Method for screening polypeptide and use thereof

Abstract

Provided is a method for screening a polypeptide. The screening method comprises S1) incubating the various polypeptides in a polypeptide library to be screened with target cells individually in the wells of a cell culture plate; S2) detecting the incubated target cells, and determining a candidate polypeptide on the basis of the changes in the target cells or the positions of the polypeptides; and optionally, S3) sequencing the candidate polypeptide to determine the sequence of candidate polypeptide. Further provided is the use of the method for screening a polypeptide drug. The provided screening method enables the sorting of a single polypeptide molecule and realizes one-by-one high-throughput screening of a polypeptide library on cells, and has the advantages of short cycle, low cost, high accuracy, etc.

Description

Screening method of polypeptide and application thereof

[0001] The present application claims priority to the Chinese patent application (application number: 202411366098.2, invention title: Screening method of polypeptide and application thereof) filed on September 27, 2024, the entire contents of which are incorporated herein by reference. TECHNICAL FIELD

[0002] The present application belongs to the field of biotechnology, and particularly relates to a screening method of polypeptide and application thereof. BACKGROUND

[0003] Polypeptide drugs have higher activity and stronger selectivity, and have obvious advantages in treating complex diseases. Moreover, since polypeptides are compounds composed of amino acids, their metabolic products are amino acids, which generally have no or little side effects on the human body. Compared with protein drugs, polypeptides have the advantages of relatively good stability, high purity, low production cost, low or no immunogenicity, and the quality control level can be close to that of traditional small molecule chemical drugs. In the drug research and development stage, the affinity, solubility, pharmacokinetic properties, toxicity, etc. of drug candidates can be improved through chemical modification. In summary, polypeptide drugs combine the advantages of small molecule chemical drugs and protein drugs.

[0004] At present, the main methods for building and screening polypeptide drug active molecules include phage display, mRNA display, one-bead-one-compound (OBOC library), etc. Among them, phage display and mRNA display are biological methods, which have limitations for screening non-natural amino acid peptide libraries. Although OBOC library can screen non-natural amino acid peptide libraries, it often binds to target proteins non-specifically during the screening process, resulting in a high false positive rate. Therefore, subsequent operations such as solid-phase synthesis, chromatography purification, and freeze-drying are required to obtain the sequences screened by the target protein, and further experiments such as surface plasmon resonance (SPR) and activity determination are required to determine the binding ability of the screened sequences to the target protein to reduce false positives, so that the entire polypeptide sequence screening cycle is long and the cost is high. SUMMARY

[0005] In view of the deficiencies in the prior art, the present disclosure provides a high-throughput screening method of polypeptide molecules based on cell level, which is particularly suitable for polypeptide drug screening of intracellular protein targets.

[0006] According to some embodiments of the present disclosure, a screening method of polypeptide is provided, which comprises the following steps:

[0007] S1) incubating a plurality of polypeptides in a polypeptide library to be screened with target cells in one or more wells of a cell culture plate, respectively;

[0008] S2) detecting the target cells after the incubation, and determining one or more candidate polypeptides according to the changes of the target cells or the positions of the plurality of polypeptides.

[0009] In some embodiments, the screening method further comprises S3) sequencing the one or more candidate polypeptides to determine the sequences of the one or more candidate polypeptides.

[0010] In some embodiments, the one or more candidate polypeptides have the ability to interact with the target cells.

[0011] In some embodiments, the interaction comprises one or more of binding of the one or more candidate polypeptides to the surface of the target cells, entry of the one or more candidate polypeptides into the target cells, binding of the one or more candidate polypeptides to target proteins in the target cells, or killing of the target cells by the one or more candidate polypeptides.

[0012] In some embodiments, the target cells can be selected from different types of cells according to the screening purpose, including but not limited to tumor cells, etc.

[0013] In some embodiments, the plurality of polypeptides in the polypeptide library to be screened have an imaging tag. In some embodiments, the imaging tag is connected to the N-terminus of the polypeptide.

[0014] In some embodiments, the imaging tag comprises one or more of biotin or fluorescent markers.

[0015] In some embodiments, the imaging tag is a fluorescent marker.

[0016] In some embodiments, the fluorescent marker is selected from one or more of fluorescein isothiocyanate (FITC), carboxyfluorescein (FAM), rhodamine red X, X-rhodamine, Alexa Fluor 647, Alexa Fluro 546, Alexa Fluor 488, Cy5, Cy7, cyanine dye, Atto dye, or Janelia Fluor dye.

[0017] In some embodiments, in step S1), each polypeptide in the polypeptide library to be screened is prepared by OBOC method.

[0018] In some embodiments, in step S1), the number of polypeptide species in each well of the cell culture plate is 1-3, preferably 1.

[0019] In some embodiments, the proportion of one polypeptide in each well of the cell culture plate is not less than 50%, preferably not less than 80%, more preferably not less than 90%.

[0020] In some embodiments, the total proportion of two polypeptides and three polypeptides in each well of the cell culture plate is not more than 50%, preferably not more than 20%, more preferably not more than 10%.

[0021] In some embodiments, the cell culture plate includes, but is not limited to, a 384 plate or a 1536 plate, etc.

[0022] In some embodiments, the polypeptide positioned on the cell membrane of the target cell, the polypeptide positioned in the target cell and / or the polypeptide killing the target cell in step S2) are determined as candidate polypeptides.

[0023] In some embodiments, the candidate polypeptide is determined according to the imaging changes of the target cell, for example, by means of cell staining, fluorescence development or high-content photography. In some embodiments, if the candidate polypeptide is a transmembrane peptide, it can be determined by observing whether the fluorescently labeled peptide segment enters the cell, i.e., whether the cell has a transmembrane pore. In other embodiments, if the candidate polypeptide is a polypeptide that binds to a cell surface protein, it can be determined by observing whether the fluorescent label appears on the cell membrane surface. In yet other embodiments, if the candidate polypeptide is a polypeptide that binds to a cell surface protein, it can also be determined by the staining results of cell staining.

[0024] In some embodiments, the polypeptide positioned in the target cell is determined as a candidate polypeptide by using the bright field image and the FITC image of the high-content imager.

[0025] In some embodiments, the candidate polypeptide positioned in the target cell is determined by observing the transmembrane phenomenon pore using the bright field image and the FITC image of the high-content imager. In some embodiments, the software provided with the high-content imager can achieve a certain degree of automatic judgment.

[0026] In some embodiments, the step S1) comprises the following steps: (1) placing a plurality of polypeptides in a polypeptide library to be screened in one or more wells of a well plate, so that the number of microbeads in one or more wells of the well plate is 1-3; the polypeptide library to be screened includes a plurality of polypeptides coupled to different microbeads according to different peptide segment sequences;

[0027] (2) disconnecting the microbeads in the one or more wells of the well plate from the polypeptides thereon;

[0028] (3) taking out a part of the polypeptide in one or more wells of the well plate, and separately incubating the part of the polypeptide with the target cells in one or more wells of the cell culture plate.

[0029] In some embodiments, the step S3) comprises sequencing the remaining part of the polypeptide in the well plate corresponding to the candidate polypeptide in step (3), and determining the sequence of the candidate polypeptide.

[0030] In some embodiments, before the step S1), the library of polypeptides to be screened is prepared by an OBOC method.

[0031] In some embodiments, in the library of polypeptides to be screened, the length of the polypeptide is 5-15 amino acids, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids.

[0032] In some embodiments, each of the amino acids in the polypeptide is independently a natural amino acid or a non-natural amino acid.

[0033] In some embodiments, in the OBOC method, TentaGel S NH2 resin microbeads are used.

[0034] In some embodiments, in the OBOC method, Fmoc-Met-OH or Fmoc-ANP-OH is used to couple with the microbeads to form the library of polypeptides to be screened.

[0035] In some embodiments, in step (1), in the well plate, the proportion of the wells in which the number of microbeads in each well is 1 is not less than 50%, preferably not less than 80%, and more preferably not less than 90%.

[0036] In some embodiments, in step (1), in the well plate, the proportion of the total number of wells in which the number of microbeads in each well is 2-3 is not more than 50%, preferably not more than 20%, and more preferably not more than 10%.

[0037] In some embodiments, step (2) comprises: disconnecting the microbeads in each well of the well plate from the polypeptide thereon by cyanogen bromide cleavage method or photolysis method.

[0038] In some embodiments, Fmoc-Met-OH is used in the OBOC method to couple with the microbeads to form the library of polypeptides to be screened, and then the cyanogen bromide cleavage method is used to disconnect the microbeads from the polypeptide thereon.

[0039] In some embodiments, Fmoc-ANP-OH is used in the OBOC method to couple with the microbeads to form the library of polypeptides to be screened, and then the photolysis method is used to disconnect the microbeads from the polypeptide thereon.

[0040] In some embodiments, the cyanogen bromide cleavage method comprises the following steps:

[0041] M1: adding cyanogen bromide cleavage solution into each well of the hole plate to react, so as to disconnect the polypeptide coupled on the microbead from the microbead;

[0042] M2: drying the hole plate to obtain the polypeptide to be screened.

[0043] In some embodiments, in step M1, the cyanogen bromide cleavage solution comprises a protonating agent and a solvent, the protonating agent comprises any one of hydrochloric acid, acetic acid or formic acid, and the solvent comprises acetonitrile and / or water. In some embodiments, the concentration of hydrochloric acid is 0.1N.

[0044] In some embodiments, in step M2, the drying treatment comprises removing the cyanogen bromide cleavage solution from the hole plate under the action of nitrogen flow.

[0045] In some embodiments, the photolysis method comprises the following steps:

[0046] N1: treating the hole plate with UV irradiation to disconnect the polypeptide coupled on the microbead from the microbead;

[0047] N2: drying the hole plate to obtain the polypeptide to be screened.

[0048] In some embodiments, in step N1, the polypeptide is suspended in PBS buffer.

[0049] In some embodiments, in step N1, the UV irradiation treatment is irradiation under the condition of 35-38W, 360-370nm for 6-10h.

[0050] In some embodiments, in step N2, the drying treatment comprises removing the liquid from the hole plate under the action of nitrogen flow.

[0051] In some embodiments, in step (3), the number of cells in each well of the cell culture plate is 1000-1400.

[0052] In some embodiments, the target cells are in logarithmic growth phase.

[0053] In some embodiments, the incubation time is 4-6h.

[0054] In some embodiments, the number of polypeptide species in each well of the cell culture plate is 1-3.

[0055] In some embodiments, the proportion of polypeptide in each well of the cell culture plate is not less than 50%, preferably not less than 80%, and more preferably not less than 90%.

[0056] In some embodiments, the total proportion of the 2 polypeptides and the 3 polypeptides in each well of the cell culture plate is no more than 50%, preferably no more than 20%, more preferably no more than 10%.

[0057] In some embodiments, in step S2), before detecting the target cells after incubation, the method further comprises the following step: washing the cells after incubation.

[0058] In some embodiments, in step S3), the sequencing is performed by liquid chromatography-mass spectrometry.

[0059] According to another aspect of the present disclosure, there is provided a use of the screening method of the present disclosure in screening of polypeptide drugs.

[0060] In some embodiments, the screening is high-throughput screening.

[0061] In some embodiments, the polypeptide drugs comprise polypeptide drugs of intracellular protein targets.

[0062] According to another aspect of the present disclosure, there is provided a polypeptide obtained by the screening method of the present disclosure.

[0063] In some embodiments, the polypeptide is a cell-penetrating peptide.

[0064] In some embodiments, the cell-penetrating peptide has an amino acid sequence as shown in any one of SEQ ID NOs: 1-9.

[0065] According to another aspect of the present disclosure, there is provided a cell-penetrating peptide having an amino acid sequence as shown in any one of SEQ ID NOs: 1-9.

[0066] In some embodiments, the cell-penetrating peptide has an amino acid sequence as shown in any one of SEQ ID NOs: 1-9.

[0067] In some embodiments, the cell-penetrating peptide has an amino acid sequence as shown in any one of SEQ ID NOs: 1, 2, 6 and 8.

[0068] In some embodiments, the cell-penetrating peptide has an amino acid sequence as shown in SEQ ID NO: 1 or 6.

[0069] According to another aspect of the present disclosure, there is provided a use of the cell-penetrating peptide of the present disclosure in drug delivery.

[0070] According to another aspect of the present disclosure, there is provided a conjugate comprising the cell-penetrating peptide of the present disclosure and a pharmaceutically active molecule coupled to the cell-penetrating peptide.

[0071] In some embodiments, the pharmaceutically active molecule comprises one or more of a nucleic acid, a plasmid, a small molecule compound, a polypeptide, and a protein.

[0072] In some embodiments, the nucleic acid is selected from DNA, mRNA, siRNA, rRNA, tRNA, snRNA, miRNA, or a combination thereof.

[0073] According to another aspect of the present disclosure, there is provided a pharmaceutical composition comprising the polypeptide, the cell-penetrating peptide, or the conjugate as described in the present disclosure, and a pharmaceutically acceptable carrier.

[0074] The present disclosure provides a brand new method to accomplish the sorting of single polypeptide molecules, and realizes the one-by-one high-throughput screening of polypeptide libraries on cells. The screening method realizes the feasibility of screening intracellular protein targets based on polypeptide libraries, reduces the false positive rate, and eliminates the step of re-verification of synthesized peptide segments in the intermediate process, and has the advantages of short cycle, low cost, high accuracy, etc. BRIEF DESCRIPTION OF DRAWINGS

[0075] FIG. 1 shows the high-content photographing result of positive well 1 according to some embodiments of the present disclosure.

[0076] FIG. 2 shows the high-content photographing result of positive well 2 according to some embodiments of the present disclosure.

[0077] FIG. 3 shows the high-content photographing result of positive well 3 according to some embodiments of the present disclosure.

[0078] FIG. 4 shows the high-content photographing result of positive well 4 according to some embodiments of the present disclosure.

[0079] FIG. 5 shows the high-content photographing result of positive well 5 according to some embodiments of the present disclosure.

[0080] FIG. 6 shows the high-content photographing result of positive well 6 according to some embodiments of the present disclosure.

[0081] FIG. 7 shows the high-content photographing result of a cell experiment of a polypeptide of SEQ ID NO: 1 (GLAAKRGR) according to some embodiments of the present disclosure.

[0082] FIG. 8 shows the high-content photographing result of a cell experiment of a polypeptide of SEQ ID NO: 2 (GGRLRGAK) according to some embodiments of the present disclosure.

[0083] FIG. 9 shows the high-content photographing result of a cell experiment of a polypeptide of SEQ ID NO: 6 (ALRGLKAR) according to some embodiments of the present disclosure.

[0084] Figure 10 shows high content imaging results of cell experiments performed with the polypeptide of SEQ ID NO: 8 (GKRGAKLK) according to some embodiments of the present disclosure. DETAILED DESCRIPTION

[0085] The present disclosure provides a high-throughput method for screening active polypeptide molecules at the cellular level, which mainly involves incubating peptide segments (single sequence) loaded on single-bead microbeads with cells in a cell culture plate (e.g., a 1536-well plate) (one bead corresponds to one well), observing the imaging changes of the cells by cell staining, fluorescence display or high-content imaging, etc., to determine positive wells, and further sequencing the corresponding remaining samples in the positive wells.

[0086] In some embodiments, if the candidate polypeptide is a transmembrane peptide, it can be determined by observing whether the fluorescently labeled peptide segment enters the cell. In other embodiments, if the candidate polypeptide is a polypeptide that binds to a cell surface protein, it can be determined by observing whether the fluorescent label appears on the cell membrane surface. In yet other embodiments, if the candidate polypeptide is a polypeptide that binds to a cell surface or intracellular protein, it can also be determined by the staining results of cell staining.

[0087] In some embodiments, the sequence of the candidate polypeptide can be obtained by LC-MS / MS detection and importing the obtained mass spectrometry data into MASCOT software.

[0088] In some embodiments, the method of the present disclosure mainly comprises the following steps:

[0089] Step 1: using Fmoc / tBu strategy, solid-phase synthesis method to synthesize the polypeptide library to be screened.

[0090] (1-1) using S NH2resin microbeads, coupling Fmoc-Met-OH to obtain a peptide library precursor resin;

[0091] (1-2) after removing the Fmoc protecting group (i.e., deprotection) of the peptide library precursor resin, the resin microbeads are evenly divided into N parts (N is the number of types of amino acids planned to be coupled at this site), the Fmoc-protected amino acids (i.e., Fmoc-protected amino acids planned to be coupled at this site, a total of N types) are activated and added to the evenly divided resin microbeads, respectively, for condensation reaction;

[0092] (1-3) Kaiser reagent monitoring reaction, after the reaction is completed, the above microbeads are combined and mixed, the Fmoc protecting group is removed, and steps 2) and 3) are repeated until the amino acid coupling at the last site is completed, to obtain the crude product of the polypeptide library to be screened (coupled on the resin microbeads, respectively);

[0093] (1-4) removing the side chain protecting groups of the crude product of the polypeptide library to be screened to obtain the polypeptide library to be screened (coupled on the resin microbeads respectively) which can be used for screening.

[0094] Preferably, the polypeptides in the polypeptide library to be screened have a length of 5-15 aa.

[0095] Preferably, in step (1-2), the Fmoc-protected amino acids are Fmoc-protected natural amino acids or Fmoc-protected unnatural amino acids.

[0096] Preferably, in step (1-2), the activating reagent used for activating the Fmoc-protected amino acids is a combination of N,N'-diisopropylcarbodiimide DIC and 1-hydroxybenzotriazole HOBT, or a combination of 2-(7-azabenzotriazolyl)-N,N,N',N'-tetramethyluronium hexafluorophosphate HATU and N,N-diisopropylethylamine (DIEA).

[0097] Preferably, in step (1-2), the reaction solvent used for the condensation reaction is any one or a combination of N,N-dimethylformamide (DMF), N-methylpyrrolidone (NMP), dichloromethane (DCM).

[0098] Preferably, in step (1-2), the activating reagent is a combination of 2-(7-azabenzotriazolyl)-N,N,N',N'-tetramethyluronium hexafluorophosphate HATU and N,N-diisopropylethylamine (DIEA).

[0099] Preferably, in step (1-2), the reaction solvent used for the condensation reaction is N,N-dimethylformamide (DMF).

[0100] Preferably, in step (1-3), the Kaiser reagent includes test solution 1 (indantrione: 500 mg, ethanol: 10 mL), test solution 2 (phenol: 80 g, ethanol: 20 mL), and test solution 3 (pyridine: 100 mL).

[0101] Preferably, in step (1-3), the detection condition for monitoring the reaction of the Kaiser reagent is 105°C, 5 min.

[0102] Preferably, in step (1-3), the reagent for removing the Fmoc protecting group includes a DMF solution containing piperidine (PIPE) or a DMF solution containing piperazine and 1,8-diazabicycloundec-7-ene, preferably a DMF solution containing 20% piperidine.

[0103] Preferably, in step (1-4), the reagent for removing the side chain protecting group of the crude product of the polypeptide library to be screened comprises trifluoroacetic acid (TFA), 1,2-ethanedithiol (EDT), thioanisole (TA), triisopropylsilane (TIPS) and water (H2O).

[0104] Preferably, in step (1-4), the volume ratio of TFA, EDT, TA, TIPS and H2O in the reagent for removing the side chain protecting group of the crude product of the polypeptide library to be screened is (92-96):(1-3):(0.5-1.5):(0.5-1.5):(1-3), for example, 94:2:1:1:2.

[0105] Second step: resin microbead dispensing and peptide segment preparation.

[0106] (2-1) Take the appropriate amount of resin microbeads obtained in the first step, add pure water, and suspend the resin microbeads in the solution by ultrasonic vibration;

[0107] (2-2) Transfer the resin microbead suspension to a separation tank, use a 10 μL dispensing gun to suck the resin microbead suspension, and transfer it to each well of a hole plate (for example, a 384-hole plate);

[0108] (2-3) Place the dispensed hole plate in a forced air drying oven to dry and remove water;

[0109] (2-4) Add cyanogen bromide (CNBr) lysis solution to each well of the hole plate for reaction to separate the peptide segment from the resin microbead;

[0110] (2-5) After the reaction is complete, transfer the hole plate to a nitrogen stream to evaporate the lysis solution and obtain the peptide segment to be screened;

[0111] (2-6) Add PBS (phosphate buffer solution) to each well of the hole plate, ultrasonically dissolve, and then proceed to the next cell experiment.

[0112] Preferably, in step (2-1), the concentration of the resin microbead is 0.1-0.3 mg / mL, preferably 0.2 mg / mL.

[0113] Preferably, in step (2-2), the resin microbead suspension sucked by each tip head in the dispensing gun is 8 μL in volume.

[0114] Preferably, in step (2-2), when the resin microbead suspension is transferred to the hole plate using the dispensing gun, the resin microbead in each well of the hole plate is controlled to be 1-3 particles (that is, each well of the hole plate contains only one resin microbead as much as possible).

[0115] Preferably, in step (2-3), the temperature of the air-drying oven is set to 30-40°C, preferably 35°C; the drying time is 2-4h, preferably 3h.

[0116] Preferably, in step (2-4), the CNBr cleavage solution comprises a protonating agent and a solvent, the protonating agent comprises any one of 0.1N hydrochloric acid (HCl), acetic acid (HOAc), formic acid (FA), and the solvent comprises any one of acetonitrile (ACN), water (H2O) or a mixed solution. Preferably, the protonating agent is acetic acid (HOAc), and the solvent is a mixed solution of acetonitrile (ACN) and water (H2O).

[0117] Preferably, in step (2-4), the volume ratio of ACN, HOAc and H2O in the CNBr cleavage solution is (2-6):(3-8):(1-3), for example, 4:5:2.

[0118] Preferably, in step (2-4), the ratio of CNBr to H2O in the CNBr cleavage solution is (0.1-0.3)g:1mL, for example, 0.15g:1mL.

[0119] Preferably, in step (2-4), the reaction time is 12-24h.

[0120] Preferably, in step (2-5), the drying time is 12-24h.

[0121] Preferably, in step (2-6), the PBS is 1x PBS.

[0122] Step 3: Incubate the peptide fragments with cells.

[0123] (3-1) Culture cells (different cells can be selected according to different screening purposes, for example, MDA-MB-231 cells) in a cell culture plate (for example, a 1536-well plate) so that the cells are in the logarithmic growth phase.

[0124] (3-2) Transfer part of the solution (for example, 50%, which can be determined according to the needs) of each peptide fragment obtained in step 2 to the cell culture plate, and add the same volume (for example, 4μL) of culture medium (for example, 1640 culture medium containing 1% FBS (fetal bovine serum)) at the same time, and incubate in a 37°C, 5% CO2 incubator.

[0125] (3-3) After the incubation is completed, wash the cells with FBS-free 1640 culture medium 3 times, and then add culture medium (for example, 8μL of 1% FBS 1640 culture medium) to each well to resuspend.

[0126] (3-4) After the bottom of the cell culture plate is wiped clean with alcohol, place it in the high-content imager, set up two shooting channels of bright field and FITC, use 40x Air, NA 0.6 times lens, 25 fields to shoot the sample.

[0127] (3-5) According to the comparison of the bright field image and the FITC image of high content shooting, find the well with positive transmembrane phenomenon, and determine the positive well number.

[0128] Preferably, in step (3-1), the number of cells in each well of the cell culture plate is about 1000-1400 / well, preferably about 1200 / well.

[0129] Preferably, in step (3-2), the cell culture time is 4-6h, preferably 6h.

[0130] Preferably, in step (3-5), the high-content imager (such as high-content screening analysis system, Operetta CLS) can be used to automatically identify the wells with transmembrane phenomenon, and if necessary, the bright field image and the FITC image of the positive transmembrane phenomenon well automatically identified by the technical expert can be further verified or re-judged to finally confirm the positive transmembrane phenomenon well.

[0131] Step 4: Positive peptide sequencing to obtain positive sequence.

[0132] According to the positive well number, determine the remaining sample of the peptide segment solution (the peptide segment solution transferred in step (3-2)) added in the positive well (specifically, it can be determined according to the correspondence between the positive well number and the well plate number of the peptide segment solution transferred in step (3-2)), and sequence the remaining sample by HPLC to obtain the specific amino acid sequence of the positive sequence.

[0133] Wherein, the specific amino acid sequence of the positive sequence obtained by sequencing the remaining sample by LC-MS specifically includes: transferring an appropriate amount (such as all, 50%, 4μL, etc., which can be adjusted according to the specific circumstances) of the remaining sample to an HPLC sample injection bottle, diluting with water and then detecting by LC-MS to determine the primary mass spectrum, and determining the secondary mass spectrum according to the primary mass spectrum result; after the determination, the secondary mass spectrum image is imported into the sequencing software to obtain the positive sequence structure.

[0134] Preferably, the sample injection amount for LC-MS detection is 20μL, and the volume after dilution with water before LC-MS detection is 60-100μL, preferably 80uL.

[0135] In other embodiments, the above-mentioned second step can also be replaced by a photolysis scheme, which mainly includes the following steps:

[0136] (2-1) Take the appropriate amount of resin microbeads obtained in the first step, add 1xPBS, and suspend the resin microbeads in the solution by ultrasonication and shaking;

[0137] (2-2) Transfer the resin microbead suspension to a separatory funnel, use a 10-μL syringe to aspirate the resin microbead suspension, and transfer it to each well of a well plate (e.g., a 384-well plate) so that the number of resin microbeads in each well is 1-3, preferably 1 (i.e., so that each well of the well plate contains only one resin microbead);

[0138] (2-3) Place the well plate after dispensing in a dark box ultraviolet analyzer and irradiate it under a UV lamp (36 W, 365 nm) (e.g., for 8 h);

[0139] (2-4) After the reaction is complete, transfer the well plate to a nitrogen stream and evaporate the lysis solution to obtain the peptide fragments to be screened;

[0140] (2-5) Add PBS to each well of the well plate, ultrasonicate to dissolve, and then perform the next step of the cell experiment.

[0141] It should be noted that the photolysis scheme is simpler and more efficient than the cyanogen bromide lysis scheme. However, the use of the photolysis scheme requires adaptive adjustment of the method for synthesizing the polypeptide library to be screened in the first step, so that the polypeptide library to be screened obtained contains a photolysis group (i.e., each polypeptide is connected to the resin microbead (directly or indirectly) through a photolysis group).

[0142] In some specific embodiments, when synthesizing the polypeptide library to be screened, Fmoc-ANP-OH (a photosensitive linker) is used instead of Fmoc-Met-OH in step (1-1) above, and the subsequent synthesis method is the same.

[0143] In other embodiments, the dispensing of the resin microbeads in the second step described above can be performed manually (e.g., using a multichannel pipette, such as a syringe) or using automated equipment, such as a microfluidic instrument. Automated dispensing of resin microbeads can improve the efficiency of dispensing and provide throughput.

[0144] The present disclosure provides a brand new microbead-based solid-phase polypeptide library for high-throughput screening of polypeptides at the cellular level. The method can be combined with a microbead-based solid-phase polypeptide synthesis method, and various polypeptide molecules can be separated by microbead separation, thereby realizing the screening of various polypeptides at the cellular level. Meanwhile, the method has high flexibility, and various polypeptide libraries obtained by using natural amino acids and / or unnatural amino acids are suitable for the method. The screening method realizes the feasibility of screening intracellular protein targets based on polypeptide libraries, reduces the false positive rate, avoids the step of re-synthesizing peptide segments in the polypeptide screening method based on the One Bead-One Compound (OBOC) library in the prior art, and further verifies the step through SPR and / or activity determination experiments, thereby having the advantages of short cycle, low cost, and low false positive rate.

[0145] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. For purposes of interpreting this specification, the following definitions will apply and, whenever appropriate, the singular forms of terms will also include the plural forms and vice versa.

[0146] Unless the context clearly indicates otherwise, as used herein, the term "a" and "an" include plural referents. For example, reference to "a cell" includes a plurality of such cells, as well as equivalents thereof known to those skilled in the art, and so forth.

[0147] As used herein, the term "about" means ±20% of the number it directly precedes. In some embodiments, the term "about" means ±10% of the number it directly precedes. In some embodiments, the term "about" means ±5% of the number it directly precedes.

[0148] As used herein, the terms "polypeptide" and "peptide" are used interchangeably herein to refer to a polymer of amino acids of any length.

[0149] As used herein, the term "change of the target cell" refers to a change of the cell after the polypeptide is incubated with the target cell, including but not limited to a change of the cell morphology, a decrease of the activity of the cell, or death of the cell, and the like.

[0150] As used herein, the term "position of the polypeptide" refers to the position of the polypeptide relative to the target cell after the polypeptide is incubated with the target cell, including but not limited to that the polypeptide enters the inside of the target cell, the polypeptide is located on the surface of the target cell, the polypeptide is enriched near a specific organelle of the target cell, or the polypeptide is bound to a target protein in the target cell, and the like.

[0151] As used herein, the term "imaging tag" is a substance capable of labeling a target object (e.g., a polypeptide, a cell, and the like), so that a person or a machine can observe the labeled object through imaging technology.

[0152] The term "bright field image" as used herein refers to a picture taken by the bright field channel of the high content imager.

[0153] The term "FITC image" as used herein refers to a picture taken by the fluorescence channel (e.g. green fluorescence channel: Fluorescein) of the high content imager.

[0154] The term "one bead one compound (OBOC)" library as used herein consists of structurally related compounds (e.g. peptides) covalently attached to a solid support (e.g. resin beads), each resin bead bearing a unique compound.

[0155] In order to make the objects, technical solutions and advantages of the present application clearer, the following further describes the present application in detail in conjunction with the accompanying drawings and examples. The specific examples described herein are only used to explain the present application and do not constitute any limitation on the present application. In addition, in the following description, the description of well-known structures and technologies is omitted to avoid unnecessary confusion of the concept of the present disclosure. Such structures and technologies are also described in many publications.

[0156] The reagents and / or kits used in the following examples are commercially available or can be synthesized by known methods.

[0157] Example 1: Establishment of OBOC polypeptide library

[0158] This example utilizes the OBOC (One Bead One Compound) method to synthesize a polypeptide library of the following sequence on resin beads, with the following steps:

[0159] X8-X7-X6-X5-X4-X3-X2-X1-Met-Beads

[0160] X1-X8 are each independently selected from the amino acids Arg, Lys, Leu, Ala and Gly, a total of X1-X8 is 8 replaceable sites.

[0161] The specific steps are as follows:

[0162] 1) Weigh 0.5 g of NH2 resin (Sub = 0.26 mmol / g, 0.13 mmol) into a solid-phase synthesis tube, add 5 mL of DMF, and swell the resin for 30 min.

[0163] 2) 15 mL centrifuge tube, Fmoc-Met-OH 0.1332 g, HATU 0.1977 g, DMF 5 mL, ultrasonic dissolution, add DIEA 180 μL, mix well after 1-2 min, add to the solid phase synthesis tube, put into the shaking bed reaction 1-2 h, after the reaction is completed, the reaction solution is dried under vacuum, washed with DMF 6 times, take a small amount of resin microbeads (50-100 particles) into a 1.5 mL EP tube, add Kaiser detection reagent 2 drops each, put into a dry constant temperature oven (105 ℃, 5 min), observe the color of the resin, the resin is colorless and transparent, the reaction is complete. Among them, the Kaiser detection reagent includes test solution 1 (indanetrione: 500 mg, ethanol: 10 mL), test solution 2 (phenol: 80 g, ethanol: 20 mL) and test solution 3 (pyridine: 100 mL).

[0164] 3) Add 20% PIPE / DMF solution, remove Fmoc protecting group, react for 20 min, after the reaction is completed, wash with DMF 6 times, take a small amount of resin Kaiser detection, the resin is blue, blue-violet or red-brown, indicating that the Fmoc removal is complete.

[0165] 4) Divide the resin into 5 parts (0.026 mmol each), put into 2.5 mL EP tubes respectively, numbered 1, 2, 3, 4, 5, add Fmoc-Arg-OH 0.0670 g, Fmoc-Lys-OH 0.0487 g, Fmoc-Leu-OH 0.0368 g, Fmoc-Ala-OH 0.0324 g, Fmoc-Gly-OH 0.0309 g respectively, add HATU 0.0395 g respectively, add DMF 1 mL respectively, ultrasonic dissolution, add DIEA 36 μL respectively after dissolution, shake and react for 1-2 h, Kaiser monitor each reaction, after the reaction is completed, combine all the resins, wash with DMF 5 mL 6 times.

[0166] 5) Based on the product of step 4), repeat operation steps 3) and 4) until all X1-X8 sites are coupled.

[0167] 6) Take 5-FAM 0.1957 g, HOBT 0.0703 g into the reaction tube, add DMF 5 mL to dissolve, add DIC 80 μL, react for 12-24 h, then dry the reaction solution, wash with DMF 6 times, dry after washing with DCM 6 times, and let the resin dry at room temperature overnight.

[0168] 7) Add TFA 9.4 mL, EDT 0.2 mL, TA 0.1 mL, H2O 0.2 mL, TIPS 0.1 mL to the reaction bottle respectively, mix well, then add to the solid phase reaction tube, react at room temperature for 3 h, after the reaction is completed, wash with DCM 5 mL 6 times, then dry, and let the resin dry at room temperature overnight.

[0169] Example 2: Microbead Dispensing and Peptide Segment Preparation

[0170] The specific steps are as follows:

[0171] 1) Weigh 4 mg of the resin microbead of the synthetic polypeptide prepared in Example 1 into a 50 mL centrifuge tube, add 20 mL of purified water, and ultrasonically disperse and suspend the resin microbead in water to obtain a resin microbead suspension, with a resin microbead concentration of 0.2 mg / mL.

[0172] 2) Transfer the resin microbead suspension to a separation tank, use a 10 μL dispensing gun to take up the resin microbead suspension and transfer it to each well of a 384-well plate, 8 μL / well, until all the wells are dispensed; use a microscope (200x) to observe the number of resins in each well, which should be 1-3, and if there are individual wells with no resin or >3 resins, use a 10 μL pipette to adjust to 1-3.

[0173] 3) Place the 384-well plate in a forced air drying oven and dry at 35°C for 3 h to evaporate the water.

[0174] 4) Prepare cyanogen bromide (CNBr) cleavage solution: cyanogen bromide (CNBr) 150 mg, acetonitrile (CAN) 2 mL, glacial acetic acid (HAc) 2.5 mL, purified water (H2O) 1 mL, dissolve and mix, then use a dispensing gun to take up the cyanogen bromide (CNBr) cleavage solution and add it to each well of the 384-well plate obtained in step 3), 10 μL / well, seal and avoid light for 16 h.

[0175] 5) After the reaction is complete, place the plate in a nitrogen stream and dry for 16 h, and under a microscope, there should be no liquid residue in the plate.

[0176] 6) Add 1x PBS 10 μL to the plate obtained in step 5), after which seal the plate and ultrasonicate it in an ultrasonic instrument for 1 min to obtain a peptide segment solution.

[0177] Example 3: Incubation of Peptide Segments with Cells

[0178] The specific steps are as follows:

[0179] 1) Culture MDA-MB-231 cells (human breast cancer cells) in an incubator at 37°C with 5% CO2 to a confluence of 80% (culture time is about 24 h) and good cell condition, so that the cells are in the logarithmic growth phase.

[0180] 2) Remove the culture solution from the culture flask, wash the cells with D-PBS (Dulbecco's phosphate buffered saline), add Trypsin (trypsin), and transfer the cells to a centrifuge tube after digestion and detachment.

[0181] 3) After centrifugation at room temperature for 5 min, discard the supernatant, add 1640 culture medium containing 10% FBS (fetal bovine serum), and resuspend gently for 3 times.

[0182] 4) Take 20 μL of the cell suspension into 3 0.5 mL centrifuge tubes, respectively, add 0.08% trypan blue, mix, take 20 μL into a cell counting plate, count, and take the average of three times (cell viability ≥ 95%, use the density of living cells) as the final cell density.

[0183] 5) Take the cell suspension into a 1536-well plate, ensure that the number of cells in each well is about 1200; after centrifugation, place it in an incubator (37°C, 5% CO2) for 24 h.

[0184] 6) After the cell culture is completed, aspirate the culture medium in the well plate; take half of each of the peptide segment solutions obtained in step 6) of Example 2 (4 μL) and an equal volume (4 μL) of 1640 culture medium containing 1% FBS, and add them to each well of the well plate obtained in step 5) (equivalent to adding only 1-3 kinds of polypeptides / peptide segments in each well where cells are cultured).

[0185] 7) Centrifuge the well plate (800 rpm, 2 min).

[0186] 8) Place the well plate in a 37°C, 5% CO2 incubator for 6 h.

[0187] 9) After the culture is completed, aspirate the culture medium in each well of the well plate, centrifuge, and wash the cells with 1640 culture medium for 3 times; after washing, add 1% FBS 1640 culture medium to each well for standby.

[0188] 10) After wiping the bottom of the well plate clean with alcohol, place it in a high-content imager, set two shooting channels of bright field and FITC, use 40x Air, NA 0.6x lens, and 25 fields to shoot the sample, and observe the position of the FITC bright spot after fusing with the bright field cell picture to determine whether it is a positive candidate peptide segment. In this test, 6 positive wells were measured, as shown in Figures 1-6.

[0189] Example 4: Sequencing of positive peptide segments to obtain positive sequence information

[0190] The specific steps are as follows:

[0191] 1) Find the microbead well plate number corresponding to the positive sequence, transfer the remaining half of the sample to an HPLC sample bottle, dilute with water to 80 μL, and then detect LC-MS, the sample injection amount is 20 μL, measure the primary mass spectrum, according to the primary mass spectrum result, set the target molecular weight, and measure the secondary mass spectrum again by injecting 20 μL.

[0192] 2) After the determination is completed, the secondary mass spectrum is introduced into the MASCOT sequencing software to obtain a positive sequence structure, and the measured sequence structure is shown in Table 1 below.

[0193] Table 1: Positive well sequencing results

[0194] Note: The well plate position is counted from left to right and from top to bottom; the polypeptide sequences in each positive well are labeled with 5-FAM at the N-terminal.

[0195] Example 5: Re-verification of positive sequences with membrane penetration ability

[0196] In order to verify whether the obtained positive membrane penetrating peptide sequence has the membrane penetrating ability and exclude the possibility of false positives, four peptides (SEQ ID NO: 1, 2, 6 and 8) were randomly selected for synthesis, preparation, and verification of cell experiments according to the method in Example 3, and the specific results are shown in Figures 7-10.

[0197] Conclusion: As can be seen from Figures 7-10, the four randomly selected peptides all have membrane penetrating ability, and the false positive rate is 0, indicating that the method of the present disclosure realizes the screening of intracellular protein targets at the cell level based on the polypeptide library, and reduces the false positive rate.

[0198] The technical solutions of the present application are not limited to the limitations of the above specific examples, and any technical variations made according to the technical solutions of the present application fall within the protection scope of the present application.

Claims

1. A method for screening polypeptides, comprising the following steps: S1) incubating each polypeptide in a polypeptide library to be screened with a target cell in each well of a cell culture plate respectively; S2) detecting the target cell after incubation, and determining a candidate polypeptide according to the change of the target cell or the position of the polypeptide; and Optionally, S3) sequencing the candidate polypeptide to determine the sequence of the candidate polypeptide. The candidate polypeptide has the ability to interact with the target cell; 2. The screening method according to claim 1, characterized in that, Preferably, the interaction includes one or more of the following: the candidate polypeptide binds to the surface of the target cell, the candidate polypeptide enters the target cell, the candidate polypeptide binds to a target protein in the target cell, or the candidate polypeptide kills the target cell. Each polypeptide in the polypeptide library to be screened has an imaging tag; 3. The screening method according to claim 1 or 2, characterized in that, Preferably, the imaging tag includes one or more of biotin or fluorescent markers; Preferably, the imaging tag is a fluorescent marker; Preferably, the fluorescent marker is selected from one or more of fluorescein isothiocyanate (FITC), carboxyfluorescein (FAM), rhodamine red X, X-rhodamine, Alexa Fluor 647, Alexa Fluor 546, Alexa Fluor 488, Cy5, Cy7, cyanine dye, Atto dye, or Janelia Fluor dye. In step S1), the number of polypeptide species in each well of the cell culture plate is 1-3; preferably, the proportion of polypeptides with 1 species in each well of the cell culture plate is not less than 50%, preferably not less than 80%, more preferably not less than 90%; preferably, the total proportion of polypeptides with 2 species and 3 species in each well of the cell culture plate is not more than 50%, preferably not more than 20%, more preferably not more than 10%; 4. The screening method according to any one of claims 1 to 3, characterized in that, And / or In step S2), the polypeptide located on the cell membrane of the target cell, the polypeptide located in the target cell, and / or the polypeptide killing the target cell are determined as the candidate polypeptide; preferably, the candidate polypeptide is determined according to the imaging change of the target cell; more preferably, the candidate polypeptide is determined by cell staining, fluorescence development, or high-content photography; more preferably, the polypeptide located in the target cell is determined as the candidate polypeptide by using the bright field map and the FITC map of the high-content imager. The step S1) comprises the following steps:

5. The screening method according to any one of claims 1 to 4, characterized in that, (1) placing each polypeptide in the polypeptide library to be screened in each well of the well plate, so that the number of microbeads in each well of the well plate is 1-3; the polypeptide library to be screened includes a plurality of polypeptides coupled to different microbeads according to different peptide sequences; (2) disconnecting the microbeads in each well of the well plate from the polypeptides thereon; (3) taking out a part of the polypeptides in each well of the well plate and incubating them with the target cells in each well of the cell culture plate respectively; And / or The step S3) comprises sequencing the remaining part of the polypeptide in the well plate corresponding to the candidate polypeptide in step (3) to determine the sequence of the candidate polypeptide. Before step S1), the polypeptide library to be screened is prepared by OBOC method.

6. The screening method according to claim 5, characterized in that, ​ Preferably, in the polypeptide library to be screened, the length of the polypeptide is 5-15 amino acids, and / or each amino acid in the polypeptide is independently a natural amino acid or a non-natural amino acid; Preferably, in the OBOC method, TentaGel S NH2 resin microbeads are used; Preferably, in the OBOC method, Fmoc-Met-OH or Fmoc-ANP-OH is used to couple with the microbeads; and / or In step (1), in the well plate, the proportion of wells in which the number of microbeads in each well is 1 is not less than 50%, preferably not less than 80%, and more preferably not less than 90%; the proportion of wells in which the number of microbeads in each well is 2-3 is not more than 50%, preferably not more than 20%, and more preferably not more than 10%. and / or Step (2) comprises: disconnecting the microbeads in each well of the well plate from the polypeptides thereon by cyanogen bromide cleavage or photolysis; Preferably, the cyanogen bromide cleavage comprises the following steps: M1: adding cyanogen bromide cleavage solution in each well of the well plate to react, so as to disconnect the polypeptides coupled on the microbeads from the microbeads; M2: drying the well plate to obtain polypeptides to be screened; More preferably, in step M1, the cyanogen bromide cleavage solution comprises a protonating agent and a solvent, the protonating agent comprises any one of hydrochloric acid, acetic acid or formic acid, and the solvent comprises acetonitrile and / or water; More preferably, in step M2, the drying treatment comprises removing the cyanogen bromide cleavage solution under the action of nitrogen flow; Preferably, the photolysis comprises the following steps: N1: UV irradiation treatment is performed on the well plate to disconnect the polypeptides coupled on the microbeads from the microbeads; N2: drying the well plate to obtain polypeptides to be screened; More preferably, in step N1, the polypeptides are suspended in PBS buffer; More preferably, in step N1, the UV irradiation treatment is irradiation under the condition of 35-38 W, 360-370 nm for 6-10 h; More preferably, in step N2, the drying treatment comprises removing the liquid under the action of nitrogen flow; and / or In step (3), in the cell culture plate, the number of cells in each well is 1000-1400; Preferably, the target cells are in the logarithmic growth phase; Preferably, the incubation time is 4-6 h; Preferably, the number of polypeptide species in each well of the cell culture plate is 1-3; more preferably, the proportion of wells containing 1 polypeptide is not less than 50%, preferably not less than 80%, and more preferably not less than 90%; the total proportion of wells containing 2 polypeptides and 3 polypeptides is not more than 50%, preferably not more than 20%, and more preferably not more than 10%.

7. The screening method according to any one of claims 1 to 6, characterized in that, In step S2), before detecting the incubated target cells, the following step is further included: washing the incubated cells; and / or In step S3), the sequencing is performed by liquid chromatography-mass spectrometry.

8. Use of the screening method of any one of claims 1-7 in polypeptide drug screening; Preferably, the screening is high-throughput screening.

9. A polypeptide obtained by the screening method of any one of claims 1-7.

10. A cell-penetrating peptide having an amino acid sequence as set forth in any one of SEQ ID NOs: 1-9. Preferably, the amino acid sequence of the cell-penetrating peptide is as set forth in any one of SEQ ID NOs: 1-9. More preferably, the amino acid sequence of the cell-penetrating peptide is as set forth in any one of SEQ ID NOs: 1, 2, 6 and 8. Further preferably, the amino acid sequence of the cell-penetrating peptide is as set forth in SEQ ID NO: 1 or 6.

11. Use of the cell-penetrating peptide of claim 10 in drug delivery.

12. A conjugate comprising the cell-penetrating peptide of claim 10 and a pharmaceutically active molecule conjugated to the cell-penetrating peptide. Preferably, the pharmaceutically active molecule comprises one or more of a nucleic acid, a plasmid, a small molecule compound, a polypeptide and a protein. More preferably, the nucleic acid is selected from DNA, mRNA, siRNA, rRNA, tRNA, snRNA, miRNA or a combination thereof.

13. A pharmaceutical composition comprising the polypeptide of claim 9, the cell-penetrating peptide of claim 10 or the conjugate of claim 12, and a pharmaceutically acceptable carrier.

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