CN113307849A - Stapler peptide of targeting tumor stem cell marker CD133 and application thereof - Google Patents
Stapler peptide of targeting tumor stem cell marker CD133 and application thereof Download PDFInfo
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
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Abstract
The invention relates to a stapler peptide of a targeted tumor stem cell marker CD133 and application thereof, belonging to the fields of biomedical detection and medicinal chemistry. Polypeptide amino acid sequence SKDEEX1X2X3X4X5X6X7X8X9X10X11X12X13X14X15Stapling, the resulting stapled peptide comprising two unnatural amino acids X cross-linked at the i and i + n (n ═ 3, 4, 5, 6, 7, 8, 9) positions by a macrocycle-forming linker. Book (I)The stapled peptide of the invention can generate the in-situ induced self-assembly of the CD133 tumor stem cell marker. The stapled peptide has more stable alpha-helix conformation, stronger self-assembly capability and greatly enhanced plasma stability to a certain extent. The stapled peptide can play a targeting role on CD133 positive cells, and can be used as a targeting material for specifically targeting cancer cells.
Description
Technical Field
The invention relates to a stapler peptide of a targeted tumor stem cell marker CD133 and application thereof, in particular to screening of a tumor stem cell marker CD133 specific targeted polypeptide and stapling of a whole carbon chain, and application thereof as a drug carrier, belonging to the fields of biomedical detection and pharmaceutical chemistry.
Background
In recent years, malignant tumors have become important diseases that endanger human health, and the morbidity and mortality of the malignant tumors increase year by year. At present, most malignant tumors can be killed by adopting methods such as surgery, radiotherapy and chemotherapy, targeting, immunotherapy and the like, but most tumors cannot be cured radically and are easy to relapse and transfer. In 2001, the concept of tumor stem cells (CSCs) was formally proposed. Tumor stem cells are the cells with self-renewal, unlimited proliferation capacity and multipotentiality present in very small amounts (1%) of tumor tissues, and are the root cause of tumorigenesis, development, invasion and metastasis. Thus, elimination of CSCs is necessary for successful tumor eradication. Noninvasive imaging methods of CSCs can have profound effects on tumor diagnosis and therapy monitoring. However, the lack of detection, localization and non-invasive quantitative detection techniques has hampered the investigation of CSCs in clinical patients. In particular, the successful non-invasive imaging of CSCs with clinically relevant imaging probes (e.g., antibodies or other ligands that bind to CSC-specific cell surface proteins) has not been reported. Therefore, current work is focused on developing a variety of assays to efficiently analyze as many CSCs as possible and target them for treatment, which may be of great significance for the current diagnosis, prevention and treatment of tumors. The search for specific surface markers by which to sort tumor stem cells is key to further study tumorigenesis, metastasis, recurrence and prognosis.
CD133 is a transmembrane glycoprotein antigen found on human stem/progenitor cell membranes, often expressed on neural progenitors, epithelial/endothelial progenitor cell lineages, hematopoietic stem/progenitor cells, etc., and is currently the most commonly used membrane surface marker for labeling or sorting stem/progenitor cells. There is increasing evidence that CD133 has become a specific marker molecule on the surface of many tumor stem cells. Research shows that the survival time of the liver cancer patient with high CD133 expression is obviously shorter than that of the liver cancer patient with low CD133 expression group, and cytoplasmic CD133 expression is a very significant risk factor influencing the overall survival of the liver cancer patient. The method detects the expression of the peripheral blood CD133 of the liver cancer patient, analyzes the interrelation of the expression with clinical pathological characteristics, tumor distant metastasis and the like, and has important clinical significance. Therefore, the CD133 tumor marker plays a great role in tumor targeted therapy, and the CD133 is taken as a target point of the tumor targeted therapy, so that the CD133 tumor marker can possibly bring about a great breakthrough for the tumor therapy. At present, various biomolecules, including antibodies, nucleic acids, polypeptides and the like, capable of specifically recognizing CD133 have been reported. Compared with antibodies and nucleic acid molecules, the polypeptide molecules have the advantages of diversity, penetrability, easiness in chemical modification and good biocompatibility, so that the polypeptide molecules have a wide prospect in tumor targeted diagnosis and treatment. So far, many reports about the use of targeting polypeptides in tumor diagnosis and treatment have been reported, including small molecule polypeptides and polypeptide nano self-assembly materials, but still face many problems, such as low affinity and insufficient stability of small molecule polypeptides; polypeptide self-assemblies are mostly in a 'building block' form (formed by splicing an identification unit, a signal unit and a hydrophilic or hydrophobic unit), and can form an ordered assembly under the condition of strictly controlling various conditions (solution purity, concentration, acidity and the like) in vitro, but are difficult to play a good synergistic effect in vivo; furthermore, linear polypeptides are susceptible to proteolytic degradation and are difficult to maintain in their native conformation, resulting in inefficient cell penetration. Therefore, in view of the above, there is a need to develop a targeting, specific and in vivo stable CD133 targeted polypeptide for diagnosis and treatment.
Disclosure of Invention
The invention aims to provide a stapled peptide of a targeted tumor stem cell marker CD133 and application thereof, namely targeted polypeptide screening and stapling of the tumor stem cell marker CD133, application of the stapled peptide and near-infrared two-region fluorescent molecule connection to realize in-vivo noninvasive molecular imaging and application of a drug-loaded nano system for self-assembly of the stapled peptide and encapsulation of chemotherapeutic drug combination. Aiming at the defects in the prior art, the invention provides a stable-living-environment staple peptide targeting CD133 for targeting tumor stem cells, the staple peptide is connected with near-infrared two-region fluorescent molecules to target a CD133 tumor marker, and the in-vivo noninvasive imaging is realized; in addition, the stapled peptide can be self-assembled into nanofibers to entrap chemotherapeutic drugs to prepare a CD133 stapled peptide nanofiber-chemotherapeutic drug loaded nano system, and the CD133 stapled peptide is utilized to improve the targeted penetration capability of the drugs on tumor tissues. The invention provides a simple, convenient, rapid, economic and accurate detection means for monitoring the peripheral blood CD133 expression of a patient and the correlation of CD133 positive tumor stem cells in tumor recurrence, metastasis, drug tolerance and prognosis in real time, so as to adjust a treatment scheme in time and improve the prognosis of the patient. The stapler peptide has the advantages of high specificity and affinity, stable living environment, simple preparation method, low cost and strong practicability.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides a polypeptide of a targeted tumor stem cell marker CD133, which can be combined with CD133 protein, and the polypeptide comprises the amino acid sequence: SKDEEX1X2X3X4X5X6X7X8X9X10X11X12X13X14X15；
Wherein S is serine, K is lysine, D is aspartic acid, E is glutamic acid, and X is1Is phenylalanine, tryptophan or tyrosine; x2Is lysine, histidine, arginine or leucine; x3Is isoleucine or leucine; x4Is phenylalanine, tryptophan or tyrosine; x5Is isoleucine or leucine; x6Is lysine, histidine or arginine; x7Is lysine, histidine or arginine; x8Is lysine, histidine or arginine; x9Is glutamic acid, aspartic acid, serine, glutamine, asparagine or threonine; x10Is tyrosine, glutamic acid or aspartic acid; x11Is glutamic acid, aspartic acid, glutamine or asparagine; x12Is leucine or isoleucine; x13Is glutamic acid, aspartic acid, glutamine or asparagine; x14Is lysine, histidine, arginine, glycine or alanine; x15Is a mixture of lysine, histidine,arginine or glycine.
The amino acid residue of the present invention may be in the L-form, the D-form, or a mixture of the L-and D-forms.
As a preferred technical scheme, the amino acid sequence of the polypeptide of the invention is shown as follows: NH (NH)2-SKDEEWHIWIKRHSYNLEGR-Ac(CCP)。
The amino acid sequence shown by the polypeptide has high specific affinity to CD 133.
The screening method of the polypeptide comprises the following steps: three-wheel solid-liquid phase combined screening
The first round of screening was solid phase screening: in the present invention, peptide libraries were designed and constructed based on the protein structural characteristics and molecular recognition theory of human CD133, as reported by Yin et al (Blood, 1997, 90 (12): 5002). Amino-modified TentaGel resin is used as a solid phase carrier, and Fmoc synthesis strategy is utilized to carry out mixing and equipartition to synthesize a library with the capacity of 108The one-bead-one-peptide library of (1). The method of fluorescence labeling magnetic balls and micro-fluidic chips is used for high-throughput one-bead one-object peptide library screening, and positive peptide beads are identified by MALDI-TOF-MS to obtain a series of active polypeptides capable of specifically binding CD 133.
The second round of screening was solid phase screening: the nitrogen end of the active polypeptide which is screened in the first round and can specifically bind to CD133 is connected with a novel molecule DBT (4, 7-bis (5-bromo-2-thienyl) -2, 1, 3-benzothiadiazole) with aggregation-induced emission (AIE) property, and the molecule is a biocompatible environment response molecule. Unlike conventional AIE molecules, DBT does not exhibit fluorescence enhancement due to simple aggregation, and only triggers strong fluorescence when specific hydrophobic cavities (e.g., fibers, vesicles, etc.) are formed. In the free state, DBT is in a freely dispersed state at the end of the peptide chain and does not emit light, whereas it is fluorescently "turned on" if the peptide chain interacts with the target during recognition to achieve ordered self-aggregation. The CD133 positive active tumor tissue suspension is interacted with peptide beads, the fluorescence intensity is observed by an inverted fluorescence microscope (OLYMPUS IX71), the fluorescence positive beads are sequenced by MALDI-TOF-MS, and the CD133 active polypeptide capable of carrying out in-situ induced self-assembly of CD133 is obtained on the basis of the first round of screening.
The third round of screening is liquid phase screening: after the two rounds of screening, 100 positive polypeptides which can not only target CD133 but also realize target induced self-assembly are obtained. The third round of screening was liquid phase screening, using Fmoc SPPS (solid phase peptide synthesis) strategy with Wang resin as solid support, the 100 polypeptides were synthesized and purified by HPLC (high performance liquid chromatography). During synthesis, 100 polypeptides were coupled to DBT-Br and then transferred to 96-well plates. Fresh ex vivo CD133 active tissue suspension was added to each well of the plate. DBT responds to different fluorescent signals, indicating the strength of the CD 133-induced self-assembly ability. Finally, we screened the final peptide sequence based on the different fluorescence intensities.
The method of targeting a CD133 polypeptide staple: introducing two unnatural amino acids X containing alpha-alkenyl, and performing olefin metathesis cyclization between the two unnatural amino acids X to form an all-carbon macrocyclic scaffold; the i-th and i + n-th positions of the amino acid sequence of the polypeptide are stapled (n is 3, 4, 5, 6, 7, 8, 9). Obtaining stapled peptide (SCCP) after stapling; the stapler peptide CD133 tumor stem cell in-situ induced self-assembly capacity is enhanced, and the stapler peptide CD133 tumor stem cell becomes a nano material, has a drug-carrying capacity and acts on CD133 in a targeted manner.
In a preferred embodiment, the present invention provides the stapled peptide as the stapling site i and i + 7.
The stapled peptide has increased alpha-helix conformation, enhanced stability, enhanced self-assembly ability, enhanced targeting ability to CD133, and enhanced membrane penetration ability.
Preferably, the resulting stapled peptide is linked to a linker molecule to form a bivalent or multivalent body, allowing for visualization of targeting of the stapled peptide;
the connection includes: covalently linked or non-covalently linked;
when the stapled peptide is covalently linked to a linker molecule to form a bivalent or multivalent body, the linker molecule is Cy5 fluorochrome, Dibenzothiophene (DBT), 7-nitrobenzo-2-oxa-1, 3-oxadiazolyl (NBD), 6-tertbutoxycarbohydrazinonicotinic acid (HYNIC), 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide (EDC), or N-hydroxysuccinimide (NHS); the connecting molecule is a novel non-toxic cross-linking agent with good biocompatibility.
When the stapled peptide is non-covalently linked to a linker molecule to form a di-or polyvalent entity, the linker molecule is a multimer; the multimer includes: one or a mixture of at least two of polyethylene glycol (PEG), polyvinyl alcohol (PVA), cyclodextrin, polyamidoamine dendrimer (PAMAM), polylactic acid (PLA), and polylactic-co-ethanolamine (PLGA).
The combination of the stapled peptide and the bivalent or multivalent body with a cancer treatment pharmaceutical preparation can target CD133 and better kill cancer cells;
the medicinal preparation is a chemical medicament, a biological medicament, a nano medicament, a radioactive medicament, a photo-thermal treatment medicament or a photodynamic treatment medicament which can kill cancer cells.
Further preferably, the preparation is any one of an alkylating agent, an antimetabolite, an antineoplastic natural drug, an antineoplastic antibiotic, a hormone and metal complex or a tumor radiation targeting marker.
The stapled peptide and the bivalent body or the multivalent body are conjugated or mixed with an imaging preparation, so that the CD133 can be targeted, in vivo imaging is realized, and the position of the CD133 in vivo, namely the position of cancer cells, can be accurately determined;
the imaging preparation is any one of radionuclide, radionuclide marker or molecular imaging preparation.
Advantageous effects
1. The polypeptide sequence is screened in a peptide library with a certain library volume, and the peptide library is designed and constructed based on the protein structure characteristics and the molecular recognition theory of human CD133, so that the polypeptide has a specific structure capable of specifically recognizing the CD133 marker.
2. The polypeptide has certain innovativeness on a screening method, and is obtained by a solid-liquid phase combined multiple-round screening method. On the basis of the prior solid-phase screening, a liquid-phase screening step is added. The in vivo environment is simulated by the fresh in vitro tissue suspension, the in vivo tissue suspension interacts with the peptide library, a polypeptide sequence which can finally generate in vivo tissue induced self-assembly is screened out, and the stability of the targeted polypeptide molecule in the in vivo circulation is further improved.
3. After the linear polypeptide sequence is obtained, the linear polypeptide is stapled to obtain stapled peptide, so that the alpha-helix of the polypeptide is increased, the hydrolysis resistance and the enzymolysis resistance in a living body are enhanced, and the stability is enhanced; meanwhile, compared with linear polypeptide, the membrane penetrating capability of the stapled peptide is enhanced, and the targeting capability of the stapled peptide is further improved.
4. The targeting stapled peptide aiming at the CD133 is the most common tumor targeting stapled peptide at present, and no related report exists, the targeting stapled peptide can be covalently or non-covalently connected with small molecules or polymers such as fluorescent molecules and the like to form a bivalent body or a multivalent body, and can also contain a drug preparation to prepare a pharmaceutical composition. Therefore, in practical application, the stapled peptide can be used as a molecular probe or a pharmaceutical composition preparation to realize targeted diagnosis and treatment on CD133 positive tumors. Meanwhile, a simple, convenient, rapid, economic and accurate detection means is provided for monitoring the correlation between the expression of CD133 in peripheral blood of a patient and the tumor stem cells of CD133 in tumor recurrence, metastasis, drug tolerance and prognosis in real time, so that a treatment scheme is adjusted in time, and the prognosis of the patient is improved.
Drawings
Figure 1 CD133 targeted polypeptide microchip screening figure: FIG. a is a schematic diagram of the principle of screening for CD133 targeting polypeptides; panel b is a schematic representation of positive peptide beads (positive beads within the dashed box).
FIG. 2 Surface Plasmon Resonance (SPRi) assay for affinity of the positive polypeptides CCP and SCCP for human CD133 protein: FIG. a is CCP; and figure b is SCCP.
FIG. 3 experiment of CCP interaction with cells: FIG. a is a schematic diagram of the interaction between CCP and U87 cells with over-expression CD133 and 293T cells with under-expression CD 133; FIG. b is the interaction diagram of CCP and CD133 plasmid transfected 293T cell and CD133 siRNA transfected U87 cell.
FIG. 4NSCCP interaction with CD133 overexpressing U87 cells and uptake in vitro: figure a isNSchematic 5min of SCCP interaction with U87 cells; figure b isNSCCP interacts with U87 cells for 20 min.
Detailed Description
The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Experimental example 1 construction and screening of the CD133 Targeted polypeptide screening System of the present invention
1) Laboratory instruments and materials
N-methylmorpholine (NMM), piperidine, trifluoroacetic acid (TFA), Dichloromethane (DCM), ninhydrin, vitamin C, phenol, tetramethyluronium Hexafluorophosphate (HBTU), piperidine, Triisopropylsilane (TIS), Ethanedithiol (EDT), N, N Dimethylformamide (DMF), dehydrated ether, resin, methanol, various Fmoc-protected amino acids, 4, 7-bis (5-bromo-2-thienyl) -2, 1, 3-benzothiadiazole (DBT), dichloroethane (EDC), Streptavidin magnetic beads (MB-Streptavidin), polypeptide synthesis tubes, a shaker, a vacuum pump, a rotary evaporator, a laser confocal microscope (Olyus FV1000-IX81), all of which are commercially available.
2) Synthesis of CD133 "one-bead-one-substance" polypeptide library
The polypeptide library is synthesized by adopting an Fmoc solid-phase peptide synthesis method, which comprises the following steps: weighing 200mg of Tentagel-NH2Resin, which is circulated according to the solid phase polypeptide synthesis program, sequentially adds Lys, His, Arg and Gly for reaction, and performs mixed homogeneous synthesis after deprotection;
(1) after the reaction is finished, equally dividing 5 parts of resin, respectively adding Lys, His, Arg, Gly and Ala into each tube to perform coupling with an equal amount of HBTU, after the coupling is finished, deprotecting the 5 tubes of resin, mixing, correspondingly equally dividing the resin into a plurality of parts, and sequentially and circularly synthesizing;
(2) glu, Asp, Gln and Asn are added respectively for coupling in the same way as in the step (1);
(3) coupling by adding Ile and Leu respectively in the same way as (1);
(4) glu, Asp, Gln and Asn are added respectively for coupling in the same way as in the step (1);
(5) the coupling method of adding Tyr, Glu and Asp is the same as that of (1);
(6) glu, Asp, Ser, Gln, Asn and Thr are added respectively for coupling in the same way as in (1);
(7) the coupling method is the same as that of (1) by adding Lys, His and Arg respectively;
(8) the coupling method is the same as that of (1) by adding Lys, His and Arg respectively;
(9) the coupling method is the same as that of (1) by adding Lys, His and Arg respectively;
(10) coupling by adding Ile and Leu respectively in the same way as (1);
(11) adding Phe, Try and Tyr respectively to couple as in (1);
(12) coupling by adding Ile and Leu respectively in the same way as (1);
(13) the coupling method is the same as that of (1) by adding Lys, His, Arg and Leu respectively;
(14) adding Phe, Try and Tyr respectively to couple as in (1);
(15) glu, Asp, Lys and Ser are added into three synthesis tubes in five steps respectively, the coupling method is the same as that in the step (1), and after the coupling is finished, resin TFA is deprotected and then mixed. And (3) performing methanol replacement and shrinkage steps, and performing vacuum pumping to obtain the dry resin loaded with the peptide library for later use.
3) Screening for CD133 Positive Polypeptides
(1) Washing the dried peptide library with 1 × PBS for 3 times, adding 5% skimmed milk, sealing the surface of the peptide beads on a vortex mixer at 37 deg.C for 2h, and washing with 1 × PBS for 3 times;
(2) labeling PD-L1 protein according to a biotin labeling kit, mixing the PD-L1 protein labeled by biotin (biotin) with a polypeptide library, incubating at 37 ℃ for 2h, and washing with 1 × PBS for 3 times;
(3) then 100. mu.L of MB-Streptavidin was added to the peptide library and incubated for 2h on a vortex mixer at 37 ℃ in the dark. After incubation, the EP tube containing the polypeptide library was placed on a magnetic frame. The positive polypeptide is magnetically attracted to the side wall of the EP tube, while the negative polypeptide settles to the bottom of the EP tube due to gravity.
FIG. 1a is a schematic diagram of the principle of screening CD133 targeting polypeptides, wherein after positive polypeptide beads are incubated with biotin-labeled receptor protein, the positive peptide beads specifically recognize the protein, and streptavidin-labeled magnetic beads recognize the positive peptide beads by recognizing biotin. In FIG. 1b, within the dotted line, are positive peptide beads. The surface of the positive peptide bead is coated with a layer of magnetic beads which have magnetism and are captured by a magnetic field. Further, the second round of screening was to screen the positive polypeptides for polypeptide sequences capable of inducing self-assembly by CD133 in situ. And selecting the positive polypeptide with the most coated magnetic beads and stronger acting force. CD133 positive tumor tissue was added to tissue lysis buffer to generate a tissue suspension. Peptide beads were incubated with fresh CD133 tissue suspension at 37 ℃ for 2 h. All beads were loaded into a 10 × 10 microarray in a single bead per well fashion. The microarray was then observed by an inverted fluorescence microscope (OLYMPUS IX71) at 40-fold magnification. The fluorescence positive beads were sequenced by MALDI-TOF-MS. The third step is liquid phase screening, 100 polypeptide sequences which can target CD133 and generate CD133 in-situ induced self-assembly are obtained after the first round and the second round of screening, 100 polypeptides are synthesized again according to the sequences, and DBT fluorescent molecules are connected to the 100 polypeptides one by one (DBT has Aggregation Induced Emission (AIE) property, and only when the molecules are assembled to form a hydrophobic cavity, the DBT can emit light). Then, adding the CD133 living tissue suspension into a 96-well plate, adding positive polypeptides connected with DBT, screening polypeptide sequences with good self-assembly performance according to fluorescence intensity in each well, performing secondary mass spectrometry identification through cyanogen bromide cracking, and then solving corresponding sequence information by using a Mascot database. The positive polypeptide was re-synthesized by sequence, partially labeled with fluorescence, identified by MALDI-TOF-MS and purified by HPLC for subsequent experiments. The amino acid sequence of the polypeptide is prepared by chemical synthesis and is as follows: NH (NH)2-SKDEEWHIWIKRHSYNLEGR-Ac(CCP)。
Experimental example 2 stapled peptides were generated and synthesized by stapling i and i +7 sites of CCP by introducing two unnatural amino acids containing an α -alkenyl group to make a loop.
The two unnatural amino acids introduced into the linear peptide chain of the invention are Fmoc-2- (7 '-octenyl) alanine and Fmoc-2- (4' -pentenyl) alanine(ii) an amino acid. Preferably, the synthetic stapled peptide sequence is represented as NH2SKDEEW R8 HIWIKR S5 HSYNLEGR-Ac (SCCP). Wherein R8 and S5 are Fmoc-2- (7 '-octenyl) alanine and Fmoc-2- (4' -pentenyl) alanine, respectively. The synthesis of linear peptides was in accordance with the procedure of Experimental example 1. Upon completion of the synthesis of the linear peptide, a Grubbs (Grubbs) -catalyzed ring-closing olefin metathesis (RCM) was performed to cyclize with the side chain linking R8 and S5. Thereafter, cleavage reagent (87.5% TFA + 5% thioanisole + 2.5% phenol + 2.5% EDT + 2.5% H) was used2O) cleavage of the peptide from Wang resin with a cleavage time of 2 h. After obtaining the stapled peptide, it was identified by MALDI-TOF and purified by HPLC for subsequent experiments.
Experimental example 3 the affinity effect of the CD133 positive polypeptides CCP and SCCP with the CD133 protein was examined by the Surface Plasmon Resonance (SPRi) method.
Respectively spotting CCP (total protein concentration) of 1mg/mL, SCCP (SCCP control protein) and 1 XPBS (phosphate buffer solution) on a chip, incubating overnight at the temperature of 4 ℃ under a humid condition, washing for 10min by 10 XPBS, washing for 10min by 1 XPBS, washing for 2 times by deionized water, soaking for 10min each time in 1 XPBS containing 5% milk, incubating overnight at the temperature of 4 ℃, washing for 10min by 10 XPBS, washing for 10min by 1 XPBS, washing for 10min by deionized water, washing for 2 times by 10min each time, drying by nitrogen, and loading on a chip (plexiera HT surface plasmon resonance imaging system).
The mobile phase was sequentially purified from human CD133 by 1 XPBS, 2 XPBS, 0.78. mu.g/mL, 1.56. mu.g/mL, 3.125. mu.g/mL, 6.25. mu.g/mL, 12.5. mu.g/mL and 25. mu.g/mL, and analyzed for SPRi signal by recording.
As can be seen from FIG. 2, the SPRi signals of CCP (FIG. 2a) and SCCP (FIG. 2b) are gradually increased with increasing protein concentration, indicating that the CD 133-positive polypeptides of the present invention strongly bind to CD133, and that the affinity dissociation constants CCP and SCCP reach 7.10X 10-8M，1.10×10-9M, binding peptide SCCP affinity enhancement after stapling, close to that of antibodies. Can be used as a probe to target the tumor stem cells of CD133 and is used for related detection research and targeted therapy application.
Experimental example 4 CCP interacts with U87 cells with high expression of CD133 and 293T cells with low expression of CD133, respectively.
Human glioma cell line U87(CD133 positive) and human renal epithelial cell line 293T cells (CD133 negative) were incubated in a 37 ℃ incubator (5% CO) with DMEM/High glucose medium containing 10% fetal bovine serum and 1% penicillin and streptomycin2) Culturing in medium.
At 1 × 105Cell concentration per mL in round glass-bottom Petri dishes (35mm), 37 ℃, 5% CO2Culturing for 24h in a cell culture box, discarding culture solution, adding 1 mu mol/L Hoechst 33342 into the two cells respectively, incubating for 15min at 4 ℃ in the dark, washing for 2 times by precooling 1 XPBS, adding 50 mu mol/L FITC labeled polypeptide CCP respectively, incubating for 20min at 4 ℃ in the dark, and washing for 3 times by precooling 1 XPBS. Fluorescence distribution in cells was examined by laser scanning confocal microscopy (Olympus FV1000-IX 81).
As shown in FIG. 3a, strong green fluorescence was observed on the cell membrane of U87 to which CCP was added, whereas 293T cells did not fluoresce. The result shows that CCP polypeptide is combined on the cell membrane of the positive cell, has specificity with the recognition of a human CD133 positive cell line, has positive correlation with the expression quantity of the target protein in specificity, and can be used as a targeting molecule for relevant diagnosis and detection. In contrast, the CCP polypeptide did not observe green fluorescent and antibody signals for negative cells 293T with low CD133 expression, thereby further illustrating that they are specific targeting CD133, and further validating the reliability of the SPRi data in fig. 3.
Experimental example 5 Gene knock-out experiments and plasmid overexpression experiments.
U87 cells were cultured in DMEM/High glucose medium containing 10% fetal bovine serum and 1% penicillin and streptomycin and allowed to grow overnight. The medium was then changed to DMEM/High glucose medium (without antibiotics) containing 10% fetal bovine serum. 0.0025nmol/L of CD133 siRNA was diluted with 50. mu.L of Opti-MEM medium and then allowed to stand at room temperature for 5 min. 2.0. mu.L Lipofectamine was diluted with 50. mu.L Opti-MEMTM2000 (transfection of liposomes) and the mixture was left at room temperature for 5 min. The transfection reagent and siRNA dilutions were mixed and left to stand at room temperature for 20 min. Immediately thereafter, the transfection complex was added to a petri dish containing U87 cells, containing 5% CO at 37 ℃2For 6 h. Thereafter, the medium containing the transfection reagent was discarded and cells were trypsinized. The siRNA-transfected U87 cells were then cultured in DMEM/High glucose medium containing 10% fetal bovine serum and 1% penicillin and streptomycin.
293T cells were grown overnight in DMEM/High glucose medium containing 10% fetal bovine serum and 1% penicillin and streptomycin. The medium was then changed to DMEM/High glucose medium (without antibiotics) containing 10% fetal bovine serum. 0.8. mu.g of the CD133 plasmid DNA was diluted with 50. mu.L of Opti-MEM culture medium and then allowed to stand at room temperature for 5 min. 2.0. mu.L Lipofectamine was diluted with 50. mu.L Opti-MEMTM2000 (transfected liposomes) and then 2. mu. L P3000 addedTMAnd the mixture was allowed to stand at room temperature for 5 min. The transfection reagent and plasmid DNA were mixed and left to stand at room temperature for 20 min. The transfection complex was then immediately added to the culture dish containing 293T cells at 37 ℃ with 5% CO2For 6 h. Thereafter, the medium containing the transfection reagent was discarded and cells were trypsinized. The 293T cells transfected with the CD133 plasmid were then cultured in DMEM/High glucose medium containing 10% fetal bovine serum and 1% penicillin and streptomycin.
Experimental example 6 CCP interacts with 293T, high expressing CD133 cells transfected with CD133 plasmid and low expressing CD133 cells transfected with CD133 siRNA, respectively, U87.
CD133 siRNA transfected U87 cells and CD133 plasmid transfected 293T cells were incubated in a 37 ℃ incubator (5% CO) with DMEM/High glucose medium containing 10% fetal bovine serum and 1% penicillin and streptomycin2) Culturing in medium.
At 1 × 105Cell concentration per mL in round glass-bottom Petri dishes (35mm), 37 ℃, 5% CO2Culturing for 24h in a cell culture box, discarding culture solution, adding 1 mu mol/L Hoechst 33342 into the two cells respectively, incubating for 15min at 4 ℃ in the dark, washing for 2 times by precooling 1 XPBS, adding 50 mu mol/L FITC labeled polypeptide CCP respectively, incubating for 20min at 4 ℃ in the dark, and washing for 3 times by precooling 1 XPBS. Fluorescence fraction in cells was detected by laser scanning confocal microscope (Olympus FV1000-IX81)And (3) cloth.
As shown in FIG. 3b, strong green fluorescence was observed on 293T cell membranes transfected with CD133 plasmid, whereas U87 cells transfected with CD133 siRNA did not fluoresce. This further demonstrates that CCP is specifically targeted to CD 133.
Experimental example 7 preparation of conjugates of SCCP and fluorescent molecules.
The near infrared two-zone dye Nz is used for conjugation to SCCP. SCCP, Nz and Ethylene Dichloride (EDC) (molar ratio 1: 4: 40) were dissolved in Phosphate Buffered Saline (PBS) pH 5.8 overnight. The coupling reaction was almost completely achieved by HPLC monitoring the binding between the peptide and Nz. The reaction mixture was purified by dialysis with a molecular weight cut-off of 3,500Da and then lyophilized. The product was analyzed by MALDI-TOF-MS. Then purified Nz conjugated SCCP (C:)NSCCP) was dissolved in the aqueous solution and left for 48h for subsequent reactions.
Experimental example 8NSCCP interacts with CD133 overexpressing U87 cells and uptake in vitro.
Human glioma cell line U87(CD133 positive) was incubated in a 37 ℃ incubator (5% CO) with DMEM/High glucose medium containing 10% fetal bovine serum and 1% penicillin and streptomycin2) Culturing in medium.
At 1 × 105Cell concentration per mL in round glass-bottom Petri dishes (35mm), 37 ℃, 5% CO2Culturing for 24h in cell culture box, discarding culture solution, adding 1 μmol/L Hoechst 33342 into cells, incubating at 4 deg.C in dark for 15min, washing with precooled 1 × PBS for 2 times, adding 200 μ L15 μ g/mLNSCCP, incubated at 4 ℃ in the dark for 20min, and washed 3 times with pre-cooled 1 XPBS. Fluorescence distribution in cells was examined by laser scanning confocal microscopy (Olympus FV1000-IX 81). Confocal images were acquired at 5min and 20min time points, respectively.
The results are shown in FIG. 4, and FIG. 4a shows that after 5min incubation,NSCCP can bind significantly to cell membranes. Figure 4b shows that after 20min,NSCCP has penetrated the membrane and distributed throughout the cytoplasm and even reached the nuclear region, indicating thatNThe affinity of SCCP for CD133 is enhanced and the cellular permeability of the polypeptide upon stapling is enhanced.
In conclusion, it can be obtained from experimental examples 1 to 8 that the stapled peptide of the present invention has the characteristics of targeted expression of CD133 positive tumor cells and tumor stem cells, and compared with linear polypeptides, the stapled peptide has enhanced targeting ability and enhanced cell permeability, and has clinical application values for diagnosis and treatment of relevant tumors. Therefore, in practical application, the stapled peptide of the present invention can be used as a targeting polypeptide, conjugated or mixed with a signal unit or an agent capable of killing cancer cells, and used for tumor imaging, targeted therapy and detection of an immunotherapy response marker.
The present invention is illustrated by the above examples, but the present invention is not limited to the above process steps, i.e., it is not meant to imply that the present invention must rely on the above process steps to be practiced. It will be apparent to those skilled in the art that any modification of the present invention, equivalent substitutions of selected materials and additions of auxiliary components, selection of specific modes and the like, which are within the scope and disclosure of the present invention, are contemplated by the present invention.
Claims (10)
1. A polypeptide targeting tumor stem cell marker CD133, characterized by: the general formula is as follows:
SKDEEX1X2X3X4X5X6X7X8X9X10X11X12X13X14X15
wherein S is serine, K is lysine, D is aspartic acid, E is glutamic acid, and X is1Is phenylalanine, tryptophan or tyrosine; x2Is lysine, histidine, arginine or leucine; x3Is isoleucine or leucine; x4Is phenylalanine, tryptophan or tyrosine; x5Is isoleucine or leucine; x6Is lysine, histidine or arginine; x7Is lysine, histidine or arginine;X8is lysine, histidine or arginine; x9Is glutamic acid, aspartic acid, serine, glutamine, asparagine or threonine; x10Is tyrosine, glutamic acid or aspartic acid; x11Is glutamic acid, aspartic acid, glutamine or asparagine; x12Is leucine or isoleucine; x13Is glutamic acid, aspartic acid, glutamine or asparagine; x14Is lysine, histidine, arginine, glycine or alanine; x15Is lysine, histidine, arginine or glycine.
2. The polypeptide of claim 1, wherein: the amino acid residues include: l-form, D-form, or a mixture of L-and D-forms;
preferably, the amino acid sequence of the polypeptide is: NH (NH)2-SKDEEWHIWIKRHSYNLEGR-Ac(CCP)。
3. A DNA fragment characterized by: comprising a nucleotide sequence encoding the polypeptide of claim 1 or 2.
4. An expression vector, characterized in that: comprising at least one copy of the DNA fragment of claim 3.
5. A host cell, characterized in that: comprising the expression vector of claim 4.
6. The method for screening the polypeptide according to claim 1 or 2, comprising three rounds of screening combined solid phase screening-liquid phase screening, wherein: in the liquid phase screening, a Fmoc SPPS (solid phase peptide synthesis) strategy is used, Wang resin is used as a solid phase carrier, polypeptide obtained by solid phase is synthesized, and the polypeptide is purified by HPLC (high performance liquid chromatography); during synthesis, the polypeptides were all coupled to DBT-Br, and the whole was then transferred to 96-well plates (one polypeptide per well); adding a suspension of fresh ex vivo CD133 active tissue to each well of the plate; DBT reacts different fluorescent signals, which shows the strength of the self-assembly capacity induced by CD 133; finally, the final peptide sequence was selected based on the different fluorescence intensities.
7. Stapling the polypeptide of claim 1 or 2 to produce a stapled peptide, wherein: introducing two unnatural amino acids X containing alpha-alkenyl, and performing olefin metathesis cyclization between the two unnatural amino acids X to form an all-carbon macrocyclic scaffold; stapling at the i-th and i + n-th position of the amino acid sequence of said polypeptide (n-3, 4, 5, 6, 7, 8, 9); obtaining stapled peptide (SCCP) after stapling; the stapler peptide CD133 tumor stem cell in-situ induced self-assembly capacity is enhanced, and the stapler peptide CD133 tumor stem cell becomes a nano material, has a drug-carrying capacity and acts on CD133 in a targeted manner. (ii) a
In a preferred embodiment, the present invention provides the stapled peptide as the stapling site i and i + 7.
8. The stapled peptide of claim 7 wherein said peptide is selected from the group consisting of: when the stapled peptide is covalently linked to a linker molecule to form a bivalent or multivalent body, the linker molecule is Cy5 fluorochrome, Dibenzothiophene (DBT), 7-nitrobenzo-2-oxa-1, 3-oxadiazolyl (NBD), 6-tertbutoxycarbohydrazinonicotinic acid (HYNIC), 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide (EDC), or N-hydroxysuccinimide (NHS); the connecting molecule is a novel non-toxic cross-linking agent with good biocompatibility;
when the stapled peptide is non-covalently linked to a linker molecule to form a di-or polyvalent entity, the linker molecule is a multimer; the multimer includes: one or a mixture of at least two of polyethylene glycol (PEG), polyvinyl alcohol (PVA), cyclodextrin, polyamidoamine dendrimer (PAMAM), polylactic acid (PLA), and polylactic-co-ethanolamine (PLGA).
9. A pharmaceutical combination characterized by: a cancer therapeutic pharmaceutical formulation comprising a polypeptide according to claim 1 or 2, or a stapled peptide according to claim 7, or a bivalent or multivalent body according to claim 8, and a cancer cell killing agent; the medicinal composition can target CD133, and better plays a role in killing cancer cells;
the medicinal preparation is a chemical medicament, a biological medicament, a nano medicament, a radioactive medicament, a photo-thermal treatment medicament or a photodynamic treatment medicament which can kill cancer cells;
preferably, the agent is any one of an alkylating agent, an antimetabolite, an antineoplastic natural drug, an antineoplastic antibiotic, a hormone and metal complex or a tumor radiotargeting marker.
10. A pharmaceutical combination characterized by: comprising the polypeptide of claim 1 or 2, or the stapled peptide of claim 7, or the bivalent or multivalent body of claim 8, and an imaging agent;
the polypeptide, the stapled peptide, the bivalent body or the multivalent body is conjugated or mixed with an imaging preparation, so that the CD133 can be targeted, in-vivo imaging is realized, and the position of the CD133 in vivo, namely the position of cancer cells, can be accurately determined;
the imaging preparation is any one of radionuclide, radionuclide marker or molecular imaging preparation.
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