WO2023221234A1 - 共价抑制剂的体外筛选方法及其应用 - Google Patents

共价抑制剂的体外筛选方法及其应用 Download PDF

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WO2023221234A1
WO2023221234A1 PCT/CN2022/100847 CN2022100847W WO2023221234A1 WO 2023221234 A1 WO2023221234 A1 WO 2023221234A1 CN 2022100847 W CN2022100847 W CN 2022100847W WO 2023221234 A1 WO2023221234 A1 WO 2023221234A1
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covalent
oligonucleotide
modification
covalent reaction
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French (fr)
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向宇
童爱军
秦梓宸
张恺宁
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清华大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/16—Aptamers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates
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    • C12N2330/00—Production
    • C12N2330/30—Production chemically synthesised
    • C12N2330/31—Libraries, arrays

Definitions

  • the present disclosure belongs to the field of biotechnology, and relates to in vitro screening methods for covalent inhibitors and their applications; specifically, the screening method of the present disclosure is compatible with covalent reaction modifications, and through this method, covalent screening of various drug target proteins can be carried out. Screening and discovery of candidate drug molecules for valent inhibitors; this disclosure also provides oligonucleotide molecules that covalently inhibit the spike protein of the new coronavirus obtained by applying this screening method.
  • Disease-related proteins are important drug targets, such as the PD-1/PD-L1 protein interaction protein targeted by cancer immune checkpoint therapy, and the spike protein used by the new coronavirus to recognize host cells (SARS-CoV- 2 spike protein, S protein), etc.
  • monoclonal antibodies Monoclonal antibodies, mAb
  • the mechanism of action is that the mAb binds non-covalently to the target protein, thereby inhibiting the function of the target protein.
  • monoclonal antibodies are expensive. For example, the price of a single treatment for the new coronavirus spike protein neutralizing antibody therapy for severe COVID-19 is more than 100,000 or even hundreds of thousands. It is of great significance to develop more affordable and efficient drugs.
  • chemically synthesized peptides and short-chain nucleic acid drug molecules are relatively cheap, their affinity for target proteins based on non-covalent binding is significantly lower than that of monoclonal antibodies, and there are disadvantages such as excessive renal clearance in the body.
  • one purpose of the embodiments of the present disclosure is to provide an in vitro screening method for covalent inhibitors, which can design and develop covalent inhibitors for various target proteins.
  • Another purpose of the embodiments of the present disclosure is to obtain covalent inhibitors by screening the new coronavirus spike protein as the target protein through the above method, which can covalently inhibit the activity of the new coronavirus spike protein.
  • An embodiment of the first aspect of the present disclosure provides an in vitro screening method for covalent inhibitors, including the following steps:
  • the covalent reaction modification reagent includes at least one covalent reaction modification group, and the covalent reaction modification group is suitable for covalent binding to the target protein;
  • the covalent reaction modification oligonucleotide library Contact the target protein, separate and collect the covalent reaction modified oligonucleotides that bind to the target protein, and then remove the target protein and the covalent reaction modification group to construct a secondary library; (4) Use the secondary library as Template, repeat steps (1)-(3), and after multiple rounds, perform sequencing analysis on the library obtained in the last round of screening, and screen to obtain covalent inhibitors.
  • the step of constructing a phosphorothioate oligonucleotide library uses a nucleoside triphosphate mixture as a substrate, and the substrate includes at least one nucleoside phosphorothioate triphosphate, and is performed using a nucleic acid polymerase amplification reaction.
  • the nucleoside thiotriphosphate is nucleoside [alpha-thio]-triphosphate.
  • the initial library for constructing a phosphorothioate oligonucleotide library is a random library, using the random library as a template, using dNTPs as a substrate, and at least one monomer in the substrate It is deoxynucleoside thiophosphoric acid and performs PCR amplification reaction under the action of DNA polymerase.
  • the deoxynucleoside thiotriphosphate is selected from the group consisting of 5′-deoxyadenosine [ ⁇ -thio]-triphosphate, 5′-deoxyguanosine [ ⁇ -thio]-triphosphate, 5 Any of '-deoxythymidine [ ⁇ -thio]-triphosphate, 5'-deoxycytidine [ ⁇ -thio]-triphosphate and their analogs and derivatives.
  • the covalent reaction modification group is the following unsubstituted or optionally substituted group: sulfonyl fluoride group, sulfonyl fluoride group, sulfonamide group, sulfonate ester group, ⁇ , ⁇ -unsubstituted group. Saturated carbonyl or fluorine substituted phenyl.
  • the covalent reaction modifying group is selected from the following structures:
  • R 1 is -H, -OH, -NO 2 , -COOH, halogen or -R';
  • R 3 is -H or -R'
  • R 4 is -H or -R'
  • n 1 or 2 or 3 or 4 or 5;
  • R' is C 1 -C 12 alkyl, C 1 -C 12 alkoxy, C 2 -C 12 alkenyl, C 2 -C 12 alkynyl, C 3 -C 12 cycloalkyl, C 3 -C 12 Cycloalkenyl, C 6 -C 12 aryl or C 5 -C 12 heteroaryl;
  • Halogen is F, Cl, Br or I.
  • the covalent reaction modification reagent further includes a functional group capable of reacting with a phosphorothioate group, and the functional group is an unsubstituted or optionally substituted following group: halogen methylene or halogen carboxyethyl base, the halogen is F, Cl, Br or I.
  • the functional group capable of reacting with the phosphorothioate group is selected from the following structures:
  • R 5 and R 6 are independently H, C 1 -C 12 alkyl, C 1 -C 12 alkoxy, C 2 -C 12 alkenyl, C 2 -C 12 alkynyl, C 3 -C 12 ring Alkyl, C 3 -C 12 cycloalkenyl, C 6 -C 12 aryl or C 5 -C 12 heteroaryl; R 7 is Cl, Br or I.
  • the number of repetitions of steps (1)-(3) is 1-25 times.
  • step (3) the covalent reaction-modified oligonucleotide bound to the target protein is contacted with an alkaline solution to remove the target protein and the covalent reaction-modifying group.
  • the target protein is a disease-related protein or a drug protein.
  • Embodiments of the second aspect of the present disclosure also provide covalent inhibitors.
  • the covalent inhibitors are screened by the method described in any embodiment of the first aspect.
  • the covalent inhibitors include at least two Covalent reaction modification group; the covalent reaction modification group is the following unsubstituted or optionally substituted group: sulfonyl fluoride group, sulfonyl fluoride group, sulfonamide group, sulfonate ester group, ⁇ , ⁇ - Unsaturated carbonyl or fluorine substituted phenyl.
  • the covalent reaction modifying group is selected from the following structures:
  • R 1 is -H, -OH, -NO 2 , -COOH, halogen or -R';
  • R 3 is -H or -R'
  • R 4 is -H or -R'
  • n 1 or 2 or 3 or 4 or 5;
  • R' is C 1 -C 12 alkyl, C 1 -C 12 alkoxy, C 2 -C 12 alkenyl, C 2 -C 12 alkynyl, C 3 -C 12 cycloalkyl, C 3 -C 12 Cycloalkenyl, C 6 -C 12 aryl or C 5 -C 12 heteroaryl;
  • Halogen is F, Cl, Br or I.
  • the covalent inhibitor uses the new coronavirus spike protein as the target protein.
  • Embodiments of the third aspect of the present disclosure provide oligonucleotide molecules comprising the following sequence of formula (I);
  • Z 8 , Z 9 and Z 10 are phosphorothioate nucleotides modified by covalent reaction; each is independently selected from one of A, T, C or G; the covalent reaction The modification is to modify with the following unsubstituted or optionally substituted groups: sulfonyl fluoride group, sulfonyl fluoride group, sulfonamide group, sulfonate ester group, ⁇ , ⁇ -unsaturated carbonyl group or fluorine-substituted phenyl group;
  • Z 4 , Z 5 , Z 11 , Z 12 , and Z 15 are independently A, T, C, G or no base;
  • Z 1 , Z 2 , Z 3 , Z 6 , Z 7 and Z 18 , Z 17 , Z 16 , Z 14 and Z 13 are respectively selected from A, T, C and G, and Z 1 and Z 18 form a base pair , Z 2 and Z 17 form a base pair, Z 3 and Z 16 form a base pair, Z 6 and Z 14 form a base pair, Z 7 and Z 13 form a base pair.
  • the covalent reaction modification is phenylsulfonyl fluoride modification, phenylsulfonyl fluoride modification or pentafluorophenyl modification.
  • Z 1 , Z 2 , Z 3 , Z 6 , Z 7 and Z 18 , Z 17 , Z 16 , Z 14 , Z 13 are respectively selected from C, G, or G, T, and wherein C and G form a base pair, and G and T form a base pair.
  • the oligonucleotide molecule further includes at least one base at the 5' end and/or 3' end of the structure of formula (I), and the bases are each independently selected from A, T, One of C or G.
  • the oligonucleotide molecule is further modified to include at least one chemical modification, wherein the chemical modification is at a sugar position, a phosphate position, and/or a base position of the oligonucleotide molecule. chemical substitution at the position.
  • the oligonucleotide molecule comprises the following nucleotide sequence
  • a * and T * are respectively unsubstituted or optionally substituted sulfonyl fluoride modified phosphorothioate adenosine and thymidine.
  • the oligonucleotide molecule further includes any one of the following four sequences:
  • An embodiment of the fourth aspect of the present disclosure also provides a vector comprising the above-mentioned oligonucleotide molecule.
  • the vector is an expression vector.
  • Embodiments of the fifth aspect of the present disclosure also provide nucleic acid molecules encoded by the above-mentioned oligonucleotide molecules.
  • Embodiments of the sixth aspect of the present disclosure also provide host cells comprising the above-mentioned nucleic acid molecules.
  • Embodiments of the seventh aspect of the present disclosure also provide pharmaceutical compositions comprising the above-mentioned oligonucleotide molecule, the above-mentioned vector, the above-mentioned nucleic acid molecule, or the above-mentioned host cell.
  • the pharmaceutical composition further includes a chemotherapeutic agent.
  • Embodiments of the eighth aspect of the present disclosure also provide the oligonucleotide molecules described in any embodiment of the third aspect, the vectors described in any embodiment of the fourth aspect, and the vectors described in any embodiment of the fifth aspect.
  • Embodiments of the ninth aspect of the present disclosure also provide a method for preventing and/or treating diseases related to the expression of the new coronavirus spike protein, including administering to the patient a therapeutically effective amount of the oligosaccharide according to any embodiment of the third aspect.
  • Nucleotide molecules, vectors according to any embodiment of the fourth aspect, nucleic acid molecules according to any embodiment of the fifth aspect, host cells according to any embodiment of the sixth aspect, or any embodiment of the seventh aspect The pharmaceutical composition.
  • Embodiments of the tenth aspect of the present disclosure also provide the oligonucleotide molecules described in any embodiment of the third aspect, the vectors described in any embodiment of the fourth aspect, and the nucleic acids described in any embodiment of the fifth aspect.
  • the molecule, the host cell according to any embodiment of the sixth aspect, or the pharmaceutical composition according to any embodiment of the seventh aspect is used as a medicine.
  • the oligonucleotide molecule described in any embodiment of the third aspect, the vector described in any embodiment of the fourth aspect, the nucleic acid molecule described in any embodiment of the fifth aspect, the sixth aspect is used to target the new coronavirus spike protein.
  • the screening method of the embodiments of the present disclosure overcomes the problem that all covalent inhibitors of target proteins in related technologies are developed through rational design guided by structure.
  • the types of target proteins that can be targeted are limited, and the activity effect of the obtained covalent inhibitors is limited. Limited by the performance of the non-covalent inhibitor used.
  • This disclosure realizes for the first time an in vitro screening technology that is compatible with covalent reaction modification, thereby enabling the development of covalent inhibitors for any target protein. Based on large-scale random molecular library screening, the obtained highly active covalent inhibitors have great potential. .
  • Figure 1 is a schematic diagram of an in vitro screening method for covalent inhibitors according to an embodiment of the present disclosure
  • Figure 2 is a schematic diagram of a nucleophilic substitution reaction between a phosphorothioate structure and a covalent chemical modification reagent to perform covalent reaction modification on an oligonucleotide according to an embodiment of the present disclosure
  • Figure 3 is a urea gel electrophoresis diagram of the primer raw material (left) and the product oligonucleotide (right) of the polymerase chain reaction in the embodiment of the present disclosure;
  • Figure 4 is a schematic diagram of the sequence of sequence X and the mutations of M1-M8 of sequence X according to an embodiment of the present disclosure
  • Figure 5 is an SDS-PAGE diagram of the reaction between the disclosed sequence X and its mutants and the new coronavirus spike protein
  • Figure 6 is a liquid chromatography-tandem mass spectrometry analysis of the conjugation of the sequence X+TG to the new coronavirus spike protein in the embodiment of the present disclosure;
  • Figure 6 shows that one amino acid position of the sequence X+TG covalently conjugated to the new coronavirus spike protein is The K458 amino acid residue of the new coronavirus spike protein;
  • Figure 7 is a liquid chromatography-tandem mass spectrometry analysis of sequence X+TG conjugated to the new coronavirus spike protein in an embodiment of the present disclosure;
  • Figure 7 shows another amino acid site of sequence X+TG covalently conjugated to the new coronavirus spike protein. It is the Y421 amino acid residue of the new coronavirus spike protein;
  • Figure 8 shows the inhibitory activity experiment of sequence X and sequence X' against the new coronavirus pseudovirus according to the embodiment of the present disclosure.
  • Disease-related proteins are important drug targets, such as the PD-1/PD-L1 protein interaction protein targeted by cancer immune checkpoint therapy, and the spike protein used by the new coronavirus to recognize host cells (SARS-CoV- 2 spike protein, S protein), etc.
  • monoclonal antibodies Monoclonal antibodies, mAb
  • the mechanism of action is that the mAb binds non-covalently to the target protein, thereby inhibiting the function of the target protein.
  • monoclonal antibodies are expensive. For example, the price of a single treatment for the new coronavirus spike protein neutralizing antibody therapy for severe COVID-19 is more than 100,000 or even hundreds of thousands. It is of great significance to develop more affordable and efficient drugs.
  • chemically synthesized peptides and short-chain nucleic acid drug molecules are relatively cheap, their affinity for target proteins based on non-covalent binding is significantly lower than that of monoclonal antibodies, and there are disadvantages such as excessive renal clearance in the body.
  • covalent inhibitors for target proteins first non-covalently binds to the target protein, and then forms covalent bonds with the target protein through the inert reactive functional groups contained in its molecules, thereby inhibiting the target protein with high specificity and permanently.
  • These covalent inhibitors of target proteins have two significant advantages over traditional monoclonal antibody inhibitors: first, highly specific and permanent inhibition, with inhibitory capabilities close to or exceeding monoclonal antibodies, and their activity is not affected by rapid renal clearance. Negative effects; secondly, because these covalent inhibitors usually have only one-tenth or less of the molecular weight of monoclonal antibodies, the dosage used is significantly smaller than that of monoclonal antibodies.
  • covalent reactive modification also known as: “covalent warhead modification (covalent warhead modification)”; refers to chemical modifications that can be used to covalently bind to a target protein of interest.
  • Covalent bonding refers to the formation of a chemical bond between atoms through the sharing of pairs of electrons.
  • amplification refers to any means by which at least a partial sequence of at least one target nucleic acid or its sequence complement is produced, usually in a template-dependent manner, including, but not limited to, various techniques for amplifying nucleic acid sequences, Non-limiting examples of amplification methods include polymerase chain reaction (PCR), reverse transcriptase PCR, and the like.
  • PCR polymerase chain reaction
  • reverse transcriptase PCR reverse transcriptase PCR
  • nucleic acid polymerase refers to an enzyme that catalyzes the incorporation of nucleotides into nucleic acids, including DNA polymerase, RNA polymerase, reverse transcriptase, and the like.
  • in vitro screening refers to obtaining molecules with specific activity from a random library of nucleic acids through repeated screening outside the cell.
  • nucleotide generally refers to a compound in which a nucleoside is linked to an acidic molecule or group through an ester bond, such as a phosphate ester of a nucleoside, usually with one, two or three phosphate groups covalently attached At position 5 of the sugar group of the nucleoside.
  • ester bond such as a phosphate ester of a nucleoside
  • nucleoside usually with one, two or three phosphate groups covalently attached At position 5 of the sugar group of the nucleoside.
  • nucleotide also includes some nucleotide analogs or derivatives thereof. Analogs refer to compounds that are structurally similar to naturally occurring nucleotides.
  • Non-limiting examples of analogs include: oxyguanosine, pseudouridine, isocytidine, isoguanosine, etc.; derivatives refer to compounds with modifications
  • the nucleotide base moiety, the modified pentose sugar moiety and/or the modified phosphate ester moiety, non-limiting examples of the modified phosphate ester moiety include: phosphorothioate, phosphorodithioate, phosphoselenate , Phosphate diselenate, aniline phosphate thiol ester, aniline phosphate, phosphoramidate, etc.
  • Non-limiting examples of exemplary modified nucleotide base parts include: 5-methylcytosine (5mC);, C -5-propynyl-C and C-5-propynyl-U; 2,6-diaminopurine, hypoxanthine, pseudouridine, 2-thiopyrimidine, isocytosine (isoC), 5-methyl isoC and isoguanine.
  • Non-limiting examples of exemplary modified pentose moieties include: 2'- or 3'-modification, wherein the 2'- or 3'-position is hydrogen, hydroxyl, alkoxy (e.g., methoxy, ethoxy, iso propoxy, butoxy, isobutoxy), azido, amino, alkylamino, fluorine, chlorine or bromine.
  • 2'- or 3'-position is hydrogen, hydroxyl, alkoxy (e.g., methoxy, ethoxy, iso propoxy, butoxy, isobutoxy), azido, amino, alkylamino, fluorine, chlorine or bromine.
  • nucleoside triphosphate generally refers to three phosphate groups covalently linked to the 5th position of the sugar group of the nucleoside.
  • the definition of nucleoside triphosphate also includes some typical nucleoside triphosphates. Analogs and derivatives of phosphoric acid.
  • nucleoside [ ⁇ -thio]-triphosphate include: 5'-deoxyadenosine [ ⁇ -thio]-triphosphate, 5'-adenosine [ ⁇ -thio]-triphosphate , 5′-deoxyguanosine [ ⁇ -thio]-triphosphate, 5′-guanosine [ ⁇ -thio]-triphosphate, 5′-deoxythymidine [ ⁇ -thio]-triphosphate, 5′ -Thymidine [ ⁇ -thio]-triphosphate, 5′-deoxyuridine [ ⁇ -thio]-triphosphate, 5′-uridine [ ⁇ -thio]-triphosphate, 5′-deoxycytidine [ ⁇ -thio]-triphosphate, 5′-cytidine [ ⁇ -thio]-triphosphate, 5′-deoxyinosine [ ⁇ -thio]-triphosphate, 5′-inosine [ ⁇ -thio]-triphosphate generation]-tri
  • nucleic acid short nucleic acid
  • polynucleotide polynucleotide
  • oligonucleotide oligonucleotide
  • oligonucleotide refers to linear or circular configurations as well as single-stranded or double-stranded Polymers of deoxyribonucleotides or ribonucleotides in the form of chains, and the term may include known analogs of natural nucleotides, as well as those in the base, sugar, and/or phosphate moieties (e.g., phosphorothioate backbones). ) modified nucleotides.
  • these terms should not be construed as limitations on polymer length.
  • sequence refers to a sequence of nucleotides of any length, which may be DNA or RNA. Can be linear, circular or branched, and can be single or double stranded.
  • homologous refers to sequence similarity to a target nucleic acid sequence.
  • Homology includes nucleotide sequences that are 80% or higher, 85% or higher, 90% or higher, 95% or higher homologous to a target nucleic acid sequence or a core sequence thereof. Homology can be assessed visually or with computer software.
  • label refers to a moiety that is attached (covalently or non-covalently) to a nucleic acid molecule and capable of providing information about the molecule.
  • exemplary labels include fluorescent labels and the like.
  • new coronavirus spike protein refers to the protein with a trimeric structure located in the outermost layer of the new coronavirus, also known as the S protein, which is a structural protein derived from coronaviruses including SARS-CoV-2.
  • optional substituent group may be substituted at each substitutable position of the group.
  • substituents may be identically or differently substituted at each position.
  • Non-limiting examples of "optionally substituted" substituents include fluorine, chlorine, bromine, iodine, hydroxyl, nitro, amino, carboxyl, alkyl, alkoxy, alkenyl, alkynyl, aryloxy, Heteroaryloxy, heterocyclyloxy, arylalkoxy, heteroarylalkoxy, heterocyclylalkoxy, cycloalkylalkoxy, alkylamino, alkylaminoalkyl, cycloalkyl Amino, cycloalkylalkylamino, haloalkyl, haloalkoxy, hydroxyl-substituted alkyl, hydroxyl-substituted alkylamino, amino-substituted alkyl, alkylacyl, heteroalkyl, cycloalkyl, cycloalkenyl, cyclo Alkylalkyl, heterocyclyl, heterocyclylalkyl, heterocycly
  • alkyl may be "C 1 -C 12 alkyl".
  • C 1 -C 12 alkyl means a saturated linear or branched monovalent hydrocarbon group containing 1 to 12 carbon atoms; in one embodiment, containing 1 to 8 carbon atoms; in another embodiment , contains 1-6 carbon atoms; in yet another embodiment, contains 1-4 carbon atoms; in yet another embodiment, contains 1-3 carbon atoms.
  • C 1 -C 12 alkyl include: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 2-pentyl, 3-pentyl, 2-methyl-2-butyl, 3-methyl-2-butyl, 3-methyl-1-butyl, 2-methyl-1-butyl, etc. wait.
  • alkenyl may be "C 2 -C 12 alkenyl".
  • C 2 -C 12 alkenyl means a linear or branched monovalent hydrocarbon group containing 2 to 12 carbon atoms, which has at least one unsaturated site, that is, a carbon-carbon sp2 double bond. In one embodiment, 2-8 carbon atoms are included; in another embodiment, 2-6 carbon atoms are included; in yet another embodiment, 2-4 carbon atoms are included.
  • Non-limiting examples of "C 2 -C 12 alkenyl” include vinyl, allyl, propenyl, and the like.
  • alkynyl may be "C 2 -C 12 alkynyl”.
  • C 2 -C 12 alkynyl means a linear or branched monovalent hydrocarbon group containing 2 to 12 carbon atoms, in which there is at least one carbon-carbon sp triple bond.
  • alkoxy may be "C 1 -C 12 alkoxy”.
  • C 1 -C 12 alkoxy means “C 1 -C 12 alkyl” attached to the remainder of the molecule through an oxygen atom, where "C 1 -C 12 alkyl” has the meaning as described herein.
  • Non-limiting examples of “C 1 -C 12 alkoxy” include: methoxy, ethoxy, 1-propoxy, 2-propoxy and the like.
  • cycloalkyl may be "C 3 -C 12 cycloalkyl".
  • C 3 -C 12 cycloalkyl means a monovalent or multivalent saturated monocyclic, bicyclic or tricyclic ring system containing 3 to 12 carbon atoms. In one embodiment, 3-10 carbon atoms are included; in another embodiment, 3-8 carbon atoms are included; in yet another embodiment, 3-6 carbon atoms are included.
  • C 3 -C 12 cycloalkyl include: cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecanyl, cyclodecyl Monoalkyl, cyclododecyl, etc.
  • cycloalkenyl may be "C 3 -C 12 cycloalkenyl".
  • C 3 -C 12 cycloalkenyl means a monovalent or multivalent monocyclic, bicyclic or tricyclic ring system containing 3-12 carbon atoms, containing at least one carbon-carbon double bond, and the ring system is non-aromatic .
  • 3-10 carbon atoms are included; in another embodiment, 3-8 carbon atoms are included; in yet another embodiment, 3-6 carbon atoms are included.
  • Non-limiting examples of "C 3 -C 12 cycloalkenyl” include: cyclobutenyl, cyclopentenyl, cyclohexenyl, cyclohexadienyl and the like.
  • aryl may be "C 6 -C 12 aryl".
  • C 6 -C 12 aryl means a monocyclic, bicyclic and tricyclic carbocyclic ring system containing 6-12 ring atoms, in which at least one ring system is aromatic, "C 6 -C 12 aryl” Non-limiting examples include: phenyl, indenyl, naphthyl and the like.
  • heteroaryl may be "C 5 -C 12 heteroaryl”.
  • C 5 -C 12 heteroaryl means monocyclic, bicyclic and tricyclic ring systems containing 5 to 12 ring atoms, in which at least one ring system is aromatic and at least one ring system contains one or more heteroatoms , "Heteroatom” is selected from O, S, N, P or Si.
  • the embodiment of the present disclosure provides an in vitro screening method for covalent inhibitors, including the following steps:
  • the valence reaction modification reagent includes at least one covalent reaction modification group, and the covalent reaction modification group is suitable for covalent binding to the target protein;
  • the in vitro screening method for covalent inhibitors in the embodiments of the present disclosure can obtain oligonucleotide molecules that can covalently inhibit target proteins; the covalent reaction modified oligonucleotides are introduced after nucleic acid polymerase amplification and are used in the next step. The cyclic polymerase is removed before amplification, so that the covalent reaction modification does not affect the nucleic acid polymerase reaction, making it compatible with in vitro screening. After multiple screening cycles and sequencing, oligos that can covalently inhibit the target protein can be obtained. Nucleotide sequence information and the location on the oligonucleotide containing the covalently reactive modification are determined by the type of phosphorothioate nucleotide used.
  • the in vitro screening method for covalent inhibitors disclosed in the present disclosure can use disease-related proteins as target proteins to screen and discover candidate drug molecules for covalent inhibitors.
  • the in vitro screening method for covalent inhibitors disclosed in the present disclosure can also use drug proteins as target proteins, non-limiting examples, such as monoclonal antibodies, hormonal drugs (such as insulin) and other drugs whose molecular forms are proteins. Modification of Drugs.
  • the step of constructing a phosphorothioate oligonucleotide library uses a nucleoside triphosphate mixture as a substrate, and the substrate includes at least one nucleoside phosphorothioate triphosphate, and a nucleic acid polymerase is used for amplification reaction.
  • the initial library for constructing a phosphorothioate oligonucleotide library is a random library, using the random library as a template, using dNTPs as a substrate, and at least one monomer in the substrate is deoxynucleoside thio Triphosphate, under the action of DNA polymerase, performs PCR amplification reaction.
  • the deoxynucleoside thiotriphosphate is selected from the group consisting of 5′-deoxyadenosine [ ⁇ -thio]-triphosphate, 5′-deoxyguanosine [ ⁇ -thio]-triphosphate, 5′- Any of deoxythymidine [ ⁇ -thio]-triphosphate, 5′-deoxycytidine [ ⁇ -thio]-triphosphate and their analogs and derivatives.
  • the covalent reaction modification group is the following unsubstituted or optionally substituted group: sulfonyl fluoride group, sulfonyl fluoride group (also known as fluorosulfate ester group), sulfonamide group, sulfonate ester group, ⁇ , ⁇ -unsaturated carbonyl group or fluorine-substituted phenyl group.
  • the covalently reactive modifying group is selected from the following structures:
  • R 1 is -H, -OH, -NO 2 , -COOH, halogen or -R';
  • R 3 is -H or -R'
  • R 4 is -H or -R'
  • n 1 or 2 or 3 or 4 or 5;
  • R' is C 1 -C 12 alkyl, C 1 -C 12 alkoxy, C 2 -C 12 alkenyl, C 2 -C 12 alkynyl, C 3 -C 12 cycloalkyl, C 3 -C 12 Cycloalkenyl, C 6 -C 12 aryl or C 5 -C 12 heteroaryl;
  • Halogen is F, Cl, Br or I.
  • the covalent reaction modification reagent further includes a functional group capable of reacting with a phosphorothioate group.
  • the functional group is an unsubstituted or optionally substituted group as follows: halogen methylene or halogen carboxyethyl, and the halogen is F, Cl, Br or I.
  • the functional group capable of reacting with the phosphorothioate group is selected from the following structures:
  • R 5 and R 6 are independently H, C 1 -C 12 alkyl, C 1 -C 12 alkoxy, C 2 -C 12 alkenyl, C 2 -C 12 alkynyl, C 3 -C 12 ring Alkyl, C 3 -C 12 cycloalkenyl, C 6 -C 12 aryl or C 5 -C 12 heteroaryl; R 7 is Cl, Br or I.
  • Figure 2 shows a schematic example of a covalent reaction modification of an oligonucleotide through a nucleophilic substitution reaction between a covalent chemical modification reagent and a phosphorothioate structure.
  • the number of repetitions of steps (1)-(3) is 1-25 times.
  • step (3) the covalent reaction-modified oligonucleotide bound to the target protein is contacted with an alkaline solution to remove the target protein and the covalent reaction-modifying group.
  • the pH of the alkaline solution is >9, further preferably, pH>10; further preferably, pH>11; further preferably, pH>12.
  • the alkaline solution is NaOH solution or KOH solution.
  • the embodiments of the present disclosure also provide covalent inhibitors.
  • the covalent inhibitors are screened by the above-mentioned methods.
  • the covalent inhibitors include at least two covalent reaction modification groups; the covalent reaction modification groups are not The following groups are substituted or optionally substituted: sulfonyl fluoride group, sulfonyl fluoride group, sulfonamide group, sulfonate ester group, ⁇ , ⁇ -unsaturated carbonyl group or fluorine-substituted phenyl group.
  • the covalent reaction modifying group is selected from the following structures:
  • R 1 is -H, -OH, -NO 2 , -COOH, halogen or -R';
  • R 3 is -H or -R'
  • R 4 is -H or -R'
  • n 1 or 2 or 3 or 4 or 5;
  • R' is C 1 -C 12 alkyl, C 1 -C 12 alkoxy, C 2 -C 12 alkenyl, C 2 -C 12 alkynyl, C 3 -C 12 cycloalkyl, C 3 -C 12 Cycloalkenyl, C 6 -C 12 aryl or C 5 -C 12 heteroaryl;
  • Halogen is F, Cl, Br or I.
  • the embodiments of the present disclosure also provide oligonucleotide molecules that covalently inhibit the spike protein of the new coronavirus and are screened using the in vitro screening method of the present disclosure.
  • the spike protein of the new coronavirus in the embodiment of the present disclosure is derived from the original strain of the new coronavirus or its mutant strain (such as Delta, Omicron).
  • Embodiments of the present disclosure provide oligonucleotide molecules, including sequences of the following formula (I);
  • At least two of Z 8 , Z 9 and Z 10 are phosphorothioate nucleotides modified by covalent reaction; each is independently selected from one of A, T, C or G; the covalent reaction modification is Use unsubstituted or optionally substituted following groups for modification: sulfonyl fluoride group, sulfonyl fluoride group, sulfonamide group, sulfonate ester group, ⁇ , ⁇ -unsaturated carbonyl group or fluorine-substituted phenyl group;
  • Z 4 , Z 5 , Z 11 , Z 12 , and Z 15 are independently A, T, C, G or no base;
  • Z 1 , Z 2 , Z 3 , Z 6 , Z 7 and Z 18 , Z 17 , Z 16 , Z 14 and Z 13 are respectively selected from A, T, C and G, and Z 1 and Z 18 form a base pair , Z 2 and Z 17 form a base pair, Z 3 and Z 16 form a base pair, Z 6 and Z 14 form a base pair, Z 7 and Z 13 form a base pair.
  • the covalently reactive modification is a phenylsulfonyl fluoride modification, a phenylsulfonyl fluoride modification, or a pentafluorophenyl modification.
  • Z 4 , Z 5 , Z 11 , Z 12 , and Z 15 do not participate in forming an active secondary folding structure, and any one of them can be replaced by A, T, G, C or no base.
  • Z 4 is T
  • Z 5 is T
  • Z 11 is G
  • Z 12 is G
  • Z 15 is T.
  • Z 1 , Z 2 , Z 3 , Z 6 , Z 7 and Z 18 , Z 17 , Z 16 , Z 14 , Z 13 are respectively selected from C, G, or G, T, and wherein C and G form a base pair, and G and T form a base pair.
  • sequence extensions can be carried out at 5′ and/or 3′ based on the above sequence. These sequences can naturally have similar functions to formula (I), for example, extending a T and a T at the 5′ end. /Or extend a G at the 3′ end.
  • the oligonucleotide molecule can further add various chemical modifications and/or labels at the 5' end and/or 3' end; for example, add Cy3 fluorophore, Cy5 fluorophore, tripolyethylene glycol, etc.
  • the oligonucleotide molecule comprises the following nucleotide sequence:
  • a * and T * are respectively unsubstituted or optionally substituted sulfonyl fluoride modified phosphorothioate adenosine and thymidine.
  • a * and T * are phenylsulfonyl fluoride-modified phosphorothioate adenosine and thymidine respectively.
  • the oligonucleotide molecule further includes any one or more of the following:
  • the oligonucleotide molecule further includes any one of the following four sequences:
  • Embodiments of the present disclosure also provide vectors comprising the above-mentioned oligonucleotide molecules.
  • the vector is an expression vector.
  • Embodiments of the present disclosure also provide nucleic acid molecules encoded by the above-mentioned oligonucleotide molecules.
  • Embodiments of the present disclosure also provide host cells containing the above-mentioned nucleic acid molecules.
  • Embodiments of the present disclosure also provide pharmaceutical compositions, including the above-mentioned oligonucleotide molecules, the above-mentioned vectors, the above-mentioned nucleic acid molecules, or the above-mentioned host cells; for example, protein antigen vaccines.
  • the pharmaceutical composition further includes a chemotherapeutic agent.
  • the embodiments of the present disclosure also provide the use of the above-mentioned oligonucleotide molecules, the above-mentioned vectors, the above-mentioned nucleic acid molecules, the above-mentioned host cells or the above-mentioned pharmaceutical compositions in the preparation of medicines for targeting the new coronavirus spike. protein.
  • the embodiments of the present disclosure also provide a method for preventing and/or treating diseases related to the expression of the new coronavirus spike protein, including administering to the patient a therapeutically effective amount of the above-mentioned oligonucleotide molecule, the above-mentioned vector, and the above-mentioned nucleic acid.
  • a therapeutically effective amount of the above-mentioned oligonucleotide molecule, the above-mentioned vector, and the above-mentioned nucleic acid including administering to the patient a therapeutically effective amount of the above-mentioned oligonucleotide molecule, the above-mentioned vector, and the above-mentioned nucleic acid.
  • Embodiments of the present disclosure also provide the above-mentioned oligonucleotide molecule, the above-mentioned vector, the above-mentioned nucleic acid molecule, the above-mentioned host cell or the above-mentioned pharmaceutical composition for use as a medicine.
  • the embodiments of the present disclosure also provide the above-mentioned oligonucleotide molecule, the above-mentioned vector, the above-mentioned nucleic acid molecule, the above-mentioned host cell or the above-mentioned pharmaceutical composition for use in targeting the new coronavirus spike protein.
  • N 33 is 33 random nucleosides (the probability of A, T, G, and C is 25% respectively):
  • the solution formula of polymerase chain reaction is: 645 ⁇ L water, 80 ⁇ L 10 ⁇ PCR buffer, 20 ⁇ L 10mM nucleoside triphosphate monomer mixture (containing dATP- ⁇ -S (5′-deoxyadenosine [ ⁇ -sulfide] [generation]-triphosphate), dTTP, dGTP and dCTP), 40 ⁇ L 10 ⁇ M primer, 5 ⁇ L hot start Taq DNA polymerase, 10 ⁇ L 1 ⁇ M template.
  • the above two primers are:
  • the steps of polymerase chain reaction are: 95°C for 30 seconds, 20 amplification cycles (each cycle contains 95°C for 20 seconds, 45°C for 20 seconds, 68°C for 25 seconds), 68°C for 300 seconds, 4°C save.
  • the gel electrophoresis diagram shown in Figure 3 shows the primer raw materials for polymerase chain reaction on the left and the urea gel electrophoresis diagram of the product oligonucleotide on the right. Cut the gel to purify and separate the product.
  • covalent reaction modification is introduced through the nucleophilic substitution reaction of phosphorothioate to form a covalent reaction modified oligonucleotide library;
  • Reaction solution formula 25 ⁇ L 10 ⁇ M phosphorothioate oligonucleotide library, 15 ⁇ L water, 10 ⁇ L 100mM phosphate buffer solution pH 6, 50 ⁇ L 30mM bromide N,N-dimethylformamide solution.
  • the bromide here is 4-bromomethylbenzenesulfonyl fluoride, 4-bromomethylbenzenesulfonic acid fluoride, or 4-bromomethylpentafluorobenzene.
  • the molecular weight of the oligonucleotides in the random library is not uniform, and it is difficult to directly verify the efficiency of the modification method through mass spectrometry. Therefore, the sequence 5′-TGGCTTGCA*A*A*GGGCTGCTG-3′ (SEQ ID NO: 5) is taken as an example. , add 20 T's to the 3' to approximate the length of the oligonucleotide in the random sequence library. After modification by the above covalent reaction, sequences modified by phenylsulfonyl fluoride, phenylsulfonyl fluoride and pentafluorobenzene were obtained respectively.
  • the molecular ion peaks characterized by soft ionization mass spectrometry are 22573, 22622, and 22598, which are consistent with the theoretical values of 22572, 22620, and 22596.
  • Screening solution recipe 20 ⁇ L 5 ⁇ M oligonucleotide library with covalent reaction modification, 50 ⁇ L 2 ⁇ reaction buffer (containing 2 ⁇ phosphate buffered saline solution and 4mM MgCl 2 ), 10 ⁇ L 250mg/L target protein, 20 ⁇ L water.
  • Covalent conjugate separation method Use gel electrophoresis to separate.
  • the gel is 15% polyacrylamide hydrogel (containing 8M urea).
  • the buffer used for electrophoresis is 1 ⁇ Tris-boric acid-EDTA buffer.
  • the electrophoresis voltage is 300V, and the electrophoresis time is 1 hour.
  • Thermo Fisher’s 1 ⁇ SYBR Gold to stain the above gel so that the part containing nucleic acids on the gel produces fluorescence that can be observed under ultraviolet light, and remove the conjugate strip that moves very slowly during electrophoresis. Extract by cutting.
  • Example 4 Perform dissociation reaction, amplify and sequence oligonucleotides
  • Dissociation solution formula 0.1M sodium hydroxide aqueous solution.
  • the spike protein RBD comes from Yiqiao Shenzhou Company product number 40592-V08H, and its amino acid sequence is:
  • One set of characteristic sequences includes at least the following similar sequences (from left to right, 5′ to 3′):
  • sequences were shortened at both ends to find the shortest active sequence that can best covalently inhibit the spike protein, which is a 20-nucleotide sequence X and a mutant sequence of sequence X: as shown in Figure 4;
  • a * and T * are sulfonyl fluoride-modified phosphorothioate adenosine and thymidine respectively.
  • the base site forms a characteristic mass spectrometry tag, and then is decomposed by trypsin and then liquid chromatography, so that a series of peptide segments are analyzed by tandem mass spectrometry, and finally the site information of the covalent modification on the RBD of the spike protein is obtained.
  • sequence Sample pretreatment 2 ⁇ M Sequence Desalt
  • sequence Sample pretreatment 2 ⁇ M Sequence Desalt
  • the imine was treated at 37°C for 1 hour, and finally desalted using an Amicon ultrafilter.
  • the resulting protein was degraded by trypsin in Tris-HCl buffer at pH 8.3, and then used for liquid chromatography-tandem mass spectrometry to analyze which amino acid in the polypeptide fragments had been covalently reacted by sequence X.
  • the measured result was the spike protein of the new coronavirus. Y421 and K458 amino acid residues.
  • Z 8 , Z 9 and Z 10 are phosphorothioate nucleotides modified by covalent reaction; each is independently selected from one of A, T, C or G; the covalent reaction The modification is to modify with the following unsubstituted or optionally substituted groups: sulfonyl fluoride group, sulfonyl fluoride group, sulfonamide group, sulfonate ester group, ⁇ , ⁇ -unsaturated carbonyl group or fluorine-substituted phenyl group;
  • Z 4 , Z 5 , Z 11 , Z 12 , and Z 15 are independently A, T, C, G or no base;
  • Z 1 , Z 2 , Z 3 , Z 6 , Z 7 and Z 18 , Z 17 , Z 16 , Z 14 and Z 13 are respectively selected from A, T, C and G, and Z 1 and Z 18 form a base pair , Z 2 and Z 17 form a base pair, Z 3 and Z 16 form a base pair, Z 6 and Z 14 form a base pair, Z 7 and Z 13 form a base pair.
  • the bold Z 4 , Z 5 , Z 11 , Z 12 and Z 15 can be selected from A, T, G, C or no base;
  • Z 8 , Z 9 , and Z 10 are three consecutive nucleotides containing unsubstituted or optionally substituted sulfonyl fluoride modified phosphorothioate at the 5' end.
  • the bases can be selected from A, T, G, and C. ; Y 3 and Y 18 can be selected from T and C.
  • the cells were lysed, and the luciferase activity was quantified using Beyotime's RG055 kit.
  • the detection instrument was a 96-well microplate reader. If more people are infected with the new coronavirus pseudovirus, more luciferase-expressing RNA will be brought into HEK 293T cells, which will produce a stronger luciferase activity signal. That is, the inhibitory effect of sequence X on the new coronavirus pseudovirus will be reduced after cell lysis. luciferase activity.
  • the terms “one embodiment,” “some embodiments,” “example,” “specific examples,” or “some examples” or the like mean that a particular feature, structure, material, or other feature is described in connection with the embodiment or example.
  • Features are included in at least one embodiment or example of the disclosure.
  • the schematic expressions of the above terms are not necessarily directed to the same embodiment or example.
  • the specific features, structures, materials or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
  • those skilled in the art may combine and combine different embodiments or examples and features of different embodiments or examples described in this specification unless they are inconsistent with each other.

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Abstract

提供了共价抑制剂的体外筛选方法及其应用，该方法包括如下步骤：构建硫代磷酸酯寡核苷酸文库；将硫代磷酸酯寡核苷酸文库与至少一种共价反应修饰试剂接触，构建共价反应修饰寡核苷酸文库；共价反应修饰试剂包括至少一个共价反应修饰基团，共价反应修饰基团适于与靶蛋白共价结合；将共价反应修饰寡核苷酸文库与靶蛋白接触，分离并收集与靶蛋白结合的共价反应修饰寡核苷酸，再脱除靶蛋白和共价反应修饰基团，构建次级文库；以次级文库为模板，重复上述步骤，多轮循环后，对最后一轮筛选得到的文库进行测序分析，筛选得到共价抑制剂。

Description

共价抑制剂的体外筛选方法及其应用
相关申请的交叉引用
本申请要求在2022年05月18日在中国提交的中国专利申请号202210542182.X的优先权，其全部内容通过引用并入本文。
技术领域
本公开属于生物技术领域，本公开涉及共价抑制剂的体外筛选方法及其应用；具体地，本公开筛选方法可以兼容共价反应修饰，通过该方法，可以针对各类药物靶点蛋白进行共价抑制剂候选药物分子的筛选与发现；本公开还提供了应用该筛选方法得到的共价抑制新冠病毒刺突蛋白的寡核苷酸分子。
背景技术
与疾病相关的蛋白质是重要的药物靶点，例如，癌症免疫检查点疗法针对的PD-1/PD-L1蛋白互作的蛋白、新冠病毒用于识别宿主细胞的刺突蛋白(SARS-CoV-2 spike protein，S protein)等。针对这些蛋白药物靶点，已有单克隆抗体(单抗，Monoclonal antibody，mAb)作为药物被批准使用，其作用机制是mAb与靶蛋白非共价结合，从而抑制靶蛋白的功能。然而，单克隆抗体价格昂贵。例如，针对新冠重症的新冠病毒刺突蛋白中和抗体疗法，一次治疗价格高达十多万甚至几十万。开发价格更加低廉的高效药物具有重要的意义。尽管可化学合成的多肽和短链核酸类药物分子价格相对低廉，但是它们基于非共价结合对靶蛋白的亲和力显著低于单抗，且在体内存在肾脏清除过快等不利因素。
发明内容
为此，本公开实施例的一个目的是提供了共价抑制剂的体外筛选方法，可以针对各种靶蛋白进行共价抑制剂的设计开发。
本公开实施例的另一个目的通过上述方法以新冠病毒刺突蛋白为靶蛋白筛选得到了共价抑制剂，能够共价抑制新冠病毒刺突蛋白的活性。
本公开第一方面的实施例提供了共价抑制剂的体外筛选方法，包括如下步骤：
(1)构建硫代磷酸酯寡核苷酸文库；(2)将所述硫代磷酸酯寡核苷酸文库与至少一种共价反应修饰试剂接触，构建共价反应修饰寡核苷酸文库；所述共价反应修饰试剂包括至少一个共价反应修饰基团，所述共价反应修饰基团适于与靶蛋白共价结合；(3)将所述共价反应修饰寡核苷酸文库与靶蛋白接触，分离并收集与靶蛋白结合的共价反应修饰寡核苷酸，再脱除靶蛋白和共价反应修饰基团，构建次级文库；(4)以所述次级文库为模板，重复步骤(1)-(3)，多轮循环后，对最后一轮筛选得到的文库进行测序分析，筛选得到共 价抑制剂。
在一些实施例中，所述构建硫代磷酸酯寡核苷酸文库的步骤以核苷三磷酸混合物为底物，且底物中至少包括一种核苷硫代三磷酸，采用核酸聚合酶进行扩增反应。
在一些实施例中，所述核苷硫代三磷酸为核苷[α-硫代]-三磷酸。
在一些实施例中，所述构建硫代磷酸酯寡核苷酸文库的初始文库为随机文库，以所述随机文库为模板，以dNTPs为底物，且所述底物中至少一种单体为脱氧核苷硫代三磷酸，在DNA聚合酶的作用下，进行PCR扩增反应。
在一些实施例中，所述脱氧核苷硫代三磷酸选自5′-脱氧腺苷[α-硫代]-三磷酸、5′-脱氧鸟苷[α-硫代]-三磷酸、5′-脱氧胸苷[α-硫代]-三磷酸、5′-脱氧胞苷[α-硫代]-三磷酸及其类似物、衍生物中的任一种。
在一些实施例中，所述共价反应修饰基团为未取代或任选取代的如下基团：磺酰氟基、硫酰氟基、磺酰胺基、磺酸酯基、α,β-不饱和羰基或氟取代苯基。
在一些实施例中，所述共价反应修饰基团选自以下结构：
Figure PCTCN2022100847-appb-000001
其中R 1为-H、-OH、-NO 2、-COOH、卤素或-R’；
R 2为-H、-OH、-R’或-C(＝O)R’；
R 3为-H或-R’；
R 4为-H或-R’；
n为1或2或3或4或5；
R’为C 1-C 12烷基、C 1-C 12烷氧基、C 2-C 12烯基、C 2-C 12炔基、C 3-C 12环烷基、C 3-C 12环烯基、C 6-C 12芳基或C 5-C 12杂芳基；
卤素为F、Cl、Br或I。
在一些实施例中，所述共价反应修饰试剂还包括一个能与硫代磷酸酯基反应的官能团，所述官能团为未取代或任选取代的如下基团：卤素亚甲基或卤素羧乙基，所述卤素为F、Cl、Br或I。
在一些实施例中，所述能与硫代磷酸酯基反应的官能团选自以下结构：
Figure PCTCN2022100847-appb-000002
其中R 5、R 6独立地为H、C 1-C 12烷基、C 1-C 12烷氧基、C 2-C 12烯基、C 2-C 12炔基、C 3-C 12环烷基、C 3-C 12环烯基、C 6-C 12芳基或C 5-C 12杂芳基；R 7为Cl、Br或I。
在一些实施例中，所述重复步骤(1)-(3)的重复次数为1-25次。
在一些实施例中，步骤(3)中将靶蛋白结合的共价反应修饰寡核苷酸与碱性溶液接触，脱除靶蛋白和共价反应修饰基团。
在一些实施例中，所述靶蛋白为疾病相关蛋白或药物蛋白。
本公开第二方面的实施例还提供了共价抑制剂，所述共价抑制剂由第一方面任一实施例所述的所述的方法筛选得到，所述共价抑制剂至少包含二个共价反应修饰基团；所述共价反应修饰基团为未取代或任选取代的如下基团：磺酰氟基、硫酰氟基、磺酰胺基、磺酸酯基、α,β-不饱和羰基或氟取代苯基。
在一些实施例中，所述共价反应修饰基团选自以下结构：
Figure PCTCN2022100847-appb-000003
其中R 1为-H、-OH、-NO 2、-COOH、卤素或-R’；
R 2为-H、-OH、-R’或-C(＝O)R’；
R 3为-H或-R’；
R 4为-H或-R’；
n为1或2或3或4或5；
R’为C 1-C 12烷基、C 1-C 12烷氧基、C 2-C 12烯基、C 2-C 12炔基、C 3-C 12环烷基、C 3-C 12环烯基、C 6-C 12芳基或C 5-C 12杂芳基；
卤素为F、Cl、Br或I。
在一些实施例中，所述共价抑制剂以新冠病毒刺突蛋白为靶蛋白。本公开第三方面的实施例提供了寡核苷酸分子，包括如下式(I)的序列；
5′-Z 1Z 2Z 3Z 4Z 5Z 6Z 7Z 8Z 9Z 10Z 11Z 12Z 13Z 14Z 15Z 16Z 17Z 18-3′   式(I)
其中，Z 8、Z 9、Z 10中至少两个为共价反应修饰的硫代磷酸酯核苷酸；各自独立地选自A、T、C或G中的一种；所述共价反应修饰为采用未取代或任选取代的如下基团进行修饰：磺酰氟基、硫酰氟基、磺酰胺基、磺酸酯基、α,β-不饱和羰基或氟取代苯基；
Z 4、Z 5、Z 11、Z 12、Z 15为各自独立的A、T、C、G或无碱基；
Z 1、Z 2、Z 3、Z 6、Z 7和Z 18、Z 17、Z 16、Z 14、Z 13分别选自A、T、C、G，且Z 1与Z 18形成碱基对，Z 2与Z 17形成碱基对，Z 3和Z 16形成碱基对，Z 6与Z 14形成碱基对，Z 7和Z 13形成碱基对。
在一些实施例中，所述共价反应修饰为苯磺酰氟基修饰、苯硫酰氟基修饰或五氟苯基修饰。
在一些实施例中，Z 1、Z 2、Z 3、Z 6、Z 7和Z 18、Z 17、Z 16、Z 14、Z 13分别选自C、G，或者是G、T，并且其中C和G形成碱基对，G和T形成碱基对。
在一些实施例中，所述的寡核苷酸分子在式(I)结构的5′端和/或3′端至少还包括一个 碱基，所述碱基各自独立地选自A、T、C或G中的一种。
在一些实施例中，所述寡核苷酸分子进一步被修饰以包含至少一种化学修饰，其中所述化学修饰是在选自寡核苷酸分子的糖位置、磷酸位置和/或碱基位置的位置处的化学取代。
在一些实施例中，所述寡核苷酸分子包含如下的核苷酸序列；
5′-GGCTTGCA*A*A*GGGCTGCT-3′；(SEQ ID NO：1)
5′-GGTTTGCA*A*A*GGGCTGCT-3′；(SEQ ID NO：2)
5′-GGCTTGCA*A*A*GGGCTGCC-3′；(SEQ ID NO：3)
5′-GGCTTGCT*T*T*GGGCTGCT-3′。(SEQ ID NO：4)
5′-TGGCTTGCA*A*A*GGGCTGCTG-3′；(SEQ ID NO：5)
5′-TGGTTTGCA*A*A*GGGCTGCTG-3′；(SEQ ID NO：6)
5′-TGGCTTGCA*A*A*GGGCTGCCG-3′；(SEQ ID NO：7)
5′-TGGCTTGCT*T*T*GGGCTGCTG-3′；(SEQ ID NO：8)
5′-TGGCTTGCA*A*AGGGCTGCTG-3′；(SEQ ID NO：9)
5′-TGGCTTGCA*AA*GGGCTGCTG-3′；(SEQ ID NO：10)或
5′-TGGCTTGCAA*A*GGGCTGCTG-3′；(SEQ ID NO：11)。
其中，A *、T *分别为未取代或任选取代磺酰氟基修饰的硫代磷酸酯腺苷、胸苷。
在一些实施例中，所述寡核苷酸分子还包括以下四种序列中的任意一种：
(1)与式(I)的核苷酸序列的同源性在80％以上；
(2)与式(I)的核苷酸序列进行杂交的序列；
(3)式(I)的核苷酸序列转录的RNA序列；
(4)与式(I)的核苷酸序列相比具有一个或多个核苷酸的替换、缺失和/或添加。
本公开第四方面的实施例还提供了载体，包含上述的寡核苷酸分子。
在一些实施例中，所述载体是表达载体。
本公开第五方面的实施例还提供了核酸分子，由上述的寡核苷酸分子编码。
本公开第六方面的实施例还提供了宿主细胞，包含上述的核酸分子。
本公开第七方面的实施例还提供了药物组合物，包含上述寡核苷酸分子、上述的载体、上述的核酸分子、或上述的宿主细胞。
在一些实施例中，所述药物组合物还包括化学治疗剂。
本公开第八方面的实施例还提供了上第三方面任一实施例所述的寡核苷酸分子、第四方面任一实施例所述的载体、第五方面任一实施例所述的核酸分子、第六方面任一实施例所述的宿主细胞或者第七方面任一实施例所述的药物组合物在制备药物中的用途，所述药物用于靶向新冠病毒刺突蛋白。
本公开第九方面的实施例还提供了一种预防和/或治疗与新冠病毒刺突蛋白表达相关的疾病的方法，包括给患者施用治疗有效量的第三方面任一实施例所述的寡核苷酸分子、第四方面任一实施例所述的载体、第五方面任一实施例所述的核酸分子、第六方面任一实 施例所述的宿主细胞或者第七方面任一实施例所述的药物组合物。
本公开第十方面的实施例还提供了第三方面任一实施例所述的寡核苷酸分子、第四方面任一实施例所述的载体、第五方面任一实施例所述的核酸分子、第六方面任一实施例所述的宿主细胞或者第七方面任一实施例所述的药物组合物用于作为药物使用。
在一些实施例中，第三方面任一实施例所述的寡核苷酸分子、第四方面任一实施例所述的载体、第五方面任一实施例所述的核酸分子、第六方面任一实施例所述的宿主细胞或者第七方面任一实施例所述的药物组合物用于靶向新冠病毒刺突蛋白。
本公开实施例具有如下有益效果：
(1)本公开实施例筛选方法克服了相关技术中所有靶蛋白的共价抑制剂均为结构指导的理性设计开发得到，能够针对的靶蛋白种类有限，且得到的共价抑制剂活力效果受限于所用的非共价抑制剂性能。本公开首次实现了兼容共价反应修饰的体外筛选技术，从而使得能够针对任意一种靶蛋白开发共价抑制剂，且基于大规模随机分子库筛选，得到的高活力共价抑制剂的潜力巨大。
(2)基于本公开之前的相关技术中新冠病毒刺突蛋白抑制剂均基于非共价，本公开首次发现了能够高活力共价抑制新冠病毒刺突蛋白的共价抑制剂。
附图说明
本公开的上述和/或附加的方面和优点从结合下面附图对实施例的描述中将变得明显和容易理解，其中：
图1为本公开实施例共价抑制剂的体外筛选方法示意图；
图2为本公开实施例一种硫代磷酸酯结构与一种共价化学修饰试剂发生亲核取代反应来对寡核苷酸进行共价反应修饰的示意图；
图3为本公开实施例聚合酶链式反应的引物原料(左)和产物寡聚核苷酸(右)的尿素凝胶电泳图；
图4为本公开实施例序列X的序列以及序列X的M1-M8的突变示意图；
图5为本公开序列X及其突变体与新冠刺突蛋白反应的SDS-PAGE图；图5中
1、新冠刺突蛋白；2、新冠刺突蛋白与序列X反应；3、新冠刺突蛋白与序列X的M5突变反应；4～6、新冠刺突蛋白与只含1个苯磺酰氟修饰的序列X的反应；7～9、新冠刺突蛋白与只含2个苯磺酰氟修饰的序列X的反应；10、新冠刺突蛋白与序列X反应；11、新冠刺突蛋白与序列X的M1突变反应；12、新冠刺突蛋白与序列X的M2突变反应；13、新冠刺突蛋白与序列X的M3突变反应；14、新冠刺突蛋白与序列X的M4突变反应；15、新冠刺突蛋白与序列X的M6突变反应；16、新冠刺突蛋白与序列X的M7突变反应；17、新冠刺突蛋白与序列X的M8突变反应；
图6为本公开实施例序列X+TG与新冠病毒刺突蛋白缀合的液相色谱-串联质谱分析；图6显示序列X+TG共价缀合新冠病毒刺突蛋白的一个氨基酸位点为新冠病毒刺突蛋白的K458氨基酸残基；
图7为本公开实施例序列X+TG与新冠病毒刺突蛋白缀合的液相色谱-串联质谱分析；图7显示序列X+TG共价缀合新冠病毒刺突蛋白的另一个氨基酸位点为新冠病毒刺突蛋白的Y421氨基酸残基；
图8为本公开实施例序列X，序列X’对新冠假病毒的抑制活力实验。
具体实施方式
与疾病相关的蛋白质是重要的药物靶点，例如，癌症免疫检查点疗法针对的PD-1/PD-L1蛋白互作的蛋白、新冠病毒用于识别宿主细胞的刺突蛋白(SARS-CoV-2 spike protein，S protein)等。针对这些蛋白药物靶点，已有单克隆抗体(单抗，Monoclonal antibody，mAb)作为药物被批准使用，其作用机制是mAb与靶蛋白非共价结合，从而抑制靶蛋白的功能。然而，单克隆抗体价格昂贵。例如，针对新冠重症的新冠病毒刺突蛋白中和抗体疗法，一次治疗价格高达十多万甚至几十万。开发价格更加低廉的高效药物具有重要的意义。尽管可化学合成的多肽和短链核酸类药物分子价格相对低廉，但是它们基于非共价结合对靶蛋白的亲和力显著低于单抗，且在体内存在肾脏清除过快等不利因素。
相关技术中，针对靶蛋白的共价抑制剂研究首先非共价结合靶蛋白，然后通过其分子中含有的惰性反应官能团与靶蛋白形成共价键，从而高特异性、永久性的抑制靶蛋白。这些针对靶蛋白的共价抑制剂比传统的单抗类抑制剂具备两方面显著优势：其一，高特异的永久性抑制，抑制能力接近或超越单抗，且活性不受肾脏清除较快的负面影响；其二，由于这些共价抑制剂通常分子量仅为单抗的十分之一或更小，使用剂量显著小于单抗。
尽管如此，至今所有已知的靶蛋白共价抑制剂的开发方法均为结构指导的理性设计(Structure-guided design)。这种开发方法需要明确的蛋白互作结构信息，并且依赖于试错模式来找寻靶蛋白上可被共价攻击的氨基酸残基，因此导致目前共价抑制剂靶向蛋白的种类极其有限。仍旧缺乏一种新的药物发现技术，来针对各种靶蛋白进行共价抑制剂开发。
传统的体外筛选技术(In vitro selection)是一种发现非共价结合靶蛋白的短链核酸序列的方法，尽管其原理可以用于对各种靶蛋白进行非共价机制抑制剂的开发，但仍然无法进行共价抑制剂的筛选。原因是，共价反应化学修饰将抑制体外筛选技术中涉及到的聚合酶，且共价抑制剂短链核酸序列与靶蛋白形成的缀合物无法通过测序获取序列信息。
公开于该背景技术部分的信息仅仅旨在增加对本公开的总体背景的理解，而不应当被视为承认或以任何形式暗示该信息构成已为本领域一般技术人员所公知的现有技术。
以下为术语或词语说明，且除非另外定义，否则本文中使用的全部技术和科学术语具有本公开所属领域的熟练技术人员通常理解的含义。
在本文中，词语“包含”和“包括”及其各种变体意指可能包含允许但没有具体描述的其它要素或整体。
在本文中，术语：“共价反应修饰”，亦称：“共价弹头修饰(covalent warhead modification)”；是指可用于与目标靶蛋白进行共价结合的化学修饰。
术语：“共价结合”是指化学键的形成，即原子间通过共用电子对所形成的键。
术语“扩增”是指通过其生产至少一个靶核酸或其序列互补物的至少部分序列的任何手段，通常以模板依赖性方式，包括但不限于，用于扩增核酸序列的各种技术，扩增方法的非限制性实例包括聚合酶链反应(PCR)、逆转录酶PCR等。
术语“核酸聚合酶”是指催化核苷酸掺入至核酸中的酶，包括DNA聚合酶、RNA聚合酶、反转录酶等。
术语“体外筛选”是指在细胞外通过反复的筛选从核酸的随机文库中得到具有特殊活性的分子。
术语“核苷酸”一般是指一个核苷通过酯键与一个酸性分子或基团相连而形成的化合物，例如核苷的磷酸酯，通常有一个、两个或三个磷酸基团共价连接在核苷的糖基团的5号位上。在一些情况下，核苷酸的定义还包括一些核苷酸的类似物或其衍生物。类似物是指在结构上类似于天然存在的核苷酸的化合物，类似物的非限制性举例包括：氧化鸟苷、假尿苷、异胞苷、异鸟苷等；衍生物是指具有修饰的核苷酸碱基部分、修饰的戊糖部分和/或修饰的磷酸酯部分，示例性修饰的磷酸酯部分非限制性举例包括：硫代磷酸酯、二硫代磷酸酯、磷酸硒酸酯、磷酸二硒酸酯、苯胺磷酸硫醇酯、苯胺磷酸酯、氨基磷酸酯等，示例性修饰的核苷酸碱基部分非限制性举例包括：5-甲基胞嘧啶(5mC)；、C-5丙炔基-C和C-5丙炔基-U；2,6-二氨基嘌呤、次黄嘌呤、假尿苷、2-硫代嘧啶、异胞嘧啶(isoC)、5-甲基isoC和异鸟嘌呤。示例性修饰的戊糖部分非限制性举例包括：2'-或3'-修饰，其中2'-或3'-位为氢、羟基、烷氧基(例如甲氧基、乙氧基、异丙氧基、丁氧基、异丁氧基)、叠氮基、氨基、烷基氨基、氟、氯或溴。
例如术语“核苷三磷酸”一般是指有三个磷酸基团共价连接在核苷的糖基团的5号位上，在一些情况下，核苷三磷酸的定义还包括一些典型核苷三磷酸的类似物、衍生物。
术语“核苷[α-硫代]-三磷酸”非限制性举例包括：5′-脱氧腺苷[α-硫代]-三磷酸、5′-腺苷[α-硫代]-三磷酸、5′-脱氧鸟苷[α-硫代]-三磷酸、5′-鸟苷[α-硫代]-三磷酸、5′-脱氧胸苷[α-硫代]-三磷酸、5′-胸苷[α-硫代]-三磷酸、5′-脱氧尿苷[α-硫代]-三磷酸、5′-尿苷[α-硫代]-三磷酸、5′-脱氧胞苷[α-硫代]-三磷酸、5′-胞苷[α-硫代]-三磷酸、5′-脱氧肌苷[α-硫代]-三磷酸、5′-肌苷[α-硫代]-三磷酸；以及其非限制性举例的甲基化衍生、酰化衍生、卤素化衍生的衍生物；以及其非限制性举例：氧化鸟苷替代鸟苷、假尿苷替代尿苷、异胞苷替代胞苷、异鸟苷替代鸟苷。
术语“核酸”，“短链核酸”，“多核苷酸”、“寡核苷酸”和“寡聚核苷酸”可互换使用，并且是指线性或环状构型以及单链或双链形式的脱氧核糖核苷酸或核糖核苷酸聚合物，且该术语可包括天然核苷酸的已知类似物，以及在碱基、糖和/或磷酸部分(例如硫代磷酸酯主链)中修饰的核苷酸。为了本公开公开的目的，这些术语不应被解释为对聚合物长度的限制。且出于用作本公开较大应用价值的寡聚核苷酸的用途，通常指10-40个核苷酸，优选10-25个核苷酸的长度合成寡聚核苷酸。
术语“序列”是指任何长度的核苷酸序列,其可以是DNA或RNA。可以是线性，圆形或分支的，并且可以是单链或双链的。
术语“同源性”指与目标核酸序列的序列相似性。“同源性”包括与目标核酸序列或其核心序列具有80％或更高，85％或更高，90％或更高，95％或更高同源性的核苷酸序列。同源性可以用肉眼或计算机软件进行评价。
术语“标记”指的是连接(共价地或非共价地)至核酸分子，并且能够提供关于分子的信息的部分，示例性的标记包括荧光标记等。
术语“新冠病毒刺突蛋白”是指位于新冠病毒最外层的三聚体结构的蛋白，也称为S蛋白，其是来源于包括SARS-CoV-2在内的冠状病毒的结构蛋白。
术语“任选取代的”表示所给结构中的一个或多个氢原子被具体取代基所取代。除非其他方面表明，一个任选的取代基团可以在基团各个可取代的位置进行取代。当所给出的结构式中不只一个位置能被选自具体基团的一个或多个取代基所取代，那么取代基可以相同或不同地在各个位置取代。“任选取代的”的取代基的非限制性举例包括，氟，氯，溴，碘，羟基，硝基，氨基，羧基，烷基，烷氧基，烯基、炔基、芳氧基，杂芳基氧基，杂环基氧基，芳基烷氧基，杂芳基烷氧基，杂环基烷氧基，环烷基烷氧基，烷氨基，烷氨基烷基，环烷基氨基，环烷基烷氨基，卤代烷基，卤代烷氧基，羟基取代的烷基，羟基取代的烷氨基，氨基取代的烷基，烷基酰基，杂烷基，环烷基，环烯基，环烷基烷基，杂环基，杂环基烷基，杂环基酰基，芳基，芳基烷基，芳氨基，杂芳基，杂芳基烷基，杂芳基氨基，酰胺基，磺酰基，氨基磺酰基等等。
例如：
术语“烷基”可以是“C 1-C 12烷基”。“C 1-C 12烷基”表示含有1-12个碳原子，饱和的直链或支链一价烃基基团；在一实施方案中，含有1-8个碳原子；在另一实施方案中，包含1-6个碳原子；在又一实施方案中，包含1-4个碳原子；还在一实施方案中，包含1-3个碳原子。“C 1-C 12烷基”的非限制性举例包括：甲基、乙基、正丙基、异丙基、正丁基、异丁基、仲丁基、叔丁基、正戊基、2-戊基、3-戊基、2-甲基-2-丁基、3-甲基-2-丁基、3-甲基-1-丁基、2-甲基-1-丁基等等。
术语“烯基”可以是“C 2-C 12烯基”。“C 2-C 12烯基”表示含有2-12个碳原子的直链或支链一价烃基，其中至少有一个不饱和位点，即有一个碳-碳sp2双键。在一实施方案中，包含2-8个碳原子；在另一实施方案中，包含2-6个碳原子；在又一实施方案中，包含2-4个碳原子。“C 2-C 12烯基”非限制性举例包括：乙烯基、烯丙基、丙烯基等等。
术语“炔基”可以是“C 2-C 12炔基”。“C 2-C 12炔基”表示含有2-12个碳原子的直链或支链一价烃基，其中至少有一个碳-碳sp三键。
术语“烷氧基”可以是“C 1-C 12烷氧基”。“C 1-C 12烷氧基”表示“C 1-C 12烷基”通过氧原子与分子其余部分相连，其中“C 1-C 12烷基”具有如本文所述的含义。“C 1-C 12烷氧基”的非限制性举例包括：甲氧基、乙氧基、1-丙氧基、2-丙氧基等等。
术语“环烷基”可以是“C 3-C 12环烷基”。“C 3-C 12环烷基”表示含有3-12个碳原子的，单价或多价的饱和单环、双环或三环体系。在一实施方案中，包含3-10个碳原子；在另一实施方案中，包含3-8个碳原子；在又一实施方案中，包含3-6个碳原子。“C 3-C 12环烷基”的非 限制性举例包括：环丙基，环丁基，环戊基，环己基，环庚基，环辛基，环壬基，环癸基，环十一烷基，环十二烷基等等。
术语“环烯基”可以是“C 3-C 12环烯基”。“C 3-C 12环烯基”表示含有3-12个碳原子的，单价或多价的单环、双环或三环体系，至少包含一个碳碳双键，且环体系为非芳香性的。在一实施方案中，包含3-10个碳原子；在另一实施方案中，包含3-8个碳原子；在又一实施方案中，包含3-6个碳原子。“C 3-C 12环烯基”的非限制举例包括：环丁烯基，环戊烯基，环己烯基，环己二烯基等等。
术语“芳基”可以是“C 6-C 12芳基”。“C 6-C 12芳基”表示含有6-12个环原子的单环、双环和三环的碳环体系，其中，至少一个环体系是芳香族的，“C 6-C 12芳基”的非限制举例包括：苯基，茚基，萘基等等。
术语“杂芳基”可以是“C 5-C 12杂芳基”。“C 5-C 12杂芳基”表示含有5-12个环原子的单环、双环和三环体系，其中至少一个环体系是芳香族的，且至少一个环体系包含一个或多个杂原子，“杂原子”选自O、S、N、P或Si。
下面详细描述本公开的实施例。下面描述的实施例是示例性的，仅用于解释本公开，而不能理解为对本公开的限制。
如图1所示，本公开实施例提供了一种共价抑制剂的体外筛选方法，包括如下步骤：
(1)构建硫代磷酸酯寡核苷酸文库；(2)将硫代磷酸酯寡核苷酸文库与至少一种共价反应修饰试剂接触，构建共价反应修饰寡核苷酸文库；共价反应修饰试剂包括至少一个共价反应修饰基团，共价反应修饰基团适于与靶蛋白共价结合；(3)将共价反应修饰寡核苷酸文库与靶蛋白接触，分离并收集与靶蛋白结合的共价反应修饰寡核苷酸，再脱除靶蛋白和共价反应修饰基团，构建次级文库；(4)以次级文库为模板，重复步骤(1)-(3)，多轮循环后，对最后一轮筛选得到的文库进行测序分析，筛选得到共价抑制剂。
本公开实施例共价抑制剂的体外筛选方法，可以得到可以共价抑制靶蛋白的寡核苷酸分子；共价反应修饰的寡聚核苷酸在核酸聚合酶扩增后引入，并在下一个循环的聚合酶扩增前移除，从而使得共价反应修饰不影响核酸聚合酶反应，使之兼容于体外筛选，经过多次筛选循环后测序，则可得到可以共价抑制靶蛋白的寡聚核苷酸序列信息，并通过所使用的硫代磷酸酯核苷酸种类来确定寡聚核苷酸上包含共价反应修饰的位置信息。该方法与已知的核酸适配体筛选技术的显著不同之处在于：引入了共价反应修饰，且确保该修饰在适当的步骤中引入和移除，从而不干扰核酸聚合酶活性，使得筛选可以共价抑制靶蛋白的含有共价反应修饰的寡聚核苷酸序列成为可能。
可以理解的是：本公开共价抑制剂的体外筛选方法，可以以疾病相关蛋白为靶蛋白，用于共价抑制剂候选药物分子的筛选和发现。同时，本公开共价抑制剂的体外筛选方法，还可以药物蛋白为靶蛋白，非限制性的举例如单克隆抗体、激素类药物(如胰岛素)等分子形式为蛋白的药物，进行已知蛋白药物的改造。
在一些实施例中，构建硫代磷酸酯寡核苷酸文库的步骤以核苷三磷酸混合物为底物，且底物中至少包括一种核苷硫代三磷酸，采用核酸聚合酶进行扩增反应。
在一些实施例中，构建硫代磷酸酯寡核苷酸文库的初始文库为随机文库，以随机文库为模板，以dNTPs为底物，且底物中至少一种单体为脱氧核苷硫代三磷酸，在DNA聚合酶的作用下，进行PCR扩增反应。
在一些实施例中，脱氧核苷硫代三磷酸选自5′-脱氧腺苷[α-硫代]-三磷酸、5′-脱氧鸟苷[α-硫代]-三磷酸、5′-脱氧胸苷[α-硫代]-三磷酸、5′-脱氧胞苷[α-硫代]-三磷酸及其类似物、衍生物中的任一种。
在一些实施例中，共价反应修饰基团为未取代或任选取代的如下基团：磺酰氟基、硫酰氟基(也称，氟硫酸酯基)、磺酰胺基、磺酸酯基、α,β-不饱和羰基或氟取代苯基。
在一些实施例中，共价反应修饰基团选自以下结构：
Figure PCTCN2022100847-appb-000004
其中R 1为-H、-OH、-NO 2、-COOH、卤素或-R’；
R 2为-H、-OH、-R’或-C(＝O)R’；
R 3为-H或-R’；
R 4为-H或-R’；
n为1或2或3或4或5；
R’为C 1-C 12烷基、C 1-C 12烷氧基、C 2-C 12烯基、C 2-C 12炔基、C 3-C 12环烷基、C 3-C 12环烯基、C 6-C 12芳基或C 5-C 12杂芳基；
卤素为F、Cl、Br或I。
在一些实施例中，共价反应修饰试剂还包括一个能与硫代磷酸酯基反应的官能团，官能团为未取代或任选取代的如下基团：卤素亚甲基或卤素羧乙基，卤素为F、Cl、Br或I。
在一些实施例中，能与硫代磷酸酯基反应的官能团选自以下结构：
Figure PCTCN2022100847-appb-000005
其中R 5、R 6独立地为H、C 1-C 12烷基、C 1-C 12烷氧基、C 2-C 12烯基、C 2-C 12炔基、C 3-C 12环烷基、C 3-C 12环烯基、C 6-C 12芳基或C 5-C 12杂芳基；R 7为Cl、Br或I。
图2示出了一种共价化学修饰试剂与一种硫代磷酸酯结构发生亲核取代反应来对寡核苷酸进行共价反应修饰的示意举例。
在一些实施例中，重复步骤(1)-(3)的重复次数为1-25次。
在一些实施例中，步骤(3)中将靶蛋白结合的共价反应修饰寡核苷酸与碱性溶液接触，脱除靶蛋白和共价反应修饰基团。
在一些实施例中，碱性溶液的pH>9，进一步优选地，pH>10；进一步优选地，pH>11； 进一步优选地，pH>12。
在一些实施例中，碱性溶液NaOH溶液或KOH溶液。
本公开实施例还提供了共价抑制剂，该共价抑制剂由上述的方法筛选得到，该共价抑制剂中，至少包括二个共价反应修饰基团；共价反应修饰基团为未取代或任选取代的如下基团：磺酰氟基、硫酰氟基、磺酰胺基、磺酸酯基、α,β-不饱和羰基或氟取代苯基。
在共价抑制剂的一些实施例中，共价反应修饰基团选自以下结构：
Figure PCTCN2022100847-appb-000006
其中R 1为-H、-OH、-NO 2、-COOH、卤素或-R’；
R 2为-H、-OH、-R’或-C(＝O)R’；
R 3为-H或-R’；
R 4为-H或-R’；
n为1或2或3或4或5；
R’为C 1-C 12烷基、C 1-C 12烷氧基、C 2-C 12烯基、C 2-C 12炔基、C 3-C 12环烷基、C 3-C 12环烯基、C 6-C 12芳基或C 5-C 12杂芳基；
卤素为F、Cl、Br或I。
本公开实施例还提供了应用本公开的体外筛选方法筛选得到的共价抑制新冠病毒刺突蛋白的寡核苷酸分子。
本公开实施例的新冠病毒刺突蛋白来源于新冠病毒原始株或其突变株(例如德尔塔(Delta)、奥密克戎(Omicron))。
本公开实施例提供了寡核苷酸分子，包括如下式(I)的序列；
5′-Z 1Z 2Z 3Z 4Z 5Z 6Z 7Z 8Z 9Z 10Z 11Z 12Z 13Z 14Z 15Z 16Z 17Z 18-3′    式(I)
其中，Z 8、Z 9、Z 10中至少两个为共价反应修饰的硫代磷酸酯核苷酸；各自独立地选自A、T、C或G中的一种；共价反应修饰为采用未取代或任选取代的如下基团进行修饰：磺酰氟基、硫酰氟基、磺酰胺基、磺酸酯基、α,β-不饱和羰基或氟取代苯基；
Z 4、Z 5、Z 11、Z 12、Z 15为各自独立的A、T、C、G或无碱基；
Z 1、Z 2、Z 3、Z 6、Z 7和Z 18、Z 17、Z 16、Z 14、Z 13分别选自A、T、C、G，且Z 1与Z 18形成碱基对，Z 2与Z 17形成碱基对，Z 3和Z 16形成碱基对，Z 6与Z 14形成碱基对，Z 7和Z 13形成碱基对。
在寡核苷酸分子的一些实施例中，共价反应修饰为苯磺酰氟基修饰、苯硫酰氟基修饰或五氟苯基修饰。
在寡核苷酸分子的一些实施例中，Z 4、Z 5、Z 11、Z 12、Z 15并不参与形成具有活性的二级折叠结构，其中任意一个可以替换为A、T、G、C或无碱基。在一些实施例中，Z 4为T， 和/或Z 5为T，和/或Z 11为G，和/或Z 12为G，和/或Z 15为T。
在一些实施例中，Z 1、Z 2、Z 3、Z 6、Z 7和Z 18、Z 17、Z 16、Z 14、Z 13分别选自C、G，或者是G、T，并且其中C和G形成碱基对，G和T形成碱基对。
在一些具体实施例中，在上述序列的基础上可以在5′和/或3′进行序列延展，这些序列很自然能具备与式(I)类似的功能，例如在5′端延长一个T和/或3′端延长一个G。
在一些具体的实施例中，寡核苷酸分子可以进一步在5′端和/或3′增加各种化学修饰和/或标记；例如在5′端和/或3′端增加Cy3荧光团、Cy5荧光团、三聚乙二醇等。
在寡核苷酸分子的一些实施例中，寡核苷酸分子包含如下的核苷酸序列:
5′-GGCTTGCA*A*A*GGGCTGCT-3′；(SEQ ID NO：1)
5′-GGTTTGCA*A*A*GGGCTGCT-3′；(SEQ ID NO：2)
5′-GGCTTGCA*A*A*GGGCTGCC-3′；(SEQ ID NO：3)
5′-GGCTTGCT*T*T*GGGCTGCT-3′。(SEQ ID NO：4)
5′-TGGCTTGCA*A*A*GGGCTGCTG-3′；(SEQ ID NO：5)
5′-TGGTTTGCA*A*A*GGGCTGCTG-3′；(SEQ ID NO：6)
5′-TGGCTTGCA*A*A*GGGCTGCCG-3′；(SEQ ID NO：7)
5′-TGGCTTGCT*T*T*GGGCTGCTG-3′；(SEQ ID NO：8)
5′-TGGCTTGCA*A*AGGGCTGCTG-3′；(SEQ ID NO：9)
5′-TGGCTTGCA*AA*GGGCTGCTG-3′；(SEQ ID NO：10)或
5′-TGGCTTGCAA*A*GGGCTGCTG-3′；(SEQ ID NO：11)。
其中，A *、T *分别为未取代或任选取代磺酰氟基修饰的硫代磷酸酯腺苷、胸苷。
在一些实施例中，A *、T *分别为苯磺酰氟基修饰的硫代磷酸酯腺苷、胸苷。
在寡核苷酸分子的一些实施例中，寡核苷酸分子进一步包括如下的任一项或多种：
将胞嘧啶C变为5-甲基胞嘧啶5mC或5-羟甲基胞嘧啶5hmC、
或者将胸腺嘧啶T变为尿嘧啶U或假尿嘧Ψ、
或者将腺嘌呤A变为6-甲基腺嘌呤6mA、
或者将鸟嘌呤G变为8-羟基鸟嘌呤8-oxoG；
或者将脱氧核糖2′的H替换为羟基(OH)、烷氧基(甲氧基、乙氧基等)、氟(F)，
或者将磷酸二酯替换为硫代磷酸二酯。
在寡核苷酸分子的一些实施例中，寡核苷酸分子还包括以下四种序列中的任意一种：
(1)与式(I)的核苷酸序列的同源性在80％以上；
(2)与式(I)的核苷酸序列进行杂交的序列；
(3)式(I)的核苷酸序列转录的RNA序列。
(4)与式(I)的核苷酸序列相比具有一个或多个核苷酸的替换、缺失和/或添加。
本公开实施例还提供了载体，包含上述的寡核苷酸分子。
在一些实施例中，载体是表达载体。
本公开实施例还提供了核酸分子，由上述的寡核苷酸分子编码。
本公开实施例还提供了宿主细胞，包含上述的核酸分子。
本公开实施例还提供了药物组合物，包含上述寡核苷酸分子、上述的载体、上述的核酸分子、或上述的宿主细胞；例如蛋白抗原疫苗。
在一些实施例中，药物组合物还包括化学治疗剂。
本公开实施例还提供了上述的寡核苷酸分子、上述的载体、上述的核酸分子、上述的宿主细胞或者上述的药物组合物在制备药物中的用途，药物用于靶向新冠病毒刺突蛋白。
本公开实施例还提供了一种预防和/或治疗与新冠病毒刺突蛋白表达相关的疾病的方法，包括给患者施用治疗有效量的上述的寡核苷酸分子、上述的载体、上述的核酸分子、上述的宿主细胞或者上述的药物组合物。
本公开实施例还提供了上述的寡核苷酸分子、上述的载体、上述的核酸分子、上述的宿主细胞或者上述的药物组合物用于作为药物使用。
本公开实施例还提供了上述的寡核苷酸分子、上述的载体、上述的核酸分子、上述的宿主细胞或者上述的药物组合物用于在靶向新冠病毒刺突蛋白中使用。
实施例1构建硫代磷酸酯寡核苷酸文库
(1)使用以下随机文库作为模板，进行聚合酶链式反应，其中N 33为33个随机核苷(A、T、G、C的概率分别为25％)：
TACACAGGGGAGTAACGAAT N 33 TGGGTGTTTGTTCTTCTCT。(SEQ ID NO：12)
(2)聚合酶链式反应的溶液配方为：645μL水、80μL 10×PCR缓冲液、20μL 10mM核苷三磷酸单体混合物(包含dATP-α-S(5′-脱氧腺苷[α-硫代]-三磷酸),dTTP,dGTP and dCTP)、40μL 10μM引物、5μL热启动Taq DNA聚合酶、10μL 1μM模板。上述两条引物为：
TACACAGGGGAGTAACGAAT；(SEQ ID NO：13)
AGAGAAGAACAAACACCCA。(SEQ ID NO：14)
(3)聚合酶链式反应的步骤为：95℃30秒、20个扩增循环(每个循环含有95℃20秒、45℃20秒、68℃25秒)、68度300秒、4℃保存。
(4)使用凝胶电泳进行分离，如图3所示的凝胶电泳图，左侧为聚合酶链式反应的引物原料，右侧为产物寡聚核苷酸的尿素凝胶电泳图，通过切胶进行产物提纯分离。
实施例2构建共价反应修饰寡核苷酸文库
在实施例1硫代磷酸酯寡核苷酸文库中，通过硫代磷酸酯的亲核取代反应引入共价反应修饰，形成共价反应修饰寡核苷酸文库；
(1)反应溶液配方：25μL 10μM硫代磷酸酯寡核苷酸文库、15μL水、10μL 100mM磷酸盐缓冲溶液pH 6、50μL 30mM溴代物的N,N-二甲基甲酰胺溶液。此处的溴代物是4-溴甲基苯磺酰氟，4-溴甲基苯硫酸酰氟，或4-溴甲基五氟苯。
(2)反应的条件：37℃反应12小时。
(3)分离方法：使用默克生命科学的Aimcon超滤器进行脱盐处理，得到共价反应修 饰寡核苷酸文库。
(4)随机文库的寡核苷酸分子量不均一，难以直接通过质谱验证修饰方法的效率，因此，以序列5′-TGGCTTGCA*A*A*GGGCTGCTG-3′(SEQ ID NO：5)为例，将其在3′增加20个T以接近随机序列库中寡核苷酸的长度，经过上述共价反应修饰后分别得到苯磺酰氟、苯硫酰氟和五氟苯修饰的序列，其软电离质谱表征得到的分子离子峰分别为22573、22622、22598，与理论值22572、22620、22596相符。
实施例3用共价反应修饰寡核苷酸文库进行靶蛋白的体外筛选
(1)筛选溶液配方：20μL 5μM带有共价反应修饰寡核苷酸文库、50μL 2×反应缓冲液(含有2×磷酸盐缓冲盐溶液和4mM MgCl 2)、10μL 250mg/L的靶蛋白、20μL水。
(2)反应条件：37℃反应2小时。
(3)共价缀合物分离方法：使用凝胶电泳分离，凝胶为15％的聚丙烯酰胺水凝胶(含8M尿素)，电泳使用的缓冲液为1×Tris-硼酸-EDTA缓冲液，电泳电压为300V，电泳时间为1小时。
(4)使用赛默飞世尔的1×SYBR Gold对上述凝胶进行染色，使得凝胶上含有核酸的部分产生可在紫外灯下观察到的荧光，将电泳移动很慢的缀合物条带切割提取出来。
实施例4进行解离反应，并进行寡核苷酸的扩增与测序
(1)解离溶液配方：0.1M氢氧化钠水溶液。
(2)离解反应条件：400μL 0.1M氢氧化钠水溶液浸泡切割提取出来的凝胶，50℃侵泡2小时。
(3)提取溶液配方：400μL 0.1M氢氧化钠水溶液侵泡体系中加入10μL 4M盐酸和40μL 10×Tris-硼酸-EDTA缓冲液。
(4)提取反应条件：50℃浸泡12小时。
(5)分离方法：对上述提取反应得到的溶液使用默克生命科学的Aimcon超滤器进行脱盐处理，得到可以进行聚合酶链式反应扩增的寡聚核苷酸序列。
实施例5通过上述筛选方法得到的新冠病毒刺突蛋白共价抑制剂分子及其性质表征
(1)使用新冠病毒刺突蛋白的受体结合域RBD作为靶蛋白进行正筛选，并以人血清作为基质进行负筛。刺突蛋白RBD来源于义翘神州公司产品编号40592-V08H，其氨基酸序列为：
Figure PCTCN2022100847-appb-000007
按照上述实施例1～4的步骤依次进行并重复13次，并对最终得到的寡聚核苷酸序列进行二代测序(使用Illumina二代测序仪)，通过测序读出得到了多个序列，其中一组特征序列至少包括以下几个相似序列(从左至右为5′至3′)：
Figure PCTCN2022100847-appb-000008
对这些序列经过两端缩短找寻最短且能最佳共价抑制刺突蛋白的活性序列为一个20个核苷酸序列X以及序列X的突变序列：如图4所示；
序列X:5′-TGGCTTGCA*A*A*GGGCTGCTG-3′；(SEQ ID NO：5)
序列X的M1突变：5′-GGCTTGCA*A*A*GGGCTGCT-3′；(SEQ ID NO：1)
序列X的M2突变：5′-CGGCTTGCA*A*A*GGGCTGCTG-3′；(SEQ ID NO：21)
序列X的M3突变：5′-TGGCTTGCA*A*A*GGGCTGCCG-3′；(SEQ ID NO：7)
序列X的M4突变：5′-CGGCTTGCA*A*A*GGGCTGCCG-3′；(SEQ ID NO：22)
序列X的M5突变：5′-TGGCTTGCT*T*T*GGGCTGCTG-3′；(SEQ ID NO：8)
序列X的M6突变：5′-TGGCTTGCA*A*A*GGGCTGTTG-3′；(SEQ ID NO：23)
序列X的M7突变：5′-TGGTTTGCA*A*A*GGGCTGCTG-3′；(SEQ ID NO：6)
序列X的M8突变：5′-TGGTTTGCA*A*A*GGGCTGTTG-3′；(SEQ ID NO：24)
A *、T *分别为磺酰氟基修饰的硫代磷酸酯腺苷、胸苷。
(2)通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)验证序列X能够与新冠病毒刺突蛋白形成共价缀合物(图5)。使用6％的SDS-PAGE对序列X与新冠病毒刺突蛋白反应后的产物进行分析，发现形成了明显的缀合物带。反应溶液配方：2.5μL 20μM序列X、2μL 250mg/L新冠病毒刺突蛋白、10μL 2×反应缓冲液(含有2×磷酸盐缓冲盐溶液和4mM MgCl 2)、5.5μL水。反应条件：37度3小时。SDS-PAGE条件：1×Tris-Glycine电泳缓冲液，200V电压、1.5小时。用考马斯亮蓝对完成电泳的SDS-PAGE进行染胶，处于更高的条带代表缀合物形成。
(3)通过液相色谱-串联质谱分析技术对解离且酶解后的上述共价缀合物进行分析。首先将缀合物用0.1M NaOH处理来移除蛋白质上的寡聚核酸，然后通过二硫苏糖醇和N-甲基马来酰亚胺将修饰位点上的巯基加帽，从而在修饰残基位点形成特征的质谱标签，进而通过胰酶分解后再液相色谱，使得一系列的肽段分别进行串联质谱分析，最终获得刺突蛋白的RBD上发生了共价修饰的位点信息。利用该方法，发现序列X+TG共价缀合新冠病毒刺突蛋白的氨基酸位点为新冠病毒刺突蛋白的Y421和K458氨基酸残基(如图6、图7所示)。样品前处理：2μM序列X与50mg/L新冠病毒刺突蛋白在400μL 1×反应缓冲液(含 有1×磷酸盐缓冲盐溶液和2mM MgCl 2)中于37度反应3小时，用Amicon超滤器脱盐，然后用0.1M氢氧化钠水溶液于25度处理0.5小时，后用盐酸中和至中性，再依次用2mM二硫苏糖醇于37度处理1小时、10mM N-乙基马来酰亚胺于37度处理1小时，最后用Amicon超滤器再脱盐。所得蛋白质在pH 8.3的Tris-盐酸缓冲液中被胰酶降解，然后用于液相色谱-串联质谱分析多肽碎片中哪个氨基酸被序列X共价反应过，测得结果为新冠病毒刺突蛋白的Y421和K458氨基酸残基。
(4)通过对序列X进行一系列的类似序列转变后，发现其共价缀合新冠病毒刺突蛋白的活性在许多序列X的类似序列中仍旧存在。如图4所示，对序列X进行的突变得到的类似序列主要包括茎环结构的茎和环两类，这些序列中有些活性降低，有些活性保留，表明序列X存在一定的保守结构以支撑其共价缀合新冠病毒刺突蛋白的能力。基于此，根据发生突变位点的重要性和推测的序列X折叠结构，归纳其可以具备新冠病毒刺突蛋白共价抑制活性的最小特征序列为：
5′-Z 1Z 2Z 3Z 4Z 5Z 6Z 7Z 8Z 9Z 10Z 11Z 12Z 13Z 14Z 15Z 16Z 17Z 18-3′   式(I)
其中，Z 8、Z 9、Z 10中至少两个为共价反应修饰的硫代磷酸酯核苷酸；各自独立地选自A、T、C或G中的一种；所述共价反应修饰为采用未取代或任选取代的如下基团进行修饰：磺酰氟基、硫酰氟基、磺酰胺基、磺酸酯基、α,β-不饱和羰基或氟取代苯基；
Z 4、Z 5、Z 11、Z 12、Z 15为各自独立的A、T、C、G或无碱基；
Z 1、Z 2、Z 3、Z 6、Z 7和Z 18、Z 17、Z 16、Z 14、Z 13分别选自A、T、C、G，且Z 1与Z 18形成碱基对，Z 2与Z 17形成碱基对，Z 3和Z 16形成碱基对，Z 6与Z 14形成碱基对，Z 7和Z 13形成碱基对。
举例如，5′-GGY 3Z 4Z 5GCZ 8Z 9Z 10Z 11Z 12GCZ 15GCY 18-3′
其中加粗的Z 4、Z 5、Z 11、Z 12、Z 15可以选自A、T、G、C或无碱基；
其中Z 8、Z 9、Z 10为3个连续的含有5′端未取代或任选取代的磺酰氟修饰硫代磷酸酯的核苷酸，碱基可以选自A、T、G、C；Y 3、Y 18可以选自T、C。
(5)使用成熟的新冠假病毒抑制活力试验方法(参见Nie,J.H.et al.Quantification of SARS-CoV-2 neutralizing antibody by a pseudotyped virus-based assay.Nat.Protoc.15,3699-3715(2020))，具体为：使用的新冠假病毒来源于义翘神州(批号PSV001)，使用浓度为10 8～10 9拷贝每毫升，培养基为Dulbecco改良Eagle培养基(含有10％小牛血清蛋白)。将序列X或其无磺酰氟修饰的序列X’与新冠假病毒作用3小时后(序列X和序列X’的试验浓度范围为0.025nM至2000nM)，用于感染表达受体蛋白的HEK 293T细胞6个小时，然后更换HEK 293T细胞除去假病毒，再培养48小时候，裂解细胞，用碧云天的RG055试剂盒对荧光素酶活性进行定量，检测仪器为96孔板酶标仪。新冠假病毒感染约多，将带入更多表达荧光素酶的RNA到HEK 293T细胞中，将产生更强的荧光素酶活性信号，即序列X对新冠假病毒的抑制作用将减少细胞裂解后的荧光素酶活性。通过上述假病毒抑制活力测试(图8所示)，得到序列X对新冠假病毒感染宿主细胞的半抑制浓度为0.95nM，而缺少共价反应化学修饰的序列X’半抑制浓度为110nM，表明共价化学修饰产生的共价抑 制效果对序列X抑制新冠假病毒活力的重要作用。因此，序列X能够抑制新冠病毒假病毒对表达受体蛋白的HEK 293T细胞的感染，半抑制浓度达到0.95nM。
在本公开中，术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本公开的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不必须针对的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外，在不相互矛盾的情况下，本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。
尽管上面已经示出和描述了本公开的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本公开的限制，本领域的普通技术人员在本公开的范围内可以对上述实施例进行变化、修改、替换和变型。

Claims (30)

  1. 共价抑制剂的体外筛选方法，包括如下步骤：
    (1)构建硫代磷酸酯寡核苷酸文库；(2)将所述硫代磷酸酯寡核苷酸文库与至少一种共价反应修饰试剂接触，构建共价反应修饰寡核苷酸文库；所述共价反应修饰试剂包括至少一个共价反应修饰基团，所述共价反应修饰基团适于与靶蛋白共价结合；(3)将所述共价反应修饰寡核苷酸文库与靶蛋白接触，分离并收集与靶蛋白结合的共价反应修饰寡核苷酸，再脱除靶蛋白和共价反应修饰基团，构建次级文库；(4)以所述次级文库为模板，重复步骤(1)-(3)，多轮循环后，对最后一轮筛选得到的文库进行测序分析，筛选得到共价抑制剂。
  2. 根据权利要求1所述的体外筛选方法，其中所述构建硫代磷酸酯寡核苷酸文库的步骤以核苷三磷酸混合物为底物，且底物中至少包括一种核苷硫代三磷酸，采用核酸聚合酶进行扩增反应。
  3. 根据权利要求1或2所述的体外筛选方法，其中所述构建硫代磷酸酯寡核苷酸文库的初始文库为随机文库，以所述随机文库为模板，以dNTPs为底物，且所述底物中至少一种单体为脱氧核苷硫代三磷酸，在DNA聚合酶的作用下，进行PCR扩增反应。
  4. 根据权利要求3所述的体外筛选方法，其中所述脱氧核苷硫代三磷酸选自5′-脱氧腺苷[α-硫代]-三磷酸、5′-脱氧鸟苷[α-硫代]-三磷酸、5′-脱氧胸苷[α-硫代]-三磷酸、5′-脱氧胞苷[α-硫代]-三磷酸及其类似物、衍生物中的任一种。
  5. 根据权利要求1-4中任一项所述的体外筛选方法，其中所述共价反应修饰基团为未取代或任选取代的如下基团：磺酰氟基、硫酰氟基、磺酰胺基、磺酸酯基、α,β-不饱和羰基或氟取代苯基。
  6. 根据权利要求1-5中任一项所述的体外筛选方法，其中所述共价反应修饰基团选自以下结构：
    Figure PCTCN2022100847-appb-100001
    其中R 1为-H、-OH、-NO 2、-COOH、卤素或-R’；
    R 2为-H、-OH、-R’或-C(＝O)R’；
    R 3为-H或-R’；
    R 4为-H或-R’；
    n为1或2或3或4或5；
    R’为C 1-C 12烷基、C 1-C 12烷氧基、C 2-C 12烯基、C 2-C 12炔基、C 3-C 12环烷基、C 3-C 12环烯基、C 6-C 12芳基或C 5-C 12杂芳基；
    卤素为F、Cl、Br或I。
  7. 根据权利要求1-6中任一项所述的体外筛选方法，其中所述共价反应修饰试剂还包括一个能与硫代磷酸酯基反应的官能团，所述官能团为未取代或任选取代的如下基团：卤素亚甲基或卤素羧乙基，所述卤素为F、Cl、Br或I。
  8. 根据权利要求7所述的体外筛选方法，其中所述能与硫代磷酸酯基反应的官能团选自以下结构：
    Figure PCTCN2022100847-appb-100002
    其中R 5、R 6独立地为H、C 1-C 12烷基、C 1-C 12烷氧基、C 2-C 12烯基、C 2-C 12炔基、C 3-C 12环烷基、C 3-C 12环烯基、C 6-C 12芳基或C 5-C 12杂芳基；R 7为Cl、Br或I。
  9. 根据权利要求1-8中任一项所述的体外筛选方法，其中所述重复步骤(1)-(3)的重复次数为1-25次。
  10. 根据权利要求1-9中任一项所述的体外筛选方法，其中步骤(3)中将靶蛋白结合的共价反应修饰寡核苷酸与碱性溶液接触，脱除靶蛋白和共价反应修饰基团。
  11. 根据权利要求1-10中任一项所述的体外筛选方法，其中所述靶蛋白为疾病相关蛋白或药物蛋白。
  12. 共价抑制剂，由权利要求1-11中任一项所述的方法筛选得到，所述共价抑制剂至少包含二个共价反应修饰基团；所述共价反应修饰基团为未取代或任选取代的如下基团：磺酰氟基、硫酰氟基、磺酰胺基、磺酸酯基、α,β-不饱和羰基或氟取代苯基。
  13. 根据权利要求12所述的共价抑制剂，其中所述共价反应修饰基团选自以下结构：
    Figure PCTCN2022100847-appb-100003
    其中R 1为-H、-OH、-NO 2、-COOH、卤素或-R’；
    R 2为-H、-OH、-R’或-C(＝O)R’；
    R 3为-H或-R’；
    R 4为-H或-R’；
    n为1或2或3或4或5；
    R’为C 1-C 12烷基、C 1-C 12烷氧基、C 2-C 12烯基、C 2-C 12炔基、C 3-C 12环烷基、C 3-C 12环烯基、C 6-C 12芳基或C 5-C 12杂芳基；
    卤素为F、Cl、Br或I。
  14. 寡核苷酸分子，包含如下式(I)的序列；
    5′-Z 1Z 2Z 3Z 4Z 5Z 6Z 7Z 8Z 9Z 10Z 11Z 12Z 13Z 14Z 15Z 16Z 17Z 18-3′  式(I)
    其中，Z 8、Z 9、Z 10中至少两个为共价反应修饰的硫代磷酸酯核苷酸；各自独立地选自A、T、C或G中的一种；所述共价反应修饰为采用未取代或任选取代的如下基团进行修饰：磺酰氟基、硫酰氟基、磺酰胺基、磺酸酯基、α,β-不饱和羰基或氟取代苯基；
    Z 4、Z 5、Z 11、Z 12、Z 15为各自独立的A、T、C、G或无碱基；
    Z 1、Z 2、Z 3、Z 6、Z 7和Z 18、Z 17、Z 16、Z 14、Z 13分别选自A、T、C、G，且Z 1与Z 18形成碱基对，Z 2与Z 17形成碱基对，Z 3和Z 16形成碱基对，Z 6与Z 14形成碱基对，Z 7和Z 13形成碱基对。
  15. 根据权利要求14所述的寡核苷酸分子，其中所述共价反应修饰为苯磺酰氟基修饰、苯硫酰氟基修饰或五氟苯基修饰。
  16. 根据权利要求14或15所述的寡核苷酸分子，其中Z 1、Z 2、Z 3、Z 6、Z 7和Z 18、Z 17、Z 16、Z 14、Z 13分别选自C、G，或者是G、T，并且其中C和G形成碱基对，G和T形成碱基对。
  17. 根据权利要求14-16中任一项所述的寡核苷酸分子，其中所述寡核苷酸分子在式(I)结构的5′端和/或3′端至少还包括一个碱基，所述碱基各自独立地选自A、T、C或G中的一种。
  18. 根据权利要求14-17中任一项所述的寡核苷酸分子，其中所述寡核苷酸分子进一步被修饰以包含至少一种化学修饰，其中所述化学修饰是在选自寡核苷酸分子的糖位置、磷酸位置和/或碱基位置的位置处的化学取代。
  19. 根据权利要求14-18中任一项所述的寡核苷酸分子，包含如下的核苷酸序列；
    5′-GGCTTGCA*A*A*GGGCTGCT-3′；(SEQ ID NO：1)
    5′-GGTTTGCA*A*A*GGGCTGCT-3′；(SEQ ID NO：2)
    5′-GGCTTGCA*A*A*GGGCTGCC-3′；(SEQ ID NO：3)
    5′-GGCTTGCT*T*T*GGGCTGCT-3′。(SEQ ID NO：4)
    5′-TGGCTTGCA*A*A*GGGCTGCTG-3′；(SEQ ID NO：5)
    5′-TGGTTTGCA*A*A*GGGCTGCTG-3′；(SEQ ID NO：6)
    5′-TGGCTTGCA*A*A*GGGCTGCCG-3′；(SEQ ID NO：7)
    5′-TGGCTTGCT*T*T*GGGCTGCTG-3′；(SEQ ID NO：8)
    5′-TGGCTTGCA*A*AGGGCTGCTG-3′；(SEQ ID NO：9)
    5′-TGGCTTGCA*AA*GGGCTGCTG-3′；(SEQ ID NO：10)或
    5′-TGGCTTGCAA*A*GGGCTGCTG-3′；(SEQ ID NO：11)
    其中，A *、T *分别为未取代或任选取代的磺酰氟基修饰的硫代磷酸酯腺苷、胸苷。
  20. 根据权利要求14-19中任一项所述的寡核苷酸分子，其中所述寡核苷酸分子还包括以下四种序列中的任意一种：
    (1)与式(I)的核苷酸序列的同源性在80％以上；
    (2)与式(I)的核苷酸序列进行杂交的序列；
    (3)式(I)的核苷酸序列转录的RNA序列；
    (4)与式(I)的核苷酸序列相比具有一个或多个核苷酸的替换、缺失和/或添加。
  21. 载体，包含如权利要求14-20中任一项所述的寡核苷酸分子。
  22. 根据权利要求21所述的载体，其中所述载体是表达载体。
  23. 核酸分子，由如权利要求14-20中任一项所述的寡核苷酸分子编码。
  24. 宿主细胞，包含如权利要求23所述的核酸分子。
  25. 药物组合物，包含如权利要求14-20中任一项所述寡核苷酸分子、权利要求21或22所述的载体、权利要求23所述的核酸分子、或权利要求24所述的宿主细胞。
  26. 根据权利要求25所述的药物组合物，还包括化学治疗剂。
  27. 权利要求14-20中任一项所述的寡核苷酸分子、权利要求21或22所述的载体、权利要求23所述的核酸分子、权利要求24所述的宿主细胞或者权利要求25所述的药物组合物在制备药物中的用途，其中所述药物用于靶向新冠病毒刺突蛋白。
  28. 一种预防和/或治疗与新冠病毒刺突蛋白表达相关的疾病的方法，包括给患者施用治疗有效量的权利要求14-20中任一项所述的寡核苷酸分子、权利要求21或22所述的载体、权利要求23所述的核酸分子、权利要求24所述的宿主细胞或者权利要求25所述的药物组合物。
  29. 权利要求14-20中任一项所述的寡核苷酸分子、权利要求21或22所述的载体、权利要求23所述的核酸分子、权利要求24所述的宿主细胞或者权利要求25所述的药物组合物用于作为药物使用。
  30. 权利要求14-20中任一项所述的寡核苷酸分子、权利要求21或22所述的载体、权利要求23所述的核酸分子、权利要求24所述的宿主细胞或者权利要求25所述的药物组合物用于在靶向新冠病毒刺突蛋白中使用。
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