A peptide targeting parp1 protein, peptide-based protac and application thereof

Abstract

The application discloses a peptide targeting a PARP1 protein, a peptide PROTC and application thereof, and belongs to the technical field of biological medicines, wherein the amino acid sequence of the peptide targeting the PARP1 protein is fdklGsGsG, and the targeting peptide of the application is helpful to realize targeted treatment of tumors.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a peptide targeting PARP1 protein, a peptide-like PROTAC, and its applications. Background Technology

[0002] DNA damage is a crucial mechanism for killing tumor cells during chemotherapy and radiotherapy. However, some tumor cells can resist treatment by activating intracellular DNA damage repair mechanisms, leading to drug resistance and reduced treatment efficacy. Therefore, blocking the DNA repair pathway in tumor cells is a key breakthrough for improving the effectiveness of tumor treatment. Poly(ADP-ribose) polymerase (PARP) is an important class of DNA damage repair enzymes, with PARP1 being the core member of this family, responsible for over 90% of the functions of the PARP family. It plays a crucial role in maintaining genomic stability and mediating DNA single-strand break repair. PARP1 protein is highly expressed in various tumors, including breast cancer, and is a key target for tumor targeted therapy. Traditional small-molecule PARP inhibitors, such as olaparib and niraparib, have been approved for the treatment of BRCA-mutated TNBC and have achieved significant clinical efficacy. However, similar to conventional chemotherapy drugs, PARP inhibitors also have issues such as hematologic side effects and drug resistance in clinical applications. Furthermore, the efficacy of PARP inhibitors may be limited for patients with non-BRCA-mutated TNBC. Therefore, developing highly specific and effective PARP1-targeting drugs is an urgent problem that needs to be solved.

[0003] Proteolytic targeted chimera (PROTAC) technology is a novel strategy for targeting protein degradation. It involves linking a target protein ligand with an E3 ubiquitin ligase ligand, inducing the formation of a "target protein-PROTAC-E3 ubiquitin ligase" ternary complex, which ubiquitinates the target protein and allows for proteasome degradation. This technology overcomes the resistance problem of small molecule inhibitors, achieving selective degradation of pathogenic proteins. When designing PROTACs, small molecule inhibitors are typically chosen as target protein ligands; however, these suffer from non-specific binding, resistance, and toxic side effects. Peptide PROTACs (P-PROTACs) exhibit high selectivity and affinity, enabling more precise targeting of specific proteins, thereby improving therapeutic specificity and reducing toxicity, especially for targets that have developed resistance to traditional small molecule inhibitors. However, peptides suffer from poor bioavailability and stability, and are easily degraded by proteases, resulting in unsatisfactory pharmacokinetic properties. Therefore, screening and designing suitable P-PROTACs has significant clinical application potential in the targeted therapy of PARP1-overexpressing tumors. Summary of the Invention

[0004] In view of this, the object of the present invention is to provide a peptide targeting PARP1 protein, P-PROTAC, and its applications. To achieve the above-mentioned object, the present invention adopts the following technical solution: This invention relates in one aspect to a peptide targeting the PARP1 protein, wherein the amino acid sequence of the peptide is fdklGsGsG. Preferably, all amino acids fdkls in the peptide sequence are of the D configuration. By using D-configured amino acids, the stability of the peptide sequence in vivo can be improved.

[0005] Another aspect of the present invention relates to a P-PROTAC targeting the PARP1 protein, wherein the amino acid sequence of the chimera is fdklGsGsGLA(Hyp)YI.

[0006] Another aspect of the present invention relates to a micelle containing the aforementioned P-PROTAC. In a preferred embodiment of the present invention, the micelle is in the form of DSPE-PEG. 2000 The micelles serve as a carrier. Preferably, the average particle size of the micelles is 150-250 nm.

[0007] Another aspect of the present invention relates to a method for preparing the above-mentioned micelles, which includes the following steps: DSPE-PEG... 2000 The chimera is dissolved in chloroform and then dissolved in purified water. After mixing, the mixture is sonicated in an ice-water bath to form an emulsion. The chloroform is then removed by rotary evaporation under water bath conditions to obtain micelles.

[0008] Another aspect of the present invention relates to a combined micelle comprising the above-described micelles containing P-PROTAC and micelles composed of DSPE-PEG. 2000 Enclosed DOX.

[0009] Another aspect of the present invention relates to the use of the above-mentioned micelles containing P-PROTAC or the above-mentioned combination micelles in the preparation of medicaments for treating tumors.

[0010] The present invention has achieved the following beneficial technical effects: This invention employs a one-bead-one-compound high-throughput screening method to screen D-type polypeptide sequences that can specifically bind to PARP1 protein from a polypeptide library constructed from D-type amino acids. These sequences are then linked with E3 ubiquitin ligase polypeptides to design proteolytic targeting chimeric molecules, which are then delivered to tumors via a drug delivery system to achieve targeted tumor therapy. Attached Figure Description

[0011] Figure 1 Fluorescence micrograph of resin beads that specifically bind to PARP1 protein.

[0012] Figure 2Results of cytotoxicity studies of different P-PROTACs on 4T1 cells.

[0013] Figure 3 Western blotting was used to detect the degradation level of PARP1 protein by P-PROTAC in 4T1 cells.

[0014] Figure 4 a) Particle size distribution of DOX@MI; b) Particle size distribution of P2P@MI.

[0015] Figure 5 (a) Transmission electron micrograph of DOX@MI; (b) Transmission electron micrograph of P2P@MI.

[0016] Figure 6 (a) Stability of Dox@MI; (b) Stability of P2P@MI.

[0017] Figure 7 (a) Dox@MI cytotoxicity; (b) P2P@MI cytotoxicity; (c) Dox@MI+P2P@MI cytotoxicity.

[0018] Figure 8 Western blotting was used to detect the degradation level of PARP1 protein in 4T1 cells by nanomicelles.

[0019] Figure 9 (a) Curves showing changes in tumor volume in each group of mice; (b) Changes in body weight in each group of mice; (c) Expression of PARP1 protein in mouse tumors. Figure 10 Images of hematologic and epithelial growth factors (H&E) in various organs of mice in each drug-treated group. Scale bar: 50 μm. Detailed Implementation

[0020] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments, but the scope of protection of the present invention is not limited to the following embodiments.

[0021] Example 1: (1) Establishment of D-type polypeptide library Weigh 1.0 g of TentaGel resin (substitution rate 0.25 mmol / g) and place it in a reaction tube. Add 25 mL of DMF to the reaction tube containing the resin and mix thoroughly to allow swelling for 4 h. Weigh 149.2 mg of Fmoc-methionine and dissolve it completely in 2 mL of DMF. Add 5 mL each of coupling reagents HBTU (0.2 mmol / mL) and DIEA (0.2 mmol / mL), mix thoroughly with the resin for 60 min, drain the reaction tube, and wash the resin three times with a small amount of DMF. Take a small amount of resin beads in an EP tube, add ninhydrin reagent, and heat at 100 °C for 3 min. The resin beads turn colorless, indicating that the amino acid has been successfully coupled to the resin beads. Deprotect the resin beads with 10 mL of 20% piperidine solution, repeating twice. Weigh 75.1 mg of glycine (Gly), dissolve it in 2 mL of DMF, add the coupling reagent, mix thoroughly for 60 min, wash away the residual solvent with DMF, and then detect the reaction. The linker sGsG in the peptide sequence was coupled using the same method. The resin was then dried with dichloromethane, weighed, and divided into 18 equal portions, each approximately 65 mg. Following an amino acid:resin molar ratio of 3:1, the following 18 Fmoc-protected D-type amino acids (alanine, valine, leucine, isoleucine, phenylalanine, tryptophan, tyrosine, aspartic acid, asparagine, glutamic acid, lysine, glutamine, serine, threonine, proline, histidine, arginine) and glycine were calculated and weighed. Each was mixed with one of the 18 resin portions, and coupling reagents HBTU and DIEA were added for coupling. After the reaction, the 18 resin portions were mixed again for Fmoc group removal; this was the first round of "resolution-mixing" steps. After completing five rounds of "resolution-mixing" steps, the resulting resin underwent amino acid group removal to obtain the nonapeptide library. (2) Fluorescent labeling of target proteins Dissolve 10 μg of PARP1 protein in 1 mL of PBS. Dissolve 1 mg of fluorescein isothiocyanate (FITC) in 200 μL LDMSO and dilute to a concentration of 0.013 mmol / mL. Slowly add the FITC solution to the protein solution while stirring. Stir for 24 h in the dark. Centrifuge the mixture using an ultrafiltration tube, wash with PBS, and centrifuge to remove unlabeled protein and unreacted FITC.

[0022] (3) Screening of P-PROTAC After deprotection and side-chain deprotection of the established peptide library, 10 μg of fluorescently labeled PARP1 protein was co-incubated with resin beads for 24 h in a shaker at 37°C and 200 rpm. Microscopic observation revealed that some resin beads bound to the protein, exhibiting bright green fluorescence (e.g., ...). Figure 1 (As shown). Select the resin beads with bright green fluorescence and place them in 1 mL of PBS solution for later use.

[0023] Weigh 2.9 mg of guanidine hydrochloride and dissolve it in 5 mL of water. Mix 1 mL of the guanidine hydrochloride solution with the selected resin beads, vortex at 1500 rpm for 5 min, centrifuge, remove the supernatant, and repeat 3 times. Wash with PBS to remove residual guanidine hydrochloride. Weigh 30 mg of cyanogen bromide and dissolve it in a mixture of 500 μL acetonitrile, 400 μL glacial acetic acid, and 100 μL water to obtain a lysis buffer. Add the lysis buffer to the resin, vortex overnight, and centrifuge. The supernatant obtained is the mixed peptide solution to be detected. Peptide sequence analysis was performed by Beijing Biotech Biotechnology Co., Ltd. First, the obtained mixed solution was subjected to reductive alkylation and desalting treatment, and then detected by liquid chromatography-mass spectrometry (LC-MS / MS) to obtain 7 target peptides that bind to PARP1 (as shown in Table 1 below).

[0024] Table 1. Peptide sequences obtained through screening Note: Lowercase letters in the sequence indicate that the corresponding amino acid is a D-type amino acid.

[0025] (4) Study on the cytotoxicity detection of P-PROTAC molecules P-PROTAC molecules that can degrade PARP1 were obtained by covalently coupling the peptide ligand LA(Hyp)YI (where Hyp is hydroxyproline) of the E3 ubiquitin ligase of VHL to the target peptides shown in Table 1 using a solid-phase synthesis method. The peptides used in subsequent experiments were synthesized by Suzhou Modifu Biotechnology Co., Ltd., and the purity of the synthesis was 98% as determined by HPLC.

[0026] Seven P-PROTAC solutions were diluted with culture medium at different concentrations (4 μmol / L, 8 μmol / L, 16 μmol / L, 32 μmol / L, 64 μmol / L, and 128 μmol / L). After 4T1 cells proliferated to an appropriate density, 100 μL of the cell suspension (concentration 2 × 10⁻⁶) was added. 5 Add ( / mL) to 96-well plates. Incubate the 96-well plates in an incubator for 24 h. Then, remove the original culture medium in a sterile laminar flow hood and add pre-prepared P-PROTAC solution, continuing incubation for another 24 h. After incubation, discard the solution from each well and add 100 μL of 10% CCK-8 solution to each well, followed by another 2 h of incubation. Finally, measure the absorbance of each well using a microplate reader.

[0027] The cytotoxic effects of different concentrations of P-PROTAC on cells were detected using a CCK-8 assay. The results are as follows: Figure 2 As shown. Within the set concentration range, the peptide sequences P3P: fGlarsGsGLA(Hyp)YI, P6P: laralsGsGLA(Hyp)YI, P1P: ffaksGsGLA(Hyp)YI, and P4P: llkqasGsGLA(Hyp)YI have little effect on cells and are almost non-toxic. P2P: fdklGsGsGLA(Hyp)YI shows a gradually increasing cytotoxic effect with increasing concentration. The two peptide sequences P5P: lvkldsGsGLA(Hyp)YI and P7P: llfplsGsGLA(Hyp)YI showed some cytotoxic effect at lower concentrations, but the cytotoxic effect tended to stabilize with increasing concentration and did not change much.

[0028] (5) Cellular-level target protein degradation effect of P-PROTAC molecules Seven P-PROTAC solutions were serially diluted using culture medium to obtain concentrations of 1 μmol / L, 10 μmol / L, 25 μmol / L, 50 μmol / L, and 100 μmol / L. 1 mL of cell suspension (concentration 2×10⁻⁶) was then used. 5 P-PROTAC solution ( / mL) was added to a six-well plate, and 1 mL of culture medium was added. After culturing the cells in an incubator for 24 h, the original culture medium was removed, and different concentrations of P-PROTAC solution were added, and the cells were cultured for another 24 h. The PARP1 content level in the cells was detected by Western blot. The results are as follows: Figure 3 As shown, P2P exhibits a significant degradation effect; therefore, P2P was selected for further research.

[0029] (6) Preparation and characterization of nanomicelles Drug-loaded micelles were prepared using three different methods: thin-film dispersion, self-assembly-solvent evaporation, and emulsion-solvent evaporation. The mass ratio of drug to carrier material was set at 1:10, resulting in three different types of drug-loaded micelles. The preparation efficiency was evaluated by comparing the particle size and polydispersity index (PDI) of the micelles obtained by these three methods.

[0030] Thin-film dispersion method: 4 mg of DSPE-PEG was dispersed in the film. 2000 0.4 mg of DOX was dissolved in chloroform, and the chloroform was then removed by rotary evaporation to form a thin film. Next, purified water was added and the mixture was sonicated to obtain drug-loaded micelles.

[0031] Self-assembly-solvent evaporation method: 4 mg of DSPE-PEG 20000.4 mg of DOX was dissolved in hexafluoroisopropanol and added dropwise to water under vigorous stirring. After stirring for 1 h, the mixture was stirred for another 1 h in a fume hood to evaporate the hexafluoroisopropanol, thus obtaining drug-loaded micelles.

[0032] In the emulsification-solvent evaporation method: 4 mg of DSPE-PEG was added. 2000 0.4 mg of DOX was dissolved in purified water and mixed with chloroform. The mixture was then subjected to probe sonication in an ice-water bath for 10 minutes to form an emulsion. The chloroform was then removed by rotary evaporation in a 45°C water bath to obtain drug-loaded micelles. The most suitable method was selected by comparing the particle size and PDI of the micelles obtained by the three methods.

[0033] Subsequently, doxorubicin was replaced with P2P, and P2P-loaded micelle aqueous solutions were obtained using the same preparation process.

[0034] Particle size and distribution of micelle solutions: The particle size, distribution and zeta potential of drug-loaded micelles were determined by dynamic light scattering (DLS), and the results are shown in Tables 2 and 3.

[0035] Table 2 DOX@MI particle size and PDI Table 3 P2P@MI particle size and PDI Because the emulsion-solvent method yielded the smallest drug-loaded micelles with the lowest PDI (polydispersity index), the emulsion-solvent evaporation method was chosen as the preparation method for micelles in subsequent experiments. Dynamic light scattering (DLS) was used to determine the particle size and distribution of the drug-loaded micelles. The results are shown in Figure 4. The average particle size of DOX@MI was 174.9 nm ± 33.4 nm, the PDI was 0.064 ± 0.045, and the zeta potential was -7.2 ± 0.8 mV; the average particle size of P2P@MI was 199.6 nm ± 51.9 nm, the PDI was 0.179 ± 0.023, and the zeta potential was -14.9 ± 0.5 mV.

[0036] Morphological characterization of micelle solutions obtained by the emulsification-solvent evaporation method: The morphology of the nanomicelle particles was observed by transmission electron microscopy (TEM). A suitable amount of diluted micelle aqueous solution was carefully added dropwise to a 400-mesh copper grid and adsorbed in the dark for 10 min. Excess solution was removed, and the solution was treated with 2% phosphotungstic acid for 5 min to remove residual dye. The solution was then rinsed twice with deionized water, allowed to air dry, and observed under TEM. The results are as follows: Figure 5 As shown, the micelle particles exhibit a uniform, near-spherical structure, with dimensions consistent with those obtained by DLS.

[0037] Stability study of micelle solutions obtained by emulsification-solvent evaporation method: Micellar particle size was monitored continuously for 7 days, and the results are as follows. Figure 6 As shown, the micelle solution remained stable for 7 days, with the particle size remaining essentially unchanged, indicating that the micelle solution has good stability.

[0038] Determination of encapsulation efficiency and drug loading of micelles using the emulsification-solvent evaporation method: Take 0.1 mL of micelle solution, add it to 1 mL of methanol, and vortex until completely dissolved. HPLC is used to determine the concentration of doxorubicin, thereby calculating the drug loading (LD) and encapsulation efficiency (EE). Take 0.1 mL of micelle solution, add it to 1 mL of methanol, and vortex until completely dissolved. HPLC is used to determine the concentration of P-PROTAC, thereby calculating the drug loading (LD) and encapsulation efficiency (EE). The drug loading of DOX@MI was 4.4%, and the encapsulation efficiency was 48.6%; the drug loading of P2P@MI was 6.5%, and the encapsulation efficiency was 71.3%. Wherein, LD (%) = weight of encapsulated drug / total weight of drug-loaded micelles × 100%; EE (%) = weight of encapsulated drug / weight of added drug × 100%.

[0039] (7) Cytotoxicity test of micelles by emulsification-solvent evaporation method Dox@MI solutions (0.125 μmol / L, 0.25 μmol / L, 0.5 μmol / L, 1 μmol / L, 2 μmol / L, 4 μmol / L) and P2P@MI solutions (0.0625 μmol / L, 0.125 μmol / L, 0.25 μmol / L, 0.5 μmol / L, 1 μmol / L, 2 μmol / L) were added to 96-well plates and cultured for 24 hours to observe the effect of the drugs on cell proliferation. After culture, 100 μL of fresh medium containing 10% CCK-8 was added to each well. The plates were incubated for another 2 hours, and the absorbance of each well was measured using a microplate reader.

[0040] The results are as follows Figure 7As shown, the cytotoxic effect of P2P@MI increased with increasing concentration. At a concentration of 1 μmol / L, Dox@MI resulted in approximately 50% cell viability. The cytotoxic effect intensified with increasing concentration. In the Dox@MI+P2P@MI group, with a fixed P2P@MI concentration of 2 μM, the cytotoxic effect significantly increased with increasing Dox@MI concentration. At a concentration ratio of Dox:P2P = 4:2, cell viability decreased to approximately 17%. This may be because Dox damages DNA in tumor cells, and P2P degrades the PARP1 protein in cells, preventing it from repairing damaged DNA. The combined use of these two drugs significantly enhanced the cytotoxic effect. The synergistic effect of the two drugs was analyzed using CompuSyn software, and the combination index (CI) was calculated to be 0.184, demonstrating a significant synergistic effect when used together.

[0041] (8) The effect of emulsification-solvent evaporation method on the degradation of target proteins in cells: Dox@MI, P2P@MI, and Dox@MI+P2P@MI solutions were diluted to different concentrations (0.25 μmol / L, 0.5 μmol / L, 1 μmol / L, 2 μmol / L, 4 μmol / L). When 4T1 cells grew and proliferated to a suitable number, 1 mL of cells was injected with 2 × 10⁻⁶ cells / mL. 5 A concentration of [value] / mL was added to six-well plates. After incubation for 24 hours, the PARP1 content in cells was monitored using Western blotting. Results are as follows: Figure 8 As shown, Dox@MI at different concentrations had almost no degradation effect on PARP1 protein in cells. P2P@MI showed a significant degradation effect on the target protein at 2 μM. The combination of Dox@MI and P2P@MI at lower concentrations resulted in a more significant degradation effect.

[0042] (9) In vivo antitumor study of nanomicelles by emulsification-solvent evaporation method Construction of xenograft model: 4T1 cells in logarithmic growth phase were obtained, digested, and their concentration was adjusted to 1×10⁻⁶. 7 / mL, 100μL of cell suspension was subcutaneously injected into the right back of each Balb / c mouse to construct a xenograft tumor model. The tumor volume was increased to 100mm². 3 At approximately 10:00 AM, mice were randomly divided into four groups: PBS group, Dox@MI group, P2P@MI group, and Dox@MI+P2P@MI group.

[0043] Treatment: Mice in each group were injected via tail vein every 3 days for 3 consecutive days. The dosage was 10 mg / kg.

[0044] Indicator detection: During the administration period, the body weight and tumor volume of mice were measured every 2 days; after the treatment, the tumors and important organs of mice in each group were dissected for HE detection; the level of PARP1 protein in the tumor was detected.

[0045] like Figure 9 Figure a shows the changes in tumor volume in mice during treatment in each group. Compared with the PBS group, the average tumor volume in the Dox@MI group and the P2P@MI group increased more slowly over time, while tumor growth was significantly inhibited in the Dox@MI+P2P@MI group, and the level of PARP1 protein in the tumor tissue of the Dox@MI+P2P@MI group was the lowest. Figure 9 The results of b showed that there were no significant changes in the body weight of mice in each group, and the HE staining results (e.g.) Figure 10 As shown in the figure, no significant damage was observed to the major organs and tissues in each group, indicating that each drug administration group had low toxicity and good biosafety.

[0046] (10) Experimental conclusions This invention uses the OBOC method to screen for D-type peptides targeting PARP1 and designs PROTACs based on these peptides. To enhance therapeutic efficacy, P-PROTACs are delivered using nanomicelles and synergistically combined with chemotherapeutic drugs. This approach has significant clinical implications and application value for addressing the challenge of treating tumors with high PARP1 expression.

[0047] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A peptide targeting the PARP1 protein, wherein the amino acid sequence of the peptide is fdklGsGsG.

2. The peptide according to claim 2, wherein the amino acids fdkls in the peptide sequence are all of D configuration.

3. A peptide PROTAC targeting PARP1 protein, wherein the amino acid sequence of the PROTAC is fdklGsGsGLA(Hyp)YI.

4. A micelle containing the PROTAC of claim 3.

5. The micelles according to claim 4, wherein the micelles are in the form of DSPE-PEG 2000 As a carrier.

6. The micelles according to claim 5, wherein the average particle size of the micelles is 150-250 nm.

7. The method for preparing micelles according to claim 5 or 6, comprising the following steps: preparing DSPE-PEG... 2000 The PROTAC is dissolved in chloroform and purified water. After mixing, the mixture is sonicated in an ice-water bath to form an emulsion. The chloroform is then removed by rotary evaporation under water bath conditions to obtain micelles.

8. A composite micelle comprising the micelles of any one of claims 4-6 and a mixture of DSPE-PEG. 2000 Micelles formed by encapsulating DOX.

9. The use of the micelles according to any one of claims 4-6 in the preparation of a medicament for treating tumors.

10. The use of the combined micelles of claim 8 in the preparation of a medicament for treating tumors.

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