Peptidomimetics based on d-configuration diphenylalanine and uses thereof

Abstract

The application discloses a kind of based on D configuration diphenylalanine peptidomimetic or its pharmaceutically acceptable salt, with the following structure: Wherein X represents C, O, S, n represents the integer of 1~6.The peptidomimetic described in the application has endoplasmic reticulum targeting, and can specifically induce endoplasmic reticulum autophagy.The peptidomimetic described in the application can be used as endoplasmic reticulum inducer for cell homeostasis and disease mechanism and treatment target research, promote the research and application of drug.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of cell biology, and particularly relates to a kind of D configuration based on diphenylalanine peptidomimetic and its application. BACKGROUND

[0002] Endoplasmic reticulum is the largest membrane organelle in eukaryotic cells, responsible for the synthesis and modification of proteins, lipids, phospholipids, cholesterol and oligosaccharides, and is also an important place for calcium ion storage and detoxification of endogenous and exogenous substances. Endoplasmic reticulum is an important center for protein synthesis, protein folding and post-translational modification in cells. Misfolded polypeptides can be reversed from endoplasmic reticulum to cytoplasm, where they are degraded by ubiquitin-proteasome system, which is called "endoplasmic reticulum-associated protein degradation" (ERAD). Compared with ERAD, 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, which 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, tumors, etc. Studying endoplasmic reticulum autophagy helps to reveal the pathogenesis of these diseases and provides new targets for disease treatment. In addition, by regulating endoplasmic reticulum autophagy, it may be possible to intervene in the disease process and promote the cell to return to a healthy state.

[0003] People have made great efforts 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, etc. At present, although there are several methods and drugs to induce autophagy, such as cell starvation treatment, mTOR inhibition (rapamycin), endoplasmic reticulum stress inducer (tunicamycin, dithiothreitol) and metal drug cisplatin, etc., all the above drugs have limited specificity for endoplasmic reticulum autophagy induction, and autophagy of other organelles can also be observed, thus lacking 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

[0004] The prior art research shows that the protein capable of inducing endoplasmic reticulum autophagy in cells has the characteristics of easy mutation, not easy to be degraded, easy to fold and aggregate to form a soluble small size (~100 nm) aggregate. Diphenylalanine (Diphenylalanine, L-Phe-L-Phe, FF) is the simplest aromatic dipeptide molecule, which has the property of self-assembling into nanometer structures such as spherical vesicles, nanotubes, nanowires, microporous tubes and microrods. Based on the above characteristics of diphenylalanine, a kind of peptidomimetic with high hydrophilicity, good stability and easy to fold and aggregate to form small size aggregate is obtained by structural modification, which successfully induces endoplasmic reticulum autophagy.

[0005] The specific technical scheme of the present application is as follows:

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

[0007] Another object of the present application is to provide the use of the peptidomimetic in the preparation of endoplasmic reticulum autophagy inducer.

[0008] Specifically: the peptidomimetic with a concentration of 40-80 μg / ml is incubated with cells at 37℃ for 24 h to induce obvious endoplasmic reticulum autophagy process.

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

[0010] The peptidomimetic of the present application can be used as a pharmaceutical active substance for the treatment of tumors by using the existing technology allowed administration route. The present application has the following advantages:

[0011] The present application constructs a symmetrical L-FFcFF peptidomimetic after evaluating the physical properties of L configuration diphenylalanine (L-FF) dipeptide, and finds that it has poor biological stability and large assembly size. Further optimization of the structure changes its configuration to D-FFcFF, and replaces the single atom of the middle linker to further optimize the hydrophilicity and hydrophobicity of the peptidomimetic. It is unexpectedly found that the D configuration peptidomimetic has better water solubility, is easier to aggregate, and has smaller aggregate size (~50 nm), and has better stability in biological system. The peptidomimetic of the present application can successfully induce endoplasmic reticulum autophagy, and can be used as an endoplasmic reticulum autophagy inducer for the research of endoplasmic reticulum and cell homeostasis, and the research of the pathogenesis of diseases such as neurodegenerative diseases, cardiovascular diseases and tumors by inducing endoplasmic reticulum autophagy. In addition, the present application finds that the peptidomimetic of the present application has anti-tumor activity by inducing endoplasmic reticulum autophagy. Attached Figure Description

[0012] Figure 1 Determination of the physical properties of the target structure precursor. Design concept of the target structure (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); photographs 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, bio-transmission electron microscopy (Bio-TEM) images show that D-FFcFF aggregates are enriched in the endoplasmic reticulum.

[0015] Figure 4 D-FFxFF induces autophagy in the endoplasmic reticulum. Taking D-FFcFF as an example, after incubating MCF-7 cells with 70 μg / ml D-FFcFF for 24 h, the co-localization of lysosomes and endoplasmic reticulum was measured by fluorescence confocal microscopy. The endoplasmic reticulum and lysosomes were labeled with ER-tracker (λex = 405 nm; λem = 420-500 nm) and Lyso-tracker (λex = 633 nm; λem = 650-710 nm), respectively (Figure A). Bio-TEM images of MCF-7 cells after incubation with 70 μg / ml D-FFxFF for 24 h: ER fragments in autolysosomes are labeled with arrows: ALY, autolysosome; 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 markers LC3B and P62 in MCF-7 cells after incubation 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 5: D-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 of 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 (ribosome autophagy), FBX027 (lysosome autophagy), Golgi autophagy receptors (YIPF3 / YIPF4), lipidophagy receptors (NUFIP1), and peroxisome marker (catalase) in MCF-7 cells after incubation with 70 μg / ml of D-FFcFF for 24 h (Figure B).

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

[0018] The following detailed description of the embodiments of the present application is given on the premise of the technical solutions of the present application, and detailed implementation manners and specific operation processes are given, but the protection scope of the present application is not limited to the following examples.

[0019] Example 1 Preparation of D-configuration phenylalanine peptidomimetic (D-FFcFF) according to the present application

[0020] L-configuration of diphenylalanine (L-FF) and other chemical reagents were purchased from Shanghai Biotechmed Co. Ltd. Peptoid L-FFcFF and D-FFxFFs were synthesized by modifying the literature known method (Abbas M, Lipiński WP, Nakashima KK, Huck WTS, and Spruijt E. A short 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 pL, 6.2 mmol), 1,5-pentanediamine hydrochloride (75 mg, 0.7 mmol) were added separately, with 1 min interval, 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 rinsed with water (yield 80%) to produce Boc-D-FcF-Boc.

[0023] Boc-D-FsF-Boc was produced by replacing the diamine dihydrochloride (CDC) in the above step with 2,2'-thiodiethylamine (84.2 mg, 0.7 mmol). Boc-D-FoF-Boc was produced by replacing the diamine dihydrochloride (CDC) in the above step with 2,2'-oxydiamine (73 mg, 0.7 mmol).

[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. Ethyl 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 produced. 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 separately, 1 min apart, 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 diethyl ether. The crude product was purified by recrystallization from EtOH / H2O and collected by filtration after drying, with a yield of 80%. Boc-D-FFcFF-Boc, Boc-D-FFsFF-Boc or Boc-D-FFoFF-Boc were prepared.

[0027] 3. Synthesis of compound D-FFx Fx:

[0028] Boc-FFx Fx-Boc (0.51 mmol) was dissolved in a round bottom flask with trifluoroacetic acid (TFA) / DCM (6 mL) 1 :1 ratio, stirred at room temperature for 1 h. The solvent was evaporated on a rotary evaporator, obtaining an oily residue. Ethyl 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 (400 MHz, DMSO-d6) δ 8.83 (dd, J = 24.5, 8.4 Hz, 2H), 8.16 (d, J = 3.0 Hz, 1H), 8.04 (s, 6H), 7.26 (dt, J = 13.1, 6.9 Hz, 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, 6.8 Hz, 4H), 2.89 - 2.78 (m, 2H), 2.65 (ddd, J = 22.1, 13.7, 8.9 Hz, 2H), 1.40 - 1.20 (m, 4H), 1.15 - 0.97 (m, 2H).13C NMR (101 MHz, DMSO-d6) δ 168.13 (d, J = 5.8 Hz), 137.90 (s), 135.20 (d, J = 11.6 Hz), 130.05 (d, J = 11.3 Hz), 129.73 (d, J = 9.4 Hz), 128.71 (dd, J = 24.4, 2.4 Hz), 128.57 - 128.28 (m), 127.47 (d, J = 3.9 Hz), 126.89 (d, J = 6.6 Hz), 55.08 (s), 54.87 (s), 53.70 (d, J = 9.2 Hz), 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 = 6.7 Hz), 38.49 (d, J = 7.5 Hz), 37.19 (d, J = 9.2 Hz), 29.09 (s), 24.10 (s). HR-MS (positive mode, m / z): Calcd. 690.3893, found 691.3959 for [M+H] 1+ .

[0032]

[0033] D-FFs FF: 1H NMR (400 MHz, DMSO-d6) δ 8.92 (dd, J = 24.9, 7.8 Hz, 2H), 8.45 - 8.24 (m, 2H), 8.17 (s, 5H), 7.23 (d, J = 16.2 Hz, 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, 12.4 Hz, 3H), 2.86 (dd, J = 13.7, 8.1 Hz, 2H), 2.65 (ddd, J = 26.3, 21.8, 11.3 Hz, 2H), 2.55 - 2.49 (m, 2H).13C NMR (101 MHz, DMSO-d6) δ 171.00 (s), 170.72 (d, J = 2.9 Hz), 168.25 (s), 158.96 (s), 158.65 (s), 137.92 (d, J = 18.1 Hz), 135.18 (d, J = 12.9 Hz), 129.88 (dd, J = 30.0, 10.9 Hz), 128.87 (d, J = 4.1 Hz), 128.62 (d, J = 3.3 Hz), 127.52 (s), 126.94 (d, J = 7.6 Hz), 54.96 (s), 54.76 (s), 53.68 (d, J = 14.5 Hz), 40.58 (s), 40.47 (d, J = 21.0 Hz), 40.16 (s), 39.96 (s), 39.84 (d, J = 21.0 Hz), 39.56 (s), 39.53 (s), 39.35 - 38.75 (m), 38.49 (d, J = 21.5 Hz), 37.30 (d, J = 11.3 Hz), 30.71 (d, J = 5.3 Hz). HR-MS (positive mode, m / z): Calcd. 708.3457, found 709.3518 for [M+H] 1+ .

[0034]

[0035] D-FFoFF: 1H NMR (400 MHz, DMSO-d6) δ 8.86 (dd, J = 31.1, 8.4 Hz, 2H), 8.30 (d, J = 4.0 Hz, 1H), 8.16 (dd, J = 19.6, 14.7 Hz, 6H), 7.37 - 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 (101 MHz, DMSO-d6) δ 129.88 (dd, J = 26.7, 10.6 Hz), 128.91 (d, J = 4.0 Hz), 128.61 (d, J = 4.6 Hz), 127.56 (d, J = 4.3 Hz), 126.96 (d, J = 7.6 Hz), 69.15 (s), 54.64 (d, J = 24.6 Hz), 53.65 (d, J = 14.9 Hz), 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 = 10.3 Hz), 38.52 (s), 37.37 (d, J = 7.4 Hz). HR-MS (positive mode, m / z): Calcd. 692.3686, found 693.3740 for [M+H] 1+ .

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

[0037]

[0038] L-FFcFF:1H NMR (600 MHz, DMSO) δ 8.84 (d, J = 8.2 Hz, 2H), 8.13 (s, 5H), 8.06 (t, J = 5.5 Hz, 2H), 7.31 - 7.19 (m, 20H), 4.51 (dd, J = 14.9, 7.7 Hz, 2H), 4.09 - 3.98 (m, 2H), 3.11 (d, J = 5.3 Hz, 1H), 3.09 (d, J = 5.2 Hz, 1H), 3.03 (td, J = 13.2, 6.7 Hz, 2H), 2.94 (td, J = 13.6, 6.8 Hz, 6H), 2.86 (dd, J = 13.6, 7.9 Hz, 2H), 1.32 - 1.20 (m, 5H), 1.06 (p, J = 7.5 Hz, 2H).13C NMR (151 MHz, DMSO) δ 169.23 (s), 167.08 (s), 157.52 (s), 157.31 (s), 136.70 (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), 36.32 (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 691.3948 for [M+H] 1+ .

[0039] Example 2: Physicochemical property study of D-configuration diphenylalanine peptoid (D-FFcFF)

[0040] The critical micelle concentration of L-FF, L-FFcFF and D-FFcFF was determined using pyrene as a fluorescent probe. Different concentrations of the above compounds were prepared in saturated pyrene solution and the fluorescence spectrum of the saturated pyrene solution was determined. The fluorescence intensity of pyrene at 378 nm and 393 nm was determined using a fluorescence spectrophotometer (Horiba FluoroMax-4 spectrofluorometer), and the concentration and the fluorescence intensity at 378 nm / fluorescence intensity at 393 nm were plotted. The turning point corresponds to the minimum assembly concentration (CMC) of the compound. The results are shown in Table 1. Figure 1 B-C and Figure 2The results show that the lowest assembly concentration of L-FF is 31.6 μg / ml, indicating that it is prone to aggregation; the lowest assembly concentration of L-FFcFF structure after preliminary optimization is further improved than that of L-FF, and the lowest assembly concentration is 57.7 μg / ml, indicating that the water solubility is further increased; the target compounds D-FFcFF, D-FFoFF and D-FFoFF after further structure optimization show higher lowest assembly concentrations, respectively 97.8 μg / ml, 166.4 μg / ml and 258.2 μg / ml, indicating that the D-FFcFF after structure optimization has better water solubility.

[0041] The average hydrodynamic size of the self-assembled nanofiber was measured by dynamic light scattering method (Brookhaven, USA). The mother liquor of the above compound was diluted to 0.5 mg / mL with ultrapure water before determination. Each sample was measured three times. The results are shown in Figure 1 B-C and Figure 2 D. The results show that the hydrated kinetic diameter of L-FF is 1618.4 nm, while the hydrated kinetic diameter of L-FFcFF is 768.1 nm, indicating that the preliminary structure optimization significantly reduces the size of the peptide aggregate; the aggregate of the target compound after further structure optimization has the smallest hydrated diameter, and the hydrated kinetic diameter of D-FFcFF is 40-60 nm.

[0042] The aggregate morphology of all short peptides was analyzed by transmission electron microscopy. After the solution of the above compound in the centrifuge tube was diluted to 0.5 mg / mL, the middle and lower layer liquid (20 μL) was added to the 300 mesh carbon-coated copper grid, and then it was stained at room temperature for 2 minutes, after which the solution was removed with a water-absorbing paper. 10 μL of 3% phosphotungstic acid staining solution was added to the copper grid, and then it was negatively stained for 1 min, after which the staining solution was absorbed, and the copper grid was naturally dried overnight. Each copper grid was detected under a JEM-1011 TEM (JEOL, Ltd., Japan) transmission electron microscope. The results are shown in Figure 1 B-C and Figure 2 D. The results show that L-FF and L-FFcFF both form relatively disordered and large-sized fibrous aggregates, while the target compounds D-FFcFF after structure optimization form smaller and regular micellar aggregates.

[0043] The anti-degradation ability of L-FFcFF and D-FFcFF in protease environment was determined by HPLC. L-FFcFF and D-FFcFF were configured at 1 mg / ml in PBS buffer solution containing 1% BSA, then 1.0 mg / kg protease K was added into the solution, and the hydrolysis of the compounds in the solution was detected by HPLC (Waters XBrigged C18 reverse column; gradient: 20%-95% MeCN / H2O containing 0.1% TFA, 20 min) at 3, 6, 12 and 24 h, respectively. The results are shown in Figure 2 The results show that L-FFcFF has been completely degraded after 6 hours, while D-FFcFF has not been degraded after 24 hours, indicating that D-FFcFF has better biological stability.

[0044] In summary, L-FF is prone to aggregation 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 proteasome, and the peptidomimetic D-FFcFF described in the application is prone to aggregation, has good water solubility and is not easily degraded.

[0045] Example 3: D-configuration diphenylalanine peptidomimetic (D-FFcFF) induces endoplasmic reticulum autophagy

[0046] It is found by Bio-TEM that the short peptide is enriched on the endoplasmic reticulum and various methods show that the short peptide D-FFcFFs can induce the cell to undergo a sustained endoplasmic reticulum autophagy process. All the cells used in this example are from American Type Culture Collection (ATCC) (Manassas, VA, USA). After MCF-7 cells were treated with D-FFcFF, D-FfoFF or D-FFsFF (70 μg / ml) for 4 h, the cells were collected and fixed with 2.5% glutaraldehyde solution, then inserted into 1% agarose, fixed after osmium tetroxide, and dehydrated with acetone series. The obtained sample was embedded in resin, polymerized at 60°C for 48 h, and ultrathin sections were prepared, and negatively stained with 2% uranyl acetate and 2.6% lead citrate, and observed under an HT7800 (hitachi) electron microscope. The results are shown in Figure 3 The experimental results show that a large number of peptidomimetic aggregates are enriched on the fragments of the endoplasmic reticulum, indicating that the peptidomimetic described in the application has endoplasmic reticulum targeting ability, which provides a theoretical basis for the subsequent short peptide to induce the cell to undergo a sustained endoplasmic reticulum autophagy.

[0047] To visualize the endoplasmic reticulum autophagy process of cells after the induction of the peptidomimetic, the D-FFcFF, D-FfoFF or D-FFsFF (70 μg / ml) was incubated with the MCF-7 cells for 24 hours, and then the interaction between the endoplasmic reticulum and lysosomes was observed by using the commercial dyes ER-Tracker blue (1 μM; λex=405 nm; λem=415-500 nm; Thermo Fisher Scientific) and Lyso-Tracker red (1 μM; λex=633 nm; λem=647-710 nm; Thermo Fisher Scientific) dyes for 30 min, and then the fluorescence confocal microscope (Zeiss LSM-710) with a 60x oil lens was used. The results are shown in Figure 4 A. The experimental results show that after the peptidomimetic D-FFxFF stimulates the MCF-7 cells for 24 hours, the overlapping coefficient of the lysosomes and the endoplasmic reticulum is obviously increased. After the D-FFxFFs (70 μg / ml) is incubated with the MCF-7 cells for 24 hours, a large number of endoplasmic reticulum fragments are observed in the autophagic lysosomes by Bio-TEM. The results are shown in Figure 4 B. After the D-FFcFF, D-FfoFF or D-FFsFF (70 μg / ml) is used to treat the MCF-7 cells for 24 hours, the cells are collected by trypsin digestion, the collected cells are lysed by using the strong cell lysis RIPA (Bi Yun Tian, P0013B), centrifuged at 12000 rpm for 20 min at 4°C to remove the supernatant, and then the protein concentration is normalized by using the protein analysis reagent. The equal amount of protein is separated by using the SDS-PAGE gel, transferred to the PVDF membrane, and then the expression amount of the autophagy flux related proteins LC3 (Abeam, ab192890, 1:1000 dilution), P62 (Abeam, ab207305, 1:1000 dilution), and the endoplasmic reticulum autophagy receptor proteins SEC62 (GenXspan, GXP446863, 1:1000 dilution), RTN3L (Proteintech, 12055-2-AP, 1:1000 dilution), CCPG1 (Proteintech, 13861-1-AP, 1:1000 dilution), FAM134B (Proteintech, 21537-1-AP, 1:1000 dilution) and TEX264 (Proteintech, 25858-1-AP, 1:1000 dilution) is analyzed by using the Western blot. The results are shown in Figure 4 C. The experimental results show that the D-FFxFFs described in the present application can induce the increase of the cell autophagy flux and the high expression of the endoplasmic reticulum autophagy receptor protein, which is the marker of the endoplasmic reticulum autophagy activation, indicating that the peptidomimetic D-FFxFFs can successfully induce the endoplasmic reticulum autophagy in the cells.

[0048] Example 4: Specificity of D-configuration diphenylalanine peptidomimetic (D-FFcFF) to induce endoplasmic reticulum autophagy

[0049] As an example of D-FFcFF, MCF-7 cells were incubated with D-FFcFF (70 pg / ml) for 24 hours, and then incubated with commercial dyes Mito-Tracker green (1 mM; ex = 488 nm; em = 500-600 nm; Thermo Fisher Scientific) and Lyso-Tracker red (1 mM; ex = 633 nm; em = 647-710 nm; Thermo Fisher Scientific) for 30 min. The interaction between mitochondria and lysosomes was observed using a fluorescence confocal microscope (Zeiss LSM-710) with a 60x oil lens. The results are shown in Figure 5 A. The results show that there is no obvious overlap of fluorescence signals between mitochondria and lysosomes, indicating that the peptidomimetic D-FFcFF does not induce the process of mitochondrial autophagy. After incubating MCF-7 cells with D-FFcFF, D-FfoFF or D-FFsFF (70 pg / ml) for 24 hours, the proteins in each group were normalized by the above method, and then the proteins related to other autophagy modes were analyzed by Western blot: AIP4 (lipophagy; Abeam, ab108515, 1: 1000 dilution), NUFIP1 (ribosome autophagy; Proteintech, 12055-2-AP, 1: 1000 dilution), FBX027 (lysosome autophagy; Thermo Fisher, PA5-100523, 1: 1000 dilution), YIPF3 (aa292-321) / YIPF4 (Golgi autophagy; GenXspan, GXP539387, 1: 1000 dilution; Abeam, ab270980, 1: 1000 dilution); catalase (peroxisome autophagy; Abeam, ab209211, 1: 1000 dilution). The results are shown in Figure 5 B. The results show that the peptidomimetic of the present application does not cause other selective autophagy processes, and specifically induces endoplasmic reticulum autophagy.

[0050] Example 5: Anti-tumor effect of D-configuration diphenylalanine peptidomimetic (D-FFcFF) of the present application

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

[0052] The cytotoxicity of precursor compounds L-FF, L-FFcFF and target peptoid D-FFcFF against different tumor cell lines (MCF-7 cells were cultured in Kay's RPMI-1640 medium, A375, Hela, MDA-MB-231 and 4T1 cells were cultured in Kay's DMEM medium. All the media contained (10%, v / v) bovine serum, penicillin (100 units / mL) and streptomycin (50 units / mL), 37 °C, 5% CO2 and normal cells (NIH3T3) were evaluated.

[0053] In each well of a 96-well plate, 8 x 10 4 cells mL -1 Five different cancer cell lines Hela, A375, 4T1, MDA-MB-231, MCF-7 and one normal cell line NIH3T3 were seeded at a density of 5 x 103cells per well in a 96-well plate. After the cells were grown to 80% confluence, fresh medium containing D-FFcFF, D-FFoFF, D-FFsFF, L-FF or L-FFcFF at different concentrations (DMSO concentration was less than 0.5%) was added to each well and incubated with the cancer cells and normal cells NIH3T3 at 37 °C in the dark for 24 h. After 24 h incubation, 100 μL of freshly prepared 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-diphenyltetrazolium bromide (MTT, 2.5 mg mL -1 in PBS) solution was added to each well. After 4 h incubation at 37 °C, the solution in each well was removed and 150 μL of DMSO was added to each well, which was shaken gently at room temperature for 10 min. Cell viability was evaluated by measuring the absorbance of MTT at 570 nm using a microplate reader (Thermo Scientific Varioskan Flash). Cell survival rate (%) and IC 50 values were calculated from the data of three parallel experiments. The results are shown in Figure 6 A. The experimental results show that D-FFcFFs exhibit more obvious toxicity to tumor cells than to normal cells; and it is found that D-FFcFF exhibits stronger cytotoxicity, which may be due to the better liposolubility of D-FFcFF, making it easier to enter the cells. L-FF and L-FFcFF do not exhibit obvious cytotoxicity to tumor cells and normal cells.

[0054] A subcutaneous xenograft tumor model was established using female BALB / c mice. About 5 x 10 5 cells of 4T1 cells were suspended in PBS and injected into the right side of the mice. When the tumor volume reached 40 mm 3The mice were randomly divided into 4 groups (normal saline, D-FFxFFs (20 mg / kg)) at the same time. Different drugs were injected in situ into tumor-bearing mice, and the anti-tumor effect was evaluated (n=4 in each group). Intratumoral administration was performed every two days. The body weight of the mice was recorded, and the tumor volume was calculated every two days according to the formula (length x width 2 / 2). The tumor growth curve of each mouse in each group and the average tumor volume were plotted. The mice were sacrificed on the 18th day, and the tumors were weighed and photographed. After the treatment, the tumor tissue was rapidly frozen with the optimal cutting temperature medium. Then the tumor tissue was cut with a frozen knife and mounted on a glass slide, and the 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) were used for staining at 4 DEG C for 12 hours. The fluorescence-labeled secondary antibody (goat anti-rabbit IgG H&L Alexa Fluro647; Abeam, ab150115, 1:500 dilution) was added, and the fluorescence confocal imaging (Zeiss LSM-710) was used to observe the glass slide. The results show that the peptidomimetic D-FFxFFs has an anti-tumor effect, and the D-FFcFF has the best anti-tumor effect.

Claims

1. A peptide based on D-configuration diphenylalanine or a pharmaceutically formable salt thereof, characterized in that... The structure of the peptidomimetic is shown below: , or .

2. Use of the peptidomimetic according to claim 1 for the preparation of an antitumor drug, the tumor being selected from cervical cancer, melanoma or breast cancer.

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