WO2024005780A1 - Method for treating coronavirus infection or disease caused by coronavirus - Google Patents

Method for treating coronavirus infection or disease caused by coronavirus Download PDF

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Publication number
WO2024005780A1
WO2024005780A1 PCT/US2022/035218 US2022035218W WO2024005780A1 WO 2024005780 A1 WO2024005780 A1 WO 2024005780A1 US 2022035218 W US2022035218 W US 2022035218W WO 2024005780 A1 WO2024005780 A1 WO 2024005780A1
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sars
cov
peimine
coronavirus
doi
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PCT/US2022/035218
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French (fr)
Inventor
Mien-Chie Hung
Wei-Jan Wang
Yeh CHEN
Wen-Chi Su
Wei-Chao Chang
Chang-Hai TSAI
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China Medical University
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Priority to PCT/US2022/035218 priority Critical patent/WO2024005780A1/en
Publication of WO2024005780A1 publication Critical patent/WO2024005780A1/en

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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
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4375—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a six-membered ring having nitrogen as a ring heteroatom, e.g. quinolizines, naphthyridines, berberine, vincamine
    • 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
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/192—Carboxylic acids, e.g. valproic acid having aromatic groups, e.g. sulindac, 2-aryl-propionic acids, ethacrynic acid 
    • 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
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • 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
    • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/88—Liliopsida (monocotyledons)
    • A61K36/896—Liliaceae (Lily family), e.g. daylily, plantain lily, Hyacinth or narcissus
    • A61K36/8966—Fritillaria, e.g. checker lily or mission bells

Definitions

  • the present disclosure relates to a method for treating a coronavirus infection or a disease caused by coronavirus. More specifically, the present disclosure relates to a method for treating a coronavirus infection or a disease caused by coronavirus with peimine.
  • Coronavirus disease 2019 (CO VID- 19) is caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).
  • SARS-CoV-2 novel severe acute respiratory syndrome coronavirus 2
  • EUA emergency use authorization
  • VOCs variants of concern
  • An aspect of the present disclosure is drawn to a pharmaceutical i composition for treating a coronavirus infection or a disease caused by coronavirus, comprising: peimine and a pharmaceutically acceptable carrier.
  • Another aspect of the present disclosure is drawn to a method for treating a coronavirus infection or a disease caused by coronavirus in a subject in need thereof, comprising: administering to the subject an effective amount of peimine, or administering to the subject the aforesaid pharmaceutical composition.
  • a further aspect of the present disclosure is drawn to a use of peimine or the aforesaid pharmaceutical composition for manufacturing a medicament for treating a coronavirus infection or a disease caused by coronavirus.
  • the coronavirus may be severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).
  • SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
  • the disease may be a disease caused by SARS-CoV-2.
  • the disease caused by SARS-CoV-2 may be CO VID- 19.
  • the disease caused by SARS-CoV-2 may be atypical pneumonia.
  • the disease caused by SARS-CoV-2 may be a respiratory disease.
  • peimine or the pharmaceutical composition can be administered to a subject orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, or via an implanted reservoir.
  • parenteral as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques.
  • the pharmaceutical composition can be an oral pharmaceutical composition, which can be any orally acceptable dosage form including capsules, tablets, emulsions and aqueous suspensions, dispersions, and solutions.
  • Oral solid dosage forms can be prepared by spray dried techniques; hot melt extrusion strategy, micronization, and nano milling technologies.
  • the pharmaceutical composition can be a nasal aerosol or inhalation composition, which can be prepared according to techniques well known in the art of pharmaceutical formulation.
  • the pharmaceutical composition of the present disclosure can also be administered in the form of suppositories for rectal administration.
  • the carrier in the pharmaceutical composition must be “acceptable” in the sense that it is compatible with the active ingredient of the composition (and preferably, capable of stabilizing the active ingredient) and not deleterious to the subject to be treated.
  • treating refers to application or administration of the active ingredient to a subject with the purpose to cure, alleviate, relieve, alter, remedy, improve, or affect the disease, the symptom, or the predisposition.
  • An effective amount refers to the amount of the active ingredient which is required to confer the desired effect on the subject. Effective amounts vary, as recognized by those skilled in the art, depending on route of administration, excipient usage, and the possibility of co-usage with other therapeutic treatments such as use of other active agents.
  • the aforesaid subject may be mammal, for example, a human, a pig, a horse, a cow, a dog, a cat, a mouse or a rat.
  • FIG. 1 shows the results of screening for natural compounds with potential inhibitory activity on SARS-CoV-2 pseudo virus entry.
  • FIG. 2A shows the western blot results of overexpressing human ACE2 (hACE2) in 293T cells.
  • FIG. 2B shows the results of sciadopitysin, vanillic acid, peimine and semagacestat for blocking SARS-CoV-2 entry.
  • FIG. 2C to FIG. 2E show the results of peimine blocking SARS-CoV-2 entry.
  • FIG. 3A shows the western blot results of overexpressing human furin in Vero-E6 monkey kidney epithelial cells.
  • FIG. 3B shows the results of Vero-E6 cells with and without furin expression infected with variants of SARS-CoV-2.
  • FIG. 3C and FIG. 3D show the results of peimine inhibiting variants of SARS-CoV-2 infection in furin-overexpressing cells and 293T/hACE2 cells.
  • FIG. 4A shows the results of time-resolved FRET assay of the binding between SARS-CoV-2 Spike S 1 and human ACE2 with or without 100 pM peimine.
  • FIG. 4B shows the molecular docking result of the interaction between peimine and the spike RBD-ACE2 complex.
  • FIG. 5 shows the results of peimine docking analysis for wild type and variants of SARS-CoV-2.
  • FIG. 6 shows the results of peimine inhibiting variants of SARS-CoV-2 infection in Calu-3 cells.
  • FIG. 7A and FIG. 7B respectively show the results of the cell viability assay of peimine and hydroxychloroquine (HCQ).
  • FIG. 8 shows the western blot results of Huh-7 or 293T/hACE2 cells treated with different doses of peimine.
  • FIG. 9 shows the results of peimine inhibiting SARS-CoV-2 infection in Calu-3 cells with or without washing.
  • FIG. 10 shows the thals of Fritillaria thunbergii, Fritillaria cirrhosa D. Don and Traditional Chinese natural herbal for blocking pseudovirus infection.
  • Huh-7 and 293T cell lines were obtained from ATCC.
  • the 293T human embryonic kidney cell line, Calu-3 lung cancer cell line, Huh-7 human hepatocarcinoma cell line and Vero-E6 African green monkey kidney cell line were maintained in Dulbecco’s MEM (Gibco) containing 10% fetal bovine serum (HyClone) and 100 units of penicillin (HyClone), 100 pg of streptomycin (HyClone).
  • MEM Gibco
  • HyClone fetal bovine serum
  • penicillin HyClone
  • streptomycin HyClone
  • 293T cells stably expressed recombinant human ACE2 (293/hACE2) (S. C. Wang et al., 2020).
  • the pure compound library Natural Compound Library (Catalog # L6000, Target Molecule Corp, Inc.) was used to screen for drugs.
  • Vpp Lentiviral particles pseudotyped (Vpp) with SARS-CoV-2 spike protein infection assay
  • Vpp contains SARS-CoV-2 spike protein and a luciferase reporter (S. C. Wang et al., 2020).
  • SARS-CoV-2 variants were purchased from the National RNAi Core Facility (NRC), Academia Sinica, Taipei, Taiwan. Then, 3-5 pL of supernatant was added to the cells in a 96-well plate (MOI ⁇ 0.2) in the presence of polybrene (8 pg/mL). The plate was centrifuged at 1,200 pg for 30 min and then returned to the incubator. Twenty-four hours postinfection (hpi), the culture supernatants were replaced with fresh medium. Seventy-two hours postinfection, luciferase activity was determined according to the manufacturer’s instructions.
  • a survey scan was acquired in the mass range m/z 200-2,000.
  • the electrospray voltage was maintained at 4 kV, and the capillary temperature was set at 275 °C.
  • the peimine content in each sample was estimated based on the mass peak-area intensity of the precise molecular weight signal with the exact LC elution time as that in the peimine standard compound.
  • the RBD-ACE2 complex (PDB ID: 6VW1) was used as the template structure for the docking experiment. Initially, we replaced the valine residue at 417 with lysine in 6VW1 to generate the wild-type (WT) docking target using (PS)2V3 protein structure prediction server (T. T. Huang et al., 2015). Next, the N501Y mutation was generated to create the B.1.1.7 docking target. The B.1.351 target was established by introducing N501Y, E484K and K417N mutations into the WT target. L452R and T478K, and K417T, E484K and N501Y were introduced to generate B.1.617.2 and P l variants.
  • iGEMDOCK Hsu, Chen, Lin, & Yang, 2011
  • the best docking pose (solution) with the smallest docking score was selected for further representation.
  • the frequency distribution of the docking scores of 100 docking poses found for the WT, B.1.1.7 and B.1.351, B.1.617.2, P. l and B.1.1.529 targets are depicted. All visualizations of the docking results were visualized using PyMOL (Eriksson et al., 2021).
  • FIG. 1 shows the results of screening for natural compounds with potential inhibitory activity on SARS-CoV-2 pseudo virus entry.
  • Huh-7 cells were pretreated with 126 natural compounds at a final concentration of 10 pM for 1 h and then infected with lentivirus particles pseudotyped (Vpp) with SARS-CoV-2 spike protein (MOI-0.1). All viruses and compounds were removed 24 hpi (hours post infection). At 72 hpi, infectivity was assessed based on luciferase activity and is displayed as a Z-score chart. As shown in FIG. 1, the four compounds with the lowest Z-score are respectively sciadopitysin, vanillic acid, peimine and semagacestat, and these four compounds are used for the following experiments.
  • FIG. 2A shows the western blot results of overexpressing human ACE2 (hACE2) in 293T cells.
  • hACE2 was overexpressed in 293T cells, and the proteins were extracted from the cell lysates.
  • tubulin is the loading control. The results shown in FIG. 2A indicates that hACE2 was successfully overexpressed in 293 T cells.
  • FIG. 2B shows the results of sciadopitysin, vanillic acid, peimine and semagacestat for blocking SARS-CoV-2 entry. Statistical significance was calculated using Student’s t-test. *, p value ⁇ 0.05; ***, p value ⁇ 0.001 compared with the control.
  • 293T/hACE2 cells were pretreated with DMSO only, vanillic acid (10 pM), peimine (10 pM), sciadopitysin (10 pM) or semagacestat (10 pM) for 2 h and then inoculated with lentivirus particles pseudotyped (Vpp) with SARS-CoV-2 spike protein for 24 h.
  • peimine can be an effective natural compound of blocking SARS-CoV-2 entry.
  • FIG. 2C to FIG. 2E show the results of peimine blocking SARS-CoV-2 entry. Statistical significance was calculated using Student’s t-test. *, p value ⁇ 0.05; ***, p value ⁇ 0.001 compared with the control.
  • peimine is an effective natural compound of blocking SARS-CoV-2 entry.
  • Vpp backbones incorporate green fluorescent protein (GFP) that is expressed upon infection into target cells. Fluorescence was recorded 24 h post- infection. Magnification, 4X. Scale bar: 1000 pm. As shown in FIG. 2E, peimine is an effective natural compound of blocking SARS-CoV-2 entry.
  • GFP green fluorescent protein
  • FIG. 3A shows the western blot results of overexpressing human furin in Vero-E6 monkey kidney epithelial cells. Human furin was overexpressed in Vero-E6 monkey kidney epithelial cells, and proteins were extracted from the cell lysates. In FIG. 3A, tubulin is the loading control. The results shown in FIG. 3A indicate that human furin was successfully overexpressed in Vero-E6 cells.
  • FIG. 3B shows the results of Vero-E6 cells with and without furin expression infected with variants of SARS-CoV-2. Error bars indicate the SEM of technical triplicates. Statistical significance was calculated using Student’s t-test. *, p value ⁇ 0.05; ***, p value ⁇ 0.001 compared with the control.
  • Vero-E6 cells with and without furin expression were transfected and then inoculated with the B.1.1.7 (United Kingdom) and 501Y.V2 (South African) variants of lentivirus particles pseudotyped (Vpp) with SARS-CoV-2 mutant spike protein for 24 h.
  • Vero-E6 cells with and without furin expression were infected with the wide-type (Wildtype S), B.1.1.7 and 501Y.V2 Vpp.
  • FIG. 3C and FIG. 3D show the results ofpeimine inhibiting variants of SARS-CoV-2 infection in furin-overexpressing cells and 293T/hACE2 cells. Error bars indicate the SEM of technical triplicates. Statistical significance was calculated using Student’s t-test. *, p value ⁇ 0.05; ***, p value ⁇ 0.001 compared with the control.
  • Vero E6 cells with and without furin expression and 293T/hACE2 cells were preincubated with 10 pM peimine for 2 h and then infected with wild-type (Wildtype S, SA), B.1.1.7, or 501Y.V2, D614G or VSVG Vpp. The cells were lysed 24 h later, and Vpp transduction was measured. Experiments were performed in triplicate. As shown in FIG. 3C and FIG. 3D, peimine can effectively inhibit variants of SARS-CoV-2 infection in furin-overexpressing cells and 293T/hACE2 cells.
  • FIG. 4A shows the results of time-resolved FRET assay of the binding between SARS-CoV-2 Spike S 1 and human ACE2 with or without 100 pM peimine. The results indicate that peimine suppresses the interaction of SARS-CoV-2 Spike SI and ACE2.
  • FIG. 4B shows the molecular docking result of the interaction between peimine and the spike RBD-ACE2 complex.
  • FIG. 4B the overview of the optimal pose of the interaction between peimine and the spike RBD-ACE2 complex (PDB: 6VW1) is shown, wherein peimine is shown as sticks, and spike RBD and ACE2 are shown in the cartoon.
  • the hydrogen bonds are shown as dashed lines. The result indicates that peimine can block SARS-CoV-2 spike protein binding ACE2.
  • FIG. 5 shows the results of peimine docking analysis for wild type and variants of SARS-CoV-2, wherein one hundred docking poses of peiminewere analyzed for the targets of wild type (WT), B.1.1.7, B.1.1.529, B.1.351, B.1.617.2 and P.l variants of SARS-CoV-2_RDB-hACE2 complex.
  • WT wild type
  • B.1.1.7, B.1.1.529, B.1.351, B.1.617.2 and P.l variants of SARS-CoV-2_RDB-hACE2 complex The result shows that peimine can block SARS-CoV-2 spike protein binding ACE2, and peimine exhibits a higher binding affinity for variant spike proteins created through amino acid substitutions.
  • the contact potential of the binding pocket of SARS-CoV-2_RDB-hACE2 for different SARS-CoV-2 variants in complex with peimine was also analyzed. Even the figures are not shown, the results indicate that the environment of the binding cavity is mainly hydrophobic.
  • the binding affinity of peimine predicted for WT is -10.6 kcal/mol, -11.2 kcal/mol for B.1.1.7, -11.0 kcal/mol for B.1.351, -11.1 kcal/mol for Pl, -10.2 kcal/mol for B.1.1.529 and -11.4 kcal/mol for B.1.617.2.
  • the binding affinity values were computed using PRODI GI platform.
  • FIG. 6 shows the results of peimine inhibiting variants of SARS-CoV-2 infection in Calu-3 cells.
  • Calu-3 cells were preincubated with 1 or 10 pM peimine for 2 h and then infected with wildtype (wildtype S), B.1.1.7, B.1.351 (501Y.V2), Pl, B.1.617.2 or B.1.1.529 lentiviral particles pseudotyped (Vpp), respectively.
  • wildtype S wildtype S
  • B.1.1.7, B.1.351 501Y.V2
  • Pl Pl
  • Vpp lentiviral particles pseudotyped
  • FIG. 7A and FIG. 7B respectively show the results of the cell viability assay of peimine and hydroxychloroquine (HCQ).
  • 293T/hACE2 cells were counted after treatment with different doses of peimine (0-1000 pM) or HCQ (1-10 pM) for 24 h. Experiments were performed in triplicate. Error bars indicate the SEM of technical triplicates. The results shown in FIG. 7A and FIG. 7B indicate that peimine exhibits no toxicity on 293T cells compared to HCQ.
  • FIG. 8 shows the western blot results of Huh-7 or 293T/hACE2 cells treated with different doses of peimine.
  • Huh-7 or 293T/hACE2 cells were treated with different doses of peimine for 24 h.
  • TMPRSS2 and ACE2 expression in Huh-7 or 293T/hACE2 cells was determined through Western blot analysis. Tubulin was used as the loading control. The results indicate that peimine does not affect TMPRSS2 or ACE2 expression in Huh-7 or 293T/hACE2 cells.
  • FIG. 9 shows the results of peimine inhibiting SARS-CoV-2 infection in Calu-3 cells with or without washing, wherein n.s refers to non-significant.
  • Calu-3 cells were preincubated with 0, 1 or 10 pM peimine for 2 h, optionally washed with PBS and inoculated for 2 h in an incubator. After inoculation, the cells were infected with lentivirus particles pseudotyped (Vpp) with SARS-CoV-2 spike protein. The cells were lysed 24 h later, and Vpp transduction was measured. Experiments were performed in triplicate. Error bars indicate the SEM of technical triplicates. Statistical significance was calculated using Student’s t-test. The results shown in FIG. 9 indicate that the efficacy of peimine is not reversible.
  • Vpp lentivirus particles pseudotyped
  • FIG. 10 shows the thals of Fritillaria thunbergii, Fritillaria cirrhosa D. Don and Traditional Chinese natural herbal for blocking pseudovirus infection.
  • ACE2-expressing 293T cells were pretreated with Fritillaria thunbergia, Fritillaria cirrhosa and a commercially available Fritillaria cirrhosa -containing herbal medicines, Nim Jiom Chuanbei Pipa Gao (NJCPG) and then inoculated with lentivirus particles pseudotyped (Vpp) with SARS-CoV-2 spike protein for 24 h.
  • NJCPG Nim Jiom Chuanbei Pipa Gao
  • Vpp lentivirus particles pseudotyped
  • SARS-CoV-2 spike protein SARS-CoV-2 spike protein
  • the cells were lysed 24 h later, and Vpp transduction was measured. Experiments were performed in triplicate. Error bars indicate the SEM of technical triplicates. Statistical significance was calculated using Student’s t-test. ***, p value ⁇ 0.001 compared with the control.
  • the results shown in FIG. 10 indicate Fritillaria thunbergii, Fritillaria cir
  • Table 1 shows the selectivity index (SI) of peimine.
  • Table 2 shows the extracts from two different kinds of Fritillaria and Nin Jiom Chuanbei Pipa Gao (NJCPG). Table 2
  • the present disclosure developed a viral infection assay to screen a library of approximately 126 small molecules that may potentially protect against SARS-CoV-2 infection and serve as alternative therapies.
  • the results of the present disclosure showed that peimine inhibits VOCs viral infections in multiple cell lines.
  • FRET fluorescence resonance energy transfer
  • peimine suppresses the interaction of spike and ACE2.
  • Molecular docking analysis revealed that peimine exhibits a higher binding affinity for variant spike proteins created through amino acid substitutions and is able to form hydrogen bonds with N501Y in the spike protein.
  • peimine is capable of using in the treatment of the coronavirus infection or the disease caused by coronavirus.
  • peimine is capable of using in the treatment of the SARS-CoV-2 variant infection or the disease caused by SARS-CoV-2 variant.
  • Neuropilin- 1 is a host factor for SARS-CoV-2 infection. Science, 370(6518), 861-865. doi: 10.1126/science.abd3072
  • iGEMDOCK a graphical environment of enhancing GEMDOCK using pharmacological interactions and post-screening analysis.
  • Soya-cerebroside reduces interleukin production in human rheumatoid arthritis synovial fibroblasts by inhibiting the ERK, NF-KB and AP-1 signalling pathways.
  • Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus. Nature, 426(6965), 450-454. doi: 10.1038/nature02145
  • Soya-cerebroside reduces IL-10-induced MMP-1 production in chondrocytes and inhibits cartilage degradation: implications for the treatment of osteoarthritis. Food and Agricultural Immunology, 30(1), 620-632. doi: 10.1080/09540105.2019.1611745
  • Peimine inhibits the growth and motility of prostate cancer cells and induces apoptosis by disruption of intracellular calcium homeostasis through Ca(2+) /CaMKII/JNK pathway. J Cell Biochem, 121(1), 81-92. doi: 10.1002/jcb.28870
  • Tannic acid suppresses SARS-CoV-2 as a dual inhibitor of the viral main protease and the cellular TMPRSS2 protease. Am J Cancer Res, 79(12), 4538-4546.

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Abstract

A method for treating a coronavirus infection or a disease caused by coronavirus in a subject in need thereof comprises: administering to the subject an effective amount of peimine.

Description

Method for treating coronavirus infection or disease caused by coronavirus
BACKGROUND
1. Field
The present disclosure relates to a method for treating a coronavirus infection or a disease caused by coronavirus. More specifically, the present disclosure relates to a method for treating a coronavirus infection or a disease caused by coronavirus with peimine.
2. Description of Related Art
Coronavirus disease 2019 (CO VID- 19) is caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Currently, several vaccines against SARS-CoV-2 have been FDA approved for emergency use authorization (EUA) in the US. However, only variants of concern (VOCs), which may evade and affect vaccine protection, were tested in recent reported studies. Therefore, the development of safe and effective small-compound drugs that directly block the interaction between the viral spike glycoprotein and ACE2 is urgently needed to provide a complementary or alternative treatment for COVID-19 patients.
Therefore, it is desirable to provide a novel method for treating a coronavirus infection or a disease caused by coronavirus.
SUMMARY
An aspect of the present disclosure is drawn to a pharmaceutical i composition for treating a coronavirus infection or a disease caused by coronavirus, comprising: peimine and a pharmaceutically acceptable carrier.
Another aspect of the present disclosure is drawn to a method for treating a coronavirus infection or a disease caused by coronavirus in a subject in need thereof, comprising: administering to the subject an effective amount of peimine, or administering to the subject the aforesaid pharmaceutical composition.
A further aspect of the present disclosure is drawn to a use of peimine or the aforesaid pharmaceutical composition for manufacturing a medicament for treating a coronavirus infection or a disease caused by coronavirus.
In the present disclosure, the coronavirus may be severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).
In the present disclosure, the disease may be a disease caused by SARS-CoV-2.
In the present disclosure, the disease caused by SARS-CoV-2 may be CO VID- 19.
In the present disclosure, the disease caused by SARS-CoV-2 may be atypical pneumonia.
In the present disclosure, the disease caused by SARS-CoV-2 may be a respiratory disease.
In the present disclosure, peimine or the pharmaceutical composition can be administered to a subject orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, or via an implanted reservoir. The term “parenteral” as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques.
In the present disclosure, the pharmaceutical composition can be an oral pharmaceutical composition, which can be any orally acceptable dosage form including capsules, tablets, emulsions and aqueous suspensions, dispersions, and solutions. Oral solid dosage forms can be prepared by spray dried techniques; hot melt extrusion strategy, micronization, and nano milling technologies. Alternatively, the pharmaceutical composition can be a nasal aerosol or inhalation composition, which can be prepared according to techniques well known in the art of pharmaceutical formulation. Further alternatively, the pharmaceutical composition of the present disclosure can also be administered in the form of suppositories for rectal administration.
The carrier in the pharmaceutical composition must be “acceptable” in the sense that it is compatible with the active ingredient of the composition (and preferably, capable of stabilizing the active ingredient) and not deleterious to the subject to be treated.
The term “treating”, “treat” or “treatment” refers to application or administration of the active ingredient to a subject with the purpose to cure, alleviate, relieve, alter, remedy, improve, or affect the disease, the symptom, or the predisposition. “An effective amount” refers to the amount of the active ingredient which is required to confer the desired effect on the subject. Effective amounts vary, as recognized by those skilled in the art, depending on route of administration, excipient usage, and the possibility of co-usage with other therapeutic treatments such as use of other active agents.
In the present disclosure, the aforesaid subject may be mammal, for example, a human, a pig, a horse, a cow, a dog, a cat, a mouse or a rat.
Other novel features of the disclosure will become more apparent from the following detailed description when taken in conjunction with the accompanying drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the results of screening for natural compounds with potential inhibitory activity on SARS-CoV-2 pseudo virus entry.
FIG. 2A shows the western blot results of overexpressing human ACE2 (hACE2) in 293T cells.
FIG. 2B shows the results of sciadopitysin, vanillic acid, peimine and semagacestat for blocking SARS-CoV-2 entry.
FIG. 2C to FIG. 2E show the results of peimine blocking SARS-CoV-2 entry.
FIG. 3A shows the western blot results of overexpressing human furin in Vero-E6 monkey kidney epithelial cells.
FIG. 3B shows the results of Vero-E6 cells with and without furin expression infected with variants of SARS-CoV-2.
FIG. 3C and FIG. 3D show the results of peimine inhibiting variants of SARS-CoV-2 infection in furin-overexpressing cells and 293T/hACE2 cells.
FIG. 4A shows the results of time-resolved FRET assay of the binding between SARS-CoV-2 Spike S 1 and human ACE2 with or without 100 pM peimine.
FIG. 4B shows the molecular docking result of the interaction between peimine and the spike RBD-ACE2 complex.
FIG. 5 shows the results of peimine docking analysis for wild type and variants of SARS-CoV-2.
FIG. 6 shows the results of peimine inhibiting variants of SARS-CoV-2 infection in Calu-3 cells.
FIG. 7A and FIG. 7B respectively show the results of the cell viability assay of peimine and hydroxychloroquine (HCQ).
FIG. 8 shows the western blot results of Huh-7 or 293T/hACE2 cells treated with different doses of peimine.
FIG. 9 shows the results of peimine inhibiting SARS-CoV-2 infection in Calu-3 cells with or without washing.
FIG. 10 shows the restuls of Fritillaria thunbergii, Fritillaria cirrhosa D. Don and Traditional Chinese natural herbal for blocking pseudovirus infection.
DETAILED DESCRIPTION OF EMBODIMENT
The following embodiments when read with the accompanying drawings are made to clearly exhibit the above-mentioned and other technical contents, features and/or effects of the present disclosure. Through the exposition by means of the specific embodiments, people would further understand the technical means and effects the present disclosure adopts to achieve the above-indicated objectives. Moreover, as the contents disclosed herein should be readily understood and can be implemented by a person skilled in the art, all equivalent changes or modifications which do not depart from the concept of the present disclosure should be encompassed by the appended claims.
Materials and Methods
Cell lines and culture conditions
Huh-7 and 293T cell lines were obtained from ATCC. The 293T human embryonic kidney cell line, Calu-3 lung cancer cell line, Huh-7 human hepatocarcinoma cell line and Vero-E6 African green monkey kidney cell line were maintained in Dulbecco’s MEM (Gibco) containing 10% fetal bovine serum (HyClone) and 100 units of penicillin (HyClone), 100 pg of streptomycin (HyClone). In addition, 293T cells stably expressed recombinant human ACE2 (293/hACE2) (S. C. Wang et al., 2020).
Small-molecule compound library
The pure compound library, Natural Compound Library (Catalog # L6000, Target Molecule Corp, Inc.) was used to screen for drugs.
Lentiviral particles pseudotyped (Vpp) with SARS-CoV-2 spike protein infection assay
Vpp contains SARS-CoV-2 spike protein and a luciferase reporter (S. C. Wang et al., 2020). SARS-CoV-2 variants were purchased from the National RNAi Core Facility (NRC), Academia Sinica, Taipei, Taiwan. Then, 3-5 pL of supernatant was added to the cells in a 96-well plate (MOI ~0.2) in the presence of polybrene (8 pg/mL). The plate was centrifuged at 1,200 pg for 30 min and then returned to the incubator. Twenty-four hours postinfection (hpi), the culture supernatants were replaced with fresh medium. Seventy-two hours postinfection, luciferase activity was determined according to the manufacturer’s instructions.
Cell viability assay
Cell survival was measured using WST-8/CCK-8 (Abeam) reagent incubated with cells for 4 h. The samples were then measured spectrophotometrically at 595 nm using an ELISA plate reader. The percentage of viable and dead cells in each treatment group was calculated by normalization with data of the untreated control group.
Western blot analysis
Experimental cells were harvested and lysed with RIPA buffer, which included 1 mM PMSF, immediately before use to prepare a modified RIPA buffer, and the lysate proteins were resolved on SDS-containing 10% polyacrylamide gel, transferred to PVDF membranes and probed with specific antibodies against a-tubulin (Sigma, #T5168), TMPRSS2 (Santa Cruz Biotechnology, #sc-515727) and ACE2 (GeneTex, GTX101395). An enhanced chemiluminescence (ECL) kit was purchased from Bio-Rad.
Sample preparations
Protein samples of SARS-CoV-2 Mpro and the CFP-YFP protein substrate were prepared as previously described (Y. C. Wang et al., 2020). Time Resolved-Fluorescence Resonance Energy Transfer (FRET) assay
Interruption of the SARS-CoV-2 spike SI and human ACE2 interaction by peimine was detected using the TR-FRET assay according to the manufacturer's protocol (Catalog #79949-1, BPS Bioscience, Inc.) (S. C. Wang et al., 2020). Briefly, ACE2 and Spike SI proteins with or without 100 pM peimine were incubated at room temperature for 1 hour. TR-FRET signals were read at 665 nm.
LC/MS analysis and quantitative analysis
Quantitative analysis of peimine content was performed with LC/MS. Aqueous extracts of Fritillaria thunbergii and Fritillaria cirrhosa D. Don were analyzed using a Velos Pro dual-pressure linear ion trap mass spectrometer (Thermo Fisher Scientific, San Jose, CA) equipped with an Agilent 1100 Series binary high-performance liquid chromatography pump (Agilent Technologies, Palo Alto, CA). Briefly, the gradient program was 2% buffer B at 2 min to 98% buffer B at 20 min with a flow rate of 50 pL/min, where buffer A was 0.1% formic acid/FLO and buffer B was 0.1% formic acid/acetonitrile. A survey scan was acquired in the mass range m/z 200-2,000. The electrospray voltage was maintained at 4 kV, and the capillary temperature was set at 275 °C. The peimine content in each sample was estimated based on the mass peak-area intensity of the precise molecular weight signal with the exact LC elution time as that in the peimine standard compound.
Molecular docking
The RBD-ACE2 complex (PDB ID: 6VW1) was used as the template structure for the docking experiment. Initially, we replaced the valine residue at 417 with lysine in 6VW1 to generate the wild-type (WT) docking target using (PS)2V3 protein structure prediction server (T. T. Huang et al., 2015). Next, the N501Y mutation was generated to create the B.1.1.7 docking target. The B.1.351 target was established by introducing N501Y, E484K and K417N mutations into the WT target. L452R and T478K, and K417T, E484K and N501Y were introduced to generate B.1.617.2 and P l variants. The docking target of current major spreading variant, B.1.1.529 was also modeled by making G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H mutations. Docking tasks were performed for the WT, B.1.1.7, B.1.351, B.1.617.2, P. l and B.1.1.529 variants using iGEMDOCK (Hsu, Chen, Lin, & Yang, 2011) with the “GA Parameters” settings of Population size=300, Generation=80 and Number of solutions=100. The best docking pose (solution) with the smallest docking score was selected for further representation. The frequency distribution of the docking scores of 100 docking poses found for the WT, B.1.1.7 and B.1.351, B.1.617.2, P. l and B.1.1.529 targets are depicted. All visualizations of the docking results were visualized using PyMOL (Eriksson et al., 2021).
Statistical analyses
The statistical significance of a difference between mean values was estimated using the SigmaPlot software package for performing independent Student’s t-tests. Error bars indicate the SEM of technical triplicates. The data are expressed as the means ± SEM. P values of less than 0.05 were considered statistically significant. *, p value < 0.05; **, p value < 0.01; ***, p value < 0.001 compared with control.
Results
FIG. 1 shows the results of screening for natural compounds with potential inhibitory activity on SARS-CoV-2 pseudo virus entry.
Huh-7 cells were pretreated with 126 natural compounds at a final concentration of 10 pM for 1 h and then infected with lentivirus particles pseudotyped (Vpp) with SARS-CoV-2 spike protein (MOI-0.1). All viruses and compounds were removed 24 hpi (hours post infection). At 72 hpi, infectivity was assessed based on luciferase activity and is displayed as a Z-score chart. As shown in FIG. 1, the four compounds with the lowest Z-score are respectively sciadopitysin, vanillic acid, peimine and semagacestat, and these four compounds are used for the following experiments.
FIG. 2A shows the western blot results of overexpressing human ACE2 (hACE2) in 293T cells. hACE2 was overexpressed in 293T cells, and the proteins were extracted from the cell lysates. In FIG. 2A, tubulin is the loading control. The results shown in FIG. 2A indicates that hACE2 was successfully overexpressed in 293 T cells.
FIG. 2B shows the results of sciadopitysin, vanillic acid, peimine and semagacestat for blocking SARS-CoV-2 entry. Statistical significance was calculated using Student’s t-test. *, p value < 0.05; ***, p value < 0.001 compared with the control.
293T/hACE2 cells were pretreated with DMSO only, vanillic acid (10 pM), peimine (10 pM), sciadopitysin (10 pM) or semagacestat (10 pM) for 2 h and then inoculated with lentivirus particles pseudotyped (Vpp) with SARS-CoV-2 spike protein for 24 h. As shown in FIG. 2B, among the four compounds, peimine can be an effective natural compound of blocking SARS-CoV-2 entry.
FIG. 2C to FIG. 2E show the results of peimine blocking SARS-CoV-2 entry. Statistical significance was calculated using Student’s t-test. *, p value < 0.05; ***, p value < 0.001 compared with the control.
293T/hACE2 and Calu-3 cells were preincubated with different doses of peimine for 2 h. The cells were lysed 24 h later, and Vpp transduction was measured. Experiments were performed in triplicate. Error bars indicate the SEM of technical triplicates. As shown in FIG. 2C and FIG. 2D, peimine is an effective natural compound of blocking SARS-CoV-2 entry.
Vpp backbones incorporate green fluorescent protein (GFP) that is expressed upon infection into target cells. Fluorescence was recorded 24 h post- infection. Magnification, 4X. Scale bar: 1000 pm. As shown in FIG. 2E, peimine is an effective natural compound of blocking SARS-CoV-2 entry.
FIG. 3A shows the western blot results of overexpressing human furin in Vero-E6 monkey kidney epithelial cells. Human furin was overexpressed in Vero-E6 monkey kidney epithelial cells, and proteins were extracted from the cell lysates. In FIG. 3A, tubulin is the loading control. The results shown in FIG. 3A indicate that human furin was successfully overexpressed in Vero-E6 cells.
FIG. 3B shows the results of Vero-E6 cells with and without furin expression infected with variants of SARS-CoV-2. Error bars indicate the SEM of technical triplicates. Statistical significance was calculated using Student’s t-test. *, p value < 0.05; ***, p value < 0.001 compared with the control.
Vero-E6 cells with and without furin expression were transfected and then inoculated with the B.1.1.7 (United Kingdom) and 501Y.V2 (South African) variants of lentivirus particles pseudotyped (Vpp) with SARS-CoV-2 mutant spike protein for 24 h. As shown in FIG. 3B, Vero-E6 cells with and without furin expression were infected with the wide-type (Wildtype S), B.1.1.7 and 501Y.V2 Vpp.
FIG. 3C and FIG. 3D show the results ofpeimine inhibiting variants of SARS-CoV-2 infection in furin-overexpressing cells and 293T/hACE2 cells. Error bars indicate the SEM of technical triplicates. Statistical significance was calculated using Student’s t-test. *, p value < 0.05; ***, p value < 0.001 compared with the control.
Vero E6 cells with and without furin expression and 293T/hACE2 cells were preincubated with 10 pM peimine for 2 h and then infected with wild-type (Wildtype S, SA), B.1.1.7, or 501Y.V2, D614G or VSVG Vpp. The cells were lysed 24 h later, and Vpp transduction was measured. Experiments were performed in triplicate. As shown in FIG. 3C and FIG. 3D, peimine can effectively inhibit variants of SARS-CoV-2 infection in furin-overexpressing cells and 293T/hACE2 cells.
FIG. 4A shows the results of time-resolved FRET assay of the binding between SARS-CoV-2 Spike S 1 and human ACE2 with or without 100 pM peimine. The results indicate that peimine suppresses the interaction of SARS-CoV-2 Spike SI and ACE2.
FIG. 4B shows the molecular docking result of the interaction between peimine and the spike RBD-ACE2 complex. In FIG. 4B, the overview of the optimal pose of the interaction between peimine and the spike RBD-ACE2 complex (PDB: 6VW1) is shown, wherein peimine is shown as sticks, and spike RBD and ACE2 are shown in the cartoon. In the enlarged view of the binding mode of peimine, the hydrogen bonds are shown as dashed lines. The result indicates that peimine can block SARS-CoV-2 spike protein binding ACE2.
FIG. 5 shows the results of peimine docking analysis for wild type and variants of SARS-CoV-2, wherein one hundred docking poses of peiminewere analyzed for the targets of wild type (WT), B.1.1.7, B.1.1.529, B.1.351, B.1.617.2 and P.l variants of SARS-CoV-2_RDB-hACE2 complex. The result shows that peimine can block SARS-CoV-2 spike protein binding ACE2, and peimine exhibits a higher binding affinity for variant spike proteins created through amino acid substitutions.
The contact potential of the binding pocket of SARS-CoV-2_RDB-hACE2 for different SARS-CoV-2 variants in complex with peimine was also analyzed. Even the figures are not shown, the results indicate that the environment of the binding cavity is mainly hydrophobic. In addition, the binding affinity of peimine predicted for WT is -10.6 kcal/mol, -11.2 kcal/mol for B.1.1.7, -11.0 kcal/mol for B.1.351, -11.1 kcal/mol for Pl, -10.2 kcal/mol for B.1.1.529 and -11.4 kcal/mol for B.1.617.2. The binding affinity values were computed using PRODI GI platform. These results indicate that peimine exhibits a higher binding affinity for variant spike proteins created through amino acid substitutions.
FIG. 6 shows the results of peimine inhibiting variants of SARS-CoV-2 infection in Calu-3 cells.
Calu-3 cells were preincubated with 1 or 10 pM peimine for 2 h and then infected with wildtype (wildtype S), B.1.1.7, B.1.351 (501Y.V2), Pl, B.1.617.2 or B.1.1.529 lentiviral particles pseudotyped (Vpp), respectively. The cells were lysed 24 h later, and Vpp transduction was measured. Experiments were performed in triplicate. Error bars indicate the SEM of technical triplicates. Statistical significance was calculated using Student’s t-test. *, p value < 0.05; **, p value < 0.01; ***, p value < 0.001 compared with the control. As shown in FIG. 6, peimine can effectively inhibit variants of SARS-CoV-2 infection in Calu-3 cells.
FIG. 7A and FIG. 7B respectively show the results of the cell viability assay of peimine and hydroxychloroquine (HCQ).
293T/hACE2 cells were counted after treatment with different doses of peimine (0-1000 pM) or HCQ (1-10 pM) for 24 h. Experiments were performed in triplicate. Error bars indicate the SEM of technical triplicates. The results shown in FIG. 7A and FIG. 7B indicate that peimine exhibits no toxicity on 293T cells compared to HCQ.
FIG. 8 shows the western blot results of Huh-7 or 293T/hACE2 cells treated with different doses of peimine.
Huh-7 or 293T/hACE2 cells were treated with different doses of peimine for 24 h. TMPRSS2 and ACE2 expression in Huh-7 or 293T/hACE2 cells was determined through Western blot analysis. Tubulin was used as the loading control. The results indicate that peimine does not affect TMPRSS2 or ACE2 expression in Huh-7 or 293T/hACE2 cells.
FIG. 9 shows the results of peimine inhibiting SARS-CoV-2 infection in Calu-3 cells with or without washing, wherein n.s refers to non-significant.
Calu-3 cells were preincubated with 0, 1 or 10 pM peimine for 2 h, optionally washed with PBS and inoculated for 2 h in an incubator. After inoculation, the cells were infected with lentivirus particles pseudotyped (Vpp) with SARS-CoV-2 spike protein. The cells were lysed 24 h later, and Vpp transduction was measured. Experiments were performed in triplicate. Error bars indicate the SEM of technical triplicates. Statistical significance was calculated using Student’s t-test. The results shown in FIG. 9 indicate that the efficacy of peimine is not reversible.
FIG. 10 shows the restuls of Fritillaria thunbergii, Fritillaria cirrhosa D. Don and Traditional Chinese natural herbal for blocking pseudovirus infection.
ACE2-expressing 293T cells were pretreated with Fritillaria thunbergia, Fritillaria cirrhosa and a commercially available Fritillaria cirrhosa -containing herbal medicines, Nim Jiom Chuanbei Pipa Gao (NJCPG) and then inoculated with lentivirus particles pseudotyped (Vpp) with SARS-CoV-2 spike protein for 24 h. The cells were lysed 24 h later, and Vpp transduction was measured. Experiments were performed in triplicate. Error bars indicate the SEM of technical triplicates. Statistical significance was calculated using Student’s t-test. ***, p value < 0.001 compared with the control. The results shown in FIG. 10 indicate Fritillaria thunbergii, Fritillaria cirrhosa D. Don and Traditional Chinese natural herbal remedy block pseudovirus infection.
The following Table 1 shows the selectivity index (SI) of peimine.
Table 1
Figure imgf000016_0001
The following Table 2 shows the extracts from two different kinds of Fritillaria and Nin Jiom Chuanbei Pipa Gao (NJCPG). Table 2
Figure imgf000017_0001
The present disclosure developed a viral infection assay to screen a library of approximately 126 small molecules that may potentially protect against SARS-CoV-2 infection and serve as alternative therapies. The results of the present disclosure showed that peimine inhibits VOCs viral infections in multiple cell lines. In addition, a fluorescence resonance energy transfer (FRET) assay showed that peimine suppresses the interaction of spike and ACE2. Molecular docking analysis revealed that peimine exhibits a higher binding affinity for variant spike proteins created through amino acid substitutions and is able to form hydrogen bonds with N501Y in the spike protein. These results suggest that peimine, a compound isolated from Fritillaria, may be a potent inhibitor of SARS-CoV-2 variant infection.
In conclusion, peimine is capable of using in the treatment of the coronavirus infection or the disease caused by coronavirus. In particular, peimine is capable of using in the treatment of the SARS-CoV-2 variant infection or the disease caused by SARS-CoV-2 variant.
Although the present disclosure has been explained in relation to its embodiment, it is to be understood that many other possible modifications and variations can be made without departing from the spirit and scope of the disclosure as hereinafter claimed. References
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Claims

What is claimed is:
1. A method for treating a coronavirus infection in a subject in need thereof, comprising: administering to the subject an effective amount of peimine.
2. The method of claim 1, wherein the coronavirus is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).
3. A method for treating a disease caused by coronavirus in a subject in need thereof, comprising: administering to the subject an effective amount of peimine.
4. The method of claim 3, wherein the disease is a disease caused by SARS-CoV-2.
5. The method of claim 4, wherein the disease caused by SARS-CoV-2 is CO VID-19.
6. The method of claim 4, wherein the disease caused by SARS-CoV-2 is atypical pneumonia.
7. The method of claim 4, wherein the disease caused by SARS-CoV-2 is a respiratory disease.
26
SUBSTITUTE SHEET (RULE 26)
PCT/US2022/035218 2022-06-28 2022-06-28 Method for treating coronavirus infection or disease caused by coronavirus WO2024005780A1 (en)

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Citations (4)

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WO2007110637A1 (en) * 2006-03-28 2007-10-04 Bioniqs Limited Method for extracting target alkaloid using an ionic liquid as extracting solvent
CN104940214A (en) * 2015-05-22 2015-09-30 德清奥丽芙生物科技有限公司 Application of peimine in preparation of novel immunosuppressive agent
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