SORT1-based lysosome targeting chimera and application thereof

Abstract

The invention belongs to the technical field of chemical synthetic drugs, and particularly relates to an SORT1-based lysosome targeting chimera and application thereof, and the structure of the SORT1-based lysosome targeting chimera is shown as a formula (I). The technical problem to be solved by the invention is to provide a novel SORT1-based lysosome targeting chimera construction mode which is applied to degradation of different pathogenic target proteins. The lysosome targeting chimera provided by the invention provides a new choice for development and application of novel anti-cancer drugs and anti-Parkinson's disease drugs. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to the technical field of chemically synthesized drugs, and more particularly, to a novel lysosome-targeting chimera based on SORT1, and its preparation method and application. Background Art

[0002] Targeted protein degradation (TPD) technology is a new technology that has emerged in recent years and can downregulate pathogenic target proteins. As the most maturely developed one in the TPD technology, Proteolysis Targeting Chimeras (PROTACs) can achieve efficient degradation of pathogenic target proteins in cells by utilizing the cell's inherent ubiquitin-proteasome system (UPS). However, not all pathogenic proteins of many diseases are in cells, and a large number of pathogenic proteins are located on the cell membrane or secreted outside the cell. The effect of using PROTAC technology to downregulate pathogenic membrane proteins and extracellular proteins may be limited. In 2020, the research group of Carolyn R. Bertozzi at Stanford University reported a brand-new TPD technology, named Lysosome Targeting Chimeras (LYTACs). This technology utilizes the cell's inherent endosome / lysosome pathway and can achieve the degradation of different membrane proteins and extracellular proteins, creating new possibilities for the treatment of related diseases caused by abnormal membrane proteins or extracellular proteins.

[0003] The chemical composition of LYTAC molecules can be divided into three parts: ① ligands targeting lysosome-targeting receptors (LTRs); ② intermediate linkers; ③ ligands targeting pathogenic target proteins. In the currently publicly reported literature, the LTRs selected when designing LYTAC molecules are mostly the cation-independent mannose 6-phosphate receptor (CI-M6PR) widely expressed in human tissues and the asialoglycoprotein receptor (ASGPR) specifically expressed in liver tissues. In addition to these two LTRs, other LTRs with endocytic ability and lysosome targeting also include transferrin receptor 1 (TfR1), scavenger receptor A (SR-A), integrin receptor, and sorting protein 1 receptor (SORT1, also called Sortilin), etc. Although some LYTAC molecules have been reported currently, LYTAC technology is still in its infancy. Exploring novel LYTAC molecules can not only enrich the existing LYTAC molecular library but also provide new options for the treatment of related diseases caused by abnormal membrane proteins or extracellular proteins.

[0004] As a LTR with high transport efficiency, SORT1 protein plays an important role in transporting target molecules to lysosomal degradation and is an ideal candidate LTR for designing LYTAC molecules. Considering that there are few reports on SORT1-based LYTAC molecules, the development of such LYTAC molecules has important research value. In view of this, the present invention is specifically proposed. Summary of the Invention

[0005] One object of the present invention is to provide a lysosome-targeting chimera molecule based on SORT1, which can utilize the cell's inherent endosome / lysosome pathway to down-regulate pathogenic target proteins.

[0006] Another object of the present invention is to provide the application of the compound.

[0007] In the first aspect of the present invention, there is provided a novel lysosome-targeting chimera molecule based on SORT1, a lysosome-targeting chimera based on SORT1, which comprises: a small molecule ligand targeting the lysosome-targeting receptor SORT1, a ligand targeting a pathogenic target protein, and a linker chain, and the small molecule ligand targeting SORT1 is connected to the ligand targeting the pathogenic target protein through the linker chain.

[0008] The small molecule ligand targeting SORT1 is connected to the ligand T targeting the pathogenic target protein through the linker chain L to form a lysosome-targeting chimera, and the chimera molecule has the structure shown in formula (I):

[0009]

[0010] Wherein:

[0011] Optionally, the ligand T targeting the pathogenic target protein in the compound of formula (I) can be a small molecule, a polypeptide or an antibody;

[0012] Optionally, the linker chain L is selected from:

[0013] (n is an integer from 1 to 10), (n is an integer from 1 to 20), (n is an integer from 1 to 20).

[0014] Preferably, the linker chain L is mainly selected from:

[0015]

[0016] The pathogenic target proteins include cell surface molecules or extracellular molecules. The cell surface molecules include growth factor receptors such as epidermal growth factor receptor (EGFR), immune checkpoint molecules such as programmed death ligand 1 (PD-L1), etc.

[0017] The lysosome-targeting chimera as described above, wherein the extracellular molecule mainly refers to a secreted protein that accumulates in the disease, such as α-synuclein, etc.

[0018] In some embodiments of the present invention, the compound of formula (I) is selected from Table 1:

[0019] Table 1

[0020]

[0021]

[0022] In a second aspect of the present invention, the application of the lysosome-targeting chimera in the preparation of a drug for treating or assisting in the treatment of a disease related to the pathogenic target protein; optionally, the disease includes any one or more of lung cancer, liver cancer, and Parkinson's disease. The application of an above-mentioned compound of formula (I) in the preparation of an anti-tumor drug and an anti-Parkinson's disease drug. In some embodiments of the present invention, the tumor is lung cancer or liver cancer.

[0023] The beneficial effects of the present invention are as follows: The present invention provides a novel lysosome-targeting chimera based on SORT1. The chimera binds to the pathogenic target protein through a ligand fragment at one end of its structure and binds to the SORT1 protein through a ligand fragment at the other end of its structure, and can transport the pathogenic target protein to the lysosome for specific degradation. The lysosome-targeting chimera provided by the present invention can achieve efficient degradation of pathogenic target proteins including cell membrane proteins such as PD-L1, EGFR, and extracellular proteins such as α-synuclein, etc., providing a new option for the treatment of cancer and Parkinson's disease. Brief Description of the Drawings

[0024] Figure 1 . Schematic diagram of the mechanism of action of the lysosome-targeting chimera provided by the present invention.

[0025] Figure 2 . Mass spectrometry identification of anti-PD-L1 antibody and representative lysosome-targeting chimeras S1, S9, and S10. (a, b) MALDI-TOF MS results of anti-PD-L1 antibody and S1; (c) HRMS results of S9; (d) MALDI-TOF MS results of S10.

[0026] Figure 3.The degradation effects of the lysosome-targeting chimeras (S1-S9) provided by the present invention on membrane proteins PD-L1 and EGFR. (a-c) Degradation of PD-L1 by S1-S7 and S9 in LLC cells; (d, e) Time- and concentration-dependent degradation of EGFR by S8 in H1975 cells; (f) Degradation of PD-L1 by S2 in Hepa 1-6 cells.

[0027] Figure 4 .The lysosome-targeting chimeras (S2, S8, S10, and S11) provided by the present invention promote the localization of PD-L1, EGFR, and α-synuclein to lysosomes. (a) S2 promotes the co-localization of PD-L1 with lysosomes in LLC cells; (b) S8 promotes the co-localization of EGFR with lysosomes in H1975 cells; (c) S10 and S11 promote the co-localization of α-synuclein with lysosomes in N2a cells. Detailed implementation manners

[0028] The technical solutions of the present invention will be described in detail below in conjunction with embodiments. It should be noted that the specific embodiments below are for illustrative purposes only, and the present invention is not limited by the embodiments listed below.

[0029] Example 1 Chemical synthesis method of compound S1

[0030]

[0031] Step 1) Prepare the intermediate tert-butyl (4-(3-(4-nitrophenyl)propylamino)phenyl)carbamate (2).

[0032] Add HATU (2.92 g, 7.69 mmol, 1.5 eq), N,N-diisopropylethylamine (2.68 mL, 15.37 mmol, 3.0 eq), and DMF (10 mL) to 3-(4-nitrophenyl)propionic acid (1, 1.0 g, 5.12 mmol, 1.0 eq). The mixture is stirred at room temperature for 10 minutes. Then add tert-butyl (4-aminophenyl)carbamate (10.7 g, 5.12 mmol, 1.0 eq), and the reaction continues to stir overnight at room temperature. After the reaction is completed, the reaction mixture is extracted 3 times with ethyl acetate / water (v / v, 50 mL), the organic layer is collected and washed 3 times with saturated sodium chloride, and then dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The obtained residue is purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 1 / 1) to obtain the intermediate tert-butyl (4-(3-(4-nitrophenyl)propylamino)phenyl)carbamate (2, 1.77 g, yield: 90%). HRMS (ESI) m / z calcd for [M+Na] + 408.1530, found 408.1532.

[0033] Step 2) Preparation of intermediate N-(4-aminophenyl)-3-(4-nitrophenyl)propanamide (3)

[0034] To intermediate 2 (1.0 g, 2.59 mmol, 1.0 eq) was added trifluoroacetic acid (1 mL, 12.97 mmol, 5.0 eq) and dichloromethane (15 mL). The mixture was stirred at 0 °C for 5 h until the reaction was completed. Then the reaction mixture was concentrated under reduced pressure to obtain intermediate N-(4-aminophenyl)-3-(4-nitrophenyl)propanamide (3, 720 mg, yield: 97%), which was used directly in the next step without further purification. HRMS (ESI) m / z calcd for [M+H] + 286.1187, found 286.1187.

[0035] Step 3) Preparation of (S)-1-methyl-N-(4-((3-(4-nitrophenyl)propanamido)phenyl)pyrrolidine-2-carboxamide (4)

[0036] To intermediate 3 (500 mg, 1.75 mmol, 1.0 eq) was added HATU (1.0 g, 2.63 mmol, 1.5 eq), N,N-diisopropylethylamine (0.92 mL, 5.26 mmol, 3.0 eq) and DMF (10 mL). The mixture was stirred at room temperature for 10 min. Then N-methyl-L-proline monohydrate (257.9 mg, 1.75 mmol, 1.0 eq) was added, and the mixture was stirred at room temperature overnight. After extraction with ethyl acetate / water (v / v, 50 mL), the organic layer was washed 3 times with saturated sodium chloride and dried over anhydrous magnesium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 20 / 1) to obtain intermediate (S)-1-methyl-N-(4-((3-(4-nitrophenyl)propanamido)phenyl)pyrrolidine-2-carboxamide (4, 542 mg, yield: 78%). HRMS (ESI) m / z calcd for [M+H] + 397.1871, found 397.1868.

[0037] Step 4) Preparation of intermediate (S)-N-(4-(3-(4-aminophenyl)propylamino)phenyl)-1-methylpyrrolidine-2-carboxamide (5)

[0038] To intermediate 4 (500 mg, 1.26 mmol, 1.0 eq) was added iron powder (352 mg, 6.31 mmol, 5.0 eq) and glacial acetic acid (10 mL). The mixture was stirred overnight at room temperature. Then the iron powder was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 10 / 1) to give the intermediate (S)-N-(4-(3-(4-aminophenyl)propylamino)phenyl)-1-methylpyrrolidin-2-carboxamide (5, 406 mg, yield: 88%). 1 H NMR (400 MHz, DMSO-d6) δ ppm 9.81 (s, 1H), 9.59 (s, 1H), 7.57 (d, J = 8.8 Hz, 2H), 7.49 (d, J = 8.8 Hz, 2H), 6.88 (d, J = 8.4 Hz, 2H), 6.47 (d, J = 8.4 Hz, 2H), 4.84 (s, 2H), 3.11 - 3.07 (m, 1H), 2.90 - 2.67 (m, 4H), 2.49 - 2.47 (m, 1H), 2.35 - 2.28 (m, 4H), 2.17 - 2.09 (m, 1H), 1.81 - 1.72 (m, 3H). 13 C NMR (101 MHz, DMSO-d6) δ ppm 171.73, 170.40, 146.65, 134.96, 133.81, 128.62×2, 128.07, 119.94×2, 119.25×2, 113.97×2, 69.25, 55.99, 41.11, 38.66, 30.26×2, 23.46. HRMS (ESI) m / z calcd for [M+H] + 367.2129, found 367.2130。

[0039] Step 5) Preparation of intermediate (S)-N-(4-(3-(4-(1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3,6,9,12,15,18-hexaoxaheneicosan-21-ylamino)phenyl)propylamino)phenyl)-1-methylpyrrolidin-2-carboxamide (6)

[0040] To intermediate 5 (100 mg, 0.27 mmol, 1.0 eq), HATU (155 mg, 0.41 mmol, 1.5 eq), N,N - diisopropylethylamine (142 μL, 0.82 mmol, 3.0 eq) and DMF (10 mL) were added. The mixture was first stirred at room temperature for 10 minutes. Then maleimide - hexaethylene glycol - carboxylic acid (118 mg, 0.27 mmol, 1.0 eq) was added, and stirring was continued at room temperature overnight. After the reaction was completed, the reaction mixture was extracted 3 times with ethyl acetate / water (v / v, 50 mL). After collecting the organic layer, it was washed, dried and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 20 / 1) to obtain intermediate (S)-N-(4-(3-(4-(1-(2,5 - dioxo - 2,5 - dihydro - 1H - pyrrol - 1 - yl)-3,6,9,12,15,18 - hexaoxaicosan - 21 - amino)phenyl)propylamino)phenyl)-1 - methylpyrrolidine - 2 - carboxamide (6, 111 mg, yield: 52%). HRMS(ESI) m / z calcd for [M+Na] + 804.3791, found 804.3793.

[0041] Step 6) Preparation of compound S1

[0042] To 1×PBS solution (377 μL), anti - PD - L1 antibody (78 μL, 6.4 mg / mL) and tris(2 - carboxyethyl)phosphine hydrochloride (40 μL, 10 mg / mL in PBS) were added respectively. First, the mixture was reacted with shaking in a rotator for 50 minutes. Then intermediate 6 (5.21 μL, 10 mg / mL in DMSO) was added, and the mixture continued to react with shaking for 5 hours. After that, the reaction mixture was purified by a Sephadex G - 25 purification column to obtain the final product S1 for further biological tests. S1: MALDI - TOF MS: m / z found 150044.70. Anti - PD - L1 antibody: MALDI - TOF MS: m / z found 147777.30.

[0043] Chemical synthesis method of compound S2 in Example 2

[0044] The preparation method of compound S2 is similar to that of compound S1. S2: MALDI - TOF MS: m / z found 150138.26.

[0045] Chemical synthesis method of compound S3 in Example 3

[0046] The preparation method of compound S3 is similar to that of compound S1. S3: MALDI-TOF MS: m / z found 149317.40.

[0047] Example 4 Chemical synthesis method of compound S4

[0048] The preparation method of compound S4 is similar to that of compound S1. S4: MALDI-TOF MS: m / z found 149581.56.

[0049] Example 5 Chemical synthesis method of compound S5

[0050] The preparation method of compound S5 is similar to that of compound S1. S5: MALDI-TOF MS: m / z found 150568.05.

[0051] Example 6 Chemical synthesis method of compound S6

[0052] The preparation method of compound S6 is similar to that of compound S1. S6: MALDI-TOF MS: m / z found 149490.68.

[0053] Example 7 Chemical synthesis method of compound S7

[0054] The preparation method of compound S7 is similar to that of compound S1. S7: MALDI-TOF MS: m / z found 149793.73.

[0055] Example 8 Chemical synthesis method of compound S8

[0056] The preparation method of compound S8 is similar to that of compound S1. S8: MALDI-TOF MS: m / z found 151335.59. Anti-EGFR antibody (cetuximab, also called erlotinib): MALDI-TOF MS: m / z found 150420.36.

[0057] Example 9 Chemical synthesis method of compound S9

[0058]

[0059] Step 1) Prepare intermediate (S)-(12-((4-(3-((3-(1-methylpyrrolidin-2-carboxamido)phenyl)amino)-3-oxopropyl)phenyl)amino)-12-oxododecyl)carbamic acid tert-butyl ester (8)

[0060] To 12-(BOC-amino)dodecanoic acid (86 mg, 0.272 mmol, 1.0 eq) was added HATU (156 mg, 0.409 mmol, 1.5 eq), N,N-diisopropylethylamine (142 μL, 0.818 mmol, 3.0 eq) and DMF (10 mL). The mixture was stirred at room temperature for 10 minutes. Then the intermediate (S)-N-(3-(3-(4-aminophenyl)propylamino)phenyl)-1-methylpyrrolidin-2-carboxamide (7, 100 mg, 0.272 mmol, 1.0 eq) was added and the reaction was continued to stir overnight at room temperature. After extraction with ethyl acetate / water (v / v, 50 mL), the collected organic layer was washed with saturated sodium chloride three times, then dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 20 / 1) to give the intermediate (S)-(12-((4-(3-((3-(1-methylpyrrolidin-2-carboxamido)phenyl)amino)-3-oxopropyl)phenyl)amino)-12-oxododecyl)carbamic acid tert-butyl ester (8, 153 mg, yield: 85%). HRMS(ESI) m / z calcd for [M+H] + 664.4433, found 664.4433。

[0061] Step 2) Preparation of intermediate (S)-N-(3-(3-(4-(12-aminododecanamido)phenyl)propionamido)phenyl)-1-methylpyrrolidin-2-carboxamide (9)

[0062] To intermediate 8 (150 mg, 0.225 mmol, 1.0 eq) was added trifluoroacetic acid (84 μL, 1.13 mmol, 5.0 eq) and dichloromethane (10 mL). The mixture was stirred at 0 °C for 5 hours until the reaction was completed. Then the reaction mixture was concentrated under reduced pressure and the residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 10 / 1) to give the intermediate (S)-N-(3-(3-(4-(12-aminododecanamido)phenyl)propionamido)phenyl)-1-methylpyrrolidin-2-carboxamide (9, 121 mg, yield: 95%). 11H NMR (400 MHz, DMSO-d6) δ ppm 9.90 (s, 1H), 9.76 (s, 1H), 9.61 (s, 1H), 7.94 (s, 1H), 7.48 - 7.54 (d, J = 8.0 Hz, 1H), 7.33 - 7.13 (m, 6H), 3.10 - 3.07 (m, 1H), 2.92 - 2.88 (m, 2H), 2.83 (d, J = 7.2 Hz), 2.57 (d, J = 7.2 Hz, 2H), 2.35 - 2.11 (m, 7H), 2.00 - 1.33 (m, 10H), 1.29 - 1.22 (m, 14H).

[0063] Step 3) Preparation of Compound S9

[0064] To the raw material (S)-1-(2,6-dimethoxy-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)benzyl)piperidine-2-carboxylic acid (BMS-1, 184 mg, 0.177 mmol, 1.0 eq) was added HATU (101 mg, 0.266 mmol, 1.5 eq), N,N-diisopropylethylamine (93 μL, 0.532 mmol, 3.0 eq) and DMF (10 mL). The mixture was stirred at room temperature for 10 minutes. Then intermediate 9 (100 mg, 0.177 mmol, 1.0 eq) was added, and the reaction was continued to stir overnight at room temperature. After extraction 3 times with ethyl acetate / water (v / v, 50 mL), the organic layer was washed 3 times with saturated sodium chloride, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 15 / 1) to obtain (S)-1-(2,6-dimethoxy-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)benzyl)-N-(12-((4-((3-((S)-1-methylpyrrolidine-2-carboxamido)phenyl)amino)-3-oxopropyl)phenyl)amino)-12-oxododecyl)piperidine-2-carboxamide (S9, 135 mg, yield: 75%). 1 1H NMR (400 MHz, CD3OD) δ ppm 7.83 (s, 1H), 7.45 - 6.33 (m, 16H), 6.33 (s, 2H), 5.14 (s, 2H), 3.80 (s, 6H), 3.34 (s, 2H), 3.28 - 3.17 (m, 3H), 2.98 - 2.91 (m, 3H), 2.62 (d, J = 7.6 Hz, 2H), 2.45 - 2.19 (m, 12H), 2.01 - 1.80 (m, 4H), 1.70 - 1.51 (m, 8H), 1.39 - 1.28 (m, 16H). 1313C NMR(101MHz,CD3OD)δppm 176.4,175.0,174.6,173.6,161.6×2,159.6,144.4,143.4,140.3,139.5,138.1,138.0,136.9,135.5,131.0,130.4×2,130.1,129.7×2,129.3,129.2×2,128.0,126.5,121.5×2,117.3,117.0,113.2,96.8,92.6×2,72.0,70.8,70.5,57.5,56.3×2,52.0,41.7,40.2,39.8,38.0,32.7,32.2,31.8,30.5×3,30.4×2,30.3×2,30.2,27.9,26.9,24.9,23.7,16.4,14.4.HRMS(ESI)m / z calcd for[M+Na] + 1043.5981,found 1043.5982。

[0065] Synthetic Method of Compound S10 in Example 10

[0066]

[0067] Step 1) Preparation of Intermediate (S)-N-(3-(3-(4-(6-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)phenyl)propanamido)phenyl)-1-methylpyrrolidine-2-carboxamide (10)

[0068] To 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoic acid (58 mg, 0.272 mmol, 1.0 eq), HATU (156 mg, 0.409 mmol, 1.5 eq), N,N-diisopropylethylamine (142 μL, 0.818 mmol, 3.0 eq) and DMF (10 mL) were added. After the reaction mixture was stirred at room temperature for 10 minutes, the intermediate (S)-N-(3-(3-(4-aminophenyl)propylamino)phenyl)-1-methylpyrrolidine-2-carboxamide (7, 100 mg, 0.272 mmol, 1.0 eq) was added, and stirring was continued overnight at room temperature. After extraction 3 times with ethyl acetate / water (v / v, 50 mL), the organic layer was collected and washed 3 times with saturated sodium chloride, then dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 15 / 1) to give the intermediate (S)-N-(3-(3-(4-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)phenyl)propionamido)phenyl)-1-methylpyrrolidine-2-carboxamide (10, 101 mg, yield: 66%). HRMS(ESI) m / z calcd for [M+H] + 560.2868, found 560.2871。

[0069] Step 2) Preparation of compound S10

[0070] First, the starting polypeptide (amino acid sequence: CGVLYVGSKTR, 10 mg) was dissolved in PBS solution (5 mL), and then the intermediate 10 (5.2 mg in 5 mL THF, 1.0 eq) was added to the mixture. The reaction mixture was stirred overnight. Subsequently, THF was removed by distillation under reduced pressure, and the residue was dried in a vacuum freeze dryer for two days to obtain compound S11 (14.0 mg, yield: 95%). MALDI-TOF MS: m / z calcd for [M+H] + 1741.909, found 1741.758。

[0071] Chemical synthesis method of compound S11 in Example 11

[0072] The preparation method of compound S11 is similar to that of compound S10. S11: MALDI-TOF MS: m / z calcd for [M+H] + 1741.909, found 1741.756。

[0073] Example 12 Western blot experiment

[0074] To verify the actual degradation effect of the lysosome-targeting chimeras provided by the present invention on different membrane proteins, the present invention selected two different membrane proteins, including PD-L1 and EGFR, as the research objects, and used Western blot experiments for preliminary verification.

[0075] After the cells were treated with the compounds provided by the present invention for a certain period of time, total proteins were extracted from the cultured cells by using a strong RIPA lysis buffer (Epizyme, Shanghai, catalog number: PC101) containing protease inhibitors. The lysate was then sonicated at low temperature and centrifuged at 15,000 rpm for 15 minutes. The supernatant was quantified using a BCA protein quantification kit (Yeasen, China, catalog number: 20201ES76) and adjusted as needed. The protein samples were boiled in SDS loading buffer (Epizyme, Shanghai, catalog number: LT101) for 10 minutes. Then, the proteins were separated by polyacrylamide gel electrophoresis (PAGE), and the proteins were transferred to a 0.22 μm polyvinylidene difluoride (PVDF) membrane (Merck KGaA, Germany, catalog number: IPVH00010). The membrane was blocked with TBST buffer containing 5% non-fat milk powder at room temperature for 1 hour, and then incubated with the corresponding primary antibody overnight at 4°C. After washing three times with TBST for 10 minutes each, the membrane was incubated with an HRP-labeled secondary antibody at room temperature for 1 hour. After washing three times again with TBST for 10 minutes each, the membrane was treated with an enzyme-linked chemiluminescence (ECL) reagent (Vazyme, Shanghai, catalog number: E433-01), and the signal was immediately detected using a chemiluminescence imager (BioRad).

[0076] The results of the Western blot experiment are as Figure 3 shown, and the following conclusions can be drawn: (1) In the mouse Lewis lung cancer cell line LLC, compared with compounds S1, S3-S7, and S9, compound S2 provided by the present invention exhibited the best PD-L1 degradation effect after treating LLC cells at a concentration of 40 μM for 48 hours ( Figure 3 in a-c); (2) In the human lung adenocarcinoma cell line H1975, the degradation effect of compound S8 provided by the present invention on EGFR was time-dependent and concentration-dependent, and the effect reached the best when treating H1975 cells at a concentration of 40 μM for 48 hours ( Figure 3 in d and e); (3) In the mouse hepatoma cell line Hepa 1-6, compound S2 provided by the present invention had a strong PD-L1 degradation effect after treating at a concentration of 40 μM for 48 hours ( Figure 3 f).

[0077] Example 13 Immunofluorescence Experiment

[0078] To further verify that the lysosome-targeting chimeras provided by the present invention have the ability to mediate the entry of target proteins into lysosomes for degradation, the present invention selected representative compounds S2, S8, S10, and S11 as research objects, and used immunofluorescence experiments to investigate their uptake ability of PD-L1, EGFR, and α-synuclein respectively, and analyzed the actual co-localization of target proteins and lysosomes.

[0079] Cells were seeded in 12-well plates at a density of 80,000 cells per well 24 hours before the experiment and cultured overnight. After the cells were treated with the compounds provided by the present invention, the cell samples were washed with DPBS and treated with 4% paraformaldehyde, and fixed at room temperature for 10 minutes. Then the cell samples were further blocked with 2% bovine serum albumin at room temperature for 1 hour. The treated cell samples were incubated with the corresponding primary antibody overnight at 4°C. Subsequently, the samples were washed three times with PBS (5 minutes each time), and then incubated with the secondary antibody at room temperature for 1 hour. Finally, the cells were observed using a confocal microscope (IXplore SpinSR, Olympus). The captured images were analyzed by ImageJ software (Ver.1.52s). Among them, LLC cells were incubated with 40 nM S2 and H1975 cells were incubated with 40 nM S8 for 48 hours, and then lysosomes were fluorescently stained with anti-LAMP1 antibody; N2a cells were co-incubated with 100 nM LysoTracker, 50 nM α-Syn-Cy5, and 10 μM S10 or S11 for 1 hour, and then lysosome co-localization analysis was performed.

[0080] The results of the immunofluorescence experiment are as Figure 4 shown, and the following conclusions can be drawn: (1) In LLC cells, the incubation of compound S2 promoted the co-localization of PD-L1 and lysosomes, indicating that it can efficiently mediate the entry of PD-L1 into lysosomes; (2) In H1975 cells, the incubation of compound S8 promoted the co-localization of EGFR and lysosomes, indicating that it can efficiently mediate the entry of EGFR into lysosomes; (3) In the mouse neuroblastoma cell line N2a, both compounds S10 and S11 promoted the significant co-localization of α-synuclein and lysosomes, indicating that both of these compounds can effectively mediate the entry of α-synuclein into lysosomes.

Claims

1. A lysosomal targeting chimera based on SORT1, characterized in that: include: A small molecule ligand targeting the lysosomal targeting receptor SORT1, a ligand targeting a pathogenic target protein and a connecting chain. The small molecule ligand targeting the lysosomal targeting receptor SORT1 is connected to the ligand targeting the pathogenic target protein through the connecting chain.

2. The lysosomal targeting chimera based on SORT1 according to claim 1, characterized in that It is the structure shown in formula (I): Wherein: T is a ligand targeting a pathogenic target protein, which is a small molecule, peptide or antibody; L is a connecting chain selected from the following structures: The first connection chain: n is an integer from 1 to 10; The second connection chain: The third connection chain: The fourth type of connection chain: n is an integer from 1 to 20; The fourth type of connection chain: n is an integer from 1 to 20.

3. The lysosomal targeting chimera based on SORT1 according to claim 1 or 2, characterized in that The pathogenic target protein is a cell surface molecule or an extracellular molecule.

4. The lysosomal targeting chimera based on SORT1 according to claim 3, characterized in that The cell surface molecule is a growth factor receptor or an immune checkpoint molecule.

5. The lysosomal targeting chimera based on SORT1 according to claim 4, characterized in that The growth factor receptor is epidermal growth factor receptor, and the immune checkpoint molecule is programmed cell death-ligand 1.

6. The lysosomal targeting chimera based on SORT1 according to claim 3, characterized in that The extracellular molecule is a secreted protein that accumulates in the disease.

7. The lysosomal targeting chimera based on SORT1 according to claim 6, characterized in that The secretory protein is α-synuclein.

8. The lysosomal targeting chimera based on SORT1 according to claim 1, characterized in that A compound having the structure shown in S1 to S11; 9. Use of the SORT1-based lysosomal targeting chimera according to any one of claims 1 to 8 in the preparation of a drug for treating or assisting in the treatment of a disease associated with the pathogenic target protein.

10. The use according to claim 9, characterized in that: The disease is any one or more of lung cancer, liver cancer, and Parkinson's disease.

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