A nanobody, ligand and nanobody-targeted chimera of gpx4 and applications

Abstract

The application discloses a kind of nanobody of GPX4, ligand and nanobody targeted chimera and application, belong to medical biological engineering technical field.Nanobody of GPX4 has the amino acid sequence shown in one of SEQ ID No.2~SEQ ID No.5, on the nanobody of targeting GPX4 DBCO-linker-CRBN ligand is coupled, and the construction of nanobody targeted chimera is carried out.The nanobody targeted chimera constructed in the application can degrade GPX4 by targeting, the degree of intracellular GPX4 degradation is not less than 70%, and the iron death of tumor cell is induced by degrading GPX4, to reach the effect of anti-tumor.Experiments prove that the application is strong in targeting, and degradation efficiency is high;Construction method is novel, and reaction condition is mild;With transmembrane capacity, suitable for intracellular target;Mechanism is clear, and the effect of inducing tumor cell iron death is remarkable.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medical biological engineering, and particularly relates to a nanobody of GPX4, a DBCO-linker-CRBN ligand for binding E3 ubiquitin ligase, a nanobody targeted chimera and application. BACKGROUND

[0002] Ferroptosis is a newly discovered iron-dependent and regulated cell death mode different from apoptosis, necrosis and other forms of cell death. More and more studies have shown that ferroptosis plays an important role in tumor, neurodegenerative diseases, tissue ischemia-reperfusion injury and other pathological processes. Glutathione peroxidase 4 (GPX4) is a key regulatory factor in the process of ferroptosis, which plays an important role in maintaining lipid metabolism and redox homeostasis.

[0003] Existing studies show that the active site of GPX4 protein is located on the surface of the protein, and the structure is relatively flat, lacking a typical small molecule ligand binding pocket, which makes it difficult to design small molecule inhibitors. The currently reported GPX4 inhibitors (such as RSL3, ML162) generally have poor selectivity, low bioavailability and other problems, which cannot meet the needs of clinical application. SUMMARY

[0004] The purpose of the present application is to provide a nanobody of GPX4, a ligand targeting E3 ubiquitin ligase CRBN and a nanobody targeted chimera, and to provide specific applications of the nanobody targeted chimera to make up for the shortcomings of the prior art.

[0005] Protein targeted degradation is a new drug action mode, which uses the protein degradation system of cells to induce the degradation of target proteins. Proteolysis-targeting chimeras (PROTAC) is a new emerging strategy for disease treatment based on target protein degradation closely related to ubiquitination process, which has the characteristics of small dosage, low toxicity, targeting of traditional non-druggable targets and long-lasting efficacy. In recent years, with the introduction of biological macromolecules (such as polypeptides and antibodies) into the PROTAC system, its targeting range has been significantly expanded. GPX4 is an intracellular protein, and its degradation pathway is highly compatible with the PROTAC technology. Currently, there is no related public report on the construction of GPX4 targeted degradation agent using nanobody as a targeted recognition module.

[0006] In order to achieve the above purpose, the present application is realized by the following scheme:

[0007] A nanobody of GPX4, the nanobody having an amino acid sequence as shown in any one of SEQ ID No. 2~ SEQ ID No. 5.

[0008] Preferably, the nanobody is 4C G26pAzF -R10.

[0009] A gene encoding a nanobody, the gene having a sequence as shown in any one of SEQ ID No. 7~ SEQ ID No. 10, wherein the gene encoding a nanobody as shown in SEQ ID No. 2, the gene having a sequence as shown in SEQ ID No. 7; the gene encoding a nanobody as shown in SEQ ID No. 3, the gene having a sequence as shown in SEQ ID No. 8; the gene encoding a nanobody as shown in SEQ ID No. 4, the gene having a sequence as shown in SEQ ID No. 9; the gene encoding a nanobody as shown in SEQ ID No. 5, the gene having a sequence as shown in SEQ ID No. 10.

[0010] A recombinant vector, wherein the vector contains a gene as shown in any one of SEQ ID No. 7~ SEQ ID No. 10.

[0011] Preferably, the vector is pET28a(+).

[0012] A vector containing tRNA / tRNA synthetase, wherein the vector has a gene sequence as shown in SEQ ID No. 11.

[0013] A recombinant cell, wherein the cell contains a vector of a nanobody-encoding gene as shown in any one of SEQ ID No. 7~ SEQ ID No. 10 and a vector containing a tRNA / tRNA synthetase system as shown in SEQ ID No. 11.

[0014] Preferably, the recombinant cell is an E. coli cell.

[0015] A ligand applied to a nanobody-targeted chimera, the ligand being a DBCO-linker-CRBN ligand, being a compound as shown in Formula 1 or a pharmaceutically acceptable salt thereof:

[0016] Formula I;

[0017] wherein X is a saturated or unsaturated straight-chain hydrocarbon group of 1~16 carbon atoms, an oxygen chain, a phenyl group, a heterocyclic group, or any one of the following groups:

[0018] ;

[0019] wherein n = 0-14, m = 0-6, the heterocyclic group is one of piperazinyl, pyrrolyl, pyrazolyl, furanyl, thienyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyridyl, pyrimidinyl, pyrazinyl, or pyridazinyl;

[0020] wherein R1is an amino group, a carbon atom, an oxygen atom, a carbonyl group, a piperazinyl group, a piperidinyl group, a heterocyclic group, or any of the following groups:

[0021] ;

[0022] wherein z = 0-12, the heterocyclic group is one of piperazinyl, pyrrolyl, pyrazolyl, furanyl, thienyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyridyl, pyrimidinyl, pyrazinyl, or pyridazinyl;

[0023] wherein R2is a hydrogen atom or a halogen.

[0024] Preferably, the pharmaceutically acceptable salt is an organic acid salt or an inorganic acid salt.

[0025] Preferably, the inorganic acid is hydrochloric acid, sulfuric acid, phosphoric acid, diphosphoric acid, hydrobromic acid, or nitric acid; the organic acid is acetic acid, maleic acid, fumaric acid, tartaric acid, succinic acid, lactic acid, p-toluenesulfonic acid, salicylic acid, oxalic acid, tannic acid, citric acid, trifluoroacetic acid, malic acid, or benzenesulfonic acid salt.

[0026] Preferably, the DBCO-linker-CRBN ligand is one of:

[0027] Compound DBCO-C9: N -(3-(azadibenzocyclooctyne-1-yl)-3-oxopropyl)-10-(4-(2-(2,6-dioxopiperidin-3-yl)-6-fluoro-1,3-dioxoisoindolin-5-yl)piperazin-1-yl)-10-oxodecanamide; DBCO-C9 corresponds to linker 1; Figure 2

[0028] Compound DBCO-C16: N -(3-(azadibenzocyclooctyne-1-yl)-3-oxopropyl)-10-(4-((4-(2-(2,6-dioxopiperidin-3-yl)-6-fluoro-1,3-dioxoisoindolin-5-yl)piperazin-1-yl)methyl)piperidin-1-yl)-10-oxodecanamide; DBCO-C16 corresponds to linker 2; Figure 3

[0029] Compound DBCO-C17: N ​​- (3-(azadibenzocyclooctyne- 1 -yl)-3-oxopropyl)-12-(4-((4-(2-(2,6-dioxopiperidin-3-yl)-6-fluoro-l,3-dioxoisoindolin-5-yl)piperazin-l-yl)methyl)piperidin-l-yl)-12- oxododecanamide; DBCO-C17 corresponds to Figure 3 linker 2;

[0030] Compound DBCO-C23: N - (3-(azadibenzocyclooctyne- 1 -yl)-3-oxopropyl)-14-(4-((4-(2-(2,6-dioxopiperidin-3-yl)-6-fluoro-l,3-dioxoisoindolin-5-yl)piperazin-l-yl)methyl)piperidin-l-yl)-14- oxotetradecanamide; DBCO-C23 corresponds to Figure 3 linker 2;

[0031] Compound DBCO-C24: N - (3-(azadibenzocyclooctyne- 1 -yl)-3-oxopropyl)-16-(4-((4-(2-(2,6-dioxopiperidin-3-yl)-6-fluoro-l,3-dioxoisoindolin-5-yl)piperazin-l-yl)methyl)piperidin-l-yl)-16- oxohexadecanamide; DBCO-C24 corresponds to Figure 3 linker 2;

[0032] Compound DBCO-C25: N - (3-(azadibenzocyclooctyne- 1 -yl)-3-oxopropyl)-8-(4-(((2-(2,6-dioxopiperidin-3-yl)-l,3-dioxoisoindol-5-yl)oxy)methyl)-l H - 1,2,3-triazol- 1 -yl)octanamide; DBCO-C25 corresponds to Figure 3 linker 3;

[0033] Compound DBCO-C26: N - (3-(azadibenzocyclooctyne- 1 -yl)-3-oxopropyl)-10-(4-(((2-(2,6-dioxopiperidin-3-yl)-l,3-dioxoisoindol-5-yl)oxy)methyl)-l H - 1,2,3-triazol- 1 -yl)decanamide; DBCO-C26 corresponds to Figure 3 linker 3;

[0034] Compound DBCO-C27: N- (3-(azadibenzocyclooctyne-1-yl)-3-oxopropyl)-12-(4-(((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-5-yl)oxy)methyl)-1 H - 1,2,3-triazol-1-yl) dodecanamide; DBCO-C27 corresponds to Figure 3 linker 3;

[0035] Compound DBCO-C50: N - (3-(azadibenzocyclooctyne-1-yl)-3-oxopropyl)-14-(4-(((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-5-yl)piperidin-1-yl)methyl)amino)tetradecanamide; DBCO-C50 corresponds to Figure 4 linker 4.

[0036] The nanobody targeting chimera is a nanobody of an amino acid sequence shown in any one of SEQ ID No. 2~ SEQ ID No. 5 coupled with DBCO-linker-CRBN ligand; the preparation strategy is to introduce unnatural amino acids at specific sites of the nanobody, and then couple the azido group of the DBCO-linker-CRBN ligand and the unnatural amino acid of the nanobody.

[0037] The nanobody targeting chimera is used for preparing a GPX4 specific degradation agent.

[0038] The nanobody is used for preparing an antitumor drug.

[0039] Compared with the prior art, the present application has the following beneficial effects:

[0040] Strong targeting and high degradation efficiency: the present application uses a screened GPX4 specific nanobody, which can efficiently and specifically recognize and bind intracellular GPX4 protein, significantly improving the target protein targeting degradation efficiency, and avoiding the problems of poor selectivity and large side effects of traditional small molecule inhibitors.

[0041] Novel construction method and mild reaction conditions: the present application introduces unnatural amino acids with azido groups into the nanobody through genetic code expansion technology, and realizes efficient covalent coupling of the nanobody and the CRBN ligand by using click chemistry, avoiding the loss of protein activity and non-specific modification that may be caused by conventional chemical modification, and ensuring the functional integrity and controllability of the nanobody targeting chimera.

[0042] With the ability to cross the membrane, suitable for intracellular targets: by fusing the cell-penetrating peptide R10 at the C-terminal of the nanobody, the ability of the macromolecular chimera to cross the cell membrane is effectively realized, successfully delivered to the intracellular and induced the degradation of the target protein.

[0043] Clear mechanism of action, significant effect of inducing tumor cell ferroptosis: the nanobody targeting chimera constructed in the application can degrade GPX4 by targeted degradation, the degree of degradation of intracellular GPX4 is not less than 70%, and the ferroptosis of tumor cells is induced by degrading GPX4, so as to achieve the effect of resisting tumors. The application provides a new scheme for inducing tumor cell death, and has good pharmacological effect and application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0044] Figure 1 GPX4 nanobody SDS-PAGE diagram of amino acid sequence shown as SEQ ID No. 2~ SEQ ID No. 5; A is the SDS-PAGE diagram of the amino acid sequence shown as SEQ ID No. 2, B is the SDS-PAGE diagram of the amino acid sequence shown as SEQ ID No. 3, C is the SDS-PAGE diagram of the amino acid sequence shown as SEQ ID No. 4, and D is the SDS-PAGE diagram of the amino acid sequence shown as SEQ ID No. 5.

[0045] Figure 2 Chemical structural formula of linker 1 and DBCO-linker 1-CRBN ligand.

[0046] Figure 3 Chemical structural formula of linker 2 and DBCO-linker 2-CRBN ligand.

[0047] Figure 4 Chemical structural formula of linker 3 and DBCO-linker 3-CRBN ligand.

[0048] Figure 5 Chemical structural formula of linker 4 and DBCO-linker 4-CRBN ligand.

[0049] Figure 6 Immunoblotting diagram of GPX4 degradation by nanobody targeting chimera.

[0050] Figure 7 Cell activity data diagram of HT1080 tumor cell ferroptosis induced by nanobody targeting chimera. DETAILED DESCRIPTION

[0051] The application will be further described in conjunction with the accompanying drawings and specific embodiments, which are only used to explain the application and are not intended to limit the scope of the application.

[0052] The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents, etc. used are commercially available reagents and materials unless otherwise specified.

[0053] Example 1: 4C N××pAzF Plasmid design and protein expression purification of R10 mutants

[0054] 1. Plasmid design and transformation

[0055] 4C N××pAzF Plasmid design of R10 mutants: In the GPX4 non-binding region of Nanobody 4C-R10 (amino acid sequence as shown in SEQ ID No. 1), the codons encoding the 14th, 26th, 41st and 66th amino acids at the N-terminus were respectively mutated to a stop codon TAG (the amino acid sequences after mutation of the 14th, 26th, 41st and 66th amino acids correspond to SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, respectively; wherein U in the sequence table represents p-azidophenylalanine). The mutated nanobody plasmid was transformed into BL21 (DE3) competent cells containing pEVOL-pAzF plasmid (containing orthogonal tRNA / tRNAse system). In the culture medium added with p-azidophenylalanine, protein expression was induced by adding L-arabinose and IPTG. At this time, the aminoacyl tRNA synthetase encoded by pEVOL-pAzF can specifically recognize p-azidophenylalanine and insert it into the TAG stop codon site, thereby introducing a non-natural amino acid with an azido group at a specific site of the nanobody.

[0056] 4C N××pAzFTransformation of pEVOL-pAzF plasmid: 1 μL pEVOL-pAzF plasmid was transformed into BL21 (DE3) competent cells, and a single colony was picked and cultured in a medium containing 25 μg / mL chloramphenicol. 0.4 mL of the bacterial solution was transferred to 40 mL of liquid culture medium containing chloramphenicol resistance, and cultured at 37 °C and 220 rpm for 3 h until the OD600 value reached between 0.5 and 0.6. Then the bacterial solution was placed on ice for 10 min, centrifuged at 4 °C and 4000 rpm / min for 10 min, and the supernatant was discarded. The collected cell pellet was suspended with 10 mL of 0.1 mol / L calcium chloride in an ice bath, and the cell suspension was ice-bathed for 30 min. Then the cell suspension was centrifuged again at 4 °C and 4000 rpm / min for 10 min, and the cells were resuspended with 2 mL of 0.1 mol / L calcium chloride in an ice bath to prepare competent cells. The prepared BL21 (DE3) competent cells containing the pEVOL-pAzF plasmid were aliquoted at 100 μL per tube and stored at -80 °C.

[0057] 1 μL 4C N××pAzF The prepared BL21 (DE3) competent cells containing the pEVOL-pAzF plasmid were added with 1 μL of the R10 plasmid and ice-bathed for 30 min, and the competent cells were heat-shocked in a constant temperature water bath at 42 °C for 90 s; the heat-shocked competent cells were ice-bathed again for 2 min. 500 μL of LB liquid medium without antibiotics was added to the competent cells, and the cells were recovered at 37 °C and 220 rpm for 1 h. 100 μL of the bacterial solution was uniformly coated on LB solid medium containing ampicillin (100 μg / mL) and chloramphenicol (25 μg / mL) double resistance with a coating rod, and after the bacterial solution was completely absorbed by the medium, the culture was sealed and inverted in a constant temperature drying oven at 37 °C and cultured for 12 h.

[0058] 2, 4C N××pAzF Expression of R10 mutants

[0059] The transformed single colony was picked and inoculated in 5 mL of LB liquid medium containing ampicillin and chloramphenicol, and cultured at 37 °C and 220 rpm for 8 h. The cultured bacterial solution was expanded to 150 mL of medium at a ratio of 1:100, and cultured under the same conditions until the OD (600) value reached 0.6. The bacterial solution was ice-bathed in an ice-water mixture for 30 min, and 0.3 mM IPTG and 1 mM 4-Azido-L-phenylalanine were added for induction at 18 °C and 220 rpm for 16 h.

[0060] 3, 4C N××pAzF R10 Purification of mutants

[0061] After expression is completed, the bacteria are centrifuged at 4 °C, 8000 rpm for 10 min, the supernatant is discarded and the bacterial precipitate is collected, and the bacterial precipitate is frozen at -80 °C for 30 min. 20 mL of lysis solution (8 mM Na2HPO4, 2 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl, 10% glycerol, 1 mg / mL lysozyme, 1 mM PMSF, pH = 8.0) is added to the bacterial precipitate, mixed evenly, and the ultrasonic cell crusher is used to ultrasonically crush at a power of 100 W for 15 min. The bacterial solution after ultrasonic is centrifuged at 4 °C, 8000 rpm for 1 h, and the protein supernatant is collected.

[0062] The collected protein supernatant is reacted with Ni-NTA agarose purification resin at 4 °C for 2 h, which allows the target protein containing a poly-histidine tag to bind to the agarose purification resin. Protein purification buffer B containing different imidazole concentrations (imidazole concentrations are 0, 50, 75, 100, 200, 300, 400 mM) is prepared using protein purification buffer A (8 mM Na2HPO4, 2 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl, 10% glycerol, pH 8.0). Purification buffer with 0-75 mM imidazole is used for elution of impurities, and purification buffer with 100-400 mM imidazole is used for elution of the target protein. 12% polyacrylamide gel is used to separate and characterize the eluate, and the eluate with relatively pure target protein is selected for subsequent experiments.

[0063] Finally, nanobody non-natural amino acid mutants with a purity greater than 90% are obtained, which are named 4C A14pAzF -R10 (amino acid sequence as shown in SEQ ID No. 2), 4C G26pAzF -R10 (amino acid sequence as shown in SEQ ID No. 3), 4C P41pAzF -R10 (amino acid sequence as shown in SEQ ID No. 4), 4C G66pAzF -R10 (amino acid sequence as shown in SEQ ID No. 5), and the protein electrophoresis results are as shown in Figure 1 , wherein 4C G26pAzF -R10 nanobody has the optimal purity and expression amount.

[0064] Example 2: Synthesis of DBCO-linker-CRBN ligand

[0065] The synthesis process of the target compounds DBCO-C9 / DBCO-C16~C17 / DBCO-C23~C24 is as shown below:

[0066] The synthesis process of the target compounds DBCO-C9 / DBCO-C16~C17 / DBCO-C23~C24 is as shown below: .

[0067] Example 1. N Preparation of (3-(azadiphenylcyclooctyne-l-yl)-3-oxopropyl)-10-(4-(2-(2,6-dioxopiperidin-3-yl)-6-fluoro-l,3-dioxoisoindolin-5-yl)piperazin-l-yl)-10- oxodecanamide DBCO-C9

[0068] (1) Preparation of intermediate 2: 2-(2,6-dioxopiperidin-3-yl)-5,6-difluoroisoindoline-l,3-dione

[0069] The starting material 4,5-difluorophthalic anhydride (2.03 g, 11.03 mmol), 3-amino-2,6-piperidinedione hydrochloride (1.81 g, 11.03 mmol) and sodium acetate (1.18 g, 14.34 mmol) were dissolved in 25 mL of acetic acid and placed under reflux at 100 o C for 8 h, monitored by TLC. After the reaction was complete, it was cooled to room temperature, water was added to precipitate a large amount of solid, which was filtered, the filter cake was washed with water and dried to give a dark gray solid 2.70 g in 83% yield.

[0070] (2) Preparation of intermediate 3: 4-(2-(2,6-dioxopiperidin-3-yl)-6-fluoro-l,3-dioxoisoindolin-5-yl)piperazine-l-carboxylic acid tert-butyl ester

[0071] Intermediate 2 (2.30 g, 7.82 mmol) and mono-Boc piperazine (1.42 g, 22.60 mmol) were dissolved in 15 mL of N-methylpyrrolidone (NMP), then DIPEA (5.04 g, 39.10 mmol) was added and the mixture was placed under reflux at 90 o C for 3 h, monitored by TLC. After the reaction was complete, it was cooled to room temperature, water was added to precipitate a large amount of solid, which was filtered, the filter cake was washed with water and dried, then column chromatography was performed to give a yellow solid 2.35 g in 65% yield.

[0072] (3) Preparation of intermediate 4: 10-(4-(2-(2,6-dioxopiperidin-3-yl)-6-fluoro-l,3-dioxoisoindolin-5-yl)piperazin-l-yl)-10-oxodecanoic acid tert-butyl ester

[0073] Compound 3 (120 mg, 0.258 mmol) was dissolved in a mixed solution of DCM / TFA (4 mL / 2 mL) at room temperature, and the reaction was allowed to proceed for 1 h, which was monitored by TLC. After the reaction was completed, the solvent was evaporated. It was redissolved in DMF, and 10-(tert-butoxy)-10-oxodecanoic acid (76 mg, 0.281 mmol), HATU (148 mg, 0.389 mmol), and DIPEA (50 mg, 68 μL, 0.300 mmol) were added, and the reaction was allowed to proceed at room temperature for 4 h. After the reaction was completed, the reaction solution was extracted with EA / H2O three times, the organic layers were combined, washed with saturated sodium chloride, dried over anhydrous sodium sulfate, and evaporated to dryness. Column chromatography (DCM:MeOH = 100:3) was used to separate the product, which was obtained as a yellow solid in a yield of 112 mg (70%).

[0074] (4) Preparation of the target compound DBCO-9 (structure shown as Figure 2

[0075] Intermediate 4 (112 mg, 0.187 mmol) was dissolved in 4 mL of DCM, and 2 mL of TFA was added. The reaction was allowed to proceed at room temperature for 1 h, which was monitored by TLC. After the reaction was completed, the solvent was evaporated. It was redissolved in DMF, and 3-aminopropanoylazadiphenylcyclooctyne (56 mg, 0.205 mmol), HATU (106 mg, 0.279 mmol), and DIPEA (36 mg, 48 μL, 0.279 mmol) were added. The reaction was allowed to proceed at room temperature for 4 h. After the reaction was completed, the reaction solution was extracted with EA / H2O three times, the organic layers were combined, washed with saturated sodium chloride, dried over anhydrous sodium sulfate, and evaporated to dryness. Column chromatography (DCM:MeOH = 100:4) was used to separate the product, which was obtained as a yellow solid in a yield of 58 mg (56%).

[0076] Example 2. N Preparation of (3-(azadiphenylcyclooctyne-1-yl)-3-oxopropyl)-10-(4-((4-(2-(2,6-dioxopiperidin-3-yl)-6-fluoro-1,3-dioxoisoindolin-5-yl)piperazin-1-yl)methyl)piperidin-1-yl)-10-oxodecanamide DBCO-C16 (structure shown as Figure 3

[0077] (1) Preparation of intermediate 5: tert-butyl 4-((4-(2-(2,6-dioxopiperidin-3-yl)-6-fluoro-1,3-dioxoisoindolin-5-yl)piperazin-1-yl)methyl)piperidine-1-carboxylate

[0078] ​​Intermediate 3 (2 g, 4.346 mmol) was dissolved in DCM (6 mL), 0 o TFA (3 mL) was added slowly under ice-cold condition, 0 o The reaction was monitored by TLC. The reaction was complete, concentrated at low temperature, redissolved in DMF (4 mL), compound 4-(iodomethyl)piperidine-1-carboxylic acid tert-butyl ester (1.7 g, 5.20 mmol), K2CO3(1.8 g, 12.9 mmol) was added at room temperature, and the reaction was allowed to proceed overnight at room temperature. The reaction was complete, extracted with EA / H2O three times, the organic layer was combined, washed with saturated sodium chloride, dried over anhydrous sodium sulfate, and the solvent was evaporated. The product was isolated by column chromatography to obtain 580 mg of yellow solid with a yield of 25%.

[0079] (2) Preparation of intermediate 6a: compound 3 in Example 1(3) was replaced with compound 5, and the other steps were the same as in Example 1(3) to prepare intermediate 6a, which was a yellow solid with a yield of 65%.

[0080] (3) Preparation of target compound DBCO-C16: compound 4 in Example 1(4) was replaced with compound 6a, and the other steps were the same as in Example 1(4) to prepare compound DBCO-C16, which was a yellow solid with a yield of 55%.

[0081] Example 3. Preparation of compound DBCO-C17 (structure shown as Figure 3 in the figure): the steps were the same as in Example 2, and the yield was 57%.

[0082] Example 4. Preparation of compound DBCO-C23 (structure shown as Figure 3 in the figure): the steps were the same as in Example 2, and the yield was 58%.

[0083] Example 5. Preparation of compound DBCO-C24 (structure shown as Figure 3 in the figure): the steps were the same as in Example 2, and the yield was 62%.

[0084] The synthesis process of target compounds DBCO-C25~C27 is shown in the following figure:

[0085] .

[0086] Example 6. N (3-(azadibenzocyclooctyne-1-yl)-3-oxopropyl)-8-(4-(((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-5-yl)oxy)methyl)-1 H Preparation of compound DBCO-C25

[0087] (1) Preparation of intermediate 8: 2-(2,6-dioxopiperidin-3-yl)-5-(prop-2-yn-1-yloxy)isoindole-1,3-dione

[0088] The starting material 2-(2,6-dioxopiperidin-3-yl)-5-hydroxyisoindoline-1,3-dione (200 mg, 0.73 mmol) was dissolved in DMF (4 mL), and then K2CO3 (201 mg, 1.46 mmol) was added and stirred for 10 min. Then KI (132 mg, 0.80 mmol) and 3-bromopropyne (96 mg, 0.80 mmol) were added, and the reaction was carried out at room temperature overnight, and TLC was used for monitoring. After the reaction was completed, the reaction liquid was introduced into ice water to precipitate the solid, and the filter cake was obtained by suction filtration, dried and separated by column chromatography to prepare intermediate 8, a white solid, with a yield of 85%.

[0089] (2) Preparation of intermediate 10a: tert-butyl 8-azidocarboxylate

[0090] The starting material tert-butyl 8-bromooctanoate (500 mg, 1.79 mmol) was dissolved in DMF (3 mL), and NaN3 (240 mg, 3.58 mmol) was added, and the reaction was carried out at 60 o C for 2 h, and TLC was used for monitoring. After the reaction was completed, EA / H2O was used for extraction three times, the organic layers were combined, washed with saturated sodium chloride, dried with anhydrous sodium sulfate, and the solvent was evaporated to dryness, and column chromatography was used for separation to obtain an oily liquid 208 mg, with a yield of 48%.

[0091] (3) Preparation of intermediate 11a: tert-butyl 8-(4-(((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-5-yl)oxy)methyl)-1 H -1,2,3-triazol-1-yl)octanoate

[0092] Intermediate 8 (100 mg, 0.32 mmol) and intermediate 10a (116 mg, 0.48 mmol) were dissolved in 5 mL of DMF, and then CuI (30 mg, 0.16 mmol) and DIPEA (62 mg, 84 µL, 0.48 mmol) were added, and the reaction was carried out under nitrogen protection at 60°C for 12 h, and TLC was used for monitoring. After the reaction was completed, water was added for dilution, and ethyl acetate was used for extraction three times, and the organic layers were combined, dried with anhydrous sodium sulfate, and separated by column chromatography (DCM: MeOH = 100: 4) to obtain a yellow solid 150 mg, with a yield of 80%.

[0093] (4) Preparation of target compound DBCO-C25 (the structural formula is shown as Figure 4 follows):

[0094] Intermediate 11a (143 mg, 0.199 mmol) was dissolved in 4 mL DCM, then 2 mL TFA was added, and the reaction was stirred at room temperature for 1 h, monitored by TLC. After the reaction was completed, the solvent was evaporated to give intermediate 12a, which was used directly in the next step without purification. Intermediate 12a was dissolved in DMF, and HATU (165 mg, 0.435 mmol) and DIPEA (56 mg, 76 μL, 0.435 mmol) were added, and the reaction was stirred at room temperature for 10 min, then 3-aminopropanoylazadiphenylcyclooctyne (84 mg, 0.205 mmol) was added, and the reaction was stirred at room temperature for 4 h, monitored by TLC. After the reaction was completed, the reaction mixture was extracted with EA / H2O three times, and the organic layers were combined, washed with saturated sodium chloride, dried over anhydrous sodium sulfate, and evaporated to dryness. The residue was separated by column chromatography to give 110 mg of a white solid, with a yield of 75%.

[0095] Example 7. Preparation of compound DBCO-C26 (structure shown below): Figure 4 The procedure was the same as in Example 6, with a yield of 65%.

[0096] Example 8. Preparation of compound DBCO-C27 (structure shown below): Figure 4 The procedure was the same as in Example 6, with a yield of 70%.

[0097] The synthesis procedure of target compound DBCO-C50 is shown below:

[0098] .

[0099] Example 9. N Preparation of compound (3-(azadiphenylcyclooctyne-1-yl)-3-oxopropyl)-14-(4-(((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)piperidin-1-yl)methyl)amino)tetradecanamide DBCO-C50

[0100] (1) Preparation of intermediate 14: 2-(2,6-dioxopiperidin-3-yl)-5-(4- (hydroxymethyl)piperidin-1-yl)isoindole-1,3-dione

[0101] The starting material 2-(2,6-dioxopiperidin-3-yl)-5-fluoroisoindoline-1,3-dione (1 g, 3.623 mmol) and 4-piperidinemethanol (500.74 mg, 4.348 mmol) were dissolved in NMP (10 mL), and then DIPEA (1.17 g, 1.6 mL, 9.058 mmol) was added. The reaction was carried out at 90 °C for 4 h, and the reaction was monitored by TLC. After the reaction was complete, the mixture was extracted three times with EA / H2O, the organic layers were combined, washed with saturated sodium chloride, dried with anhydrous sodium sulfate, the solvent was evaporated, and the mixture was separated by column chromatography to give 800 mg of yellow solid, with a yield of 57%.

[0102] (2) Preparation of intermediate 15: 1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-5-yl)piperidin-4-carboxaldehyde

[0103] Intermediate 14 (800 mg, 2.156 mmol) was dissolved in DCM (10 mL), and then DMP (1.83 g, 4.312 mmol) was added. The mixture was stirred at room temperature for 2 h, and monitored by TLC. After the reaction was complete, it was quenched with a mixture of saturated NaHCO3 aqueous solution and 10% Na2S2O3 aqueous solution. After stirring for another 30 min, the resulting mixture was extracted with DCM, dried over Na2SO4, concentrated under reduced pressure, and separated by column chromatography (DCM / MeOH = 10 / 1) to give 460 mg of yellow solid, with a yield of 58%.

[0104] (3) Preparation of intermediate 16: 14-(((1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-5-yl)piperidin-4-yl)methyl)amino)tert-butyl tetradecanoate

[0105] Intermediate 15 (100 mg, 0.271 mmol) and tert-butyl 14-aminotetradecanoate (89 mg, 0.298 mmol) were dissolved in THF (15 mL), and 2 drops of acetic acid were added. The mixture was stirred at room temperature for 30 min. Then, sodium triethoxyborohydride (143 mg, 0.675 mmol) was added, and the reaction was carried out at room temperature for 4 h, monitored by TLC. After the reaction was complete, the solvent was evaporated, and the mixture was separated by column chromatography to give 100 mg of a yellow solid, with a yield of 48%.

[0106] (4) Preparation of the target compound DBCO-C50 (structural formula as follows) Figure 5 (as shown)

[0107] The procedure was the same as in Example 6(4), yielding 54 mg of yellow solid with a yield of 35%.

[0108] The structural formula and 1H NMR data of the DBCO-linker-CRBN ligand are shown in Table 1:

[0109] Table 1. Structure formula and NMR data of ligand compounds

[0110] ;

[0111] ;

[0112] ;

[0113] ;

[0114] .

[0115] Example 3: Preparation of Nanobody-targeting chimera

[0116] 4C G26pAzF -R10 respectively with DBCO-linker 1-pomalidomide, DBCO-linker 2- pomalidomide, DBCO-linker 3-pomalidomide and DBCO-linker 4-pomalidomide at a reaction ratio of 1:4 in 1xPBS buffer, at a rotation speed of 75 rpm / min, at room temperature for 3 hours. After reaction, the sample was purified using 1xPBS as purification buffer, 7 kDa ZebaTM desalting column. Free DBCO-linker 1-pomalidomide, DBCO-linker 2-pomalidomide, DBCO-linker 3-pomalidomide or DBCO-linker 4-pomalidomide can be left in the desalting column due to their small molecular weight. While 4C-R10 G26pAzF successfully coupled with 4C-R10 G26pAzF -linker-pomalidomide (named as NbTAC) can smoothly pass through the desalting column.

[0117] Example 4: Test of the ability of GPX4 nanobody-targeting chimera to degrade intracellular GPX4

[0118] HT1080 cells in logarithmic growth phase were taken, about 4,000 cells per well were seeded in a 12-well plate, 3 replicates were set for each experiment, 1 mL of complete culture medium was added to each well, and the cells were incubated in a 37 °C, 5% CO2 incubator overnight. The NbTAC with a concentration of 3, 1.5, 0.75 and 0 μM was mixed with 10 μM of TNB-R10 in each well, and the cells were incubated in a 37 °C, 5% CO2 incubator for 24 hours. The cells were lysed with IP lysis buffer at 4 °C for 30 minutes. The cell lysate was centrifuged at 4 °C, 12,000 rpm for 10 minutes, and the supernatant was used for WB analysis. In the WB analysis, the total protein amount loaded on each sample was 50 μg. The samples were separated on a 12% polyacrylamide gel, and then the proteins were transferred to a PVDF membrane. The membrane was incubated with anti-GPX4 antibody, and finally developed using a gel imager.

[0119] As shown in Figure 6 , the results of the Western blotting experiment showed that 4C G26pAzF -R10-linker 1-pomalidomide, 4C G26pAzF -R10-linker 2-pomalidomide, 4C G26pAzF -R10-linker 3-pomalidomide and 4C G26pAzF -R10-linker 4-pomalidomide all had the activity of degrading GPX4 in cells. Among them, 4C G26pAzF -R10-C50-pomalidomide had the best effect on degrading GPX4. Among them, 0.75 μM of 4C G26pAzF -R10-C50-pomalidomide could achieve 70% degradation of GPX4 in cells.

[0120] Example 5: Activity test of GPX4 nanobody targeting chimera inducing ferroptosis of HT1080 tumor cells

[0121] HT1080 cells in logarithmic growth phase were taken, about 4,000 cells per well were seeded in a 96-well plate, 2 replicates were set for each experiment, 100 μL of complete culture medium was added to each well, and the cells were incubated in a 37 °C, 5% CO2 incubator overnight. The NbTAC with a concentration of 5 μM, 1.25 μM and 0 μM was mixed with 10 μM of TNB-R10 in each well, and the cells were incubated in a 37 °C, 5% CO2 incubator for 24 hours. Cell viability was measured using a CCK-8 kit.

[0122] The results of the cell viability test showed that, as shown in Figure 7 , 4C G26pAzF -R10-linker 1-pomalidomide, 4C G26pAzF- R10-linker 2-pomalidomide, 4C G26pAzF - R10-linker 3-pomalidomide and 4C G26pAzF - R10-linker 4-pomalidomide all have the activity of inducing iron death of HT1080 tumor cells, and have the potential of anti-tumor.

[0123] Finally, it should be explained that the above examples are only used to illustrate the technical solutions of the present application, but not to limit the protection scope of the present application. For those skilled in the art, on the basis of the above description and ideas, other different forms of changes or variations can also be made, which do not need and cannot be exhausted here. Any modification, equivalent replacement and improvement made within the spirit and principle of the present application shall be included in the protection scope of the claims of the present application.

Claims

1. A GPX4 nanobody targeting chimera, characterized in that, This nanobody-targeting chimera introduces non-natural amino acids at specific sites on the GPX4 nanobody, and then couples the DBCO-linker-CRBN ligand with the azide groups of the non-natural amino acids introduced into the GPX4 nanobody; the amino acid sequence of the GPX4 nanobody is shown in SEQ ID No. 3 (4C). G26pAzF -R10, where U in the sequence listing represents p-azidophenylalanine; the DBCO-linker-CRBN ligand is one of them: Compound DBCO-C9: N -(3-(azadiphenylcyclooctyne-1-yl)-3-oxopropyl)-10-(4-(2-(2,6-dioxopiperidin-3-yl)-6-fluoro-1,3-dioxoisoindoline-5-yl)piperazin-1-yl)-10-oxodecanoamide; Compound DBCO-C16: N -(3-(azadiphenylcyclooctyne-1-yl)-3-oxopropyl)-10-(4-((4-(2-(2,6-dioxopiridine-3-yl)-6-fluoro-1,3-dioxoisoindoline-5-yl)piperazin-1-yl)methyl)piperidine-1-yl)-10-oxodecanoamide; Compound DBCO-C17: N -(3-(azadiphenylcyclooctyne-1-yl)-3-oxopropyl)-12-(4-((4-(2-(2,6-dioxopiperidin-3-yl)-6-fluoro-1,3-dioxoisoindoline-5-yl)piperazin-1-yl)methyl)piperidin-1-yl)-12-oxododecanoamide; Compound DBCO-C23: N -(3-(azadiphenylcyclooctyne-1-yl)-3-oxopropyl)-14-(4-((4-(2-(2,6-dioxopiperidin-3-yl)-6-fluoro-1,3-dioxoisoindoline-5-yl)piperazin-1-yl)methyl)piperidin-1-yl)-14-oxotetradecanoamide; Compound DBCO-C24: N -(3-(azadiphenylcyclooctyne-1-yl)-3-oxopropyl)-16-(4-((4-(2-(2,6-dioxopiperidin-3-yl)-6-fluoro-1,3-dioxoisoindoline-5-yl)piperazin-1-yl)methyl)piperidin-1-yl)-16-oxohexadecanoamide; Compound DBCO-C25: N -(3-(aza-diphenylcyclooctyn-1-yl)-3-oxopropyl)-8-(4-(((2-(2,6-dioxoperidin-3-yl)-1,3-dioxoisoindole-5-yl)oxy)methyl)-1 H -1,2,3-triazol-1-yl)octamide; Compound DBCO-C26: N -(3-(aza-diphenylcyclooctyn-1-yl)-3-oxopropyl)-10-(4-(((2-(2,6-dioxoperidin-3-yl)-1,3-dioxoisoindole-5-yl)oxy)methyl)-1 H -1,2,3-triazol-1-yl)decanoamide; Compound DBCO-C27: N -(3-(aza-diphenylcyclooctyn-1-yl)-3-oxopropyl)-12-(4-(((2-(2,6-dioxoperidin-3-yl)-1,3-dioxoisoindole-5-yl)oxy)methyl)-1 H -1,2,3-triazol-1-yl)dodecanoamide; Compound DBCO-C50: N -(3-(azadiphenylcyclooctyne-1-yl)-3-oxopropyl)-14-(4-(((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-5-yl)piperidin-1-yl)methyl)amino)tetradecanoamide.

2. The use of the nanobody targeting chimera according to claim 1 in the preparation of a drug for treating human fibrosarcoma.

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