WO2021043234A1

WO2021043234A1 - Use of berbamine or its analogue for preventing or treating rna virus infection

Info

Publication number: WO2021043234A1
Application number: PCT/CN2020/113378
Authority: WO
Inventors: Jianbo YUE; Lihong Huang; Qingru ZHANG
Original assignee: City University Of Hong Kong; 6J Biotechnology (Hong Kong) Limited
Priority date: 2019-09-04
Filing date: 2020-09-04
Publication date: 2021-03-11

Abstract

Provided herein is a method of preventing or treating a subject suffering from a flavivirus infection by administering an effective amount of berbamine or its analogue to the subject, berbamine has a structure of Formula (I), wherein the flavivirus infection is caused by Japanese encephalitis virus, Zika virus or Dengue virus, and a method of inhibiting the entry of a flavivirus, an enterovirus and/or a lentivirus into host cells, comprising contacting the host cells with an effective amount of berbamine of its analogue, berbamine has a structure of Formula (I), wherein the flavivirus is Japanese encephalitis virus, Zika virus or Dengue virus. Also provided is a method of preventing or treating a subject suffering from a coronavirus, particularly the coronavirus is SARS-CoV-2 or MERS-CoV, a method of inhibiting the entry of a coronavirus into host cells, as well as use of berbamine or its analogue in the preparation of a medicament for preventing or treating a flavivirus infection, enterovirus infection, lentivirus infection or coronavirus infection.

Description

USE OF BERBAMINE OR ITS ANALOGUE FOR PREVENTING OR TREATING RNA VIRUS INFECTION

TECHNICAL FIELD

The present invention relates to a method of preventing or treating a subject suffering from an infection caused by a RNA virus including infections caused by a positive single-stranded RNA virus. The method is useful in prevention or treatment of an infection caused by a flavivirus, an enterovirus, alentivirus or a coronavirus infection.

BACKGROUND OF THE INVENTION

RNA viruses, particularly positive single-stranded RNA viruses, such as viruses from Flaviviridae, Enterovirus and Coronavirus are expanding huge threat in public health. West Nile virus, Japanese encephalitis virus (JEV) , Zika virus (ZIKV) , Dengue virus (DENV) , and enterovirus A17 (EV-A17) are considered as the leading causes of human and animal infectious diseases in the world. The morbidity and mortality of related illness caused by these viruses have been increasing every year. West Nile virus has recently spread from the Mediterranean Basin to the Western Hemisphere and now accounts for thousands of sporadic encephalitis cases each year. Also, Japanese encephalitis have caused thousands of deaths each year in a wide range of endemic areas.

Although there are some commercially available vaccines against yellow fever, Japanese encephalitis and neonatal encephalitis, there are few or almost no effective clinical treatment against flaviviruses or enteroviruses. For example, patients suffering from serious flavivirus or enterovirus infection may only receive supportive care including administration with intravenous fluids, hospitalization, respiratory support, and prevention of secondary infections. There is currently a lack of effective remedy in treating RNA virus infection particularly caused by flaviviruses and enteroviruses.

Furthermore, a coronavirus is a kind of RNA virus that causes mild to moderate respiratory illness. Whilst most of the coronaviruses are not dangerous, with some only have mild symptoms, certain coronaviruses such as Middle East Reparatory Syndrome (MERS-CoV) , Severe Acute Respiratory Syndrome (SARS-CoV) , Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) are serious types which trigger severe symptoms. The spread of the illness can be uncontrollable. Currently, COVID-19 infections caused by SARS-CoV-2 has already reached over 24 million globally and the number of deaths increases every day. The COVID-19 pandemic also causes worldwide lockdown, causing significant economic losses worldwide.

Accordingly, there remains an urgent need for therapy, and/or novel compounds which are useful in the prevention or treatment of RNA viral infection particularly flavivirus infection and coronavirus infection.

SUMMARY OF THE INVENTION

In an aspect, the present invention pertains to a method of preventing or treating a subject suffering from a flavivirus infection by administering an effective amount of berbamine or its analogue to the subject, berbamine has a structure of Formula (I) :

wherein the flavivirus infection is caused by Japanese encephalitis virus, Zika virus or Dengue virus.

In another aspect, the present invention pertains to a method of inhibiting the entry of a flavivirus into host cells, comprising contacting the host cells with an effective amount of berbamine or its analogue, berbamine has a structure of Formula (I) :

wherein the flavivirus is Japanese encephalitis virus, Zika virus or Dengue virus.

In a further aspect, the present invention relates to a method of inhibiting the entry of an enterovirus and/or a lentivirus into host cells, comprising contacting the host cells with an effective amount of berbamine or its analogue as described above.

Still further, the present invention relates to use of berbamine or its analogue in prevention or treatment of a RNA virus infection, particularly but not exclusively a flavivirus infection, an enterovirus infection or a lentivirus infection.

Furthermore, the present invention also pertains to use of berbamine or its analogue in the preparation of a medicament for preventing or treating a RNA virus infection, particularly but not exclusively a flavivirus infection, an enterovirus infection or a lentivirus infection.

In one aspect, the present invention also pertains to a method of preventing or treating a subject suffering from a coronavirus infection, particularly infection caused by MERS-CoV and/or SARS-CoV-2, by administering an effective amount of berbamine or its analogue to the subject. It would be appreciated that the invention also relates to a method of inhibiting the entry of a coronavirus into host cells.

There is also provided use of berbamine or its analogue in the preparation of a medication for preventing or treating a coronavirus infection.

The inventors unexpectedly found that benzylisoquinoline alkaloids of the present invention, i.e. berbamine and its analogues, have an antiviral effect, particularly against RNA virus infections such as a flavivirus infection, an enterovirus infection, a lentivirus infection, and a coronavirus infection. The inventors found that berbamine and its analogues are capable of inhibiting the entry of the viruses into host cells thereby protecting the cells from being infected. Mice infected with the virus particularly flavivirus were also found to have higher survival rate after treatment with the benzylisoquinoline alkaloids. There are also experimental data indicating that berbamine is effective in inhibiting the entry of MERS-CoV and SARS-CoV-2 into host cells. Accordingly, the present invention provides effective compounds for treating and/or preventing flavivirus infection, particularly Japanese encephalitis virus infection, Zika virus infection and/or Dengue virus infection, as well as coronavirus infection.

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. The invention includes all such variations and modifications. The invention also includes all steps and features referred to or indicated in the specification, individually or collectively, and any and all combinations of the steps or features.

Other features and aspects of the invention will become apparent by consideration of the following detailed description and accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1A shows the microscopic images of HeLa cells obtained after in situ hybridization of EV-71 positive strand RNA and EV71 negative strand RNA and immunostaining of dsRNA or LAMP1, in which the HeLa cells were infected with EV-71 (MOI = 1) for 7 h and EV-71 virions were observed.

Fig. 1B is a plot prepared based on the results in Fig. 1A, demonstrating the colocalization coefficient of the EV-71 positive strand RNA and EV71 negative strand RNA in the infected cells.

Fig. 1C shows the microscopic images of A549 cells, in which the A549 cells were infected with 10 MOI of JEV for 12 h, and stained with anti-double stranded RNA antibody and antibodies against different organelle markers, including early endosome marker EEA1, cis-golgi marker GM130, tran-golgi network marker TGN46, endoplasmic reticulum marker Calnexin, and lysosome marker LAMP1.

Fig. 1D shows the microscopic images of A549 cells infected with ZIKV in the presence or absence of extracellular calcium ions.

Fig. 1E shows the microscopic images of A549 cells infected with JEV after treatment with BAPTA-AM, a calcium chelator, in which the presence of absence of JEV positive RNA strand is detected.

Fig. 1F is a Western blot pattern showing that BAPTA-AM treatment inhibited the JEV envelope protein synthesis in the A549 cells in a dose dependent manner.

Fig. 2A illustrates the high-content image assay based on DsRNA immunostaining performed by the user for the purpose of measuring viral infection in host cells.

Fig. 2B shows the high-content images of A549 cells after pretreating A549 cells with indicated dose of berbamine for 1 h and infected with 50 MOI of JEV for 18 h, and a dose responsive curve of berbamine on JEV.

Fig. 2C shows the high-content images of A549 cells after pretreating A549 cells with indicated dose of berbamine for 1 h and infected with 50 MOI of ZIKV for 18 h, and a dose responsive curve of berbamine on ZIKV.

Fig. 2D is a Western blot pattern showing the expression of JEV-NS1 in A549 cells which were pretreated with berbamine at indicated dose for 1 h and infected with ～10 MOI JEV for 10 h, and the expression of JEV-NS1 in A549 cells which were pretreated with DMSO as control group.

Fig. 2E is a Western blot pattern showing the expression of ZIKV-E in A549 cells which were pretreated with berbamine at indicated dose for 1 h and infected with ～10 MOI ZIKV for 10 h, and the expression of ZIKV-E in A549 cells which were pretreated with DMSO as control group.

Fig. 2F is a plot of JEV titer against post-treatment time, after pretreating A549 cells with berbamine, in which berbamine markedly inhibited JEV virus production.

Fig. 2G includes microscopic images of A549 cells and BHK-21 cells after JEV infection with /without berbamine pretreatment.

Fig. 3A shows the microscopic images of A549 cells after immunostaining, in which the cells were pretreated with berbamine for 1 h, then infected with 100 MOI of JEV for 80 min before fixation and staining. The encircled pattern refers to the presence of RNA genome of JEV.

Fig. 3B shows the microscopic images of A549 cells after immunostaining, in which the cells were pretreated with berbamine, then infected with 100 MOI of ZIKV on ice for 1 h followed by incubation in warm medium for the indicated time course before fixation and staining. The arrows point to the presence of ZIKV.

Fig. 4A shows the high-content images of A549 cells after immunostaining, in which the cells were pretreated with indicated doses of isotetrandrine for 1 h, then infected with about 50 MOI of JEV.

Fig. 4B shows the high-content images of A549 cells after immunostaining, in which the cells were pretreated with indicated doses of isotetrandrine for 1 h, then infected with about 50 MOI of ZIKV.

Fig. 4C is plot showing the relative infectivity of JEV in 549 cells in the treatment of isotetrandrine at different doses, wherein the EC ₅₀ of isotetrandrine against JEV in A549 cells is around 12 μM.

Fig. 5A shows the high-content images of A549 cells after immunostaining, in which the cells were pretreated with indicated doses of fangchinoline for 1 h, then infected with about 50 MOI of JEV.

Fig. 5B shows the high-content images of A549 cells after immunostaining, in which the cells were pretreated with indicated doses of fangchinoline for 1 h, then infected with about 50 MOI of ZIKV.

Fig. 5C is plot showing the relative infectivity of JEV in 549 cells in the treatment of fangchinoline at different doses, wherein the EC ₅₀ of fangchinoline against JEV in A549 cells is around 11 μM.

Fig. 6A is a Western blot pattern showing the expression of JEV-NS1 and ZIKV-E in A549 cells, in which the cells were pretreated with indicated doses of E6 berbamine for 1 h, then infected with about 50 MOI of JEV or ZIKV.

Fig. 6B shows the high-content images of JEG-3 cells after immunostaining, in which the cells were pretreated with 20 μM of berbamine, 40 μM of berbamine, 10 μM of fangchinoline, 20 μM of fangchinoline, 10 μM of isotetrandrine or 40 μM of isotetrandrine for 1 h, then infected with about 50 MOI of JEV or ZIKV for 18 h.

Fig. 7A shows the high-content images of A549 cells after immunostaining, in which the cells were pretreated with 5 μM of berbamine, 15 μM of berbamine, 30 μM of berbamine, 10 μM of fangchinoline, 20 μM of fangchinoline, 30 μM of fangchinoline, 10 μM of isotetrandrine, 20 μM of isotetrandrine or 40 μM of isotetrandrine for 1 h, then infected with about 10 MOI of DENV-2 for 18 h. High-content images of A549 cells of a control group pretreated with DMSO are also illustrated.

Fig. 7B is a plot showing the relative infectivity of DENV-2 in A549 cells, in which the cells were pretreated with indicated doses of berbamine, fangchinoline, or isotetrandrine for 1 h, then infected with about 10 MOI of DENV-2.

Fig. 8A is a plot showing the relative infectivity of JEV in A549 cells pretreated with 13 additionally identified compounds.

Fig. 8B shows the high-content images of A549 cells after immunostaining, in which the cells were pretreated with Compound #1, Compound #7, Compound #9 or Compound #11 respectively, then infected with about 50 MOI of JEV.

Fig. 9A shows the cytotoxicity of berbamine in A549 cells, BHK-21 cells, Vero cells, Hela cells, and Huh 7 cells.

Fig. 9B illustrates the animal experiment, in which mice were injected with JEV, and then administered with PBS as the control group or berbamine as the treatment group.

Fig. 9C shows the survival rate of mice after JEV challenge followed by treatment of berbamine for 15 days, in which the mock group and the mock group with 15 mg/kg of berbamine have the same survival rate.

Fig. 9D shows the body weight change of mice after JEV challenge followed by treatment of berbamine for 15 days.

Fig. 10 shows the high-content images of RD cells after immunostaining, in which the cells were pretreated with berbamine with the indicated doses, and then infected with 10 MOI of EV-71, as well as a plot of relative infectivity of EV-71 in the pretreated cells with EC ₅₀ being about 17.3 μM.

Fig. 11 shows the high-content images of A549 cells after immunostaining, in which the cells were pretreated with berbamine, and then infected with lentivirus encoding histone B-RFP; and the corresponding quantitative plot.

Fig. 12A shows that 10 μM berbamine inhibited the entry of pseudovirus particles MLV-SARS-CoV-2 S into Huh7 overexpressed HEK293T cells.

Fig. 12B shows that 10 μM berbamine inhibited the entry of pseudovirus particles MERS-SARS-CoV S into hACE2-overexpressed HEK293T cells.

Fig. 12C shows the relative intracellular MERS-CoV RNA level in supernatant of the sample after treating primary human lung fibroblast cells with berbamine at the indicated concentration, i.e. 10 μM, 20 μM or 30 μM, for 3 h, followed by infection with MERS-CoV and RT-PCR quantification.

Fig. 12D shows the relative extracellular MERS-CoV RNA level in cell lysate of the sample after treating primary human lung fibroblast cells with berbamine at the indicated concentration, i.e. 10 μM, 20 μM or 30 μM, for 3 h, followed by infection with MERS-CoV and RT-PCR quantification.

Fig. 12E is a plot showing the relative SARS-CoV-2 RNA level determined after treating Vero-E6 cells with berbamine at the indicated concentrations for 3 h, followed by infection with SARS-CoV-2. The cell lysates were collected and subjected to RT-PCR quantification of SARS-CoV-2 RNA.

Fig. 12E is a plot showing the inhibition rate determined after treating Vero-E6 cells with berbamine at the indicated concentrations for 3 h, followed by infection with SARS-CoV-2 and qRT-PCR assay.

Fig. 12F is a plot showing the inhibition rate determined by using virus titer measurement, in which Vero-E6 cells were treated with berbamine at the indicated concentrations for 3 h, followed by infection with SARS-CoV-2.

Fig. 13A shows that berbamine, denoted as BBM in the figure, significantly inhibited the GPN-induced cytosolic Ca2+ increase in Fura-2-loaded HeLa cells.

Fig. 13B shows that berbamine, denoted as BBM in the figure, significantly inhibited the ML-SA1- (B) induced cytosolic Ca2+ increase in Fura-2-loaded HeLa cells.

Fig. 13C shows the microscopic images of Huh7 cells, treated with or without berbamine (10 μM) . The cells were first incubated with an ACE2 antibody on ice for 90 min, and the internalization of the ACE2-antibody complex was then initiated at 37℃ for the indicated times, followed by ACE2, LAMP1, and DAPI staining and confocal imaging.

Fig. 13D shows that level of ACE2 in Huh7 cells which were treated with or without berbamine (10 μM) for 3h. The live cells were immunolabeled with the anti-ACE2 antibody, followed by FACS analysis to measure the cell surface ACE2 levels.

Fig. 13E shows that level of DPP4 in Huh7 cells which were treated with or without berbamine (10 μM) for 3h. The live cells were immunolabeled with the anti-DPP4 antibody, followed by FACS analysis to measure the cell surface DPP4 levels.

Fig. 13F shows the number of extracellular vesicles per cell. The EVs were collected from the culture medium of control or berbamine-treated Huh7 cells, and their concentration and distribution of sizes were determined with a nanoparticle tracking analyzer.

Fig. 13G shows the levels of ACE2, DPP4, TSG101, and CD63 in the EVs were determined by immunoblot analysis.

Fig. 14A shows that TRPMLs knockdown inhibited SARS-CoV-2 infection in Huh7 cells according to the dsRNA staining results.

Fig. 14B shows that TRPMLs knockdown significantly inhibited the cell surface ACE2 levels in Huh7 cells as determined by FACS analysis.

Fig. 14C shows that TRPMLs knockdown significantly inhibited the cell surface DPP4 (C) levels in Huh7 cells as determined by FACS analysis.

Fig. 14D is a plot showing the number of EV per cell. The EVs were collected from the culture medium of control or TRPMLs-knockdown Huh7 cells, and their concentration and distribution of sizes were determined with a nanoparticle tracking analyzer (D) .

Fig. 14E shows the levels of ACE2, DPP4, and CD63 in these EVs were determined by immunoblot analysis.

Fig. 15A shows the microscopic images of A549 cells treated with or without berbamine (50 μM) for the indicated times, followed by immunostaining of ACE2 and a plot indicating the relative ACE2 intensity.

Fig. 15B shows the microscopic images of A549 cells treated with or without berbamine (50 μM) for the indicated times, followed by immunostaining of DPP4 and a plot indicating the relative ACE2 intensity.

Fig. 15C shows the expression of ACE2, DPP4, and HSP70 in Vero-E6 cells in the presence of cycloheximide (7.5 mg/ml) and being treated with/without berbamine (50 μM) for 6 h. The levels of ACE2, DPP4, and HSP70 in the cell lysates were determined by immunoblot analysis.

Fig. 15D shows the expression of ACE2, DPP4, and HSP70 in A549 cells in the presence of cycloheximide (7.5 mg/ml) and being treated with/without berbamine (50 μM) for 6 h. The levels of ACE2, DPP4, and HSP70 in the cell lysates were determined by immunoblot analysis.

Fig. 15E shows the expression of ACE2, DPP4, and ALIX in EVs collected from the culture medium of control or berbamine-treated A549 cells. The levels of ACE2, DPP4, and ALIX were determined by immunoblot analysis.

Fig. 16 shows a table summarizing the antibodies and reagents used in flow cytometric analysis, immunofluorescence staining, and Western Blot analysis.

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one skilled in the art to which the invention belongs.

As used herein, “comprising” means including the following elements but not excluding others. “Essentially consisting of” means that the material consists of the respective element along with usually and unavoidable impurities such as side products and components usually resulting from the respective preparation or method for obtaining the material such as traces of further components or solvents. “Consisting of” means that the material solely consists of, i.e. is formed by the respective element. As used herein, the forms “a” , “an” , and “the” are intended to include the singular and plural forms unless the context clearly indicates otherwise.

The present invention in an aspect provides a method of preventing or treating a subject suffering from a RNA virus infection particularly an infection caused by a positive single-stranded RNA virus. The RNA virus may be a flavivirus, an enterovirus, a lentivirus, or a coronavirus.

In embodiments herein, the method is suitable for preventing or treating a subject suffering from a flavivirus infection by administering an effective amount of berbamine or its analogue to the subject.

In an embodiment, the flavivirus is Japanese encephalitis virus, Zika virus or Dengue virus. In an alternative embodiment, the flavivirus may be selected from the group consisting of West Nile virus, Murray Valley encephalitis virus, and Yellow Fever virus.

Berbamine and its analogue can be classified as bis-benzylisoquinoline alkaloids including two benzylisoquinoline moieties linked through diphenyl ether, benzyl phenyl ether or biphenyl bonds. Berbamine and its analogue may be artificially synthesized or may be a naturally occurring compound derived from a plant material, a fungus or the like.

Berbamine has a structure of Formula (I)

Analogues of berbamine generally share a core structure of Formula (Ib) :

wherein R ₁, R ₂, R ₃, R ₄ are independently selected from a hydrogen atom, a C1-C3 alkyl group, a halogen atom or a nitrogen containing group, and with the provision that the analogue is not tetrandrine. C1-C3 alkyl group may be any of a methyl group, an ethyl group, a propyl group, an isopropyl group, or a cyclopropyl group. The halogen atom may be selected from the group consisting of fluorine, chlorine, and bromine.

In an embodiment, the analogue of berbamine preferably has a structure of Formula (II) , (III) , or (IV) :

In an embodiment, the analogue of berbamine has a structure of Formula (IIb) , (IIIb) , or (IVb) :

The inventors found that berbamine and its analogues as disclosed above are effective against flavivirus for example by inhibiting the entry of the virus into the host cells, and/or protecting a subject from being infected at a particular dose. They are potential anti-viral agents particularly anti-flavivirus agents.

Berbamine is found to be exceptionally suitable for use in prevention and treatment of flavivirus infection. The inventors proved that berbamine has an inhibitory effect against at least JEV, ZIKV, DENV-2, EV-71 and lentivirus.

Berbamine and its analogues as disclosed herein may also inhibit the entry of an enterovirus and/or a lentivirus into host cells of the subject, thereby boosting the immunity of the subject against various types of virus. The inventors also found that their antiviral effects are not cell-specific.

It would be appreciated that salts or solvates of berbamine and its analogues are also included in the scope, and may be used for preventing or treating the same virus infection.

The method of the present invention may be used as a precautionary method to prevent a subject from suffering a RNA virus infection, e.g. a flavivirus infection, as the method is useful in boosting the immune system in an individual, inhibiting the entry of the virus into host cells, and/or inhibiting the interaction between the virus and the host cells. The method can also delay the onset or slow down the progression of a condition associated with the infection in an individual. It would be appreciated that the treatment of the infection may involve inhibition of the viral proteins in the infected subject, killing of the virus, alleviating symptoms caused by the virus, and/or inhibiting the synthesis of the virus, or the combinations thereof.

The term “subject” in particular refers to an animal or human, in particular a mammal and most preferably human. In an embodiment, the subject is susceptible to a flavivirus infection, or is suffering from a flavivirus infection. In a further embodiment, the subject is suffering from two different types of RNA virus infections. In an example embodiment, the subject is susceptible to or suffering from an enterovirus infection, lentivirus infection or a combination thereof.

The expression "effective amount" generally denotes an amount sufficient to produce therapeutically desirable results, wherein the exact nature of the result varies depending on the specific condition which is treated. Berbamine or its analogue may be contained in a composition, in particular a pharmaceutical composition, in an effective amount, i.e. an amount suitable to treat or prevent the RNA virus infection particularly flavivirus infection, enterovirus infection, lentivirus infection, or coronavirus infection in a subject, in particular a mammal, which also depends on the frequency and number of compositions to be administered. In an embodiment, the subject is a mammal and berbamine or its analogue may be administered to the subject at a dose of about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, about 30 mg/kg, about 35 mg/kg, about 40 mg/kg, about 45 mg/kg, about 50 mg/kg; or at a dose of about 15 mg/kg to about 50 mg/kg, about 20 mg/kg to about 50 mg/kg, or above. In other embodiment, the subject is human and berbamine or its analogue may be administered to the subject at a dose of about 15 mg/kg to about 50 mg/kg, or above.

When berbamine or its analogue is provided in a pharmaceutical composition to a subject, the skilled person is able to select suitable pharmaceutically tolerable excipients depending on the form of the pharmaceutical composition and is aware of methods for manufacturing pharmaceutical compositions as well as able to select a suitable method for preparing the pharmaceutical composition depending on the kind of pharmaceutically tolerable excipients and the form of the pharmaceutical composition. Berbamine or its analogue may be formulated in a liquid form such as an aqueous solution and a non-aqueous mixture; a semi-solid form such as a gel, an emulsion, acream and a paste; or a solid form such as a tablet, a capsule and powders. Berbamine or its analogue may also be formulated in the form of a prodrug so as to achieve the desired effect after administration.

In embodiments of the present invention, berbamine or its analogue as disclosed herein is administered to the subject by a route selected from a group consisting of oral delivery, intravenous delivery, intradermal delivery, intraperitoneal delivery and intramuscular delivery. The person skilled in the art is able to formulate berbamine or its analogue in a pharmaceutical composition according to the specific flavivirus infection and the disclosure herein.

In addition, berbamine or its analogue may be administered in combination with a compound selected from the group consisting of the following compounds and a derivative thereof:

The above compounds were identified and considered to have a similar 3D conformation with berbamine. Particularly, these compounds were identified using berbamine as reference and therefore it is believed that they can achieve similar or identical inhibitory effect as berbamine. It has also been determined that the above compounds have anti-viral effect, which is described in the examples and the effect is also represented in the corresponding Figs. 8A and 8B. The above compounds can be provided in any salt form suitable for administration or use.

It would be appreciated that the above compounds may be used alone for treating or preventing RNA virus infection such as flavivirus infection as they demonstrated antiviral effect particularly by inhibiting the entry of flavivirus into host cells. Therefore, the present invention also pertains to a method of preventing or treating a subject suffering from a flavivirus infection by administering the subject with an effective amount of a compound selected from the group consisting of the following compounds and a derivative thereof:

These compounds may be used to prepare a medicament for preventing or treating the infection as described above.

The present invention further pertains to a method of inhibiting the entry of a flavivirus, particularly Japanese encephalitis virus, Zika virus or Dengue virus, into host cells, comprising contacting the host cells with an effective amount of berbamine or its analogue. The flavivirus is as described above. Particularly, the flavivirus is Japanese encephalitis virus or Zika virus. Berbamine and its analogue are also as described above.

The method may comprise a step of incubating the host cells with a medium containing berbamine or its analogue for a period of time such as for at least 0.5 h, at least 1 h, at least 1.5 h, or above.

In an embodiment, berbamine or its analogue may further inhibit the entry of an enterovirus and/or a lentivirus into the host cells. Accordingly, the present invention may further relate to a method of inhibiting the entry of an enterovirus and/or a lentivirus into host cells, comprising contacting the host cells with an effective amount of berbamine or its analogue as described above. The enterovirus may be EV-A71 (also known as EV-71) , and the lentivirus may encode histone B-RFP.

The inventors further found that berbamine as described above is suitable for preventing or treating a coronavirus infection, and preventing the entry of a coronavirus into host cells. Accordingly, the present invention further pertains to a method for preventing or treating a subject suffering from a coronavirus infection. The method comprises the step of administering an effective amount of berbamine or its analogue, preferably berbamine, to the subject. Berbamine and its analogue are as described above.

In particular, the coronavirus infection is caused by Middle East respiratory syndrome coronavirus (MERS-CoV) and/or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) . The inventors found that berbamine is a potential TRPMLs inhibitor that can inhibit lysosomal calcium channel. The modulation of the calcium channel via TRPML by berbamine may compromise the trafficking of ACE2, thereby preventing the entry of the coronavirus. Berbamine is also found to be able to decrease the level of DPP4 at the plasma membrane. Based on the experimental results, berbamine is found to have potential values in coronavirus therapy and prevention.

It would be appreciated that the method may be used as a precautionary method to prevent a subject from suffering a coronavirus infection, or used in a therapy, as similar to the method for preventing or treating a flavivirus infection mentioned above. In particular, berbamine may act as a TRPMLs inhibitor to inhibit the entry of the coronavirus into host cells, and improve the immune system of the subject. The method can also delay the onset or slow down the progression of a condition associated with the infection in an individual. It would be appreciated that the treatment of coronavirus infection may involve inhibition of the viral proteins in the infected subject, killing of the virus, alleviating symptoms caused by the virus, and/or inhibiting the synthesis of the virus, or the combinations thereof.

The method is suitable for a subject, particularly an animal or human as described above. In an embodiment, the subject is susceptible to a coronavirus infection, or is suffering from a coronavirus infection. For example, the subject is an individual who is about to visit a high-risk area or an elderly people who is vulnerable to the coronavirus infection. Berbamine or its analogue, particularly berbamine is provided to the subject at the effective amount, or an amount that helps boost the immune system of the subject, and in the appropriate pharmaceutical composition or form, to produce the desired therapeutic effect.

In a particular embodiment, the subject is a mammal and the berbamine or its analogue is administered to the subject at a dose of about 15 mg/kg to about 50 mg/kg, or about 20 mg/kg to about 50 mg/kg.

In embodiments therein, there is also provided a method of inhibiting the entry of a coronavirus, particularly MERS-CoV and/or SARS-CoV-2, into host cells comprising contacting the host cells with an effective amount of berbamine or its analogue, particularly berbamine is as described above.

It would be appreciated that the present invention relates to use of berbamine or its analogue in prevention or treatment of a RNA virus infection as described above, and use in the preparation of a medicament or a composition for preventing or treating of a RNA virus infection as described above. A person having ordinary skills in the art would appreciate suitable methods to prepare a medicament for the intended purposes based on the disclosure herein. In embodiments, there are also provided use of berbamine or its analogue in the preparation of a medicament for inhibiting the entry of a RNA virus, particularly a flavivirus, an enterovirus, a lentivirus, and/or a coronavirus into host cells. The medicament is capable of inhibiting the entry of the aforesaid viruses.

EXAMPLES

I. Determination of anti-viral effect of berbamine against flavivirus, enterovirus, and lentivirus

Assay for detecting viral infection

The inventors established an assay for detecting viral infection. The inventors performed RNA in situ hybridization to detect both positive (+) and negative (-) strand virus RNA in the host cells after EV-A71 infection. As shown in Fig. 1A, both EV-A71 positive and negative RNA strands were detected after 7h virus infection of HeLa cells. EV-A71 (-) RNA strand exhibited subtle co-localization with EV-A71 (+) RNA strand. On the other hand, double-stranded RNA, as shown by double-stranded RNA (DsRNA) immunostaining, exhibited strong co-localization with virus (-) RNA strand, not LAMP1, a lysosomal marker. Besides, DsRNA and (+) RNA were accumulated in time-dependent manner after virus infection (as shown in Fig. 1B) . Thus, DsRNA immunostaining and viral specific (+) RNA hybridization can be applied to measure viral RNA replication.

Besides EV-A71, the inventors also performed the aforementioned assays to measure ZIKV, JEV and DENV infection. The inventors particularly examined the localization of the virus replication in host cells by co-immunostaining the host cells after JEV infection with DsRNA antibody and antibodies against different organelle markers. As shown in Fig. 1C, DsRNA signal exhibited weak co-localization with early endosome (anti-EEIA staining) , Golgi, endoplasmic reticulum (anti-Calnexin staining) , or lysosome (anti-LAMP1 staining) . These data suggested that JEV replication complex is located at a novel membrane structure in the host cell.

Accordingly, the inventors have established a valid immunostaining and a valid RNA hybridization assay to measure positive single strand RNA virus replication. This assay was applied to determine the antiviral effect of alkaloids against various viral infections, which is described in detail below.

Determination of the role of Ca ²⁺ signaling in viral infection

The inventors determined whether removal of extracellular Ca ²⁺ affects ZIKV infection of host cells by performing ZIKV H+RNA hybridization and anti-ZIKV envelope protein immunostaining. As shown in Fig. 1D, ZIKV +RNA was detectable inside host cells after 90 minutes of viral infection in the presence of extracellular Ca ²⁺, whereas no positive viral RNA was detected in host cells after 90 minutes of the viral infection in the absence of extracellular Ca ²⁺. Moreover, in the host cells in the absence of extracellular Ca2+, intact virirons (staining with ZIKV envelope proteins) were detected in the plasma membrane of host cells as shown in the right panel of Fig. 1D. These results suggest that Ca2+ influx is required for flavivirus entry of host cells.

With reference to Fig. 1E, in cells treated with BAPTA-AM, a calcium chelator, no ZIKV positive RNA strand was spotted inside the host cells. It was also found that BAPTA-AM treatment of host cells markedly inhibited the expression of JEV envelope protein in a dose dependent manner, as shown in Fig. 1F. Again, these date suggest that intracellular calcium signaling is involved in flavivirus infection of host cells. Similar data have also been observed for EV-A71 infection of host cells (data not shown) . These data together support the role of Ca ²⁺ influx or intracellular Ca ²⁺ in flavivirus or enterovirus infections.

High-content image based assay for measuring viral infection

To measure flavivirus or enterovirus infection of host cells, the inventors developed a high-content image assay to measure DsRNA staining of virus infected cells by an automatic fluorescence microscopy, thereby quantifying flavivirus or enterovirus infection. The scheme of the measurement is illustrated in Fig. 2A. Particularly, the cells are pre-treated with a calcium channel inhibitor before being subject to viral infection. After viral infection, the cells are fixed. DsRNA immunostaining is then performed on the fixed cells, followed by high-content screening and image analysis.

This assay was then applied to screen compounds affecting viral infection, as follows. Host cells, e.g. A549, RD, PC3, or JEG-3 cells, were seeded in 96-weIl plates in triplicates, and cells were then pretreated with different compounds at different concentrations for 1 h before being infected with about 10 to about 100 MOI of JEV, ZIKV, or EV-A71 virus. After 8 to 24 h of infection, cells were fixed with 4%PFA and subject to DsRNA immunostaining and DAPI staining. The images were finally captured by CellInsight CX7 High-Content Screening platform with a 20x objective lens, and analyzed in HCS Studio ^TM 3.0 (Thermo Fisher, Waltham, MA, USA) to quantify the percentage of infected cells versus uninfected cells.

Inhibitory effect of berbamine and its analogues against ZIKV or JEV infection

The inventors determined the anti-infection activity of berbamine, a bis-benzylisoquinoline alkaloid isolated from the traditional Chinese medicine berberis, against ZIKV or JEV infection. As shown in Fig. 2B, berbamine, the alkaloid of Formula (I) , significantly inhibited the infection of JEV in A549 cells, with EC ₅₀ being 20 μM.

Fig. 2C also demonstrates that berbamine inhibited the infection of ZIKV in A549 cells, with EC ₅₀ being 2 μM. Moreover, as shown in Fig. 2D and 2E, the pre-treatment of cells with berbamine markedly inhibited the protein expression of JEV-NSl and ZIKV-E in a dose dependent manner. The inventors further performed a virus titration assay and confirmed that pretreatment of cells with berbamine significantly inhibited the production of JEV according to the results in Fig. 2F. Fig. 2G also shows that the pretreatment of A549 cells or BHK-21 cells with berbamine markedly reduced JEV-induced cell death. These data indicated that berbamine has inhibitory effect against both JEV and ZIKV infection in host cells.

Furthermore, as demonstrated in Fig. 1D, calcium influx is required for the entry of JEV or ZIKV. The inventors further determined whether treatment of host cells with berbamine can block the entry of these viruses. To evaluate this, A549 cells were pre-treated with berbamine for 1 h, and were then infected with JEV for 80 min before fixation. Viral positive strand RNA hybridization was subsequently performed to detect the RNA genome of JEV. The results in Fig. 3A show that the positive strand JEV RNA was only detected inside control cells, not in cells pretreated with berbamine.

Also, A549 cells pretreated with or without berbamine were incubated with ZIKV on ice for 1 h, and were then incubated with warm medium at 37 ℃ for another 40 min before fixation, followed by anti-ZIKV envelope protein immunostaining. The results in Fig. 3B show that the intact ZIKV virus (encircled in the figure) was detectable on the surface of virus infected cells pretreated with or without berbamine, whereas the intact ZIKV virus were highly concentrated at the surface of virus infected cells pretreated with berbamine, not the control cells, after cells were incubated at warm medium. Accordingly, these data demonstrate that berbamine have inhibitory effect against the entry of ZIKV or JEV into host cells.

The inventors further tested three analogues of berbamine including isotetrandrine, fangchinoline, and E6 berbamine to see if they have anti-infection effect against the flavivirus particularly JEV and ZIKV.

As shown in Figs. 4A to 4C, it was found that isotetrandrine significantly inhibited both JEV and ZIKV infections of host cells. Referring to Fig. 7C, the EC50 of isotetrandrine against JEV in A549 cells is around 12 μM.

As shown in Figs. 5A to 5C, it was found that fangchinoline significantly inhibited both JEV and ZIKV infections of host cells. Referring to Fig. 8C, the EC50 of fangchinoline against JEV in A549 cells is around 11 μM.

As shown in Fig. 6A, it was found that E6-berbamme markedly inhibited the infection of JEV or ZIKV, as manifested by strong inhibition of JEV-NSl and ZIKV-Envelope protein expression in host cells treated with E6-berbamine.

Moreover, the inventors determined their effect against JEV or ZIKV infection in another host cell line to ensure their anti-viral effects are not cell type specific. Referring to Fig. 6B, treatment of JEG-3 cells with berbamine, isotetrandrine, fangchinoline, and E6 berbamine all significantly inhibited JEV or ZIKV infection as shown by DsRNA immunostaining followed by high-content image analysis. Taken together, these data demonstrate that bis-benzylisoquinoline alkaloids are potent anti-JEV or anti-ZIKV agent in vitro.

Inhibitory effect of berbamine and its analogues against DENV infection

The inventors also studied whether berbamine and its analogues have any effects on DENV infection. Briefly, A549 cells plated in triplicates in 96-well plates were pretreated with different doses of berbamine (5 μM, 15μM or 30μM) , fangchinoline (10 μM, 20μM or 30μM) or isotetrandrine (10 μM, 20μM or 40μM) for 1 h before infected with about 10 MOI of Dengue virus type 2 (DENV-2) . Cells were then fixed at 24 h. p. i. and subjected to DsRNA immunostaining to detect DENV-2 replication.

As shown in Fig. 7A and 7B, all berbamine, isotetrandrine, and fangchinoline can significantly inhibit DENV-2 infection in A549 cells in a dose dependent manner. Taken together, these data demonstrate that berbamine and its analogues are potent anti-DENV agents and may be used as pan-anti-flavivirus agents.

Identification of compounds with a similar structure by virtual screening

The inventors identified further 13 compounds via ligand-based virtual drug screening by using berbamine as reference. The identified 13 compounds are listed below.

Table 1 Identified additional compounds

As shown in Fig. 8A, all these compounds have demonstrated an inhibitory effect against JEV infection in A549 host cells. Among them, 4 compounds including Compound #1, #7, #9 and #11 exhibited highest potency. As shown in Fig. 8B, these 4 compounds can effectively block the entry of JEV into the host cells.

Antiviral effect against JEV in mice

The inventors determined the cytotoxicity of berbamine in different cell lines including A549 cells, BHK-21 cells, and RD cells. As shown in Fig. 9A and Table 2, it was found that berbamine has lowest cytotoxicity but highest therapeutic index as compared to other alkaloids (Table 3) . The inventors therefore assessed the anti-JEV effects of berbamine in a mouse model.

Table 2. Cytotoxicity of berbamine in various cell lines.

Cell Type

CC ₅₀ (μM)

Vero	114.936 ± 5.297
Hela	114.762 ± 3.596
A549	126.629 ± 3.896
Huh7	90.436 ± 2.977
BHK-21	126.841 ± 2.426

Table 3. Selectivity index

As illustrated in Fig. 9B, adult female BALB/c mice (age, 4 to 6-week) mice were randomly divided into two groups (7 mice per group) : a JEV-infected with vehicle-treated group and a JEV-infected with berbamine-treated group. For infection, mice were injected intraperitoneally with approximately 10 ^6.5 TCID ₅₀ of JEV. For the berbamine treatment group, mice were injected intraperitoneally with a dose of 15 mg/kg berbamine or PBS daily. For the control-vehicle group, mice were injected intraperitoneally with a dose of 50 mg/kg PBS daily. Survival rate and change of body weight if the mice in each group were monitored for 15 days after JEV injection.

As shown in Fig. 9C and 9D, berbamine treatment increases the survival rate of the mice, i.e. protect the mice from the lethal challenge of JEV. The higher survival rate and less weight changes in berbamine treatment group, as compared to the control group, further suggest that berbamine is a potential anti-flavivirus drug against JEV.

Inhibitory effect of berbamine and its analogues against enterovirus and lentivirus infections In addition, other than flavivirus infection, the inventors determined whether berbamine has any antiviral effect against enterovirus and lentivirus infection, by applying the same high-content image based assay as discussed above.

As shown in Fig. 10, berbamine treatment of RD cells significantly inhibited EV-A71 infection, with EC ₅₀ being around 17 μM.

Further, pretreatment of A549 cells with berbamine also abolished lentivirus infection, as shown in Fig. 11.

Based on the above experimental results, it has been demonstrated that berbamine and its analogues particularly berbamine are potential anti-RNA virus agents.

II. Determination of anti-viral effect of berbamine against coronavirus

Coronavirus is an enveloped positive-sense single-stranded ( (+) ss) RNA virus, and has four subgenusα, β, γ, and δ. The α and β coronavirus mainly infect mammals, while γ and δcoronavirus mainly infect birds, and a few of them can also infect mammals. The α and β coronavirus can cause respiratory or intestinal infections. Coronavirus has a very large (30 Kb) RNA genome but only around 10 Kb is the coding region. This coding region encodes 4 structural proteins: spike (S) protein, membrane (M) protein, envelope (E) protein, nucleocapsid (N) proteins, as well as many non-structural proteins such as RNA-dependent RNA polymerase (RdRp) and helicase (Hel) . Among them, the S protein is a trimeric glycoprotein, which can bind to the viral receptor of the host cells and is a key protein for the virus to enter the cells. Different coronaviruses may target different receptors.

SARS-CoV’s S protein binds with angiotensin converting enzyme 2 (ACE2) to infect ciliated bronchial epithelial cells and type II lung epithelial cells. MERS-CoV’s S protein interacts with dipeptidyl peptidase 4 (DPP4) to infect nonciliated bronchial epithelial cells, bronchiolar epithelial cells, alveolar epithelial cells, and endothelial cells. Recently, it has been reported that SARS-CoV-2 infects human respiratory epithelial cells via its S-protein binding to human ACE2 followed by the receptor-mediated internalization. A transmembrane serine protease TMPRSS2 and other proteases, e.g. furin, are likely involved in SARS-CoV-2 entry of cells as well.

The inventors evaluated whether berbamine has any effect on endosomal trafficking system, so as to evaluate its inhibitory effect against the coronavirus. The endosomal trafficking system is comprised of a series of dynamically interconverted membrane-enclosed vesicular structures, including early endosome (EE) , endosomal carrier vesicle (ECV) /multivesicular body (MVB) , and late endosome (LE) . When EEs mature to MVBs, the inward invagination of the membrane of MVBs forms the intraluminal vesicles (ILVs) inside the lumen of MVBs. During this process, some membrane proteins, e.g. CD81, CD63, and CD9, are incorporated into the invaginated membrane, while some cytosolic contents, e.g. proteins (TSG101, ALIX, HSP70, and HSP90) , nucleic acid (RNA and DNA) , metabolites, and amino acids, are enclosed inside ILVs. MVBs can either be delivered to lysosomes for degradation, or can fuse with plasma membrane to release its luminal ILVs. These released ILVs are called exosomes, a subset of extracellular vesicles (EVs) with sizes ranging from 40-150 nm. Another subset of EVs are the vesicles directly budding from cell plasma membrane with size ranging from 50-1000 nm. The cell-cell communications via the release and uptake of exosomes have been implicated in a number of physiological and pathological processes.

The TRPML family comprises three members: TRPML1, TRPML2 and TRPML3, and they share around ～40%amino acid sequence homology. They are non-selective cation channels, and are permeable to variety of cations, including Ca ²⁺, Na ⁺, Zn ²⁺and Fe ²⁺. Loss-of-function mutations in TRPML1 lead to mucolipidosis type IV (ML4) , a lysosomal storage disease. TRPMLs are located in the membrane of early endosomes and recycling endosomes, and they are especially rich in late endosomes and lysosomes. The activation of TRPMLs via PI (3, 5) P2 can trigger the release Ca ²⁺ from endosomes and lysosomes, which participates in various endolysosomal trafficking events, including trafficking of endosomal vesicles, fusion events between late endosomes and lysosomes, and lysosome-mediated exocytosis.

The inventors via the following experiments found that berbamine potently supressed the infection of SARS-CoV-2 and MERS-CoV by inhibiting TRPMLs to decrease the levels of ACE2 and DPP4 at cell surface and thus preventing the entry of these viruses into the host cells. Accordingly, berbamine is a potential drug for preventing or treating a coronavirus infection. For instance, it may boost the immune system of a subject, and/or help delaying the on-set of a condition associated with the coronavirus infection in the subject.

Effect of berbamine on endolysosomal trafficking

The S protein of SARS-CoV-2 or MERS-CoV binds with ACE2 or DPP4, respectively, to facilitate the viral particles entry into cells via receptor-mediated endocytosis. The inventors generated the murine leukemia virus (MLV) -based pseudotyped particles to incorporate S protein from either MERS-CoV or SARS-CoV-2 and express reporter genes, e.g. luciferase and/or RFP. MLV-SARS-CoV-2 S or MERS-SARS-CoV S pseudotyped particles effectively entered Huh7 or hACE2-overexpressed HEK293T cells, respectively, manifested by the expression of RFP and luciferase in cells.

The inventors assessed the ability of berbamine to affect the entry of MLV-SARS-CoV-2 S or MERS-SARS-CoV S pseudotyped particles into host cells, and found that berbamine indeed effectively inhibited the entry of these psudoviruses (Fig. 12A and 12B) .

The inventors subsequently assessed the activity of berbamine against MERS-CoV and SARS-CoV-2 infection in vitro. Primary human lung fibroblasts were treated with berbamine and then infected with MERS-CoV, followed by qRT-PCR to measure the amount of intra-or extracellular viral RNA. The results show that berbamine significantly decreased both the intracellular (Fig. 12C) and extracellular (Fig. 12D) level of MERs-CoV RNA. The inventors also assessed the anti-SARS-CoV-2 activity of berbamine in Vero-E6 cells, and found that berbamine significantly inhibited the viral yield, as quantified by qRT-PCR assay (EC50 = ～2.3 μM) (Fig. 12E) or a virus titration assay (EC50 = ～4.7 μM) (Fig. 12F) . In summary, these data indicate that berbamine is a potential drug against SARS-CoV-2 and MERS-CoV.

Interestingly, berbamine significantly inhibited the ability of Gly-Phe β-naphthylamide (GPN) to trigger Ca ²⁺ release from lysosomes (Fig. 13A) , which suggests that it inhibits lysosomal Ca ²⁺ channels. Since TRPMLs are one of main Ca ²⁺-permeable channels in lysosomes, the inventors assessed whether berbamine modulates TRPMLs-mediated Ca ²⁺ release from lysosomes. Treatment of cells with ML-SA1, a selective and potent TRPMLs agonist, markedly increased cytosolic Ca ²⁺ levels, and this ML-SA1-induced Ca ²⁺ increase was significantly inhibited by berbamine treatment (Fig. 13B) . These results indicated that berbamine is a potential TRPMLs inhibitor.

Since TRPMLs have been shown to participate in various endolysosomal trafficking events, it is possible that berbamine might inhibit TRPMLs to compromise the trafficking of ACE2, thereby preventing the entry of virus. The inventors, thus, examined whether berbamine changes the trafficking of ACE2. Briefly, cells were first incubated with an ACE2 antibody on ice for 90 min, and the internalization of the ACE2-antibody complex was then initiated at 37℃ for 2 h. In control cells, within 60 min, the ACE2-antibody complex had re-localized from the cell membrane to the late endosomes or lysosomes for degradation, as manifested by the co-localization of ACE2 and LAMP1, a late endosome/lysosome marker. After ～2 h, majority of the internalized ACE2-antibody complex was degraded in control cells (left panel in Fig. 13C) . In contrast, following berbamine treatment, the ACE2-antibody complex failed to be sent to lysosomes for degradation (right panels in Fig. 13C) . Thus, these data indicate that berbamine inhibits the endolysosmal degradation of ACE2. It is believed that the inhibition of ACE2 endolysosmal degradation by berbamine might affect its levels at the cell surface. By immunolabeling ACE2 in cells treated with or without berbamine followed by flow cytometric analysis or confocal imaging, the results show that berbamine indeed significantly decreased the levels of ACE2 at the plasma membrane (Figs. 13D and 15A) . Similarly, berbamine treatment significantly decreased the levels of DPP4 at plasma membrane (Figs. 13E and 15B) . These results suggest that berbamine prevents SARS-CoV-2 or MERS-CoV from entering host cells by decreasing the levels of ACE2 or DPP4 at the plasma membrane.

Interfering endolysomal trafficking has been shown to promote the exosome release, the inventors, thus, quantified the concentration of EVs in the cell culture medium of control or berbamine-treated cells using a nanoparticle analyzer. As expected, berbamine significantly promoted the secretion of EVs in Huh7 cells (Fig. 13F) . The inventors then examined whether these EVs contain elevated levels of ACE2 or DPP4 in the berbamine-treated group when compared with the control group. Thus, EVs in the culture medium from the control and berbamine-treated Huh7 cells were collected by ultracentrifugation, and the protein levels of ACE2 and DPP4 were analyzed by immunoblot analysis. The results show that the levels of ACE2 and DPP4, similar to other exosome protein markers, e.g. TSG101, CD63, and Alix, were markedly increased in EVs collected from the berbamine-treated cell culture medium, when compared with the control group (Fig. 13G) . The inventors also speculated that the increase in the secretion of ACE2 and DPP4-containing exosomes out of cells might lead to the reduced levels of these receptors in berbamine-treated cells. Indeed, when compared with the control cells, berbamine treatment markedly decreased the levels of ACE2 and DPP4 in Vero-E6 cells (Fig. 15C) . Similar results regarding the effects of berbamine on the expression of ACE2 in cells or EVs have also observed in A549 cells (Figs. 15D and 15E) . Taken together, these results suggest that berbamine inhibits the endolysosomal trafficking of ACE2 or DPP4. This leads to an increase in the level of secretion of ACE2 or DPP4 via EVs and a concomitant decrease in their levels at the plasma membrane.

Finally, the inventors assessed the role of TRPMLs in SARS-CoV-2 infection. The inventors knocked down the expression of TRPML1, 2, or 3 individually by respective siRNAs in Huh7 cells, and then infected the control or knockdown cells with SARS-CoV-2, followed by dsRNA staining. The results show that knockdown of TRPML1, TRPML2, or TRPML3 all significantly inhibited SARS-CoV-2 infection in Huh7 cells, manifested by much weaker dsRNA immunofluorescence intensity in TRPMLs-knockdown cells when compared to the control cells (Fig. 14A) . Consistently, knockdown of TRPMLs significantly increased EVs secretion in Huh7 cells (Fig. 14D) , and markedly increased the levels of ACE2, DPP4, CD63 and ALIX in exosomes collected from the knockdown cells when compared to the control cells (Fig. 14E) . Also, TRPMLs knockdown significantly decreased the levels of ACE2 and DPP4 at cell surface (Figs 14B and 14C) . In summary, these data indicate that berbamine compromises the endolysosomal trafficking of ACE2 via inhibition of TRPMLs, and this leads to a decrease in the levels of ACE2 at cell surface, thereby preventing SARS-CoV-2 from entering the host cells.

DISCUSSION

The inventors found that berbamine inhibited TRPML-mediated Ca ²⁺ release from lysosomes (Figs. 13A-13C) . Although whether berbamine directly inhibits TRPMLs remains to be determined, it did compromise the endolysosomal trafficking of ACE2 (Fig. 13D) , and promoted its secretion out of cells via EVs (Figs. 13F and 13G) . This resulted in the decreased levels of ACE2 at cell surface (Fig. 13D) , and led to the failure of SARS-CoV-2 to enter berbamine-treated host cells (Fig. 12B) . TRPLMs knockdown also induced exosome secretion (Fig. 14D) , reduced the levels of ACE2 at cell surface (Fig. 14B) , and prevented the infection of SARS-CoV-2 (Fig. 14A) . These knockdown phenotypes were similar to berbamine, suggesting that the inhibitory effect of berbamine on TRPMLs is, at least partially, responsible for its anti-viral activity. Notably, although berbamine or TRPMLs knockdown significantly decreased, but not abolished, the levels of ACE2 or DPP4 at plasma membrane (Figs. 13D, 13E, 14B, and 14C) , they did very effectively inhibit SARS-CoV-2 or MERS-CoV infection (Figs. 12C-12F, and 14A) . These results suggest that when the levels of ACE2 or DPP4 at the plasma membrane drop to certain levels, these CoVs could not effectively infect the host cells.

The inventors showed that berbamine or TRPMLs knockdown not only inhibited the endolysosomal degradation of ACE2, but also induced the secretion of exosomes containing ACE2 (Figs. 13 and 14) . Interfering endolysomal trafficking has been shown to promote the exosome release, and this is likely due to the increased formation of ILVs in the accumulated late endosomal MVBs, and/or increased fusion between MVBs and the plasma membrane, resulting in the increased release of exosomes. It is also possible that berbamine might regulate the core molecular machinery of exosome secretion or biogenesis to increase exosome secretion. It is possible that berbamine might regulate CaMKII or NF-kB, to inhibit other parts of viral life cycle in the host cells, e.g. replication, packaging, or/and release of the viral particles, in addition to the viral entry. Berbamine might also inhibit TRPMLs-induced autophagy to control SARS-CoV-2 infection.

The results herein indicate that berbamine possesses great potential to be developed into an effective therapeutic agent for the prevention and/or treatment of MERS-CoV and SARS-CoV-2 infections.

MATERIALS AND METHODS

Cell culture and virus propagation-VeroE6, Huh7 and HEK293T cells were maintained in DMEM (Gibco, 12800082) containing 10%fetal bovine serum (Gibco, 10500064) and 100 U/ml of penicillin/streptomycin.

Immunofluorescence staining-

Cells were fixed with 4%paraformaldehyde (PFA) solution, blocked with PBS containing 5%normal donkey serum and 0.3% Triton ^TM X-100, and then incubated with primary antibody followed by the appropriate fluorescent secondary antibody. To label the receptors on the plasma membrane, live cells were incubated with the primary antibody in PBS (+1%BSA) on ice for 90 min, followed by incubation with the fluorescent secondary antibody on ice. Images were captured with a Carl Zeiss LSM 880 confocal microscope using a 63×oil objective lens. The primary antibodies used in these experiments are shown in Fig. 16.

Western blot analysis-

The Bradford assay (Bio-RAD) was performed to measure the protein concentration of cell lysates. An equal amount of protein sample was loaded onto 8%-12%SDS-PAGE gels for electrophoresis. The proteins were then transferred to a PVDF membrane (Millipore) , blocked with 5%non-fat milk, and blotted with primary and secondary antibodies. The primary antibodies used for immunoblotting are shown in Fig. 16.

The anti-virus activity of drugs-

For anti-SARS-CoV-2 activity of berbamine, Vero-E6 cells were pre-treated with berbamine at a titration of different concentrations (0-75 μM) for 6 hours. Then, the cells were washed with PBS and inoculated with SARS-CoV-2 at 0.01 MOI for 2 hours. At 2 hours post infection (hpi) , the cells were washed with PBS and treated with berbamine at a titration of different concentrations (0-75 μM) . At 24 hpi, 100 μL of viral supernatant was lysed and proceed to total RNA extraction using the QIAamp viral RNA mini kit (Qiagen, Hilden, Germany) . The extracted RNA was then used to quantify the replication of SARS-CoV-2 using real-time quantitative RT-PCR (qRT-PCR) .

Purification of extracellular vesicles from the culture medium-A549 cells or Huh7 cells were grown in 15-cm dishes to ～80%confluency. The cells were then rinsed with PBS and incubated in EV-depleted complete medium containing DMSO or berbamine (25 μM) for 48 h. The supernatant was then collected and subjected to sequential centrifugation steps at different centrifugal forces (g) to remove the intact cells, dead cells or cell debris. After each centrifugation, the supernatant was transferred into a new 50 mL tube and the pellet was discarded. Finally, the supernatant was subjected to ultracentrifugation at 120,000 × g for 90 min, and the pellet (now containing the extracellular vesicles) was washed with PBS and subjected to another ultracentrifugation at 120,000 × g for 90 min. Finally, the exosome pellet collected and used for immunoblot analysis.

Intracellular Ca2+ measurements-

HeLa cells were grown in 24-well plates to ～80%confluency. The cells were then loaded with HBSS (Gibco, 14025092) containing 4 μM Fura-2 AM (Invitrogen, F1221) and 0.4% Pluronic ^TM F-127 (Invitrogen, P3000MP) at room temperature for 30 min. The cells were then washed with Ca ²⁺-free HBSS containing 2 mM EGTA and incubated in Ca ²⁺-free HBSS in the presence or absence of berbamine (10 μM) at room temperature for another 30 min. Fluorescence images were acquired at 3 s intervals by alternate excitation at 340 nm and 380 nm with emission at 510 nm using a Nikon Eclipse Ti-SCalcium imaging system. Approximately 1 min after live cell imaging, 200 μM GPN (Abcam, ab145914) or 25 μM ML-SA1 (Tocris Bioscience, 4746) was added to the cells to trigger Ca2+ release from the lysosomes.

Small interference RNA (siRNA) -

Cells were transfected with siRNAs against respective genes (Table S1) using Lipofectamine 3000 according to the manufacturer’s instructions. The knockdown efficiency was validated by immunoblot analysis or qRT-PCR.

Statistical analysis-

Data are presented as mean ± S.E.M. Statistically significant differences were determined by the Student’s t-test and P < 0.05 was considered to be statistically significant.

Claims

A method of preventing or treating a subject suffering from a flavivirus infection by administering an effective amount of berbamine or its analogue to the subject, berbamine has a structure of Formula (I) :

wherein the flavivirus infection is caused by Japanese encephalitis virus, Zika virus or Dengue virus.
The method of claim 1, wherein the analogue of berbamine has a structure of Formula (II) , (III) , or (IV) :
The method of claim 2, wherein the analogue of berbamine has a structure of Formula (IIb) , (IIIb) or (IVb) :
The method of claim 1, wherein berbamine or its analogue further inhibits the entry of an enterovirus and/or a lentivirus in host cells of the subject.
The method of claim 1, wherein berbamine or its analogue is administered to the subject by a route selected from a group consisting of oral delivery, intravenous delivery, intradermal delivery, intraperitoneal delivery and intramuscular delivery.
The method of claim 1, wherein the subject is a mammal preferably a human, and the berbamine or its analogue is administered to the subject at a dose of about 15 mg/kg to about 50 mg/kg.
The method of claim 1, wherein berbamine or its analogue is administered in combination with a compound selected from the group consisting of the following compounds and a derivative thereof:
A method of inhibiting the entry of a flavivirus into host cells, comprising contacting the host cells with an effective amount of berbamine or its analogue, berbamine has a structure of Formula (I) :

wherein the flavivirus is Japanese encephalitis virus, Zika virus or Dengue virus.
The method of claim 8, wherein the analogue of berbamine has a structure of Formula (II) , (III) , or (IV) :
The method of claim 9, wherein the analogue of berbamine has the structure of Formula (IIb) , (IIIb) , or (IVb) :
The method of claim 8, wherein berbamine of its analogue further inhibits the entry of an enterovirus and/or a lentivirus into the host cells.
A method of inhibiting the entry of an enterovirus and/or a lentivirus into host cells, comprising contacting the host cells with an effective amount of berbamine or its analogue, berbamine has a structure of Formula (I) :
[Rectified under Rule 91, 22.10.2020]
The method of claim 12, wherein the analogue of berbamine has a structure of Formula (II) , (III) , or (IV) :
[Rectified under Rule 91, 22.10.2020]
The method of claim 13, wherein the analogue of berbamine has a structure of Formula (IIb) , (IIIb) , or (IVb) :
The method of claim 12, wherein the enterovirus is EV-A71.
The method of claim 12, wherein the lentivirus encodes histone B-RFP.
A method of preventing or treating a subject suffering from a coronavirus infection by administering an effective amount of berbamine or its analogue to the subject, berbamine has a structure of Formula (I) :
The method of claim 17, wherein the coronavirus infection is caused by Middle East respiratory syndrome coronavirus and/or severe acute respiratory syndrome coronavirus 2.
The method of claim 17, wherein the subject is a mammal and the berbamine or its analogue is administered to the subject at a dose of about 20 mg/kg to about 50 mg/kg.
A method of inhibiting the entry of a coronavirus into host cells, comprising contacting the host cells with an effective amount of berbamine or its analogue, berbamine has a structure of Formula (I) :
The method of claim 20, wherein the coronavirus is Middle East respiratory syndrome coronavirus or severe acute respiratory syndrome coronavirus 2.
Use of berbamine or its analogue in the preparation of a medicament for preventing or treating a flavivirus infection, berbamine has a structure of Formula (I) :

wherein the flavivirus infection is caused by Japanese encephalitis virus, Zika virus or Dengue virus.
The use of claim 22, wherein the analogue of berbamine has a structure of Formula (II) , (III) , or (IV) :
The use of claim 23, wherein the analogue of berbamine has a structure of Formula (IIb) , (IIIb) or (IVb) :
The use of claim 22, wherein the medicament further inhibits the entry of an enterovirus and/or a lentivirus in host cells.
The use of claim 22, wherein the medicament further comprises a compound selected from the group consisting of the following compounds and a derivative thereof:
Use of berbamine or its analogue in the preparation of a medicament for inhibiting the entry of an enterovirus and/or a lentivirus into host cells, berbamine has a structure of Formula (I) :
The use of claim 27, wherein the analogue of berbamine has a structure of Formula (II) , (III) , or (IV) :
The use of claim 28, wherein the analogue of berbamine has a structure of Formula (IIb) , (IIIb) , or (IVb) :
The use of claim 27, wherein the enterovirus is EV-A71.
The use of claim 27, wherein the lentivirus encodes histone B-RFP.
Use of berbamine or its analogue in the preparation of a medicament for preventing or treating a coronavirus infection, berbamine has a structure of Formula (I) :
The use of claim 32, wherein the coronavirus infection is caused by Middle East respiratory syndrome coronavirus and/or severe acute respiratory syndrome coronavirus 2.
The use of claim 32, wherein the medicament is capable of inhibits the entry of SARS-CoV-2 or MERS-CoV in host cells of a subject.