Method for screening substances capable of inhibiting SARS-cov-2 invasion or infection of human cells

Abstract

Screening method SOLUTION: A method for screening for a substance capable of inhibiting invasion or infection of human cells with SARS-CoV-2, the method comprising the following steps (I) to (iii), wherein in the step (iii), a difference in the compared signals indicates that a candidate substance is highly likely to be a substance capable of inhibiting invasion or infection of human cells with SARS-CoV-2: (i) subjecting ACE2 to NMR spectrum measurement in the presence of a candidate substance to obtain an NMR signal (ii) subjecting ACE2 to NMR spectrum measurement in the absence of the candidate substance to obtain an NMR signal (iii) comparing signals derived from amino acid residues involved in binding between subunits of ACE2 dimers among the signals obtained in the steps (i) and (ii) SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for screening for a substance capable of inhibiting the entry or infection of SARS-CoV-2 into human cells. [Background technology]

[0002] COVID-19 (novel coronavirus disease) spread throughout the world in 2020, and there is still a threat of the emergence of mutant strains. Therefore, there is a need to develop drugs that specifically inhibit the invasion or infection of SARS-CoV-2.

[0003] Entry of the SARS-CoV-2 virus into human cells is triggered by protein-protein interaction (PPI) between the receptor-binding domain (SP-RBD) of the SARS-CoV-2 spike protein and angiotensin-converting enzyme 2 (ACE2), a viral receptor on human cells. Non-Patent Document 1 reports that mutations that modulate ACE2 dimer formation reduce the infectivity of SARS-CoV-2. Non-Patent Document 2 reports that mutations near the ACE2 dimer interface allosterically affect the PPI interface with SP-RBD. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Zhu J. et al, Frontiers in Virology Front. Virol., 04 July 2022 Sec. Antivirals and Vaccines Volume 2 - 2022. https: / / doi.org / 10.3389 / fviro.2022.916700 [Non-patent document 2] Calvaresi et al, Nature Communications volume 14, Article number: 1421 (2023). https: / / doi.org / 10.1038 / s41467-023-36745-0 Summary of the Invention [Problem to be solved by the invention]

[0005] However, the above literature does not report that inhibition of PPIs between SP-RBD and ACE2 inhibits viral entry.

[0006] Based on the changes in the NMR spectrum of ACE2 upon compound addition, the inventors identified that the binding site of compounds capable of inhibiting SARS-CoV-2 infection of human cells is not at the PPI interface with the SP-RBD, but at the amino acid site where the subunits of the ACE2 dimer are in close proximity. The inventors also successfully demonstrated that compounds capable of inhibiting SARS-CoV-2 infection of human cells bind to the ACE2 dimer interface and affect the dimer structure, thereby inhibiting infection, demonstrating a completely new mechanism of action as PPI inhibitors. This led to the development of a unique drug discovery strategy to search for compounds with this mechanism of action.

[0007] Therefore, an objective of the present invention is to provide a novel method for screening for substances that can inhibit SARS-CoV-2 entry into or infection of human cells.A further objective of the present invention is to provide a novel method for screening for substances that can inhibit SARS-CoV-2 entry into or infection of human cells by inhibiting ACE2 dimerization. [Means for solving the problem]

[0008] The present invention encompasses the embodiments described below.

[0009] Section 1. A method for screening for a substance capable of inhibiting SARS-CoV-2 entry into or infection of human cells, comprising the following steps (i) to (iii): (i) performing NMR spectroscopy on ACE2 in the presence of a candidate substance to obtain an NMR signal; (ii) performing NMR spectroscopy on ACE2 in the absence of the candidate substance to obtain an NMR signal; (iii) comparing the signals obtained in steps (i) and (ii) with those derived from amino acid residues involved in the interaction between the subunits of the ACE2 dimer; A screening method in which, if there is a difference in the compared signals in step (iii), this indicates that the candidate substance is likely to be a substance that can inhibit SARS-CoV-2 entry or infection into human cells.

[0010] Section 2. Item 2. The screening method according to Item 1, wherein the amino acid residues involved in the interaction between the subunits of the ACE2 dimer include at least one amino acid residue in the regions of residue numbers 131 to 141 and 157 to 178 of ACE2.

[0011] Section 3. Item 2. The screening method according to Item 1, wherein the amino acid residues involved in the interaction between the subunits of the ACE2 dimer include at least one selected from the group consisting of tryptophan 163, glutamine 139, and glutamine 175.

[0012] Section 4. After the step (iii), (iv) contacting the animal cells with the SARS-CoV-2 virus; (v) contacting the animal cell with the candidate substance; and (vi) measuring the amount of SARS-CoV-2 RNA in the animal cells after contact with the SARS-CoV-2 virus and the candidate substance. Item 1. The screening method according to Item 1, further comprising:

[0013] Section 5. Item 2. The screening method according to Item 1, wherein the ACE2 is human-derived ACE2.

[0014] Section 6. The ACE2 2 H and 15 Item 1. The screening method according to Item 1, wherein the ACE2 is N-labeled.

[0015] Section 7. Item 2. The screening method according to Item 1, wherein the NMR spectroscopy of ACE2 in steps (i) and (ii) is NMR spectroscopy of a solution containing ACE2.

[0016] Section 8. Item 2. The screening method according to Item 1, wherein step (i) is a step of performing NMR spectroscopy on ACE2 in the presence of multiple concentrations of the candidate substance to obtain an NMR signal.

[0017] Section 9. An inhibitor of SARS-CoV-2 entry into or infection of human cells, comprising a compound represented by the following formula (I), a derivative thereof, or a salt thereof:

[0018] [ka] [Effects of the Invention]

[0019] According to the present invention, a novel screening method for substances that can inhibit SARS-CoV-2 entry into or infection of human cells can be provided. [Brief explanation of the drawings]

[0020] [Figure 1] NMR spectrum changes of ACE2 before and after addition of compound 1. [Figure 2] Mapping the binding site of compound 1 onto the ACE2 monomer structure [Figure 3]Graph of chemical shift overlay in two-dimensional spectrum measurements before and after the addition of compound 2. [Figure 4] A graph showing the effect of adding compound 1 on inhibiting infection of various SARS-CoV-2 strains in human cells. [Figure 5] A graph showing the effect of adding compound 2 on inhibiting infection of various SARS-CoV-2 strains in human cells. [Figure 6] Experiments on reducing SARS-CoV-2 infectivity to live virus by ACE2 mutants [Figure 7] Experiments on reducing SARS-CoV-2 infectivity by ACE2 mutants DETAILED DESCRIPTION OF THE INVENTION

[0021] As used herein, "polynucleotide" can be alternatively referred to as "nucleic acid" or "nucleic acid molecule," and refers to a polymer of nucleotides. Furthermore, "base sequence" can be alternatively referred to as "nucleic acid sequence" or "nucleotide sequence," and unless otherwise specified, polynucleotides can exist in the form of DNA or RNA. Examples of DNA include cDNA or genomic DNA. DNA may be double-stranded or single-stranded. Examples of RNA include mRNA.

[0022] As used herein, the term "protein" can also be referred to as "polypeptide."

[0023] The protein described herein may be a polypeptide formed by peptide bonds between amino acids, but is not limited thereto, and may also include structures other than polypeptides. Examples of structures other than polypeptides referred to here include, but are not limited to, sugar chains and isoprenoid groups.

[0024] According to one aspect of the present invention, there is provided a method for screening for a substance capable of inhibiting SARS-CoV-2 entry into or infection of human cells, comprising the following steps (i) to (iii): (i) performing NMR spectroscopy on ACE2 in the presence of a candidate substance to obtain an NMR signal; (ii) performing NMR spectroscopy on ACE2 in the absence of the candidate substance to obtain an NMR signal; (iii) comparing the signals obtained in steps (i) and (ii) with those derived from amino acid residues involved in the interaction between the subunits of the ACE2 dimer; A screening method is provided in which, in step (iii), if there is a difference in the compared signals, this indicates that the candidate substance is likely to be a substance capable of inhibiting SARS-CoV-2 entry or infection into human cells.

[0025] As used herein, SARS-CoV-2 (Severe acute respiratory syndrome coronavirus 2) is a coronavirus belonging to the SARS-associated coronaviruses, and is an enveloped single-stranded positive-sense RNA virus.

[0026] In the present specification, the human cells are not particularly limited, and may be, for example, cells collected from biological tissues or cultured cells.

[0027] As used herein, ACE2 refers to angiotensin-converting enzyme 2. Angiotensin-converting enzyme 2 may have the sequence of a native protein or may be mutated. Such mutants include those capable of forming dimers and having the activity of angiotensin-converting enzyme 2 with 90% or more sequence identity, preferably 95% or more sequence identity, and preferably 99% or more sequence identity with the native sequence of angiotensin-converting enzyme 2. For measurement by NMR, stable isotopes ( 1 H, 2 H( 2 Also written as D), 13 C. 15 ACE2 labeled with one or more of the following: N, etc.

[0028] The amino acid sequences of natural ACE2 proteins are, for example, SEQ ID NO: 1 (Uniprot Q9BYF1) for humans, SEQ ID NO: 2 (Uniprot Q8R0I0) for mice, and SEQ ID NO: 3 (Uniprot Q5EGZ1) for rats.

[0029] Human ACE2 (SEQ ID NO: 1) MSSSSWLLLSLVAVTAAQSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQ NMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTIL NTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLY EEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVEHTFEEIKPLYEHL HAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQ AWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILM CTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKS IGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEM KREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLH KCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNK NSFVGWSTDWSPYADQSIKVRISLKSALGDKAYEWNDNEMYLFRSSVAYAMRQYFLKVKN QMILFGEEDVRVANLKPRISFNFFVTAPKNVSDIIPRTEVEKAIRMSRSRINDAFRLNDN SLEFLGIQPTLGPPNQPPVSIWLIVFGVVMGVIVVGIVILIFTGIRDRKKKNKARSGENP YASIDISKGENNPGFQNTDDVQTSF

[0030] Mouse ACE2 (SEQ ID NO: 2) MSSSSWLLLSLVAVTTTAQSLTEENAKTFLNNFNQEAEDLSYQSSLASWNYNTNITEENAQ KMSEAAAKWSAFYEEQSKTAQSFSLQEIQTPIIKRQLQALQQSGSSALSADKNKQLNTIL NTMSTIYSTGKVCNPKNPQECLLLEPGLDEIMATSTDYNSRLWAWEGWRAEVGKQLRPLY EEYVVLKNEMARANNYNDYGDYWRGDYEAEGADGYNYNRNQLIEDVERTFAEIKPLYEHL HAYVRRKLMDTYPSYISPTGCLPAHLLGDMWGRFWTNLYPLTVPFAQKPNIDVTDAMMNQ GWDAERIFQEAEKFFVSVGLPHMTQGFWANSMLTEPADGRKVVCHPTAWDLGHGDFRIKM CTKVTMDNFLTAHHEMGHIQYDMAYARQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKS IGLLPSDFQEDSETEINFLLKQALTIVGTLPFTYMLEKWRWMVFRGEIPKEQWMKKWWEM KREIVGVVEPLPHDETYCDPASLFHVSNDYSFIRYYTRTIYQFQFQEALCQAAKYNGSLH KCDISNSTEAGQKLLKMLSLGNSEPWTKALENVVGARNMDVKPLLNYFQPLFDWLKEQNR NSFVGWNTEWSPYADQSIKVRISLKSALGANAYEWTNNEMFLFRSSVAYAMRKYFSIIKN QTVPFLEEDVRVSDLKPRVSFYFFVTSPQNVSDVIPRSEVEDAIRMSRGRINDVFGLNDN SLEFLGIHPTLEPPYQPPVTIWLIIFGVVMALVVVGIIILIVTGIKGRKKKNETKREENP YDSMDIGKGESNAGFQNSDDAQTSF

[0031] Rat ACE2 (SEQ ID NO: 3) MSSSCWLLLSLVAVATAQSLIEEKAESFLNKFNQEAEDLSYQSSLASWNYNTNITEENAQ KMNEAAAKWSAFYEEQSKIAQNFSLQEIQNATIKRQLKALQQSGSSALSPDKNKQLNTIL NTMSTIYSTGKVCNSMNPQECFLLEPGLDEIMATSTDYNRRLWAWEGWRAEVGKQLRPLY EEYVVLKNEMARANNYEDYGDYWRGDYEAEGVEGYNYNRNQLIEDVENTFKEIKPLYEQL HAYVRTKLMEVYPSYISPTGCLPAHLLGDMWGRFWTNLYPLTTPFLQKPNIDVTDAMVNQ SWDAERIFKEAEKFFVSVGLPQMTPGFWTNSMLTEPGDDRKVVCHPTAWDLGHGDFRIKM CTKVTMDNFLTAHHEMGHIQYDMAYAKQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKS IGLLPSNFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFQDKIPREQWTKKWWEM KREIVGVVEPLPHDETYCDPASLFHVSNDYSFIRYYTRTIYQFQFQEALCQAAKHDGPLH KCDISNSTEAGQKLLNMLSLGNSGPWTLALENVVGSRNMDVKPLLNYFQPLFVWLKEQNR NSTVGWSTDWSPYADQSIKVRISLKSALGKNAYEWTDNEMYLFRSSVAYAMREYFSREKN QTVPFGEADVWVSDLKPRVSFNFFVTSPKNVSDIIPRSEVEEAIRMSRGRINDIFGLNDN SLEFLGIYPTLKPPYEPPVTIWLIIFGVVMGTVVVGIVILIVTGIKGRKKKNETKREENP YDSMDIGKGESNAGFQNSDDAQTSF The organism from which ACE2 is derived for use in the NMR spectroscopy of the present invention is not particularly limited as long as it forms a dimeric subunit, but is preferably a mammal, such as a human, mouse, rat, pig, monkey, rabbit, cat, dog, cow, horse, or sheep, with human origin being preferred.

[0032] Intermolecular interactions between the subunits of the ACE2 dimer include non-covalent interactions, which include hydrogen bonds, van der Waals force bonds, or both between the subunits of the ACE2 dimer.

[0033] In the present invention, the candidate substance is not particularly limited, and may be a low molecular weight compound or a high molecular weight compound.

[0034] In this specification, a low molecular weight compound refers to a compound with a molecular weight of less than 500, a medium molecular weight compound refers to a compound with a molecular weight of 500 or more and 2,000 or less, and a high molecular weight compound refers to a compound with a molecular weight of more than 2,000.

[0035] The candidate substance may be at least one selected from the group consisting of low molecular weight compounds, nucleic acids, and proteins. From the viewpoints of ease of NMR analysis, ease of synthesis, ease of availability, etc., the candidate substance is preferably a low molecular weight compound or a medium molecular weight compound.

[0036] In some embodiments, the NMR spectroscopy of ACE2 in steps (i) and (ii) is NMR spectroscopy of a solution containing ACE2.

[0037] In a preferred embodiment, the NMR spectroscopy is a two-dimensional NMR spectroscopy, and in a more preferred embodiment, 1 H and 15 This is an NMR spectrum measurement using N. 1 H and 15 By using two stable isotopes of N, 1 H- 15 In the N HSQC spectrum, only hydrogen atoms adjacent to nitrogen atoms are observed, so the positions and intensities of correlation peaks originating from the main chain amide hydrogen atoms of proteins and the side chains of some amino acids, as well as their changes, can be analyzed from the chemical structure of the amino acids. 1 H and 13 There is NMR spectroscopy using C.

[0038] In a preferred embodiment, in step (i), the NMR signal obtained in the presence of the candidate substance is 1 H and 15 The chemical shift of N is mapped two-dimensionally as the horizontal axis and the vertical axis, respectively, and in step (ii), the NMR signal obtained in the absence of the candidate substance is 1 H and 15 The chemical shifts of N are mapped two-dimensionally as the horizontal and vertical axes, respectively, and in step (iii), the two two-dimensional maps are compared, thereby making it possible to identify each amino acid residue for which a chemical shift change is observed on the map in the presence and absence of the candidate substance.

[0039] If the chemical shift of the NMR signal from a certain amino acid residue changes in the presence and absence of a candidate substance, that amino acid residue can be considered to be involved in the interaction between the subunits of the ACE2 dimer.

[0040] Candidate substances containing amino acid residues involved in the interaction between the subunits of the ACE2 dimer can then be determined to be highly likely to be substances capable of inhibiting SARS-CoV-2 entry into or infection of human cells.

[0041] The screening method of the above aspect of the present invention is innovative in that it can predict whether a candidate substance is capable of inhibiting SARS-CoV-2 entry or infection into human cells by examining whether the candidate substance binds to the ACE2 dimer interface and inhibits the formation of ACE2 dimers.

[0042] In some embodiments, step (i) involves performing NMR spectroscopy on ACE2 in the presence of multiple concentrations of the candidate substance to obtain NMR signals. That is, NMR spectroscopy is performed on ACE2 multiple times at different concentrations of the candidate substance. This allows for evaluation or estimation of the strength of the candidate substance's inhibitory ability against the interaction between subunits of the ACE2 dimer, the strength of the candidate substance's inhibitory ability against SARS-CoV-2 entry into or infection of human cells, etc.

[0043] In some embodiments, the amino acid residues involved in the interaction between the subunits of the ACE2 dimer include at least one amino acid residue in the regions of ACE2 residue numbers 131 to 141 and 157 to 178. These regions contain amino acid residues involved in the interaction between the subunits of the ACE2 dimer.

[0044] In some embodiments, the amino acid residues involved in the interaction between the subunits of the ACE2 dimer include at least one selected from the group consisting of tryptophan 163, glutamine 139, and glutamine 175.

[0045] Tryptophan 163, glutamine 139, and glutamine 175 are amino acids that are thought to inhibit ACE2 dimerization upon binding of candidate substances. It is presumed that binding of a candidate substance to tryptophan 163 of ACE2 inhibits the hydrogen bond between glutamine 139 and glutamine 175.

[0046] In some embodiments, after step (iii), the method further comprises steps of: (iv) contacting the animal cells with the SARS-CoV-2 virus; (v) contacting the animal cells with the candidate substance; and (vi) measuring the amount of SARS-CoV-2 RNA in the animal cells after contact with the SARS-CoV-2 virus and the candidate substance.

[0047] In the screening method of the above aspect of the present invention, performing steps (i) to (iii) alone can determine whether a candidate substance is likely to be a substance capable of inhibiting SARS-CoV-2 entry into or infection of human cells. However, by further performing steps (iv) to (vi), the inhibitory effect of the candidate substance on SARS-CoV-2 entry into or infection of human cells can be confirmed. The contact of SARS-CoV-2 with animal cells in step (iv) and the contact of the candidate substance with animal cells in step (v) can be performed simultaneously, or the candidate substance can be contacted after contacting the animal cells with SARS-CoV-2. If the amount of SARS-CoV-2 RNA in the animal cells after contact with the candidate substance is reduced compared to the amount of SARS-CoV-2 RNA in the animal cells before contact with the candidate substance, the candidate substance can be determined to have an inhibitory effect on SARS-CoV-2 entry into or infection of human cells. Alternatively or additionally, if the amount of SARS-CoV-2 RNA in the animal cells after contact with the candidate substance is the same as or increased compared to the amount of SARS-CoV-2 RNA in the animal cells before contact with the candidate substance, it can be determined that the candidate substance does not have an inhibitory effect on SARS-CoV-2 entry or infection into human cells.

[0048] In the present invention, SARS-CoV-2 entry or infection into human cells can be evaluated by known methods, including, but not limited to, inoculation of live virus into cells and evaluation by viral RNA quantification, inoculation of pseudovirus into cells and evaluation by reporter assay (luciferase assay), etc.

[0049] Another aspect of the present invention provides an inhibitor of SARS-CoV-2 entry or infection into human cells, comprising a compound represented by the following formula (I), a derivative thereof, or a salt thereof, selected by the screening method of the present invention. The compound of formula (I) is 6-imino-7-[2-(4-methoxyphenyl)ethyl]-N-[(4-methoxyphenyl)methyl]-2-oxo-1,7,9-triazatricyclo[8.4.0.03,8]tetradeca-3(8),4,9,11,13-pentaene-5-carboxamide.

[0050] [ka] Derivatives of the compound represented by formula (I) include, but are not limited to, derivatives in which some or all of the C=O group, hydrogen of the NH group, or -OCH3 group are substituted with other substituents (e.g., alkyl groups, halogens, etc.), and derivatives in which hydrogen attached to the carbon skeleton of the compound represented by formula (I) is substituted with other substituents (e.g., alkyl groups, halogens, etc.).

[0051] The salt of the compound represented by formula (I) or its derivative may be a pharmaceutically acceptable salt. A pharmaceutically acceptable salt is a salt having the desired pharmacological activity of the compound, and refers to a salt prepared from a pharmaceutically acceptable base or acid, including an inorganic or organic base and an inorganic or organic acid.

[0052] Examples of inorganic bases include alkali metals (e.g., Na, K) and alkaline earth metals (e.g., Ca, Mg). Examples of organic bases include triethylamine, pyridine, etc. Examples of salts with inorganic acids include salts with hydrochloric acid, hydrofluoric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid, perchloric acid, hydroiodic acid, etc. Examples of salts with organic acids include salts with formic acid, acetic acid, trifluoroacetic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, mandelic acid, ascorbic acid, lactic acid, etc.

[0053] The inhibitor of SARS-CoV-2 invasion or infection into human cells according to the above-mentioned aspect of the present invention may be used as a composition in combination with various carriers (e.g., isotonicity agents, chelating agents, stabilizers, pH adjusters, preservatives, antioxidants, solubilizers, thickeners, etc.), food materials, etc., within a range that does not impair the effects of the present invention.

[0054] Examples of isotonicity agents include sugars such as glucose, trehalose, lactose, fructose, mannitol, xylitol, and sorbitol, polyhydric alcohols such as glycerin, polyethylene glycol, and propylene glycol, and inorganic salts such as sodium chloride, potassium chloride, and calcium chloride. These isotonicity agents can be used alone or in combination of two or more.

[0055] Examples of chelating agents include edetate salts such as disodium edetate, calcium disodium edetate, trisodium edetate, tetrasodium edetate, and calcium edetate, ethylenediaminetetraacetate, nitrilotriacetic acid or a salt thereof, sodium hexametaphosphate, citric acid, etc. These chelating agents can be used alone or in combination of two or more.

[0056] The stabilizer may, for example, be sodium hydrogen sulfite.

[0057] Examples of pH adjusters include acids such as hydrochloric acid, carbonic acid, acetic acid, and citric acid, as well as alkali metal hydroxides such as sodium hydroxide and potassium hydroxide, alkali metal carbonates or hydrogen carbonates such as sodium carbonate, alkali metal acetates such as sodium acetate, alkali metal citrates such as sodium citrate, and bases such as trometamol. These pH adjusters can be used alone or in combination of two or more.

[0058] Examples of preservatives include sorbic acid, potassium sorbate, parahydroxybenzoic acid esters such as methyl parahydroxybenzoate, ethyl parahydroxybenzoate, propyl parahydroxybenzoate, and butyl parahydroxybenzoate, quaternary ammonium salts such as chlorhexidine gluconate, benzalkonium chloride, benzethonium chloride, and cetylpyridinium chloride, alkylpolyaminoethylglycine, chlorobutanol, polyquad, polyhexamethylene biguanide, and chlorhexidine. These preservatives can be used alone or in combination of two or more.

[0059] Examples of antioxidants include sodium hydrogen sulfite, dry sodium sulfite, sodium pyrosulfite, concentrated mixed tocopherols, etc. These antioxidants can be used alone or in combination of two or more.

[0060] Examples of solubilizing agents include sodium benzoate, glycerin, D-sorbitol, glucose, propylene glycol, hydroxypropylmethylcellulose, polyvinylpyrrolidone, macrogol, D-mannitol, etc. These solubilizing agents can be used alone or in combination of two or more.

[0061] Examples of thickeners include polyethylene glycol, methyl cellulose, ethyl cellulose, carmellose sodium, xanthan gum, sodium chondroitin sulfate, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, polyvinylpyrrolidone, polyvinyl alcohol, etc. These thickeners may be used alone or in combination of two or more.

[0062] The inhibitor of SARS-CoV-2 invasion or infection of human cells of the present invention can also be used as an experimental reagent in cell experiments (including tissue culture experiments) or living cell experiments. Furthermore, the method for providing the inhibitor of SARS-CoV-2 invasion or infection of human cells of the present invention is not particularly limited, and it can also be used, for example, as a pharmaceutical or quasi-drug.

[0063] The total content of the compound represented by formula (I), its derivative, or its salt, which is the active ingredient in the inhibitor of SARS-CoV-2 invasion or infection of human cells of the present invention, varies depending on the route of intake, the patient's age, weight, symptoms, etc., and cannot be generally specified, but can be set to an amount that provides a daily intake of about 1 to 5,000 mg, more preferably about 10 to 1,000 mg, for an adult. In an embodiment of the composition, the total content of the compound represented by formula (I), its derivative, or its salt in the composition is not particularly limited and can be appropriately set based on conditions such as, for example, 90% by mass or more, 70% by mass or more, 50% by mass or more, 30% by mass or more, 10% by mass or more, 5% by mass or more, or 1% by mass or more.

[0064] When the inhibitor of SARS-CoV-2 invasion or infection into human cells of the present invention is provided as a pharmaceutical or quasi-drug, the formulation is not particularly limited, and examples thereof include various formulation forms such as orally administered preparations such as tablets, pills, capsules, powders, granules, syrups, and sublingual preparations; and parenterally administered preparations such as injections (intravenous injection, intramuscular injection, local injection, etc.), mouthwashes, infusions, topical preparations (ointments, creams, patches, inhalants), and suppositories. Among the above-mentioned formulation forms, preferred are, for example, injections, orally administered preparations, and topical preparations, with inhalants (inhalant-administered preparations) being particularly preferred. [Example]

[0065] The present invention will be explained in more detail using Production Examples and Examples, but the present invention is not limited to these Examples.

[0066] Example 1. NMR spectroscopy of ACE2 in the presence or absence of candidate substances (material) The following samples were prepared.

[0067] Uniform 2 H, 15 N-labeled human ACE2 (residue number 19-615) (Expressed in E. coli and purified) Protein concentration of the measurement sample: 50uM ·Buffer 20mM MES-NaOH, pH6.5, 50mM NaCl, 10% D2O DMSO solutions of Compound 1 (compound represented by formula (I)) and Compound 2 (compound represented by formula (II)) were used as 10 mM stocks. Experiment information (Temperature 25℃, Experiment name: 1 H- 15 N TROSY, 128 cumulative times) NMR instrument Bruker AVANCE NEO 600 (with cryo probe) (Experimental Method) Compound 1 was added to the protein in amounts of 0, 0.5, 1.0, 2.0, 3.0, 4.0, and 6.0 equivalents, and NMR spectra were measured. Two-dimensional spectra were obtained by Fourier transform. The binding site was identified from the magnitude of the chemical shift change of each signal.

[0068] (result) As shown in Figure 1, the signal derived from tryptophan residue 163 was significantly different before and after the addition of compound 1. The present inventors successfully identified the binding region of compound 1 on ACE2, and it was predicted that compound 1 binds to the area around tryptophan residue 163, where the most significant chemical shift change was observed (Figure 2).

[0069] In the case of Compound 2, no difference in the signals was observed in the regions of ACE2 residue numbers 131 to 141 and 157 to 178 in the spectra before and after the addition of Compound 2 (Figure 3).

[0070] [ka]

[0071] Example 2: Verification of the ability of Compound 1 and Compound 2 to inhibit SARS-CoV-2 infection in human cells 1. Infection Inhibition Assay (material) ·Cells VeroE6 / TMPRRS2 cells (JCR cell bank, #JCRB1819) Culture medium: DMEM (Fujifilm Wako), FBS (Capricorn Scientific), G418 (Invivogen, final: 1mg / ml) SARS-CoV-2 virus Wuhan strain 2019-hCoV / Japan / TY / WK-521 / 2020), GISAID ID: EPI_ISL_408667 Delta strain B.1.617.2 (hCoV-19 / Japan / TY11-927 / 2021), GISAID ID: EPI_ISL_2158617 Omicron strain (Omicron variant) BA.1 (hCoV-19 / Japan / TY38-873 / 2021), GISAID ID: EPI_ISL_7418017 Compounds were stored as received (-20°C or 4°C) and used as 10 mM stocks (DMSO). (Experimental Method) 5.0 × 10 in 48-well plates 4 VeroE6 / TMPRRS2 cells were seeded overnight at 1000 TCID50 / cell. Each virus (diluted in 2% FBS medium to 0.002 TCID50 / cell) was mixed with an equal volume of a solution of each compound 1 or 2 (diluted in 2% FBS medium to 200 mM, serially diluted (twice the concentration used in cells), DMSO final: 1%) and incubated at 37°C for 30 minutes. The virus / compound mixture was inoculated onto the cells (500 mL / well, 0.001 TCID50 / cell) and incubated at 37°C for 2 hours. After washing three times with 10% FBS medium, the mixture was replaced with the compound solution (in 10% FBS medium, each concentration (0.1 mM)) and incubated at 37°C for 22 hours. The culture supernatant was collected, and RNA was extracted using the FavorPrep Viral DNA / RNA Kit (Favorgen Biotech Corporation). Viral RNA was quantified by real-time qRT-PCR (Light Cycler 96 (Roche Diagnostics)) using the THUNDERBIRD probe one-step qRT-PCR kit (Toyobo Co., Ltd.) with specific primers and TaqMan probes (N2 set, Eurofins Genomics).

[0072] (result) Compound 1, which showed a significant difference in the signal derived from tryptophan residue 163, was able to inhibit SARS-CoV-2 infection in human cells (Figure 4). On the other hand, compound 2, which showed no difference in the signal derived from residues 131-141 and 157-178 in Example 1, did not have the ability to inhibit infection (Figure 5).

[0073] Example 3. Experiment on reduced infectivity with dimer interface mutants (material) Cells: ACE2-WT, ACE2-Q139A, ACE2-Q175A, and ACE2-Q139A / Q175A were stably expressed in HEK293T cells using retroviral vectors (each gene was incorporated into pCX4-pur). Culture medium: DMEM, 10% FBS SARS-CoV-2 virus Wuhan strain Same as Example 2 (Experimental Method) 5.0 × 10 in 48-well plates 4 HEK293T cells were seeded at 1000 cells / well overnight. Each virus (2% FBS medium) was inoculated into the cells (500 mL / well) and treated at 37°C for 1.5 hours (0.001 TCID50 / cell). After washing three times with 10% FBS medium, the medium was replaced with 10% FBS and cultured at 37°C for 22 hours. The culture supernatant was collected, and RNA was extracted using the FavorPrep Viral DNA / RNA Kit (Favorgen Biotech Corporation). Real-time qRT-PCR was performed to quantify viral RNA levels.

[0074] (result) The amount of viral RNA in cells expressing each ACE2 mutant was reduced to approximately 1 / 100 of that in cells expressing wild-type ACE2 (ACE2-WT) (Figure 6). This indicates that the glutamine 139 and glutamine 175 mutations in ACE2 inhibit SARS-CoV-2 infection of human cells.

[0075] Example 4: Experiment on reduced infectivity with dimer interface mutants material 1. Cells HEK293T cells (ATCC, Cat# CRL-3216) 2. Cell culture medium: DMEM (Fujifilm Wako, Cat# 044-29765) + 7.5% FBS (Biowest) + Kanamycin (Fujifilm Wako, Cat# 117-00961) 3. Transfection reagent PEIpro (Polyplus-Transfection, Cat# 101000017) 4. SARS-CoV-2 pseudotype virus (Wuhan type: D614G, alpha type, delta type, omicron type: BA.1) (Prepared according to the paper by Murae et al., Biochem Biophys Res Commun. 597:30-36 (2022)) 5. Luciferase activity measurement: This was performed according to the PicaGene Meliora Star-LT Luminescence Reagent (TOYO B-NET Co. Ltd., Cat# MLT100) kit manual. This was performed according to Murae et al., Biochem Biophys Res Commun. 597:30-36 (2022).

[0076] 6. Wild-type ACE2 expression plasmid: pcDNA3.1-C-DYK-hACE2 (Genscript, Cat# MC_0101086) 7. Dimer interface mutant ACE2 expression plasmids (prepared by the Department of Cytochemistry, National Institute of Infectious Diseases): pCX4-pur-hACE2 (Q139A), pCX4-pur-hACE2 (Q175A), pCX4-pur-hACE2 (Q139A / Q175A) untagged 8. ACE2 activity measurement kit: ACE2 Activity Assay Kit, Fluorimetric, SensoLyte 390 (Funakoshi, Cat# AS-72086). Perform according to the kit manual. 9. ACE2 activity measurement equipment: SpectraMax iD3 (Molecular Devices)

[0077] (Experimental Method) (1) Experiments using pseudoviruses to examine the reduced infectivity of dimer interface mutants HEK293T cells were seeded and transfected with various ACE2 expression plasmids the following day. The following day, the cells were infected with the Wuhan pseudotyped virus and cultured for another two days. Luciferase activity in each sample was then measured using the kit's reagents. Luciferase activity was detected in the pseudotyped virus-infected cell samples, but non-ACE2-expressing cells were used as a negative control and wild-type ACE2-expressing cells were used as a positive control.

[0078] (2) Evaluation of ACE2 enzymatic activity HEK293T cells were seeded and transfected with various ACE2 expression plasmids the following day. After two more days, the ACE2 activity of each sample was measured using an ACE2 activity assay kit. Because HEK293T cells express very little endogenous ACE2, non-ACE2-transfected cells served as the negative control, and wild-type ACE2-expressing cells served as the positive control.

[0079] (result) As shown in Table 1 and Figure 7, introducing mutations into Q139, Q175, or both, which form hydrogen bonds between dimer subunits, reduced the infectivity of both SARS-CoV-2 strains. Unexpectedly, this suggests that inhibition of ACE2 dimerization suppresses infectivity, even though it does not inhibit the virus-ACE2 interface.

[0080] [Table 1]

Claims

1. A method for screening for a substance capable of inhibiting SARS-CoV-2 entry into or infection of human cells, comprising the following steps (i) to (iii): (i) performing NMR spectroscopy on ACE2 in the presence of a candidate substance to obtain an NMR signal; (ii) performing NMR spectroscopy on ACE2 in the absence of the candidate substance to obtain an NMR signal; (iii) comparing the signals obtained in steps (i) and (ii) with those derived from amino acid residues involved in the interaction between the subunits of the ACE2 dimer; A screening method in which, if there is a difference between the compared signals in step (iii), this indicates that the candidate substance is likely to be a substance capable of inhibiting SARS-CoV-2 entry into or infection of human cells.

2. The screening method according to claim 1, wherein the amino acid residues involved in the interaction between the subunits of the ACE2 dimer include at least one amino acid residue in the regions of residue numbers 131 to 141 and 157 to 178 of ACE2.

3. The screening method described in claim 1, wherein the amino acid residues involved in the interaction between the subunits of the ACE2 dimer include at least one selected from the group consisting of tryptophan at position 163, glutamine at position 139, and glutamine at position 175.

4. After the step (iii), (iv) contacting the animal cells with the SARS-CoV-2 virus; (v) contacting the animal cell with the candidate substance; and (vi) measuring the amount of SARS-CoV-2 RNA in the animal cells after contact with the SARS-CoV-2 virus and the candidate substance. The screening method according to claim 1, further comprising:

5. The screening method according to claim 1, wherein the ACE2 is human-derived ACE2.

6. The ACE2 2 H and 15 The screening method according to claim 1, wherein the ACE2 is N-labeled.

7. The screening method according to claim 1, wherein the NMR spectroscopy for ACE2 in steps (i) and (ii) is NMR spectroscopy for a solution containing ACE2.

8. 2. The screening method according to claim 1, wherein step (i) comprises performing NMR spectroscopy on ACE2 in the presence of the candidate substance at multiple concentrations to obtain an NMR signal.

9. An inhibitor of SARS-CoV-2 invasion or infection into human cells, comprising a compound represented by the following formula (I), a derivative thereof, or a salt thereof: 【Chemistry 1】

Images