WO2022177029A1 - セレノネインを含む、ｃｏｖｉｄ１９治療又は予防薬 - Google Patents

セレノネインを含む、ｃｏｖｉｄ１９治療又は予防薬 Download PDF

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WO2022177029A1
WO2022177029A1 PCT/JP2022/007397 JP2022007397W WO2022177029A1 WO 2022177029 A1 WO2022177029 A1 WO 2022177029A1 JP 2022007397 W JP2022007397 W JP 2022007397W WO 2022177029 A1 WO2022177029 A1 WO 2022177029A1
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selenoneine
protease
papain
coronavirus
pro
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French (fr)
Japanese (ja)
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紀行 鈴木
康光 小椋
泰典 福本
惠一 市川
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Kikkoman Corp
Chiba University NUC
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Kikkoman Corp
Chiba University NUC
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Priority to US18/278,142 priority Critical patent/US20250352517A1/en
Priority to JP2023500976A priority patent/JP7837513B2/ja
Publication of WO2022177029A1 publication Critical patent/WO2022177029A1/ja
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4164—1,3-Diazoles
    • A61K31/4172—Imidazole-alkanecarboxylic acids, e.g. histidine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/04—Sulfur, selenium or tellurium; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/99—Enzyme inactivation by chemical treatment
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus

Definitions

  • the present invention relates to the technical field of COVID19 treatment or prevention, including selenoneine.
  • Non-Patent Document 1 Am J Clin Nutr. 2020 Jun 1;111(6):1297-1299. doi : 10.1093/ajcn/nqaa095).
  • Coronaviruses have single-stranded RNA as their genome, and when they infect host cells, long polyproteins are translated from the RNA genome. Proper cleavage of the polyprotein allows each fragment to function as a structural protein or enzyme necessary for viral replication, resulting in viral replication.
  • Proteases that mainly catalyze polyprotein cleavage include the main protease (M pro ) and papain-like protease (PLpro), and these proteases are potential drug discovery targets. Results of computer screening of three-dimensional models by crystallography have reported that some existing drugs can function as effective M pro inhibitors (Non-Patent Document 2: Nature (2020) vol. 582(7811):289-293).
  • Ebselen which is a type of selenium compound, shows a remarkable affinity for the catalytic domain, and is expected to be developed as a therapeutic agent for COVID19 (Non-Patent Document 3: Sci. Adv. 2020 6 eadb0345).
  • Ebselen is a functional molecule containing selenium, one of the essential trace elements, in its molecule.
  • Ebselen is a molecule that potentially has a free selenol group as a tautomer.
  • the mechanism of M pro inhibition by ebselen is thought to be covalent bonding of the selenol group of ebselen to the Cys145 thiol group of the active center of the protease.
  • organoselenium compounds show high binding affinity to the main protease (M pro ) of SARS-CoV-2, suggesting that organoselenium compounds can be candidate molecules for antiviral drugs.
  • M pro main protease
  • the purpose is to provide a drug that has inhibitory activity against the protease of SARS-CoV-2, which is a target for drug discovery.
  • the present inventors focused on the fact that both the main protease (M pro ) and papain-like protease (PLpro) of SARS-CoV-2 are cysteine proteases, and established a screening system using papain inhibitory activity as an indicator. Using such a screener, selenoneine was screened as a substance having a higher inhibitory activity than ebselen, which was identified as an inhibitor of Mpro . Furthermore, they prepared M pro of SARS-CoV-2 and confirmed that protease activity is inhibited by selenoneine, leading to the present invention.
  • the invention thus relates to:
  • a coronavirus protease inhibitor comprising selenoneine or a tautomer or dimer thereof or a pharmaceutically acceptable salt thereof.
  • the protease inhibitor according to item 1 wherein the protease is main protease or papain-like protease.
  • the protease inhibitor according to item 1, wherein the coronavirus is SARS-CoV2.
  • a composition for treating or preventing coronavirus infection comprising selenoneine, a tautomer or dimer thereof, or a pharmaceutically acceptable salt thereof.
  • Selenoneine or a tautomer or dimer thereof or a pharmaceutically acceptable salt thereof for use in treating or preventing coronavirus infection.
  • the invention according to any one of items [4-1] to [4-4], wherein the coronavirus infection is COVID19.
  • [6] A method for screening therapeutic or preventive agents for coronavirus infections, using inhibitory activity against papain as an index.
  • the method according to item 6, wherein the coronavirus infection is COVID19.
  • the therapeutic or prophylactic agent inhibits main protease or papain-like protease of coronavirus.
  • Selenoneine exerts higher M pro inhibitory activity than ebselen. In addition, selenoneine exhibits higher M pro inhibitory activity than other selenium-containing compounds.
  • FIG. 1 is a three-dimensional model showing that ebselen acts on the active center of M pro .
  • the selenol group which appeared by tautomerization of ebselen, covalently bonds to cysteine 145.
  • FIG. 2 shows a comparison of the sequence and active center conformation of (1) M pro , which is a cysteine protease, and (2) papain sequence and conformation of the active center.
  • FIG. 3 shows a comparison of the sequence and active center conformation of (1) PLpro, a cysteine protease, and (2) papain sequence and conformation of the active center.
  • Figure 4 shows papain enzymatic activity in the presence or absence of ebselen or selenoneine.
  • FIG. 5 shows inhibition curves of ebselen or selenoneine on papain enzymatic activity.
  • FIG. 6 shows a schematic diagram of a plasmid carrying the M pro gene.
  • FIG. 7 shows the results of SDS-PAGE of M pro expressed in E. coli and purified with a His tag. The 33.8 kDa band corresponds to the Mpro band.
  • Figure 8 shows the protease activity of Mpro in the presence or absence of ebselen, ergothioneine or selenoneine. M pro decomposes the fluorescent substrate, resulting in an increase in fluorescence intensity over time.
  • FIG. 5 shows inhibition curves of ebselen or selenoneine on papain enzymatic activity.
  • FIG. 6 shows a schematic diagram of a plasmid carrying the M pro gene.
  • FIG. 7 shows the results of SDS-PAGE of M pro expressed in E. coli and purified with a His tag. The 33.8
  • FIG. 10 shows test compounds (selenoine, ebselen , selenocystine ((SeCys) 2 ), methylselenocysteine (MeSeCys), selenomethionine (SeMet), diphenyldiselenide (PhSeSePh), sodium selenite ( selenite)).
  • this embodiment an embodiment of the present invention (hereinafter referred to as “this embodiment”) will be described in detail, but the present invention is not limited to this, and various modifications are possible without departing from the scope of the invention. is.
  • the present invention relates to a coronavirus protease inhibitor or a composition for the treatment or prevention of coronavirus infection, containing selenoneine or a tautomer or dimer thereof, or a pharmaceutically acceptable salt thereof.
  • selenoneine has the following chemical name: 2-selenyl-N ⁇ ,N ⁇ ,N ⁇ -trimethyl - L - histidine is a compound of Specifically, the following formula (I): refers to a compound represented by The OH group, NH group, etc. in the molecule may be in a state without hydrogen atoms, that is, in an ionized state. This compound may exist as any optical isomer, geometric isomer, tautomer, or dimer, or mixtures thereof.
  • selenoneine may take any of the forms of formulas (I)-(III) below:
  • selenoneine may contain compounds in the form of formulas (I)-(III) in any proportion.
  • a dimerized compound can be reduced to a monomer by the ambient environment.
  • the composition containing the compound of formula (I) or (II) may further contain a reducing agent.
  • Any reducing agent can be used as such a reducing agent, and examples thereof include glutathione (GSH), dithiothreitol (DTT), and mercaptoethanol.
  • Selenoneine is a component that is abundantly contained in blood such as tuna, marlin, and mackerel, and is a component that is ingested on a daily basis.
  • Selenoneine can be produced as appropriate by those skilled in the art.
  • a method for producing selenoneine it can be produced by a chemical synthesis method (Angew. Chem. Int. Ed. 2019, 58, 1-6), or by extraction from a biological tissue containing selenoneine, or by fermentation with microorganisms. can be manufactured.
  • ergothioneine is produced simultaneously with selenoneine, and it is difficult to separate them.
  • the resulting transformant extract containing selenoneine may contain ergothioneine in addition to selenoneine.
  • the selenoneine is preferably purified selenoneine.
  • Selenoneine can be purified by techniques known to those skilled in the art, such as HPLC.
  • coronavirus protease preferably relates to the SARS-CoV2 protease.
  • Coronavirus proteases include the main protease ( Mpro ) and the papain-like protease (PLpro).
  • Mpro main protease
  • PLpro papain-like protease
  • SARS-CoV2 main protease (M pro ) is preferred from the viewpoint of drug discovery targets.
  • the main protease also called 3Clpro, nonstructural protein 5 (nsp5), relates to the main protease that degrades polyproteins.
  • the main protease is a cysteine protease and functions as a dimer composed of identical subunits.
  • the main protease of SARS-CoV2 has an amino acid sequence (SEQ ID NO: 1) consisting of 306 residues. An active center formed from such sequence Cys145 and His41 is known, and the selenol group of ebselen is believed to covalently bond to the thiol group of Cys145 (Fig. 1).
  • Selenoneine which is a selenium-containing compound, also has a selenol group, so that it can covalently bind to Cys145 of the active center of M pro and have M pro inhibitory activity.
  • Papain-like proteases are related to nonstructural protein 3 (nsp3) proteases and are cysteine proteases that degrade polyproteins.
  • the papain-like protease of SARS-CoV2 has an amino acid sequence (SEQ ID NO: 3) consisting of 317 residues.
  • the catalytic triad structure spanning the active center of papain-like protease is Asp286-His272-Cys111.
  • Papain is a type of cysteine protease contained in papaya and has an amino acid sequence (SEQ ID NO: 2) consisting of 345 residues.
  • a cysteine protease refers to a proteolytic enzyme that contains a cysteine in the catalytic domain of the enzyme. Cysteine thiols are deprotonated by histidines, which are normally located near cysteines in the catalytic domain, and the anionized thiol groups attack the carbonyl carbons of substrate peptides or proteins, hydrolyzing peptide bonds. Therefore, covalent binding of a protease inhibitor to the thiol groups of cysteines in the catalytic domain inhibits the enzymatic activity of cysteine proteases.
  • Both papain and the main protease or papain-like protease are cysteine proteases, and form catalytic triads or catalytic dyads characterized by amino acid residues common to the active centers.
  • Catalytic triad refers to the three coordinating amino acids found in the active site of some enzymes. The constituent coordinating amino acids differ depending on the type of enzyme.
  • Catalytic triads of cysteine proteases are composed of cysteine, histidine, and asparagine or aspartic acid as the third amino acid.
  • Such a screening method specifically includes preparing a solution containing a candidate agent, a papain-degrading fluorescent substrate, and papain, and measuring the fluorescence intensity of the solution.
  • the fluorescence intensity over time may be measured, or the papain inhibition curve of the candidate drug may be obtained by measuring the change in fluorescence intensity when the concentration of the candidate drug is changed.
  • selenonein that exhibits papain inhibitory activity may have inhibitory activity on the main protease or papain-like protease.
  • the protease inhibitor of the present invention can suppress the proliferation of coronaviruses by suppressing the degradation of polyproteins produced from coronaviruses, and can thereby be used as therapeutic and prophylactic agents. Also, the protease inhibitors of the present invention may be included in foods or food compositions.
  • composition of the present invention comprises a therapeutically effective amount of selenoneine or a tautomer or dimer thereof or a pharmaceutically acceptable salt thereof. Additionally, a pharmaceutically acceptable carrier or excipient may be included. Therefore, the composition of the present invention can also be called a pharmaceutical composition.
  • the protease inhibitors and compositions of the invention are administered to patients in need of treatment or prevention.
  • Another aspect of the present invention is the use of selenoneine or a tautomer or dimer thereof or a pharmaceutically acceptable salt thereof, a protease inhibitor according to the present invention, or a therapeutic or prophylactic pharmaceutical composition for treatment or prevention.
  • the dosage/number of doses can be appropriately selected depending on the symptoms.
  • pharmaceutically acceptable excipient includes any carrier, diluent, adjuvant or medium, preservative or antioxidant, filler, disintegrant, wetting agent, emulsifier, suspending agent. Also included are clouding agents, solvents, dispersion media, coatings, antibacterial agents, fungicides, isotonic agents and absorption retardants and the like.
  • clouding agents solvents, dispersion media, coatings, antibacterial agents, fungicides, isotonic agents and absorption retardants and the like.
  • Excipients can be used in the compositions of the present invention, except where conventional excipients are incompatible with selenoneine. Supplementary active ingredients can also be incorporated into the compositions as appropriate therapeutic combinations.
  • Subjects who need treatment or prevention include those who may be exposed to coronavirus.
  • a “therapeutically effective amount” is an amount that, when administered, is capable of inhibiting active proteases involved in pathogenesis, and which, when administered, is effective in preventing or treating the onset or exacerbation of COVID 19.
  • a therapeutically effective amount can be determined through animal experiments and human clinical trials.
  • selenoneine which is an active ingredient of the present invention, is ingested by eating fish. can also be used to determine the therapeutically effective amount.
  • selenoneine can be administered at 28 ⁇ g/kg, but is not intended to be limited to these amounts.
  • compositions of the present invention can be provided in any dosage form, including tablets, capsules, powders, nasal drops, or aerosol forms, injection solutions, drops, ointments, creams, sprays, transdermal patches. can be formulated into
  • Another aspect of the invention may relate to a food composition
  • a food composition comprising selenoneine or a tautomer or dimer thereof or a food acceptable salt thereof.
  • Such food compositions include functionally labeled foods, nutritionally functional foods, and functionally labeled foods that display functions such as prevention and resistance to coronaviruses, particularly SARS-CoV2, or functions to inhibit coronavirus protease, particularly the main protease. Or it may be a food for specified health use.
  • a food composition, a food with function claims, a food with nutrient function claims, or a food for specified health uses for example, contains 1 to 1000 ppm, preferably 10 to 100 ppm, and most preferably 30 to 70 ppm of selenoneine or a tautomer or dimeric form thereof. It may be a beverage, food, or supplement containing the polymer or its food-acceptable salt. Selenoneine is known to be abundant in fish.
  • the selenoneine content in bluefin tuna is 30 mg Se/kg, and that 100 g of fish meat is eaten and uniformly distributed in a human body weighing 60 kg, the selenoneine content in the body is theoretically about 1 ⁇ M, and the blood concentration is It can be estimated to be approximately 8 ⁇ M.
  • Fish that may contain selenoneine include tuna, marlin, mackerel, yellowtail, sea bream, pufferfish, salmon/trout, flounder/flatfish, and particularly tuna, marlin, mackerel, A lot of yellowtails are included.
  • Examples of food compositions, foods with function claims, foods with nutrient function claims, or foods for specified health uses include raw edible parts of these fish and processed foods made from fish. These fish may be wild or farmed. Since the selenoneine content in fish varies depending on the type of feed, farmed fish with increased selenonein content are more preferred.
  • Examples of processed foods made from fish include any foods made from fish, such as canned food, bottled food, food boiled in soy sauce, dried fish, dried fish, paste, pickled fish, and supplements.
  • Tuna species used in food compositions, foods with function claims, foods with nutrient function claims, or foods for specified health uses include the tuna family and the Hagatsuo family.
  • Examples of the tuna family include the genus Tuna, the genus Pomella, the genus Suma, and the genus Bonito, and examples of the genus Hagatsuo include the genus Dogtooth tuna and the genus Pomfret.
  • tuna examples include albacore tuna, bluefin tuna, southern bluefin tuna, Atlantic tuna, Atlantic bluefin tuna, yellowfin tuna, bigeye tuna, and the like; Alternatively, mention may be made of albacore, bluefin tuna, southern bluefin tuna, Atlantic tuna, Atlantic bluefin tuna, yellowfin, bigeye, longfin, bonito or trout. Preferable examples include albacore, bluefin tuna, Atlantic bluefin tuna, bluefin tuna, yellowfin, bigeye, longfin, bonito, and trout.
  • Example 1 Inhibitory Activity of Papain Activity 10 ⁇ g/ml of papain (sold by Sigma-Aldrich Japan) and 5 ⁇ M of ebselen or selenoneine were dissolved in 50 mM Tris-HCl (pH 7.4) kept at 37° C., After preincubation for 15 minutes, 10 ⁇ M Bz-Arg-MCA was added to the same reaction solution, and changes in fluorescence intensity due to cleavage of Bz-Arg-MCA were observed over time with a fluorometer. The results are shown in FIG.
  • Example 2 Preparation of M pro of SARS-CoV2 M pro DNA was synthesized according to the nucleotide sequence of NC_45512 (10055-10972: SEQ ID NO: 4) by total gene synthesis.
  • Mpro was introduced into a GST- and histidine-tagged plasmid (Fig. 6) and transformed into E. coli strain BL21(DE3).
  • E. coli BL21(DE3) strain was cultured at 37° C. to express the M pro protein.
  • Cell bodies were harvested and the M pro protein was purified using a binding agent for histidine tags.
  • M pro and human rhinovirus 3C protease HRV
  • HRV human rhinovirus 3C protease
  • a binding agent for histidine tag was used to remove undigested M pro and HRV to obtain a purified preparation of M pro protein.
  • Purified M pro protein was dissolved in a storage buffer of 20 mM Tris-HCl, 100 mM NaCl, 0.01% Triton-X-100, 50% glycerol, 1 mM EDTA, 1 mM DTT, subjected to SDS-PAGE and analyzed with Coomassie brilliant. Stained with blue (Fig. 7).
  • the purified M pro protein dissolved in a storage buffer containing DTT was dialyzed using a microdialysis cartridge Xpress Micro / Mini Dialyzer (manufactured by Funakoshi Co., Ltd.) to remove DTT and inhibit the inhibitory activity of Example 4. submitted for testing.
  • Example 3 Preparation of PLpro of SARS-CoV2 PLpro DNA is synthesized according to the base sequence of SEQ ID NO: NC_45512 (4955-5908: SEQ ID NO: 5) by total gene synthesis.
  • PLpro is introduced into a histidine-tagged plasmid and transformed into E. coli strain BL21(DE3).
  • E. coli strain BL21(DE3) is cultured to express the PLpro protein.
  • Cell bodies are harvested and the PLpro protein purified using a binding agent to the histidine tag. GST and histidine tag are removed by autolysis of PLpro and HRV, and a binder for histidine tag is used to remove undigested PLpro and HRV to obtain a purified product of PLpro protein.
  • Example 4 SARS-CoV2 inhibitory activity against M pro 2 ⁇ g/ml of M pro and 5 ⁇ M of ebselen, selenoneine, or ergothioneine were each dissolved in 50 mM Tris-HCl (pH 7.4) kept at 37°C. , After preincubation for 5 minutes, 10 ⁇ M Ac-Abu-Tle-Leu-Gln-MCA was added to the same reaction solution, and Ac-Abu-Tle-Leu-Gln-MCA was cleaved with a fluorometer. Inhibitory activity was examined by measuring changes in fluorescence intensity (Fig. 8).
  • Example 5 SARS-CoV2 inhibitory activity against M pro 2 ⁇ g/ml of M pro and 1 ⁇ M of test compound were each dissolved in 50 mM Tris-HCl (pH 7.4) kept at 37° C. and preincubated. After 5 minutes, 10 ⁇ M Ac-Abu-Tle-Leu-Gln-MCA was added to the same reaction solution, and the fluorescence intensity accompanying the cleavage of Ac-Abu-Tle-Leu-Gln-MCA was measured with a fluorometer. to measure the inhibitory activity (Fig. 10).
  • Selenoneine was about 20%, while ebselen, selenocystine ((SeCys) 2 ), methyl Selenocysteine (MeSeCys), selenomethionine (SeMet), diphenyldiselenide (PhSeSePh) and sodium selenite (selenite) were all above 90%. Therefore, it was shown that selenoneine is remarkably superior to other selenium-containing compounds, particularly in terms of M pro inhibitory activity.
  • Example 6 Growth Inhibitory Activity against SARS-CoV2 Inhibition of SARS-CoV2 infection of host cells by ebselen or selenoneine can be measured, for example, by plaque assay.
  • Host cells are grown in monolayers in a 37° C., 5% CO 2 incubator until confluent. Next, after removing the medium and washing the cell surface with PBS(-), a virus solution and a 0 to 100 ⁇ M protease inhibitor solution are added and incubated for 30 minutes. After 30 minutes, the sample solution is removed and an agar medium is overlaid. When the agar has hardened, the plates are inverted and incubated in a 37°C, 5% CO2 incubator for 2 days.
  • the layered medium is removed, the plate is dried, stained with a crystal violet staining solution for 5 minutes, washed with purified water, and air-dried. Finally, the number of plaques is counted, compared with the control group, and the infection inhibition rate of the protease inhibitor against the virus is calculated.

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