Peptidomimetic peptide based on D-configuration diphenylalanine and application thereof

Abstract

The invention discloses a D-configuration diphenylalanine-based peptidomimetic or a pharmaceutically acceptable salt thereof. The peptidomimetic has the following structure: # imgabs0 #, wherein X represents C, O and S, and n represents an integer of 1-6. The peptidomimetic disclosed by the invention has endoplasmic reticulum targeting property and can specifically induce endoplasmic reticulum autophagy. The peptidomimetic disclosed by the invention can be used as an endoplasmic reticulum inducer for researching cell homeostasis, a disease generation mechanism and a therapeutic target, and the research, development and application of medicines are promoted.

Description

Technical Field

[0001] The invention belongs to the technical field of cell biology, and specifically relates to a type of peptidomimetic based on D-configuration diphenylalanine and application thereof. Background Art

[0002] The endoplasmic reticulum is the largest membrane organelle in eukaryotic cells. It is responsible for the synthesis and modification of proteins, lipids, phospholipids, cholesterol, and oligosaccharides. It is also an important site for calcium ion storage and detoxification of endogenous and exogenous substances. The endoplasmic reticulum is an important hub for intracellular protein synthesis, protein folding, and post-translational modification. Misfolded polypeptides can be reversed from the endoplasmic reticulum to the cytoplasm, where they are degraded by the ubiquitin-proteasome system, which is called "endoplasmic reticulum-associated protein degradation" (ERAD). Compared with ERAD, the endoplasmic reticulum-lysosome-associated degradation pathway (ERLAD), also known as endoplasmic reticulum autophagy (ER-phagy), provides a proteasome-free degradation pathway for protein quality control and is essential for endoplasmic reticulum turnover and cell homeostasis. Endoplasmic reticulum autophagy is closely related to a variety of human diseases, including neurodegenerative diseases, cardiovascular diseases, and tumors. Studying endoplasmic reticulum autophagy helps to reveal the pathogenesis of these diseases and provide new targets for disease treatment. In addition, by regulating endoplasmic reticulum autophagy, it may be possible to intervene in the disease process and promote cells to return to a healthy state.

[0003] Great efforts have been made to reveal the detailed mechanism of ER-phagy, and a variety of ER-phagy receptors have been identified in mammalian cells, such as FAM134B, SEC62, RTN3L, CCPG1, ATL3 and TEX264. At present, although there are several methods and drugs to induce autophagy, such as cell starvation treatment, mTOR inhibition (rapamycin), ER stress inducers (tunicamycin, dithiothreitol) and metal drug cisplatin, etc., all of the above drugs have limited specificity for ER autophagy induction, and autophagy of other organelles can also be observed at the same time, which lacks selectivity. Therefore, the development of small molecules that selectively induce ER-phagy is of great significance for studying the exact effects of independent ER-phagy. Summary of the invention

[0004] Prior art studies have shown that proteins that can induce endoplasmic reticulum autophagy in cells have the characteristics of being easy to mutate, not easy to degrade, and easy to fold and aggregate to form soluble small-sized aggregates (~100nm). Diphenylalanine (Diphenylalanine, L-Phe-L-Phe, FF) is the simplest aromatic dipeptide molecule, which has the property of self-assembling into nanostructures such as spherical vesicles, nanotubes, nanowires, microtubes and microrods. Based on the above characteristics of diphenylalanine, the present invention obtains a class of peptidomimetics with high hydrophilicity, good stability, easy folding and aggregation to form small-sized aggregates through structural modification, which successfully induces endoplasmic reticulum autophagy.

[0005] The specific technical solutions of the present invention are as follows:

[0006] A class of peptidomimetics based on D-configuration diphenylalanine or pharmaceutically acceptable salts thereof has the following structure: Wherein X represents C, O, S, and n represents an integer of 1 to 6. Preferably, X represents C, and n represents 1, 2, or 3.

[0007] Another object of the present invention is to provide the use of the peptoid in preparing an endoplasmic reticulum autophagy inducer.

[0008] Specifically, incubating cells with a peptide mimetic at a concentration of 40-80 μg / ml at 37°C for 24 hours can induce a significant endoplasmic reticulum autophagy process.

[0009] Another object of the present invention is to provide the use of the peptidomimetic in the preparation of anti-tumor drugs.

[0010] The peptidomimetic of the present invention can be used as a pharmaceutical active substance and can be used for the treatment of tumors by using a drug administration route permitted by the prior art. Advantages of the present invention:

[0011] The present invention constructs a symmetrical L-FFcFF peptoid by evaluating the physical properties of the L-configuration diphenylalanine (L-FF) dipeptide, and finds that its biological stability is poor and the size of the assembly is large. The structure is further optimized, and its configuration is changed to D-FFcFF. At the same time, in order to further optimize the hydrophilicity of the peptoid, the intermediate linker is replaced by a single atom. It is unexpectedly found that the obtained D-configuration peptoid has better water solubility, is easier to aggregate, and has a smaller aggregate size (~50nm) than L-configuration diphenylalanine (L-FF) and L-configuration peptoid (L-FFcFF), and has better stability in biological systems and is not easily degraded. The peptoid of the present invention can successfully induce endoplasmic reticulum autophagy, and can be used as an endoplasmic reticulum autophagy inducer for the study of endoplasmic reticulum and cell homeostasis, as well as the pathogenesis of diseases such as neurodegenerative diseases, cardiovascular diseases, and tumors by inducing endoplasmic reticulum autophagy. In addition, the present invention has found that the peptoid of the present invention has anti-tumor activity by inducing endoplasmic reticulum autophagy. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 :Determination of physical properties of target structure precursors. Design ideas for target structures (Figure A); determination of the minimum assembly concentration of L-FF, the hydration diameter and distribution of L-FF aggregates, and TEM morphology (Figure B); determination of the minimum assembly concentration of L-FFcFF, the hydration diameter and distribution of L-FFcFF aggregates, and TEM morphology (Figure C).

[0013] Figure 2 :Determination of basic physical properties of D-FFxFF. LC spectra of D-FFcFF (1 mg / mL) and L-FFcFF (1 mg / mL) after incubation in proteinase K (1.0 mg / mL) and 1% BSA solution for 3, 6, 12, and 24 h (Figure A); photographic images of L-FFcFF and D-FFcFF after degradation (Figure B). Determination of the minimum assembly concentration of D-FFxFF (Figure C); hydration diameter and distribution of D-FFxFF aggregates and TEM morphology (Figure D).

[0014] Figure 3 :Taking D-FFcFF as an example, biological transmission electron microscopy (Bio-TEM) images show that D-FFcFF aggregates are enriched in the endoplasmic reticulum.

[0015] Figure 4 :D-FFxFF induces ER autophagy. Taking D-FFcFF as an example, after MCF-7 cells were incubated with 70μg / ml D-FFcFF for 24h, the co-localization of lysosomes and ER in cells was determined by fluorescence confocal microscopy. ER and lysosomes were labeled with ER-tracker (λex=405nm; λem=420-500nm) and Lyso-tracker (λex=633nm; λem=650~710nm), respectively (Figure A). Bio-TEM images of MCF-7 cells incubated with 70μg / ml D-FFxFF for 24h: ER fragments in autophagolysosomes are marked with arrows, ALy, autophagolysosome; M, mitochondria; N, nucleus; AV, autophagosome; Ly, lysosome; ER, endoplasmic reticulum; PM, cell membrane (Figure B). Western blot was used to detect the expression of autophagy flux marker proteins LC3B and P62 after MCF-7 cells were incubated with 70 μg / ml D-FFxFF for 24 h; and the expression of endoplasmic reticulum autophagy receptors SEC62, RTN3L, CCPG1, FAM134B and TEX264 (Figure C).

[0016] Figure 5D-FFxFF did not induce other selective autophagy in cells. Confocal microscopy was used to determine the co-localization of lysosomes and mitochondria in MCF-7 cells after incubation with 70 μg / ml D-FFcFF for 24 h. Mitochondria and lysosomes were labeled with Mito-tracker (λex=488 nm; λem=500-540 nm) and Lyso-tracker (λex=633 nm; λem=650-710 nm), respectively (Figure A). Western blot was used to detect the expression of AIP4 (ribosomal autophagy), FBXO27 (lysosomal autophagy), Golgi autophagy receptors (YIPF3 / YIPF4) and lipophagy receptors (NUFIP1), and peroxisomal markers (catalase) in MCF-7 cells after incubation with 70 μg / ml D-FFxFF for 24 h (Figure B).

[0017] Figure 6 :Evaluation of the antitumor effect of D-FFxFF. IC values ​​of D-FFxFF against different cancer cell lines (HeLa, A375, 4T1, MDA-MB-231, MCF-7 and normal cell line NIH3T3) 50 Values ​​(Figure A). Representative immunofluorescence staining images of endoplasmic reticulum autophagy receptors CCPG1, FAM134B, RTN3L, and TEX264 (red) in tumor tissues of different groups after in vivo D-FFxFF treatment; cell nuclei were labeled with DAPI; scale bar = 20 μm (Figure B). Volume changes of primary tumors during peptidomimetic treatment. Error bars represent standard deviations (n ​​= 4) (Figure C). Photos and weight statistics of tumors collected at the end of treatment (Figures D and E). DETAILED DESCRIPTION

[0018] The following is a detailed description of an embodiment of the present invention. This embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation method and a specific operation process are given, but the protection scope of the present invention is not limited to the following examples.

[0019] Example 1 Preparation of the D-configuration diphenylalanine peptidomimetic (D-FFxFF) of the present invention

[0020] L-diphenylalanine (L-FF) and other chemical reagents were purchased from Shanghai Bid Pharmaceutical Technology Co., Ltd. The peptidomimetics L-FFcFF and D-FFxFFs were synthesized by improved methods according to the literature (Abbas M, Lipiński WP, Nakashima KK, Huck WTS, and Spruijt E. Ashort peptide synthon for liquid-liquid phase separation. Nat. Chem. 2021.13, 1046-1054.).

[0021] 1. Synthesis of compound Boc-D-FxF-Boc:

[0022] N-(tert-butylcarbonyl)-d-phenylalanine (D-Boc-Phe-OH, 398 mg, 1.55 mmol), benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU, 557 mg, 1.47 mmol) and 1-hydroxybenzotriazole (HOBT, 199 mg, 1.47 mmol) were dissolved in DMF (3 ml) and stirred with a magnetic stirrer. N,N-diisopropylethylamine (DIPEA, 1050 μL, 6.2 mmol) and 1,5-pentanediamine hydrochloride (75 mg, 0.7 mmol) were added respectively, with an interval of 1 min and stirred at room temperature for 24 h. The reaction mixture was poured into 50 ml of water. The white precipitate was collected by filtration and washed with water (yield 80%) to prepare Boc-D-FcF-Boc.

[0023] The half-amine dihydrochloride (CDC) in the above step was replaced by 2,2'-thiobis(ethylamine) (84.2 mg, 0.7 mmol) to prepare Boc-D-FsF-Boc. The half-amine dihydrochloride (CDC) in the above step was replaced by 2,2'-oxydiamine (73 mg, 0.7 mmol) to prepare Boc-D-FoF-Boc.

[0024] 2. Synthesis of compound Boc-D-FFxFF-Boc:

[0025] The first step product Boc-D-FxF-Boc (0.51 mmol) was dissolved in a round-bottom flask with trifluoroacetic acid (TFA) / DCM (6 mL) in a ratio of 1:1 and stirred at room temperature for 1 h. The solvent was evaporated on a rotary evaporator to obtain an oily residue. Ether (40 mL) was added to the flask and stirred gently. The white precipitate was separated by centrifugation. D-FcF, D-FsF or D-FoF was obtained. The crude product was used directly in the next reaction without purification.

[0026] D-Boc-Phe-OH (297 mg, 1.12 mmol), HBTU (402 mg, 1.06 mmol) and HOBt (143 mg, 1.06 mmol) were dissolved in DMF (3 mL) and stirred with a magnetic stirrer. DIPEA (780 μL, 4.5 mmol) and D-FcF, 202 mg, 0.51 mmol, D-FsF, 212 mg, 0.51 mmol or D-FoF, 203 mg, 0.51 mmol were added respectively, with an interval of 1 min, and stirred at room temperature for 24 h. The reaction mixture was poured into 50 ml of water. The white precipitate was collected by filtration and washed with water and ether. The crude product was purified by recrystallization from EtOH / H2O, dried and collected by filtration, with a yield of 80%. Boc-D-FFcFF-Boc, Boc-D-FFsFF-Boc or Boc-D-FFoFF-Boc were obtained.

[0027] 3. Synthesis of compound D-FFxFF:

[0028] Boc-FFxFF-Boc (0.51 mmol) was dissolved in a round-bottom flask with trifluoroacetic acid (TFA) / DCM (6 mL) in a ratio of 1:1 and stirred at room temperature for 1 h. The solvent was evaporated on a rotary evaporator to obtain an oily residue. Ether (40 mL) was added to the flask and stirred gently.

[0029] A white precipitate was formed and separated by centrifugation. The final product was obtained after freeze drying (yield 60%).

[0030]

[0031] D-FFcFF:1H NMR(400MHz,DMSO-d6)δ8.83(dd,J=24.5,8.4Hz,2H),8.16(d,J=3.0Hz,1H),8.04(s,6H),7.26(dt,J=13.1,6.9Hz,18H),7.09–6.89(m,2H),4.62–4.41(m,2H),4.13–3.92(m,2H),3.14–3.02(m,3H),2.93(dd,J=13.5,6.8Hz,4H),2.89–2.78(m,2H),2.65(ddd,J=22.1,13.7,8.9Hz,2H),1.40–1.20(m,4H),1.15–0.97(m,2H).13C NMR(101MHz,DMSO-d6)δ168.13(d,J=5.8Hz),137.90(s),135.20(d,J=11.6Hz),130.05(d,J=11.3Hz),129.73(d,J=9.4Hz),128.71(dd,J=24.4,2.4Hz),128.57–128.28(m),127.47(d,J=3.9Hz),126.89(d,J=6.6Hz),55.08(s),54.87(s),53.70(d,J=9.2Hz),40.56(s),40.36(s),40.15(s),39.94(s),39.73(s),39.52(s),39.31(s),38.95(d,J=6.7Hz),38.49(d,J=7.5Hz),37.19(d,J=9.2Hz),29.09(s),24.10(s).HR-MS(positive mode,m / z):Calcd.690.3893,found 691.3959for[M+H] 1+ 。

[0032]

[0033] D-FFsFF:1H NMR(400MHz,DMSO-d6)δ8.92(dd,J=24.9,7.8Hz,2H),8.45–8.24(m,2H),8.17(s,5H),7.23(d,J=16.2Hz,18H),6.95(s,2H),4.65–4.42(m,2H),4.06(s,2H),3.45(s,3H),3.30–3.08(m,5H),2.97(dd,J=19.5,12.4Hz,3H),2.86(dd,J=13.7,8.1Hz,2H),2.65(ddd,J=26.3,21.8,11.3Hz,2H),2.55–2.49(m,2H).13C NMR(101MHz,DMSO-d6)δ171.00(s),170.72(d,J=2.9Hz),168.25(s),158.96(s),158.65(s),137.92(d,J=18.1Hz),135.18(d,J=12.9Hz),129.88(dd,J=30.0,10.9Hz),128.87(d,J=4.1Hz),128.62(d,J=3.3Hz),127.52(s),126.94(d,J=7.6Hz),54.96(s),54.76(s),53.68(d,J=14.5Hz),40.58(s),40.47(d,J=21.0Hz),40.16(s),39.96(s),39.84(d,J=21.0Hz),39.56(s),39.53(s),39.35–38.75(m),38.49(d,J=21.5Hz),37.30(d,J=11.3Hz),30.71(d,J=5.3Hz).HR-MS(positive mode,m / z):Calcd.708.3457,found 709.3518for[M+H] 1+ 。

[0034]

[0035] D-FFoFF:1H NMR (400MHz, DMSO-d6) δ8.86(dd,J=31.1,8.4Hz,2H),8.30(d,J=4.0Hz,1H),8.16(dd,J=19.6,14.7Hz,6H),7.3 7–7.11(m,18H),7.06–6.91(m,2H),4.78–4.38(m,2H),4.05(s,2H),3.40–3.06(m,10H),2.99–2.55(m,7H).13C NMR(101MHz,DMSO-d6)δ129.88(dd,J=26.7,10.6Hz),128.91(d,J=4.0Hz),128.61( d,J=4.6Hz),127.56(d,J=4.3Hz),126.96(d,J=7.6Hz),69.15(s),54.64(d,J=24.6H z),53.65(d,J=14.9Hz),40.61(s),40.40(s),40.19(s),39.98(s),39.77(s),39.56 (s),39.35(s),38.88(d,J=10.3Hz),38.52(s),37.37(d,J=7.4Hz).HR-MS(positive mode,m / z):Calcd.692.3686,found 693.3740for[M+H] 1+ .

[0036] L-FFcFF was synthesized by referring to the above method.

[0037]

[0038] L-FFcFF:1H NMR(600MHz,DMSO)δ8.84(d,J=8.2Hz,2H),8.13(s,5H),8.06(t,J=5.5Hz,2H), 7.31–7.19(m,20H),4.51(dd,J=14.9,7.7Hz,2H),4.09–3.98(m,2H),3.11(d,J= 5.3Hz, 1H), 3.09 (d, J = 5.2Hz, 1H), 3.03 (td, J = 13.2, 6.7Hz, 2H), 2.94 (td, J = 13. 6,6.8Hz,6H),2.86(dd,J=13.6,7.9Hz,2H),1.32–1.20(m,5H),1.06(p,J=7.5Hz ,2H).13CNMR(151MHz,DMSO)δ169.23(s),167.08(s),157.52(s),157.31(s),13 6.70(s),134.11(s),128.99(s),128.58(s),127.84(s),127.56(s),126.49(s) ,125.84(s),115.69(s),53.82(s),52.88(s),52.48(s),37.83(s),37.55(s),3 6.32(s),28.04(s),22.96(s),17.41(s),16.10(s),11.75(s).HR-MS(positive mode,m / z):Calcd.690.3893,found 691.3948for[M+H] 1+ .

[0039] Example 2: Study on the physicochemical properties of the D-configuration diphenylalanine peptidomimetic (D-FFxFF) of the present invention

[0040] Using pyrene as a fluorescent probe, the critical micelle concentration of L-FF, L-FFcFF and D-FFxFFs was determined. Different concentrations of the above compounds were prepared in a saturated pyrene solution and the fluorescence spectrum of the saturated pyrene solution was measured. A fluorescence spectrophotometer (Horiba FluoroMax-4 spectrofluorometer) was used to measure the fluorescence intensity of pyrene at 378nm and 393nm and the concentration was plotted against the fluorescence intensity at 378nm / fluorescence intensity at 393nm. The concentration corresponding to the turning point is the minimum assembly concentration (CMC) of the compound. The results are shown in Figure 2. Figure 1 BC and Figure 2C. The results showed that the minimum assembly concentration of L-FF was 31.6μg / ml, indicating that it was prone to aggregation; the minimum assembly concentration of the L-FFcFF structure after preliminary optimization was further improved compared with L-FF, and the minimum assembly concentration was 57.7μg / ml, indicating that its water solubility was further increased. The target compound D-FFxFF after further structural optimization showed a higher minimum assembly concentration of D-FFcFF: 97.8μg / ml; D-FFoFF: 166.4μg / ml and D-FFoFF: 258.2μg / ml, indicating that the structurally optimized D-FFxFF has better water solubility.

[0041] The average hydrodynamic size of the self-assembled nanofibers was measured by dynamic light scattering (Brookhaven, USA). Before the measurement, the mother solution of the above compounds (100 mg / mL) was diluted to 0.5 mg / mL with ultrapure water. Each sample was measured three times. The results are shown in Figure 1 BC and Figure 2 D. The results showed that the hydration kinetic diameter of L-FF was 1618.4 nm, while that of L-FFcFF was 768.1 nm, indicating that the initial structural optimization significantly reduced the size of the peptide aggregates, and the target compound aggregates after further structural optimization had the smallest hydration diameter, and the hydration kinetic diameter of D-FFxFF was 40-60 nm.

[0042] Transmission electron microscopy was used to analyze the aggregate morphology of all short peptides. After diluting the above compounds to a 0.5 mg / mL solution in a centrifuge tube, take the lower and middle layers of liquid (20 μL) and drop them on a 300-mesh carbon-coated copper grid. After staining at room temperature for 2 minutes, remove the solution with a piece of absorbent paper. Add 10 μL of 3% phosphotungstic acid staining solution to the copper grid, absorb the staining solution after negative staining for 1 minute, and let the copper grid dry naturally overnight. Each copper grid was tested under a JEM-1011TEM (JEOL, Ltd., Japan) transmission electron microscope. The results are as follows: Figure 1 BC and Figure 2 D is shown in the inset diagram. The results showed that both L-FF and L-FFcFF formed relatively disordered and large fibrous aggregates, while the target compound D-FFxFF after structural optimization formed smaller and regular micellar aggregates.

[0043] The degradation resistance of L-FFcFF and D-FFcFF in the protease environment was determined by HPLC. 1 mg / ml L-FFcFF and D-FFcFF were prepared in a PBS buffer solution containing 1% BSA, and then 1.0 mg / kg proteinase K was added to the solution. The hydrolysis of the compound in the solution was detected by HPLC (Waters XBrigged C18 reverse column; gradient: 20%-95% MeCN / H2O containing 0.1% TFA, 20 min) after 3, 6, 12 and 24 hours. The results are shown in Figure 2 As shown in A. The results showed that L-FFcFF was completely degraded after 6 hours, while D-FFcFF was still not degraded after 24 hours, indicating that D-FFcFF has better biostability.

[0044] In summary, L-FF is easy to aggregate and the aggregate size is large. The aggregate size formed by L-FFcFF is significantly smaller than that of L-FF but is easily degraded by proteasomes. The peptidomimetic D-FFxFFs of the present invention is easy to aggregate, has good water solubility and is not easily degraded.

[0045] Example 3: The D-configuration diphenylalanine peptidomimetic (D-FFxFF) of the present invention induces endoplasmic reticulum autophagy

[0046] Through Bio-TEM, it was found that short peptides were enriched on the endoplasmic reticulum, and various methods showed that the short peptide D-FFxFFs could induce cells to undergo sustained endoplasmic reticulum autophagy. All cells used in this example were from the American Type Culture Collection (ATCC) (Manassas, VA, USA). MCF-7 cells were treated with D-FFcFF, D-FfoFF or D-FFsFF (70 μg / ml) for 4 hours, and then the cells were collected and fixed with 2.5% glutaraldehyde solution and then inserted into 1% agarose, fixed with osmium tetroxide, and dehydrated in an acetone series. The obtained samples were embedded in resin, polymerized at 60°C for 48 hours, made into ultrathin sections, negatively stained with 2% uranyl acetate and 2.6% lead citrate, and observed under a HT7800 (hitachi) electron microscope. The results are as shown in Figure 3 The experimental results show that a large number of peptoid aggregates are enriched in the endoplasmic reticulum fragments, indicating that the peptoids of the present invention have the ability to target the endoplasmic reticulum, providing a theoretical basis for the subsequent short peptides to induce cells to undergo sustained endoplasmic reticulum autophagy.

[0047] To visualize the process of ER autophagy in cells induced by peptoids, D-FFcFF, D-FfoFF or D-FFsFF (70 μg / ml) were incubated with MCF-7 cells for 24 hours, and then the commercial dyes ER-Tracker blue (1 μM; λex = 405 nm; λem = 415-500 nm; Thermo Fisher Scientific Inc.) and Lyso-Tracker red (1 μM; λex = 633 nm; λem = 647-710 nm; Thermo Fisher Scientific Inc.) were used for 30 minutes to observe the interaction between ER and lysosomes using a fluorescence confocal microscope (Zeiss LSM-710) at 60× oil objective. The results are shown in Figure 4 As shown in A. The experimental results showed that after 24 hours of stimulation of MCF-7 cells with the peptide D-FFxFF, the overlap coefficient between lysosomes and endoplasmic reticulum increased significantly. After 24 hours of incubation of MCF-7 cells with D-FFxFFs (70 μg / ml), a large number of endoplasmic reticulum fragments were observed in the autophagic lysosomes in Bio-TEM. Figure 4 As shown in B. MCF-7 cells were treated with D-FFcFF, D-FfoFF or D-FFsFF (70 μg / ml) for 24 hours, and then trypsin digested and collected. The collected cells were lysed with a strong cell lysis buffer RIPA (Biyuntian, P0013B), centrifuged at 12000 rpm for 20 min at 4°C, and the supernatant was removed. The protein concentration was normalized with a protein analysis reagent, and equal amounts of proteins were separated by SDS-PAGE gel and transferred to a PVDF membrane for protein immunoblotting (Western The expression levels of autophagy flux-related proteins LC3 (Abcam, ab192890, diluted 1:1000), P62 (Abcam, ab207305, diluted 1:1000) and endoplasmic reticulum autophagy receptor proteins SEC62 (GenXspan, GXP446863, diluted 1:1000), RTN3L (Proteintech, 12055-2-AP, diluted 1:1000), CCPG1 (Proteintech, 13861-1-AP, diluted 1:1000), FAM134B (Proteintech, 21537-1-AP, diluted 1:1000) and TEX264 (Proteintech, 25858-1-AP, diluted 1:1000) were analyzed by blot. The results are shown in the figure. Figure 4 C. The experimental results show that the D-FFxFFs of the present invention can induce the increase of cell autophagy flux and the high expression of endoplasmic reticulum autophagy receptor protein, which is a marker of endoplasmic reticulum autophagy activation, indicating that the peptidomimetic D-FFxFFs can successfully induce cell endoplasmic reticulum autophagy.

[0048] Example 4: Specificity of the D-configuration diphenylalanine peptoid (D-FFxFF) of the present invention in inducing endoplasmic reticulum autophagy

[0049] Taking D-FFcFF as an example, after incubation of MCF-7 cells with D-FFcFF (70 μg / ml) for 24 hours, commercial dyes Mito-Tracker green (1 μM; λex = 488 nm; λem = 500-600 nm; Thermo Fisher Scientific Inc.) and Lyso-Tracker red (1 μM; λex = 633 nm; λem = 647-710 nm; Thermo Fisher Scientific Inc.) were used for incubation for 30 minutes, and then fluorescence confocal microscopy (Zeiss LSM-710) was used to observe the interaction between mitochondria and lysosomes at 60× oil objective. The results are shown in Figure 5 As shown in A. The results showed that there was no obvious overlap of fluorescence signals between mitochondria and lysosomes, indicating that the peptidomimetic D-FFcFF did not induce mitochondrial autophagy. After incubating MCF-7 cells with D-FFcFF, D-FfoFF or D-FFsFF (70 μg / ml) for 24 hours, the cell proteins in each group were normalized using the above-mentioned protein extraction method, and then other autophagy-related proteins were analyzed by protein immunoblotting (Western blot): AIP4 (lipophagy; Abcam, ab108515, 1:1000 dilution), NUFIP1 (ribosome autophagy; Proteintech, 12055-2-AP, 1:1000 dilution), and NUFIP2 (ribosome autophagy; Proteintech, 12055-2-AP, 1:1000 dilution). FBXO27 (lysosomal autophagy; ThermoFisher, PA5-100523, 1:1000 dilution), YIPF3 (aa292-321) / YIPF4 (Golgi autophagy; GenXspan, GXP539387, 1:1000 dilution; Abcam, ab270980, 1:1000 dilution); catalase (peroxisome autophagy; Abcam, ab209211, 1:1000 dilution). The results are shown in Figure 5 As shown in B. The results show that the peptoids of the present invention do not induce other selective autophagy processes, but specifically induce endoplasmic reticulum autophagy.

[0050] Example 5: Antitumor effect of the D-configuration diphenylalanine peptidomimetic (D-FFxFF) of the present invention

[0051] All cells used in the present invention were obtained from the American Type Culture Collection (ATCC) (Manassas, VA, USA).

[0052] First, the precursor compounds L-FF, L-FFcFF and the target peptidomimetic D-FFxFFs were evaluated for their cytotoxicity against different tumor cell lines (MCF-7 using KGI RPMI-1640 medium, A375, Hela, MDA-MB-231and 4T1 cells using KGI DMEM medium. All culture media contained (10%, v / v) bovine serum, penicillin (100 units / mL) and streptomycin (50 units / mL), 37°C, 5% CO2) and normal cells (NIH3T3).

[0053] 8 × 10 4 Cell mL -1 100 μL of cell suspension of five different cancer cells Hela, A375, 4T1, MDA-MB-231, MCF-7 and one normal cell NIH3T3 was added at a density of 100 μL. When the cells grew to 80% full, fresh culture medium containing different concentrations of D-FFcFF, D-FFoFF, D-FFsFF, L-FF or L-FFcFF was added (DMSO concentration was less than 0.5%), and incubated with cancer cells and normal cells NIH3T3 at 37°C in the dark for 24 h. After incubation for 24 h, freshly prepared 3-(4,5)-dimethylthiophene azo (-z-y1)-3,5-diphenyltetrazolium (MTT, 2.5 mg mL -1 in PBS) solution was added to each well. After incubation at 37°C for 4 h, the solution in each well was removed and 150 μL DMSO was added to each well. The cells were gently shaken at room temperature for 10 min and the absorbance of MTT at 570 nm was measured using a microplate reader (Thermo Scientific Varioskan Flash) to assess cell viability. Cell viability (%) and IC 50 The values ​​were calculated based on the data from three parallel experiments. Figure 6 As shown in A. The experimental results show that D-FFxFFs showed more obvious toxicity to tumor cells than normal cells; and D-FFcFF was found to show stronger cytotoxicity, which may be due to the better lipid solubility of D-FFcFF, making it easier to enter cells. L-FF and L-FFcFF did not show obvious cytotoxicity to tumor cells and normal cells.

[0054] Female BALB / c mice were used to establish a subcutaneous xenograft tumor model. Approximately 5×10 5 4T1 cells suspended in PBS were injected into the right flank of mice. 3The mice were randomly divided into 4 groups (normal saline, D-FFxFFs (20 mg / kg)). Different drugs were injected into the tumor of tumor-bearing mice in situ to evaluate their antitumor effects (n = 4 per group). The drug was administered intratumorally once every two days. The weight of the mice was recorded and the weight was calculated every two days according to the formula (length × width 2 / 2) Calculate the tumor volume. The tumor growth curve and average tumor volume of each mouse in each group were plotted. The mice were killed on the 18th day, and the tumors were weighed and photographed. After the treatment, the tumor tissue was taken and quickly frozen with the optimal cutting temperature medium. The tumor tissue was then cut with a freezing knife, mounted on a slide, and stained with primary antibodies CCPG1 (Proteintech, 13861-1-AP, 1:100 dilution), FAM134B (Proteintech, 21537-1-AP, 1:100 dilution), RTN3L (Proteintech, 12055-2-AP, 1:100 dilution) and TEX264 (Proteintech, 25858-1-AP, 1:100 dilution) at 4°C for 12 hours. Fluorescently labeled secondary antibodies (goat anti-rabbit IgG H&L Alexa Fluro647; Abcam, ab150115, 1:500 dilution) were added, and fluorescence confocal imaging (Zeiss LSM-710) was performed to observe the slides. The results showed that the peptidomimetic D-FFxFFs of the present invention had anti-tumor effects, among which D-FFcFF had the best anti-tumor effect.

Claims

1. A peptidomimetic based on D-diphenylalanine or a pharmaceutically acceptable salt thereof, characterized in that It has the following structure: Wherein X represents C, O, S, and n represents an integer of 1 to 6.

2. The peptidomimetic according to claim 1, characterized in that The X represents C, and n represents 1, 2, or 3.

3. Use of the peptidomimetic according to claim 1 or 2 in the preparation of an endoplasmic reticulum autophagy inducer.

4. Use of the peptidomimetic according to claim 1 or 2 in the preparation of anti-tumor drugs.

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