CN114990122A

CN114990122A - In vitro screening method of covalent inhibitor and application thereof

Info

Publication number: CN114990122A
Application number: CN202210542182.XA
Authority: CN
Inventors: 向宇; 童爱军; 秦梓宸; 张恺宁
Original assignee: Tsinghua University
Current assignee: Tsinghua University
Priority date: 2022-05-18
Filing date: 2022-05-18
Publication date: 2022-09-02
Also published as: WO2023221234A1

Abstract

The invention discloses an in vitro screening method of a covalent inhibitor and application thereof, wherein the method comprises the following steps: constructing a phosphorothioate oligonucleotide library; contacting the phosphorothioate oligonucleotide library with at least one covalent reaction modifying reagent to construct a covalent reaction modified oligonucleotide library; the covalent reactive modification reagent comprises at least one covalent reactive modification group, the covalent reactive modification group is suitable for being covalently combined with the target protein; contacting the covalent reaction modified oligonucleotide library with target protein, separating and collecting the covalent reaction modified oligonucleotide combined with the target protein, and then removing the target protein and covalent reaction modified groups to construct a secondary library; and (3) repeating the steps by taking the secondary library as a template, and after multiple cycles, performing sequencing analysis on the library obtained by the last round of screening to obtain the covalent inhibitor by screening. The invention realizes the in vitro screening technology compatible with covalent reaction modification for the first time, thereby developing the covalent inhibitor aiming at any one target protein.

Description

In vitro screening method of covalent inhibitor and application thereof

Technical Field

The invention belongs to the technical field of biology, and relates to an in vitro screening method of a covalent inhibitor and application thereof; specifically, the screening method of the invention can be compatible with covalent reaction modification, and by the method, the screening and discovery of the covalent inhibitor candidate drug molecules can be carried out aiming at various drug target proteins; the invention also provides a nucleotide molecule for covalently inhibiting the spike protein of the new coronavirus, which is obtained by applying the screening method.

Background

Proteins associated with diseases are important drug targets, for example, proteins of PD-1/PD-L1 protein interaction targeted by cancer immune checkpoint therapy, spike protein (SARS-CoV-2spike protein, S protein) for recognizing host cells by a novel coronavirus, and the like. Monoclonal antibodies (mabs) have been approved as drugs for drug targets of these proteins, and their mechanism of action is that the mabs bind to target proteins non-covalently, thereby inhibiting the function of the target proteins. However, monoclonal antibodies are expensive. For example, new coronavirus spike protein neutralizing antibody therapy against severe new corona virus is up to a cost of hundreds of thousands or even hundreds of thousands at a time. The development of high-efficiency medicaments with lower price has important significance. Although chemically synthesized polypeptides and short-chain nucleic acid drug molecules are relatively inexpensive, they have significantly lower affinity for target proteins than monoclonal antibodies based on non-covalent binding, and suffer from disadvantages such as rapid renal clearance in vivo.

Disclosure of Invention

To this end, it is an object of embodiments of the present invention to provide in vitro methods for screening for covalent inhibitors that allow for the design and development of covalent inhibitors for a variety of target proteins.

Another object of the embodiments of the present invention is to obtain a covalent inhibitor by screening the new coronavirus spike protein as a target protein by the above method, which can covalently inhibit the activity of the new coronavirus spike protein.

The embodiment of the invention provides an in vitro screening method of a covalent inhibitor, which comprises the following steps:

(1) constructing a phosphorothioate oligonucleotide library; (2) contacting the phosphorothioate oligonucleotide library with at least one covalent reaction modifying reagent to construct a covalent reaction modified oligonucleotide library; the covalent reactive modification reagent comprises at least one covalent reactive modification group adapted to covalently bind to a target protein; (3) contacting the covalent reaction modified oligonucleotide library with a target protein, separating and collecting the covalent reaction modified oligonucleotide combined with the target protein, and then removing the target protein and a covalent reaction modified group to construct a secondary library; (4) and (3) repeating the steps (1) to (3) by taking the secondary library as a template, and after multiple cycles, performing sequencing analysis on the library obtained by the last round of screening, and screening to obtain the covalent inhibitor.

In some embodiments, the step of constructing a library of phosphorothioate oligonucleotides uses a mixture of nucleoside triphosphates as substrates, and at least one nucleoside phosphothioate is included in the substrates, and the amplification reaction is performed using a nucleic acid polymerase.

In some embodiments, the nucleoside thiotriphosphate is a nucleoside [ α -thio ] -triphosphate.

In some embodiments, the constructing of the initial library of phosphorothioate oligonucleotide library is a random library, the random library is used as a template, dNTPs are used as substrates, and at least one monomer in the substrates is deoxynucleoside thiophosphate, and PCR amplification reaction is performed under the action of DNA polymerase.

In some embodiments, the deoxynucleoside thiophosphate is selected from any one of 5 '-deoxyadenosine [ α -thio ] -triphosphate, 5' -deoxyguanosine [ α -thio ] -triphosphate, 5 '-deoxythymidine [ α -thio ] -triphosphate, 5' -deoxycytidine [ α -thio ] -triphosphate and analogs, derivatives thereof.

In some embodiments, the covalent reactive modifying group is an unsubstituted or optionally substituted group selected from the group consisting of: sulfonyl fluoride, sulfonamide, sulfonate, alpha, beta-unsaturated carbonyl, or fluoro substituted phenyl.

In some embodiments, the covalently reactive modifying group is selected from the following structures:

wherein R is ₁ is-H, -OH, -NO ₂ -COOH, halogen or-R';

R ₂ is-H, -OH, -R 'or-C (═ O) R';

R ₃ is-H or-R';

R ₄ is-H or-R';

n is 1 or 2 or 3 or 4 or 5;

r' is C ₁ -C ₁₂ Alkyl radical, C ₁ -C ₁₂ Alkoxy radical, C ₂ -C ₁₂ Alkenyl radical, C ₂ -C ₁₂ Alkynyl, C ₃ -C ₁₂ Cycloalkyl radical, C ₃ -C ₁₂ Cycloalkenyl radical, C ₆ -C ₁₂ Aryl or C ₅ -C ₁₂ A heteroaryl group;

halogen is F, Cl, Br or I.

In some embodiments, the covalent reaction modifying reagent further comprises a functional group capable of reacting with a phosphorothioate group, the functional group being unsubstituted or optionally substituted as follows: halogen methylene or halogen carboxyethyl, wherein the halogen is F, Cl, Br or I.

In some embodiments, the functional group capable of reacting with a phosphorothioate group is selected from the following structures:

wherein R is ₅ 、R ₆ Independently H, C ₁ -C ₁₂ Alkyl radical, C ₁ -C ₁₂ Alkoxy radical, C ₂ -C ₁₂ Alkenyl radical, C ₂ -C ₁₂ Alkynyl, C ₃ -C ₁₂ Cycloalkyl radical, C ₃ -C ₁₂ Cycloalkenyl radical, C ₆ -C ₁₂ Aryl or C ₅ -C ₁₂ A heteroaryl group; r ₇ Is Cl, Br or I.

The number of repetition of the repeating steps (1) - (3) is 1-25.

In some embodiments, the target protein-bound covalently-modified oligonucleotide of step (3) is contacted with an alkaline solution to remove the target protein and the covalently-modified group.

In some embodiments, the target protein is a disease-associated protein or a pharmaceutical protein.

In another aspect, the embodiments of the present invention further provide a covalent inhibitor, wherein the covalent inhibitor is screened by the method of any one of claims 1 to 11, and comprises at least two covalent reactive modification groups; the covalent reaction modification group is the following unsubstituted or optionally substituted group: sulfonyl fluoride, sulfonamide, sulfonate, alpha, beta-unsaturated carbonyl, or fluoro substituted phenyl.

In some embodiments of the covalent inhibitor, the covalent reactive modifying group is selected from the following structures:

wherein R is ₁ is-H, -OH, -NO ₂ -COOH, halogen or-R';

R ₂ is-H, -OH, -R 'or-C (═ O) R';

R ₃ is-H or-R';

R ₄ is-H or-R';

n is 1 or 2 or 3 or 4 or 5;

halogen is F, Cl, Br or I.

On the other hand, the embodiment of the invention also provides a covalent inhibitor which is obtained by screening the new coronavirus spike protein serving as the target protein and adopting the screening method and aims at the new coronavirus spike protein. Namely:

the embodiments of the present invention provide nucleotide molecules, including sequences of the following formula (I);

5′-Z ₁ Z ₂ Z ₃ Z ₄ Z ₅ Z ₆ Z ₇ Z ₈ Z ₉ Z ₁₀ Z ₁₁ Z ₁₂ Z ₁₃ Z ₁₄ Z ₁₅ Z ₁₆ Z ₁₇ Z ₁₈ -3' formula (I)

Wherein, Z ₈ 、Z ₉ 、Z ₁₀ At least two of which are covalently reaction-modified phosphorothioate nucleotides; each independently selected from A, T, C or G; the covalent reaction is modified by adopting the following unsubstituted or optionally substituted groups: sulfonyl fluoride, sulfonamide, sulfonate, alpha, beta-unsaturated carbonyl, or fluoro-substituted phenyl;

Z ₄ 、Z ₅ 、Z ₁₁ 、Z ₁₂ 、Z ₁₅ a, T, C, G or no base independently of each other;

Z ₁ 、Z ₂ 、Z ₃ 、Z ₆ 、Z ₇ and Z ₁₈ 、Z ₁₇ 、Z ₁₆ 、Z ₁₄ 、Z ₁₃ Are each selected from A, T, C, G, and Z ₁ And Z ₁₈ Form base pairs, Z ₂ And Z ₁₇ Form base pairs, Z ₃ And Z ₁₆ Form base pairs, Z ₆ And Z ₁₄ Formation of base pairs, Z ₇ And Z ₁₃ Base pairs are formed.

In some embodiments of the nucleotide molecule, the covalent reactive modification is a phenylsulfonyl fluoride modification, a phenylthio fluoride modification, or a pentafluorophenyl modification.

In some embodiments of the nucleotide molecule, Z ₁ 、Z ₂ 、Z ₃ 、Z ₆ 、Z ₇ And Z ₁₈ 、Z ₁₇ 、Z ₁₆ 、Z ₁₄ 、Z ₁₃ Are independently selected from C, G, or G, T, and wherein C and G form base pairs and G and T form base pairs.

In some embodiments of the nucleotide molecule, the nucleotide molecule further comprises at least one base at the 5 'end and/or the 3' end of the structure of formula (I), each of the bases is independently selected from A, T, C or G.

In some embodiments of the nucleotide molecule, the nucleotide molecule is further modified to comprise at least one chemical modification, wherein the chemical modification is a chemical substitution at a position selected from the group consisting of a sugar position, a phosphate position, and/or a base position of the nucleotide molecule.

In some embodiments of the nucleotide molecule, the nucleotide molecule comprises a nucleotide sequence as follows;

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) or

5′-TGGCTTGCAA*A*GGGCTGCTG-3′；(SEQ ID NO：11)。

Wherein A is ^* 、T ^* Respectively, unsubstituted or optionally substituted sulfonyl fluoride modified phosphorothioate adenosine and thymidine.

In some embodiments of the nucleotide molecule, the nucleotide molecule further comprises any one of the following four sequences:

(1) the homology with the nucleotide sequence of the formula (I) is more than 80 percent;

(2) a sequence which hybridizes to a nucleotide sequence of formula (I);

(3) an RNA sequence transcribed from a nucleotide sequence of formula (I);

(4) having substitutions, deletions and/or additions of one or more nucleotides compared to the nucleotide sequence of formula (I).

The embodiment of the invention also provides a carrier, which comprises the nucleotide molecule.

In some embodiments, the vector is an expression vector.

The embodiment of the invention also provides a nucleic acid molecule which is coded by the nucleotide molecule.

Embodiments of the invention also provide host cells comprising the nucleic acid molecules described above.

The embodiment of the invention also provides a pharmaceutical composition, which comprises the nucleotide molecule, the vector, the nucleic acid molecule or the host cell.

In some embodiments, the pharmaceutical composition further comprises a chemotherapeutic agent.

The embodiment of the invention also provides the application of the nucleotide molecule, the vector, the nucleic acid molecule, the host cell or the pharmaceutical composition in preparing a medicine for targeting the new coronavirus spike protein.

The embodiment of the invention also provides a method for preventing and/or treating diseases related to the expression of the novel coronavirus spike protein, which comprises the step of administering a therapeutically effective amount of the nucleotide molecule, the vector, the nucleic acid molecule, the host cell or the pharmaceutical composition to a patient.

The embodiment of the invention has the following beneficial effects:

(1) the screening method provided by the embodiment of the invention overcomes the defects that all covalent inhibitors of target proteins in the related technology are obtained by structural-guided rational design and development, the types of the target proteins capable of being targeted are limited, and the activity effect of the obtained covalent inhibitors is limited by the performance of the used non-covalent inhibitors. The invention realizes the in vitro screening technology compatible with covalent reaction modification for the first time, so that the covalent inhibitor can be developed aiming at any target protein, and the obtained high-activity covalent inhibitor has huge potential based on large-scale random molecular library screening.

(2) Based on the fact that the novel coronavirus spike protein inhibitor in the related technology before the invention is based on non-covalent, the invention discovers the covalent inhibitor capable of covalently inhibiting the novel coronavirus spike protein with high activity for the first time.

Drawings

The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:

FIG. 1 is a schematic diagram of an in vitro method for screening for covalent inhibitors in accordance with an embodiment of the present invention;

FIG. 2 is a schematic representation of the nucleophilic substitution reaction of a phosphorothioate structure with a covalent chemical modifier to covalently modify an oligonucleotide, in accordance with embodiments of the present invention;

FIG. 3 is a urea gel electrophoresis of the primer materials (left) and product oligonucleotides (right) of the polymerase chain reaction of the embodiment of the present invention;

FIG. 4 is a schematic diagram of the sequence of sequence X and mutations of M1-M8 of sequence X in the examples of the present invention;

FIG. 5 is a SDS-PAGE picture of the inventive sequence X and its mutants reacted with the novel crown spike protein; in FIG. 5

1. A novel coronatine protein; 2. reacting the new coronary spike protein with the sequence X; 3. the new crown spike protein reacts with M5 mutation of sequence X; 4-6, reacting the new crown spike protein with a sequence X only containing 1 benzenesulfonyl fluoride modification; 7-9, reacting the new coronatine protein with a sequence X only containing 2 benzenesulfonyl fluoride modifications; 10. reacting the new coronary spike protein with the sequence X; 11. the new crown spike protein reacts with M1 mutation of sequence X; 12. the new crown spike protein reacts with M2 mutation of sequence X; 13. the new crown spike protein reacts with M3 mutation of sequence X; 14. the new crown spike protein reacts with M4 mutation of sequence X; 15. the new crown spike protein reacts with M6 mutation of sequence X; 16. the new crown spike protein reacts with M7 mutation of sequence X; 17. the new crown spike protein reacts with M8 mutation of sequence X;

FIG. 6 is a liquid chromatography-tandem mass spectrometry analysis of the conjugation of sequence X + TG to the new coronavirus spike protein of the examples of the invention; figure 6 shows that one amino acid position of the covalently conjugated new coronavirus spike protein of sequence X + TG is K458 amino acid residue of the new coronavirus spike protein;

FIG. 7 is a liquid chromatography-tandem mass spectrometry analysis of the conjugation of sequence X + TG to the new coronavirus spike protein of the examples of the invention; FIG. 7 shows another amino acid position of the covalently conjugated new coronavirus spike protein of the sequence X + TG as Y421 amino acid residue of the new coronavirus spike protein;

FIG. 8 shows the inhibitory activity of sequence X, sequence X' on the novel coronavirus according to the example of the present invention.

Detailed Description

Proteins associated with diseases are important drug targets, for example, proteins of PD-1/PD-L1 protein interaction targeted by cancer immune checkpoint therapy, spike protein (SARS-CoV-2spike protein, S protein) for recognizing host cells by a novel coronavirus, and the like. Monoclonal antibodies (mabs) have been approved as drugs for drug targets of these proteins, and their mechanism of action is that the mabs bind to target proteins non-covalently, thereby inhibiting the function of the target proteins. However, monoclonal antibodies are expensive. For example, the cost of a therapy with neutralizing antibodies against the spike protein of new coronavirus in severe new coronary disease is as high as one treatment of more than ten thousand or even hundreds of thousands. The development of high-efficiency medicaments with lower price has important significance. Although chemically synthesized polypeptides and short-chain nucleic acid drug molecules are relatively inexpensive, they have significantly lower affinity for target proteins than monoclonal antibodies based on non-covalent binding, and suffer from disadvantages such as rapid renal clearance in vivo.

In the related art, studies on covalent inhibitors of target proteins involve non-covalent binding of target proteins and then formation of covalent bonds with target proteins through inert reactive functional groups contained in the molecules, thereby inhibiting target proteins with high specificity and permanence. These covalent inhibitors against the target protein have two significant advantages over traditional monoclonal antibody inhibitors: firstly, the high specificity is permanently inhibited, the inhibition ability is close to or surpasses that of monoclonal antibody, and the activity is not influenced by the negative effect of rapid renal clearance; secondly, since these covalent inhibitors are typically only one-tenth the molecular weight of the mab or less, the dosage used is significantly less than that of the mab.

Nevertheless, all known methods for the development of covalent inhibitors of target proteins have been Structure-guided rational design. This development approach requires explicit protein interaction structural information and relies on trial and error patterns to find covalently assault amino acid residues on the target protein, thus leading to an extremely limited variety of proteins targeted by current covalent inhibitors. There is still a lack of new drug discovery techniques to develop covalent inhibitors against various target proteins.

Conventional In vitro screening techniques (In vitro selection) are a method for finding short-chain nucleic acid sequences that non-covalently bind to target proteins, and although the principles can be applied to the development of non-covalent mechanism inhibitors for various target proteins, the screening for covalent inhibitors is still impossible. The reason is that covalent reactive chemical modification will inhibit the polymerase involved in vitro screening techniques and the conjugate of the short chain nucleic acid sequence of the covalent inhibitor and the target protein cannot be sequenced to obtain sequence information.

The information disclosed in this background section is only for enhancement of understanding of the general background of the invention and should not be taken as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person skilled in the art.

The following are terms or word descriptions and, unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by one of skill in the art to which this invention belongs.

In this document, the words "comprise" and "include" and variations thereof mean that there may be additional elements or integers which are permissible but which are not specifically described.

Herein, the term: "covalent reactive modification", also known as: "covalent warhead modification"; refers to chemical modifications that can be used to covalently bind to a target protein of interest.

The terms: "covalent bonding" refers to the formation of a chemical bond, i.e., a bond between atoms formed by a common pair of electrons.

The term "amplifying" refers to any means by which at least a partial sequence of at least one target nucleic acid or its sequence complement is produced, typically 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.

The term "nucleic acid polymerase" refers to an enzyme that catalyzes the incorporation of nucleotides into nucleic acids, including DNA polymerases, RNA polymerases, reverse transcriptases, and the like.

The term "in vitro screening" refers to the repeated screening of a random library of nucleic acids outside the cell to obtain molecules with a particular activity.

The term "nucleotide" generally refers to a compound formed by linking a nucleoside to an acidic molecule or group via an ester linkage, such as a phosphate ester of a nucleoside, typically having one, two or three phosphate groups covalently attached at the 5-position of the sugar group of the nucleoside. In some cases, the definition of nucleotide also includes analogs of some nucleotides or derivatives thereof. Analogs refer to compounds that are structurally similar to naturally occurring nucleotides, non-limiting examples of which include: guanosine oxide, pseudouridine, isocytidine, isoguanosine, and the like; derivatives refer to compounds having modified nucleotide base moieties, modified pentose moieties and/or modified phosphate moieties, exemplary modified phosphate moieties include by way of non-limiting example: phosphorothioate, phosphorodithioate, selenophosphate, diselenoate phosphate, phosphoroanilidate phosphate thiolate, phosphoroanilidate, phosphoramidate, and the like, and non-limiting examples of exemplary modified nucleotide base moieties include: 5-methylcytosine (5 mC); c-5 propynyl-C and C-5 propynyl-U; 2, 6-diaminopurine, hypoxanthine, pseudouridine, 2-thiopyrimidine, isocytosine (isoC), 5-methylisoc and isoguanine. Non-limiting examples of exemplary modified pentose moieties include: 2 '-or 3' -modifications, wherein the 2 '-or 3' -position is hydrogen, hydroxy, alkoxy (e.g., methoxy, ethoxy, isopropoxy, butoxy, isobutoxy), azido, amino, alkylamino, fluoro, chloro, or bromo.

For example, the term "nucleoside triphosphate" generally refers to a nucleoside having three phosphate groups covalently attached to the 5-position of the sugar group of the nucleoside, and in some cases, the definition of nucleoside triphosphate also includes analogs, derivatives of some typical nucleoside triphosphates.

The term "nucleoside [ α -thio ] -triphosphates" includes by way of non-limiting example: 5 '-deoxyadenosine [ alpha-thio ] -triphosphate, 5' -adenosine [ alpha-thio ] -triphosphate, 5 '-deoxyguanosine [ alpha-thio ] -triphosphate, 5' -guanosine [ alpha-thio ] -triphosphate, 5 '-deoxythymidine [ alpha-thio ] -triphosphate, 5' -thymidine [ alpha-thio ] -triphosphate, 5 '-deoxyuridine [ α -thio ] -triphosphate, 5' -uridine [ α -thio ] -triphosphate, 5 '-deoxycytidine [ α -thio ] -triphosphate, 5' -cytidine [ α -thio ] -triphosphate, 5 '-deoxyinosine [ α -thio ] -triphosphate, 5' -inosine [ α -thio ] -triphosphate; and their non-limiting examples are methylated, acylated, and halogenated derivatives; and non-limiting examples thereof: guanosine is replaced by guanosine oxide, uridine is replaced by pseudouridine, cytidine is replaced by isocytidine, and guanosine is replaced by isoguanosine.

The terms "nucleic acid", "short-chain nucleic acid", "polynucleotide", "oligonucleotide" and "oligonucleotide" are used interchangeably and refer to deoxyribonucleotide or ribonucleotide polymers in either linear or circular configuration and in either single-or double-stranded form, and can include known analogs of natural nucleotides, as well as nucleotides modified in base, sugar and/or phosphate moieties (e.g., phosphorothioate backbones). For the purposes of this disclosure, these terms should not be construed as limiting the length of the polymer. And for use as oligonucleotides of greater utility in the present invention, synthetic oligonucleotides of length generally ranging from 10 to 40 nucleotides, preferably 10 to 25 nucleotides, are referred to.

The term "sequence" refers to a nucleotide sequence of any length, which may be DNA or RNA. May be linear, circular or branched and may be single-stranded or double-stranded.

The term "homology" refers to sequence similarity to a target nucleic acid sequence. "homology" includes a nucleotide sequence having 80% or more, 85% or more, 90% or more, 95% or more homology with a target nucleic acid sequence or a core sequence thereof. Homology can be assessed visually or by computer software.

The term "label" refers to a moiety that is linked (covalently or non-covalently) to a nucleic acid molecule and is capable of providing information about the molecule, exemplary labels include fluorescent labels and the like.

The term "new coronavirus spike protein" refers to a protein located in the trimeric structure of the outermost layer of new coronavirus, also called S protein, which is a structural protein derived from coronavirus including SARS-CoV-2.

The term "optionally substituted" means that one or more hydrogen atoms in a given structure are replaced with a particular substituent. Unless otherwise indicated, an optional substituent group may be substituted at each substitutable position of the group. When more than one position in a given formula can be substituted with one or more substituents selected from a particular group, the substituents may be substituted at each position, identically or differently. Non-limiting examples of substituents that are "optionally substituted" include, fluorine, chlorine, bromine, iodine, hydroxyl, nitro, amino, carboxyl, alkyl, alkoxy, alkenyl, alkynyl, aryloxy, heteroaryloxy, heterocyclyloxy, arylalkoxy, heteroarylalkoxy, heterocyclylalkoxy, cycloalkylalkoxy, alkylamino, alkylaminoalkyl, cycloalkylamino, cycloalkylalkylamino, haloalkyl, haloalkoxy, hydroxyl-substituted alkyl, hydroxyl-substituted alkylamino, amino-substituted alkyl, alkanoyl, heteroalkyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, heterocyclylacyl, aryl, arylalkyl, arylamino, heteroaryl, heteroarylalkyl, heteroarylamino, amide, sulfonyl, aminosulfonyl, and the like.

For example:

the term "alkyl" may be "C ₁ -C ₁₂ Alkyl groups ". ' C ₁ -C ₁₂ Alkyl "represents a saturated, straight or branched chain, monovalent hydrocarbon radical containing from 1 to 12 carbon atoms; in one embodiment, from 1 to 8 carbon atoms; in another embodiment, from 1 to 6 carbon atoms; in yet another embodiment, from 1 to 4 carbon atoms; and also in an implementationIn the scheme, 1 to 3 carbon atoms are contained. "C ₁ -C ₁₂ Non-limiting examples of 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-1-butyl, 2-methyl-1-butyl and the like.

The term "alkenyl" may be "C ₂ -C ₁₂ Alkenyl ". "C ₂ -C ₁₂ Alkenyl "means a straight or branched chain monovalent hydrocarbon radical containing 2 to 12 carbon atoms, wherein there is at least one site of unsaturation, i.e., a carbon-carbon sp2 double bond. In one embodiment, from 2 to 8 carbon atoms; in another embodiment, from 2 to 6 carbon atoms; in yet another embodiment, from 2 to 4 carbon atoms. "C ₂ -C ₁₂ Non-limiting examples of alkenyl groups include: vinyl, allyl, propenyl, and the like.

The term "alkynyl" may be "C ₂ -C ₁₂ Alkynyl ". "C ₂ -C ₁₂ Alkynyl "means a straight or branched chain monovalent hydrocarbon radical containing 2 to 12 carbon atoms, wherein there is at least one carbon-carbon sp triple bond.

The term "alkoxy" may be "C ₁ -C ₁₂ Alkoxy ". "C ₁ -C ₁₂ Alkoxy "denotes" C ₁ -C ₁₂ Alkyl "is attached to the rest of the molecule through an oxygen atom, wherein" C ₁ -C ₁₂ Alkyl "has the meaning as described herein. "C ₁ -C ₁₂ Non-limiting examples of alkoxy "include: methoxy, ethoxy, 1-propoxy, 2-propoxy, and the like.

The term "cycloalkyl" may be "C ₃ -C ₁₂ Cycloalkyl groups ". "C ₃ -C ₁₂ Cycloalkyl "denotes a monovalent or polyvalent saturated monocyclic, bicyclic or tricyclic ring system containing from 3 to 12 carbon atoms. In one embodiment, from 3 to 10 carbon atoms; in another embodiment, from 3 to 8 carbon atoms; in yet another embodiment, from 3 to 6 carbon atoms. "C ₃ -C ₁₂ Non-limiting examples of cycloalkyl "include: the alkyl group of the cyclopropyl group, the cyclobutyl group,cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, cycloundecyl, cyclododecyl and the like.

The term "cycloalkenyl" can be "C ₃ -C ₁₂ Cycloalkenyl groups ". "C ₃ -C ₁₂ Cycloalkenyl "denotes a mono-, bi-or tricyclic ring system containing 3 to 12 carbon atoms, mono-, or polyvalent, comprising at least one carbon-carbon double bond, and the ring system is nonaromatic. In one embodiment, from 3 to 10 carbon atoms; in another embodiment, from 3 to 8 carbon atoms; in yet another embodiment, from 3 to 6 carbon atoms. "C ₃ -C ₁₂ Non-limiting examples of cycloalkenyl "include: cyclobutenyl, cyclopentenyl, cyclohexenyl, cyclohexadienyl, and the like.

The term "aryl" may be "C ₆ -C ₁₂ Aryl ". "C ₆ -C ₁₂ Aryl "denotes monocyclic, bicyclic and tricyclic carbocyclic ring systems containing 6 to 12 ring atoms, wherein at least one ring system is aromatic and" C "is ₆ -C ₁₂ Non-limiting examples of aryl "include: phenyl, indenyl, naphthyl and the like.

The term "heteroaryl" may be "C ₅ -C ₁₂ Heteroaryl group ". "C ₅ -C ₁₂ Heteroaryl "denotes monocyclic, bicyclic and tricyclic ring systems containing 5 to 12 ring atoms, wherein at least one ring system is aromatic and at least one ring system contains one or more heteroatoms, selected from O, S, N, P or Si.

The following describes embodiments of the present invention in detail. The following examples are illustrative only and are not to be construed as limiting the invention.

As shown in fig. 1, the embodiment of the present invention provides an in vitro screening method of a covalent inhibitor, comprising the following steps:

(1) constructing a phosphorothioate oligonucleotide library; (2) contacting the phosphorothioate oligonucleotide library with at least one covalent reaction modifying reagent to construct a covalent reaction modified oligonucleotide library; the covalent reactive modification reagent comprises at least one covalent reactive modification group, the covalent reactive modification group is suitable for being covalently combined with the target protein; (3) contacting the covalent reaction modified oligonucleotide library with target protein, separating and collecting the covalent reaction modified oligonucleotide combined with the target protein, and then removing the target protein and covalent reaction modified groups to construct a secondary library; (4) and (3) repeating the steps (1) to (3) by taking the secondary library as a template, and after multiple cycles, performing sequencing analysis on the library obtained by the last round of screening, and screening to obtain the covalent inhibitor.

The in vitro screening method of the covalent inhibitor can obtain oligonucleotide molecules capable of covalently inhibiting the target protein; the oligonucleotide modified by covalent reaction is introduced after the amplification of nucleic acid polymerase and removed before the amplification of polymerase of the next cycle, so that the covalent reaction modification does not influence the nucleic acid polymerase reaction, the nucleic acid polymerase reaction is compatible with in vitro screening, the sequence information of the oligonucleotide capable of covalently inhibiting the target protein can be obtained by sequencing after multiple screening cycles, and the position information of covalent reaction modification contained in the oligonucleotide is determined by the type of the phosphorothioate nucleotide used. This method differs significantly from known aptamer screening techniques in that: covalent reaction modifications are introduced and it is ensured that the modifications are introduced and removed in appropriate steps so as not to interfere with the nucleic acid polymerase activity, making it possible to screen oligonucleotide sequences containing covalent reaction modifications that can covalently inhibit the target protein.

It can be understood that: the in vitro screening method of the covalent inhibitor can take the protein related to diseases as the target protein and is used for screening and discovering the candidate drug molecules of the covalent inhibitor. Meanwhile, the in vitro screening method of the covalent inhibitor can also use drug protein as target protein, such as monoclonal antibody, hormone drug (such as insulin) and other drugs with molecular form of protein, and modify the known protein drug.

In some embodiments, the PCR amplification reaction is performed by DNA polymerase using the initial library of phosphorothioate oligonucleotide libraries as a random library, the random library as a template, dNTPs as a substrate, and at least one monomer in the substrate as deoxynucleoside phosphothioate.

In some embodiments, the deoxynucleoside thiophosphoric acid is selected from any one of 5 '-deoxyadenosine [ α -thio ] -triphosphate, 5' -deoxyguanosine [ α -thio ] -triphosphate, 5 '-deoxythymidine [ α -thio ] -triphosphate, 5' -deoxycytidine [ α -thio ] -triphosphate, and analogs, derivatives thereof.

In some embodiments, the covalent reactive modifying group is an unsubstituted or optionally substituted group selected from: sulfonyl fluoride, sulfonyl fluoride (also known as fluorosulfate), sulfonamide, sulfonate, α, β -unsaturated carbonyl, or fluoro substituted phenyl.

In some embodiments, the covalent reactive modifying group is selected from the following structures:

wherein R is ₁ is-H, -OH, -NO ₂ -COOH, halogen or-R';

R ₂ is-H, -OH, -R 'or-C (═ O) R';

R ₃ is-H or-R';

R ₄ is-H or-R';

n is 1 or 2 or 3 or 4 or 5;

halogen is F, Cl, Br or I.

FIG. 2 shows a schematic example of a nucleophilic substitution reaction of a covalent chemical modification reagent with a phosphorothioate structure to covalently modify an oligonucleotide.

In some embodiments, the number of repetitions of steps (1) - (3) is 1-25.

In some embodiments, the pH of the alkaline solution is >9, further preferably, pH > 10; further preferably, pH > 11; further preferably, the pH is > 12.

In some embodiments, the alkaline solution is a NaOH solution or a KOH solution.

The embodiment of the invention also provides a covalent inhibitor, which is obtained by screening by the method, wherein the covalent inhibitor at least comprises two covalent reaction modifying groups; the covalent reactive modifying group is the following unsubstituted or optionally substituted group: sulfonyl fluoride, sulfonamide, sulfonate, alpha, beta-unsaturated carbonyl, or fluoro substituted phenyl.

wherein R is ₁ is-H, -OH, -NO ₂ -COOH, halogen or-R';

R ₂ is-H, -OH, -R 'or-C (═ O) R';

R ₃ is-H or-R';

R ₄ is-H or-R';

n is 1 or 2 or 3 or 4 or 5;

halogen is F, Cl, Br or I.

The embodiment of the invention also provides the nucleotide molecules which are obtained by screening through the in vitro screening method and covalently inhibit the spike protein of the new coronavirus.

The novel coronavirus spike protein of the embodiments of the invention is derived from the original strain of the novel coronavirus or a mutant strain thereof (e.g., Deltay, Omicronron).

Wherein Z is ₈ 、Z ₉ 、Z ₁₀ At least two of which are covalently reaction-modified phosphorothioate nucleotides; each independently selected from A, T, C or G; in totalThe valency reaction is modified with the following unsubstituted or optionally substituted groups: sulfonyl fluoride, sulfonamide, sulfonate, alpha, beta-unsaturated carbonyl, or fluoro-substituted phenyl;

Z ₁ 、Z ₂ 、Z ₃ 、Z ₆ 、Z ₇ and Z ₁₈ 、Z ₁₇ 、Z ₁₆ 、Z ₁₄ 、Z ₁₃ Are each selected from A, T, C, G, and Z ₁ And Z ₁₈ Form base pairs, Z ₂ And Z ₁₇ Form base pairs, Z ₃ And Z ₁₆ Formation of base pairs, Z ₆ And Z ₁₄ Formation of base pairs, Z ₇ And Z ₁₃ Forming a base pair.

In some embodiments of the nucleotide molecule, the covalent reaction modification is a phenylsulfonyl fluoro modification, a phenylthio fluoro modification, or a pentafluorophenyl modification.

In some embodiments of the nucleotide molecule, Z ₄ 、Z ₅ 、Z ₁₁ 、Z ₁₂ 、Z ₁₅ And do not participate in the formation of active secondary folding structures, either of which may be replaced with A, T, G, C or be free of bases. In some embodiments, Z ₄ Is T, and/or Z ₅ Is T, and/or Z ₁₁ Is G, and/or Z ₁₂ Is G, and/or Z ₁₅ Is T.

In some embodiments, Z ₁ 、Z ₂ 、Z ₃ 、Z ₆ 、Z ₇ And Z ₁₈ 、Z ₁₇ 、Z ₁₆ 、Z ₁₄ 、Z ₁₃ Each selected from C, G, or G, T, and wherein C and G form a base pair and G and T form a base pair.

In some embodiments, 5 'and/or 3' sequence extensions may be made on the basis of the above sequences, which naturally have a similar function as in formula (I), e.g.a T extension at the 5 'end and/or a G extension at the 3' end.

In some specific embodiments, the nucleotide molecule may further be provided with various chemical modifications and/or labels at the 5 'end and/or 3'; for example, a Cy3 fluorophore, a Cy5 fluorophore, a triethylene glycol, etc. is added to the 5 'end and/or the 3' end.

In some embodiments of the nucleotide molecule, the nucleotide molecule comprises a nucleotide sequence as follows:

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) or

5′-TGGCTTGCAA*A*GGGCTGCTG-3′；(SEQ ID NO：11)。

Wherein A is ^* 、T ^* Respectively unsubstituted or optionally substituted sulfonyl fluoride modified phosphorothioate adenosine and thymidine.

In some embodiments, A ^* 、T ^* Respectively, the phosphorothioate adenosine and thymidine modified by benzenesulfonyl fluoro group.

In some embodiments of the nucleotide molecule, the nucleotide molecule further comprises any one or more of:

changing cytosine C into 5-methylcytosine 5mC or 5-hydroxymethylcytosine 5hmC,

Or changing thymine T into uracil U or pseudouracil Ψ,

Or changing adenine A into 6-methyladenine 6mA,

Or changing guanine G to 8-hydroxyguanine 8-oxoG;

or replacing H of deoxyribose 2' with hydroxyl (OH), alkoxy (methoxy, ethoxy, etc.), fluorine (F),

or replacing the phosphodiester with a phosphorothioate diester.

(1) homology with the nucleotide sequence of the formula (I) is more than 80 percent;

(2) a sequence which hybridises to the nucleotide sequence of formula (I);

(3) an RNA sequence transcribed from the nucleotide sequence of formula (I).

In some embodiments, the vector is an expression vector.

The embodiment of the invention also provides a pharmaceutical composition, which comprises the nucleotide molecule, the vector, the nucleic acid molecule or the host cell; such as protein antigen vaccines.

The embodiment of the invention also provides the application of the nucleotide molecule, the vector, the nucleic acid molecule, the host cell or the pharmaceutical composition in preparing a medicament for targeting the new coronavirus spike protein.

Example 1 construction of phosphorothioate oligonucleotide library

(1) Polymerase chain reaction was performed using the following random library as template, where N ₃₃ 33 random nucleosides (A, T, G, C with a probability of 25% each):

TACACAGGGGAGTAACGAAT N ₃₃ TGGGTGTTTGTTCTTCTCT。(SEQ ID NO：12)

(2) the solution formula of the polymerase chain reaction is as follows: 645 μ L of water, 80 μ L of 10 XPCR buffer, 20 μ L of 10mM nucleoside triphosphate monomer mix (containing dATP-. alpha. -S (5' -deoxyadenosine (. alpha. -thio ] -triphosphate), dTTP, dGTP and dCTP), 40 μ L of 10 μ M primer, 5 μ L of hot start Taq DNA polymerase, 10 μ L of 1 μ M template. The two primers are as follows:

TACACAGGGGAGTAACGAAT；(SEQ ID NO：13)

AGAGAAGAACAAACACCCA。(SEQ ID NO：14)

(3) the steps of the polymerase chain reaction are: 30 seconds at 95 ℃ and 20 amplification cycles (each cycle comprising 20 seconds at 95 ℃, 20 seconds at 45 ℃ and 25 seconds at 68 ℃), 300 seconds at 68 ℃ and 4 ℃.

(4) The separation is performed by gel electrophoresis, as shown in FIG. 3, the left side is the PCR primer material, the right side is the product oligonucleotide urea gel electrophoresis, and the product purification separation is performed by gel cutting.

Example 2 construction of a library of covalently reactive modified oligonucleotides

In the phosphorothioate oligonucleotide library of example 1, covalent reaction modification was introduced by nucleophilic substitution reaction of phosphorothioate to form a covalent reaction modified oligonucleotide library;

(1) the reaction solution formula is as follows: mu.L of 10. mu.M phosphorothioate oligonucleotide library, 15. mu.L water, 10. mu.L of 100mM phosphate buffer solution pH 6, 50. mu.L of 30mM bromide in N, N-dimethylformamide. The bromide here is 4-bromomethylbenzenesulfonyl fluoride, 4-bromomethylbenzenesulfonyl fluoride or 4-bromomethylpentafluorobenzene.

(2) The reaction conditions are as follows: the reaction was carried out at 37 ℃ for 12 hours.

(3) The separation method comprises the following steps: desalting treatment was performed using an Aimcon ultrafilter of Merck Life sciences to obtain a library of covalently-reacted modified oligonucleotides.

(4) Therefore, taking the sequence 5 ' -TGGCTTGCA A GGGCTGCTG-3 ' (SEQ ID NO: 5) as an example, 20T is added at the 3' to approach the length of the oligonucleotide in the random sequence library, and the sequence is modified by the covalent reaction to obtain benzenesulfonyl fluoride, thiophenyl fluoride and pentafluorobenzene modified sequences respectively, and the soft ionization mass spectrometry of the modified sequences respectively obtains molecular ion peaks of 22573, 22622 and 22598, which are consistent with theoretical values of 22572, 22620 and 22596.

Example 3 in vitro screening of target proteins with covalently modified oligonucleotide libraries

(1) Screening solution formula: 20 μ L of 5 μ M library of oligonucleotides modified by covalent reaction, 50 μ L of 2 × reaction buffer (containing 2 × phosphate buffered saline and 4mM MgCl) ₂ ) 10. mu.L of 250mg/L of target protein, and 20. mu.L of water.

(2) Reaction conditions are as follows: the reaction was carried out at 37 ℃ for 2 hours.

(3) Covalent conjugate separation methods: gel electrophoresis separation is carried out, wherein the gel is 15% polyacrylamide hydrogel (containing 8M urea), the buffer solution used for electrophoresis is 1 xTris-boric acid-EDTA buffer solution, the electrophoresis voltage is 300V, and the electrophoresis time is 1 hour.

(4) The gel was stained with 1 × SYBR Gold from Silmer Fielder, so that the nucleic acid-containing portion of the gel produced fluorescence that was visible under UV light, and the conjugate bands that were electrophoretically slow to move were cleaved and extracted.

Example 4 dissociation reactions, amplification and sequencing of oligonucleotides

(1) The dissociation solution formula is as follows: 0.1M aqueous sodium hydroxide solution.

(2) Dissociation reaction conditions: the extracted gel was cut by soaking in 400. mu.L of 0.1M aqueous sodium hydroxide solution and soaked at 50 ℃ for 2 hours.

(3) The formula of the extraction solution is as follows: mu.L of 0.1M aqueous sodium hydroxide solution was added to the soaked system 10. mu.L of 4M hydrochloric acid and 40. mu.L of 10 XTTris-borate-EDTA buffer.

(4) Extraction reaction conditions are as follows: soaking at 50 deg.C for 12 hr.

(5) The separation method comprises the following steps: desalting the solution obtained by the above extraction reaction by using an Aimcon ultrafilter of Merck bioscience to obtain an oligonucleotide sequence capable of being amplified by polymerase chain reaction.

Example 5 novel molecules of covalent inhibitors of the coronavirus spike protein obtained by the above-described screening method and characterization of their properties

(1) The receptor binding domain RBD of the novel coronavirus spike protein was used as a target protein for positive screening, and human serum was used as a matrix for negative screening. The spike protein RBD is from the product number 40592-V08H of Yinqiao Shenzhou company, and the amino acid sequence is as follows:

RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF (numbered 319 to 541 from left to right amino acids). (SEQ ID NO: 15)

The steps of examples 1-4 above were performed sequentially and repeated 13 times, and the resulting oligonucleotide sequences were subjected to secondary sequencing (using Illumina secondary sequencer) to obtain a plurality of sequences by sequencing read, wherein a set of signature sequences at least includes the following similar sequences (5 'to 3' from left to right):

TACACAGGGGAGTAACGAATGTTCTCTTGGCTTGCAAAGGGCTGCTGTCTGGGTGGGTGTTTGTTCTTCTCT(SEQ ID NO：16)

TACACAGGGGAGTAACGAATGTTCTCCTGGCTTGCAAAGGGCTGCTGTCTGGGTGGGTGTTTGTTCTTCTCT(SEQ ID NO：17)

TACACAGGGGAGTAACGAATGCTCTCTTGGCTTGCAAAGGGCTGCTGTCTGGGTGGGTGTTTGTTCTTCTCT(SEQ ID NO：18)

TACACAGGGGAGTAACGAATGTTCTCGTGGCTTGCAAAGGGCTGCTGTCTGGGTGGGTGTTTGTTCTTCTCT(SEQ ID NO：19)

TACACAGGGGAGTAACGAATGTGCTCTTGGCTTGCAAAGGGCTGCTGTCTGGGTGGGTGTTTGTTCTTCTCT(SEQ ID NO：20)

the active sequence which is shortest and can optimally and covalently inhibit the spike protein is found by shortening the two ends of the sequences and is a 20 nucleotide sequence X and a mutant sequence of the sequence X: as shown in fig. 4;

the sequence X5 '-TGGCTTGCA A GGGCTGCTG-3'; (SEQ ID NO: 5)

M1 mutation of sequence X:5 '-GGCTTGCA a GGGCTGCT-3'; (SEQ ID NO: 1)

M2 mutation of sequence X:5 '-CGGCTTGCA a GGGCTGCTG-3'; (SEQ ID NO: 21)

M3 mutation of sequence X:5 '-TGGCTTGCA a GGGCTGCCG-3'; (SEQ ID NO: 7)

M4 mutation of sequence X:5 '-CGGCTTGCA a GGGCTGCCG-3'; (SEQ ID NO: 22)

M5 mutation of sequence X:5 '-TGGCTTGCT T GGGCTGCTG-3'; (SEQ ID NO: 8)

M6 mutation of sequence X:5 '-TGGCTTGCA a GGGCTGTTG-3'; (SEQ ID NO: 23)

M7 mutation of sequence X:5 '-TGGTTTGCA a GGGCTGCTG-3'; (SEQ ID NO: 6)

M8 mutation of sequence X:5 '-TGGTTTGCA a GGGCTGTTG-3'; (SEQ ID NO: 24)

A ^* 、T ^* Respectively, the phosphorothioate adenosine and thymidine modified by sulfonyl fluoride.

(2) Sequence X was able to form a covalent conjugate with the new coronavirus spike protein as verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (fig. 5). Analysis of the product of the reaction of sequence X with the new coronavirus spike protein using 6% SDS-PAGE revealed the formation of a distinct conjugate band. The reaction solution formula is as follows: 2.5 μ L of 20 μ M sequence X, 2 μ L of 250mg/L Neocoronaviral spike protein, 10 μ L of 2 Xreaction buffer (containing 2 Xphosphate buffered saline and 4mM MgCl ₂ ) 5.5. mu.L of water. Reaction conditions are as follows: 3 hours at 37 degrees. SDS-PAGE conditions: 1 × Tris-Glycine electrophoresis buffer, 200V voltage, 1.5 hours. The SDS-PAGE which completed the electrophoresis was stained with Coomassie Brilliant blue, the higher band representing conjugate formation.

(3) By liquid chromatography-tandem massThe above-mentioned covalent conjugates after dissociation and enzymatic hydrolysis are analyzed by spectroscopic analysis techniques. Firstly, the conjugate is treated by 0.1M NaOH to remove the oligonucleotide on the protein, then the thiol group on the modification site is capped by dithiothreitol and N-methylmaleimide, so that a characteristic mass spectrum label is formed on the modification residue site, and then a series of peptide segments are respectively subjected to tandem mass spectrum analysis by pancreatin decomposition and liquid chromatography, and finally the site information of covalent modification on the RBD of the spike protein is obtained. Using this approach, the amino acid sites of the sequence X + TG covalently conjugated to the novel coronavirus spike protein were found to be the Y421 and K458 amino acid residues of the novel coronavirus spike protein (as shown in fig. 6, 7). Sample pretreatment: mu.M sequence X with 50mg/L of the Neocoronavirus spike protein in 400. mu.L of 1 × reaction buffer (containing 1 × phosphate buffered saline and 2mM MgCl) ₂ ) The reaction was performed at 37 ℃ for 3 hours, desalted using an Amicon ultrafilter, then treated with 0.1M aqueous sodium hydroxide solution at 25 ℃ for 0.5 hour, then neutralized to neutrality with hydrochloric acid, then treated with 2mM dithiothreitol at 37 ℃ for 1 hour, 10mM N-ethylmaleimide at 37 ℃ for 1 hour, and finally desalted again using an Amicon ultrafilter. The protein obtained is degraded by pancreatin in Tris-hydrochloric acid buffer solution with pH 8.3, and then is used for analyzing which amino acid in the polypeptide fragment is subjected to covalent reaction by sequence X by liquid chromatography-tandem mass spectrometry, and the results are determined to be Y421 and K458 amino acid residues of the new coronavirus spike protein.

(4) After a series of similar sequence transformations of sequence X, it was found that its activity of covalently conjugating the new coronavirus spike protein was still present in many of the similar sequences of sequence X. As shown in FIG. 4, the mutations in sequence X resulted in similar sequences that predominantly included both stem and loop types of stem-loop structures, with some of these sequences having reduced activity and some remaining, indicating that sequence X has some conserved structure to support its ability to covalently conjugate a new coronavirus spike protein. Based on the importance of the mutation site and the presumed folding structure of the sequence X, the minimal characteristic sequence which can have the covalent inhibitory activity of the spike protein of the new coronavirus is as follows:

Z ₄ 、Z ₅ 、Z ₁₁ 、Z ₁₂ 、Z ₁₅ a, T, C, G or no base independently;

For example, 5' -GGY ₃ Z ₄ Z ₅ GCZ ₈ Z ₉ Z ₁₀ Z ₁₁ Z ₁₂ GCZ ₁₅ GCY ₁₈ -3′

In which the bold Z ₄ 、Z ₅ 、Z ₁₁ 、Z ₁₂ 、Z ₁₅ May be selected from A, T, G, C or no base;

wherein Z ₈ 、Z ₉ 、Z ₁₀ Is 3 consecutive nucleotides containing sulfonyl fluoride modified phosphorothioate with unsubstituted or substituted 5' end, and the base can be selected from A, T, G, C; y is ₃ 、Y ₁₈ May be selected from T, C.

(5) Assay for inhibitory Activity Using mature New coronavirus (see Nie, J.H.et al.Quanentification of SARS-CoV-2 neutralling antibody by a pseudotyp)The product is prepared from the following components in parts by weight, Nat. Protoc.15,3699-3715 (2020): the new coronavirus was originated from Shenzhou Yinqiao (batch PSV001) and was used at a concentration of 10 ⁸ ～10 ⁹ The medium was Dulbecco's modified Eagle's Medium (containing 10% calf serum protein) per ml. After the sequence X or the sequence X ' without sulfonyl fluoride modification and the new corona pseudovirus act for 3 hours (the test concentration range of the sequence X and the sequence X ' is 0.025nM to 2000nM), the sequence X and the sequence X ' are used for infecting HEK 293T cells expressing receptor protein for 6 hours, then the HEK 293T cells are replaced to remove the pseudovirus, the cells are cultured for 48 hours again, the cells are lysed, the luciferase activity is quantified by an RG055 kit in Biyun, and a detection instrument is a 96-well plate microplate reader. The new corona pseudovirus is infected by about more times, more RNA expressing luciferase is brought into HEK 293T cells, and a stronger luciferase activity signal is generated, namely the luciferase activity after cell lysis is reduced by the inhibition effect of the sequence X on the new corona pseudovirus. Through the pseudovirus inhibition activity test (shown in figure 8), the semi-inhibition concentration of the sequence X to the host cell infected by the new corona pseudovirus is 0.95nM, and the semi-inhibition concentration of the sequence X' lacking covalent reaction chemical modification is 110nM, which shows that the covalent inhibition effect generated by the covalent chemical modification plays an important role in the inhibition of the sequence X to the activity of the new corona pseudovirus. Therefore, the sequence X can inhibit the infection of the new coronavirus pseudovirus to HEK 293T cells expressing the receptor protein, and the half-inhibitory concentration reaches 0.95 nM.

In the present disclosure, the terms "one embodiment," "some embodiments," "an example," "a specific example," or "some examples" and the like mean that a specific feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present disclosure. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.

Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.

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<211> 72

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

tacacagggg agtaacgaat gttctcctgg cttgcaaagg gctgctgtct gggtgggtgt 60

ttgttcttct ct 72

<210> 18

<211> 72

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

tacacagggg agtaacgaat gctctcttgg cttgcaaagg gctgctgtct gggtgggtgt 60

ttgttcttct ct 72

<210> 19

<211> 72

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

tacacagggg agtaacgaat gttctcgtgg cttgcaaagg gctgctgtct gggtgggtgt 60

ttgttcttct ct 72

<210> 20

<211> 72

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

tacacagggg agtaacgaat gtgctcttgg cttgcaaagg gctgctgtct gggtgggtgt 60

ttgttcttct ct 72

<210> 21

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (9)..(11)

<223> covalent reaction modification

<400> 21

cggcttgcaa agggctgctg 20

<210> 22

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (9)..(11)

<223> covalent reaction modification

<400> 22

cggcttgcaa agggctgccg 20

<210> 23

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (9)..(11)

<223> covalent reaction modification

<400> 23

tggcttgcaa agggctgttg 20

<210> 24

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (9)..(11)

<223> covalent reaction modification

<400> 24

tggtttgcaa agggctgttg 20

Claims

1. An in vitro method for screening for a covalent inhibitor, comprising the steps of:

(1) constructing a phosphorothioate oligonucleotide library; (2) contacting the phosphorothioate oligonucleotide library with at least one covalent reaction modifying reagent to construct a library of covalent reaction modifying oligonucleotides; the covalent reactive modification reagent comprises at least one covalent reactive modification group adapted to covalently bind to a target protein; (3) contacting the covalent reaction modified oligonucleotide library with target protein, separating and collecting the covalent reaction modified oligonucleotide combined with the target protein, and then removing the target protein and covalent reaction modified groups to construct a secondary library; (4) and (4) repeating the steps (1) to (3) by taking the secondary library as a template, and after multiple cycles, performing sequencing analysis on the library obtained by the last round of screening, and screening to obtain the covalent inhibitor.

2. The in vitro screening method of claim 1, wherein the step of constructing the library of phosphorothioate oligonucleotides comprises performing an amplification reaction with a nucleic acid polymerase using a mixture of nucleoside triphosphates as substrates, wherein the substrates comprise at least one nucleoside phosphothioate.

3. The in vitro screening method of claim 2, wherein the initial library for constructing the phosphorothioate oligonucleotide library is a random library, the random library is used as a template, dNTPs are used as substrates, and at least one monomer in the substrates is deoxynucleoside thiophosphate, and the PCR amplification reaction is performed under the action of DNA polymerase.

4. The in vitro screening method according to claim 3, wherein said deoxynucleoside thiophosphate is selected from any one of 5 '-deoxyadenosine [ α -thio ] -triphosphate, 5' -deoxyguanosine [ α -thio ] -triphosphate, 5 '-deoxythymidine [ α -thio ] -triphosphate, 5' -deoxycytidine [ α -thio ] -triphosphate and analogues and derivatives thereof.

5. The in vitro screening method of claim 1, wherein said covalent reaction modifying group is unsubstituted or optionally substituted as follows: sulfonyl fluoride, sulfonamide, sulfonate, alpha, beta-unsaturated carbonyl, or fluoro substituted phenyl.

6. The in vitro screening method of claim 5, wherein said covalent reaction modifying group is selected from the following structures:

wherein R is ₁ is-H, -OH, -NO ₂ -COOH, halogen or-R';

R ₂ is-H, -OH, -R 'or-C (═ O) R';

R ₃ is-H or-R';

R ₄ is-H or-R';

n is 1 or 2 or 3 or 4 or 5;

r' is C ₁ -C ₁₂ Alkyl radical, C ₁ -C ₁₂ Alkoxy radical, C ₂ -C ₁₂ Alkenyl radical, C ₂ -C ₁₂ Alkynyl, C ₃ -C ₁₂ Cycloalkyl, C ₃ -C ₁₂ Cycloalkenyl radical, C ₆ -C ₁₂ Aryl radicalsOr C ₅ -C ₁₂ A heteroaryl group;

halogen is F, Cl, Br or I.

7. The in vitro screening method of claim 1, wherein said covalent reaction modification reagent further comprises a functional group capable of reacting with a phosphorothioate group, said functional group being unsubstituted or optionally substituted as follows: halogen methylene or halogen carboxyethyl, wherein the halogen is F, Cl, Br or I.

8. The in vitro screening method of claim 7, wherein said functional group capable of reacting with a phosphorothioate group is selected from the following structures:

9. The in vitro screening method of claim 1, wherein the number of repetitions of steps (1) - (3) is 1-25.

10. The in vitro screening method of claim 1, wherein the target protein-bound covalently-reacted modified oligonucleotide is contacted with an alkaline solution in step (3) to remove the target protein and the covalently-reacted modifying group.

11. The in vitro screening method of any one of claims 1 to 10, wherein the target protein is a disease-associated protein or a pharmaceutical protein.

12. A covalent inhibitor characterized by: the covalent inhibitor is screened by the method of any one of claims 1 to 11, and comprises at least two covalent reaction modifying groups; the covalent reaction modification group is the following unsubstituted or optionally substituted group: sulfonyl fluoride, sulfonamide, sulfonate, alpha, beta-unsaturated carbonyl, or fluoro substituted phenyl.

13. The covalent inhibitor of claim 12, wherein said covalent reactive modifying group is selected from the following structures:

wherein R is ₁ is-H, -OH, -NO ₂ -COOH, halogen or-R';

R ₂ is-H, -OH, -R 'or-C (═ O) R';

R ₃ is-H or-R';

R ₄ is-H or-R';

n is 1 or 2 or 3 or 4 or 5;

halogen is F, Cl, Br or I.

14. A nucleotide molecule comprising a sequence of formula (I) below;

Wherein Z is ₈ 、Z ₉ 、Z ₁₀ At least two of which are covalently reaction-modified phosphorothioate nucleotides; each independently selected from A, T, C or G; the covalent reaction is modified by adopting the following unsubstituted or optionally substituted groups: sulfonyl fluoride, sulfonamide, sulfonate, alpha, beta-unsaturated carbonyl, or fluoro-substituted phenyl;

Z ₁ 、Z ₂ 、Z ₃ 、Z ₆ 、Z ₇ and Z ₁₈ 、Z ₁₇ 、Z ₁₆ 、Z ₁₄ 、Z ₁₃ Are each selected from A, T, C, G, and Z ₁ And Z ₁₈ Form base pairs, Z ₂ And Z ₁₇ Form base pairs, Z ₃ And Z ₁₆ Form base pairs, Z ₆ And Z ₁₄ Form base pairs, Z ₇ And Z ₁₃ Forming a base pair.

15. The nucleotide molecule of claim 14, wherein the covalent reactive modification is a phenylsulfonyl fluoro modification, a thiophenyl fluoro modification, or a pentafluorophenyl modification.

16. The nucleotide molecule of claim 14, wherein Z is ₁ 、Z ₂ 、Z ₃ 、Z ₆ 、Z ₇ And Z ₁₈ 、Z ₁₇ 、Z ₁₆ 、Z ₁₄ 、Z ₁₃ Are independently selected from C, G, or G, T, and wherein C and G form base pairs and G and T form base pairs.

17. The nucleotide molecule of claim 14, further comprising at least one base at the 5 'end and/or the 3' end of the structure of formula (I), wherein each base is independently selected from A, T, C or G.

18. The nucleotide molecule of claim 14, wherein the nucleotide molecule is further modified to comprise at least one chemical modification, wherein the chemical modification is a chemical substitution at a position selected from the group consisting of a sugar position, a phosphate position, and/or a base position of the nucleotide molecule.

19. The nucleotide molecule according to claim 14, comprising a nucleotide sequence;

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) or

5′-TGGCTTGCAA*A*GGGCTGCTG-3′；(SEQ ID NO：11)

20. The nucleotide molecule of any one of claims 14-19, further comprising any one of the following four sequences:

(2) a sequence which hybridizes to a nucleotide sequence of formula (I);

(3) an RNA sequence transcribed from a nucleotide sequence of formula (I);

(4) having one or more nucleotide substitutions, deletions and/or additions compared to the nucleotide sequence of formula (I).

21. A vector comprising the nucleotide molecule of any one of claims 14-20.

22. The vector of claim 21, wherein the vector is an expression vector.

23. A nucleic acid molecule encoded by the nucleotide molecule of any one of claims 14-20.

24. A host cell comprising the nucleic acid molecule of claim 23.

25. A pharmaceutical composition comprising the nucleotide molecule of any one of claims 14-20, the vector of claim 21 or 22, the nucleic acid molecule of claim 23, or the host cell of claim 24.

26. The pharmaceutical composition of claim 25, further comprising a chemotherapeutic agent.

27. Use of the nucleotide molecule of any one of claims 14-20, the vector of claim 21 or 22, the nucleic acid molecule of claim 23, the host cell of claim 24, or the pharmaceutical composition of claim 25 for the preparation of a medicament for targeting a neocoronavirus spike protein.