A medication for the treatment or prevention of COVID-19, including selenoneine.

Abstract

The purpose of the present invention is to provide a composition for treating or preventing an infection (COVID19) caused by SARS-CoV-2. A method for screening for a drug having an inhibitory activity against a protease of SARS-CoV2 is established, and it is found that selenoneine has an inhibitory activity against the protease of SARS-CoV2. On the basis of this finding, a composition for treating or preventing COVID19 which contains selenoneine is provided.

Description

[Technical Field]

[0001] The present invention relates to the technology of treatment or prevention of COVID-19, comprising selenonein. [Background technology]

[0002] The development of vaccines and treatments for COVID-19, caused by the SARS-CoV-2 virus that triggered the pandemic from autumn 2019 to 2022, is urgently needed. Several vaccines that have shown efficacy have been developed and are being used. On the other hand, epidemiological studies have shown that the severity and mortality rates of COVID-19 patients in Asian countries, including Japan, are lower than in European countries, suggesting that some factor may be conferring resistance to COVID-19. Research is being conducted on potential such factors. It is known that selenium deficiency in the body increases the virulence of RNA viruses such as coxsackievirus and influenza virus. Against this backdrop, an association between selenium status in cities in China and the outcomes of COVID-19 patients has been reported (Non-patent Literature 1: Am J Clin Nutr. 2020 Jun 1;111(6):1297-1299. doi: 10.1093 / ajcn / nqaa095).

[0003] Coronaviruses have single-stranded RNA as their genome, and upon infecting host cells, long polyproteins are translated from the RNA genome. Proper cleavage of these polyproteins allows each fragment to function as a structural protein or enzyme necessary for viral replication, thus enabling viral growth. The main protease that catalyzes polyprotein cleavage is the main protease (M pro Examples include papain-like proteases (PLpro), and these proteases are promising drug targets. Computer screening of three-dimensional models using crystallography has shown that several existing drugs are effective against M proIt has been reported that it can function as an inhibitor (Non-patent Literature 2: Nature (2020) vol. 582(7811):289-293). In particular, ebselen, a type of selenium compound, shows remarkable affinity for the catalytic region, and is therefore expected to be developed as a therapeutic agent for COVID-19 (Non-patent Literature 3: Sci. Adv. 2020 6. eadb0345). Ebselen is a functional molecule that contains selenium, one of the essential trace elements, within its molecule. Ebselen is a molecule that potentially has a free selenoyl group as a tautomer. pro The inhibition mechanism is thought to involve the covalent bonding of the selenool group of ebselenium to the Cys145 thiol group of the protease's active site. Furthermore, in silico analysis, it was found that the organoselenite compound inhibits the main protease of SARS-CoV-2 (M pro Because they exhibit high binding affinity to ), organoselenide compounds have been shown to be potential candidate molecules for antiviral drugs (Non-Patent Literature 4). [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Am J Clin Nutr. 2020 Jun 1;111(6):1297-1299. doi: 10.1093 / ajcn / nqaa095 [Non-Patent Document 2] Nature (2020) vol. 582(7811):289-293 [Non-Patent Document 3] Sci. Adv. 2020 6. eadb0345 [Non-Patent Document 4] Chemrexiv (2020-07-02) DOI:10.26434 / chemrxiv.12594134 [Overview of the project] [Problems that the invention aims to solve]

[0005] An object is to provide a drug having an inhibitory activity against a protease of SARS-CoV-2, which is a drug target. [Means for Solving the Problems]

[0006] The present inventors focused on the fact that both the main protease (M pro ) and the papain-like protease (PLpro) of SARS-CoV-2 are cysteine proteases, and established a screening system using papain inhibitory activity as an index. By such screening, selenonine was screened as a substance having a higher inhibitory activity than ebselen, which was identified as an inhibitor of M pro . Furthermore, M of SARS-CoV-2 pro was prepared, and it was confirmed that the protease activity was inhibited by selenonine, leading to the present invention. Therefore, the present invention relates to the following:

[0007] [1] A protease inhibitor of a coronavirus comprising selenonine or a tautomer or dimer thereof or a pharmaceutically acceptable salt thereof. [2] The protease inhibitor according to item 1, wherein the protease is a main protease or a papain-like protease. [3] The protease inhibitor according to item 1, wherein the coronavirus is SARS-CoV2. [4-1] A composition for treating or preventing coronavirus infection comprising selenonine or a tautomer or dimer thereof or a pharmaceutically acceptable salt thereof. [4-2] Selenonine or a tautomer or dimer thereof or a pharmaceutically acceptable salt thereof for use in the treatment or prevention of coronavirus infection. [4-3] A method for treating or preventing coronavirus infection in a subject who needs treatment or prevention of infection by a coronavirus, the method comprising administering selenonine or a tautomer or dimer thereof or a pharmaceutically acceptable salt thereof to the subject. Use of selenoneine or its tautomer or dimer or a pharmaceutically acceptable salt thereof for the manufacture of a therapeutic or prophylactic agent against coronavirus. [5] The invention according to any one of items [4-1] to [4-4], wherein the coronavirus infection is COVID-19.

[0008] [6] A method for screening a therapeutic or prophylactic agent against coronavirus infection, using the inhibitory activity against papain as an index. [7] The method according to item 6, wherein the coronavirus infection is COVID-19. [8] The method according to item 6 or 7, wherein the therapeutic or prophylactic agent inhibits the main protease or papain-like protease of coronavirus. [Effect of the Invention]

[0009] Selenoneine exhibits higher M pro inhibitory activity than ebselen. Also, selenoneine exhibits higher M pro inhibitory activity compared to other selenium-containing compounds. [Brief Description of the Drawings]

[0010] [Figure 1] Figure 1 is a diagram showing, in a three-dimensional model, the action of ebselen on the active center of Mpro. The selenol group that appears due to the tautomerization of ebselen forms a covalent bond with cysteine 145. [Figure 2] Figure 2 shows a comparison between the sequence and three-dimensional structure of the active center of (1) Mpro, which is a cysteine protease, and the sequence and three-dimensional structure of the active center of (2) papain. [Figure 3] Figure 3 shows a comparison between the sequence and three-dimensional structure of the active center of (1) PLpro, which is a cysteine protease, and the sequence and three-dimensional structure of the active center of (2) papain. [Figure 4]Figure 4 shows papain enzyme activity in the presence or absence of ebselen or selenoneine. Papain enzyme activity degrades the fluorescent substrate, causing the fluorescence intensity to increase over time. [Figure 5] Figure 5 shows the inhibition curves of ebselen or selenonein against papain enzyme activity. [Figure 6] Figure 6 shows a schematic diagram of a plasmid containing the Mpro gene. [Figure 7] Figure 7 shows the results of SDS-PAGE analysis of Mpro expressed in E. coli and purified with a His tag. The 33.8 kDa band corresponds to the Mpro band. [Figure 8] Figure 8 shows the protease activity of Mpro in the presence or absence of ebselen, ergothioneine, or selenoneine. The fluorescence intensity increases over time as the fluorescent substrate is degraded by Mpro. [Figure 9] Figure 9 shows the inhibition curves of Mpro protease activity by ebselen, ergothioneine, or selenoneine. [Figure 10] Figure 10 shows the inhibitory activity of the test compounds (selenonenein, ebselen, selenocystine ((SeCys)2), methylselenocysteine ​​(MeSeCys), selenomethionine (SeMet), diphenyl diselenide (PhSeSePh), sodium selenite (selenite)) against Mpro. [Modes for carrying out the invention]

[0011] The following describes in detail embodiments of the present invention (hereinafter referred to as "this embodiment"), but the present invention is not limited thereto, and various modifications are possible without departing from its spirit.

[0012] The present invention relates to a coronavirus protease inhibitor or a composition for the treatment or prevention of coronavirus infection, comprising selenonein or its tautomers or dimers or pharmaceutically acceptable salts thereof.

[0013] In the present invention, selenoneine has the following chemical name: 2-Selenyl-N α ,N α ,N α -Trimethyl-L-histidine (2-selenyl-N α ,N α ,N α It is a compound of the form (-trimethyl-L-histidine). Specifically, the following formula (I): [ka] This refers to the compound represented by [formula]. The OH group, NH group, etc., in the molecule may be in a state without hydrogen atoms, i.e., an ionized state. This compound may exist as any optical isomer, geometric isomer, tautomer, or dimer, or as a mixture thereof. As a result of tautomerization and dimerization, selenoneine may take any of the following forms (I) to (III): [ka] In the present invention, selenonein may contain compounds of the form of formulas (I) to (III) in any proportion. The dimerized compound can be reduced to a monomer by the surrounding environment. Furthermore, in order to maintain the monomer, the composition containing the compound represented by formula (I) or (II) may further contain a reducing agent. Any reducing agent can be used as such, but examples include glutathione (GSH), dithiothreitol (DTT), and mercaptoethanol. Selenonein is a component found in large quantities in the dark meat of tuna, swordfish, mackerel, and other fish, and is a component that is consumed on a daily basis.

[0014] Selenonein can be manufactured as appropriate by those skilled in the art. Methods for producing selenonein include chemical synthesis (Angew. Chem. Int. Ed. 2019, 58, 1 - 6), extraction from biological tissues containing selenonein, and fermentation by microorganisms. For example, extraction from tissues of squid, fish, birds, mammals, etc., as described in Japanese Patent Publication No. 5669056; and Schizosaccharomyces pombe, a fission yeast into which genes involved in ergothioneine biosynthesis have been introduced, as described in the literature by Pluskal T et al. (Pluskal T et al., PLoS One 2014 May 14;9(5):e97774). Schizosaccharomyces pombe A method using ); described in International Publication No. 2017 / 026173, histidine and selenium compounds are obtained by overexpressing the gene encoding selenoneine synthase in Aspergillus soybean ( Aspergillus sojae ), Aspergillus oryzae ( Aspergillus oryzae ), Aspergillus niger ( Aspergillus niger ) and other microorganisms of the Aspergillus genus and Escherichia coli ( Escherichia coli Methods utilizing transformants such as ) are also mentioned. Of these, when producing selenonein on an industrial scale, the method described in International Publication No. 2017 / 026173 is preferred from the viewpoint of producing selenonein in high yield.

[0015] Furthermore, in the method using a transformant that overexpresses selenoneine synthase as described in International Publication No. 2017 / 026173, ergothioneine is simultaneously produced along with selenoneine, and it is difficult to separate them. Therefore, the resulting transformant extract containing selenoneine may also contain ergothioneine in addition to selenoneine. If available, purified selenoneine is preferred. Selenoneine can be purified by methods known to those skilled in the art, such as HPLC.

[0016] Coronavirus proteases preferably relate to SARS-CoV-2 proteases. Coronavirus proteases include the main protease (M pro Examples include the main protease of SARS-CoV-2 (M pro ) is preferable.

[0017] The main protease, also known as 3Clpro or non-structural protein 5 (nsp5), is the primary protease that degrades polyproteins. The main protease is a cysteine ​​protease and functions as a dimer composed of the same subunit. The main protease of SARS-CoV-2 has an amino acid sequence of 306 residues (SEQ ID NO: 1). The active site formed from Cys145 and His41 in this sequence is known, and it is thought that the selenool group of ebselenium is covalently bonded to the thiol group of Cys145 (Figure 1). Since selenoneine, a compound containing selenium, also has a selenool group, M pro It covalently binds to Cys145 at the active site of M pro It may have inhibitory activity.

[0018] Papain-like proteases are cysteine ​​proteases that degrade polyproteins, particularly those of non-structural protein 3 (nsp3). The papain-like protease of SARS-CoV-2 has an amino acid sequence (SEQ ID NO: 3) consisting of 317 residues. The catalytic triad structure of the active site of the papain-like protease is Asp286-His272-Cys111.

[0019] Papain is a type of cysteine ​​protease found in papaya, possessing an amino acid sequence of 345 residues (SEQ ID NO: 2). Cysteine ​​proteases are proteolytic enzymes that contain cysteine ​​in their catalytic region. Normally, histidine present near the cysteine ​​in the catalytic region deprotonates the thiol group of cysteine, and the resulting anionic thiol group attacks the carbonyl carbon of the substrate peptide or protein, hydrolyzing the peptide bond. Therefore, the enzymatic activity of cysteine ​​protease is suppressed when a protease inhibitor covalently bonds to the thiol group of cysteine ​​in the catalytic region.

[0020] Papain and its main protease or papain-like protease are both cysteine ​​proteases, and they share a common amino acid residue at their active site, forming a characteristic catalytic triad or catalytic dyad. A catalytic triad refers to three coordinating amino acids found in the active site of several enzymes. The constituent coordinating amino acids differ depending on the type of enzyme. The catalytic triad of cysteine ​​proteases consists of cysteine, histidine, and asparagine or aspartic acid as the third amino acid. Of these, cysteine ​​and histidine, which contributes to the deprotonation of the thiol of cysteine, are essential components, but asparagine, as seen in papain, has little effect on activity, in which case it is also called a catalytic dyad consisting of cysteine ​​and histidine. Therefore, by selecting a substance that has inhibitory activity against papain, it is possible to screen for inhibitors of the main protease or papain-like protease. Another aspect of the present invention relates to a screening method for inhibitors of main proteases or papain-like proteases or for the treatment or prevention of coronavirus, using inhibitory activity against papain as an indicator. Specifically, such a screening method includes preparing a solution containing a candidate drug, a papain-degrading fluorescent substrate, and papain, and measuring the fluorescence intensity of the solution. The fluorescence intensity may be measured over time, or a 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. From the viewpoint of having common features in a catalytic triad structure or a catalytic dyad structure, selenoneines that exhibit papain inhibitory activity may have inhibitory activity against main proteases or papain-like proteases.

[0021] The protease inhibitor of the present invention can suppress the proliferation of coronavirus by inhibiting the degradation of polyproteins produced from coronavirus, and can therefore be used as a therapeutic and preventive agent. Furthermore, the protease inhibitor of the present invention may be included in food or food composition.

[0022] The composition of the present invention comprises a therapeutically effective amount of selenonein or its tautomers or dimers or pharmaceutically acceptable salts thereof. Furthermore, it may also contain pharmaceutically acceptable carriers or excipients. Therefore, the composition of the present invention can also be referred to as a pharmaceutical composition. The protease inhibitors and compositions of the present invention are administered to patients in need of treatment or prevention.

[0023] Another aspect of the present invention involves administering selenonein or its tautomers or dimers or pharmaceutically acceptable salts thereof, protease inhibitors according to the present invention, or therapeutic or prophylactic pharmaceutical compositions to a subject in need of treatment or prevention. This may be administered orally or parenterally. Examples of parenteral administration include intraperitoneal, intramuscular, intravenous, intra-arterial, nasal, oral, pulmonary, and local administration. The dosage / frequency can be appropriately selected according to the symptoms.

[0024] As used herein, “pharmaceutically acceptable excipients” include any carrier, diluent, auxiliary or medium, preservative or antioxidant, filler, disintegrant, wetting agent, emulsifier, suspending agent, solvent, dispersion medium, coating, antimicrobial agent, fungicide, isobaric agent, and absorption retarder. The use of these excipients with active ingredients is well known in the art. Excipients may be used in the compositions of the present invention unless conventional excipients are incompatible with selenonein. Auxiliary active ingredients may also be incorporated into the compositions as appropriate therapeutic combinations.

[0025] Those who require treatment or prevention include individuals who may have been exposed to the coronavirus.

[0026] The "therapeutic dose" refers to the amount of the compound / agent according to the present invention that, when administered, is capable of inhibiting the active protease involved in disease development and is effective in preventing or treating the onset or exacerbation of COVID-19 when administered. The therapeutic dose can be determined through animal experiments or clinical trials in humans. On the other hand, selenonein, the active ingredient of the present invention, is obtained through fish consumption, and can be used without safety concerns at doses of up to 1.7 mg per day, for example, and the therapeutic dose can also be determined taking such amounts into consideration. As an example, selenonein can be administered at 28 μg / kg, but it is not intended that the amount is limited to these figures.

[0027] The compositions of the present invention can be provided in any dosage form, and may be in the form of tablets, capsules, powders, nasal drops, or aerosols, or in the form of injections, infusions, ointments, creams, sprays, or transdermal patches.

[0028] Another aspect of the present invention may relate to a food composition comprising selenonein or its tautomers or dimers or food-acceptable salts thereof. Such a food composition may be a functional food, nutrient functional food, or food for specified health uses that displays a function such as prevention or resistance to coronavirus, particularly SARS-CoV-2, or a function of inhibiting coronavirus proteases, particularly main proteases. The food composition, functional food, nutrient functional food, or food for specified health uses may, for example, be a beverage, food, or supplement containing selenonine or its tautomers or dimers or food-acceptable salts thereof in an amount of about 1 to 1000 ppm, preferably 10 to 100, most preferably 30 to 70 ppm. Selenonein is known to be abundant in fish. For example, assuming that bluefin tuna contains 30 mg Se / kg of selenonein, and that 100 g of fish meat is consumed and uniformly distributed in a 60 kg human body, theoretically, the selenonein content in the body can be estimated to be approximately 1 μM, and the blood concentration approximately 8 μM. Fish that may contain selenonein include tuna, swordfish, mackerel, yellowtail, sea bream, pufferfish, salmon and trout, and flounder and sole, with tuna, swordfish, mackerel, and yellowtail containing particularly high amounts. Examples of food compositions, functional foods, nutritional functional foods, or foods for specified health uses include the raw edible parts of these fish or processed foods made from fish. These fish may be wild or farmed. Since the selenonein content in fish varies depending on the type of feed, farmed fish with increased selenonein content are more preferable. Examples of processed foods made from fish include canned goods, bottled goods, tsukudani (simmered seafood or vegetables), dried fish, dried fish products, processed fish products, pickled fish, supplements, and any other foods made from fish.

[0029] Examples of tuna species used in food compositions, functional foods, nutritional functional foods, or foods for specified health uses include the Thunnus tribe and the Skipjack genus. Examples of Thunnus species include the genera Thunnus, Frigate Tuna, Skipjack Tuna, and Bonito, while examples of Skipjack genus include the genera Dogtooth Tuna and Skipjack Tuna. Examples of tuna species include albacore, bluefin tuna, southern bluefin tuna, Atlantic bluefin tuna, yellowfin, bigeye tuna, and longtail tuna of the Thunnus genus, skipjack tuna of the Frigate Tuna genus, frigate tuna and bullet tuna of the Frigate Tuna genus, skipjack tuna of the Frigate Tuna genus, and Skipjack tuna of the Skipjack genus, or albacore, bluefin tuna, southern bluefin tuna, Atlantic bluefin tuna, yellowfin, bigeye tuna, longtail tuna, Skipjack tuna, or skipjack tuna. Preferably, the species include albacore tuna, bluefin tuna, Atlantic bluefin tuna, yellowfin tuna, bigeye tuna, longtail tuna, skipjack tuna, or skipjack tuna.

[0030] All references made herein are incorporated herein by citation in their entirety.

[0031] The embodiments of the present invention described below are for illustrative purposes only and do not limit the technical scope of the invention. The technical scope of the invention is limited solely by the claims. Modifications to the invention, such as additions, deletions, and substitutions of constituent elements of the invention, can be made without departing from the spirit of the invention. [Examples]

[0032] Example 1: Inhibitory activity of papain 10 μg / ml papain (distributed by Sigma-Aldrich Japan) and 5 μM ebselenium or selenoneine were dissolved in 50 mM Tris-HCl (pH 7.4) maintained at 37°C. After pre-incubation for 15 minutes, 10 μM Bz-Arg-MCA was added to the reaction solution, and the change in fluorescence intensity accompanying the cleavage of Bz-Arg-MCA was observed over time using a fluorometer. The results are shown in Figure 4.

[0033] 10 μg / ml papain (distributed by Sigma-Aldrich Japan) and 0 to 125 μM ebselen or 0 to 5 μM selenonein were dissolved in 50 mM Tris-HCl (pH 7.4) maintained at 37°C. After pre-incubation for 15 minutes, 10 μM Bz-Arg-MCA was added to the reaction mixture, and the inhibitory activity was investigated by measuring the change in fluorescence intensity associated with the cleavage of Bz-Arg-MCA using a fluorometer. The results are shown in Figure 5. From the results in Figure 5, the inhibitory activity of selenonein against papain (IC) was determined. 50 =0.25 μM), and the inhibitory activity of ebselen (IC) 50 We calculated (=5.0 μM).

[0034] Example 2: M of SARS-CoV-2 pro Preparation Through total gene synthesis, M is created according to the base sequence of NC_45512 (10055-10972: SEQ ID NO: 4). pro The DNA was synthesized. pro The plasmid was introduced into a plasmid tagged with GST and histidine (Figure 6) and transformed into E. coli BL21(DE3) strain. The E. coli BL21(DE3) strain was cultured at 37°C and Mpro Protein expression was performed. The cell bodies were harvested and, using a binder for histidine tags, M pro The protein was purified. pro GST and histidine tags are removed by autodigestion and human rhinovirus 3C protease (HRV), and undigested M is removed using a binder for the histidine tags. pro Remove HRV and M pro A purified protein sample was obtained. pro The protein was dissolved in a storage buffer consisting of 20 mM Tris-HCl, 100 mM NaCl, 0.01% Triton-X-100, 50% glycerol, 1 mM EDTA, and 1 mM DTT, subjected to SDS-PAGE, and stained with Coomassie brilliant blue (Figure 7). Purified M dissolved in the storage buffer containing DTT pro The protein was dialysis-treated to remove DTT using a micro-dialysis cartridge, Xpress Micro / Mini Dialyzer (distributed by Funakoshi Co., Ltd.), and then subjected to the inhibitory activity test of Example 4.

[0035] Example 3: Preparation of PLpro from SARS-CoV-2 PLpro DNA is synthesized by total gene synthesis according to the nucleotide sequence of sequence number NC_45512 (4955-5908: sequence number 5). PLpro is introduced into a histidine-tagged plasmid and transformed into E. coli BL21(DE3) strain. E. coli BL21(DE3) strain is cultured to express PLpro protein. The cell bodies are harvested and the PLpro protein is purified using a histidine tag binder. GST and the histidine tag are removed by autodigestion of PLpro and HRV, and undigested PLpro and HRV are removed using a histidine tag binder to obtain a purified PLpro protein preparation.

[0036] Example 4: M of SARS-CoV-2 pro Inhibitory activity against 2 μg / ml M pro5 μM ebselen, selenonein, or ergothioneine were each dissolved in 50 mM Tris-HCl (pH 7.4) maintained at 37°C, and after pre-incubation for 5 minutes, 10 μM Ac-Abu-Tle-Leu-Gln-MCA was added to the reaction solution, and the change in fluorescence intensity accompanying the cleavage of Ac-Abu-Tle-Leu-Gln-MCA was measured using a fluorometer to investigate the inhibitory activity (Figure 8). As a control, the change in fluorescence intensity was measured under conditions that differed only in that no inhibitory compound was added. Ac-Abu-Tle-Leu-Gln-MCA is M pro It is decomposed by the following and generates a fluorescent substance, M pro The generation of fluorescent substances is suppressed by the action of inhibitory compounds. [ka] The changes in fluorescence intensity when ebselen and ergothioneine were added were equivalent to those of the control without addition. On the other hand, when selenonein was added, the fluorescence intensity was kept lower compared to the control without addition, indicating that selenonein was M pro It was shown that it inhibits the activity of [the substance].

[0037] 2 μg / ml M pro Then, 0-25 μM ebselen, 0-25 μM ergothioneine, or 0-5 μM selenoneine were each dissolved in 50 mM Tris-HCl (pH 7.4) maintained at 37°C, and after pre-incubation for 5 minutes, 10 μM Ac-Abu-Tle-Leu-Gln-MCA was added to the reaction solution, and the fluorescence intensity change accompanying the cleavage of Ac-Abu-Tle-Leu-Gln-MCA was measured using a fluorometer, and the fluorescence intensity was measured to obtain the inhibition curve (Figure 8). From the regression curve, IC 50 The values ​​were calculated and are shown in the table below: [Table 1]

[0038] Example 5: SARS-CoV-2 M pro Inhibitory activity against 2 μg / ml M pro Each 1 μM test compound was dissolved in 50 mM Tris-HCl (pH 7.4) maintained at 37°C, and after pre-incubation for 5 minutes, 10 μM Ac-Abu-Tle-Leu-Gln-MCA was added to the reaction solution, and the fluorescence intensity associated with the cleavage of Ac-Abu-Tle-Leu-Gln-MCA was measured using a fluorometer to determine the inhibitory activity (Figure 10). The test compounds used were selenonein, ebselenium, selenocystine ((SeCys)2), methylselenocysteine ​​(MeSeCys), selenomethionine (SeMet), diphenyl diselenide (PhSeSePh), and sodium selenite. These serine-containing compounds, excluding sodium selenite, showed M pro This compound was predicted to have inhibitory activity (Non-Patent Literature 4). When the fluorescence intensity one minute after adding the test compound was measured, with the fluorescence intensity without the test compound set to 100, selenonein was approximately 20%, while ebselenium, selenocystine ((SeCys)2), methylselenocysteine ​​(MeSeCys), selenomethionine (SeMet), diphenyl diselenide (PhSeSePh), and sodium selenite (selenite) were all over 90%. Therefore, selenonein was predicted to have inhibitory activity compared to other selenium-containing compounds, especially M pro It was shown to be remarkably superior in terms of inhibitory activity.

[0039] Example 6: Inhibitory activity against SARS-CoV-2 proliferation The inhibition of SARS-CoV-2 infection in host cells by ebselen or selenonein can be measured, for example, by a plaque assay. Host cells are cultured in a monolayer at 37°C and 5% CO2 until confluence. The culture medium is then removed, the cell surface is washed with PBS(-), and the virus solution and a 0 to 100 μM protease inhibitor solution are added and incubated for 30 minutes. After 30 minutes, the sample solution is removed and an agar plate is overlaid. Once the agar has solidified, the plate is inverted and incubated at 37°C and 5% CO2 for 2 days. The overlaid medium is then removed, the plate is dried, stained with crystal violet for 5 minutes, washed with purified water, and air-dried. Finally, the number of plaques is counted and compared with the control group to calculate the infection inhibition rate of the protease inhibitor against the virus.

Claims

1. The tautomer contains selenoneine, or its tautomers or dimers, or a pharmaceutically acceptable salt thereof, wherein the tautomer has the following chemical formula: 【Chemistry 1】 This is the main protease inhibitor of SARS-CoV-2, represented by [formula].

2. The tautomer contains selenoneine or its tautomer or dimer or a pharmaceutically acceptable salt thereof, wherein the tautomer has the following chemical formula: 【Chemistry 2】 A composition for the treatment or prevention of COVID-19, represented by [the specified symbol].

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