Covalent protease inhibitors and uses thereof

Abstract

The present application relates to a covalent protein inhibitor and its application in neutralizing the novel coronavirus. Specifically, it relates to a binding compound which can specifically bind to a virus or a mutant strain thereof and covalently bind to the virus or the mutant strain thereof.

Description

Technical Field

[0001] This application relates to the field of biomedicine, specifically to a covalent protein inhibitor and its uses. Background Technology

[0002] Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), also known as the novel coronavirus, is the culprit behind the global pandemic of Coronavirus Disease 2019 (COVID-19). Countries around the world have been working to develop vaccines against the novel coronavirus, and while these efforts have initially helped to curb its spread, the fact that the novel coronavirus genome is more prone to mutation than other RNA viruses, resulting in immune-evading mutants that are often more infectious, has severely hampered the global pandemic response.

[0003] Therefore, there is an urgent need in this field for a universal neutralizing agent that can overcome viral mutation escape. Summary of the Invention

[0004] This application provides a covalent protein inhibitor and its use. In one aspect, this application provides a binding compound capable of specifically binding to a virus or a mutant strain thereof and covalently binding to the virus or its mutant strain.

[0005] On the one hand, this application provides a nucleic acid molecule that encodes the binding compound of this application.

[0006] On the one hand, this application provides a carrier containing the nucleic acid molecule of this application.

[0007] On the one hand, this application provides an immunoconjugate comprising the binding compound of this application.

[0008] On the one hand, this application provides a cell comprising the binding compound of this application, the nucleic acid molecule of this application, the vector of this application, and / or the immunoconjugate of this application.

[0009] On one hand, this application provides a composition comprising the binding compound of this application, the nucleic acid molecule of this application, the carrier of this application, the immunoconjugate of this application, and / or the cell of this application, and optionally a pharmaceutically acceptable carrier.

[0010] On one hand, this application provides a kit comprising the binding compound of this application, the nucleic acid molecule of this application, the vector of this application, the immunoconjugate of this application, the cells of this application, and / or the composition of this application.

[0011] On the one hand, this application provides the use of the binding compounds of this application, the nucleic acid molecules of this application, the vectors of this application, the immunoconjugates of this application, the cells of this application, the compositions of this application, and / or the kits of this application in the preparation of medicaments for diagnosing, preventing and / or treating diseases.

[0012] On one hand, this application provides a method for determining the presence and / or content of a virus or its mutant strain, the method comprising administering the binding compound of this application, the nucleic acid molecule of this application, the vector of this application, the immunoconjugate of this application, the cell of this application, the composition of this application, and / or the kit of this application.

[0013] On one hand, this application provides a method for reducing the infection of cells by a virus or its mutant strain, the method comprising administering the binding compound of this application, the nucleic acid molecule of this application, the vector of this application, the immunoconjugate of this application, the cells of this application, the composition of this application, and / or the kit of this application.

[0014] The covalently bound compounds provided in this application can irreversibly inhibit the target site and enhance the retention of the bound compounds at the target site, thereby minimizing drug resistance caused by target mutations. In this application, a covalently bound protein inhibitor targeting the escape mutation of SARS-CoV-2 was prepared and characterized by converting the interaction between a non-covalent protein inhibitor and the spike S protein into a covalent bond. The non-natural amino acid-modified covalently bound protein inhibitor shows a significantly improved virus neutralization efficiency relative to its wild-type form.

[0015] Other aspects and advantages of this application will readily be apparent to those skilled in the art from the detailed description below. Only exemplary embodiments of this application are shown and described in the following detailed description. As will be appreciated by those skilled in the art, the content of this application enables them to make modifications to the disclosed specific embodiments without departing from the spirit and scope of the invention to which this application pertains. Accordingly, the descriptions in the accompanying drawings and specification of this application are merely exemplary and not restrictive. Attached Figure Description

[0016] The specific features of the invention involved in this application are shown in the appended claims. The features and advantages of the invention can be better understood by referring to the exemplary embodiments and drawings described in detail below. A brief description of the drawings is as follows:

[0017] Figure 1 The diagram shown is a schematic of the covalent protein inhibitor described in this application targeting and neutralizing the SARS-CoV-2 virus.

[0018] Figure 2 The diagram shows the covalent chemical reaction between the non-natural amino acids of the covalent protein inhibitor and the nucleophilic amino acids of the spike protein.

[0019] Figure 3 A-3B show the complex structures of the wild-type SARS-CoV-2 spike protein receptor-binding domain (RBD) and the non-covalent protein inhibitor LCB1. (A) Complex structure of wild-type RBD (top) and the non-covalent protein inhibitor LCB1 (bottom), with labeled nucleophilic amino acids on the RBD facing the interaction interface, including K417, Y421, Y449, Y453, Y473, Y489, and Y505. PDB: 7JZU; (B) Complex structure of wild-type RBD (top) and the non-covalent protein inhibitor LCB3 (bottom), with labeled nucleophilic amino acids on the RBD facing the interaction interface, including K417, Y421, Y449, Y453, Y473, Y489, and Y505. PDB: 7JZN.

[0020] Figure 4 A-4B shows five candidate sites on the non-covalent protein inhibitor LCB1. (A) L6, Q7, and K37; (B) A22 and D30.

[0021] Figure 5 A-5B shows four candidate sites on the non-covalent protein inhibitor LCB3. (A) D3, Y40; (B) K26, F30.

[0022] Figure 6 A-6B shows the primary mass spectrum of a non-covalent protein inhibitor. (A) Non-covalent protein inhibitor LCB1 (A) Primary mass spectrometry of the non-covalent protein inhibitor LCB3, with a theoretical value of 7876 Da and a measured value of 7876 Da; (B) Primary mass spectrometry of the non-covalent protein inhibitor LCB3, with a theoretical value of 9501 Da and a measured value of 9504 Da.

[0023] Figure 7 A-7F show the characterization results of LCB1-based candidate covalent protein inhibitors. (A) L6, (B) Q7, (C) A22, (D) D30, (E) Molecular weight of the purified candidate covalent protein inhibitor with the non-natural amino acid FSY inserted at position K37 in primary mass spectrometry. (F) SDS-PAGE gel electrophoresis characterizes the purity of the LCB1-based candidate covalent protein inhibitors.

[0024] Figure 8A-8E show the molecular weights of LCB3-based candidate covalent protein inhibitors after purification by primary mass spectrometry for (A) D3, (B) K26, (C) D30, and (D) Y40 insertion of the non-natural amino acid FSY. (E) SDS-PAGE gel electrophoresis characterizes the purity of the LCB3-based candidate covalent protein inhibitors.

[0025] Figure 9 A-9E show the results of denaturing SDS-PAGE gel electrophoresis analysis of the cross-linking reaction between five candidate covalent protein inhibitors of LCB1 and the SARS-CoV-2 RBD protein. (A) In vitro cross-linking results with wild-type RBD protein; (B) In vitro cross-linking results with Beta mutant RBD protein; (C) In vitro cross-linking results with Delta mutant RBD protein; (D) In ​​vitro cross-linking results with Kappa mutant RBD protein; (E) In vitro cross-linking results with Omicron mutant RBD protein.

[0026] Figure 10 A-10E shows the results of denaturing SDS-PAGE gel electrophoresis analysis of cross-linking reactions between four candidate covalent protein inhibitors based on LCB3 and the SARS-CoV-2 RBD protein. (A) In vitro cross-linking results with wild-type RBD protein; (B) In vitro cross-linking results with Beta mutant RBD protein; (C) In vitro cross-linking results with Delta mutant RBD protein; (D) In ​​vitro cross-linking results with Kappa mutant RBD protein; (E) In vitro cross-linking results with Omicron mutant RBD protein.

[0027] Figure 11 A-11E shows the relationship between the efficiency of the cross-linking reaction between covalent LCB3 and the SARS-CoV-2 RBD protein and time and concentration, analyzed by denaturing SDS-PAGE gel electrophoresis. (A) Cross-linking results with wild-type RBD protein; (B) Cross-linking results with Beta mutant RBD protein; (C) Cross-linking results with Delta mutant RBD protein; (D) Cross-linking results with Kappa mutant RBD protein; (E) Cross-linking results with Omicron mutant RBD protein.

[0028] Figure 12 A-12C shows the tandem mass spectrum of the cross-linked peptide after the reaction of covalent LCB3 with RBD protein. (A) Wild-type RBD protein; (B) Delta mutant RBD protein; (C) Tandem mass spectra of the cross-linked product of Delta mutant RBD protein and covalent LCB3 all show that the non-natural amino acid FSY at position 30 of covalent LCB3 forms a covalent bond with tyrosine residue 473 of RBD protein, where U represents the non-natural amino acid FSY.

[0029] Figure 13 A-13F shows that the insertion of the non-natural amino acid FSY does not affect the binding affinity of the protein inhibitor to the RBD protein. The dissociation constant K of non-covalent LCB3 and SARS-CoV-2 (A) wild-type RBD is also shown. D = 0.12 nM, (C) Dissociation constant K of Delta mutant RBD D = 0.11 nM, and the dissociation constant K of the (E) Kappa mutant RBD. D = 0.52 nM; Dissociation constant K of covalent LCB3 and wild-type RBD of SARS-CoV-2 (B) D = 0.12 nM, (D) Dissociation constant K of Delta mutant RBD D =0.15 nM, and the dissociation constant K of (F) Kappa mutant RBD. D = 0.20 nM.

[0030] Figure 14 A-14C shows that, compared to non-covalent LCB3, covalent LCB3 has a stronger blocking ability against the interaction of SARS-CoV-2 with (A) wild-type RBD, (B) Delta mutant RBD, and (C) Kappa mutant RBD, and human ACE2, with a half-inhibitory concentration (IC50). 50 The value is reduced to 1 / 20 to 1 / 6 of that of non-covalent LCB3.

[0031] Figure 15 The graph shows the results demonstrating that covalent LCB3 exhibits a stronger neutralizing ability against the SARS-CoV-2 Delta mutant compared to non-covalent LCB3. The IC50 of covalent LCB3 is also shown. 50 The value was 0.019 ± 0.003 nM; the IC50 of non-covalent LCB3 was 0.019 ± 0.003 nM. 50 The value is 0.121 ± 0.022 nM.

[0032] Figure 16 Figure A-16F shows that covalent LCB3 retained its activity after atomization and lyophilization, exhibiting the same stability results as non-covalent LCB3. Biomembrane interferometry analysis showed that (A, C) non-covalent LCB3 and (B, D) covalent LCB3, compared with the untreated control, retained high binding affinity to the RBD protein of the SARS-CoV-2 Delta mutant after atomization or lyophilization. Detailed Implementation

[0033] The following specific embodiments illustrate the implementation of the invention. Those skilled in the art can easily understand other advantages and effects of the invention from the content disclosed in this specification.

[0034] Terminology Definition

[0035] In this application, the term "mutant strain" generally refers to a lineage that carries a mutation in its genome. For example, if a viral strain has one or more mutations relative to the natural form of SARS-CoV-2, it can be considered a mutant strain of SARS-CoV-2.

[0036] In this application, the term "SARS-CoV-2" generally refers to Severe Acute Respiratory Syndrome Coronavirus 2. SARS-CoV-2 belongs to the genus *Betacoronavirus*, subgenus *Sarbecovirus*, family *Coronaviridae*. SARS-CoV-2 is an enveloped, non-segmented, positive-sense, single-stranded RNA virus. SARS-CoV-2 can cause infection with the novel coronavirus (COVID-19). In this application, SARS-CoV-2 may include the S protein (spike protein). In some implementations, the SARS-CoV-2 mutant has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleotide sequence of Genome Reference Sequence NC_045512.

[0037] In this application, the term "structural protein" generally refers to the protein components that make up a virion. For example, a structural protein can be a capsid protein, matrix protein, or envelope protein of the virion.

[0038] In this application, the term "spike protein" generally refers to a protein of a viral envelope. For example, viruses can use spike proteins to promote the fusion of the viral envelope with the cell membrane.

[0039] In this application, the term "receptor binding domain (RBD)" generally refers to the receptor binding domain (RBD) of the coronavirus S protein. The RBD plays a crucial role in the binding of the virus to angiotensin-converting enzyme 2 (ACE2) on the surface of host cells and its entry into host cells. The RBD exhibits good accuracy and specificity for the novel coronavirus and can be used for the detection of SARS-CoV-2; simultaneously, the RBD plays a role in the invasion of SARS-CoV-2 into cells, and recognizing and specifically binding to the RBD could be used for the treatment of diseases caused by SARS-CoV-2.

[0040] In this application, the term "nucleophile" generally refers to a group with a negative charge or a lone pair of electrons. For example, a nucleophile may include an amino group, an imidazole group, a hydroxyl group, and derivatives thereof. In this application, the term "amino" generally refers to -NH₂. For example, the amino group may be substituted in any way. In this application, the term "imidazole" generally refers to 1,3-diazolyl. For example, the imidazole group may be substituted in any way. In this application, the term "hydroxyl" generally refers to -OH. For example, the hydroxyl group may be substituted in any way.

[0041] In this application, the term "compound" generally refers to a substance having two or more different elements. For example, the compound in this application can be an organic compound, or a compound with a molecular weight of less than 500, less than 1000, more than 1000, or more than 10,000 or 100,000. In this application, a compound can also refer to a compound linked by chemical bonds, such as a compound in which one or more molecules with a molecular weight of less than 1000 are linked by chemical bonds to a biological macromolecule, which can be a polysaccharide, protein, nucleic acid, polypeptide, etc. For example, the compound in this application can include a compound in which a protein is linked to one or more molecules with a molecular weight of less than 1000, a compound in which a protein is linked to one or more molecules with a molecular weight of less than 10,000, or a compound in which a protein is linked to one or more molecules with a molecular weight of less than 100,000. Unless otherwise specified, the structures described in this application can also include compounds that differ only in the presence or absence of one or more isotopically enriched atoms. For example, compounds whose structure is identical to that of this application except that hydrogen atoms are replaced by deuterium or tritium, or carbon atoms are replaced by carbon-13 or carbon-14, are all within the scope of this application.

[0042] In this application, the term "covalent bonding" generally refers to the connection of two molecules or groups by a covalent bond. For example, two molecules or groups may interact through the sharing of electron pairs.

[0043] In this application, the term "bonded interface" generally refers to the interface where two molecules or groups are in direct contact when they interact. For example, the composition of the groups at the bonded interface can be analyzed using three-dimensional structural analysis software known in the art.

[0044] In this application, the term "distance" generally refers to the distance between two groups. For example, the distance in this application may refer to the average distance between two groups, or the shortest distance between two groups. For example, the theoretically possible distance between two groups can be analyzed using three-dimensional structural analysis software known in the art.

[0045] In this application, the term "pharmaceutically acceptable carrier" generally refers to a pharmaceutically acceptable carrier, excipient, or stabilizer that is non-toxic to cells or mammals exposed to it at the doses and concentrations used. Physiologically acceptable carriers may include suitable substances. A pharmaceutically acceptable carrier is generally not the same substance as a vector used in genetic engineering to insert nucleic acids.

[0046] In this application, the terms "comprising" or "including" generally mean including the expressly specified features, but do not exclude other elements. In some cases, "comprising" or "including" also covers the meaning of "is" or "consisting of".

[0047] In this application, the term "about" generally refers to a variation within a range of 0.5% to 10% above or below a specified value, such as a variation within a range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below a specified value. Invention Details

[0049] This application provides a covalent protein inhibitor and its use. In one aspect, this application provides a binding compound capable of specifically binding to a virus or a mutant strain thereof and covalently binding to the virus or its mutant strain.

[0050] The novel coronavirus enters host cells through the interaction between its spike protein (S protein) and human angiotensin-converting enzyme 2 (huACE2). Therefore, developing neutralizing antibodies to block this interaction is a key preventative and therapeutic strategy against SARS-CoV-2 infection. The spike protein consists of two regions, S1 and S2, with the key domain interacting with human ACE2 called the receptor-binding domain (RBD). Many neutralizing antibodies bind to the RBD domain of the S protein. However, under the selective pressure of potent treatments, the novel coronavirus has evolved various immune escape mutations concentrated in the RBD domain to survive, thus acquiring stronger infectivity and lethality. On the other hand, extensive clinical experience indicates that effective treatment of SARS-CoV-2 infection requires high doses of neutralizing antibodies. This is because traditional neutralizing antibodies have a low inhibitory density, meaning fewer atoms of the same molecular weight are used to inhibit the virus, and low doses of antibodies can cause antibody-dependent enhancement (ADE). Many traditional antibody-based treatment strategies, including convalescent plasma therapy, have shown little effectiveness against SARS-CoV-2 escape mutants.

[0051] On the other hand, protein inhibitors (minibinders) are a class of designed or screened antigen-neutralizing agents. They have broad application prospects due to their small size, high stability, high inhibition density, and absence of the ADE effect that may occur with the Fc region of monoclonal antibodies. Furthermore, the nasal administration capability of protein inhibitors is particularly valuable for the prevention and treatment of respiratory viruses. For example, the protein inhibitors (LCB1 and LCB3) of this application combine a series of modes of interaction with the RBD domain of the SARS-CoV-2 virus, significantly improving the inhibition density of the neutralizing agent and achieving neutralization capacity at the picomolar level of the real virus. However, this application finds that the blocking effect of current protein inhibitors against SARS-CoV-2 still depends on their non-covalent interaction with specific residues (receptor binding motif, RBM) in the RBD domain; therefore, escape mutations on the RBM will inevitably reduce or even eliminate their neutralizing capacity. Therefore, there is an urgent need in the art to develop a universal neutralizing agent with high inhibition density that can overcome viral mutation escape.

[0052] For example, this application provides a reagent that can overcome viral mutation escape and efficiently neutralize the virus. For example, this application selects peptides capable of interacting with viral proteins (e.g., non-covalent binding), and achieves efficient neutralization of the virus by mutating candidate interfacial amino acids to amino acids with covalent reactivity. For example, based on the reactivity of the covalent amino acid FSY, nucleophilic amino acids Nu on the SARS-CoV-2 RBD that can react with FSY, such as lysine (Lys(K), histidine (His(H)), and tyrosine (Tyr(Y), can be selected; in the protein complex structure of RBD-LCB1 or RBD-LCB3, the nucleophilic amino acids facing the binding interface can be determined using known three-dimensional structural analysis tools in the art: K417, Y421, Y449, Y453, Y473, ... Y489 and Y505; these 7 nucleophilic amino acids can be used as the origin, within a radius of 10 Å to determine possible LCB1 or LCB3 sites. On LCB1, these sites are K2, E3, W4, I5, L6, Q7, K8, I9, Y10, E11, I12, M13, R14, L15, L16, D17, E18, L19, G20, H21, A22, E23, A24, S25, M26, R27, V28, S29, D30, L... 31, I32, Y33, E34, F35, M36, K37, K38, E46, L50 (39 in total); on LCB3, they are N1, D2, D3, E4, L5, H6, M7, L8, M9, T10, D11, L12, V13, Y14, E15, A16, L17, H18, F19, E23, I25, K38, Y40, K41, N43, V52. K56 (27 sites in total); these 66 sites were then mutated one by one to tyrosine Y using a three-dimensional structure analysis tool, adjusting the spatial orientation after mutation to ensure it did not affect the original protein structure and was aligned with the 7 nucleophilic amino acids on the SARS-CoV-2 RBD; the linear distance d between the phenolic hydroxyl oxygen atom O of the mutated and adjusted tyrosine Y side chain and the nucleophilic atoms of the 7 nucleophilic amino acids (amino nitrogen atom N of lysine, imidazole nitrogen atom N of histidine, and phenolic hydroxyl oxygen atom O of tyrosine) on the SARS-CoV-2 RBD was measured using a three-dimensional structure analysis tool; 5 candidate mutation sites on LCB1 (L6, Q7, A22, D30, K37) and 4 candidate mutation sites on LCB3 (D3, K26, F30, Y40) were screened using in vitro in situ cross-linking to verify whether the above candidate mutation sites, after being mutated to the covalently binding amino acid FSY, have the ability to efficiently neutralize the virus. The method of this application can be used to determine candidate mutation sites for any wild-type virus. The method described in this application can also be used to identify candidate mutation sites for any variant.

[0053]

[0054] On the one hand, this application provides a binding compound that can specifically bind to a virus or its mutant strain and covalently bind to the virus or its mutant strain.

[0055] For example, the virus or its mutant strain may comprise coronaviruses or mutant strains thereof. For example, the virus or its mutant strain may comprise SARS-CoV-2 or its mutant strains. For example, the mutant strain of SARS-CoV-2 may be selected from the group consisting of: Beta variants, Delta variants, Kappa variants, and Omicron variants.

[0056] For example, the binding compound can specifically bind to the virus or its mutant strain or variants. For example, the binding compound can specifically bind to the spike protein of the virus or its mutant strain or variants. For example, the binding compound can specifically bind to the receptor binding domain (RBD) of the spike protein of the virus or its mutant strain.

[0057] For example, when the binding compound binds to the virus or its mutant strain, the virus or its mutant strain may contain nucleophilic groups at the binding interface.

[0058] For example, when the binding compound binds to the virus or its mutant strain, the virus or its mutant strain may contain a nucleophilic amino acid at the binding interface, and the nucleophilic amino acid may contain a side group having a nucleophilic group. For example, the nucleophilic amino acid may be selected from the group consisting of lysine (K), histidine (H), and tyrosine (Y).

[0059] For example, the binding compound can react with nucleophilic groups at the interface of the virus or its mutant strain to form a covalent bond.

[0060] For example, the binding compound may contain a covalently reactive group. For example, when the binding compound binds to the virus or its mutant strain, the covalently reactive group has the ability to bind to the nucleophilic group of the virus or its mutant strain. For example, when the binding compound is near the virus or its mutant strain, the binding compound may react with the virus or its mutant strain to form a covalent bond. For example, the binding compound may undergo a cross-linking reaction with the virus or its mutant strain, for example, a substitution reaction and / or an addition reaction. For example, the binding compound and the virus or its mutant strain may be covalently linked through a substitution reaction and / or an addition reaction. For example, when the binding compound is near the virus or its mutant strain through an antibody-antigen non-covalent interaction, the covalently reactive group of the binding compound may react with the nucleophilic group of the virus or its mutant strain at the binding interface to form a covalent bond. For example, when the binding compound is near the virus or its mutant strain, the distance between the backbone of the binding compound and the backbone of the virus or its mutant strain may be approximately equal to the sum of the side chain length of the covalently reactive group of the binding compound and the side chain length of the nucleophilic group of the virus or its mutant strain at the binding interface. For example, the orientation of the covalent reactive group of the binding compound and the nucleophilic group of the virus or its mutant at the binding interface can be head-to-head, or the side chain of the covalent reactive group of the binding compound and the side chain of the nucleophilic group of the virus or its mutant at the binding interface can be rotated to achieve a head-to-head orientation. For example, in certain states, when the distance between the covalent reactive group of the binding compound and the nucleophilic group of the virus or its mutant at the binding interface is less than about 10 angstroms (10E-10 meters), the covalent reactive group of the binding compound can react with the nucleophilic group of the virus or its mutant at the binding interface to form a covalent bond. For example, the distance between the covalent reactive group of the binding compound and the nucleophilic group of the virus or its mutant at the binding interface can be achieved by adjusting the length of the side chain of the binding compound with the covalent reactive group of the binding compound.

[0061] For example, when the distance between the covalent reactive group and the nucleophilic group is less than about 10E-10 meters, the covalent reactive group can react with the nucleophilic group to form a covalent bond.

[0062] For example, when the binding compound binds to the virus or its mutant strain, the distance between the nucleophilic amino acid at the binding interface and the covalent reactive group of the binding compound can be less than about 10E-10 meters.

[0063] For example, the covalent reactive group may include: For example, the combined compound may contain residues of a non-natural amino acid, which may contain the covalently reactive group, and the non-natural amino acid may contain... Or its derivatives. For example, the combined compound may contain residues of a non-natural amino acid, the non-natural amino acid may contain the covalently reactive group, and the non-natural amino acid may contain... Or its derivatives. For example, the conjugated compound may contain residues of a non-natural amino acid, the non-natural amino acid may contain the covalently reactive group, and the non-natural amino acid may contain fluorosulfate-L-tyrosine (FSY) or its derivatives.

[0064] For example, the binding compound may comprise a polypeptide or a variant thereof. In this application, the polypeptide encompasses variants thereof, which include amino acid sequences of the polypeptide that have been substituted, deleted, and / or added with one or more amino acids. Examples include 1-30, 1-20, or 1-10 amino acids, or, for example, 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid substitutions, deletions, and / or insertions; homologs are also included, which can be amino acid sequences having at least about 85% (e.g., having at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or higher) sequence homology to the amino acid sequence of the polypeptide.

[0065] For example, the binding compound may comprise a polypeptide or a variant thereof capable of non-covalently binding to the virus or a mutant thereof. For example, the polypeptide or a variant thereof may bind to the receptor domain of the spike protein of the virus or a mutant thereof with a Kd value of less than about 1 nM. For example, the binding compound may be derived from a polypeptide or a variant thereof selected from the group consisting of LCB1 shown in SEQ ID NO: 1 and LCB3 shown in SEQ ID NO: 3.

[0066] For example, the binding compound may contain an amino acid sequence similar to SEQ ID. Compared to the corresponding amino acid sequence shown in NO:1, the amino acids at positions 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 46, and / or 50 may contain covalently reactive groups.

[0067] For example, the amino acid sequence contained in the binding compound may differ from the corresponding amino acid sequence shown in SEQ ID NO: 1, specifically the amino acid sequences at positions 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, and 22. The amino acids at positions 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 46, and / or 50 are replaced by non-natural amino acids, which may contain covalently reactive groups.

[0068] For example, the amino acid sequence contained in the combined compound may contain covalent reactive groups at positions 6, 7, 22, 30, and / or 37, compared to the corresponding amino acid sequence shown in SEQ ID NO: 1.

[0069] For example, the amino acid sequence contained in the combined compound may be different from the corresponding amino acid sequence shown in SEQ ID NO: 1, in which the 6th, 7th, 22nd, 30th, and / or 37th amino acids of the combined compound are replaced by non-natural amino acids, which may contain covalent reactive groups.

[0070] For example, the amino acid sequence contained in the combined compound may include a covalently reactive group at position 22 compared to the corresponding amino acid sequence shown in SEQ ID NO: 1.

[0071] For example, the amino acid sequence of the bound compound may be different from the corresponding amino acid sequence shown in SEQ ID NO: 1, in which the 22nd amino acid of the bound compound is replaced by a non-natural amino acid, which may contain a covalently reactive group.

[0072] For example, the amino acid sequence contained in the combined compound may contain covalently reactive groups at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 23, 25, 38, 40, 41, 43, 52, and 56, compared to the corresponding amino acid sequence shown in SEQ ID NO: 3.

[0073] For example, the amino acid sequence contained in the combined compound may be different from the corresponding amino acid sequence shown in SEQ ID NO: 3, except that the amino acids at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 23, 25, 38, 40, 41, 43, 52, and 56 are replaced by non-natural amino acids, which may contain covalently reactive groups.

[0074] For example, the amino acid sequence contained in the combined compound may contain covalent reactive groups at positions 3, 26, 30, and / or 40, compared to the corresponding amino acid sequence shown in SEQ ID NO: 3.

[0075] For example, the amino acid sequence contained in the combined compound may be different from the corresponding amino acid sequence shown in SEQ ID NO: 3, in which the 3rd, 26th, 30th, and / or 40th amino acids of the combined compound are replaced by non-natural amino acids, which may contain covalent reactive groups.

[0076] For example, the amino acid sequence of the binding compound may contain a covalently reactive group at amino acid position 26 and / or amino acid position 30, compared to the corresponding amino acid sequence shown in SEQ ID NO: 3. For example, the binding compound may covalently bind to wild-type SARS-CoV-2 or its Delta or Kappa mutant strains.

[0077] For example, the binding compound may contain an amino acid sequence that, compared to the corresponding amino acid sequence shown in SEQ ID NO: 3, has amino acid positions 26 and / or 30 replaced by non-natural amino acids, which may contain covalently reactive groups. For example, the binding compound may covalently bind to wild-type SARS-CoV-2 or its Delta or Kappa mutant strains.

[0078] For example, the amino acid sequence of the binding compound may contain a covalently reactive group at position 30, compared to the corresponding amino acid sequence shown in SEQ ID NO: 3. For example, the binding compound can covalently bind to SARS-CoV-2 Beta and Omicron mutant strains.

[0079] For example, the binding compound may contain an amino acid sequence that, compared to the corresponding amino acid sequence shown in SEQ ID NO: 3, has its 30th amino acid replaced by a non-natural amino acid, which may contain a covalently reactive group. For example, the binding compound can covalently bind to SARS-CoV-2 Beta or Omicron mutant strains.

[0080] On the one hand, this application provides a nucleic acid molecule that can encode the binding compound of this application. On the other hand, this application provides an isolated nucleic acid molecule that can encode the binding compound described in this application. For example, it can be generated or synthesized by: (i) in vitro amplification, such as by polymerase chain reaction (PCR); (ii) by clonal recombination; (iii) purification, such as by enzyme digestion and gel electrophoresis fractionation; or (iv) synthesis, such as by chemical synthesis.

[0081] On the one hand, this application provides a vector that can contain the nucleic acid molecule of this application. On the other hand, this application provides a vector that can contain the nucleic acid molecule described in this application. Furthermore, the vector may also contain other genes, such as marker genes that allow selection of the vector in appropriate host cells and under appropriate conditions. Additionally, the vector may also contain expression control elements that allow the coding region to be correctly expressed in an appropriate host. Such control elements are well known to those skilled in the art and may include, for example, promoters, ribosome binding sites, enhancers, and other control elements regulating gene transcription or mRNA translation. The vector can be transformed, transduced, or transfected into host cells to express the genetic material elements it carries within the host cells. The vector may include, for example, plasmids, granules, viruses, bacteriophages, or other vectors commonly used in, for example, genetic engineering. For example, the vector may be an expression vector. Furthermore, the vector may also include components that facilitate its entry into the cell, such as viral particles, liposomes, or protein coats.

[0082] On the one hand, this application provides an immunoconjugate that may contain the binding compound of this application.

[0083] On the one hand, this application provides a cell that may contain the binding compound of this application, the nucleic acid molecule of this application, the vector of this application, and / or the immunoconjugate of this application. On the other hand, this application provides a cell that may contain the nucleic acid molecule or the vector described in this application. In some embodiments, each or every host cell may contain one or more of the nucleic acid molecules or vectors described in this application. In some embodiments, each or every host cell may contain multiple (e.g., two or more) or more types (e.g., two or more) of the nucleic acid molecules or vectors described in this application. For example, the vector described in this application may be introduced into the host cell, such as a eukaryotic cell, such as a plant cell, fungal cell, or yeast cell. In some embodiments, the cell may be a bacterial cell (e.g., Escherichia coli), a yeast cell, or other eukaryotic cells, such as COS cells, Chinese hamster ovary (CHO) cells, CHO-K1 cells, LNCAP cells, HeLa cells, 293T cells, COS-1 cells, SP2 / 0 cells, NSO cells, or myeloma cells. The vector described in this application can be introduced into the host cell by methods known in the art, such as thermal conversion, electroporation, viral infection, Ca ion conversion, lipofectine transfection, lipofectamin transfection, or other transfection reagents.

[0084] On one hand, this application provides a composition that may comprise the binding compound of this application, the nucleic acid molecule of this application, the carrier of this application, the immunoconjugate of this application, and / or the cell of this application, and optionally a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition may also comprise suitable formulations of one or more (pharmaceutically effective) adjuvants, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers, and / or preservatives. The acceptable components of the composition may be non-toxic to the recipient at the doses and concentrations used. The pharmaceutical compositions of the present invention may include, but are not limited to, liquid, freeze-dried, and lyophilized compositions.

[0085] In some embodiments, the pharmaceutical composition may also contain more than one active compound, typically those with complementary activities that do not adversely affect each other. The type and effective amount of such a drug may depend, for example, on the amount and type of antagonist present in the formulation, and on the clinical parameters of the subject.

[0086] In some embodiments, the pharmaceutically acceptable carrier may include any and all solvents, dispersion media, coatings, isotonic agents, and absorption delay agents that are compatible with drug administration and are generally safe and non-toxic.

[0087] In some embodiments, the pharmaceutical composition may be administered parenterally, percutaneously, intracavitarily, intra-arterially, intrathecally, and / or intranasally, or directly injected into tissues. For example, the pharmaceutical composition may be administered to a patient or subject by infusion or injection. In some embodiments, the pharmaceutical composition may be administered in various ways, such as intravenously, intraperitoneally, subcutaneously, intramuscularly, locally, or intradermally. In some embodiments, the pharmaceutical composition may be administered continuously. This continuous (or uninterrupted) administration may be achieved using a small pump system worn by the patient to measure the amount of therapeutic agent flowing into the patient's body.

[0088] On the one hand, this application provides a kit that may contain the binding compound of this application, the nucleic acid molecule of this application, the vector of this application, the immunoconjugate of this application, the cells of this application, and / or the composition of this application.

[0089] On one hand, this application provides the use of the conjugating compound of this application, the nucleic acid molecule of this application, the vector of this application, the immunoconjugate of this application, the cell of this application, the composition of this application, and / or the kit of this application in the preparation of medicaments for diagnosing, preventing, and / or treating diseases. For example, the disease may include an infection. For example, the disease may include a viral infection.

[0090] On one hand, this application provides a binding compound of this application, a nucleic acid molecule of this application, a vector of this application, an immunoconjugate of this application, a cell of this application, a composition of this application, and / or a kit of this application for the diagnosis, prevention, and / or treatment of diseases. For example, the disease may include an infection. For example, the disease may include a viral infection.

[0091] On one hand, this application provides a method for diagnosing, preventing, and / or treating a disease, the method comprising administering a binding compound of this application, a nucleic acid molecule of this application, a vector of this application, an immunoconjugate of this application, a cell of this application, a composition of this application, and / or a kit of this application. For example, the disease may comprise an infection. For example, the disease may comprise a viral infection.

[0092] On one hand, this application provides a method for determining the presence and / or quantity of a virus or its mutant strain, the method which may include administering the binding compound of this application, the nucleic acid molecule of this application, the vector of this application, the immunoconjugate of this application, the cells of this application, the composition of this application, and / or the kit of this application. For example, the virus or its mutant strain may comprise a coronavirus or a mutant strain thereof. For example, the virus or its mutant strain may comprise SARS-CoV-2 or a mutant strain thereof.

[0093] On one hand, this application provides a method for reducing the infection of cells by a virus or its mutant strain, the method comprising administering a binding compound of this application, a nucleic acid molecule of this application, a vector of this application, an immunoconjugate of this application, cells of this application, a composition of this application, and / or a kit of this application. For example, the virus or its mutant strain may comprise a coronavirus or a mutant strain thereof. For example, the virus or its mutant strain may comprise SARS-CoV-2 or a mutant strain thereof.

[0094] The embodiments described below are not intended to be limited by any theory, but are merely for illustrating the products, preparation methods and uses of this application, and are not intended to limit the scope of the invention.

[0095] Example

[0096] The terminology used in this application may be those commonly used in the art, such as receptor-binding domain (RBD), enzyme-linked immunosorbent assay (ELISA), ultra-high performance liquid chromatography-mass spectrometry (LC-MS), sodium dodecylbenzenesulfonate-polyacrylamide gel electrophoresis (SDS-PAGE), angiotensin-converting enzyme 2 (ACE2,3,3′,5,5′-tetramethylbenzidine), and half-inhibitory concentration (IC50). 50 .

[0097] Example 1

[0098] Select and identify covalent protein inhibitors targeting the SARS-CoV-2 RBD, such as GlueBinder (covalent LCB3).

[0099] The binding principle of the compound in this application is roughly as shown in the schematic diagram: Figure 1 The diagram shown is a schematic of the covalent protein inhibitor described in this application targeting and neutralizing the SARS-CoV-2 virus. Figure 2 The diagram shows the covalent chemical reaction between the non-natural amino acids of the covalent protein inhibitor and the nucleophilic amino acids of the spike protein.

[0100] This application selects nucleophilic amino acids of the virus-binding protein to determine candidate covalent binding sites. Figure 3A-3B show the complex structure of the wild-type SARS-CoV-2 spike protein receptor-binding domain (RBD) with the non-covalent protein inhibitor LCB1. (A) Complex structure of wild-type RBD (top) and non-covalent protein inhibitor LCB1 (bottom), with nucleophilic amino acids labeled on the RBD facing the interaction interface, including K417, Y421, Y449, Y453, Y473, Y489, and Y505. PDB: 7JZU; (B) Complex structure of wild-type RBD (top) and non-covalent protein inhibitor LCB3 (bottom), with nucleophilic amino acids labeled on the RBD facing the interaction interface, including K417, Y421, Y449, Y453, Y473, Y489, and Y505. PDB: 7JZN.

[0101] Computer-aided selection of non-natural amino acid insertion sites

[0102] Using PyMOL software, each amino acid in the non-covalent protein inhibitors LCB1 and LCB3 sequences can be mutated one by one to a tyrosine residue (Tyr), adjusting their spatial orientation to face the seven nucleophilic amino acids selected on the RBD. The distance between the phenolic hydroxyl oxygen atom of the tyrosine side chain and the nucleophilic atoms of the aforementioned nucleophilic amino acids (the amino nitrogen atom of lysine, the imidazole nitrogen atom of histidine, and the phenolic hydroxyl oxygen atom of tyrosine) after mutation is measured. When this distance is less than the crosslinking radius of the non-natural amino acid FSY, the corresponding site is selected as a candidate insertion site. By traversing all amino acids in the LCB1 and LCB3 sequences, five sites were selected for LCB1: L6, Q7, A22, D30, K37, and four sites for LCB3: D3, K26, F30, Y40.

[0103] Figure 4 A-4B shows five candidate sites on the non-covalent protein inhibitor LCB1. (A) L6, Q7, and K37; (B) A22 and D30.

[0104] Figure 5 A-5B shows four candidate sites on the non-covalent protein inhibitor LCB3. (A) D3, Y40; (B) K26, F30.

[0105] Expression and purification of non-covalent protein inhibitors

[0106] The pET-20b(+) plasmid containing the non-covalent protein inhibitors LCB1 or LCB3 was transformed into the expression host bacterium BL 21(DE3) (TIANGEN, Cat. No. CB105-02) via heat shock. The transformed bacterium was plated on solid medium containing ampicillin (100 µg / mL) and incubated overnight at 37 °C. Single colonies were selected for inoculation and cultured overnight. The next day, the overnight culture was transferred at a 1:100 ratio to fresh LB medium (1 L contains 10 g peptone, 5 g yeast extract, and 10 g sodium chloride). The culture was incubated at 37 °C with shaking until the OD value reached 0.6-0.8. Isopropyl-β-D-thiogalactoside (IPTG) was added to a final concentration of 1 mM for induction, and the culture was transferred to a 27 °C shaking incubator for 12-16 h. The cells were collected by centrifugation at 4000 rpm, discarding the supernatant. Resuspend the microbial sludge at a ratio of 15 mL of lysis solution (20 mM phosphate buffer, 150 mM sodium chloride, pH 7.4) per 1 g of sludge, and sonicate on ice for 20 min. Centrifuge at 17,000 rpm for 40 min at 4 °C to remove the precipitate and collect the supernatant. Then, purify the protein using a nickel affinity chromatography column (Sangon Biotech, Cat. No. C600792-0505), and further purify LCB1 and LCB3 using size exclusion chromatography (GE Healthcare). The purified protein was concentrated and stored in phosphate buffer containing glycerol (pH 7.4), and the purification effect was characterized by UPLC-MS.

[0107] Figure 6 A-6B shows the primary mass spectrum of a non-covalent protein inhibitor. (A) Non-covalent protein inhibitor LCB1 (A) The theoretical value of the mass spectrometer for the non-covalent protein inhibitor LCB3 was 7876 Da, and the measured value was 7876 Da; (B) The theoretical value of the mass spectrometer for the non-covalent protein inhibitor LCB3 was 9501 Da, and the measured value was 9504 Da. The results indicate that the molecular weight of the non-covalent protein inhibitor is correct and the purity is above 95%.

[0108] Non-covalent protein inhibitor LCB1:

[0109] Amino acid sequence (SEQ ID NO: 1)

[0110] DKEWILQKIYEIMRLLDELGHAEASMRVSDLIYEFMKKGDERLLEEAERLLEEVER

[0111] Encoding DNA sequence (SEQ ID NO: 2)

[0112] ATGGCAGACAAGGAATGGATTCTGCAGAAAATTTATGAAATTATGAGATTACTTGATGAGCTGGGCCATGCAGAAGCAAGCATGAGAGTGAGTGATCTGATTTATGAATTTATGAAAAAAGGTGATGAGAGACTGCTGGAAGAGGCAGAAAGACTGTTGGAAGAAGTGGAAAGAGGTGGTGGCCATCATCATCATCATCATTAA

[0113] Non-covalent protein inhibitor LCB3:

[0114] Amino acid sequence (SEQ ID NO: 3)

[0115] NDDELHMLMTDLVYEALHFAKDEEIKKRVFQLFELADKAYKNNDRQKLEKVVEELKELLERLLS

[0116] Encoding DNA sequence (SEQ ID NO: 4)

[0117] ATGGCAAATGACGACGAATTGCATATGTTAATGACTGATTTAGTATATGAGGCCCTGCACTTCGCAAAAGATGAAGAAATTAAAAAACGGGTGTTCCAGCTCTTTGAACTTGCTGATAAAGCGTACAAGAATAATGACCGCCAAAAGCTTGAAAAAGTCGTTGAAGAATTAAAGGAACTTCTCGAACGCCTCTTATCTGGTGGCGGTGGGAGCCTCCCGGAAACGGGCGGGCACCACCATCACCACCATTAA

[0118] Construction of amber codon mutant plasmids

[0119] Double-stranded nucleic acids (SEQ ID NO: 2 or 4) containing expression sequences of non-covalent protein inhibitors LCB1 and LCB3 were synthesized and cloned into the pBAD / His (Thermo, Cat. No. V43001) vector by double digestion with endonucleases NcoI and XhoI. Sequencing was performed using pBAD-Forward as the sequencing primer to obtain the correct vector for subsequent expression and construction of single-domain antibodies.

[0120] The PCR polymerase (Novazia, Cat. No. P505-d1) was used to introduce the amber codon TAG mutation into the expression sequence of a non-covalent protein inhibitor via overlay linear amplification PCR for the insertion of non-natural amino acids.

[0121] The primers used for PCR are as follows:

[0122]

[0123] Add 3 µL of the amplification product to 100 µL of DH5α (TIANGEN, Cat. No. CB101-02) chemocompetent cells, heat shock at 42 °C for 1 min, then incubate on ice for recovery. Finally, plate the product onto a plate containing ampicillin (100 µg / mL) and incubate overnight at 37 °C. Randomly select at least three single colonies and sequence them using pBAD-Forward as the sequencing primer. Select the correct construct for the expression and purification of the non-natural amino acid-intercalated single-domain antibody.

[0124] Expression and purification of non-natural amino acid-intercalated single-domain antibodies

[0125] Protein inhibitor expression vector plasmids with amber codon mutations were co-transformed with aminoacyl-tRNA synthetase / tRNA vector plasmids into host bacteria DH10B (Zhuangmeng Biotechnology, Cat. No. ZC112-2) via heat shock. The transformed plasmids were plated on solid media containing ampicillin (100 µg / mL) and chloramphenicol (34 µg / mL) and incubated overnight at 37 °C. A single colony from each plate was inoculated into LB medium containing antibiotics and cultured at 37 °C, 220 rpm for 12 h. Subsequently, the colonies were transferred 1:100 to fresh LB medium and cultured at 37 °C with shaking until the OD value reached 0.6-0.8. Then, 1 mM of the non-natural amino acid FSY was added, and the culture was continued at 37 °C for 30 min. Finally, 0.2% arabinose was added to induce protein expression, and the culture was transferred to a shaker at 27 °C for 12-16 h. Centrifuge at 4000 rpm, discard the supernatant culture medium, and collect the bacterial cells. Resuspend in lysis buffer (20 mM phosphate buffer, 150 mM sodium chloride, pH 7.4), sonicate on wet ice, centrifuge at 17000 rpm for 40 min at 4 ℃, remove the precipitate, and collect the supernatant. Then, purify the protein using a nickel affinity chromatography column (Sangon Biotech, Cat. No. C600792-0505), and further purify the protein inhibitors using size exclusion chromatography (GE Healthcare). The purified protein is stored in glycerol-containing phosphate buffer (pH 7.4), and the purification effect is characterized by UPLC-MS and SDS-PAGE.

[0126] Figure 7 A-7F show the characterization results of LCB1-based candidate covalent protein inhibitors. (A) L6, (B) Q7, (C) A22, (D) D30, (E) Molecular weight of the purified candidate covalent protein inhibitor with the non-natural amino acid FSY inserted at position K37 in primary mass spectrometry. (F) SDS-PAGE gel electrophoresis characterizes the purity of the LCB1-based candidate covalent protein inhibitors.

[0127] Figure 8 A-8E show the molecular weights of LCB3-based candidate covalent protein inhibitors after purification by primary mass spectrometry for (A) D3, (B) K26, (C) D30, and (D) Y40 insertion of the non-natural amino acid FSY. (E) SDS-PAGE gel electrophoresis characterizes the purity of the LCB3-based candidate covalent protein inhibitors.

[0128] In vitro cross-linking of non-naturally intercalated amino acid protein inhibitors with RBD proteins: Protein inhibitors with intercalated non-natural amino acid FSY were incubated with SARS-CoV-2 RBD proteins (including wild-type, Beta mutant, Delta mutant, Kappa mutant, and Omicron mutant) in phosphate buffer (pH 7.4) at a molar ratio of 10:1 at 37°C for 12 h. The mass of RBD was 2 μg. 5× protein loading buffer (Kangwei Century, Cat. No. CW0027) was added to the reaction system, and the mixture was heated at 95°C for 10 min. Samples were separated by 8–16% SDS-PAGE gel electrophoresis and then stained with Coomassie Brilliant Blue. Destaining was performed using destaining solution to identify cross-linking.

[0129] Figure 9 A-9E show the results of denaturing SDS-PAGE gel electrophoresis analysis of the cross-linking reaction between five candidate covalent protein inhibitors of LCB1 and the SARS-CoV-2 RBD protein. (A) In vitro cross-linking results with wild-type RBD protein; (B) In vitro cross-linking results with Beta mutant RBD protein; (C) In vitro cross-linking results with Delta mutant RBD protein; (D) In ​​vitro cross-linking results with Kappa mutant RBD protein; (E) In vitro cross-linking results with Omicron mutant RBD protein.

[0130] Compared to the non-covalent protein inhibitor LCB1, the covalent protein inhibitor with the non-natural amino acid FSY inserted at position A22 produced a new high-molecular-weight band in the system after incubation with RBD protein, indicating that a new covalent interaction was formed between the protein inhibitor and RBD protein. This covalent binding was not affected by the denaturing agent SDS.

[0131] Figure 10 A-10E shows the results of denaturing SDS-PAGE gel electrophoresis analysis of cross-linking reactions between four candidate covalent protein inhibitors based on LCB3 and the SARS-CoV-2 RBD protein. (A) In vitro cross-linking results with wild-type RBD protein; (B) In vitro cross-linking results with Beta mutant RBD protein; (C) In vitro cross-linking results with Delta mutant RBD protein; (D) In ​​vitro cross-linking results with Kappa mutant RBD protein; (E) In vitro cross-linking results with Omicron mutant RBD protein.

[0132] Compared to the non-covalent protein inhibitor LCB3, the covalent protein inhibitor with the non-natural amino acid FSY inserted at the F30 position produced new high-molecular-weight bands after incubation with RBD proteins, indicating that a new covalent interaction was formed between the protein inhibitor and the RBD protein. This covalent binding was unaffected by the denaturing agent SDS. The insertion of the non-natural amino acid FSY at the F30 position in the protein inhibitor LCB3 showed the highest covalent cross-linking efficiency for various RBD proteins; therefore, it was renamed the covalent protein inhibitor LCB3 (covalent LCB3), or GlueBinder.

[0133] Time and concentration dependence of the cross-linking reaction between covalent protein inhibitors and RBD proteins

[0134] Covalently coupled LCB3 was incubated with SARS-CoV-2 RBD proteins (including wild-type, Beta mutant, Delta mutant, Kappa mutant, and Omicron mutant) in phosphate buffer (pH 7.4) at molar ratios of 1:1, 2:1, 5:1, and 10:1 at 37°C for 2 h, 5 h, and 12 h, respectively. The mass of RBD was 2 μg. 5× protein loading buffer (Kangwei Century, Cat. No. CW0027) was added to the reaction system, and the mixture was heated at 95°C for 10 min. Samples were separated by 8–16% SDS-PAGE gel electrophoresis and then stained with Coomassie Brilliant Blue. Destaining was performed to identify cross-linking.

[0135] Figure 11 A-11E shows the relationship between the efficiency of the cross-linking reaction between covalent LCB3 and the SARS-CoV-2 RBD protein and time and concentration, analyzed by denaturing SDS-PAGE gel electrophoresis. (A) Cross-linking results with wild-type RBD protein; (B) Cross-linking results with Beta mutant RBD protein; (C) Cross-linking results with Delta mutant RBD protein; (D) Cross-linking results with Kappa mutant RBD protein; (E) Cross-linking results with Omicron mutant RBD protein.

[0136] Covalent LCB3 can efficiently undergo covalent cross-linking reactions with various RBD proteins, thus possessing the ability to resist viral mutations.

[0137] Multistage mass spectrometry identifies the cross-linking sites between covalently bound LCB3 and RBD protein.

[0138] On clean plastic wrap, using a new blade, the cross-linked bands on the gel were cut into 1 mm square pieces. The gel particles were treated three times with 50% acetonitrile (Fisher, Cat. No. 955-4) aqueous solution to remove residual Coomassie Brilliant Blue staining solution, followed by dehydration with pure acetonitrile. Subsequently, disulfide bonds in the protein were reduced to a free state with dithiothreitol, and the reduced cysteine ​​sulfhydryl groups were modified with iodoacetyl. The water in the gel particles was again removed with pure acetonitrile. The particles were then proteased into peptides using chymotrypsin (Promega, Cat. No. V1061), extracted with 0.1% formic acid, desalted, and then analyzed by tandem mass spectrometry. Tandem mass spectrometry was performed using a Thermo QE Plus or LUMOS triplet mass spectrometer, with a mobile phase of 0.1% formic acid aqueous solution and 0.1% formic acid acetonitrile, and a gradient time of 90 min. The cross-linked peptides identified by pLink2 software were searched, and the amino acid sites on the RBD protein that can form covalent bonds with the covalently cross-linked LCB3 were determined.

[0139] Figure 12 A-12C shows the tandem mass spectrum of the cross-linked peptide after the reaction of covalent LCB3 with RBD protein. (A) Wild-type RBD protein; (B) Delta mutant RBD protein; (C) Tandem mass spectra of the cross-linked product of Delta mutant RBD protein and covalent LCB3 all show that the non-natural amino acid FSY at position 30 of covalent LCB3 forms a covalent bond with tyrosine residue 473 of RBD protein, where U represents the non-natural amino acid FSY.

[0140] Example 2

[0141] In vitro characterization of covalent protein inhibitors

[0142] This embodiment provides the binding affinity test results of covalent protein inhibitors to various SARS-CoV-2 RBD proteins and the results of blocking human ACE2-RBD protein interactions. Compared with non-covalent LCB3, the transient binding affinity of covalent LCB3 to RBD proteins is not affected by non-natural amino acid insertions, and it has an enhanced ability to block ACE2-RBD protein interactions.

[0143] Determination of the binding affinity between covalent LCB3 and RBD protein

[0144] The parameters of the Fortebio Octet Red 96 biomembrane interference molecular interaction analyzer were set to 30 °C and 1000 rpm oscillation. Streptavidin probes (Fortebio, Cat. No. 18-5020) were used in the assay. Wild-type RBD, Delta mutant RBD, and Kappa mutant RBD were first biotinylated using 5 equivalents of biotinylate succinimide active ester. Excess biotin-NHS was then removed by desalting using a desalting column (Bio-rad, Cat. No. 7326221), and the biotinylated RBD was diluted to 8 μg / ml using equilibration buffer (10 mM phosphate buffer, 0.05% Tween-20, 0.5% bovine serum albumin).

[0145] The test samples (non-covalent LCB3 and covalent LCB3) were diluted with equilibration buffer to 5 nM, 2.5 nM, 0.63 nM, and 0.31 nM. Dissociation constant K. D The assay was performed as follows: (1) Sensor check: Immerse the sensor in equilibration buffer for 60 seconds; (2) RBD loading: Immerse the sensor in biotinylated RBD solution for 400 seconds; (3) Baseline determination: Immerse the sensor in equilibration buffer for 120 seconds; (4) Binding: Immerse the sensor in different concentrations of the test sample for 1800 seconds; (5) Dissociation: Immerse the sensor in equilibration buffer for 1000 seconds; (6) Washing: Immerse the sensor in washing buffer 1 (0.1 M glycine, pH 2.2) for 5 seconds, then in washing buffer 2 (10 mM phosphate buffer, 0.05% Tween-20, 0.5% bovine serum albumin) for 5 seconds, for 5 cycles. Curve fitting was performed using Fortebio Octet Data analysis software.

[0146] Figure 13 A-13F shows that the insertion of the non-natural amino acid FSY does not affect the binding affinity of the protein inhibitor to the RBD protein. The dissociation constant K of non-covalent LCB3 and SARS-CoV-2 (A) wild-type RBD is also shown. D = 0.12 nM, (C) Dissociation constant K of Delta mutant RBD D = 0.11 nM, and the dissociation constant K of the (E) Kappa mutant RBD. D = 0.52 nM; Dissociation constant K of covalent LCB3 and wild-type RBD of SARS-CoV-2 (B) D = 0.12 nM, (D) Dissociation constant K of Delta mutant RBD D =0.15 nM, and the dissociation constant K of (F) Kappa mutant RBD.D = 0.20 nM.

[0147] Determination of the ability of covalent LCB3 to block RBD-ACE2 interaction

[0148] The SARS-CoV-2 RBD protein (wild-type, Delta mutant, and Kappa mutant) was added to 96-well microplates (Corning, Cat. No. 3590) at a concentration of 2 μg / mL, 200 μL per well, and incubated overnight at 4 °C to coat the microplates with the RBD protein. The plates were then washed three times with phosphate-buffered saline (PPS). Next, each well was blocked with 300 μL of 2% bovine serum albumin in PPS for 2 hours at room temperature, followed by one wash with PPS.

[0149] The test samples (non-covalent LCB3 and covalent LCB3) were serially diluted with assay buffer (0.1% bovine serum albumin, 0.05% Tween-20). The diluted sample (50 μL / well) was mixed with 50 μL of 0.5 μg / mL human ACE2-mouse Fc protein (Sinochem, Cat. No. 10108-H05H) and incubated at room temperature for 3.5 hours. The plate was washed three times with assay buffer. 100 μL of horseradish peroxidase-conjugated goat anti-mouse secondary antibody (1:2500 dilution) was added to each well and incubated at room temperature for 1 hour. The plate was then washed four times with phosphate buffer, and 100 μL of TMB solution was added to each well under dark conditions and incubated at room temperature. At the desired time, 50 μL of 2 M sulfuric acid was added to terminate the reaction. The absorbance at 450 nm was immediately measured using a microwell detector. A valid assay value requires an absorbance greater than 2.1 times the background absorbance.

[0150] Figure 14 A-14C shows that, compared to non-covalent LCB3, covalent LCB3 has a stronger blocking ability against the interaction of SARS-CoV-2 with (A) wild-type RBD, (B) Delta mutant RBD, and (C) Kappa mutant RBD, and human ACE2, with a half-inhibitory concentration (IC50). 50 The value is reduced to 1 / 10 to 1 / 8 of that of non-covalent LCB3.

[0151] Example 3

[0152] Neutralization of live viruses by covalent protein inhibitors

[0153] Vero E6 cells were cultured in MEM medium (Gibco, Invitrogen) containing 2% fetal bovine serum at a constant temperature of 37°C and 5% CO2. Cells were spaced at 1 × 10⁶ cells per well. 5Cells were seeded at a density of [number] cells / well in 48-well cell culture plates. Neutralization reagents (non-covalent and covalent LCB3) were serially diluted and incubated with the SARS-CoV-2 Delta mutant strain (provided by the National Virus Resource Center, CSTR.16698.06.NPRC 6.CCPM-BV-049-2105-8) at 37 °C for 2 hours. The original culture medium in the wells was discarded, and the cells were infected with the virus-neutralization reagent mixture at a multiplicity of infection (MOI) of 0.05. After incubation at 37 °C for 1 hour, the supernatant was discarded, cells were washed twice with phosphate-buffered saline, and cultured in fresh culture medium. The cell supernatant was then collected for RNA extraction and real-time PCR quantification. All infection experiments were performed in a biosafety level 3 (BSL-3) laboratory.

[0154] Figure 15 The graph shows the results demonstrating that covalent LCB3 exhibits a stronger neutralizing ability against the SARS-CoV-2 Delta mutant compared to non-covalent LCB3. The IC50 of covalent LCB3 is also shown. 50 The value was 0.019 ± 0.003 nM; the IC50 of non-covalent LCB3 was 0.019 ± 0.003 nM. 50 The value is 0.121 ± 0.022 nM.

[0155] Example 4

[0156] The starting concentration used to evaluate the stability of covalent and non-covalent LCB3 to atomization, lyophilization, and heat treatment was 0.5 mg / mL. For atomization, both covalent and non-covalent LCB3 were atomized using a portable mesh nebulizer (Yuwell) to produce 3–8 µm particles. The resulting aerosols were collected after cooling on ice using a condenser. For lyophilization, both covalent and non-covalent LCB3 were rapidly frozen in liquid nitrogen using a lyophilization protectant (containing 5% trehalose, 10% mannitol, and 0.01% Tween-80), followed by complete drying under vacuum. The resulting lyophilized proteins were resuspended in phosphate buffer. For heat stability testing, both covalent and non-covalent LCB3 were incubated at 4 °C, 25 °C, 37 °C, and 60 °C for 12 hours, respectively. The binding of both to the Delta mutant RBD protein was assessed using a biomembrane interferometer.

[0157] Figure 16 Figure A-16F shows that covalent LCB3 retained its activity after atomization and lyophilization, exhibiting the same stability results as non-covalent LCB3. Biomembrane interferometry analysis showed that (A, C) non-covalent LCB3 and (B, D) covalent LCB3, compared with the untreated control, retained high binding affinity to the RBD protein of the SARS-CoV-2 Delta mutant after atomization or lyophilization.

[0158]

[0159] As shown in the table above, covalent LCB3 can maintain its activity after heat treatment and has higher stability than non-covalent LCB3 at 60 °C.

[0160] The foregoing detailed description is provided by way of explanation and example and is not intended to limit the scope of the appended claims. Various variations of the embodiments listed herein will be apparent to those skilled in the art and are reserved within the scope of the appended claims and their equivalents.

Claims

1. A binding compound capable of specifically binding to a virus or a mutant strain thereof and covalently binding to the virus or the mutant strain thereof, wherein, the virus or the mutant strain thereof is SARS-CoV-2 or a mutant strain thereof selected from the group consisting of: Beta variant, Delta variant, Kappa variant, and Omicron variant; the amino acid sequence of the binding compound comprises a fluorosulfate-L-tyrosine (FSY) residue at the 22nd amino acid compared to the corresponding amino acid sequence set forth in SEQ ID NO: 1, or the amino acid sequence of the binding compound comprises a fluorosulfate-L-tyrosine (FSY) residue at the 30th amino acid compared to the corresponding amino acid sequence set forth in SEQ ID NO:

3.

2. The binding compound of claim 1, capable of specifically binding to a spike protein or a variant thereof of the virus or the mutant strain thereof.

3. The binding compound of claim 1, capable of specifically binding to a receptor binding domain (RBD) of a spike protein of the virus or the mutant strain thereof.

4. The binding compound of claim 1, capable of reacting with a nucleophilic group at the interface of the binding to form a covalent bond.

5. The binding compound of claim 1, capable of reacting with a nucleophilic group to form a covalent bond when the distance between the covalent reactive group and the nucleophilic group is less than 10E-10 meters.

6. The binding compound of claim 1, wherein the distance between a nucleophilic amino acid at the interface of the binding and the covalent reactive group of the binding compound is less than 10E-10 meters when the binding compound binds to the virus or the mutant strain thereof.

7. The binding compound of claim 1, derived from a polypeptide capable of non-covalently binding to the virus or the mutant strain thereof.

8. The binding compound of claim 1, wherein the polypeptide is capable of binding to a receptor binding domain of a spike protein of the virus or the mutant strain thereof with a KD value of less than 1 nM.

9. A nucleic acid molecule encoding the binding compound of any one of claims 1-8.

10. A vector comprising the nucleic acid molecule of claim 9.

11. A cell comprising the binding compound of any one of claims 1-8, the nucleic acid molecule of claim 9, and / or the vector of claim 10.

12. A composition comprising the binding compound of any one of claims 1-8, the nucleic acid molecule of claim 9, the vector of claim 10, and / or the cell of claim 11, and a pharmaceutically acceptable carrier.

13. A kit comprising the binding compound of any one of claims 1-8, the nucleic acid molecule of claim 9, the vector of claim 10, the cell of claim 11, and / or the composition of claim 12.

14. Use of the binding compound of any one of claims 1-8, the nucleic acid molecule of claim 9, the vector of claim 10, the cell of claim 11, the composition of claim 12, and / or the kit of claim 13 in the manufacture of a medicament for the diagnosis, prevention, and / or treatment of a disease, wherein, The disease is a viral infection, the virus is SARS-CoV-2 or a mutant strain thereof selected from the group consisting of: Beta variant, Delta variant, Kappa variant and Omicron variant.

15. A method of determining the presence and / or amount of a virus or mutant strain thereof, said method comprising administering the binding compound of any one of claims 1-8, the nucleic acid molecule of claim 9, the vector of claim 10, the cell of claim 11, the composition of claim 12, and / or the kit of claim 13, said method being a method for non-therapeutic and non-diagnostic purposes, wherein, The virus or the mutant strain thereof is SARS-CoV-2 or a mutant strain thereof selected from the group consisting of: Beta variant, Delta variant, Kappa variant and Omicron variant.

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