting programmed cell death of tumor cells.
[0014] Preferably, the tumor cells include at least one of HCT116, MCF7, and MDA-MB-231.
[0015] Preferably, the tumor includes at least one of the following: colon cancer, breast cancer, prostate cancer, head and neck tumors, stomach cancer, lung cancer, skin cancer, esophageal cancer, bladder cancer, cervical cancer, and hematopoietic system malignancies.
[0016] The sulfonyl-γ-AA peptide provided by this invention offers a series of peptide-mimicking inhibitors based on the sulfonyl-γ-AA peptide-mimicking backbone that inhibit the interaction of eIF4E / eIF4G. These molecules successfully mimic the conserved binding sequences of eIF4G and 4E-BP1, exhibiting strong translational inhibition capabilities in vitro. By linking cell-penetrating peptides and lysosomal sorting sequences, they can effectively enter cells and inhibit intracellular translation, thereby inhibiting tumor cell proliferation and mediating programmed cell death (apoptosis and ferroptosis), thus providing a new approach for the development of anti-tumor drugs. Attached Figure Description
[0017] Figure 1 The diagram shows the design principle of the eIF4E / eIF4G protein interaction inhibitor; a is the co-crystal structure of eIF4G and eIF4E; b is the right-handed helical structure in the D-sulfonyl-γ-AA peptide. Figure 2 Synthetic route 1 for sulfonyl-γ-AA peptide structural units B1-B5; Figure 3 Synthetic route 2 for sulfonyl-γ-AA peptide structural units B6-B9; Figure 4 Synthetic route 3 for sulfonyl-γ-AA peptide structural units B10-B14; Figure 5 Synthetic route 4 for sulfonyl-γ-AA peptide structural unit B15; Figure 6 Synthetic route 5 for sulfonyl-γ-AA peptide 1A; Figure 7 Characterization results for sulfonyl-γ-AA peptide 11; Figure 8 Characterization results for sulfonyl-γ-AA peptide 12; Figure 9 Characterization results for sulfonyl-γ-AA peptide 13; Figure 10 Characterization results for sulfonyl-γ-AA peptide 15; Figure 11 The results show the inhibition of in vitro translation levels of 15 sulfonyl-γ-AA peptides; Figure 12 The results of circular dichroism analysis of 15 sulfonyl-γ-AA peptides; Figure 13 Synthetic route 6 for CPP-sulfonyl-γ-AA peptide 16; Figure 14 Characterization results for CPP-sulfonyl-γ-AA peptide 16; Figure 15 Characterization results for CPP-sulfonyl-γ-AA peptide 17; Figure 16 Characterization results for CPP-sulfonyl-γ-AA peptide 18; Figure 17 Characterization results for CPP-sulfonyl-γ-AA peptide 19; Figure 18 Synthetic route 7 for intermediate Mc-Val-Cit-PABC-11; Figure 19 Synthetic route 8 for CPP-LSS-sulfonyl-γ-AA peptide 20 Figure 20 Characterization results for CPP-LSS-sulfonyl-γ-AA peptide 20; Figure 21 The inhibitory effect of CPP-sulfonyl-γ-AA peptide 16 to CPP-LSS-sulfonyl-γ-AA peptide 20 on the mRNA translation level in colorectal cancer cells HCT116 was investigated. Figure 22 CPP-sulfonyl-γ-AA peptide 17 exhibits proliferative inhibitory effects on various tumor cells. Figure 23 CPP-LSS-sulfonyl-γ-AA peptide 20 exhibits proliferative inhibitory effects on various tumor cells. Figure 24 The inhibitory effect of CPP-LSS-sulfonyl-γ-AA peptide 20 on the proliferation of HCT116 cells; Figure 25 The inhibitory effect of CPP-LSS-sulfonyl-γ-AA peptide 20 on the proliferation of DLD-1 cells; Figure 26 The results show the effect of CPP-LSS-sulfonyl-γ-AA peptide 20 on the inhibition of HCT116 proliferation by inducing ferroptosis. Figure 27 The effect of CPP-LSS-sulfonyl-γ-AA peptide 20 on apoptosis in HCT116 cells. Detailed Implementation
[0018] The eutectic structure of eIF4G and eIF4E is as follows: Figure 1As shown in Figure a, only nine amino acid residues (Asp 613–Gln 621) in the conserved binding sequence of eIF4G bind to the conserved hydrophobic region on the dorsal surface of eIF4E using an α-helix structure, while in 4E-BP1, these correspond to amino acid residues Asp 55–Arg 63. The residues at LΦ in the corresponding shared sequence YXXXXLΦ form similar hydrophobic interactions with V69, W73, and L135 residues in eIF4E; the shared Tyr(Y) residue interacts with V69 residue on eIF4E via van der Waals forces, while the phenolic hydroxyl group forms hydrogen bonds with the conserved backbone of the eIF4E sequence H37-P38-L39. The typical α-helix sequences of eIF4G and 4E-BP1 contain Arg / Gln / Lys residues at positions 2 and 9, allowing the shared binding sequence to be extended to YX(R / K)X2LΦX2(R / K / Q). These residues facilitate interaction with eIF4E, possibly by protecting the hydrophobic regions on the eIF4E surface from solvent exposure. Furthermore, the Arg residue at position 2 further stabilizes the binding of the typical helical structure by forming a salt bridge with the acidic residue E132 in eIF4E; while the Arg residue at position 9 on 4E-BP1 can interact with the N77 residue on eIF4E. Therefore, this invention designs novel compounds to inhibit the eIF4E / eIF4G protein interaction by designing peptide-like molecules capable of targeting the binding sites on eIF4E. This invention utilizes the α-helical mimicry ability of sulfonyl-γ-AA peptides, employing D-sulfonyl-γ-AA structural units to construct peptides that mimic a right-handed helical structure. In the D-sulfonyl-γ-AA peptide, chiral side chains 2a and 4a are on the same face of the helical scaffold, and sulfonyl side chains 3b and 5b are located on adjacent faces (e.g., ...). Figure 1 As shown in b), these side chains were selected to mimic residues at R614, L617, L618, and Q621 in eIF4G. For highly conserved Tyr residues, chiral side chain 1a was used for mimicry. For residues that do not directly participate in binding (such as E615 and G619 in eIF4G and K57 and E61 in 4E-BP1), positively or negatively charged hydrophilic side chains were used to improve the solubility and cell permeability of the peptide mimicry.
[0019] This invention provides a sulfonyl-γ-AA peptide having the following general formula; ; Where n = 0 or 1, and R1 represents a hydrogen atom, an acetyl group, or one of the following structural formulas: R2 represents one of ethyl, 2-aminoethyl, or carboxymethyl; R3 represents one of 4-aminobutyl, 4-guanidinobutyl, and 3-guanidinopropyl; R4 represents either 2-aminoethyl or ethyl; R5 represents one of benzyl or phenethyl; R6 represents one of isobutyl or 2-methylthioethyl; R7 represents one of 2-aminoethyl or ethyl; R8 represents one of (R)-benzyl or (L)-benzyl; R9 represents either ethyl or isobutyl; R10 represents a hydrogen atom or one of the following structural formulas: .
[0020] In this invention, R1 preferably represents a hydrogen atom or the following structural formula; .
[0021] R2 represents ethyl or 2-aminoethyl; R3 represents 4-aminobutyl, 4-guanidinobutyl or 3-guanidinopropyl; R4 represents 2-aminoethyl; R5 stands for benzyl; R6 represents isobutyl or 2-methylthioethyl; R7 represents 2-aminoethyl; R8 represents (R)-benzyl; R9 represents isobutyl; R10 represents a hydrogen atom or one of the following structural formulas; , .
[0022] In this invention, the sulfonyl-γ-AA peptide preferably comprises having the following structural formula; sulfonyl-γ-AA peptide 3A, 8. Sulfonyl-γ-AA peptide 11. Sulfonyl-γ-AA peptide sulfonyl-γ-AA peptide 12 13. Sulfonyl-γ-AA peptide 15. Sulfonyl-γ-AA peptide 17. Sulfonyl-γ-AA peptide 18. Sulfonyl-γ-AA peptide Sulfonyl-γ-AA peptide 20.
[0023] The present invention provides an eIF4E / eIF4G interaction inhibitor comprising the sulfonyl-γ-AA peptide.
[0024] In this invention, the sulfonyl-γ-AA peptide has the following characteristics: The peptide molecules with a backbone that mimics the structure of natural polypeptides are a series of peptide-mimicking folds based on a sulfonyl-γ-AA peptide backbone that inhibit the eIF4E / eIF4G interaction. These helical folds successfully mimic the conserved binding sequences of eIF4G and 4E-BP1, exhibiting strong translational repression capabilities in vitro. By linking cell-penetrating peptides and lysosomal sorting sequences, they can effectively enter cells and inhibit intracellular translation, thereby inhibiting tumor cell proliferation and mediating programmed cell death in tumor cells.
[0025] In this invention, the sulfonyl-γ-AA peptide may also include a pharmaceutically acceptable salt of the sulfonyl-γ-AA peptide.
[0026] The present invention provides an anticancer drug comprising the sulfonyl-γ-AA peptide or the eIF4E / eIF4G interaction inhibitor.
[0027] In this invention, the anticancer drug preferably also includes pharmaceutically acceptable excipients.
[0028] This invention provides the use of the sulfonyl-γ-AA peptide or the eIF4E / eIF4G interaction inhibitor in the preparation of antitumor drugs for prevention and / or treatment.
[0029] In this invention, the antitumor effect preferably includes inhibiting the translational level and proliferation of tumor cells and promoting programmed cell death of tumor cells. The tumor cells preferably include at least one of HCT116, MCF7, and MDA-MB-231.
[0030] In this invention, the tumor preferably includes at least one of the following: colon cancer, breast cancer, prostate cancer, head and neck tumors, stomach cancer, lung cancer, skin cancer, esophageal cancer, bladder cancer, cervical cancer, and hematopoietic system malignant tumors.
[0031] In this invention, sulfonyl-γ-AA peptide 20 inhibits tumor growth by inducing ferroptosis. In one embodiment of this invention, ferroptosis inhibitor (Fer-1) and sulfonyl-γ-AA peptide 20 were used together to act on tumor cells. The results showed that the ferroptosis inhibitor antagonized the inhibitory effect of sulfonyl-γ-AA peptide 20 on tumor cell growth.
[0032] The following detailed description, in conjunction with embodiments, illustrates an eIF4E / eIF4G interaction inhibitor provided by the present invention and its applications, but these descriptions should not be construed as limiting the scope of protection of the present invention.
[0033] The structural units used in the examples to synthesize sulfonyl-γ-AA peptides are as follows: Starting with Fmoc-protected amino acids, sulfonyl-γ-AA peptide structural units B1-B15 were synthesized. B1-B5 were obtained according to synthetic route 1, B6-B9 according to synthetic route 2, B10-B14 according to synthetic route 3, and B15 according to synthetic route 4.
[0034] See Synthesis Route 1 Figure 2 Specific synthesis steps: Add 6g of Fmoc-protected amino acids and 200 mL of [unspecified substance] to a 1 L round-bottom flask. N,N - Dimethylformamide (DMF). Stir the mixture in an ice bath and add 1-hydroxybenzotriazole (HOBt, 1.2 equivalents), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 1.2 equivalents), and... N,N - Diisopropylethylamine (DIPEA, 1.2 equivalents). After stirring for 15 minutes, dimethylhydroxylamine hydrochloride (1.2 equivalents) and DIPEA (1.2 equivalents) were added, and the solution was stirred at 0 °C for 2 hours. After the reaction was complete, 200 mL of water was added to the solution, and then the mixture was extracted with ethyl acetate (150 mL × 3). The organic layers were combined and washed with 1M dilute hydrochloric acid solution (200 mL × 3), saturated sodium bicarbonate solution (200 mL × 1), and saturated brine solution (200 mL × 1), respectively. After drying with anhydrous sodium sulfate and concentrating under reduced pressure, the desired Weinreb amide I-1a was obtained. The crude product was used directly in the next reaction.
[0035] Lithium aluminum hydride (1.1 equivalents) was added to an anhydrous tetrahydrofuran solution (150 mL) of I-1a (1 equivalent) at -20 °C. After stirring at this temperature for 30 minutes, the reaction was quenched by adding 1 M dilute hydrochloric acid solution (200 mL). The tetrahydrofuran in the reaction solution was rotary evaporated and extracted with ethyl acetate (150 mL × 3). The organic layers were combined and washed with saturated saline solution (200 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain aldehyde I-1b, which could be used for the next step without purification.
[0036] A methanol solution (150 mL) of I-1b (1 equivalent) was stirred in an ice bath for 15 minutes, followed by the addition of a methanol solution (20 mL) of glycine tert-butyl hydrochloride (1.1 equivalent) and triethylamine (1.1 equivalent), and stirring was continued at 0 °C for 10 minutes. Acetic acid (1.5 equivalent) and sodium cyanoborohydride (2 equivalent) were then added, and the reaction was continued for 2 hours. After the reaction was complete, the solvent was removed by rotary evaporation, and the residue was extracted with 200 mL of saturated sodium bicarbonate solution and ethyl acetate (150 mL × 3). The combined organic layers were dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography with ethyl acetate / petroleum ether to obtain the secondary amine intermediate I-1c.
[0037] Pyridine (10 equivalents) was added to a 150 mL solution of I-1c in dichloromethane, and the mixture was stirred in an ice bath for 10 minutes. Then, sulfonyl chloride (2 equivalents) was added, and stirring continued at 0 °C for 1 hour, followed by stirring overnight at room temperature. After the reaction was complete, the solvent was removed by rotary evaporation, and the mixture was purified by column chromatography using dichloromethane / ethyl acetate to obtain pure intermediate I-1d.
[0038] The 1-day residue was treated with 50 mL of trifluoroacetic acid / dichloromethane (1:1) solution at room temperature for 2 hours. The solvent was removed by rotary evaporation, and the residue was purified by column chromatography with dichloromethane / methanol to obtain pure sulfonyl-γ-AA peptide structural unit B1-B5.
[0039] See synthesis route 2 Figure 3 Specific synthetic steps: Intermediate I-2a was obtained from the corresponding Fmoc-protected amino acid, following the synthetic procedure of I-1a. A methanol solution (150 mL) of I-2a (1 equivalent) was stirred in an ice bath for 15 minutes, followed by the addition of a methanol solution (20 mL) of glycine benzyl ester hydrochloride (1.1 equivalent) and triethylamine (1.1 equivalent), and stirring was continued at 0 °C for 10 minutes. Acetic acid (1.5 equivalent) and sodium cyanoborohydride (2 equivalent) were then added, and the reaction was continued for 2 hours. After the reaction was complete, the solvent was removed by rotary evaporation, and the residue was extracted with saturated sodium bicarbonate solution (200 mL) and ethyl acetate (150 mL × 3). The organic layers were combined, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography with ethyl acetate / petroleum ether to obtain the secondary amine intermediate I-2b.
[0040] Pyridine (10 equivalents) was added to a 150 mL solution of I-2b in dichloromethane, and the mixture was stirred in an ice bath for 10 minutes. Then, sulfonyl chloride (2 equivalents) was added, and stirring continued at 0 °C for 1 hour, followed by stirring overnight at room temperature. After the reaction was complete, the solvent was removed by rotary evaporation, and the mixture was purified by column chromatography using dichloromethane / ethyl acetate to obtain pure intermediate I-2c.
[0041] I-2c (1 equivalent) was dissolved in 150 mL of dichloromethane / methanol (1:1) solution, and 10% palladium on carbon was added. The mixture was purged three times under hydrogen atmosphere, and the reaction was carried out at room temperature for 4 hours. After the reaction was complete, the solvent was removed by rotary evaporation, and the residue was purified by column chromatography with dichloromethane / methanol to obtain pure sulfonyl-γ-AA peptide structural units B6-B9.
[0042] See synthesis route 3 Figure 4 Specific synthetic steps: Intermediate I-3a was obtained from the corresponding Fmoc-protected amino acid, following the synthetic procedure of I-2b. Pyridine (10 equivalents) was added to a 150 mL solution of I-3a in dichloromethane, and the mixture was stirred in an ice bath for 10 minutes. Subsequently, Cbz-protected aminoethanesulfonyl chloride (2 equivalents) was added, and stirring continued at 0 °C for 1 hour, followed by overnight stirring at room temperature. After the reaction was complete, the solvent was removed by rotary evaporation, and the mixture was purified by column chromatography using dichloromethane / ethyl acetate to obtain pure intermediate I-3b.
[0043] I-3b (1 equivalent) was dissolved in 150 mL of dichloromethane / methanol (1:1) solution, and 10% palladium on carbon was added. The mixture was purged three times under hydrogen atmosphere, and the reaction was carried out at room temperature for 4 hours. After the reaction was complete, the mixture was filtered through diatomaceous earth, and the solvent was removed from the filtrate by rotary evaporation. The resulting intermediate I-3c was directly added to the next step without further purification.
[0044] I-3c (1 equivalent) was dissolved in 150 mL of sodium bicarbonate (2 equivalents) aqueous solution, and 50 mL of tetrahydrofuran solution of di-tert-butyl dicarbonate (2 equivalents) was added with stirring. The reaction was continued at room temperature for 2 hours. After the reaction was completed, 1 M citric acid solution (150 mL) was added to the reaction solution and extracted with ethyl acetate (150 mL x 3). The organic layers were combined, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography with dichloromethane / methanol to obtain pure sulfonyl-γ-AA peptide structural units B10-B14.
[0045] See synthesis route 4 Figure 5 Specific synthetic steps: Intermediate I-4a was obtained from Fmoc-protected D-lysine following the same synthetic procedure as I-3b. I-4a was treated with 50 mL of trifluoroacetic acid / dichloromethane (1:1) solution at room temperature for 2 hours. The solvent was removed by rotary evaporation, and the resulting intermediate I-4b was used directly in the next step. Triethylamine (2 equivalents) was added to a 150 mL solution of I-4b (1 equivalent) in dichloromethane, followed by... N,N' -bis(tert-butyloxycarbonyl)-1 H-Pyrazole-1-formamidinium (1.5 equivalents) was reacted at room temperature for 2 hours. After the reaction was complete, the solvent was removed by rotary evaporation, and the intermediate I-4c was purified by column chromatography using dichloromethane / ethyl acetate to obtain pure intermediate I-4c.
[0046] I-4c (1 equivalent) was dissolved in 150 mL of dichloromethane / methanol (1:1) solution, and 10% palladium on carbon was added. The mixture was purged three times under hydrogen atmosphere, and the reaction was carried out at room temperature for 4 hours. After the reaction was complete, the mixture was filtered through diatomaceous earth, and the solvent was removed from the filtrate by rotary evaporation. The resulting intermediate I-4d was directly added to the next step without further purification.
[0047] I-4d (1 equivalent) was dissolved in 150 mL of sodium bicarbonate (2 equivalents) aqueous solution, and 50 mL of tetrahydrofuran solution of di-tert-butyl dicarbonate (2 equivalents) was added with stirring. The reaction was continued at room temperature for 2 hours. After the reaction was completed, 1 M citric acid solution (150 mL) was added to the reaction solution and extracted with ethyl acetate (150 mL x 3). The organic layers were combined, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography with dichloromethane / methanol to obtain pure sulfonyl-γ-AA peptide structural unit B15.
[0048] Table 1. Proton NMR spectral data of sulfonyl-γ-AA peptide structural units
[0049] Example 1 Synthetic method of sulfonyl-γ-AA peptide 1A Sulfonyl-γ-AA peptide 1A was obtained through solid-phase synthesis, and the specific steps are shown in synthetic route 5. Figure 6As shown: 100 mg of Rink Amide-MBHA resin (0.631 mmol / g) was used in a 20 mL peptide synthesis tube for synthesis. Before use, the resin was swollen in DMF for 5 minutes, then treated with 20% piperidine / DMF solution (2 mL) for 15 minutes (×2) to remove the Fmoc protecting group, followed by washing three times each with DCM and DMF. The Fmoc-protected sulfonyl-γ-AA peptide structural unit B1 (2 equivalents), HOBt (4 equivalents), and DIC (4 equivalents) were dissolved in 2 mL of DMF, shaken for 10 minutes to activate the starting material, then added to the resin and aerated for 4 hours to complete the coupling reaction. After washing with DCM and DMF, the resin was treated with 20% piperidine / DMF solution (2 mL) for 15 minutes (×2). The second Fmoc-protected sulfonyl-γ-AA peptide structural unit B12 (2 equivalents) was attached to the resin according to the procedure in the first coupling step. The above steps were repeated sequentially to couple sulfonyl-γ-AA peptide structural units B1, B11, and B6 onto the resin to obtain the desired sulfonyl-γ-AA peptide. The mixture was then treated with 20% piperidine / DMF solution (2 mL) for 15 minutes (×2), followed by washing three times each with DCM and DMF. The resin was then treated with 4 mL of 50% TFA / DCM solution for 2 hours to lyse the peptide from the resin. The lysate was collected, and the solvent was removed by rotary evaporation. The residue was precipitated with diethyl ether, and the supernatant was removed by centrifugation to obtain the crude product. The crude product was purified by preparative HPLC to obtain sulfonyl-γ-AA peptide 1A (structural formula shown in Formula I).
[0050] Formula I.
[0051] Sulfonyl-γ-AA peptide 1A: LC-MS (ESI) [M+H] 1+ Calc. for C 63 H 108 N 14 O 16 S5: 1476.67, found: 493.56 [M+3H] 3+ 739.69 [M+2H] 2+ .
[0052] Example 2 The synthetic route of sulfonyl-γ-AA peptide 1B is the same as that in Example 1A, except that: after removing the N-terminal Fmoc protecting group, the resin is treated with acetic anhydride (1 mL) and pyridine (2 mL) solution for 15 minutes, and then the pseudopeptide is cleaved from the resin to obtain sulfonyl-γ-AA peptide 1B (structural formula is shown in Formula II).
[0053] Formula II.
[0054] LC-MS (ESI) [M+H] 1+ Calc. for C 65 H 110 N 14 O 17 S5: 1518.68, found: 507.56 [M+3H] 3+ 760.88 [M+2H] 2+ .
[0055] Example 3 The synthetic route for sulfonyl-γ-AA peptide 2A is the same as that in 1A. The sulfonyl-γ-AA peptide structural units used are B2, B12, B1, B11 and B6 in sequence to obtain sulfonyl-γ-AA peptide 2A (structural formula is shown in Formula III).
[0056] Formula III.
[0057] LC-MS (ESI) of sulfonyl-γ-AA peptide 2A [M+H] 1+ Calc. for C 61 H 104 N 14 O 16 S5: 1448.64, found: 484.21 [M+3H] 3+ 725.67 [M+2H] 2+ .
[0058] Example 4 The synthetic route for sulfonyl-γ-AA peptide 2B is the same as that in 1B. The sulfonyl-γ-AA peptide structural units used are B2, B12, B1, B11 and B6 in sequence to obtain sulfonyl-γ-AA peptide 2B (structural formula is shown in Formula IV).
[0059] Formula IV LC-MS (ESI) of sulfonyl-γ-AA peptide 2B [M+H] + Calc. for C 63 H 106 N 14 O 17 S5: 1490.65, found: 498.21 [M+3H] 3+ 746.70 [M+2H] 2+ . Example 5 The synthetic route for sulfonyl-γ-AA peptide 3A is the same as that in 1A. The sulfonyl-γ-AA peptide structural units used are B1, B12, B1, B11 and B10 in sequence to obtain sulfonyl-γ-AA peptide 3A (structural formula is shown in Formula V).
[0060] Formula V LC-MS (ESI) [M+H] + Calc. for C 63 H 109 N 15 O 16 S5: 1491.68, found: 374.27 [M+4H] 4+ , 498.42 [M+3H] 3+ . Example 6 The synthetic route for sulfonyl-γ-AA peptide 3B is the same as that in 1B. The sulfonyl-γ-AA peptide structural units used are B1, B12, B1, B11 and B10 in sequence to obtain sulfonyl-γ-AA peptide 3B (structural formula is shown in Formula VI).
[0061] Style VI LC-MS (ESI) of sulfonyl-γ-AA peptide 3B [M+H] + Calc. for C 65 H 111 N 15 O 17 S5: 1533.69, found: 512.52 [M+3H] 3+ [M+2H] 2+ . Example 7 The synthetic route for sulfonyl-γ-AA peptide 4A is the same as that in 1A. The sulfonyl-γ-AA peptide structural units used are B2, B7, B2, B12, B1, B11 and B6 in sequence, to obtain sulfonyl-γ-AA peptide 4A (structural formula VII).
[0062] Equation VII Sulfonyl-γ-AA peptide 4A LC-MS (ESI) [M+H] + Calc. for C 84 H 143 N 19 O 22 S7: 1993.87, found: 400.14 [M+5H] 5+ 499.79 [M+4H] 4+, 666.03 [M+3H] 3+ . Example 8 The synthetic route for sulfonyl-γ-AA peptide 4B is the same as that in 1B. The sulfonyl-γ-AA peptide structural units used are B2, B7, B2, B12, B1, B11 and B6 in sequence to obtain sulfonyl-γ-AA peptide 4B (see structural formula VI).
[0063] Formula VIII LC-MS (ESI) of sulfonyl-γ-AA peptide 4B [M+H] + Calc. for C 86 H 145 N 19 O 23 S7: 2035.88, found: 510.34 [M+4H] 4+ , 680.11 [M+3H] 3+ , 1019.35 [M+2H] 2+ . Example 9 The synthetic route for sulfonyl-γ-AA peptide 5A is as described in 1A. The sulfonyl-γ-AA peptide structural units used are B8, B7, B2, B12, B1, B11 and B6 in sequence to obtain sulfonyl-γ-AA peptide 5A (see structural formula IX).
[0064] Formula IX LC-MS (ESI) of sulfonyl-γ-AA peptide 5A [M+H] + Calc. for C 78 H 139 N 19 O 23 S7: 1933.83, found: 388.11 [M+5H] 5+ 484.79 [M+4H] 4+ , 645.91 [M+3H] 3+ . Example 10 The synthetic route for sulfonyl-γ-AA peptide 5B is the same as that in 1B. The sulfonyl-γ-AA peptide structural units used are B8, B7, B2, B12, B1, B11 and B6 in sequence, to obtain sulfonyl-γ-AA peptide 5B (structural formula X).
[0065] Formula X LC-MS (ESI) of sulfonyl-γ-AA peptide 5B [M+H] +Calc. for C 80 H 141 N 19 O 24 S7: 1975.84, found: 495.27 [M+4H] 4+ , 659.96 [M+3H] 3+ . Example 11 The synthetic route for sulfonyl-γ-AA peptide 6 is as described in 1A. The sulfonyl-γ-AA peptide structural units used are B1, B12, B1, B7 and B10 in sequence, to obtain sulfonyl-γ-AA peptide 6 (structural formula XI).
[0066] Formula XI LC-MS (ESI) of acyl-γ-AA peptide 6 [M+H] + Calc. for C 63 H 108 N 14 O 16 S5: 1476.67, found: 493.57 [M+3H] 3+ , 739.37 [M+2H] 2+ . Example 12 The synthetic route for sulfonyl-γ-AA peptide 7 is as described in 1A. The sulfonyl-γ-AA peptide structural units used are B1, B3, B1, B11 and B10 in sequence, to obtain sulfonyl-γ-AA peptide 7 (structural formula XII).
[0067] Formula XII LC-MS (ESI) of sulfonyl-γ-AA peptide 7 [M+H] + Calc. for C 63 H 108 N 14 O 16 S5: 1476.67, found: 493.52 [M+3H] 3+ 739.69 [M+2H] 2+ . Example 13 The synthetic route for sulfonyl-γ-AA peptide 8 is as described in 1A. The sulfonyl-γ-AA peptide structural units used are B12, B1, B11 and B10 in sequence, to obtain sulfonyl-γ-AA peptide 8 (structural formula XIII).
[0068] Structural XIII LC-MS (ESI) of sulfonyl-γ-AA peptide 8 [M+H] + Calc. for C 48 H 87 N 13 O 13 S4: 1181.54, found: 395.24 [M+3H] 3+ 591.94 [M+2H] 2+ . Example 14 The synthetic route for sulfonyl-γ-AA peptide 9 is as described in 1A. The sulfonyl-γ-AA peptide structural units used are B4, B12, B1, B11 and B6 in sequence, to obtain sulfonyl-γ-AA peptide 9 (structural formula XIV).
[0069] Formula XIV LC-MS (ESI) of sulfonyl-γ-AA peptide 9 [M+H] + Calc. for C 63 H 108 N 14 O 16 S5: 1476.67, found: 493.65 [M+3H] 3+ . Example 15 The synthetic route for sulfonyl-γ-AA peptide 10 is as described in 1A. The sulfonyl-γ-AA peptide structural units used are B5, B12, B1, B11 and B6 in sequence, to obtain sulfonyl-γ-AA peptide 10 (structural formula XV).
[0070] XV LC-MS (ESI) of sulfonyl-γ-AA peptide 10 [M+H] + Calc. for C 61 H 104 N 14 O 16 S5: 1448.64, found: 484.35 [M+3H] 3+ . Example 16 The synthetic route for sulfonyl-γ-AA peptide 11 is as described in 1A. The sulfonyl-γ-AA peptide structural units used are B4, B12, B1, B11 and B10 in sequence, to obtain sulfonyl-γ-AA peptide 10 (structural formula XVI).
[0071] XVI See results Figure 7LC-MS (ESI) of sulfonyl-γ-AA peptide 10 [M+H] + Calc. for C 63 H 109 N 15 O 16 S5:1491.68, found: 374.31 [M+4H] 4+ , 498.52 [M+3H] 3+ . Example 17 The synthetic route for sulfonyl-γ-AA peptide 12 is as described in 1A. The sulfonyl-γ-AA peptide structural units used are B4, B12, B1, B14 and B10 in sequence, to obtain sulfonyl-γ-AA peptide 12 (structural formula XVII).
[0072] Formula XVII The results are as follows Figure 8 LC-MS (ESI) of sulfonyl-γ-AA peptide 12 [M+H] + Calc. for C 63 H 109 N 17 O 16 S5:1519.68, found: 381.28 [M+4H] 4+ 507.97 [M+3H] 3+ , 761.24 [M+2H] 2+ . Example 18 The synthetic route for sulfonyl-γ-AA peptide 13 is as described in 1A. The sulfonyl-γ-AA peptide structural units used are B4, B12, B1, B15 and B10 in sequence, to obtain sulfonyl-γ-AA peptide 13 (structural formula XVIII).
[0073] Formula XVIII The results are as follows Figure 9 LC-MS (ESI) of sulfonyl-γ-AA peptide 13 [M+H] + Calc. for C 64 H 111 N 17 O 16 S5:1533.70, found: 384.83 [M+4H] 4+ 512.65 [M+3H] 3+ 768.30 [M+2H] 2+ . Example 19 The synthetic route for sulfonyl-γ-AA peptide 14 is as described in 1A. The sulfonyl-γ-AA peptide structural units used are B4, B12, B1, B11 and B9 in sequence, to obtain sulfonyl-γ-AA peptide 14 (structural formula IXX).
[0074] Formula IXX The results are as follows Figure 10 LC-MS (ESI) of sulfonyl-γ-AA peptide 14 [M+H] + Calc. for C 63 H 106 N 14 O 18 S5:1506.64, found: 503.55 [M+3H] 3+ 754.65 [M+2H] 2+ . Example 20 The synthetic route for sulfonyl-γ-AA peptide 15 is as described in 1A. The sulfonyl-γ-AA peptide structural units used are B4, B13, B1, B11 and B10 in sequence, to obtain sulfonyl-γ-AA peptide 15 (structural formula XX).
[0075] Formula XX The results are as follows Figure 11 LC-MS (ESI) of sulfonyl-γ-AA peptide 15 [M+H] + Calc. for C 62 H 107 N 15 O 16 S6:1509.63, found: 504.56 [M+3H] 3+ , 756.24 [M+2H] 2+ . Example 21 Evaluation of in vitro translational ability of different sulfonyl-γ-AA peptides The inhibition of the eIF4E / eIF4G interaction by the sulfonyl-γ-AA peptides prepared in Examples 1-20 led to a decrease in translational levels. The effects of the sulfonyl-γ-AA peptides were evaluated using an in vitro translation system. The specific steps are as follows: In vitro translation was performed using a nuclease-treated rabbit reticulocyte lysate (RRL) system (Promega L4960). A reporter plasmid containing the T7 promoter and downstream of a firefly luciferase sequence was linearized by PCR and then transcribed in vitro using a MEGAscriptTMT7 kit (Thermo AM1334). The plasmid was incubated with in vitro transcriptase in a buffer containing ATP, UTP, GTP, and CTP at 37°C for 6–8 h, and RNA was extracted and purified to obtain an RNA fragment containing the firefly luciferase sequence of the T7 promoter. The reporter RNA was then capped in vitro using a vaccinia virus capping enzyme (NEB M2080S). The resulting plasmid, along with the treated rabbit reticulocyte lysate and sulfonyl-γ-AA peptide 1A–sulfonyl-γ-AA peptide 15 (final concentration 10 μM), was incubated at 30°C for 90 min to complete the in vitro translation reaction and generate the firefly luciferase protein. After the translation reaction was completed, the same concentration of luciferin substrate was added to the reaction system. Firefly luciferase catalyzed the enzymatic luminescence reaction of luciferin in the presence of ATP and oxygen, generating a corresponding luminescent signal. The luminescence intensity of each reaction system was measured using a luciferase reporter gene detection system (Promega E1500) and a multi-functional microplate reader. The reaction system without the added peptide served as a blank control group, and its fluorescence intensity was measured. The ratio of the fluorescence intensity of each peptide-treated group to that of the control group was calculated, and the resulting ratio was used to characterize the inhibition rate of the peptide on the in vitro translation process. The degree of decrease in fluorescence intensity reflects the inhibitory effect of the tested peptide on translational activity.
[0076] The results are as follows Figure 11 As shown in Table 2, all 20 peptides, from sulfonyl-γ-AA peptide 1A to sulfonyl-γ-AA peptide 15, can inhibit in vitro translation levels, with compound 11 showing the strongest effect, exhibiting an inhibition rate of 98% at 10 μM.
[0077] Table 2 In vitro translation capabilities
[0078] Example 22 Circular dichroism analysis of sulfonyl-γ-AA peptide Homogeneous D-sulfonyl-γ-AA peptides can form a well-defined right-handed helical structure in solution. Specific procedures: Sulfonyl-γ-AA peptides with different structures were dissolved in PBS buffer to prepare solutions with concentrations ranging from 250 μM to 500 μM. These solutions were then measured in a 1 mm quartz cuvette using a Jasco J-1500 spectrometer at a wavelength between 190 nm and 260 nm. PBS buffer was used as a blank control. Each sample was scanned three times, and the average value was taken. The average residue ellipticity [θ] was calculated using the following formula.
[0079] [θ] = θobs / (n × l ×c ×10) Formula I; θobs = the ellipticity (in millimeters) obtained from the test; n = the number of side chains of the sulfonyl-γ-AA peptide; l = path length (0.1cm); c = Sample concentration (M).
[0080] like Figure 12 As shown, homogeneous D-sulfonyl-γ-AA peptides 1A, 2A, 3A, 4A, and 5A exhibit a significant negative Cotton effect between 205 nm and 215 nm, indicating that these peptides possess a right-handed helical structure. D-sulfonyl-γ-AA peptides 9, 10, and 11, as D-sulfonyl-γ-AA peptides with conformational inversions at the 1A, 2A, and 3A carbon ends, also exhibit a significant right-handed helical characteristic, although the strength is weaker than that of the homogeneous D-sulfonyl-γ-AA peptides. This further demonstrates the stability of the sulfonyl-γ-AA peptide backbone.
[0081] Example 23 The specific steps for synthesizing CPP-sulfonyl-γ-AA peptide 16 are shown in synthetic route 6. Figure 13As shown, the transmembrane peptide was synthesized via solid-phase synthesis: 100 mg of Rink Amide-MBHA resin (0.631 mmol / g) was synthesized in a 20 mL peptide synthesis tube. Before use, the resin was swollen in DMF for 5 minutes, then treated with 20% piperidine / DMF solution (2 mL) for 15 minutes (×2) to remove the Fmoc protecting group, followed by washing three times each with DCM and DMF. The Fmoc-protected natural amino acid (4 equivalents), HOBt (8 equivalents), and DIC (8 equivalents) were dissolved in 2 mL of DMF, shaken for 10 minutes to activate the starting material, then added to the resin and aerated for 4 hours to complete the coupling reaction. After washing with DCM and DMF, the resin was treated with 20% piperidine / DMF solution (2 mL) for 15 minutes (x2). Another Fmoc-protected natural amino acid (4 equivalents) was coupled to the resin following the same steps, and the reaction cycle was repeated until the desired transmembrane peptide was obtained. The Fmoc protecting group was then removed by treatment with 20% piperidine / DMF solution (2 mL) for 15 minutes (×2), followed by washing three times each with DCM and DMF. Subsequently, following synthetic route 5, the desired Fmoc-protected sulfonyl-γ-AA peptide structural units were sequentially coupled onto the resin to obtain the desired CPP-sulfonyl-γ-AA peptide. The resin was treated with 4 mL of TFA:TIS:H2O (95:2.5:2.5) solution for 2 hours to cleave the CPP-sulfonyl-γ-AA peptide from the resin. The lysate was collected, and the solvent was removed by rotary evaporation. The residue was precipitated with diethyl ether, and the supernatant was removed by centrifugation to obtain the crude product. The crude product was purified by preparative HPLC to obtain CPP-sulfonyl-γ-AA peptide 16 (structural formula XXI).
[0082] Formula XXI See results Figure 14 CPP-sulfonyl-γ-AA peptide 16: LC-MS (ESI) [M+H] + Calc. forC 118 H 216 N 46 O 27 S5: 2869.55, found: 411.33 [M+7H] 7+ 479.66 [M+6H] 6+ , 575.42 [M+5H] 5+ 718.90 [M+4H] 4+ . Example 24 The synthetic route for CPP-sulfonyl-γ-AA peptide 17 is the same as in 16, except that a transmembrane peptide with the sequence RRRRRRRRR is synthesized. Then, under the same conditions, Fmoc-protected β-alanine is coupled to the resin as a linker to obtain CPP-sulfonyl-γ-AA peptide 18 (formula XXII).
[0083] Formula XXII See results Figure 15 CPP-sulfonyl-γ-AA peptide 17: LC-MS (ESI) [M+H] + Calc. forC 120 H 222 N 52 O 26 S5: 2967.63, found: 425.27 [M+7H] 7+ 495.93 [M+6H] 6+ 594.85 [M+5H] 5+ . Example 25 The synthetic route for CPP-sulfonyl-γ-AA peptide 18 is the same as in 16, except that the transmembrane peptide with the sequence RQIKIWFQNRRMKWKK is synthesized. Then, under the same conditions, the Fmoc-protected β-alanine is coupled to the resin as a linker to obtain CPP-sulfonyl-γ-AA peptide 18 (Formula XXIII).
[0084] Formula XXIII See results Figure 16 LC-MS (ESI) [M+H] + Calc. for C 170 H 280 N 50 O 36 S6: 3789.99, found:422.56 [M+9H] 9+ 475.23 [M+8H] 8+ 542.88 [M+7H] 7+ , 633.10 [M+6H] 6+ , 948.89 [M+4H] 4+ . Example 26 The synthetic route for CPP-sulfonyl-γ-AA peptide 19 is the same as in 16, except that a transmembrane peptide with the sequence RKKRRQRRRGYK is synthesized. Then, under the same conditions, an Fmoc-protected alanine is coupled to a resin as a linker to obtain CPP-sulfonyl-γ-AA peptide 19 (Formula XXIV).
[0085] Formula XXIV See results Figure 17 CPP-sulfonyl-γ-AA peptide 19: LC-MS (ESI) [M+H] + Calc. forC 135 H 240 N 50 O 31 S5: 3217.73, found: 403.58 [M+8H] 8+ , 461.11 [M+7H] 7+ 537.67 [M+6H] 6+ , 645.06 [M+5H] 5+ . Example 27 Synthesis method of CPP-LSS-sulfonyl-γ-AA peptide 20 1. Intermediate Mc-Val-Cit-PABC-11 is synthesized via route 7 ( Figure 18 As shown in Figure 5, the corresponding sulfonyl-γ-AA peptide was synthesized according to synthetic route 5. The Fmoc protecting group was then removed by treatment with 20% piperidine / DMF solution (2 mL) for 15 minutes (x2), followed by washing three times each with DCM and DMF. Fmoc-Val-Cit-PAB-PNP (2 equivalents), HOBt (2 equivalents), and DIPEA (4 equivalents) were then dissolved in 2 mL of DMF and added to the resin, followed by aeration for 4 hours to complete the reaction. The Fmoc protecting group was then removed by treatment with 20% piperidine / DMF solution (2 mL) for 15 minutes (x2), followed by washing three times each with DCM and DMF. 6-maleimide hexanoic acid (4 equivalents), HOBt (8 equivalents), and DIC (8 equivalents) were activated in 2 mL of DMF solution for 10 minutes, added to the resin, and aerated for 4 hours to complete the coupling reaction. The peptide was lysed from the resin by treating it with 4 mL of 50% TFA / DCM solution for 2 hours. The lysate was collected, and the solvent was removed by rotary evaporation. The residue was precipitated with diethyl ether, and the supernatant was removed by centrifugation to obtain the crude product. The crude product was purified by preparative HPLC to obtain Mc-Val-Cit-PABC-11.
[0086] 2. CPP-LSS-sulfonyl-γ-AA peptide 20 was synthesized via route 8 ( Figure 19 As shown in the diagram, the intermediate CPP-LSS peptide Ac-CGGGRRRRRRRRRNPGY was obtained following the CPP synthesis procedure in synthetic route 6. After removing the N-terminal Fmoc protecting group, the resin was treated with a solution of acetic anhydride (1 mL) and pyridine (2 mL) for 15 minutes. The CPP-LSS peptide was then cleaved from the resin, and the lysate was collected. The solvent was removed by rotary evaporation. The residue was precipitated with diethyl ether, and the supernatant was removed by centrifugation to obtain the crude product. The crude product was purified by preparative HPLC to obtain the CPP-LSS peptide.
[0087] Intermediate Mc-Val-Cit-PABC-11 (1 equivalent) and CPP-LSS peptide Ac-CGGGRRRRRRRRRNPGY (1.1 equivalent) were added to 2 mL of 0.01 M PBS buffer and stirred for 30 minutes. After the reaction was completed, the reaction solution was purified by preparative HPLC to obtain compound CPP-LSS-sulfonyl-γ-AA peptide 20 (structural formula XXV). Formula XXV
[0088] See results Figure 20 CPP-LSS-sulfonyl-γ-AA peptide 20: LC-MS (ESI) [M+H] + Calc. forC 177 H 299 N 67 O 44 S6: 4259.15, found: 474.50 [M+9H] 9+ 533.77 [M+8H] 8+ 609.85 [M+7H] 7+ , 711.26 [M+6H] 6+ . Example 28 Evaluation of intracellular translation inhibition in tumor cells To improve the cell membrane permeability of sulfonyl-γ-AA peptides, sulfonyl-γ-AA peptides 16-20, linked with CPP or CPP-LSS, were synthesized based on sulfonyl-γ-AA peptide 11. Their inhibitory effect on mRNA translation in colorectal cancer cells HCT116 was evaluated. Specific procedures: The compounds were co-incubated with HCT116 colorectal cancer cells, with a blank control or a small molecule inhibitor (4EGI) interacting with 50 μM eIF4E-4G used as a positive control. Then, 10 μg / mL puromycin was added, and the cells were cultured at 37 °C for 15 min. Cells were scraped from 6-well plates, centrifuged at 800 g for 3 min to collect the cell pellet, and then lysed with an appropriate amount of RIPA lysis buffer on ice for 30 min. After centrifugation at 16000 g for 10 min at 4 °C, the supernatant was collected, and BCA protein was quantified. Protein samples of the same concentration were prepared according to the protein concentration. Western blotting was then performed. The gel was run at a constant voltage of 80V until protein marker separation, followed by a constant voltage of 140V until completion. The PVDF membrane was activated with methanol and transferred at a constant current of 300mA on ice for 75 min. It was blocked with 5% milk (prepared with PBST) at room temperature for 1 h, washed three times with 5% PBST for 5 min each time, and then incubated overnight at 4°C with the corresponding primary antibodies (puromycin and β-actin). The next day, after washing, the corresponding mouse secondary antibody was added, and the membrane was incubated at room temperature for 1 h, washed three times with 5% PBST for 5 min each time, and then exposed with a 1:1 ECL developing solution. Gray-scale analysis was performed, and changes in translational activity were statistically analyzed.
[0089] The results are as follows Figure 21 As shown, sulfonyl-γ-AA peptide 20 exhibits the best inhibitory effect on intracellular translation, and at a concentration of 10 μM, it can significantly inhibit the translation level in tumor cells.
[0090] Example 29 Evaluation of tumor cell proliferation inhibition effect To investigate whether sulfonyl-γ-AA peptide 17 and sulfonyl-γ-AA peptide 20 affect tumor cell proliferation, 150 μL (4000-10000 cells) of cells were seeded in 96-well plates. After 24 h, sulfonyl-γ-AA peptide 17 and sulfonyl-γ-AA peptide 20 were added at concentrations of 0, 0.625, 1.25, 2.5, 5, 10, and 20 μM for 72 h. Cells were fixed in each well with 150 μL of pre-chilled 10% trichloroacetic acid (TCA) solution, followed by 100 μL of 0.2% SRB solution (prepared with 1% glacial acetic acid) and staining on a shaker at room temperature for 30 min. After staining, the cells were rinsed with freshly prepared 1% glacial acetic acid until no excess SRB staining solution remained in the wells before drying in an oven. The dye was dissolved in 100 μL of 10 mM Tris-HCl pH 10.5 solution per well, shaken on a shaker at room temperature for 5 min, and the absorbance was measured at 515 nm.
[0091] To examine the inhibitory effect of the ferroptosis inhibitor Fer-1 on the growth of tumor cells by antagonizing sulfonyl-γ-AA peptide 20, 1 μM Ferrostatin-1 (Fer-1) was added to the above concentration gradient of sulfonyl-γ-AA peptide 20, and the cells were treated for 72 h. The fixation and staining methods were the same as above, and the absorbance was measured at 515 nm.
[0092] 2 mL (500 cells in total) of cells were seeded in 6-well plates. After 24 h, peptide 20 was added at a concentration of 0.5 μM sulfonyl-γ-AA peptide 20 for 14 days. The drug and medium were changed every two days. The cells were washed twice with 1× PBS, fixed with 4% paraformaldehyde (PBS) for 30 min, washed twice with 1× PBS, stained with crystal violet for 20 min, and finally washed three times with 1× PBS. The cells were then dried and photographed under white light.
[0093] like Figure 22 and Figure 23 The results showed that sulfonyl-γ-AA peptide 17 and sulfonyl-γ-AA peptide 20 had inhibitory effects on the proliferation of different tumor cells.
[0094] like Figure 24 and Figure 25 The results showed that sulfonyl-γ-AA peptide 20 had an inhibitory effect on the proliferation of tumor cells.
[0095] like Figure 26 The results showed that the ferroptosis inhibitor Fer-1 antagonized the inhibitory effect of sulfonyl-γ-AA peptide 20 on tumor cell growth, suggesting that inducing ferroptosis is one of the mechanisms by which sulfonyl-γ-AA peptide 20 inhibits tumor growth.
[0096] Example 30 Evaluation of tumor cell apoptosis mediating activity (sulfonyl-γ-AA peptide 20 is the core compound of this patent, and only the tumor cell apoptosis mediating activity of sulfonyl-γ-AA peptide 20 was evaluated here). HCT116 cells were treated with 5 μM peptide 20, DMSO, or control group CTL for 12 h. Upon collection, the culture medium, washed PBS, and digested cell suspension were all collected into a centrifuge tube. After centrifugation at 800 g for 3 min, the cells were washed twice with pre-cooled 1x PBS, and then resuspended in 100 μL 1x PBS. 5 μL Annexin V-FITC and 5 μL LPI dye were added, and the mixture was incubated at room temperature in the dark for 10 min. The cells were then resuspended in 400 μL Binding Buffer and analyzed by flow cytometry within 1 h after staining. The FITC and PE channels were set up to detect the apoptosis rate.
[0097] See results Figure 27 20 sulfonyl-γ-AA peptide 20 can significantly induce tumor cell apoptosis.
[0098] The above description is only a preferred embodiment of 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 principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.
Claims
1. A sulfonyl-γ-AA peptide, characterized in that, It has the following general formula; ； Where n = 0 or 1, and R1 represents a hydrogen atom, an acetyl group, or one of the following structural formulas: R2 represents one of ethyl, 2-aminoethyl, or carboxymethyl. R3 represents one of 4-aminobutyl, 4-guanidinobutyl, and 3-guanidinopropyl; R4 represents either 2-aminoethyl or ethyl; R5 represents one of benzyl or phenethyl; R6 represents one of isobutyl or 2-methylthioethyl; R7 represents one of 2-aminoethyl or ethyl; R8 represents one of (R)-benzyl or (L)-benzyl; R9 represents either ethyl or isobutyl; R10 represents a hydrogen atom or one of the following structural formulas: 。 2. The sulfonyl-γ-AA peptide according to claim 1, characterized in that, R1 represents a hydrogen atom or the following structural formula; ； R2 represents ethyl or 2-aminoethyl; R3 represents 4-aminobutyl, 4-guanidinobutyl or 3-guanidinopropyl; R4 represents 2-aminoethyl; R5 stands for benzyl; R6 represents isobutyl or 2-methylthioethyl; R7 represents 2-aminoethyl; R8 represents (R)-benzyl; R9 represents isobutyl; R10 represents a hydrogen atom or one of the following structural formulas; 、 。 3. The sulfonyl-γ-AA peptide according to claim 1 or 2, characterized in that, Including those with the following structural formula; 、 、 、 、 、 、 。 4. An inhibitor of eIF4E / eIF4G interaction, characterized in that, Includes the sulfonyl-γ-AA peptide according to any one of claims 1 to 3.
5. An anticancer drug, characterized in that, Includes the sulfonyl-γ-AA peptide according to any one of claims 1 to 3 or the eIF4E / eIF4G interaction inhibitor according to claim 4.
6. The anticancer drug according to claim 5, characterized in that, It also includes pharmaceutically acceptable excipients.
7. The use of the sulfonyl-γ-AA peptide according to any one of claims 1 to 3 or the eIF4E / eIF4G interaction inhibitor according to claim 4 in the preparation of antitumor drugs for prevention and / or treatment.
8. The application according to claim 7, characterized in that, The anti-tumor effects include inhibiting the translation level and proliferation of tumor cells and promoting programmed cell death in tumor cells.
9. The application according to claim 7, characterized in that, The tumor cells include at least one of HCT116, MCF7, and MDA-MB-231.
10. The application according to any one of claims 7 to 9, characterized in that, The tumors include at least one of the following: colon cancer, breast cancer, prostate cancer, head and neck tumors, stomach cancer, lung cancer, skin cancer, esophageal cancer, bladder cancer, cervical cancer, and hematopoietic system malignancies.