CN115724917A - PD-1 targeted polypeptide-PROTAC and application thereof - Google Patents

Abstract

The invention discloses PD-1 targeted polypeptide-PROTAC and application thereof, belonging to the technical field of antitumor drugs. The PD-1 targeted degradation polypeptide-PROTAC comprises a transmembrane peptide sequence, a Linker sequence, an E3 recruitment peptide sequence and a targeted PD-1 protein recognition peptide sequence, and the amino acid sequence of the polypeptide-PROTAC is shown as SEQ ID No:1 is shown. The polypeptide-PROTAC can be combined with PD-1 protein in a targeted manner to degrade PD-1 protein and block a PD-1/PD-L1 signal channel, and the immunosuppression effect is eliminated, so that PD-1 positive cells are specifically killed, and an effective solution is provided for tumor immunotherapy.

Description

PD-1 targeted polypeptide-PROTAC and application thereof

Technical Field

The invention relates to the technical field of antitumor drugs, and particularly relates to PD-1-targeting polypeptide-PROTAC and application thereof.

Background

PD-1/PD-L1 is used as an important component marker participating in the immune regulation process of body tumors, plays an important role in various tumors, is used as an immune suppression site for regulating the effect function of T lymphocytes, exists in the whole tumor immune process in the form of a co-suppression molecule, is combined with PD-1 after the T lymphocytes receive a stimulation signal emitted by PD-1 ligand PD-L1 on the surface of tumor cells, immediately plays a function of monitoring the marker in the immune environment, and helps the tumor cells avoid immune surveillance. Blocks PD-1/PD-L1 signal channel, recovers the immune killing power of T cells and can effectively kill tumor cells.

With the rapid development of biotechnology, the design and development speed of new drugs is greatly improved, but when drugs are taken in vivo, the multi-organ distribution of the drugs is still a great problem for the clinical entry of many drugs from laboratories. The non-specific distribution of the medicine in other tissues or organs not only increases the dosage of the medicine in vivo and increases the cost of the medicine, but also damages normal tissues and organs and causes toxic and side effects.

The PROTAC (protein-targeting chimeras) is a drug development technology for degrading target proteins by using Ubiquitin-Proteasome System (UPS). Structurally, PROTAC comprises three parts: (1) E3 ubiquitin ligase ligands; (3) a target protein ligand; (3) a linker connecting the two ligands. The active form of the triplet "ProTAC" is ultimately formed. PROTACs mediate degradation of the desired target protein by hijacking the activity of the E3 ubiquitin ligase for target protein ubiquitination and subsequent degradation by the 26S proteasome. Binding of proteins to ubiquitin, a small protein modifier, is essential for the 26S proteasome to regulate protein degradation.

As a new promising technology, PROTAC presents a huge opportunity in the following respects. First, proTAC is particularly sensitive to drug-resistant targets. Traditionally, chemotherapy has been the primary method of cancer treatment. Acquisition of chemotherapy drug resistance hinders clinical use and leads to disease recurrence. With the advance of new targets and new drug discovery technologies, another powerful strategy is to inhibit the function of carcinogens or receptors directly through small molecules. The discovery of kinase inhibitors does not address the issue of cancer therapy and eliminates whole target PROTAC. In addition, PROTAC is not sensitive to increase target expression and target protein mutation because they have catalytic effects, and thus low dose of PROTAC is required to exert good effects

The invention content is as follows:

aiming at the defects of the prior art, the invention aims to provide a polypeptide-PROTAC targeting PD-1 and application thereof.

In order to achieve the purpose, the technical scheme of the invention is as follows:

in a first aspect, the present invention provides a PD-1 targeted degradation polypeptide-PROTAC, characterized in that: the amino acid sequence of the PD-1 targeted degradation polypeptide-PROTAC is shown as SEQ ID No:1 is shown.

Preferably, the polypeptide-PROTAC has a molecular structural formula:

SKSYGESQGKEILHK-GSGS-ALAPYIP-RRRRRRRR。

further, the polypeptide-PROTAC can specifically bind to PD-1 and specifically bind to PD-1, induce the degradation of PD-1 and inhibit the downstream signal path of PD-1.

In a second aspect, the invention provides an application of the PD-1 targeted degradation polypeptide-PROTAC in screening/preparing a medicine for treating cervical cancer.

Preferably, the polypeptide-PROTAC can inhibit tumor cell proliferation by blocking a signal pathway of PD-1, and can be used as a biological degradation medicament of a PD-1 binding site for treating tumors.

In a third aspect, the present invention provides a polynucleotide characterized by: encoding a targeted PD-1 degrading polypeptide as described in any one of the above.

In particular, the targeted degradation PD-1 polypeptide-procac of the present invention includes the above-described PD-1 degrading polypeptides and drugs. The linker also comprises a connecting group which connects the PD-1 degradation polypeptide with a drug.

The targeted degradation PD-1 polypeptide-PROTAC is obtained by adopting a known method in the prior art, and can be chemically synthesized by using an automatic polypeptide synthesizer; the nucleotide sequence is deduced through the short peptide sequence, then the nucleotide sequence is cloned into a vector for biosynthesis, and then the nucleotide sequence is connected with a drug; it can also be extracted and purified in large quantities from existing organisms, and then subjected to drug conjugation.

The application of the compound in preparing an anti-tumor medicament refers to the application of targeted PD-1 degradation small molecule polypeptide-PROTAC in preparing a medicament for inhibiting the proliferation of tumor cells with high expression of PD-1 and promoting the apoptosis.

The tumor with high expression of PD-1 is exemplified by cervical cancer. A pharmaceutical composition comprising the targeted PD-1 degrading polypeptide-PROTAC described above; also contains one or more pharmaceutically acceptable carriers.

The pharmaceutically acceptable carrier includes diluent, excipient, filler, binder, wetting agent, disintegrant, absorption enhancer, adsorption carrier, surfactant or lubricant.

The medicine composition can be prepared into various forms such as tablets, granules, capsules, oral liquid or injection, and the medicines of various formulations can be prepared according to the conventional method in the pharmaceutical field.

The invention has the following advantages and beneficial effects:

the PD-1 targeted degradation polypeptide-PROTAC can prevent the combination of PD-1 and other proteins or genes and induce the degradation of PD-1 protein to further influence downstream related genes and signal paths after the PD-1 targeted degradation polypeptide-PROTAC is combined with PD-1, and the PD-1 targeted degradation polypeptide-PROTAC has important significance in the aspects of targeted inhibition of tumor cell proliferation, promotion of tumor cell apoptosis and the like

The polypeptide-PROTAC for targeted degradation of PD-1 can inhibit tumor cell proliferation by blocking a signal path of PD-1, can be used as a biological degradation small molecule drug of a PD-1 binding site, and can be used for preparing drugs for treating tumors. Can be widely applied in the medical and biological fields and can generate huge social and economic benefits.

Drawings

FIG. 1 shows the prediction of the binding strength of protein ligands;

the protein structure prediction visualization results are shown in fig. 2.

FIG. 3 is the amino acid structure of polypeptide-PROTAC.

FIG. 4 shows the quantitative information of the target protein and peptide fragment;

FIGS. 5 and 6 show the difference in signal between the IP group to which peptide1 was exogenously added and the control group to which no polypeptide was added.

Fig. 7, 8, and 9 are the integrated peak areas of the target protein and peptide fragment.

FIG. 10 shows the result of PD-1 degradation by different polypeptide-PROTAC concentrations;

FIG. 11 shows the result of PD-1 degradation by different polypeptide-PROTAC reaction times.

FIG. 12 is a flow cytometry result of observing the entry of polypeptide-PROTAC into cells at different polypeptide-PROTAC concentrations;

FIG. 13 shows the results of flow cytometry for the observation of polypeptide-PROTAC entry at different polypeptide-PROTAC reaction times.

FIG. 14 shows the immunofluorescence results of the polypeptide-PROTAC induced degradation of PD-1 protein.

FIG. 15 shows the results of Ki-67 staining of cells with the polypeptide-PROTAC.

FIG. 16 shows the TUNEL results of the polypeptide-PROTAC on cells.

Detailed Description

The technical solution of the present invention is further explained in detail with reference to the accompanying drawings and specific embodiments.

The examples are for illustration only and do not limit the invention in any way.

In the examples, each raw reagent material is commercially available, and the experimental method without specifying specific conditions is a conventional method and conditions well known in each field, or conditions as recommended by the instrument manufacturer.

The polypeptide-PROTAC sequence of the targeted degradation PD-1 in the following examples is SEQ ID No:1.

the invention is shown in SEQ ID NO:1: SKSYGESQKEILHK-GSGS-RRRG-RRRRRRRR

In the present invention, the amino acid structure of the polypeptide-PROTAC is shown in FIG. 1.

Example 1 design of polypeptide-ProTAC.

An open-source protein structure prediction tool is constructed by using Alphafold 2 and Robeta combined deep learning methods to analyze the PD-1 target structure, AI drug screening software SWISS-Mode is used to perform homologous modeling and computer virtual screening, and a polypeptide fragment with high affinity with PD-1 is obtained by screening. Virtual screening of protein ligand binding strength and structure prediction was performed using Maesrto 11.1 molecular docking software, and the visualization results are shown in fig. 2.

Example 2 Synthesis of polypeptide-PROTAC.

Firstly, a cross-peptide sequence, a Linker sequence and an E3 recruitment peptide sequence are synthesized and connected with a known target protein recognition peptide sequence to finally form the high-purity target PD-1 polypeptide-PROTAC. The amino acid structure of polypeptide-PROTAC is shown in FIG. 3.

Example 3PRM method detects the interaction of a polypeptide with a protein.

1) And (3) immunoprecipitation:

1.1 Add 10-50. Mu.g of cell lysate and appropriate amount of antibody to the microcentrifuge tube. The sample was mixed with the antibody and placed on a magnetic rotator for 10 hours incubation with rotation at 4 ℃. Agarose beads were prepared. The powdered beads were incubated first at 100mg in 1mL0.1MPBS, washed for 1 hour and centrifuged to remove the supernatant. 0.1% BSA prepared in 1ml PBS was added to the above precipitate, mixed for 1 hour using a rotator, and then washed twice in PBS. The supernatant was removed and 400. Mu.L of a buffer made with protease inhibitor (same as lysis buffer) was added. Preparing a sample solution which can be preserved at 4 ℃; the beads were stored for a long time in PBS containing 0.02% azide.

1.2 the sample fluid was mixed well and 80. Mu.L of beads were added to each sample. The lysed bead mixture was incubated at 4 ℃ for 4 hours with rotary stirring. The tube was removed, the supernatant aspirated from the beads and discarded. The beads were washed 3 times with wash buffer or lysis buffer to remove non-specific binding. For each wash, the beads were gently mixed with wash buffer, centrifuged at 4 ℃ and the supernatant discarded. Carefully remove as much wash buffer from the beads as possible.

1.3 the complexes were eluted from the beads by acidification using a buffer containing 0.1-0.2M glycine. Interaction between antibodies is impaired by virtue of the low pH of glycine. And (4) recovering the beads. The eluted sample was immediately neutralized with Tris. The beads (50. Mu.L) were eluted with 3X 50. Mu.L 0.2M glycine by incubating the samples for 10 min with frequent stirring and then centrifugation at low speed. The eluates were combined and neutralized by adding an equal volume of Tris (pH 8.0). Elution and neutralization were repeated once. Wash 2 times with 150 μ L lysis buffer. Gel electrophoresis was used to detect protein precipitation.

2) Sample treatment:

2.1 reaction solution (1% SDC/100mM Tris-HCLpH =8.5/10mM TCEP/40mM CAA) was added to the sample and incubated at 60 ℃ for 1h one-step method for protein denaturation, reduction and alkylation. After diluting with ultrapure water of the same volume, trypsin was added according to the mass ratio of the enzyme to the protein 1, and the mixture was incubated at 37 ℃ overnight with shaking for enzyme digestion.

2.2 the next day TFA was added to stop the cleavage, 16000g were centrifuged and the supernatant was desalted by an in-house SDB desalting column. After being pumped out, the mixture is frozen and stored at the temperature of minus 20 ℃ for standby.

3) Mass spectrum detection:

3.1 Mass Spectrometry analysis was performed using Exploris 480 LC-MS system from Thermo. The peptide fragment sample was aspirated by an autosampler, bound to a C18 capture column, and then eluted to an analytical column (75 μm x 250mm,3 μ M particulate size,

pore size, acclaim PepMap C18 column, thermo). Using two mobile phases (mobile phase A:0.1%80% ACN, 0.1%.

3.2 the flow rate of the liquid phase was set at 300nL/min. Mass spectrometry PRM mode analysis, one MS full scan (R =60k, agc =3e6, max it =50ms, scan range =300-1200 m/z) and several PRM MS2 scans for selected peptide fragments (R =15k, agc =2e5, max it = 50ms) were included in each scan cycle. The HCD collision energy was set at 28. The screening window of the quadrupole was set to 1.6Da.

4) And (3) data analysis:

4.1PRM method establishment and protein quantification are carried out by using Skyline software to obtain quantitative information of target protein and peptide fragment.

4.2 the quantitative values of the protein are from a plurality of peptide fragment parent ions (precorsor), and the quantitative information of the precorsor is from the accumulation of the integrated peak areas of 3-5 daughter ions as shown in FIGS. 7, 8 and 9.

4.3 the protein quantitation value obtained is used for subsequent quantitative comparison after normalization of the total TIC signal between different samples. Quantitative information is shown in figure 4, signals of an IP group added with peptide1 and a control group without added polypeptide are obviously different, and as shown in figures 5 and 6, the exogenous added polypeptide is suggested to have an interaction relation with PD-1 protein.

EXAMPLE 4 polypeptide-PROTAC degradation PD-1 assay

1.1 MOLT-4 cells at 5X 10 ⁵ Inoculating each cell/well in a 6-well cell culture plate, wherein the volume of a culture medium in each well is 2mL, and culturing for 24 hours; adding polypeptide with different concentration gradients (0 μ M,2 μ M,5 μ M,10 μ M,20 μ M,30 μ M,50 μ M), culturing for 16 hr, collecting cells, adding lysate, and collecting protein; adding polypeptide with the same concentration gradient (20 mu M) for culturing for different time (0h, 2h,4h,8h,12h and 24h), collecting cells, adding lysate and collecting protein;

1.2 preparing SDS-PAGE gel with the concentration of 10% according to an elegant enzyme gel preparation kit; SDS-PAGE protein loading buffer 5 XSample bf was added to the collected protein samples and mixed. The protein sample was mixed with sample bf and centrifuged at 12000rpm for 10 seconds at room temperature. And (3) putting the protein mixed solution into a water bath kettle, and carrying out water bath for 5-10 minutes at the temperature of 100 ℃ to fully denature the protein. And (3) centrifuging the protein after cracking at the rotating speed of 12000rpm for 60s at room temperature, and cooling at room temperature. After cooling to room temperature, the protein samples were loaded directly into SDS-PAGE gel loading wells. And aligning the sample loading groove according to the designed sequence, and slowly adding the sample and the pre-dyed protein Marker. When electrophoresis is carried out, the bromophenol blue reaches the position close to the bottom end of the gel, and then the electrophoresis is stopped. The cassette was prepared and filled with methanol, transfer bf, and the gel glass plate was removed from the electrophoresis apparatus. The gel was removed and the concentrated gel and bromophenol blue at the lower end were cut off with a razor blade. The length and width of the glue was measured with a ruler. Two pieces of filter paper of the same size and one piece of PVDF membrane were cut out. The membrane was shaken for 1 min by treatment with methanol and then washed 5 times with RO water, 1 min/1 time. And then soaked with transferbf for more than 10 minutes. Filter paper and glue were also soaked in transferbf. The cotton blocks in the film converter are cleaned, dried and wetted by a transfer bf, and the film converter blackboard is arranged for self according to the sequence of sponge-filter paper-gel-PVDF film-filter paper-sponge. The air bubbles can be removed by lightly rolling the glass tube back and forth with a smearing bar or a 15ml glass centrifuge tube. 260mA voltage for 100min. The membrane was removed with forceps, protein side up, and blocked in 5% skim milk powder blocking solution for 90min. With reference to the specification of the primary antibody, 10mL of the hybridization system was selected. The membrane was incubated on a shaker at 4 ℃ overnight. Washed 3 times with TBST, 5 min/time, and washed on a shaker with slow shaking. The secondary antibodies were diluted in the appropriate proportions, with reference to the secondary antibody specifications. Incubate for one hour on a shaker with slow shaking. Washed 5 times with TBST for 5 min/time and slowly shaken on a shaker.

1.3 detection of proteins using ECLWestern fluorescence detection reagents. As shown in FIGS. 10 and 11, the polypeptide degrades PD-1 more over time, and the higher the concentration of the polypeptide, the more PD-1 degrades over the same time.

Example 4 flow cytometry Observation of polypeptide-PROTAC Envelopment

MOLT-4 cells were treated with rhodamine-labeled drugs at different concentrations and at different time gradients. The cells were then washed 2 times with PBS, digested with 0.25% trypsin, and the fluorescent cells were counted using a flow cytometer. As a result, as shown in fig. 12 and 13, the concentration gradient increased and the number of fluorescent cells gradually increased with the increase of the treatment time. Indicating that the amount of the drug entering the cells has a time-dependent and concentration-dependent relationship.

Example 5 mechanism of polypeptide-PROTAC to induce degradation of PD-1 protein

1.1 degradation of PD1 protein was observed without/with proteasome inhibitors. The drug itself carries a rhodamine red fluorescent label and localizes the target protein by immunofluorescence staining. The experimental procedure was protected from light. Cells were fixed in 4% paraformaldehyde for 15 minutes at room temperature and fixed with 0.3% Triton-X100 permeabilities for 15 minutes. After 30min incubation with 1% Bovine Serum Albumin (BSA), cells were incubated with primary anti-PD-1 (1. After multiple washes with PBS, cells were incubated with 1% bsa secondary antibody for 1 hour at room temperature and nuclei were counterstained with DAPI.

1.2 slides were mounted with anti-fluorescence quenching slides. Cells were observed and imaged using a confocal microscope. As a result, it was found that protease inhibitors could prevent polypeptide-mediated degradation of PD-1, as shown in FIG. 14.

Example 6 the polypeptide-ProTAC increases cisplatin sensitivity in cervical cancer cells.

1) KI67 detection of cell proliferation

The confluent cell was washed 2 times with PBS, fixed in 4% formaldehyde for 15min, and washed with PBS. Triton X-100 (0.3% assay) was added and reacted for 15 minutes, followed by incubation with BSA at 37 ℃ for 30 minutes. Incubate with anti-ki 67 antibody (1. After washing 2 times with PBS, nuclei were counterstained with DAPI, incubated with Cy 3-coupled goat anti-rabbit antibody. Slides were mounted with an anti-fluorescence quenching slide. The fluorescence signal was observed under a fluorescence microscope. As shown in FIG. 15, cisplatin inhibited C33a proliferation and promoted apoptosis in tumor cells, and the polypeptide-PROTAC promoted this effect, suggesting that the polypeptide-PROTAC could enhance the sensitivity of tumor cells to cisplatin.

2) TUNEL detection of apoptosis

Confluent cell-confluent slides were washed 2 times with PBS, fixed in 4% formaldehyde for 15min, and washed with PBS. TUNEL detection of cervical cancer cells was performed using a one-step TUNEL apoptosis assay kit according to the manufacturer's instructions. The sample was incubated with TUNEL reaction mixture for 1h at 37 ℃ in the dark, then washed 2 times with PBS. Condensed or fragmented nuclei of apoptotic cells were observed under 200-fold microscope. As shown in FIG. 16, cisplatin promotes C33a apoptosis and increased cell debris, and the polypeptide-PROTAC promotes this effect, further suggesting that the polypeptide-PROTAC may enhance the sensitivity of tumor cells to cisplatin.

Claims (6)

1. A PD-1-targeted degrading polypeptide-procac, characterized by: the amino acid sequence of the PD-1 targeted degradation polypeptide-PROTAC is shown as SEQ ID No:1 is shown.

2. The PD-1 targeted degradation polypeptide-PROTAC of claim 1, wherein: the molecular structural formula of the polypeptide-PROTAC is as follows: SKSYGESQKEILHK-GSGS-ALAPYIP-RRRRRRRR.

3. The PD-1 targeted degradation polypeptide-PROTAC of claim 2, wherein: the polypeptide-PROTAC can be specifically combined with PD-1 and PD-1, induce the degradation of PD-1 and inhibit the downstream signal path of PD-1.

4. Use of the PD-1 targeted degrading polypeptide-PROTAC according to any one of claims 1 to 3 in the screening/preparation of a medicament for the treatment of cervical cancer.

5. Use according to claim 4, characterized in that: the polypeptide-PROTAC can inhibit tumor cell proliferation by blocking a signal path of PD-1, and can be used as a biological degradation drug of a PD-1 binding site for treating tumors.

6. A polynucleotide, wherein: encoding the targeted PD-1 degrading polypeptide of any one of claims 1 to 3.

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