Use of plpro protein inhibitors in the manufacture of a medicament for treating or preventing a novel coronavirus infection

Abstract

The present invention relates to the use of a PLpro protein inhibitor for the preparation of a medicament for the treatment or prevention of a disease associated with a novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and provides a pharmaceutical composition comprising a PLpro inhibitor for the preparation of a medicament for the treatment or prevention of a disease associated with a novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection.

Description

Technical Field

[0001] This invention relates to the use of PLpro protein inhibitors in the preparation of medicaments for the treatment or prevention of diseases related to novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, and provides pharmaceutical compositions comprising PLpro inhibitors for the preparation of medicaments for the treatment or prevention of diseases related to novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Background Technology

[0002] Coronaviruses are a class of positive-sense RNA viruses that pose significant health risks. Genetically, coronaviruses are divided into four main genera: Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus. The first two genera primarily infect mammals, while the latter two primarily infect birds. Prior to the discovery of the novel Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), six human coronaviruses had been identified, including HCoV-NL63 and HCoV-229E, which belong to the Alphacoronavirus family; and HCoV-OC43, HCoV-HKU1, SARS-CoV, and MERS-CoV, which belong to the Betacoronavirus family. SARS-CoV and MERS-CoV are considered highly pathogenic. The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, which causes COVID-19) is the seventh known coronavirus that can infect humans.

[0003] There is an urgent need to develop novel antiviral drugs targeting SARS-CoV-2.

[0004] Tioguanine (also known as thioguanine, 6-thioguanine, or 6-thioguanine, abbreviated as 6-TG) has the following structural formula:

[0005]

[0006] Thioguanine (6-TG) is a known chemotherapy drug used to treat acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), and chronic myeloid leukemia (CML). It can be administered orally. Common side effects include bone marrow suppression, liver damage, and oral inflammation. Individuals with thioguanine methyltransferase deficiency have a higher risk of side effects. Thioguanine is an antimetabolite, an analogue of guanine, that disrupts DNA and RNA synthesis.

[0007] Among the many derivatives of thioguanine, 6-mercaptopurine (abbreviated 6-TG-5) is also a known chemotherapy drug used to treat acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), and chronic myeloid leukemia (CML). It is metabolized into thioguanine in the body, thus exerting a similar effect. In addition, other derivatives of thioguanine are all known compounds. Summary of the Invention

[0008] This invention relates to the use of PLpro protein inhibitors in the preparation of medicaments for the treatment or prevention of diseases related to COVID-19 infection; in one aspect of the invention, the PLpro protein inhibitor is thioguanine and its derivatives or pharmaceutically acceptable salts, esters, stereoisomers, tautomers, polymorphs, solvates, metabolites or isotopically labeled compounds thereof.

[0009] The present invention also provides pharmaceutical compositions for treating or preventing diseases related to COVID-19 infection, comprising a PLpro protein inhibitor as an active ingredient; in one aspect of the invention, the PLpro protein inhibitor is thioguanine and its derivatives or pharmaceutically acceptable salts, esters, stereoisomers, tautomers, polymorphs, solvates, metabolites or isotopically labeled compounds thereof.

[0010] The present invention also provides a method for treating or preventing diseases related to COVID-19 infection, comprising administering an effective dose of a pharmaceutical composition containing a PLpro protein inhibitor as an active ingredient to patients exhibiting symptoms of COVID-19 infection, suspected COVID-19 patients, and individuals at high risk of COVID-19 infection. Attached Figure Description

[0011] Figure 1A Protein purification of PLpro from SARS-CoV-2: Coomassie brilliant blue staining image of PLpro from SARS-CoV-2

[0012] Figure 1B Protein purification of PLpro from SARS-CoV-2: Size exclusion chromatogram of PLpro protein, showing the protein eluting in a homogeneous monomeric state.

[0013] Figure 2 Assay for the inhibitory activity of thioguanine against PLpro enzyme of SARS-CoV-2: In vitro inhibitory activity of 6-TG against PLpro cleavage substrate Ub-AMC.

[0014] Figure 3A and 3B Inhibitory activity of thioguanine against SARS-CoV-2 in cells (A: 48h; B: 72h)

[0015] Figure 4A and 4B Inhibitory activity assays of thioguanine and its derivatives against PLpro: (A) Chemical structure of the derivatives and activity assays at single concentration points; (B) In vitro inhibitory activity of the derivatives against PLpro protein (IC50). Detailed Implementation

[0016] Due to the complexity of SARS-CoV-2 replication, many processes are considered essential to the novel coronavirus life cycle, thus providing numerous potential targets for inhibiting viral replication. The coronavirus genome is between 26,000 and 32,000 bases in size and includes two large open reading frames (ORFs). The first ORF, comprising approximately 67% of the entire genome, encodes 16 non-structural proteins (nsp), while the remaining ORFs encode accessory and structural proteins. The first ORF encodes a polyprotein containing the 16 non-structural proteins. Two cysteine ​​proteases residing in this polyprotein, namely a papain-like protease (PLpro) and a 3C-like protease (3CLpro), catalyze the release of themselves and other Nsps from the polyprotein, further initiating virus-mediated RNA replication. PLpro cleaves SARS-CoV-2 ORF1a / 1ab at three sites to release itself (nsp3), nsp1, and nsp2, while the remainder of the polyprotein is cleaved by 3CLpro. Studies have shown that PLpro, in addition to its important role in viral peptide cleavage, also functions in deubiquitination and ISG15 deactivation. By cleaving ubiquitin and ISG15, the PLpro protease exerts a negative regulatory effect on the host's innate antiviral immune response, facilitating viral evasion of the host's immune system. Therefore, the papain-like protease (PLpro) of SARS-CoV-2 is crucial for viral replication. This protease is not only responsible for processing viral polyproteins into their functional units but also plays a vital role in helping SARS-CoV-2 evade the human immune system. Based on this, the inventors hypothesize that inhibiting PLpro (SARS-CoV-2) has the potential to treat this highly lethal respiratory disease (COVID-19).

[0017] In addition, the inventors discovered that thioguanine (6TG) can inhibit the cleavage activity of PLpro (SARS-CoV-2) in vitro. Further cell experiments also showed that 6-TG can efficiently inhibit the replication of the novel coronavirus in Vero cells (EC50 = 2.6-3.5 μM, see Figure 3), thus serving as a treatment or preventive measure for diseases related to novel coronavirus infection.

[0018] Furthermore, through further research on the inhibitory activity of 6TG and its derivatives against the target protein PLpro, the inventors discovered the structure-activity relationship of 6TG and its derivatives. As shown in Figure 4, because the thiocarbonyl group (C=S) at position 6 of both 6-TG and 6-TG-6 is replaced by a carbonyl group (C=O), 6-TG-4 and 6-TG-6 have no inhibitory activity; while the other 6-TG-1 / 2 / 3 / 5 all have inhibitory activity against the PLpro protein. Therefore, the inventors found that the thiocarbonyl group at position 6 of thioguanine is indispensable for the inhibitory activity of 6-TG and its derivatives against the target protein PLpro.

[0019] The thioguanine and its derivatives involved in this invention are all compounds known in the art, and the compounds themselves and their synthesis methods are known to those skilled in the art.

[0020] One aspect of the present invention relates to the use of PLpro protein inhibitors in the preparation of medicaments for the treatment or prevention of diseases associated with novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection.

[0021] In one specific embodiment, thioguanine and its derivatives are: 6-thio-2-hydroxypurine, 6-thioguanine nucleoside, 2,6-dimercaptopurine, 6-mercaptopurine, or thioguanine. Their structural formulas are as follows:

[0022]

[0023] in:

[0024] R3 is selected from H and C. 1-6 Alkyl, Halogenated C 1-6 Alkyl and C 3-6 cycloalkyl;

[0025] R5 is selected from H, halogens, -OH, -COOH, -CN, -NO2, -N(R)2, C 1-6 Alkyl, Halogenated C 1-6 Alkyl, -WC 1-6 Alkyl, -C 1-6 Alkylene-WR, -WC 1-6 Alkylene -W'-R, -WC 2-6 alkenyl, -C 2-6alkenyl-WR, -WC 2-6 alkenyl-W'-R and C 3-6 cycloalkyl, wherein the alkylene and alkenyl groups are optionally further separated by one or more W groups;

[0026] Each time W and W' appear, they independently choose O, C(=O), C(=O)O, NR, NC(=O), N(S=O), NS(=O)2, S, S=O and S(=O)2;

[0027] Each time R appears, it is independently selected from H and C. 1-6 Alkyl and C 3-6 Cycloalkyl.

[0028] In a preferred embodiment, thioguanine and its derivatives are 6-thioguanine nucleoside.

[0029] Another aspect of the invention provides pharmaceutical compositions for treating or preventing diseases associated with novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, these pharmaceutical compositions comprising a PLpro protein inhibitor.

[0030] Another aspect of the invention provides pharmaceutical compositions for treating or preventing diseases associated with novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, comprising thioguanine and its derivatives as disclosed in this invention, or pharmaceutically acceptable salts, esters, stereoisomers, tautomers, polymorphs, solvates, metabolites, or isotopically labeled compounds thereof.

[0031] In a preferred embodiment, thioguanine and its derivatives are selected from 6-thio-2-hydroxypurine, 6-thioguanine nucleoside, 2,6-dimercaptopurine, 6-mercaptopurine or thioguanine; particularly preferred are 6-thioguanine nucleoside or thioguanine.

[0032] Unless otherwise defined below, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The term "technique" as used herein refers to technology commonly understood in the art, including variations or equivalents of technology as understood by one of ordinary skill in the art. While it is believed that the following terms will be understood by one of ordinary skill in the art, the following definitions are set forth to better explain the invention.

[0033] This invention covers all possible crystalline forms or polymorphs of the compounds of this invention, which may be a single polymorph or a mixture of more than one polymorph in any proportion.

[0034] It should also be understood that the compounds of the present invention may exist in their free form for therapeutic purposes, or, where appropriate, in their pharmaceutically acceptable derivative forms. In the present invention, pharmaceutically acceptable derivatives include, but are not limited to, pharmaceutically acceptable salts, esters, solvates, metabolites, isotopically labeled compounds, or prodrugs, which, upon administration to a patient in need, can directly or indirectly provide the compounds of the present invention or their metabolites or residues. Therefore, when referring to "compounds of the present invention" herein, it is also intended to cover the various derivative forms of the compounds described above.

[0035] Pharmaceutically acceptable salts of the compounds of the present invention include their acid addition salts and base addition salts.

[0036] Suitable acid addition salts are formed from acids that form pharmaceutically acceptable salts, including appropriate inorganic and organic acids. Examples include aspartate, benzoate, bicarbonate / carbonate, bisulfate / sulfate, fumarate, glucohepanoate, glucuronate, glucuronate, hexafluorophosphate, hydrobromide / bromide, hydroiodate / iodide, maleate, malonate, methyl sulfate, naphthylcarbamate, nicotinate, nitrate, orotate, oxalate, palmitate, and other similar salts.

[0037] Suitable base addition salts are formed from bases that form pharmaceutically acceptable salts, including appropriate inorganic and organic bases. Examples include aluminum salts, arginine salts, choline salts, diethylamine salts, lysine salts, magnesium salts, meglumine salts, potassium salts, and other similar salts.

[0038] For a review of suitable salts, see Stahl and Wermuth's "Handbook of Pharmaceutical Salts: Properties, Selection, and Use" (Wiley-VCH, 2002). Methods for preparing pharmaceutically acceptable salts for the compounds of the present invention are known to those skilled in the art.

[0039] As used herein, the term "ester" means an ester derived from the various general formula compounds of this application, including physiologically hydrolyzable esters, which can be hydrolyzed under physiological conditions to release the compounds of the present invention in the form of free acids or alcohols. The compounds of the present invention may themselves also be esters.

[0040] The compounds of the present invention may exist as solvates (e.g., hydrates), wherein the compounds of the present invention contain a polar solvent, particularly water, methanol, or ethanol, as a structural element of the lattice of the compound. The amount of the polar solvent, particularly water, may be stoichiometric or non-stoichiometric.

[0041] The scope of this invention also includes metabolites of the compounds of this invention, i.e., substances formed in the body when the compounds of this invention are administered. Such products can be generated, for example, by oxidation, reduction, hydrolysis, amidation, deamidation, esterification, enzymatic hydrolysis, etc., of the administered compound. Therefore, this invention includes metabolites of the compounds of this invention, including compounds obtained by methods that expose the compounds of this invention to mammals for a time sufficient to produce their metabolites.

[0042] Severe acute respiratory syndrome (SARS-CoV-2) infection-related illness is mainly characterized by fever, dry cough, and fatigue. A few patients may also experience nasal congestion, runny nose, sore throat, muscle pain, and diarrhea. Some patients may have severe radiographic pneumonia but lack clinical symptoms. Severe cases typically develop dyspnea and / or hypoxemia one week after onset (but some patients may develop it after 20 days, or even during two episodes). Severe cases can rapidly progress to ARDS, septic shock, uncorrectable metabolic acidosis, coagulation disorders, and multiple organ failure. It is noteworthy that severe and critically ill patients may experience low-grade fever or even no fever during the course of the illness.

[0043] The present invention also provides pharmaceutical compositions comprising a preventatively or therapeutically effective amount of the compound of the present invention or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, metabolite, or isotopically labeled compound thereof. In one embodiment, the pharmaceutical composition further comprises one or more non-PLpro protein inhibitors. For example, other therapeutic agents for the prevention or treatment of viral diseases. In one embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0044] In this invention, "pharmaceutically acceptable carrier" refers to a diluent, excipient, vehicle, or medium that is administered co-administered with a therapeutic agent and is suitable, to the extent of reasonable medical judgment, for contact with human and / or other animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications commensurate with a reasonable benefit / risk ratio.

[0045] Pharmaceutically acceptable carriers that can be used in the pharmaceutical compositions of the present invention include, but are not limited to, sterile liquids such as water and oils, including those of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Water is an exemplary carrier when the pharmaceutical composition is administered intravenously. Physiological saline and aqueous solutions of glucose and glycerol can also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, maltose, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene glycol, water, ethanol, etc. The compositions may also contain small amounts of wetting agents, emulsifiers, or pH buffers as needed. Oral formulations may contain standard carriers such as pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutically acceptable carriers are described in Remington's Pharmaceutical Sciences (1990).

[0046] The pharmaceutical compositions of the present invention can act systemically and / or locally. For this purpose, they can be administered via suitable routes, such as by injection (e.g., intravenous, intra-arterial, subcutaneous, intraperitoneal, intramuscular injection, including infusion) or transdermal administration; or by oral, sublingual, nasal, transmucosal, topical, ophthalmic formulation, or inhalation administration. For these routes of administration, suitable dosage forms can be employed.

[0047] The dosage forms include, but are not limited to, tablets, capsules, lozenges, hard candies, powders, sprays, creams, ointments, suppositories, gels, pastes, lotions, ointments, aqueous suspensions, injectable solutions, elixirs, and syrups.

[0048] As used herein, the term "effective dose" refers to the amount of a compound that, when administered, provides some relief for one or more symptoms of the treated condition. Dosing regimens can be adjusted to provide the optimal desired response. For example, a single dose may be administered, several fractions may be given over time, or the dose may be proportionally reduced or increased depending on treatment progress. It should be noted that dose values ​​can vary depending on the type and severity of the condition and may include single or multiple doses. To further understand, for any given individual, the specific dosing regimen should be adjusted over time based on individual needs.

[0049] The amount of the compounds of the present invention administered will depend on the individual being treated, the severity of the condition or illness, the frequency of administration, the disposal of the compounds, and the prescribing physician's judgment. Generally, the effective dose is from about 0.01 to about 400 mg per kg of body weight per day, for example, from about 0.02 to about 200 mg / kg / day (single or divided doses). For a 70 kg person, this would total from about 0.7 mg / day to about 28,000 mg / day, for example, from about 1.4 mg / day to about 14,000 mg / day. In some cases, dose levels not exceeding the lower limit of the foregoing range may be sufficient, while in other cases, larger doses may still be used without causing any harmful side effects, provided that the larger dose is first divided into several smaller doses administered throughout the day.

[0050] The content or amount of the compound of the present invention in the pharmaceutical composition may be from about 0.01 mg to about 10,000 mg, suitably 0.1-5,000 mg, preferably 0.5-3,000 mg, more preferably 1-1,500 mg, particularly preferably 1-500 mg, such as 1.5 mg, 2 mg, 4 mg, 10 mg, 25 mg, 100 mg, 200 mg, etc.

[0051] Unless otherwise stated, as used herein, the term “treatment” means to reverse, alleviate, or inhibit the progression of a disease or condition or one or more symptoms of such a disease or condition to which such term is applied, or to prevent such a disease or condition or one or more symptoms of such a disease or condition.

[0052] Example 1: PLpro protein induction expression and purification

[0053] The PLpro prokaryotic expression plasmid (the amino acid sequence of the expressed PLpro protein is shown in SEQ ID NO.1) was transformed into BL21(DE3) E. coli competent cells and grown in LB medium at 37°C until the OD600 reached 0.6-0.8. Protein expression was induced by adding 0.5 mM IPTG and 1 mM zinc chloride (ZnCl2), and then E. coli was grown overnight at 18°C. After centrifugation, the cell pellet was resuspended in a buffer solution of 50 mM Tris-HCl, 150 mM NaCl, 10 mM Imidazole, 2 mM DTT, and pH 8.5. The cells were then sonicated and centrifuged at 18,000 rpm, and the supernatant was collected. Purification was performed using a His-TRAP™ column, followed by elution with buffer (50 mM Tris-HCl, 150 mM NaCl, 250 mM Imidazole, 1 mM DTT, pH 7.4). Further purification was then carried out on a gel chromatography column (Superdex 20016 / 60, GE) using SEC buffer (20 mM Tris-HCl, 100 mM NaCl, 1 mM DTT, pH 7.4). The purified PLpro protein was concentrated to 5-10 mg / mL and stored for crystallization. For the purification results of PLpro protein from the novel coronavirus, please see [link to relevant documentation]. Figure 1A and Figure 1B , Figure 1A The image shows a distinct and unique staining band at the location where the molecular weight of the novel coronavirus is consistent; Figure 1B The results show that PLpro protein eluted in a uniform state as a monomer.

[0054] Example 2: In vitro assay to determine the inhibitory activity (IC50) of thioguanine (6-TG) on PLpro protein.

[0055] The reaction system consisted of 50 μL, containing 30 nM PLpro, 250 nM Ub-AMC (Boston Biochem) substrate, 50 mM Hepes (pH 7.5) reaction buffer, 0.01% Trixton X-100, 0.1 mg / ml BSA, and 2 mM DTT. Gradual concentrations (100, 50, 25, 12.5, 6.25, 3.125, 1.56, 0.78 μM) of the inhibitor thioguanine (6-TG) were added. Fluorescence emission intensity (excitation: 340 nm; emission: 430 nm) was measured using a microplate reader. The IC50 of thioguanine (6-TG) inhibiting PLpro enzyme activity was calculated based on the fluorescence intensity. The inhibition curves were fitted using GraphPad Prism. The fitting results are shown below. Figure 2 .

[0056] Example 3: P3 laboratory test of the inhibitory activity of thioguanine (6-TG) against SARS-CoV-2

[0057] (1) Compound cytotoxicity test:

[0058] 1×10 4 Vero cells (derived from the Cell Resource Center of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences) were seeded in 96-well plates with three replicates. Cells were incubated at 37°C for 20-24 hours. The culture medium was then removed, and 100 μL of fresh culture medium containing different concentrations of the compound (100, 50, 25, 12.5, 6.25, 3.125, 1.56, 0 μM) was added. Cell viability was measured using MTS / CCK8 reagent (purchased from Beijing Solarbio Science & Technology Co., Ltd., catalog number CK04-500T) with DMSO as a control. The cytotoxicity of the compound (CC50) was then calculated.

[0059] (2) Compound inhibition of viral activity test:

[0060] 1×10 4Vero cells were seeded in 96-well plates with three replicates and incubated at 37°C for 20-24 hours. Different concentrations of candidate compounds (100, 50, 25, 12.5, 6.25, 3.125, 1.56, 0 μM) were pretreated for 1 hour. Then, the target cells were infected with the SARS-CoV-2 Shenzhen clinical isolate BetaCoV / Shenzhen / SZTH-003 / 2020 at a multiplicity of infection (MOI) of 0.01. After incubation for 2 hours, the virus-compound mixture was removed, and fresh medium containing the candidate compounds (100, 50, 25, 12.5, 6.25, 3.125, 1.56, 0 μM) was added. The cells were incubated for another 48 hours, with DMSO treatment as a negative control. Viral RNA was extracted from the supernatant and reverse transcribed to obtain transcripts. Linearized plasmids containing the S gene of the COVID-19 virus (its corresponding nucleotide sequence is shown in SEQ ID NO.2) were transcribed in vitro to create a standard curve and quantify viral copy number. Primer and probe information: TaqMan primers for COVID-19 virus: 5'TCCTGGTGATTCTTCTTCAGG-3' and 5'-TCTGAGAGAGGGTCAAGTGC-3', and the COVID-19 virus probe 5'-FAM-AGCTGCAGCACCAGCTGTCCA-BHQ1-3'. Data analysis was performed to obtain data on the inhibition of viral replication by candidate compounds. The inhibitory activity data of thioguanine (6-TG) obtained after data processing against the PLpro cleavage substrate Ub-AMC at 48 and 72 hours are shown in Figure 3. sequence list <110> Peking University Shenzhen Graduate School <120> Application of PLpro protein inhibitors in drugs for the treatment or prevention of novel coronavirus infection <130> NTD-6699 <160> 2 <170> PatentIn version 3.5 <210> 1 <211> 317 <212> PRT <213> Severe acute respiratory syndrome coronavirus‑2 <400> 1 Glu Val Arg Thr Ile Lys Val Phe Thr Thr Val Asp Asn Ile Asn Leu 1 5 10 15 His Thr Gln Val Val Asp Met Ser Met Thr Tyr Gly Gln Gln Phe Gly 20 25 30 Pro Thr Tyr Leu Asp Gly Ala Asp Val Thr Lys Ile Lys Pro His Asn 35 40 45 Ser His Glu Gly Lys Thr Phe Tyr Val Leu Pro Asn Asp Asp Thr Leu 50 55 60 Arg Val Glu Ala Phe Glu Tyr Tyr His Thr Thr Asp Pro Ser Phe Leu 65 70 75 80 Gly Arg Tyr Met Ser Ala Leu Asn His Thr Lys Lys Trp Lys Tyr Pro 85 90 95 Gln Val Asn Gly Leu Thr Ser Ile Lys Trp Ala Asp Asn Asn Cys Tyr 100 105 110 Leu Ala Thr Ala Leu Leu Thr Leu Gln Gln Ile Glu Leu Lys Phe Asn 115 120 125 Pro Pro Ala Leu Gln Asp Ala Tyr Tyr Arg Ala Arg Ala Gly Glu Ala 130 135 140 Ala Asn Phe Cys Ala Leu Ile Leu Ala Tyr Cys Asn Lys Thr Val Gly 145 150 155 160 Glu Leu Gly Asp Val Arg Glu Thr Met Ser Tyr Leu Phe Gln His Ala 165 170 175 Asn Leu Asp Ser Cys Lys Arg Val Leu Asn Val Val Cys Lys Thr Cys 180 185 190 Gly Gln Gln Gln Thr Thr Leu Lys Gly Val Glu Ala Val Met Tyr Met 195 200 205 Gly Thr Leu Ser Tyr Glu Gln Phe Lys Lys Gly Val Gln Ile Pro Cys 210 215 220 Thr Cys Gly Lys Gln Ala Thr Lys Tyr Leu Val Gln Gln Glu Ser Pro 225 230 235 240 Phe Val Met Met Ser Ala Pro Pro Ala Gln Tyr Glu Leu Lys His Gly 245 250 255 Thr Phe Thr Cys Ala Ser Glu Tyr Thr Gly Asn Tyr Gln Cys Gly His 260 265 270 Tyr Lys His Ile Thr Ser Lys Glu Thr Leu Tyr Cys Ile Asp Gly Ala 275 280 285 Leu Leu Thr Lys Ser Ser Glu Tyr Lys Gly Pro Ile Thr Asp Val Phe 290 295 300 Tyr Lys Glu Asn Ser Tyr Thr Thr Thr Ile Lys Ala Ala 305 310 315 <210> 2 <211> 3822 <212> DNA <213> Severe acute respiratory syndrome coronavirus‐2 <400> 2 atgtttgtttt ttctgtttt attgccacta gtctctagtc atgtgttaa tcttacaacc 60 agaactcaat taccccctgc atacactaat tctttcacac gtggtgttta taccctgac 120 aaagttttca catcctcagttcattca actcaggact tgttcttch ttctttca 180 aatgttactt ggttccatgc tatacatgtc tctgggacca atggtactaa gaggtttgat 240 aaccctgtcc taccatta tgatgtgtt tattttgctt ccactgagaa gtctaacata 300 ataagaggct ggatttttgg tactacttta gattcgaaga cccagtccct acttattgtt 360 ataacgcta ctaatgttgt tattaaagtc tgtgaatttc aattttgtaa tgatccattt 420 ttgggtgttt attaccacaaaacaaaa agttggatgg aaagtgagtt cagagtttat 480 tctagtgcga atattgcac ttttgaatat gtctctcagc cttttcttat ggaccttgaa 540 ggaaaacagg gtaatttca aaatcttagg gatttgtgt ttaagaat tgatggttat 600 tttaaaatat attctaagca cacgcctatt aatttagtgc gtgatctccc tcagggtttt 660 tcggctttag aaccattggt agatttgcca ataggtatta acatcactag gtttcaaact 720 ttacttgctt tacatagaag ttattgact cctggtgatt cttcttcagg ttggacagct 780 ggtgctgcag cttattatgt gggttatctt caacctagga cttttctatt aaatataat 840 gaaaatggaa ccattacaga tgctgtagac tgtgcacttg accctctc agaaacaaag 900 tgtacgttga aatccttcac tgtagaaaaa ggaatctatc aaacttctaa ctttagagtc 960 caaccaacag aatctattgt tagatttcct aatattacaa acttgtgccc ttttggtgaa 1020 gttttaacg ccaccagatt tgcatctgtt tatgcttgga acaggaagag aatcagcaac 1080 tgtgttgctg attattctgt cctatataat tccgcatcat tttccacttt taagtgttat 1140 ggagtgtctc ctactaaatt aaatgatctc tgctttacta atgtctatgc agattcattt 1200 gtaattagag gtgatgaagt cagacaaatc gctccagggc aaactggaaa gattgctgat 1260 tataattata aattaccaga tgattttaca ggctgcgtta tagcttggaa ttctaacaat 1320 cttgattcta aggttggtgg taattataat tacctgtata gattgtttag gaagtctaat 1380 ctcaaacctt ttgagagaga tatttcaact gaaatctatc aggccggtag cacaccttgt 1440 aatggtgttg aaggttttaa ttgttacttt cctttacaat catatggttt ccaacccact 1500 aatggtgttg gttaccaacc atacagagta gtagtacttt cttttgaact tctacatgca 1560 ccagcaactg tttgtggacc taaaaagtct actaatttgg ttaaaaacaa atgtgtcaat 1620 ttcaacttca atggtttaac aggcacaggt gttcttactg agtctaacaa aaagtttctg 1680 cctttccaac aatttggcag agacattgct gacactactg atgctgtccg tgatccacag 1740 acacttgaga ttcttgacat tacaccatgt tcttttggtg gtgtcagtgt tataacacca 1800 ggaacaaata cttctaacca ggttgctgtt ctttatcagg atgttaactg cacagaagtc 1860 cctgttgcta ttcatgcaga tcaacttact cctacttggc gtgtttattc tacaggttct 1920 aatgtttttc aaacacgtgc aggctgttta ataggggctg aacatgtcaa caactcatat 1980 gagtgtgaca tacccattgg tgcaggtata tgcgctagtt atcagactca gactaattct 2040 cctcggcggg cacgtagtgt agctagtcaa tccatcattg cctacactat gtcacttggt 2100 gcagaaaatt cagttgctta ctctaataac tctattgcca tacccacaaa ttttactatt 2160 agtgttacca cagaaattct accagtgtct atgaccaaga catcagtaga ttgtacaatg 2220 tacatttgtg gtgattcaac tgaatgcagc aatcttttgt tgcaatatgg cagtttttgt 2280 acacaattaa accgtgcttt aactggaata gctgttgaac aagacaaaaa cacccaagaa 2340 gtttttgcac aagtcaaaca aatttacaaa acaccaccaa ttaaagattt tggtggtttt 2400 aatttttcac aaatattacc agatccatca aaaccaagca agaggtcatt tattgaagat 2460 ctacttttca acaaagtgac acttgcagat gctggcttca tcaaacaata tggtgattgc 2520 cttggtgata ttgctgctag agacctcatt tgtgcacaaa agtttaacgg ccttactgtt 2580 ttgccacctt tgctcacaga tgaaatgatt gctcaataca cttctgcact gttagcgggt 2640 acaatcactt ctggttggac ctttggtgca ggtgctgcat tacaaatacc atttgctatg 2700 caaatggctt ataggtttaa tggtattgga gttacacaga atgttctcta tgagaaccaa 2760 aaattgattg ccaaccaatt taatagtgct attggcaaaa ttcaagactc actttcttcc 2820 acagcaagtg cacttggaaa acttcaagat gtggtcaacc aaaatgcaca agctttaaac 2880 acgcttgtta aacaacttag ctccaatttt ggtgcaattt caagtgtttt aaatgatatc 2940 ctttcacgtc ttgacaaagt tgaggctgaa gtgcaaattg ataggttgat cacaggcaga 3000 cttcaaagtt tgcagacata tgtgactcaa caattaatta gagctgcaga aatcagagct 3060 tctgctaatc ttgctgctac taaaatgtca gagtgtgtac ttggacaatc aaaaagagtt 3120 gatttttgtg gaaagggcta tcatcttatg tccttccctc agtcagcacc tcatggtgta 3180 gtcttcttgc atgtgactta tgtccctgca caagaaaaga acttcacaac tgctcctgcc 3240 atttgtcatg atggaaaagc acactttcct cgtgaaggtg tctttgtttc aaatggcaca 3300 cactggtttg taacacaaag gaatttttat gaaccacaaa tcattactac agacaacaca 3360 tttgtgtctg gtaactgtga tgttgtaata ggaattgtca acaacacagt ttatgatcct 3420 ttgcaacctg aattagactc attcaaggag gagttagata aatattttaa gaatcataca 3480 tcaccagatg ttgatttagg tgacatctct ggcattaatg cttcagttgt aaacattcaa 3540 aaagaaattg accgcctcaa tgaggttgcc aagaatttaa atgaatctct catcgatctc 3600 caagaacttg gaaagtatga gcagtatata aaatggccat ggtacatttg gctaggtttt 3660 atagctggct tgattgccat agtaatggtg acaattatgc tttgctgtat gaccagttgc 3720 tgtagttgtc tcaagggctg ttgttcttgt ggatcctgct gcaaatttga tgaagacgac 3780 tctgagccag tgctcaaagg agtcaaatta cattacacat aa 3822

Claims

1. Use of a PLpro protein inhibitor in the preparation of a medicament for the treatment or prevention of novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, wherein the PLpro protein inhibitor is a 6-thioguanine nucleoside having the structure of formula (IV). in: R3 is H; R5 is -NH2.

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