CN116199746A - High affinity Trop2 targeting multi-cyclic peptide molecular framework - Google Patents

High affinity Trop2 targeting multi-cyclic peptide molecular framework Download PDF

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CN116199746A
CN116199746A CN202211508910.1A CN202211508910A CN116199746A CN 116199746 A CN116199746 A CN 116199746A CN 202211508910 A CN202211508910 A CN 202211508910A CN 116199746 A CN116199746 A CN 116199746A
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trop2
peptide
polypeptide
cyclic peptide
phage display
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吴川六
刘洪潭
董萌
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Xiamen University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1037—Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

A high affinity targeted Trop2 multi-cyclic peptide molecular framework belongs to the technical field of biology. Ligand screening is carried out on human trophoblast surface glycoprotein antigen 2 (Trop 2) by utilizing phage display polycyclic peptide library, and a series of polycyclic peptide molecular frameworks capable of combining with Trop2 with high affinity are obtained. On the basis, the obtained specific cyclopeptide ligand is further optimized, so that more polypeptide ligands with pharmaceutical properties are obtained. Can be used for constructing various polypeptide-drug coupling bodies and provides a targeting recognition element for developing a novel disease diagnosis and treatment method. The advantage of CPPC motif orthogonal pairing is utilized to obtain the multi-element cyclic peptide with obvious oxidation main product and structural rigidity. Compared with the common linear peptide and unit cyclic peptide, the constructed multi-membered cyclic peptide has larger binding interface, more complex rigid structure and better binding affinity with a target.

Description

High affinity Trop2 targeting multi-cyclic peptide molecular framework
Technical Field
The invention belongs to the technical field of biology, in particular to a high-affinity targeted Trop2 multi-cyclic peptide molecular framework, which is based on phage display technology to obtain a series of polypeptide frameworks with high affinity for human trophoblast surface glycoprotein antigen 2 (Trop 2), and can be used for screening, synthesizing, purifying and affinity characterizing ternary cyclic peptide molecular ligands capable of binding Trop2 proteins with high affinity.
Background
Human trophoblast surface antigen 2 (Trop 2) is a cell surface glycoprotein that is overexpressed on a variety of cancer cells, but is expressed poorly or not in normal tissues. Trop2 regulates extracellular regulated protein kinase (ERK), mitogen Activated Protein Kinase (MAPK), inositol triphosphate (IP) 3 ) Signal paths such as calcium ions and the like promote proliferation and growth of tumor cells through various action mechanisms. In addition, the overexpression of Trop2 also reduces cell adhesion and promotes metastasis of tumor cells (Michelle Shen, shiqin Liu, tanya Stoyanova. The roll of Trop2 in prostate cancer: an oncogene, boom, and therapeutic target [ J ]]AmJ Clin Exp Urol,2021,9 (1): 73-87.). Based on the Trop2 serving as a molecular marker for clinically detecting the malignancy degree of tumors and a tumor treatment target, the development of a drug targeting the Trop2 has significance for the treatment of various cancers of human beings. Trodelvy is the only antibody coupled drug (ADC) targeting Trop2 marketed in batches at present for treating the triple negative breast cancer, and a certain therapeutic effect is achieved.
Three common therapeutic agents are small molecules, polypeptides and biological macromolecules. The specificity of the small molecular medicine is relatively poor, off-target or cytotoxicity is easy to cause, the biological macromolecular medicine is easy to cause immunogenicity, and the polypeptide medicine can well make up the defects between the two. In addition, polypeptides may mimic some of the characteristics of a protein interface in structure, and may act on a protein-protein interaction interface in combination with the advantages of its molecular size. The polypeptide has better biocompatibility and lower biotoxicity, and is a very potential drug molecule. Cyclic peptides also have a number of advantages over conventional linear polypeptides. The cyclic peptide has higher receptor affinity and selectivity, has rigid conformation, low sensitivity to proteolytic enzyme and high stability.
At present, the development of a plurality of medicaments starts from peptide ligands serving as lead compounds, and the development of display technologies such as phage and the like also provides new technical support for the discovery of polypeptide ligands aiming at targets, so that the development process of the polypeptide medicaments is quickened. The phage display technology is characterized in that phage display polypeptides which specifically bind to target proteins can be rapidly and directionally screened. Meanwhile, by virtue of the advantages of cyclic peptides, a conformational restricted cyclic peptide library can be constructed to screen for a macromolecular target of interest, and new drug leads and new reagents for chemical biology can be developed.
The invention constructs a series of cyclopeptide ligands capable of specifically binding with Trop2 based on phage display technology. The cyclopeptide ligand has the advantages of simple synthesis, low preparation cost and high affinity and stability, and provides a brand new drug lead molecule for diseases taking Trop2 as a therapeutic target.
Disclosure of Invention
The invention aims at providing a high-affinity Trop 2-targeting multi-membered cyclic peptide molecular framework, and the polypeptide molecules have three binding rings which interact with targets, and have a larger binding interface compared with a single cyclic peptide, so that better binding affinity and specificity can be provided, and the polypeptide molecules are expected to become Trop 2-targeting drug lead molecules.
It is another object of the present invention to provide a method for constructing a high affinity Trop2 targeting multi-cyclic peptide molecular framework.
A third object of the present invention is to provide the use of the above-mentioned framework of multi-cyclic peptide molecules for developing drugs/reagents/drug lead molecules for the treatment and diagnosis of related diseases caused by Trop2 signaling pathway.
The high-affinity Trop 2-targeted multi-cyclic peptide molecular framework is obtained by phage display technology and constructing a secondary polypeptide library aiming at target protein Trop2, and the sequence of the multi-cyclic peptide molecular framework comprises the following characteristics:
CPPC(X a )XW(X b )EC(X c )(X d )(X e )(X f )(X g )C(X h )(X i )(X j )(X k )(X l )CPPC；
wherein the amino acids are L amino acids, and X represents any natural amino acidAn acid; (X) k ) D, W, H, T, Y, V, E, S, F, G, M, N, I, preferably D; (X) f ) F, V, L, N, A, D, S, Y, M, I, T, W, preferably F; (X) d ) D, G, S, N, R, T, Q, E, A, preferably G; (X) e ) E, F, L, D, Y, M, preferably E; (X) j ) I, V, L, M, F, R, T, K, preferably V; (X) l ) F, W, G, M, I, Y, L, V, preferably F; (X) g ) Is any amino acid, preferably N; (X) h ) Is any amino acid, preferably V; (X) c ) Is any amino acid, preferably S; (X) i ) V, A, I, P, L, A, E, T, D, S, F, Y, M, preferably V; (X) a ) G or S, preferably G; (X) b ) Is L or I, preferably L;
d is aspartic acid; w is tryptophan; h is histidine; t is threonine; y is tyrosine; v is valine; e is glutamic acid; s is serine; f is phenylalanine; g is glycine; m is methionine; n is asparagine; i is isoleucine; l is leucine; a is alanine; r is arginine; k is lysine; p is proline; c is cysteine; q is glutamine.
In particular, ligand sequences of the high affinity Trop 2-targeting multi-cyclic peptide molecular framework include, but are not limited to:
CPPCGRWLECDSFTNCWELLTCPPC(peptide 1)
CPPCGAWLECDSFTNCWELLTCPPC(peptide 2)
CPPCGSWIECDSFTNCWELLTCPPC(peptide 3)
CPPCGAWIECDSFTNCWELLTCPPC(peptide 4)
CPPCSEWIECDSFTNCWELLTCPPC(peptide 5)
CPPCGRWLECSDENLCAIIDWCPPC(peptide 6)
CPPCGRWLECYDFNECELIDWCPPC(peptide 7)
CPPCGRWLECWGMFNCQEFQGCPPC(peptide 8)
CPPCGRWLECWTHIDCQFIDWCPPC(peptide 9)
CPPCGRWLECSNDFDCDYLYFCPPC(peptide 10)
CPPCGRWLECLDEFRCTLIFHCPPC(peptide 11)
CPPCGRWLECYDEFSCEPFWFCPPC(peptide 12)
CPPCGRWLECSDGFTCNAVDFCPPC(peptide 13)
CPPCGRWLECSSPFTCTIFIGCPPC(peptide 14)
CPPCGRWLECSEDFLCDLRTFCPPC(peptide 15)。
the construction method of the high-affinity targeted Trop2 multi-cyclic peptide molecular framework comprises the following specific steps:
1) Screening a Trop2 specific targeting ligand by using a phage display ternary cyclic peptide library to construct a phage display polypeptide library1;
2) Screening by utilizing a phage display polypeptide library1 aiming at a target protein Trop2, and selecting a monoclonal after 3 rounds of screening for sequencing analysis to obtain a polypeptide sequence shown in sequence table SEQ ID No. 1-5;
3) Selecting the polypeptide peptide1 with the highest enrichment amount according to the screening result, constructing a phage display polypeptide library 2 according to the polypeptide sequence of the polypeptide peptide1, and screening the Trop2 protein by utilizing the phage display polypeptide library 2; and 3 rounds of phage sequencing analysis after screening to obtain polypeptide sequences shown in sequence table SEQ ID No. 6-15.
In step 1), the phage display polypeptide library1, the N-terminal to C-terminal polypeptide sequence backbone is as follows:
CPPC(X) 5-10 CDSFTNCWELLTCPPC(library 1)
wherein, the amino acids are L-type amino acids, X represents any natural amino acid, and the subscript represents the number containing X.
In step 3), the phage display polypeptide library 2, the N-terminal to C-terminal polypeptide sequence backbone is as follows:
CPPCGRWLEC(X) 5 C(X) 5 CPPC(library 2)
wherein, the amino acids are L-type amino acids, X represents any natural amino acid, and the subscript represents the number containing X.
In the step 3), constructing a phage display polypeptide library 2, taking a polypeptide sequence skeleton as a template, carrying out fusion expression on polypeptides and p III proteins of phage, and displaying the fusion expression on the surface of the phage to construct a phage library for displaying different polypeptide sequences; phage display polypeptide library 2 is a secondary library constructed on the screening result of phage display polypeptide library1 against target protein Trop2, and the partial conserved sequences obtained by screening phage display polypeptide library1 against target protein Trop2 are immobilized to randomize the amino acids of the other two binding loops.
The high-affinity Trop 2-targeted multi-cyclic peptide molecular framework can be applied to construction of various polypeptide-drug coupling bodies.
The high-affinity Trop 2-targeting multi-cyclic peptide molecular framework can be used as a targeting recognition motif.
The high affinity Trop 2-targeting multi-cyclic peptide molecular framework can be used for developing drugs/reagents/drug lead molecules for the treatment and diagnosis of related diseases caused by Trop2 signaling pathways.
Compared with the prior art, the invention has the following technical effects and advantages:
a. the invention utilizes the advantages of CPPC motif orthogonal pairing, and can obtain the multi-element cyclic peptide with obvious oxidation main product and structural rigidity. Compared with the common linear peptide and unit cyclic peptide, the multi-element cyclic peptide constructed by the invention has larger binding interface, more complex rigid structure and better binding affinity with a target.
b. The polypeptide of the invention is composed of natural amino acids, is simple to synthesize, and can be prepared in a large quantity by solid phase synthesis or recombinant expression.
c. According to the invention, a series of polypeptide frames with high specific binding with the target protein Trop2 are obtained through phage display technology and construction of a secondary polypeptide library aiming at the target protein Trop 2. The obtained specific cyclopeptide ligand can be further optimized on the basis of the tendency and conservation of amino acids at different positions, so that more polypeptide ligands with pharmaceutical properties can be obtained.
Drawings
FIG. 1 shows the results of monoclonal sequencing obtained by library screening of library 1.
FIG. 2 shows the top 50 sequences with the highest enrichment for library screening of library 2.
FIG. 3 is a chromatogram after reduction and oxidative purification of peptide 1.
FIG. 4 is a reduction and oxidation (after purification) chromatogram of peptide 6.
FIG. 5 is a reduction and oxidation (after purification) chromatogram of peptide 7.
FIG. 6 is a reduction and oxidation (after purification) chromatogram of peptide 8.
FIG. 7 shows a peptide1 (oxidized form) Surface Plasmon Resonance (SPR) curve.
FIG. 8 is a peptide 6 (oxidized form) Surface Plasmon Resonance (SPR) curve.
FIG. 9 shows a peptide7 (oxidized form) Surface Plasmon Resonance (SPR) curve.
FIG. 10 shows a peptide8 (oxidized form) Surface Plasmon Resonance (SPR) curve.
FIG. 11 is a schematic illustration of the experimental procedure for screening high affinity polypeptide ligands by constructing phage display polypeptide libraries through a framework of polycyclic peptide molecules.
Detailed Description
The invention will be further illustrated by the following examples in conjunction with the accompanying drawings
According to the embodiment of the invention, a series of high-affinity target Trop2 multi-cyclic peptide molecular frameworks are obtained by a phage display technology and a secondary polypeptide library aiming at target protein Trop2 is constructed, and the sequences of the multi-cyclic peptide molecular frameworks are as follows:
CPPC(X a )XW(X b )EC(X c )(X d )(X e )(X f )(X g )C(X h )(X i )(X j )(X k )(X l )CPPC；
wherein, the amino acids are L amino acids, X represents any natural amino acid; (X) k ) D, W, H, T, Y, V, E, S, F, G, M, N, I, preferably D; (X) f ) F, V, L, N, A, D, S, Y, M, I, T, W, preferably F; (X) d ) D, G, S, N, R, T, Q, E, A, preferably G; (X) e ) E, F, L, D, Y, M, preferably E; (X) j ) I, V, L, M, F, R, T, K, preferably V; (X) l ) F, W, G, M, I is a,Y, L, V, preferably F; (X) g ) Is any amino acid, preferably N; (X) h ) Is any amino acid, preferably V; (X) c ) Is any amino acid, preferably S; (X) i ) V, A, I, P, L, A, E, T, D, S, F, Y, M, preferably V; (X) a ) G or S, preferably G; (X) b ) Is L or I, preferably L;
d is aspartic acid; w is tryptophan; h is histidine; t is threonine; y is tyrosine; v is valine; e is glutamic acid; s is serine; f is phenylalanine; g is glycine; m is methionine; n is asparagine; i is isoleucine; l is leucine; a is alanine; r is arginine; k is lysine; p is proline; c is cysteine; q is glutamine.
The high-affinity Trop 2-targeted multi-cyclic peptide molecular framework can be constructed by adopting the following method:
1) Screening a phage display polypeptide library for a Trop2 specific targeting ligand;
(1) Phage display polypeptide libraries were designed to construct different backbones, the polypeptide sequence backbones (from N-terminus to C-terminus) were as follows:
CPPC(X) 5-10 CDSFTNCWELLTCPPC(library 1)
CPPCGRWLEC(X) 5 C(X) 5 CPPC(library 2)
wherein, the amino acids are L-type amino acids, X represents any natural amino acid, and the subscript represents the number containing X.
(2) Taking a polypeptide sequence skeleton as a template, carrying out fusion expression on the polypeptide and p III proteins of phage, and displaying the fusion expression on the surface of phage to construct a phage library for displaying different polypeptide sequences; phage display polypeptide library 2 is a secondary library constructed on the screening result of library1 against target protein Trop2, and the partial conserved sequence obtained by screening of phage display polypeptide library1 against target protein Trop2 is immobilized to randomize the amino acids of the other two binding loops.
2) Screening by utilizing a phage display polypeptide library1 aiming at a target protein Trop2, and after 3 rounds of screening, selecting a monoclonal to carry out sequencing analysis to obtain polypeptide sequences peptide 1-5 (sequence table SEQ ID No. 1-5) including but not limited to the following:
CPPCGRWLECDSFTNCWELLTCPPC(peptide 1)
CPPCGAWLECDSFTNCWELLTCPPC(peptide 2)
CPPCGSWIECDSFTNCWELLTCPPC(peptide 3)
CPPCGAWIECDSFTNCWELLTCPPC(peptide 4)
CPPCSEWIECDSFTNCWELLTCPPC(peptide 5)
3) Selecting the polypeptide peptide1 with the highest enrichment amount according to the screening result of the phage display polypeptide library1 aiming at the target protein Trop2, constructing a phage display polypeptide library 2 according to the polypeptide sequence of the polypeptide peptide1, and screening aiming at the Trop2 protein; phage sequencing analysis after 3 rounds of screening was performed to obtain polypeptide sequences peptides 6-15 (sequence listing SEQ ID Nos. 6-15) including but not limited to:
CPPCGRWLECSDENLCAIIDWCPPC(peptide 6)
CPPCGRWLECYDFNECELIDWCPPC(peptide 7)
CPPCGRWLECWGMFNCQEFQGCPPC(peptide 8)
CPPCGRWLECWTHIDCQFIDWCPPC(peptide 9)
CPPCGRWLECSNDFDCDYLYFCPPC(peptide 10)
CPPCGRWLECLDEFRCTLIFHCPPC(peptide 11)
CPPCGRWLECYDEFSCEPFWFCPPC(peptide 12)
CPPCGRWLECSDGFTCNAVDFCPPC(peptide 13)
CPPCGRWLECSSPFTCTIFIGCPPC(peptide 14)
CPPCGRWLECSEDFLCDLRTFCPPC(peptide 15)
4) According to the enriched polypeptide sequence obtained after sequencing, synthesizing several polypeptides of peptide1, peptide 6, peptide7 and peptide8 by using a solid-phase polypeptide synthesizer, carrying out affinity characterization on the corresponding oxidation products by Surface Plasmon Resonance (SPR) after oxidation and folding, and verifying the effectiveness of the screening result.
According to the invention, a phage display ternary cyclopeptide library is constructed by virtue of the CPPC motif orthogonal pairing advantages, then a liquid phase screening method of phage display technology is utilized to successfully obtain the specific cyclopeptide ligand of Trop2, and a secondary phage polypeptide library is constructed to perform ligand affinity optimization, so that a series of ternary cyclopeptide ligands with higher affinity with Trop2 are obtained. The multi-membered cyclic peptide ligand can be used for constructing various polypeptide-drug coupling bodies and provides a targeting recognition primitive for developing a novel disease diagnosis and treatment method. The ternary cyclopeptide ligand molecules can be used for developing drugs/reagents/drug lead molecules for treating and diagnosing related diseases caused by Trop2 signal paths.
The construction of the framework of the polycyclic peptide molecule comprises the following steps: screening Trop2 specific utilization cyclic peptide ligands by phage display ternary cyclic peptide library; synthesis, oxidation, and affinity characterization of the cyclopeptide ligand to the target protein; affinity optimization of cyclic peptide ligands.
Specific examples are given below:
example 1
3 rounds of screening enrichment were performed on Trop2 protein using library of library1 phage display polypeptides. The biotinylated proteins were immobilized on magnetic beads using avidin-coated magnetic beads as solid phase carriers for biopanning. Magnetic beads for fixing target proteins are used as an experimental group, and magnetic beads are used as a control group. And respectively adding the phage after equivalent blocking for incubation and combination for a period of time, washing unbound phage, and eluting the bound phage. The ratio of phage eluted from the experimental group to the control group was defined as the enrichment. Whether screening for the target protein is successful is judged by whether the enrichment degree is obviously improved. After three rounds of screening, the monoclone of the experimental group is randomly selected for gene sequencing and polypeptide sequences are analyzed. The sequencing results are shown in FIG. 1.
And (3) selecting the sequence peptide1 with the greatest enrichment number to construct a phage polypeptide library 2, and carrying out ligand affinity optimization on the target protein Trop 2. Enriched sequences were also obtained by 3 rounds of screening (FIG. 2).
Example 2
And (5) synthesizing the polypeptide. And synthesizing the polypeptide peptide1,peptide 6,peptide 7 obtained by gene sequencing by using a full-automatic microwave polypeptide synthesizer solid-phase synthesis method. The synthesis strategy of Fmoc protected amino acid was used to synthesize the amino acid by means of dehydration condensation starting from the C-terminus. 50mg of Rink amide MBHA resin was weighed and 10mL of N, N-Dimethylformamide (DMF) was added to the reaction vessel to swell for 30min, and then automatic synthesis was started. The removal of Fmoc protecting groups takes place in DMF containing 20% piperidine, each coupling reaction being carried out with the addition of activators (Oxyma and DIC). After the completion of the polypeptide synthesis, the mixture was washed with 10mL of diethyl ether (2 times) to obtain a drier polypeptide resin. After cleavage of the polypeptide resin, purification by chromatography and lyophilization into powder. Polypeptide powder was dissolved in DMSO as a mother solution, and a certain amount of the polypeptide mother solution was taken in a buffer solution (100 mm, ph=7.4) containing 20% (v/v) DMSO and 80% (v/v) PB, and 10-fold excess oxidized glutathione (GSSG) was added to perform polypeptide oxidation. The final concentration of the polypeptide in the oxidation reaction system was 100. Mu.M, and reacted in a shaking table at 37℃for 6 hours. And separating and purifying the oxidized and folded product by utilizing an analytical High Performance Liquid Chromatography (HPLC), and freeze-drying the product after the product is characterized by mass spectrum. After dissolution, the solution can be used for measuring the binding force. The oxidative folding chromatograms of the polypeptide peptide1,peptide 6,peptide 7,peptide 8 are shown in figures 3-6 respectively.
Example 3
The affinity of the polypeptide peptide1 to the target protein Trop2 was examined by Surface Plasmon Resonance (SPR). The test uses a CAP sensing chip and a Biacore T200 molecular interaction system. CAP contains a carboxymethylated dextran matrix to which a single stranded DNA molecule is immobilized. The Biotin CAPTURE reagent (containing a complementary single-stranded DNA molecule that binds streptavidin) is injected onto the CAP chip surface and hybridized to it, followed by injection and CAPture of the Biotin-labeled target protein in the channel of the experimental set, followed by reinjecting the polypeptide sample to be tested. The polypeptides (peptides 1:200-3200 nM) at different concentrations were combined with Trop2 protein on the chip to give corresponding curves (FIG. 7), and fitted to give corresponding affinities.
The affinity of peptide 6,peptide 7,peptide 8 to the target protein Trop2 was examined by Surface Plasmon Resonance (SPR). The CM5 sensor chip and Biacore T200 molecular interaction system are used for testing. The CM5 chip has exposed carboxyl groups, the carboxyl groups are activated by N-hydroxysuccinimide (NHS) and 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC.HCl), the experimental group is injected with Trop2 protein, the amino groups of the Trop2 protein react with the activated carboxyl groups to be coupled to the surface of the CM5 chip, and then the polypeptide sample to be tested is injected. The polypeptides (peptides 6:6.25-100 nM, peptides 7:1-16nM,peptide 8:6.25-100 nM) at different concentrations were combined with Trop2 protein on the chip to give the corresponding curves (FIGS. 8-10), and fitted to give the corresponding affinities.
FIG. 11 shows a schematic of the experimental procedure for screening high affinity polypeptide ligands by constructing phage display polypeptide libraries through a framework of multi-cyclic peptide molecules.
In conclusion, the affinity test of the serial ternary cyclopeptide ligand of the specific target Trop2 and the target protein Trop2 shows that the polypeptide obtained by the mode has higher affinity with the target protein. The series of polypeptides are rich in multiple pairs of disulfide bonds, so that the polypeptides have more complex rigid structures, the stability of the polypeptides is improved, and a larger interaction interface is provided. Meanwhile, the affinity of the polypeptide ligand is effectively improved by constructing a secondary library, and affinity optimization of the ligand is successfully carried out. The affinities of the Peptide 6 and the Peptide8 and the target protein are about 10-20 nM, and the affinity of the Peptide7 is 1nM, so that a novel drug lead molecule is provided for the development of a target Trop2 polypeptide therapeutic drug, and the target treatment of tumors taking Trop2 as a surface marker is realized.

Claims (10)

1. A high affinity Trop2 targeting multi-cyclic peptide molecular framework characterized in that it is obtained by phage display technology and construction of a secondary polypeptide library directed against the target protein Trop2, the sequence of said multi-cyclic peptide molecular framework comprising the following features:
CPPC(X a )XW(X b )EC(X c )(X d )(X e )(X f )(X g )C(X h )(X i )(X j )(X k )(X l )CPPC；
wherein, the amino acids are L amino acids, X represents any natural amino acid; (X) k ) D, W, H, T, Y, V, E, S, F, G, M, N, I; (X) f ) F, V, L, N, A, D, S, Y, M, I, T, W; (X) d ) D, G, S, N, R, T, Q, E, A; (X e ) E, F, L, D, Y, M; (X) j ) I, V, L, M, F, R, T, K; (X) l ) F, W, G, M, I, Y, L, V; (X) g ) Is any amino acid; (X) h ) Is any amino acid; (X) c ) Is any amino acid; (X) i ) V, A, I, P, L, A, E, T, D, S, F, Y, M; (X) a ) Is G or S; (X) b ) Is L or I;
d is aspartic acid; w is tryptophan; h is histidine; t is threonine; y is tyrosine; v is valine; e is glutamic acid; s is serine; f is phenylalanine; g is glycine; m is methionine; n is asparagine; i is isoleucine; l is leucine; a is alanine; r is arginine; k is lysine; p is proline; c is cysteine; q is glutamine.
2. The high affinity Trop2 targeting multi-cyclic peptide molecular framework of claim 1, characterized by (X k ) Is D; (X) f ) F is the same as F; (X) d ) G is G; (X) e ) E is; (X) j ) V is the number; (X) l ) F is the same as F; (X) g ) Is N; (X) h ) V is the number; (X) c ) S is the same as the original formula; (X) i ) V is the number; (X) a ) G is G; (X) b ) Is L.
3. The high affinity Trop2 targeting multi-cyclic peptide molecular framework of claim 1 or 2, characterized in that its ligand sequence includes but is not limited to:
CPPCGRWLECDSFTNCWELLTCPPC(peptide 1)
CPPCGAWLECDSFTNCWELLTCPPC(peptide 2)
CPPCGSWIECDSFTNCWELLTCPPC(peptide 3)
CPPCGAWIECDSFTNCWELLTCPPC(peptide 4)
CPPCSEWIECDSFTNCWELLTCPPC(peptide 5)
CPPCGRWLECSDENLCAIIDWCPPC(peptide 6)
CPPCGRWLECYDFNECELIDWCPPC(peptide 7)
CPPCGRWLECWGMFNCQEFQGCPPC(peptide 8)
CPPCGRWLECWTHIDCQFIDWCPPC(peptide 9)
CPPCGRWLECSNDFDCDYLYFCPPC(peptide 10)
CPPCGRWLECLDEFRCTLIFHCPPC(peptide 11)
CPPCGRWLECYDEFSCEPFWFCPPC(peptide 12)
CPPCGRWLECSDGFTCNAVDFCPPC(peptide 13)
CPPCGRWLECSSPFTCTIFIGCPPC(peptide 14)
CPPCGRWLECSEDFLCDLRTFCPPC(peptide 15)。
4. a method of constructing a high affinity Trop2 targeting multi-cyclic peptide molecular framework according to claim 1 or 2 or 3, characterized in that the specific steps thereof comprise:
1) Screening a Trop2 specific targeting ligand by using a phage display ternary cyclic peptide library to construct a phage display polypeptide library1;
2) Screening by utilizing a phage display polypeptide library1 aiming at a target protein Trop2, and selecting a monoclonal after 3 rounds of screening for sequencing analysis to obtain a polypeptide sequence shown in sequence table SEQ ID No. 1-5;
3) Selecting the polypeptide peptide1 with the highest enrichment amount according to the screening result, constructing a phage display polypeptide library 2 according to the polypeptide sequence of the polypeptide peptide1, and screening the Trop2 protein by utilizing the phage display polypeptide library 2; and 3 rounds of phage sequencing analysis after screening to obtain polypeptide sequences shown in sequence table SEQ ID No. 6-15.
5. The method of constructing a high affinity Trop2 targeting multi-cyclic peptide molecular frame according to claim 4, wherein in step 1), the phage display polypeptide library1 has the following polypeptide sequence skeleton from N-terminus to C-terminus:
CPPC(X) 5-10 CDSFTNCWELLTCPPC(library 1)
wherein, the amino acids are L-type amino acids, X represents any natural amino acid, and the subscript represents the number containing X.
6. The method of constructing a high affinity Trop2 targeting multi-cyclic peptide molecular frame according to claim 4, wherein in step 3), said phage display polypeptide library 2 has the following polypeptide sequence backbone from N-terminus to C-terminus:
CPPCGRWLEC(X) 5 C(X) 5 CPPC(library 2)
wherein, the amino acids are L-type amino acids, X represents any natural amino acid, and the subscript represents the number containing X.
7. The method for constructing a high affinity targeted Trop2 multi-cyclic peptide molecular frame according to claim 4, wherein in the step 3), a phage display polypeptide library 2 is constructed, a polypeptide sequence skeleton is taken as a template, a polypeptide and a p III protein of a phage are fused and expressed and displayed on the surface of the phage, and a phage library displaying different polypeptide sequences is constructed; phage display polypeptide library 2 is a secondary library constructed on the screening result of phage display polypeptide library1 against target protein Trop2, and the partial conserved sequences obtained by screening phage display polypeptide library1 against target protein Trop2 are immobilized to randomize the amino acids of the other two binding loops.
8. The use of the high affinity Trop2 targeting multi-cyclic peptide molecular framework of claim 1 for the construction of various classes of polypeptide-drug conjugates.
9. Use of the high affinity Trop2 targeting multi-cyclic peptide molecular framework of claim 1 as a targeting recognition motif.
10. The high affinity Trop2 targeted multi-cyclic peptide molecular framework of claim 1 useful for the development of drugs/reagents/drug lead molecules for the treatment and diagnosis of related diseases caused by Trop2 signaling pathways.
CN202211508910.1A 2022-11-29 2022-11-29 High affinity Trop2 targeting multi-cyclic peptide molecular framework Pending CN116199746A (en)

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