A peptide targeting IDO1 and a chimera targeting IDO1 protein degradation

Abstract

The present invention belongs to the field of biopharmaceutical technology, and specifically relates to a polypeptide targeting IDO1 and an IDO1 protein degradation targeting chimera. The polypeptide targeting IDO1 provided by the present invention is a high-affinity and targeted IDO1 extracellular domain protein binding polypeptide successfully selected using phage display technology. The polypeptide has a strong binding force with the IDO1 extracellular domain structural protein, and has the characteristics of high sensitivity, high recognition efficiency and small molecular weight. The present invention further combines the above-mentioned polypeptide with the E3 ubiquitin ligase ligand thalidomide through rational design to obtain PROTAC, which has good cell entry ability and IDO1 targeted degradation ability, can make the therapeutic drug for cancer disease more selective, and provide higher therapeutic efficiency while reducing systemic toxicity. Therefore, the present invention can provide a new treatment strategy and effective technical support for the development of new tumor targeted drugs and the treatment of related cancers.

Description

Technical Field

[0001] The present invention belongs to the technical field of biopharmaceuticals, and particularly relates to a polypeptide targeting IDO1 and an IDO1 protein degradation targeting chimera. Background Art

[0002] Indoleamine 2,3-dioxygenase 1 (IDO1) is an oxidoreductase composed of 403 amino acids and heme. It contains a single active site and is the rate-limiting enzyme in the kynurenine pathway. Through the kynurenine pathway, IDO1 catalyzes the metabolism of tryptophan to produce cytotoxic metabolites such as kynurenine and quinolinic acid. Normally, IDO1 is expressed at low levels in vivo. Recent studies have shown that IDO1 is expressed at elevated levels in various tumor tissues and positively correlated with poor prognosis in various cancers. Furthermore, IDO1 has been implicated in tumor immune evasion and is closely associated with several major human diseases, such as cataracts and Alzheimer's disease. Further studies have demonstrated that IDO1 plays an important role in immunomodulation, including suppressing T cell immunity, anti-tumor immunity, and tumor immune escape. Therefore, screening affinity peptides using purified IDO1 protein as a drug target is of great significance for the prevention and treatment of tumors.

[0003] The proteolysis targeting chimera (PROTAC) technology is a bifunctional molecule, also known as a heterobifunctional degrader, which consists of three parts: a ligand that binds to the target protein, an E3 ubiquitin ligase ligand, and a linker connecting the two. PROTAC can bind to the target protein at one end and to the E3 ligase at the other end to form a ternary complex. The E3 ligase and the target protein are spatially adjacent to each other, mediating the ubiquitination of the target protein by the E2 conjugating enzyme; subsequently, the proteasome recognizes and degrades the ubiquitinated target protein. In theory, the PROTAC molecule only provides binding activity and is an "event-driven" mode of action. It does not require long-term, high-intensity binding to the target protein and can therefore be recycled repeatedly. Compared with traditional “occupancy-driven” drugs, PROTAC molecules have the following advantages: (1) providing a new rapid and reversible chemical knockout method; (2) making difficult-to-drug targets druggable; (3) reducing drug toxicity and side effects; (4) maintaining the sensitivity of targets that are prone to drug-resistant mutations in the clinic for a long time; and (5) affecting non-enzyme activity functions by degrading the entire protein.

[0004] As such, protein degradation-targeted chimeras, as an emerging therapeutic approach, offer numerous advantages over traditional small molecule drugs. However, currently, no protein degradation-targeted chimeras have been established that can effectively target IDO1 for degradation. Therefore, identifying a high-affinity IDO1 ectodomain-binding peptide and successfully constructing a protein hydrolysis-targeted chimera for IDO1 degradation remain key challenges in this field. Summary of the Invention

[0005] In order to solve the problems of the above-mentioned prior art, the first object of the present invention is to provide a polypeptide targeting IDO1. The polypeptide is an affinity polypeptide targeting IDO1 that is successfully selected by phage display technology in the present invention and can effectively bind to E3 ubiquitin ligase ligands, thereby being used for the preparation of anti-tumor drugs targeting IDO1.

[0006] The second object of the present invention is to provide the use of the above-mentioned polypeptide targeting IDO1.

[0007] The third object of the present invention is to provide a chimera targeting IDO1 protein degradation.

[0008] The fourth object of the present invention is to provide the use of the above-mentioned IDO1 protein degradation targeting chimera.

[0009] The above technical objectives of the present invention are achieved by adopting the following technical solutions:

[0010] A polypeptide targeting IDO1, wherein the amino acid sequence of the polypeptide targeting IDO1 is shown in SEQ ID No. 1. Specifically, the amino acid sequence shown in SEQ ID No. 1 is PISFDLAQPSGQ.

[0011] The polypeptide targeting IDO1 provided by the present invention is a high-affinity and targeted IDO1 extracellular domain protein-binding polypeptide successfully selected by phage display technology. Phage display technology is a technology in which an exogenous DNA fragment encoding a target protein or polypeptide is fused with a gene encoding a phage surface protein, and then presented on the surface of the phage in the form of a fusion protein. The displayed protein or polypeptide can maintain a relative spatial structure and biological activity and be displayed on the surface of the phage. The present invention is screened by phage display technology to obtain phage clones with high affinity for IDO1 extracellular domain proteins, and then enriched by two rounds of panning, DNA extracted and sequenced, and a 12-peptide was successfully obtained, the amino acid sequence of which is: PISFDLAQPSGQ, named Q1. The polypeptide can be combined with therapeutic drugs as a targeting functional group, making therapeutic drugs for cancer diseases more selective, while reducing systemic toxicity and providing higher therapeutic efficiency.

[0012] An application of the above-mentioned polypeptide targeting IDO1 in the preparation of anti-tumor drugs.

[0013] As a preferred solution, the anti-tumor drug is an anti-gastric cancer drug.

[0014] An IDO1 protein degradation targeting chimera is mainly prepared from the IDO1 targeting polypeptide, thalidomide, a linker and a cell-penetrating peptide R8.

[0015] The present invention screens polypeptides with high affinity and targeting to IDO1, uses them as target protein ligands for IDO1, and rationally designs them with a specific E3 ubiquitin ligase ligand (thalidomide). The IDO1 protein-targeting chimera (PROTAC) is synthesized for the first time, and its activity is evaluated in cells. It is confirmed that the constructed PROTAC can be effectively used for the preparation of anti-tumor drugs, especially anti-gastric cancer drugs, targeting IDO1.

[0016] As a preferred embodiment, the linker is PEG2. The length and composition of the linker play a key role in the biological activity and physicochemical properties of PROTAC. The linker is crucial for helping the spatial orientation and positioning of the E3 ligase and the target protein to ensure favorable protein-protein interactions (PPIs). The present invention uses two polyethylene glycol groups as the linker for the following reasons: 1) amphiphilic polyethylene glycol (PEG) is selected as the linker because it has good ductility in both hydrophobic and hydrophilic properties. 2) In addition, SAR data show that the active site of IDO1 is located in a deep cavity, and the distance from the amino group at the connection site to the external interface is >10A / cm. Differences in binding modes may affect the trajectory and optimal length of the linker motif. In the IDO1-PROTAC of the present invention, polypeptide Q1 is used as an IDO1 targeting ligand. Compared with small molecules, the polypeptide itself is larger in size. If the linker remains longer, the PROTAC volume increases, and the expansion of the molecule often inevitably leads to a significant decrease in affinity for IDO1. In order to make the PROTAC with thalidomide as a ligand for the CRBNE3 ligase closer to the "drug-like" space, its average hydrogen bond donor and clogP value fall within the drug-like rule of 5 (Rule of 5) due to the unique starting properties of the E3 ligase warhead. Therefore, the present invention shortened the linker to 2 PEG after the experiment.

[0017] As a preferred solution, the raw materials used in the preparation of the IDO1 protein degradation targeted chimera also include lysine.

[0018] As a preferred solution, the structure of the cell-penetrating peptide R8 is:

[0019]

[0020] As a preferred solution, the structure of the IDO1 protein degradation targeting chimera is:

[0021]

[0022] The application of the above-mentioned IDO1 protein degradation targeting chimera in the preparation of anti-tumor drugs.

[0023] As a preferred solution, the anti-tumor drug is an anti-gastric cancer drug.

[0024] Compared with the prior art, the beneficial effects of the present invention are mainly:

[0025] The present invention uses phage display technology for the first time to screen out a high-affinity IDO1 extracellular domain protein-binding polypeptide Q1, which has strong binding force to the IDO1 extracellular domain structural protein and has the characteristics of low cost, high sensitivity, high recognition efficiency and small molecular weight.

[0026] Furthermore, the present invention rationally designs the above-mentioned polypeptide Q1 and the E3 ubiquitin ligase ligand thalidomide to obtain PROTAC. Among them, since the molecular weight of the CRBN ligand is smaller than that of the VHL ligand, the number of rotatable bonds and hydrogen bond donors is smaller, it has better pharmacokinetic (PK) characteristics and higher blood-brain barrier (BBB) ​​permeability, and the present invention selects the CRBN ligand thalidomide as the target head at one end of the PROTAC. The CRBN-E3 ligase-based PROTAC designed by the present invention with thalidomide or its derivatives as the E3 ligase ligand is closer to the "drug-like" space, and its average hydrogen bond donor and clogP value fall within the boundaries of Lipinski's "rule of 5", thereby giving the E3 ligase warhead unique starting properties. In addition, the present invention shortens the linker to 2 PEG units, wherein the high-affinity polypeptide Q1 serves as the IDO1 ligand, and the branched chain increases the ability of the membrane-penetrating peptide to enter the cell. Experiments have confirmed that the PROTAC designed by the present invention has good intracellular entry ability and IDO1 targeted degradation ability, which can provide new treatment strategies and effective technical support for the development of new tumor-targeted drugs, especially anti-gastric cancer drugs, and the treatment of related cancers. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a schematic diagram of the structure of the IDO1 protein hydrolysis targeting chimera provided in Example 2 of the present invention;

[0028] Figure 2 This is the ELISA test result of the bacteriophage in Experimental Example 1 of the present invention;

[0029] Figure 3aThis is the binding and dissociation curve of polypeptide Q1 and IDO1 extracellular domain protein verified by BLI biomembrane interferometry in Experimental Example 2 of the present invention;

[0030] Figure 3b The KD value and fitting curve constant R calculated by the BLI biomembrane interference technology to verify the binding and dissociation curve of polypeptide Q1 and IDO1 extracellular domain protein in Experimental Example 2 of the present invention 2 ;

[0031] Figure 3c This is the binding and dissociation curve of polypeptide Q8 and IDO1 extracellular domain protein verified by BLI biomembrane interferometry in Experimental Example 2 of the present invention;

[0032] Figure 3d The KD value and fitting curve constant R calculated by the BLI biomembrane interference technology to verify the binding and dissociation curve of polypeptide Q8 and IDO1 extracellular domain protein in Experimental Example 2 of the present invention 2 ;

[0033] Figure 4 This is the IP verification result of the binding between polypeptide Q1 and IDO1 protein in Experimental Example 3 of the present invention;

[0034] Figure 5 This is the concentration-dependent test result of the IDO1-PROTAC biological activity evaluation in Experimental Example 4 of the present invention;

[0035] Figure 6 This is the time-dependent test result of the IDO1-PROTAC biological activity evaluation in Experimental Example 4 of the present invention. DETAILED DESCRIPTION

[0036] In order to make the technical purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with the accompanying drawings and Examples. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the scope of protection of the present invention. In the following examples, the raw materials or reagents involved are conventional materials or reagents that can be obtained through commercial channels unless otherwise specified.

[0037] Among them, the materials such as thalidomide, cell-penetrating peptide R8, and lysine involved in the following examples were from Shanghai Chupeptide Biotechnology Co., Ltd.

[0038] The LB liquid culture medium involved in the following examples was prepared as follows: 15 g of sodium chloride, 15 g of tryptone, and 7.5 g of yeast extract were weighed into a 2 L conical flask, and 1.5 L of pure water was added. After autoclaving at 121°C, the flask was taken out and placed in a cold room for later use.

[0039] The 20% polyethylene glycol / 2.5 M sodium chloride solution involved in the following examples was prepared as follows: 10 g of PEG-8000 and 7.3 g of sodium chloride powder were accurately weighed, 50 mL of ultrapure water was added, and the solution was sterilized under high pressure. While the solution was still warm, the solution was inverted to mix the ingredients evenly, and then stored at room temperature.

[0040] The methods for preparing the IDO1 protein involved in the following examples include obtaining the IDO1 gene sequence (the IDO1 gene is located at nucleotides 39913891 to 39928790 of NC_000008; Homo sapiens chromosome 8, GRCh38.p14 Primary Assembly-Nucleotide-NCBI (nih.gov)), constructing a pET-28a(+)-IDO1 prokaryotic expression vector containing a histone (His) tag, and then transfecting the vector into an E. coli BL21(DE3) expression strain to obtain a strain that highly expresses IDO1. Positive clones were selected and amplified on a kanamycin-resistant culture plate, and IPTG was added to induce IDO1 expression. Total protein in the strain was then extracted using a cell ultrasonic disruptor. Finally, based on the properties of the His tag, an imidazole-nickel affinity column purification system was used to separate and purify the IDO1 protein with a high purity (concentration of 6.66 μg / μL) for subsequent experiments.

[0041] Example 1

[0042] This embodiment provides a polypeptide targeting IDO1, the amino acid sequence of which is PISFDLAQPSGQ (as shown in SEQ ID No. 1).

[0043] The above-mentioned IDO1-targeting peptide was screened and obtained by the following method:

[0044] 1. Enrichment of phage

[0045] (1) Immobilization of IDO1 protein

[0046] Step 1-1: Prepare IDO1 protein using 0.1M NaHCO3 solution (pH 8.6) to a final concentration of 10-100 μg / mL. Prepare a sterile ELISA plate (maximum volume per well: 200 μL), add 150 μL of the diluted IDO1 protein solution, seal the plate, and incubate overnight at 4°C with gentle shaking.

[0047] Step 1-2: Prepare sterile paper towels, turn the incubated sterile ELISA plate upside down and gently tap it to remove residual NaHCO3 solution;

[0048] Step 1-3: Prepare TBST buffer (TBS + 0.1% (v / v) Tween-20) and quickly wash the wells 6 times.

[0049] (2) First round of selection

[0050] Step 2-1: Prepare PH.D TM .-12 phage display peptide library kit (New England Biolabs, USA), 1 μL of phage was diluted into 99 μL of TBST buffer and incubated at room temperature for 30 min in an empty well without protein immobilization. The solution was aspirated and added to the wells with fixed IDO1 protein and washed, and incubated at room temperature for 30 min.

[0051] Step 2-2: Invert the plate onto a sterile paper towel to remove unbound phage. Wash the plate 10 times with 150 μL of TBST buffer as described in Steps 1-3. Use a clean paper towel each time to prevent cross-contamination.

[0052] Step 2-3: Prepare elution buffer (0.2 M glycine-HCl buffer, pH 2.2), pipette 100 uL into the well treated in the previous step, shake gently for 10 minutes, pipette the eluate into a 200 uL centrifuge tube, and neutralize with 30 uL neutralization buffer (1 M Tris-HCl, pH 9.1).

[0053] (3) Titer determination before the first round of panning amplification

[0054] Step 3-1: Use sterilized LB liquid medium (added with 5 μg / mL tetracycline) to gradiently dilute the first round of panning eluate obtained in step 2-3, with the dilution multiples of 10 1 , 10 2 , 10 3 , 10 4 ;

[0055] Step 3-2: Take 10uL of each gradient dilution and 200uL of E. coli ER2738 strain (OD 600 = 0.5) cultures were mixed and incubated for 1-5 min, 3 mL of 45°C top agar solution (supplemented with 5 μg / mL tetracycline) was added, mixed evenly, and plated on IPTG / Xgal titration plates. The plates were incubated in the dark for 14-16 h. The results are shown in Table 1.

[0056] Table 1. Titer determination before the first round of panning amplification of IDO1-phage screening

[0057]

[0058] During each round of phage panning, the eluted and amplified phage solutions are titered to determine the amount of phage to be added for the next round. During titering, only when the multiplicity of infection (MOI) is significantly less than 1—that is, when there is a large excess of host cells—will the number of phage plaques formed during phage infection increase linearly with the addition of phage. Therefore, the present invention dilutes the phage solution before infecting E. coli, ensuring that the resulting phage plaques contain only a single DNA sequence.

[0059] The lacZ gene is a structural gene of the lac operon in Escherichia coli and is responsible for expressing β-galactosidase. The gene encoding the α fragment of β-galactosidase in ER2738 Escherichia coli is deleted, resulting in a loss of β-galactosidase activity. When a phage carrying the lacZ (α fragment) of the lac operon infects ER2738, under the action of IPTG, the α fragment of β-galactosidase is successfully expressed, and the activity of β-galactosidase is restored, capable of decomposing X-gal to produce blue metabolites. Therefore, after the modification, the M13 phage carrying the dodecapeptide will form blue plaques on IPTG / X-gal plates after infecting the ER2738 host bacteria. The titer of the phage solution is determined by counting the blue plaques on the plate, while the wild-type phage does not have this effect and forms white plaques on the IPTG / X-gal plates. White plaques are a key factor in quality control during the entire panning process of the present invention.

[0060] The optimal dilution for titer determination was 100 blue plaques on the solid culture medium. As shown in Table 1, the titer before amplification was 1.3×10 4 pfu / μL.

[0061] (4) First round of phage amplification and purification

[0062] Step 4-1: Add 20 mL of Escherichia coli ER2738 strain D to the first round of eluate 600 =0.01-0.05) cultures were incubated at 37°C and 220 rpm for 4.5 h;

[0063] Step 4-2: Transfer the culture to a 10 mL centrifuge tube and centrifuge at 12,000 g for 10 min at 4°C. Transfer the supernatant to a new centrifuge tube and centrifuge at 12,000 g for 10 min at 4°C.

[0064] Step 4-3: Transfer 80% of the supernatant to a new centrifuge tube, add 1 / 6 volume of 20% polyethylene glycol / 2.5 M sodium chloride solution, and incubate at 4°C overnight;

[0065] Step 4-4: Incubate the polyethylene glycol precipitate obtained in step 4-3 on ice for 15-60 minutes. Centrifuge at 12,000 g for 15 minutes at 4°C. Discard the supernatant, resuspend, and completely discard the remaining supernatant. A white film on the wall of the centrifuge tube represents the phage particles.

[0066] Step 4-5: Suspend the phage particles obtained in the previous step in 200 μL of TBS buffer and transfer to a 200 μL centrifuge tube to obtain the eluate after the first round of amplification.

[0067] (5) Titer determination after the first round of panning amplification

[0068] Step 5-1: Use sterilized LB liquid medium (add 5 μg / mL tetracycline) to gradient dilute the first round of panning eluate, the dilution multiples are 10 9 , 10 10 , 10 11 , 10 12 ;

[0069] Step 5-2: Take 10uL of each gradient dilution and mix with 200uL of E. coli ER2738 strain (OD 600 = 0.5) culture was mixed and incubated for 1-5 min. 3 mL of 45°C top agar solution (supplemented with 5 μg / mL tetracycline) was added. After mixing evenly, the solution was spread on an IPTG / Xgal titration plate and incubated in the dark for 14-16 h. The results are shown in Table 2. This marked the end of the first round of panning and enrichment.

[0070] Table 2. Titer determination after the first round of panning and amplification of IDO1-phage screening

[0071]

[0072] After obtaining the titer after the first round of amplification, the volume of phage of the same order of magnitude as in the first round was calculated based on the titer to carry out the second round of panning. The purpose of the gradient dilution is to ensure that the number of blue spots on the solid culture medium can be within 100, because within 100 is the optimal dilution and the titer is most accurate. As shown in Table 2, the titer after the first round of amplification is 1.08×10 12 pfu / μL, according to the first round of amplification before the input 1×10 10 In terms of pfu phage, the second round requires 1×10 10 / 1.08×10 12 = 0.00925 μL of amplified phage eluate.

[0073] (6) Second round of selection

[0074] Step 6-1: Count the blue plaques on the IPTG / Xgal titer plate and determine the titer of the amplified phage. The titer after the first round of amplification was 1.08×10 12 pfu / μL. Use this value to calculate the input volume corresponding to the input titer in step 2-1, add it to the wells with immobilized IDO1 protein, and incubate at room temperature for 30-60 minutes. Step 6-2: Same as step 2-2; Step 6-3: Same as step 2-3.

[0075] (7) Titer determination before the second round of panning amplification

[0076] Step 7-1: Use sterilized liquid culture medium to gradient dilute the second round of panning eluate, the dilution factor is 10 1 , 10 2 , 10 3 , 10 4 Step 7-2: Same as step 3-2. The titer determination results are shown in Table 3.

[0077] Table 3. Titer determination before the second round of panning amplification of IDO1-phage screening

[0078]

[0079] As shown in Table 3, the titer before amplification was 5 pfu / μL.

[0080] (8) Second round of phage amplification and purification

[0081] Step 8-1: Add 20 mL of Escherichia coli ER2738 (OD 600 =0.01-0.05), culture at 37°C, 220 rpm for 4.5h; Steps 8-2, 8-3, 8.4: the same as steps 4-2, 4-3, 4-4; Step 8-5: suspend the phage particles in 200 μL of TBS phage, transfer to a 200 μL centrifuge tube, and obtain the eluate after the second round of amplification.

[0082] (9) Titer determination after the second round of panning amplification

[0083] Step 9-1: Use sterilized liquid culture medium to gradient dilute the second round of panning eluate, the dilution factor is 10 9 , 10 10 , 10 11 , 10 12 ;

[0084] Step 9-2: Same as step 5-2. The titer test results are shown in Table 4. This concludes the second round of panning and enrichment.

[0085] Table 4. Titer determination after the second round of panning and amplification of IDO1-phage screening

[0086]

[0087] As shown in Table 4, the titer after the first round of amplification was 1.33×10 10 pfu / μL.

[0088] (10) Select blue spots and sequence

[0089] Step 10-1: Add 300 μL of Escherichia coli ER2738 strain (D600 = 0.01-0.05) culture to a 1.5 mL centrifuge tube. Use a sterile pipette tip to pick up 10 blue spots after the second round of panning amplification and add them to the centrifuge tube separately, stirring. Amplify for 4.5 hours until the solution becomes turbid. Then, use high-throughput sequencing to identify the phage dodecapeptide that specifically binds to USP7.

[0090] Step 10-2: Analyze the sequencing results. A large number of repeated sequences prove that the phage has been initially enriched.

[0091] The process of high-throughput sequencing to identify USP7 specifically binding to the phage twelve peptide is as follows:

[0092] After two rounds of panning with the random 12-peptide phage library, DNA was extracted from the final round of elution. DNA fragments containing the specific 12-peptide sequence were amplified by PCR using designed primers. The amplified target fragments were then sent to Beijing Qingke Biotechnology for library construction and high-throughput sequencing. The high-throughput sequencing results were further analyzed, and the sequencing results are shown in Table 5. In Table 5, "blank" indicates the peptide sequence resolved from the background spot (monoclonal colony), which represents the nonspecific binding 12-peptide sequence.

[0093] Table 5. Amino acid sequences of IDO1-specifically binding phage dodecapeptides

[0094]

[0095] As shown in Table 5, a total of 62,720 dodecapeptide sequences that specifically bind to IDO1 were obtained, among which the sequence with the highest frequency of specific binding was the Q1 sequence, followed by the Q8 sequence.

[0096] During the above screening process, the present invention selects the extracellular domain IDO1 as the panning source, and uses a phage random 12-peptide library to pan for specific binding polypeptides of the immune checkpoint IDO1. The specifically bound polypeptides are obtained by continuous bio-panning and enrichment. Bio-panning refers to the incubation of the phage library with the target protein molecule for a certain period of time, washing away the unbound free phages, and competing for the receptor or acid elution of the phages bound to the target molecule. After the eluted phage infects the host, it undergoes the next round of elution after reproduction and amplification. After 3-5 rounds of "adsorption, elution-amplification", the phages that specifically bind to the target molecule are highly enriched. During the above screening process of the present invention, strict sterility must be ensured. The ER2738 strain is tetracycline-resistant. In order to ensure the growth of specific strains rather than wild-type strains, thereby screening out polypeptides with strong characteristics and affinity, tetracycline is added to the culture medium used in the screening process of the present invention. Subinhibitory concentrations of tetracycline are used to continuously induce resistance in wild-type Escherichia coli (sensitive strain) to prevent phages from infecting non-target strains and causing false positive results.

[0097] Example 2

[0098] This example provides a chimera targeting IDO1 protein degradation, denoted as IDO1-PROTAC, with a structural schematic diagram as shown in FIG. Figure 1 As shown, it is mainly prepared from the IDO1-targeting polypeptide of Example 1, thalidomide, a linker, a cell-penetrating peptide R8, and lysine. Among them, the linker is PEG2. When synthesizing IDO1-PROTAC in this embodiment of the present invention, it was handed over to Shanghai Chupeptide Biotechnology Co., Ltd. for biosynthesis according to the following structure.

[0099] The specific structure of the IDO1 protein degradation targeting chimera is as follows:

[0100]

[0101] For ease of understanding, the positions of the IDO1-targeting peptide, thalidomide, linker, transmembrane peptide R8, and lysine in IDO1-PROTAC are shown in the following formula:

[0102]

[0103] Experimental Example 1: Preliminary identification of the binding of peptide Q1 to IDO1 protein using enzyme-linked immunosorbent assay (ELISA)

[0104] The test method is:

[0105] 1) Immobilize IDO1 protein on an ELISA plate, as in step 1-1;

[0106] 2) Count the blue plaques on the titer plate and determine the phage titer at 1013-14 pfu / mL, dilute the eluate after the second round of panning and amplification (containing phage enriched with peptide Q1) to the same order of magnitude, use this value to calculate the input volume corresponding to the ELISA input titer, incubate at room temperature for 1-2 hours, and quickly wash each plate 6 times with TBST buffer (TBS + 0.1% (v / v) Tween-20), pouring the solution upside down with a clean paper towel each time;

[0107] 3) Add 100 μL of 0.1-0.4 μg / mL phage antibody to each well and incubate at room temperature for 1 hour. Wash each well quickly with TBST buffer 6 times, pouring the solution out of the well face down with a clean paper towel each time.

[0108] 4) Add 100 μL of color development solution to each well and incubate at room temperature in the dark for 30 minutes;

[0109] 5) Add 50uL of stop solution to each well and measure the value with a microplate reader at a wavelength of 450nm. The result is as follows Figure 2 shown.

[0110] The present invention uses Elisa test to preliminarily detect whether the polypeptide Q1 in the complex mixture enriched after two rounds of panning binds to the IDO1 protein. In the figure, Target seq refers to the polypeptide Q1 sequence. Figure 2 It can be seen that compared with the control, the peptide Q1 in the complex mixture enriched by two rounds of panning binds to IDO1.

[0111] Experimental Example 2: Using BLI Biomembrane Interferometry to Explore the Binding Kinetics of Peptides Q1 and Q8 to IDO1

[0112] Bio-layer interferometry (BLI) is an experimental method for detecting molecular interactions based on the displacement changes of interference spectra. When different biomolecules combine with the ligands on the surface of the sensor, biofilms of different thicknesses and densities will be formed on the surface of the sensor. The interference spectrum generated by visible light on the surface of the membrane will also change accordingly, thereby accurately measuring the interaction process between the molecules to be measured. In order to evaluate the binding affinity of Q1 and Q8 polypeptides with IDO1 protein at the molecular level, the present invention modified the N-termini of Q1 and Q8 polypeptides with biotin, solidified the Q1 polypeptide and Q8 polypeptide on the SA sensor, and passed the sensor through a concentration gradient of IDO1 protein solution, that is, performed binding kinetics experiments on the BLI molecular interaction instrument.

[0113] The experimental process of BLI biofilm interferometry technology is as follows:

[0114] 1) Place the SA sensor in a pre-wetted plate for 5 minutes. Dilute the synthesized biotin-tagged Q1 peptide to 100 nmol / L with 0.1 mol / L NaHCO3, pH 8.6. Add 200 μL to a black 96-well plate. Also set up a blank SA probe and a negative control group without peptide buffer. Set the BLI molecular interaction instrument to run (Baseline 60s, Loading 3600s, Baseline 60s) to allow the biotin-tagged Q1 peptide to solidify on the SA sensor.

[0115] 2) In a 0.1 mol / L NaHCO3, pH 8.6 solution, 0.02% Tween-20 was added. The purified IDO1 catalytic domain protein was diluted 10-fold downward from 50 μmol / L in this solution into five concentration gradients. The diluted proteins were added to a black 96-well plate in ascending order of concentration. A negative control group without peptide buffer was also set up. The BLI molecular interaction instrument was programmed (Baseline 60 s, Association 180 s, Disassociation 180 s), with one cycle for each concentration.

[0116] 3) Data processing: export the results to Graphpad to calculate the KD value and draw the binding and dissociation curve. The results are as follows Figures 3a to 3d shown.

[0117] in, Figure 3a and Figure 3c The results show that the binding and dissociation curves between the two peptides show a trend of fast binding and slow dissociation when Q1 peptide binds to IDO1. The average response signals obtained at different concentrations were further fitted with curves. Figure 3b and Figure 3d KD value and fitting curve constant R calculated in Graphpad 2 The above experimental results show that the Q1 polypeptide has good binding affinity with the IDO1 protein in vitro. The Q8 polypeptide has relatively weak binding affinity with the IDO1 protein.

[0118] Experimental Example 3: IP experiment verifies the binding of peptide to IDO1 extracellular domain protein

[0119] Although Q1 peptides have previously been shown to have good binding affinity, their targeting of IDO1 still requires further exploration. To demonstrate the targeting of Q1 peptides to IDO1, Q1 peptides with biotin tags were conjugated to streptavidin magnetic beads, acting as "bait" to bind to IDO1.

[0120] Experimental steps: 1) Take 20 μL of streptavidin magnetic beads, wash them three times with RIPA lysis buffer, and use a magnetic stand to perform magnetic separation to clean the magnetic bead protective solution. 2) Add 10 μM Q1 polypeptide with a biotin tag, incubate on a rotating shaker at room temperature for two hours to couple the Q1 polypeptide to the streptavidin magnetic beads for subsequent target protein capture. After the incubation is completed, discard the supernatant, wash the magnetic beads three times with RIPA lysis buffer to remove unbound Q1 polypeptide. 3) One group added purified IDO1 catalytic domain protein solution, and the other group added MGC803 gastric cancer cell lysate, incubated on a rotating shaker at room temperature overnight, discarded the supernatant, washed the magnetic beads three times with RIPA lysis buffer to remove unbound proteins. 4) Add 50 μL of pH 3.0 eluent, perform magnetic separation using a magnetic stand, transfer to a new EP tube, add SDS-PAGE protein loading buffer for denaturation, and use Western blot to analyze the experimental results. The results are as follows. Figure 4 shown.

[0121] Depend on Figure 4 As shown, the Q1 peptide successfully binds to the IDO1 catalytic domain, demonstrating its molecular targeting of IDO1. Furthermore, incubation of MGC803 gastric cancer cell lysate treated with Q1 peptide with streptavidin magnetic beads confirmed that the Q1 peptide also targets IDO1 within gastric cancer cells. IgG was used as a negative control antibody to detect nonspecific binding. Ideally, IgG should not bind to any target protein, resulting in a very weak or absent band in the IgG lane.

[0122] Experimental Example 4: Evaluation of IDO1-PROTAC in vitro biological activity

[0123] This study demonstrates that the Q1 polypeptide exhibits excellent binding affinity and targeting properties for IDO1 at both the molecular and cellular levels. Based on this, the IDO1-PROTAC, an IDO1-targeting peptide degrader described in Example 2, was designed and synthesized using the Q1 polypeptide as a target protein ligand for IDO1. Its bioactivity was evaluated in MGC803 gastric cancer cells.

[0124] The protein immunoblotting method (Western blot) was used to verify the IDO1-PROTAC concentration-dependent degradation experiment. The specific steps are as follows: 1) Gastric cancer cells MGC803 in the logarithmic growth phase were taken and 7×10 6Cells were seeded into 6-well plates at 100 μg / well. Different concentrations of the test compound diluted with complete medium were added to a final volume of 2 mL (final concentrations were 2 μM, 4 μM, 6 μM, 8 μM, 16 μM, and 32 μM). The same volume of DMSO was used as a negative control group. After culturing for 24 hours, the cells were collected and diluted to 1.5 mL. 1) Place the cells in an EP tube and centrifuge at 3000 rpm for 5 minutes. Wash the cell pellet with PBS. 2) Add protein lysis buffer and lyse on ice for 30 minutes. Then centrifuge at 4°C and 12000 rpm for 15 minutes. Take the supernatant to a new EP tube and quantify the protein using BCA. Add SDS-PAGE protein loading buffer (6X) to the remaining supernatant and boil it in a metal bath at 100°C for 15 minutes. 3) After the sample is cooled, perform SDS-PAGE electrophoresis, transfer to a nitrocellulose membrane, block with 5% skim milk powder at room temperature for 2 hours, add IDO1 and GAPDH antibodies (diluted to 1000 times with 5% BSA, respectively), incubate at 4°C overnight, recover the antibodies, wash (3×10 minutes), add goat anti-rabbit IgG (diluted to 5000 times with PBST, respectively), incubate at room temperature for 1 hour, wash (3×10 minutes), add ECL developer, and expose the gel imager. The results are as follows: Figure 5 shown.

[0125] Depend on Figure 5 Western blot results showed that IDO1-PROTAC showed a concentration-dependent degradation of IDO1 in MGC803 gastric cancer cells, that is, IDO1-PROTAC degraded IDO1 in a concentration-dependent manner, and the degradation was obvious at 8 μM.

[0126] Furthermore, the protein immunoblotting method was used to verify the time-dependent degradation experiment of IDO1-PROTAC. The specific steps are as follows: 1) Gastric cancer cells MGC803 in the logarithmic growth phase were taken and 7×10 61) Cells were seeded into 6-well plates at 100 μg / well. The test compound was diluted in complete culture medium to a final concentration of 8 μM. At different time points (4 h, 8 h, 16 h, 24 h, and 48 h), the same volume of DMSO was used as a negative control group. The cells were collected into 1.5 EP tubes, centrifuged at 3000 rpm for 5 min, and the cell pellet was washed with PBS. 2) Protein lysis buffer was added and the cells were lysed on ice for 30 min, then centrifuged at 4°C and 12000 rpm for 15 min. The supernatant was transferred to a new EP tube and quantified by BCA protein. The remaining supernatant was added to 6× loading buffer and boiled in a metal bath at 100°C for 15 min. 3) After cooling, the sample was subjected to SDS-PAGE electrophoresis and transferred to a nitrocellulose membrane. The membrane was blocked with 5% skim milk powder at room temperature for 2 hours. IDO1 and GAPDH antibodies (diluted to 1000-fold with 5% BSA) were added and incubated overnight at 4°C. The antibodies were recovered and washed (3 × 10 minutes). After that, goat anti-rabbit IgG (diluted to 5000-fold with PBST) was added and incubated at room temperature for 1 hour. After washing (3 × 10 minutes), ECL developer was added and the membrane was exposed on a gel imager. The results are shown in Figure 2. Figure 6 shown.

[0127] Figure 6 Western blot results showed that IDO1-PROTAC degradation of IDO1 in MGC803 gastric cancer cells was time-dependent, and degradation was obvious within 48 hours.

[0128] In summary, the polypeptide targeting IDO1 provided by the present invention is a high-affinity and targeted IDO1 extracellular domain protein binding polypeptide successfully selected using phage display technology. The polypeptide has strong binding force with the IDO1 extracellular domain structural protein, and has the characteristics of low cost, high sensitivity, high recognition efficiency and small molecular weight. The present invention further combines the above-mentioned polypeptide with the E3 ubiquitin ligase ligand thalidomide through rational design to obtain PROTAC, which has good cell entry ability and IDO1 targeted degradation ability, and can make the treatment of cancer diseases such as gastric cancer more selective, while reducing systemic toxicity and providing higher treatment efficiency. Therefore, the present invention can provide a new treatment strategy and effective technical support for the development of new tumor-targeted drugs and the treatment of related cancers such as gastric cancer.

Claims

1. A polypeptide targeting IDO1, characterized in that The amino acid sequence of the polypeptide targeting IDO1 is shown in SEQ ID No.

1.

2. An IDO1 protein degradation targeting chimera, characterized in that: The structure of the IDO1 protein degradation targeting chimera is:

3. A use of the IDO1 protein degradation targeting chimera according to claim 2, characterized in that: Application in the preparation of anti-tumor drugs; the anti-tumor drugs are anti-gastric cancer drugs.

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