Use of Disulfiram in Antiviral Therapy Against Coronavirus

Abstract

The present invention discloses the use of disulfiram in anti-coronavirus treatment, specifically in the manufacture of a medicament for treating and / or preventing coronavirus diseases. In vitro enzyme activity experiments have demonstrated that disulfiram can effectively inhibit the activity of the main protease in coronaviruses, thereby resolving the lack of existing technologies for treating coronavirus diseases.

Description

Detailed Description of the Invention

[0001] [Cross - Reference to Related Applications] This application claims the priority of Chinese Application No. 2020101216884 with a filing date of February 26, 2020. This application incorporates by reference the entire text of the Chinese patent application.

[0002] [Technical Field] The present invention belongs to the technical field of biopharmaceuticals, and relates to the use of disulfiram in anti - coronavirus, specifically, the use of disulfiram in the manufacture of drugs for treating and / or preventing diseases caused by coronaviruses.

[0003] [Background Art] Coronaviruses are viruses that are closely related to humans and animals. Coronaviruses HCoV-229E and HCoV-OC43 cause common colds (van der Hoek, L., Pyrc, K., Jebbink, M. et al. Identification of a new human coronavirus. Nat Med 2004, 10, 368-373). During 2002 to 2003, severe acute respiratory syndrome (SARS) caused by the SARS coronavirus infected 8,098 people worldwide (Stadler, K., Masignani, V., Eickmann, M. et al. SARS - beginning to understand a new virus. Nat Rev Microbiol 2003, 1, 209-218). HCoV-NL63 identified in 2004 also causes respiratory diseases similar to colds (van der Hoek, L., Pyrc, K., Jebbink, M. et al. Identification of a new human coronavirus. Nat Med 2004, 10, 368-373). The Middle East respiratory syndrome coronavirus (MERS-CoV) emerged in 2012, and by April 26, 2016, there were 1,728 infections in 27 countries (de Wit, E., van Doremalen, N., Falzarano, D. et al. SARS and MERS: recent insights into emerging coronaviruses. Nat Rev Microbiol 2016, 14, 523-534.). The recently prevalent SARS-CoV-2 virus causes COVID-19, and the clinical findings are fever, dry cough, and dyspnea (Jeannette Guarner, MD, Three Emerging Coronaviruses in Two Decades: The Story of SARS, MERS, and Now COVID-19, American Journal of Clinical Pathology,, aqaa029).The coronavirus has a great impact on the livestock industry. Porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), and porcine delta coronavirus (PDCoV, also called deltavirus) cause severe gastroenteritis, diarrhea, vomiting, and dehydration in pigs, resulting in huge losses to the pig farming industry (Akimkin V, Beer M, Blome S, et al. New Chimeric Porcine Coronavirus in Swine Feces, Germany, 2012. Emerg Infect Dis. 2016,22(7):1314-1315). Feline infectious peritonitis virus (FIPV) causes a lethal disease in felids. Infectious bronchitis virus (IBV) infects birds and is a widely distributed poultry disease that has a great negative impact on the poultry industry.

[0004] The coronavirus belongs to the order Nidovirales, family Coronaviridae, subfamily Orthocoronavirinae in phylogenetic classification. Currently, the genomic sequence of the SARS-CoV-2 coronavirus has already been disclosed. The nucleotide similarity of this sequence to SARS-Cov is approximately 90%, and the protein sequence has approximately 80% sequence similarity to SARS-CoV. The genome of the coronavirus is single-stranded sense RNA, about 28 kb in length, and mainly encodes the structural proteins necessary for virus packaging and the non-structural proteins related to replication and transcription. The development of drugs and vaccines for treating coronavirus-related diseases mainly targets the above two types of proteins. Two-thirds of the viral genome mainly encodes non-structural proteins. The virus encodes two types of polymerase proteins, pp1a and pp1ab, which are involved in the virus replication process. Pp1a and pp1ab can be cleaved into 16 non-structural proteins nsp1-16 by papain and main protease encoded by two types of viruses. If these functional subunits are not cleaved into independent protein units by the protease encoded by the virus, the virus cannot complete its normal transcription and replication functions, and further assemble into a replication and transcription complex to complete virus replication and transcription. Among them, papain has three enzyme cleavage sites, and the main protease has 11 enzyme cleavage sites in pp1a and pp1ab.Since it has been found that the main protease plays an important regulatory role in the process of virus transcription and replication, it has become the focus of research (Ziebuhr, J.; Snijder, E. J.; Gorbalenya, A. E. Virus-encoded proteinases and proteolytic processing in the Nidovirales. J Gen Virol 2000, 81, 853-79.;Ziebuhr, J. Molecular biology of severe acute respiratory syndrome coronavirus. Curr Opin Microbiol 2004, 7, 412-9.). Due to the importance of the main protease for the amplification and replication of coronaviruses, the search for inhibitors with strong specificity and high safety for the catalytic site of the main protease is particularly important for drug development.

[0005] Disulfiram (CAS number: 97-77-8) is a specific aldehyde dehydrogenase (ALDH1) inhibitor (Liu, Xinwei, et al. “Targeting ALDH1A1 by disulfiram / copper complex inhibits non-small cell lung cancer recurrence driven by ALDH-positive cancer stem cells.” Oncotarget 7.36 (2016): 58516.), which is used in the treatment of chronic alcoholism and causes acute sensitivity to alcohol. In addition, disulfiram acts on the dopaminergic system, and both disulfiram and its metabolite carbon disulfide act on dopamine-β-hydroxylase (DBH) to increase dopamine levels. This leads to some neuropsychiatric symptoms such as delirium, paranoia, memory impairment, ataxia, dysarthria, and frontal release signs. Besides such effects, disulfiram inhibits dopamine-β-hydroxylase, resulting in an increase in dopamine concentration and a decrease in norepinephrine in the brain, suggesting the anti-dependence effect of disulfiram in alcohol dependence.

[0006] In 2017, the anti-tumor activity of disulfiram was reported in the journal Nature (Skrott, Zdenek, et al. “Alcohol-abuse drug disulfiram targets cancer via p97 segregase adaptor NPL4.” Nature 552.7684 (2017): 194-199.). The study found that in the body, disulfiram is metabolized into a small molecule that promotes the aggregation of the natural protein NPL4 and binds to its ligand, the p97 enzymes. This process inhibits the function of the NPL4-p97 complex, which promotes tumor growth, and induces the death of cancer cells. In addition, disulfiram has been found to have specific activity against zinc finger and ring finger E3 ubiquitin ligases, which play important roles in cancer progression (R Kona, F., D. A. M. B. Buac, and A. M Burger. “Disulfiram, and disulfiram derivatives as novel potential anticancer drugs targeting the ubiquitin proteasome system in both preclinical and clinical studies.” Current cancer drug targets 11.3 (2011): 338-346.). The related patent application is WO2017077336A1. In the existing technology, there is still no drug that can be used to treat related diseases caused by the coronavirus with disulfiram.

[0007] [Disclosure of the Invention] The technical problem to be solved by the present invention is to address the defect that there is still no drug in the existing technology that can effectively treat and / or prevent diseases caused by the coronavirus, and to provide the use of disulfiram in anti-coronavirus, specifically, the use of disulfiram in the manufacture of drugs for treating and / or preventing diseases caused by the coronavirus.

[0008] The present invention mainly solves the above technical problems by the following technical solutions. The present invention provides the use of disulfiram (CAS number: 97-77-8) in the manufacture of a drug for treating and / or preventing diseases caused by coronaviruses.

[0009] Preferably, the disease is a disease of mammals or birds. Preferably, the mammals include humans, pigs, and cats. The coronavirus described in the present invention is well-known in the art in terms of definition and belongs to the order Nidovirales, family Coronaviridae, subfamily Orthocoronavirinae in phylogenetic classification. Coronaviruses have an envelope, are single-stranded sense-strand RNA viruses with genomes, and are a type of virus widely present in nature.

[0010] The object of the present invention is to provide a potential treatment plan for diseases caused by coronavirus infection. The coronavirus described in the present invention preferably belongs to the subfamily Orthocoronavirinae, more preferably to the genus Alphacoronavirus, Betacoronavirus, Gammacoronavirus, or Deltacoronavirus.

[0011] In one preferred embodiment of the present invention, the disulfiram can be used to treat important infectious diseases caused by other coronaviruses such as SARS-CoV (beta coronavirus genus) and MERS-CoV in addition to treating diseases caused by SARS-CoV-2 (beta coronavirus genus). As an ordinary cold medicine, it can also be used to treat diseases caused by coronaviruses such as HCoV-HKU1 (Human coronavirus HKU1; beta coronavirus genus), HCoV-NL63 (Human coronavirus NL63; alpha coronavirus genus), HCoV-OC43 (Human coronavirus OC43), and HCoV-229E (Human coronavirus 22E; alpha coronavirus genus). Furthermore, as a veterinary medicine, it can be used to treat animal diseases such as Transmissible gastroenteritis virus (TGEV; alpha coronavirus genus), Porcine epidemic diarrhea virus (PEDV; alpha coronavirus genus), Porcine delta coronavirus (PDCoV; delta coronavirus genus), Feline infectious peritonitis virus (FIPV; alpha coronavirus genus), and Infectious bronchitis virus (IBV; gamma coronavirus genus).

[0012] Therefore, the coronavirus described in the present invention is preferably selected from SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-HKU1, HCoV-NL63, HCoV-OC43, HCoV-229E, TGEV, PEDV, PDCoV, FIPV, or IBV.

[0013] In one embodiment, the disulfiram of the present invention or a pharmaceutically acceptable salt thereof is present in the form of a drug composition containing the same. Preferably, the drug composition contains disulfiram and / or a pharmaceutically acceptable salt thereof as the sole active ingredient of the drug composition, and / or the drug composition further contains a pharmaceutically acceptable carrier, such as a pharmaceutically acceptable pharmaceutical adjuvant.

[0014] In one embodiment, the disulfiram of the present invention or a pharmaceutically acceptable salt thereof is present in the form of a kit containing the same. The kit further contains a drug for treating a disease associated with a coronavirus and / or a drug for treating a disease caused by another virus.

[0015] The drug compositions using disulfiram of the present invention as the active substance can all be produced by methods known in the art. They can be produced by formulating the compounds of the present invention into any dosage form suitable for human or animal use. The weight content of the compounds of the present invention in their drug compositions is usually 0.1 - 99.0%.

[0016] The above-mentioned pharmaceutically acceptable carrier is a conventional carrier in the art, and the carrier may be any suitable physiologically or pharmaceutically acceptable pharmaceutical adjuvant. The above-mentioned pharmaceutical adjuvants are conventional pharmaceutical adjuvants in the art, preferably including pharmaceutically acceptable excipients, fillers, diluents, etc. More preferably, the drug composition contains 0.01 - 99.99% of the above protein and / or the above antibody-drug conjugate and 0.01 - 99.99% of a pharmaceutical carrier, and the percentages are mass percentages in the drug composition.

[0017] The disulfiram in the present invention or a drug composition containing the same may be administered in the form of a unit dosage, and the administration route may be enteral or parenteral, such as oral, intravenous injection, intramuscular injection, subcutaneous injection, nasal cavity, oral mucosa, eyes, lungs and airways, skin, vagina, rectum, etc.

[0018] The dosage form may be a liquid dosage form, a solid dosage form, or a semi-solid dosage form. The liquid dosage form may be a solution (including a true solution and a colloidal solution), an emulsion (o / w type, w / o type, and multiple emulsions), a suspension, an injection (including a water injection, a powder injection, and an infusion solution), an eye drop, a nasal drop, a lotion, and a liniment, etc. The solid dosage form may be a tablet (ordinary tablet, enteric-coated tablet, troche, dispersible tablet, chewable tablet, effervescent tablet, orally disintegrating tablet), a capsule tablet (hard capsule, soft capsule, enteric-coated capsule), a granule, a powder, a micropellet, a dripping pill, a suppository, a film, an adhesive sheet, an aerosol (powder inhalation) agent, a spray agent, etc. The semi-solid dosage form may be an ointment, a gel, a paste, etc.

[0019] Disulfiram in the present invention can be manufactured into ordinary preparations, sustained-release preparations, release-controlled preparations, targeted preparations, and various microparticle administration systems. The term "pharmaceutically acceptable" means that salts, solvents, adjuvants, etc. are generally non-toxic, safe, and suitable for use by patients. The above-mentioned "patient" is preferably a mammal, more preferably a human.

[0020] The term "pharmaceutically acceptable salt" refers to a salt produced by a pharmaceutically acceptable acid or base that is relatively non-toxic compared to disulfiram in the present invention. On the premise of conforming to the common general knowledge in this field, by arbitrarily combining the above-mentioned various preferred conditions, each preferred example of the present invention can be obtained.

[0021] All the reagents and raw materials used in the present invention can be obtained as commercially available products. The positive progressive effects of the present invention are as follows. Through in vitro enzyme activity experiments, the present invention found that disulfiram can preferably inhibit the activity of the main protease in coronaviruses, compensating for the deficiency that existing technologies lack agents for treating diseases caused by coronaviruses. Disulfiram can be further studied, modified, and then applied.

Brief Description of the Drawings

[0022]

Figure 1

Figure 2

Figure 3

Mode for Carrying Out the Invention

[0023] Hereinafter, the present invention will be further described in the form of examples, but the present invention is not limited to the scope of the described examples. In the following examples, experimental methods for which specific conditions are not described were selected according to ordinary methods and conditions, or the product manuals.

[0024] [Example 1] High-throughput screening was performed from different compound libraries, and all the obtained disulfiram had good inhibitory effects on the main protease of the novel coronavirus. The conditions for the enzyme activity experiment are specifically as follows.

[0025] The fluorescent substrate for the enzyme activity experiment is MCA-AVLQSGFR-Lys(Dnp)-Lys-NH2, and the excitation wavelength and emission wavelength of the fluorescent substrate are 320 nm and 405 nm, respectively. The enzyme activity reaction buffer is 50 mM Tris-HCl (pH 7.3) and 1 mM EDTA, and the enzyme activity reaction temperature is 30°C. The reaction is initiated by the addition of protease, with the final concentration of protease reaction being 0.2 μM, the substrate concentration being 20 μM, and the disulfiram concentration being 1.5 μM (Xue X, Yang H, Shen W, et al. Production of authentic SARS-CoV Mpro with enhanced activity: application as a novel tag-cleavage endopeptidase for protein overproduction[J]. Journal of molecular biology, 2007, 366(3): 965-975.).

[0026] The positive control used is an N3 inhibitor, which can effectively inactivate the main protease of coronavirus (Yang Haitao, Xie Weiqing, Xiaoyu X, et al. Design of wide-spectrum inhibitors targeting coronavirus main proteases[J]. Organic Chemistry of Life, 2005, 3(10):1742-1752.), and its structural formula is shown below.

[0027] [Chemical Structure]

[0028] From Figure 1, it can be seen that the enzyme activity experimental curve of disulfiram is clearly different from that of the negative control, and it has obvious inhibitory activity against the main protease of coronavirus.

[0029] [Example 2] The fluorescent substrate used for measuring the enzyme activity inhibition curve is MCA-AVLQSGFR-Lys(Dnp)-Lys-NH2, and the excitation wavelength and emission wavelength of the fluorescent substrate are 320 nm and 405 nm respectively.

[0030] The buffer for measuring the enzyme activity inhibition curve is 50 mM Tris-HCl (pH 7.3) and 1 mM EDTA, and the reaction temperature is 30 °C. The reaction is initiated by the addition of protease, the final concentration of the protease reaction is 0.2 μM, the substrate concentration is 20 μM, and the results measured with 11 different concentrations of disulfiram showed that the IC 50 value was 9.35 μM (Figure 2) (Jin Z, Du X, Xu Y, et al. Structure of Mpro from COVID-19 virus and discovery of its inhibitors[J]. Nature, 2020, 582(7811):1-9.).

[0031] [Example 3] It was detected by the qRT-PCR method in the in vitro cell antiviral experiment. Vero cells were inoculated into a 96-well plate and incubated at 37 °C for 20 - 24 h, and then the cells were treated with 10 μM disulfiram for 1 h. Then, cells infected with SARS-CoV-2 were added for 2 h, and the MOI (multiplicity of infection) was 0.01. Then, the virus and drug mixture were removed, and after washing, the cells were further cultured in fresh medium containing the drug. After culturing for 72 h, viral RNA was extracted from the culture supernatant with the QIAamp viral RNA mini kit (Qiagen) and finally detected by qRT-PCR (Jin Z, Du X, Xu Y, et al. Structure of Mpro from COVID-19 virus and discovery of its inhibitors[J]. Nature, 2020, 582(7811):1-9.).

[0032] In the in vitro cell antiviral experiment, it was found that disulfiram inhibits the replication of the novel coronavirus. Therefore, disulfiram can be used for the treatment of related diseases caused by the coronavirus. The specific embodiments of the present invention have been described above. For those skilled in the art, these are only illustrative explanations, and various changes and modifications can be made to these embodiments on the premise that they do not contravene the principle and essence of the present invention. Therefore, the protection scope of the present invention is limited by the appended claims.

Claims

1. Use of disulfiram in the manufacture of a drug for the treatment and / or prevention of a disease caused by SARS-CoV-2.

2. The use according to claim 1, wherein the disease is a disease of mammals or birds.

3. The use according to claim 2, wherein the mammals include humans, pigs and cats.

4. The use according to claim 1, wherein the disulfiram is the sole active ingredient of the drug composition.

5. The use according to claim 4, wherein the drug composition further comprises a pharmaceutically acceptable carrier.

6. The use according to claim 5, wherein the drug composition further comprises a pharmaceutically acceptable pharmaceutical adjuvant.

7. The disulfiram is present in the form of a kit containing the same, and the kit further comprises a drug for treating a disease associated with a coronavirus and / or a drug for treating a disease caused by another virus. The use according to any one of claims 1 to 6.

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