[go: up one dir, main page]

US20130035328A1 - Antiviral compounds and methods - Google Patents

Antiviral compounds and methods Download PDF

Info

Publication number
US20130035328A1
US20130035328A1 US13/553,239 US201213553239A US2013035328A1 US 20130035328 A1 US20130035328 A1 US 20130035328A1 US 201213553239 A US201213553239 A US 201213553239A US 2013035328 A1 US2013035328 A1 US 2013035328A1
Authority
US
United States
Prior art keywords
guanidine
hydrogen
phenyl
cinnamoylguanidine
virus
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
US13/553,239
Other languages
English (en)
Inventor
Peter William Gage
Gary Dinneen Ewart
Lauren Elizabeth Wilson
Wayne Best
Anita Premkumar
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Individual
Original Assignee
Individual
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from AU2003903251A external-priority patent/AU2003903251A0/en
Priority claimed from AU2003903850A external-priority patent/AU2003903850A0/en
Priority claimed from AU2003904692A external-priority patent/AU2003904692A0/en
Application filed by Individual filed Critical Individual
Priority to US13/553,239 priority Critical patent/US20130035328A1/en
Publication of US20130035328A1 publication Critical patent/US20130035328A1/en
Priority to US14/615,616 priority patent/US20150313909A1/en
Priority to US15/602,526 priority patent/US10472332B2/en
Priority to US16/554,990 priority patent/US11192863B2/en
Abandoned legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D241/00—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings
    • C07D241/02—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings not condensed with other rings
    • C07D241/10—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings not condensed with other rings having three double bonds between ring members or between ring members and non-ring members
    • C07D241/14—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings not condensed with other rings having three double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D241/24—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
    • C07D241/26—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals with nitrogen atoms directly attached to ring carbon atoms
    • C07D241/28—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals with nitrogen atoms directly attached to ring carbon atoms in which said hetero-bound carbon atoms have double bonds to oxygen, sulfur or nitrogen atoms
    • C07D241/34—(Amino-pyrazine carbonamido) guanidines
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D277/00—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings
    • C07D277/02—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings
    • C07D277/20—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C07D277/32—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D277/38—Nitrogen atoms
    • C07D277/44—Acylated amino or imino radicals
    • C07D277/46—Acylated amino or imino radicals by carboxylic acids, or sulfur or nitrogen analogues thereof
    • 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
    • A61K31/13—Amines
    • A61K31/15—Oximes (>C=N—O—); Hydrazines (>N—N<); Hydrazones (>N—N=) ; Imines (C—N=C)
    • 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
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/165—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide
    • 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
    • A61K31/38—Heterocyclic compounds having sulfur as a ring hetero atom
    • A61K31/381—Heterocyclic compounds having sulfur as a ring hetero atom having five-membered rings
    • 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
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4406—Non condensed pyridines; Hydrogenated derivatives thereof only substituted in position 3, e.g. zimeldine
    • 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
    • A61K31/47—Quinolines; Isoquinolines
    • 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
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/4965—Non-condensed pyrazines
    • 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
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C279/00—Derivatives of guanidine, i.e. compounds containing the group, the singly-bound nitrogen atoms not being part of nitro or nitroso groups
    • C07C279/20—Derivatives of guanidine, i.e. compounds containing the group, the singly-bound nitrogen atoms not being part of nitro or nitroso groups containing any of the groups, X being a hetero atom, Y being any atom, e.g. acylguanidines
    • C07C279/22—Y being a hydrogen or a carbon atom, e.g. benzoylguanidines
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to methods for retarding, reducing or otherwise inhibiting viral growth and/or functional activity.
  • the invention also relates to compounds and compositions suitable for use in the methods.
  • PCT application PCT/AU99/00872 describes the use of compounds 5-(N,N-hexamethylene)-amiloride and 5-(N,N-dimethyl)-amiloride in the treatment of HIV infection.
  • HCV Hepatitis C virus
  • Coronaviruses are enveloped positive-straned RNA viruses that bud from the endoplasmic reticulum-Golgi intermediate compartment or the cis-Golgi network (Fischer, Stegen et al. 1998; Maeda, Maeda et al. 1999; Cores and Machamer 2000; Maeda, Repass et al. 2001; Kuo and Masters 2003).
  • Coronaviruses infest humans and animals and it is thought that there could be a coronavirus that infests every animal.
  • the two human coronaviruses, 229E and OC43 are known to be the major causes of the common cold and can occasionally cause pneumonia in older adults, neonates, or immunocompromised patients (Peiris, Lai et al. 2003).
  • Animal coronaviruses can cause respiratory, gastrointestinal, neurological, or hepatic diseases in their host (Peiris, Lai et al. 2003).
  • Several animal coronavirus are significant veterinary pathogens (Rots, Oberste et al. 2003).
  • Severe acute respiratory syndrome is caused by a newly identified virus.
  • SARS is a rsespiratory illness that has recently been reported in Asia, North America, and Europe (Peiris, Lai et al. 2003).
  • the causative agent of SARS was identified as a coronavirus. (Drosten, Gunther et al. 2003; Ksiazek, Erdman et al. 2003; Peiris, Lai et al. 2003).
  • the World Health Organization reports that the cumulative number of reported probable cases of SARS from 1 Nov. 2002 to the 11 Jul. 2003 is 8,5437 with 813 deaths, nearly a 10% death rats. It is believed that SARS will not be eradicated, but will cause seasonal epidemics like the cold or influenza viruses (Vogel 2003).
  • the inventors have surprisingly found that certain compounds that fall under the classification of substituted acylguasidines have antiviral activity against viruses from a range of different virus families. Without intending to be bound by any particular theory or mechanism of action, and despite current dogma, it appears possible that viral replication can be retarded by inibiting otherwise down-regulating the activity of ion channels expressed in the host cell.
  • the negative impact of the compounds of the present invention on viral replication may be mediated by the inhibition or otherwise down-regulation of membrane ion channel relied upon by the virus for replication.
  • This membrane ion channel may be a viral membrane ion channel (exogenous to the host cell) or a host cell ion channel induced as a result of viral infection (endogenous to the host cell).
  • the compounds of the present invention may inhibit Vpu or p7 function and thereby inhibit the continuation of the respective HIV or HCV life cycle.
  • the SARS virus encodes an E protein which is shown for the first time, by the presaat inventors, to act as an ion channel.
  • E proteins are present in other coronaviruses, the compounds, compositions and methods of the present invention would have utility in the inhibition and/or treatment of infections by other coronaviruses.
  • the present invention is concerned with novel antiviral compounds that fall under the classification of substituted acylguanidines. It does not include in its scope the use of compounds 5-(N,N-hexsamethylene)amiloride and 5-(N,N-dimethyl)-amiloride for retarding, reducing or otherwise inhibiting viral growth and/or functional activity of HIV.
  • a first aspect of the present invention provides an acylguanidine with antiviral activity.
  • the present invention provides an antiviral componnd of Formula I
  • the present invention provides an antiviral compound of Formula I
  • the compounds of the invention include the following:
  • EIPA 5-(N-ethyl-N-isopropyl)amiloride
  • N-amidino-3,5-diamino-6-phenyl-2-pyrazinecarboxamide comprising the structure
  • Bodipy-FL Amiloride comprising the structure
  • N-(2-napthoyl)-N′-phenylguanidine comprising the structure
  • N,N′-bis(2-napthoyl)guanidine comprising the structure
  • N,N′-bis(1-napthoyl)guanidine comprising the structure
  • N-Cinnamoyl-N′,N′-dimethylguanidine comprising the structure
  • trans-3-furanacryoylguanidine comprising the strusture
  • N,N′-Bis(amidimo)napthalene-2,6-dicarboxamide comprising the structure
  • N′′-Cinnamoyl-N,N′-diphenylguanidine comprising the structure
  • N,N′-Bis(3-phenylpropanoyl)guanidine comprising the structure
  • the compounds of the invention are capable of reducing, retarding or otherwise inhibiting viral growth and/or replication.
  • the antiviral activity of the compounds of the invention is against viruses such as those belonging to the Lentivirus family, and the Coronovirus family family of viruses.
  • viruses such as Human Immunodeficiency Virus (HIV), Severe Acute Respiratory Syndrome virus (SARS), Mouse Hepatitis virus ( ), and Hepatitis C virus (HCV).
  • HIV Human Immunodeficiency Virus
  • SARS Severe Acute Respiratory Syndrome virus
  • HCV Hepatitis C virus
  • a pharmaceutical composition comprising an antiviral compound according to any one of the first, second or third aspects, and optionally one or more pharmaceutical acceptable carriers or derivatives, wherein said compound is capable of reducing, retarding or otherwise inhibiting viral growth and/or replication.
  • the antiviral activity of the compounds of the invention is against viruses such as those belonging to the Lentivirus family, and the Coronovirus family of viruses.
  • viruses such as Human Immunodeficency Virus (HIV), Severe Acute Respiratory Syndrome virus (SARS), Human Coronavirus 229E, Human Coronavirus OC43, Mouse Hepatitis virus (MHV), Bovine Coronavirus (BCV), Porcine Respiratory Coronavirus (PRCV), Hepatitis C virus (HCV) and Equine Arteritis Virus (EAV).
  • viruses such as Human Immunodeficency Virus (HIV), Severe Acute Respiratory Syndrome virus (SARS), Human Coronavirus 229E, Human Coronavirus OC43, Mouse Hepatitis virus (MHV), Bovine Coronavirus (BCV), Porcine Respiratory Coronavirus (PRCV), Hepatitis C virus (HCV) and Equine Arteritis Virus (EAV).
  • Coronaviruses which can be inhibited or their infections treated by the compounds of the invention are those listed in Table 1.
  • compositions of the invention may further comprise one or more known antiviral compounds or molecules.
  • a method for reducing, retarding or otherwise inhibiting growth and/or replication of a virus comprising contacting a cell infected with said virus or exposed to said virus with a compound according to any one of the first, second or third aspects.
  • the virus is from the Lentivirus family, or the Coronavirus family. More preferably, the virus is Human Immunodeficiency Virus (HIV), Severe Respiratory Syndrome virus (SARS), Human Coronavirus 229E, Human Coronavirus OC43, Mouse Hepatitis virus (MHV), Bovine Coronavirus (BCV), Poreiae Respiratory Ceronavims (PRCV), Mouse Hepatitis virus (MBV), Hepatitis C virus (HCV), or Equine Arteritis Virus (EAV). Most preferably, the virus is HIV-1, HIV-2, the SARS virus, Coronaviruse 229E, Coronavirus OC43, PRCV, BCV, HCV, or EAV.
  • HIV Human Immunodeficiency Virus
  • SARS Severe Respiratory Syndrome virus
  • MHV Mouse Hepatitis virus
  • BCV Bovine Coronavirus
  • PRCV Poreiae Respiratory Ceronavims
  • MMV Mouse Hepatitis virus
  • HCV Hepatitis C
  • Coronaviruses which can be inhibited or their infections treated by the compounds of the invention are those listed in Table 1.
  • a method for preventing the infection of a cell exposed to a virus comprising contacting said cell with a compound according to any one of the first, second or third aspects.
  • the virus is from the Lentivirus family, or the Coronavirus family. More preferably, the virus is Human immunodeficiency Virus (HIV), Severe Respiratory Syndrome virus (SARS), Human Coronavirus 229E, Human Coronavirus OC43, Mouse Hepatitis virus (MHV), Bovine Coronavirus (BCV), Porcine Respiratory Corobavirus (PRCV), Mouse Hepatitis virus (MHV), Hepatitis C virus (HCV), or Equine Arteritis Virus (EAV). Most preferably, the virus is HIV-1, HIV-2, the SARS virus, Coronavirus 229E, Coronavirus OC43, PRCV, BCV, HCV, EAV.
  • Coronaviruses which can be inhibited or their infections treated by the compounds of the invention are those listed in Table 1.
  • a method for the therapeutic or prophylactic treatment of a subject infected with or exposed to a virus comprising the administration of a compound according to any one of the first, second or thitd aspects, to a subject in need of said treatment.
  • infection with a virus or exposure to a virus occurs with viruses belonging to the Lentivirus family, or the Coronovirus family. More preferably, infection or exposure occurs with HIV, SARS, Human Coronavirus 229E, Human Coronavirus OC43, Mouse Hepatitis virus (MHV), Bovine Coronavirus (BCV), Porcine Respiratory Coronavirus (FRCV), Hepatitis C virus (HCV), or Equine Arteritis Virus (EAV). Most preferably, infection or exposure occurs with HIV-1, HIV-2, SARS, Human Coronavirus 229E, Human Coronavirus OC43, Hepatitis C virus (HCV), or Equine Arteritis Virus (EAV).
  • Coranaviruses which can be inhibited or their infections treated by the compounds of the invention are those listed in Table 1.
  • the subject of the viral inibition is generally a mammal such as but not limited to human, primate, livestock animal (e.g. sheep, cow, horse, donkey, pig), companion animal (e.g. dog, cat), laboratory test animal (e.g. mouse, rabbit, rat, guinea pig, hamster), captive wild animal (e.g. fox, deer).
  • livestock animal e.g. sheep, cow, horse, donkey, pig
  • companion animal e.g. dog, cat
  • laboratory test animal e.g. mouse, rabbit, rat, guinea pig, hamster
  • captive wild animal e.g. fox, deer.
  • the subject is a primate, or horse.
  • the subject is a human.
  • a method of down regulating a membrane ion channel functional activity in a cell infected with a virus comprising contacting said cell with a compound according to any one of the first, second or third aspects.
  • the membrane ion channel may be endogenous to the cell or exogenous to the cell.
  • the membrane ion channel of which functional activity is down regulated is that which Lentiviruses, and Coronaviruses utilise for mediating viral replication and include, for example, the HIV membrane ion channel Vpu, the HCV membrane ion channel P7, the Coronavirus E protein membrane ion channel, and the SARS E protein membrane ion channel.
  • infection with a virus or exposure to a virus occurs with viruses belonging to the Lentivirus family, or the Coronovirus family. More preferably, infection or exposure occurs with HIV, SARS, Human Coronavirus 229E, Human Coronavirus OC43, Mouse Hepatitis virus (MEV), Bovine Coronsvirus (BCV), Porcine Respiratory Coronavirus (PRCV), Hepatitis C virus (HCV), or Equine Arteritis Virus (EAV). Most preferably, infection or exposure occurs with HIV-1, HIV-2, SARS, Human Coronavirus 229E, Human Coronavirus OC43s Hepatitis C virus (HCV), or Equine Arteritis Virus (EAV).
  • a ninth aspect of the present invention there is provided a method of reducing, retarding or otherwise inhibiting growth and/or replication of a virus that has infected a cell, said method comprising contacting said infected cell with a compound according to any one of the first, second or third aspects, wherein said compound down regulates functional activity of a membrane ion channel derived from said virus and expressed in said infected cell.
  • infection occurs with a virus belonging to the Lentivirus family, or the Coronovirus family. More preferably, infection or exposure occurs with HIV, SARS, Human Coronavirus 229E, Human Coronavirus OC43, Mouse Hepatitis virus (MHV), Bovine Coronavirus (BCV), Porcine Respiratory Coronavirus (PRCV), Hepatitis C virus (HCV), or Equine Arteritis Virus (EAV). Most preferably, infection or exposure occurs with HIV-1, HIV-2, SARS, Human Coronavirus 229E, Human Coronavirus OC43, Hepatitis C virus (HCV), or Equine Arteritis Virus (EAV).
  • HIV-1 HIV-2, SARS, Human Coronavirus 229E, Human Coronavirus OC43, Hepatitis C virus (HCV), or Equine Arteritis Virus (EAV).
  • Coronaviruses which can be inhibited or their infections treated by the compounds of the invention are those listed in Table 1.
  • the membrane ion channel of which functional activity is down regulated is that which Lentiviruses, and Coronaviruses utilise for mediating viral replication and include, for example, the HIV membrane ion channel Vpu, the HCV membrane ion channel P7, and the Coronavirus E protein membrane ion channel.
  • the present invention provides a method of reducing, retarding or otherwise inhibiting growth and/or replication of a virus that has infected a cell in a mammal, said method comprising administering to said mammal a compoand according to any one of the first, second or third aspects, or a pharmaceutical composition according to the fourth aspect, wherein said compound or said composition down regulates functional activity of a membrane ion channel expressed in said infected cell.
  • infection occurs with a viruss belonging to the Lentivirus family, or the Coronovirus family. More preferably, infection or exposure occurs with HIV, SARS, Human Coronavirus 229E, Human Coronavirus OC43, Mouse Hepatitis virus (MHV), Bovine Coronavirus (BCV), Porcine Respiratory Coronavirus (PRCV), Hepatitis C virus (HCV), or Equine Arteritis Virus (EAV). Most preferably, infection or exposure occurs with HIV-1, HIV-2, SARS, Human Coronavirus 229E, Human Coronavirus OC43, Hepatitis C virus (HCV), or Equine Arteritis Virus (EAV).
  • HIV-1 HIV-2, SARS, Human Coronavirus 229E, Human Coronavirus OC43, Hepatitis C virus (HCV), or Equine Arteritis Virus (EAV).
  • Coronaviruses which can he inhibited or their infections treated by the compounds of the invention are those listed in Table 1.
  • the membrane ion channel of which functional activity is down regulated is that which Lentiviruses, and Coronaviruses utilise for mediating viral replication and include, for example, the HIV membrane ion channel Vpu, the HCV membrane ion channel P7, and the Coronavirus E protein membrane ion channel.
  • the subject of the viral inhibition is generally a mammal such as but not limited to human, primate, livestock animal (e.g. sheep, cow, horse, donkey, pig), companion animal (e.g. dog, cat), laboratory test animal (e.g. mouse, rabbit, rat, guinea pig, hamster), captive wild animal (e.g. fox, deer).
  • livestock animal e.g. sheep, cow, horse, donkey, pig
  • companion animal e.g. dog, cat
  • laboratory test animal e.g. mouse, rabbit, rat, guinea pig, hamster
  • captive wild animal e.g. fox, deer.
  • the subject is a primate, or horse.
  • the subject is a human.
  • the present invention provides a method for the therapeutic or prophylactic treatment of a subject infected with or exposed to a virus comprising administering to said subject a compound according to any one of the first, second or third aspects, or a pharmaceutical composition according to the fourth aspect, wherein said compound or said composition down-regulates functional activity of a membrane ion channel derived from said virus.
  • infection occurs with a virus belonging to the Lentivirus family, or the Coronovirus family of viruses. More preferably, infection or exposure occurs with HIV, SARS, Human Coronavirus 229E, Human Coronavirus OC43, Mouse Hepatitis virus (MHV), Bovine Coronavirus (BCV), Porcine Respiratory Coronavirus (PRCV), Hepatitis C virus (HCV), or Equine Arteritis Virus (EAV). Most preferably, infection or exposure occurs with HIV-1, HIV-2, SARS, Human Coronavirus 229E, Human Coronavirus OC43, Hepatitis C virus (HCV), or Equine Arteritis Virus (EAV).
  • HIV-1 HIV-2, SARS, Human Coronavirus 229E, Human Coronavirus OC43, Hepatitis C virus (HCV), or Equine Arteritis Virus (EAV).
  • Coronaviruses which can bo inhibited or their infections treated by the compounds of the invention are those listed in Table 1.
  • the membrane ion channel of which functional activity is down regulated is that which Lentiviruses, and Coronaviruses utilise for mediating viral replication and include, for example, the HIV membrane ion channel Vpu, the HCV membrane ion channel P7, and the Coronavirus E protein membrane ion channel.
  • the subject of the viral inhibition is generally a mammal such as but not limited to human, primate, livestock animal (e.g. sheep, cow, horse, donkey, pig), companion animal (e.g. dog, cat), laboratory test animal (e.g. mouse, rabbit, rat, guinea pig, hamster), captive wild animal (e.g. fox, deer).
  • livestock animal e.g. sheep, cow, horse, donkey, pig
  • companion animal e.g. dog, cat
  • laboratory test animal e.g. mouse, rabbit, rat, guinea pig, hamster
  • captive wild animal e.g. fox, deer.
  • the subject is a primate, or horse.
  • the subject is a human.
  • the invention provides an antiviral compound selected from the group consisting of:
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a compound according to the twelfth aspect, and optionally one or mors pharmaceutical acceptable carriers or derivatives.
  • the pharmaceutical composition may further comprise one or more known antiviral compounds or molecules.
  • FIG. 1 is a schematic representation of plasmids used for expression of Vpu in E. coli .
  • A The amino acid sequence ( ⁇ 400>1) encoded by the vpu open reading frame (ORF) generated by PCR from an HIV-1 strain HXB2 oDNA clone.
  • the vpu ORF was cloned in-frame at the 3′ end of the GST gene in p2GBX to generate p2GEXVpu (B). It was subsequently cloned into pPL451 to produce the plasmid pPL+Vpu (C).
  • FIG. 2 is a photographic representation of the expression and purification of Vpu in E. coli .
  • A Western blotting after SDS-PAGE was used to detect expressed Vpu in E. coli extracts. Lanes 1-4 contain samples, at various stages of purity, of Vpu expressed from p2GEXVpu: lane 1, GST-Vpu flasiors protein isolated by glutathione-agarose affinity chromatography; lane 2, Vpu liberated from the fusion protein by treatment with thrombin; lane 35 Vpu purified by HPLC anion exchange chromatography; lane 4, Vpu after passage through the immunoaffinity column. Lanes 5 and 6, membrane vesicles prepared from 42′C induced cells containing pPL+Vpu or pPL451, respectively.
  • B Silver stained SDS-PAGE gel: lane 1, Vpu purified by HPLC anion exchange chromatography; lane 2, Vpu after passage through the immunoaffinity column.
  • FIG. 3 is a graphical representation of ion channel activity observed after exposure of lipid bilayers to aliquots containing purified Vpu.
  • the CIS chamber contained 500 mM NaCl and the TRANS chamber contained 50 mM NaCl; both solutions were buffered at pH 6.0 with 10 mM MES.
  • B shows a current virus voltage curve generated from data similar to that shown in A.
  • FIG. 4 is a photographic representation of bacterial cross-feeding assays.
  • the Met ⁇ , Pro ⁇ auxotrophic strain waa used to seed a soft agar overlay.
  • Plates A and B contain minimal drop-out medium minus proline; in plate C the medium was minus methionine.
  • the discs labelled P and M contained added proline or methionine, respectively.
  • the discs labelled C and V were inoculated with Met + , Pro + E. coli cells containing the plasmids pPL451, or pPL+Vpu, respectively. Plates were incubated at 37° C. (A and C) or 30° C.
  • FIG. 5 is a graphical representation of the screening of drugs for potential Vpu channel blockers.
  • the photograph shows a section of a minimal medium-lacking adenine-agarose plate onto which a lawn of XL-1-blue E. coli cells containing the Vpu expression plasmid pPLVpu has been seeded. Numbers 6-11 are located at the sites of application of various drugs being tested, which were applied in 3 ⁇ l drops and allowed to soak into the agarose. The plate was then incubated at 37° C. for 48 hr prior to being photographed. The background grey shade corresponds to areas of no bacterial growth.
  • the bright circular area around “10” represents bacterial cell growth as a result of application of adenine at that location (positive control).
  • the smaller halo of bacterial growth around “9” is due to the application of 5-(N,N-hexamethylene)-amiloride at that location.
  • FIG. 6 SARS E protein ion channel activity observed in NaCl solutions after exposure of lipid bilayer to 3-10 ⁇ g of E protein.
  • the CIS chamber contained 50 mM NaCl in 5 mM HEPES buffer pH 7.2
  • the TRANS chamber contained 500 mM NaCl in 5 mM HEPES buffer pH 7.2.
  • the CIS chamber was earthed and the TRANS chamber was held at various potentials between ⁇ 100 to +100 mV.
  • FIG. 7 SARS E protein ion channel activity observed in NaCl solutions after exposure of lipid bilayer to 3-10 ⁇ g of E protein.
  • the CIS chamber contained 50 mM NaCl in 5 mM HEPES buffer pH 7.2.
  • the TRANS chamber contained 500 mM NaCl in 5 mM HEPES buffer pH 7.2.
  • the CIS chamber was earthed and the TRANS chamber was held at various potentials between ⁇ 100 to +100 mV.
  • FIG. 8 Cinnamoylguanidine (Bit036) inhibits SARS E protein ion channel activity in NaCl solution.
  • C Average current (pA), before formation of E protein ion channel, E protein ion channel activity and after addition of 100 ⁇ pM Bit036.
  • FIG. 9 229E protein ion channel activity in lipid bilayers in KCl solutions.
  • FIG. 10 Part A shows raw currents generated by the 229E-E protein ion channel in a planar lipid bilayer. The top trace shows current activity prior to drug addition and the lower trace shows the effect of addition of 100 ⁇ M cinnamoylguanidine on channel activity. Part B is a graphical representation of the average current flowing across the bilayer (in arbitrary units), before and after addition of cinnamoylguanidine.
  • FIG. 11 MHV E protein Ion channel activity in lipid bilayars NaCl solutions.
  • FIG. 12 Part A shows raw currents generated by the MHV-E protein ion channel in a planar lipid bilayer. The top trace shows current activity prior to drug addition and the lower trace shows the effect of addition of 100 ⁇ M cinnamoylguanidine on channel activity. Part B is a graphical representation of the average current flowing across the bilayer (in arbitrary units), before and after addition of cinnamoylguanidine.
  • the present invention is based, in part, on the surprising determination that certain compounds that fall under the classification of substituted acylguanidines have antiviral activity against viruses from a range of different virus families.
  • the negative impact of the compounds of the present invention on viral replication may be mediated by the inhibition or otherwise down-regulation of a membrane ion channel relied upon by the virus for replication.
  • This membrane ion channel may be a viral membrane ion channel (exogenous to the host cell) or a host cell ion channel induced as a result of viral infection (endogenous to the host cell).
  • the compounds of the present invention may inhibit Vpu or p7 function and thereby inhibit the continuation of the respective HIV or HCV life cycle.
  • the SARS virus encodes an E protein which is shown for the first time, by the present inventors, to act as an ion channel.
  • E proteins are present in other coronaviruses, the compounds, compositions and methods of the present invention would have utility in the inhibition and/or treatment of infections by other coronaviruses.
  • While the present invention is concerned with novel antiviral compounds falling under the classification of substituted acylguanidines, it does not include in its scope the use of compounds 5-(N,N-hexamethlene)amiloride and 5-(N,N-dimethyl)-amiloride for retarding, reducing or otherwise inhibiting viral growth and/or fractional activity of HIV.
  • the compounds of the invention may be administered in the form of a composition or formulation comprising pharmaceutically acceptable carriers and excipients.
  • the pharmaceutical compositions of the invention may further comprise one or more known antiviral compounds or molecules.
  • the known antiviral compounds are selected from the group consisting of Vidarabine, Acyclovir, Ganciclovir, Valganciclovir, Valacyclovir, Cidofovir, Famciclovir, Ribavirin, Amantadina, Riantadine, Interferon, Oseltamivir, Palivizumab, Rimantadine, Zanamivir, nucleoside-analog reverse transcriptase inhibitors (NRTI) such as Zidovudine, Didanosine, Zalcitabine, Stavudine, Lamivudine and Abacavir, non-nucleoside reverse transcriptase inhibitors (NNRTI) such as Nevirapine, Delavirdine and Efavirenz, protease inhibitors such as Sequinavir, Ritonavir, Indinavir, Nelfinavir, Amprenavir, and
  • the methods and compositions of the present invention may be particularly effective against viruses which rely on ion channel formation for their replication, however it will be understood that this is not the only mechanism relied on by viruses for replication and that the compounds and methods of the present invention are not limited to agents which exert their action by retarding or inhibiting the function of ion channels.
  • membrane ion channel should be understood as a reference to a structure which transports ions across a membrane.
  • the present invention extends to ion channels which may function by means such as passive, osmotic, active or exchange transport.
  • the ion channel may be formed by intracellular or extracellular means.
  • the ion channel may be an ion channel which is naturally formed by a cell to facilitate its normal functioning.
  • the ion channel may be formed by extracellular means. Extracellular means would include, for example, the formation of ion channels due to introduced chemicals, drugs or other agents such as ionophores or due to the functional activity of viral proteins encoded by a virus which has entered a cell.
  • the ion channels which are the subject of certain embodiments of the present invention facilitate the transport of ions across membranes.
  • Said membrane may be any membrane and is not limited to the outer cell wall plasma membrane.
  • membrane encompasses the membrane surrounding any cellular organelle, snsh as the Golgi apparatus and endoplasmic reticulum, the outer cell membrane, the membrane surrounding any foreign antigen which is located within the cell (for example, a viral envelope) or the membrane of a foreign organism which is located extraceliularly.
  • the membrane is typically, but not necessarily, composed of a fluid lipid bilayer.
  • the subject ion channel may be of any structure.
  • Vpu ion channel is formed by Vpu which is an integral membrane protein encoded by HIV-1 which associates with, for example, the Golgi and endoplasmic reticulum membranes of infected cells.
  • Vpu ion channels is a reference to all related ion channels for example P7 HCV and M2 of influenze and the like.
  • HIV HIV
  • SARS Session Virus
  • Coronavirus Coronavirus
  • HCV HCV
  • references to the “functional activity” of an ion channel should be understood as a reference to any one or more of the functions which an ion channel performs or is involved in.
  • the Vpu protean encoded ion channel in addition to facilitating the transportation of Na + , K + , Cl + and PO 4 3+ , also plays a role in the degradation of the CD4 molecule in the endoplasmic reticulum.
  • the Vpu protein encoded ion channel is also thought to play a role in mediating the HIV life cycle.
  • the present invention is not limited to treating HIV infection via the mechanism, of inhibiting the HIV life cycle and, in particular, HIV replication. Rather, the present invention should be understood to encompass any mechanism by which the compounds of the present invention exert their anti-viral activity and may include hihibition of HIV viability or functional activity. This also applies to HCV, Coronaviruses, and to other viruses.
  • references to the “functional activity” of a virus should be understood as a reference to any one or more of the functions which a virus performs or is involved in.
  • Ion channel mediation of viral replication may be by direct or indirect means. Said ion channel mediation is by direct means if the ion channel interacts directly with the virion at any one or more of its life cycle stages. Said ion channel mediation is indirect if it interacts with a molecule other than those of the virion, which othsr molecule either directly or indirectly modulates any one or more aspects or stages of the viral life cycle. Accordingly, the method of the present invention encompasses the mediation of viral replication via the induction of a cascade of steps which lead to the mediation of any one or more aspects or stages of the viral life cycle.
  • a suitable agent may interact directly with an ion channel to prevent replication of a virus or, alternatively, may act indirectly to prevent said replication by, for example, interacting with a molecule other than an ion channel.
  • a further alternative is that said other molecule interacts with and inhibits the activity of the ion channel.
  • references to a “cell” infected with a virus should bo understood as a reference to any cell, prokaryotic or eukaryotlc, which has been infected with a virus. This includes, for example, immortal or primary cell lines, bacterial cultures and cells in situ. In a suitable screening system for antiviral compounds, the preferred infected cells would be macrophages/monocytes or hepatocytes/lymphoid cells infected with either HIV or HCV respectively.
  • the compounds of the present invention are thought to inhibit viral replication or virion release from cells by causing ion channels, namely VPU of HIV, the E protein of SARS and other Coronaviruses, or P7 of HCV to become blocked.
  • the present invention encompasses antiviral compounds that are substituted acylguanidines.
  • the present invention also includes the use of compounds 5-(N,N-hexamethylene)amiloride and 5-(N,N-dimethyl)-amiloride is the control of viral replication and/or growth other than HIV.
  • the subject of the viral irthibition is generally a mammal such as but not limited to human, primate, livestock animal (e.g. sheep, cow, horse, donkey, pig), companion animal (e.g. dog, cat), laboratory test animal (e.g. mouse, rabbit, rat, guinea pig, hamster), captive wild animal (e.g. fox, deer).
  • livestock animal e.g. sheep, cow, horse, donkey, pig
  • companion animal e.g. dog, cat
  • laboratory test animal e.g. mouse, rabbit, rat, guinea pig, hamster
  • captive wild animal e.g. fox, deer
  • the method of the present invention is useful in the treatment and prophylaxis of viral infection such as, for example, but not limited to HIV infection, HCV infection and other viral infections.
  • the antiviral activity may be effected in subjects known to be infected with HIV in order to prevent replication of HIV thereby preventing the onset of AIDS.
  • the method of the present invention may be used to reduce serum viral load or to alleviate viral infection symptoms.
  • antiviral treatment may be effected in subjects known to be infected with, for example, HCV, is order to prevent replication of HCV, thereby preventing the further hspatocyte involvement and the ultimate degeneration of liver tissue.
  • the method of the present invention may be particularly useful either in the early stages of viral infection to prevent the establishment of a viral reservoir in affected cells or as a prophylactic treatment to be applied immediately prior to or for a period after eKpoaurc to a possible source of virus.
  • therapy and prophylaxis include amelioration of the symptoms of a particular condition or preventing or otherwise reducing the risk of developing & particular condition.
  • prophylaxis may be considered as reducing the severity of onset of a particular condition. Therapy may also reduce the severity of as existing condition or the frequency of acute attacks.
  • more than one compound or composition may be co-administered with one or more other compounds, such as knows anti-viral compounds or molecules.
  • co-administered is meant simultaneous admirdstration in the same formulation or in two different formulations via the same or different routes or sequential administration by the same or different routes.
  • sequential administration is meant a time difference of from seconds, minutes, hours or days between the adminstration of the two or more separate compounds.
  • the subject antiviral compounds may be administered in any order.
  • Routes of administration include but are not limited to intravenously, intraperitionealy, subcutaneously, intracramialy, intradermally, intramuscularly, intraocularly, intrathecaly, intracerebrally, intranasally, transmucosally, by infusion, orally, rectally, via iv drip, patch and implant. Intravenous routes are particularly preferred.
  • compositions suitable for injectable use include sterile aqueous solutions (where water soluble) and sterile powders for the extemporaneous preparation of sterile injectable solutions.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like), suitable mixtures thereof and vegetable oils.
  • the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thirmerosal and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by for example, filter sterilisation or sterilization by other appropriate means.
  • Dispersions are also contemplated and these may be prepared by incorporating the various sterilised active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • a preferred method of preparation includes vacuum drying and the freeze-drying technique which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution.
  • the active ingredients When the active ingredients are suitably protected, they may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsule, or it may be compressed into tablets.
  • the active compound may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.
  • Such compositions and preparations should contain at least 0.01% by weight, more preferably 0.1% by weight, even more preferably 1% by weight of active compound.
  • compositions and preparations may, of course, be varied and may conveniently be between, about 1 to about 99%, more preferably about 2 to about 90%, even more preferably about 5 to about 80% of the weight of the unit.
  • the amount of active compound in such therapeutically useful compositions in such that a suitable dosage will be obtained.
  • Preferred compositions or preparations according to the present invention are prepared so that an oral dosage unit form contains between about 0.1 ng and 2000 mg of active compound.
  • the tablets, troches, pills, capsules and the like may also contain the components as listed hereafter.
  • a binder such as gum, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, lactose or saccharin may be added or a flavouring agent such as peppermint, oil of wintergreen, or cherry flavouring.
  • the dosage unit form When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit.
  • tablets, pills, or capsules may be coated with shellac, sugar or both.
  • a syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavouring such as cherry or orange flavour. Any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed.
  • the active compound(s) may be incorporated into sustained-release preparations and formulations.
  • the present invention also extends to forms suitable for topical application such as creams, lotions and gels.
  • the anti-clotting peptides may need to be modified to permit penetration of the surface barrier.
  • Procedures for the preparation of dosage unit forms and topical preparations are readily available to those skilled in the art from texts such as Pharmaceutical Handbook. A Martindale Companion Volume Ed. Ainley Wade Nineteenth Edition The Pharmaceutical Press London,
  • Pharmaceutically acceptable carriers and/or diluents include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like.
  • the use of such media and agents for pharmaceutically active substances is well knows in the art. Except insofar as any conventional media or agent is incompatable with the active ingredient, use thereof in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the novel dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active material and the particular therapeutic effect to be achieved and (b) the limitations inherent in the art of compounding.
  • Effective amounts contemplated by the present invention will vary depending on the severity of the pain and the health and age of the recipient. In general terms, effective amounts may vary from 0.01 ng/kg body weight to about 100 mg/kg body weight.
  • Alternative amounts include for about 0.1 ng/kg body weight about 100 mg/kg body weight or from 1.0 ng/kg body weight to about 80 mg/kg body weight.
  • the subject of the viral inhibition is generally a mammal such as but not limited to human, primate, livestock animal (e.g. sheep, cow, horse, donkey, pig), companion animal (e.g. dog, cat), laboratory test animal (e.g. mouse, rabbit, rat, guinea pig, hamster), captive wild animal (e.g. fox, deer).
  • livestock animal e.g. sheep, cow, horse, donkey, pig
  • companion animal e.g. dog, cat
  • laboratory test animal e.g. mouse, rabbit, rat, guinea pig, hamster
  • captive wild animal e.g. fox, deer
  • the methods of the present invention is useful in the treatment and prophylaxis of viral infection such as, for example, but not limited to HIV infection, HCV infection and other viral infections.
  • the antiviral activity may be effected in subjects known to be infected with HIV in order to prevent replication of HIV thereby preventing the onset of AIDS.
  • the methods of the present invention may be used to reduce serum viral load or to alleviate viral infection symptoms.
  • antiviral treatment may be effected in subjects known to be infected, with, for example, HCV, hi order to prevent replication of HCV, thereby preventing the further hepatocyte involvement and the ultimate degeneration of liver tissue.
  • the methods of the present invention may be particularly useful either in the early stages of viral infection to prevent the establishment of a viral reservoir in affected cells or as a prophylactic treatment to be applied immediately prior to or for a period after exposure to a possible source of virus.
  • the compounds of the present invention may be made from the corresponding acid chlorides or methyl esters as shown in Scheme 1. Both of these methods are wall described in the literature.
  • trans-cinnamic acid (1.50 g, 10.12 mmol) in dry benzene (30 mL) containing a drop of N,N-dimethylformaniide was added oxalyl chloride (5.14 g, 40.5 mmol) causing the solution to effervesce. After refluxing 2 h, the solution was evaporated to dryness under reduced pressure. The resulting solid was dissolved in dry tetrahydfotaran (20 mL) and added slowly to a solution of guanidine hydrochloride in 2M aqueous sodium hydroxide (25 mL).
  • Complimentary DNA (cDNA) fragments for the various viral proteins listed in Table 2 were obtained either by PCR amplication from a parental virus genome clone, or by direct chemical synthesis of the polynucleotide sequence.
  • Vpu open reading frame encoding Vpu ( FIG. 1 a ) was amplified by PCR from a cDNA clone of an Nde I fragment of tbe HIV-1 genome (isolate HXB2, McFarlane Burnet Centre, Melbourne, Australia) as follows: Native Pfu DNA polymerase (Stratagene; 0.035 U//II) was chosen to catalyse the VCR reaction to minimise possible PCR introduced errors by virtue of the enzyme's proofreading activity.
  • Native Pfu DNA polymerase (Stratagene; 0.035 U//II) was chosen to catalyse the VCR reaction to minimise possible PCR introduced errors by virtue of the enzyme's proofreading activity.
  • the 5′, sense, primer AGTA GGATCC ATGCAACCTATACC ( ⁇ 400>2) introduces a BamHI site (underlined) for cloning in-frame with the 3′ end of the GST gene in p2GEX (41).
  • This primer also repairs ths start codon (bold T replaces a Q of the vpn gene which is a threonine codon in the HXB2 isolate.
  • the 3′, antisense, primer TCT GGAATT LTACAGATCAT CAAC introduces an EcoRI site (underlined) to the other end of the PGR product to facilitate cloning. After 30 cycles of 94° C. for 45 sec, 55° C. for 1 min and 72° C.
  • FIG. 1 b The entire Vpu open reading frame and the BamHI and EcoRI ligation sites were sequenced by cycle sequencing, using the Applied Biosystems dye-terminator kit, to confirm the DNA sequence.
  • Other cDNAs were synthesissd for us using state of the art methods by GenScript Corporation (New Jersey, USA). Codon sequences were optimised for expression in bacterial, insect or mammalian cells, as appropriate.
  • Restriction endonuclease enzyme recognition sites were incorporated at the 5′ and 3′ ends of the synthetic cDNAs to facilitate cloning into plasmid expression vectors, pcDNA3.1, pFastBac and pPL451 for expression of the encoded virus proteins in mammalian, insect or bacterial cells, respectively.
  • p2GEXVpu was first digested with BamHI and the 5′ base overhang was filled in the Klenow DNA polymerase in the presence of dNTPs.
  • the Vpn-encoding fragment was then liberated by digestion with EcoRI, purified from an agarose gel and ligated into pPL451 which had been digested with HpaI and EcoRI.
  • Western blots subsequently confirmed that the pPLVpu construct ( FIG. 1 c ) expressed Vpu after induction of cultures at 42° C. to inactivate the cI857 repressor of the PR and PL promoters.
  • E. coli strain XLI-blue cells containing p2GEXVpu were grown at 30° C. with vigors aeration in LB medium supplemented with glucose (6 g/L) and ampicillin (50 mg/L) to a density of approximately 250 Klett units, at which time IPTG was added to a final concentration of 0.01 mM and growth was contained for a further 4 hr.
  • the final culture density was approximately 280 Klett units.
  • the osmotically sensitised cells were pelleted at 12,000 g and sesuspended to the original volume in water to burst the cells.
  • the suspension was then made up to 1xMTEBS/DTT using a 10 ⁇ buffer stock and the ghosts were isolated by centrifugation and resuspendsd in MTPBS/DTT to which was then sequentially added glycerol (to 20 % wt/vol) and CHAPS (to 2 % wt/vol) to give a final volume of one quarter the original volume.
  • This mixture was stirred on ice for 1 hr and then centrifuged at 400,000 g for 1 hr to remove insoluble material.
  • the GST-Vpu fusion protein was purified from the detergent extract by affinity chromatography on a glutathione agarose resin (Sigma). The resin was thoroughly washed in 50 mM Tris pH 7.5 containing glycerol (5 %), DTT (1 nM), and CHAPS (0.5 %) (Buffer A) and then the Vpu portion of the fusion protein was liberated and eluted from the resin-bound GST by treatment of a 50% (v/v) suspension of the beads with human thrombm (100U/ml; 37° C. for 1 hr). PMSF (0.5 mM) was added to the eluant to eliminate any remaking thrombin activity. This Vpu fraction was farther purified on a column of MA7Q anion exchange resin attached to a BioRad HPLC and eluted with a linear NaCl gradient (0-2M) in buffer A.
  • Vpu was purified to homogeneity—as determined on silver stained gels—on an immunoaffinity column as follows: HPLC factions containing Vpu were desalted on a NAP 25 column (Pharmacia) into buffer A and then mixed with the antibody-agarose beads for 1 hr at room temperature. The beads were washed thoroughly and Vpu was eluted by increasing the salt concentration to 2M. Protein was quantitated using the BioRad dye binding assay.
  • the plasmid p2GEXVpn ( FIG. 1 ) was constructed to create an in-frame gene fusion between the GST and Vpu open-reading frames. This system enabled IPTG-inducible expression of the Vpu polypeptide fused to the C-terminus of GST and allowed purification of the fusion protein by affinity chromatography on glutathione agarose.
  • Optimal levels of GST-Vpu expression were obtained by growing the cultures at 30° C. to a cell density of approximately 250-300 Klett units and inducing with low levels of IPTG (0.01 mM).
  • a combined cellular fraction containing the cell debris aad plasma membrane was prepared by lysozyme treatment of the induced cells followed by a low-speed centrifugation. Approximately 50% of the GST-Vpu protein could be solubilised from this fraction using the zwitterionic detergent CHAPS.
  • Vpu Vpu was the major protein visible on silver stained gels ( FIG. 2B , lane 1). Finally, Vpu was purified to apparent homogeneity on an immunoaffinity column ( FIG. 2B , lane 2).
  • the N-terminal amino acid sequence of the protein band (excised from SDS-PAGE gels) corresponding to the immunidetected protein confirmed its identity as Vpu.
  • Proteoliposomes containing Vpu were prepared by the detergent dilution method (New, 1990). A mixture of lipids (PE:PC:PS; 5:3:2; 1 mg total lipid) dissolved in chloroform was dried under a stream of nitrogen gas and resuspended in 0.1 ml of potassium phosphate buffer (50 mM pH 7.4) containing DTT (1 mM). A 25 ⁇ l aliquot containing purified Vpu was added, followed by octylglucoside to a final concentration of 1.25 % (wt/vol).
  • Vpu was tested for its ability to induce channel activity in planar lipid bilayers using standard techniques as described elsewhere (Miller, 1986; and Piller et al, 1996).
  • the solutions in the CIS and TRANS chambers were separated by a DelrinTM plastic wall containing a small circular hole of approximately 100 ⁇ m diameter across which a lipid bilayer was painted so as to form a higk resistance electrical seal.
  • Bilayers were painted from a mixture (8:2) of palmtoyl-oleoly-phosphatidyl-ethanolamine and palmitoyl-oleolyphosphatidyl-choline (Avanti Polar Lipids, Alabaster, Ala.) in n-decane.
  • Ths solutions in the two chambers contained MES buffer (10 mM, pH 6.0) to which various NaCl or KCl concentrations were added. Currents were recorded with an AxopatchTM 200 amplifier. The electrical potential between the two chambers could be manipulated between +/ ⁇ 200 mV (TRANS relative to grounded CIS). Aliquots containing Vpu were added to the CIS chamber either as a detergent solution or after incorporation of the protein into phospholipid vesicles. The chamber was stirred until currents were observed.
  • Channel activity was observed in over 40 individual experiments with Vpu samples prepared from five independent purifications. In different experiments, the amplitude of the entrants varied over a large range and, again, seemed to approximately correlate with the amount of protein added. The smallest and largest channels measured had conductance of 14 pS and 280 pS, respectively. The channels were consistently smaller when lipid vesicles containing Vpu were prepared and fused to the bilayer rather than when purified protein in detergent solution was added. This may be because the former method included treatment with high concentrations of detergent and a dilution step that may have favoured the breakdown of large aggregates into monomers.
  • This bio-assay is based on the observation that expression of Vpn in E. coli results in an active Vpu channel located is the plasmalemma that dissipates the transmembrane sodium gradient.
  • Vpu channel activity metabolites whose accumulation within the cells is mediated by & sodium dependent co-transporter (for example proline or adenine) leak out of the cell faster than they can be synthesised so that the metabolites' intracellular levels become limiting for growth of the cell.
  • & sodium dependent co-transporter for example proline or adenine
  • the vpu open-reading frame was cloned into the plasmid pPL451 to create the recombinant plasmid pFL-Vpu ( FIG. 1 b ).
  • the strong P L and P R lambda promoters are used to drive expression of Vpu under control of the temperature sensitive c1857 represser, such that when grown at 30° C. expression is tightly repressed and can be induced by raising the temperature to between 37° C. and 42° C.
  • the temperature sensitive c1857 represser such that when grown at 30° C. expression is tightly repressed and can be induced by raising the temperature to between 37° C. and 42° C.
  • On agar plates cells containing pPL-Vpu grew when incubated at 30° C. and 37° C. but not at 42° C., while control strains grew well at 42° C.
  • the plasma membrane fraction was prepared and western blotting, using an antibody that specifically binds to the C-terminus of Vpu, detected a single band at approximately 16 kDa, indicating that Vpu was expressed and associated with the membranes ( FIG. 2A , lane 5).
  • E. coli Uptake of proline by E. coli is well characterised and active transport of the amino acid into the cells is known to use the sodium gradient as the energy source (Yamato et al, 1994).
  • the following cross-feeing assay was used: A lawn of an E. coli strain auxotrophic for proline and methionine (Met ⁇ Pro ⁇ ), was seeded and poured as a soft agar overlay on minimal drop-out media plates lacking proline but containing methionine. Sterile porous filter discs were inoculated with a Met + Pro + strain (XL-1 blue) containing either the pPL451 control plasmid or pPL-Vpu and placed onto the soft agar.
  • Met + Pro + strain XL-1 blue
  • the E. coli methionine permease is known to belong to the ABC transporter family (Rosen, 1987) and hence be energised by ATP. Identical crossfeeding experiments to those described above were set us except that the Met ⁇ Pro ⁇ strain was spread on minimal drop-out plates lacking methionine but containing proline. No growth of this strain was evident around any of the discs ( FIG. 4C ), indicating that methionine was not leaking out of the XL-1 blue cells even when Vpu was being expressed.
  • Vpu N-terminal peptide (residues 1-32) dissolved in trifluoroethanol was added to the CIS chamber of the bilayer apparatus and the solutions was stirred until ion currents were observed, indicating incorporation of one or more Vpu ion channels into the bilayer.
  • drugs were added to the solutions in the CIS and TRANS chambers—with stirring—to a final concentration of 100 ⁇ M.
  • Channel activity was then recorded for at least a further three minutes and the effect of drug addition on ion current was detennined by comparing the channel activity before and after drug addition.
  • Table 4 lists the scores for inhibition of Vpn protein in the bacterial bio-assay.
  • Human monocytes were isolated from peripheral blood and cultured either for 24 hr (one day old monocytes) or for 7 days to allow differentiation into monocyte derived macrophages (MDM). These cells were then exposed to cell-free preparations of HIV isolates and allowed to absorb for 2 hr before complete aspiration of the medium, washing once with virus-free medium and resuspension in fresh medium. The cells were exposed to various concentration of compound either 24 hr prior to infection or after infection. Subsequent HIV replication, at various times after infection, was compared in cells exposed to drugs and in cells not exposed to drugs (controls). The progression and extent of viral replication was assayed using either an HIV DNA PCR method (Fear et al, 1998) or an ELISA method to quantitate p24 in culture supernatants (Kelly et al, 1998).
  • Table 5 provides examples of results obtained using this assay and test antiviral compounds.
  • a peptide corresponding to the full-length SARS-CoV (isolate Tox2 and Urbani) E protein (MYSFVSEETGTLIVNSVLLFLAFVVFLLVTLAILTALRLCA YCCNIVNVSLVKPTVYVYSRVKNLNSSBGVPDLLV) and a second peptide comprising the first 40 amino acids of the full length E protein which correspond to the transmembrane domain (MYSFVSEETGTLIVNSVLLFLAFVVF LLVTLAILTALRLC) were synthesized manually using FMOC chemistry and solid phase peptide synthesis The synthesis was done at the Biomolecular Resource Facility (John Curtin School of Medical Research, ANU, Australia) using a Symphony R Peptide Synthesiser from Protein Technologies Inc. (Tucson, Ariz., USA) according to the manufacturers instructions.
  • the insoluble material containing the E protein was dried using Speedvac an the weight of the final product was used to calculate the yield.
  • the purified peptide was analysed by Bruker Onmiflex MALDI-TOF mass spectrometry in HABA matrix at 2.5 mg/ml in methanol at a 1:1 ratio and spectra were obtained in the positive linear mode. A clear peat at m/z ratio of 8,360.1 was seen as expected for the calculated molecular weight of full-length E protein and 4422.3 for the N-terminal E protein.
  • the SARS virus B protein was resuspended at 1 mg/ml in 2,2,2-trifluoroethanol.
  • the SARS virus E protein's ability to form ion channels was tested on a Warner (Warner instruments, Inc. 1125 Dixwell Avenue, Hamden, Conn. 06514) bilayar rig as follows; A lipid mix of 3:1,1, 1-Palmitoyl-2-oleolyl phosphatidyl Ethanolamine: 1-Palmitoyl-2-oleolyl phosphatidyl Serine: 1-Palmitoyl-2-oleolyl phosphatidyl choline in CHCl 3 was dried under N 2 gas and suspended to 50 mg/ml in n-decane.
  • Bilayers were painted across a circular hole of approximately 100 ⁇ m diameter in a DelrinTM cup separating aqueous solution in the CIS and TRANS chambers.
  • the CIS chamber contained a solution of 500 mM NaCl or KCl, in a 5 mM HEPES buffer pH 7.2
  • the TRANS chamber contained a solution of 50 mM NaCl or KCl, in a 5 mM HEPES buffer pH 7.2.
  • Silver electrodes coated in chloride with 2% agarose bridges are placed in the CIS and TRANS chamber solutions.
  • the SARS E protein full-length or N-terminal peptides (3-10 ng) were added to the CIS chamber, which was stirred until channel activity was detected.
  • the CIS chamber was earthed and the TRANS chamber was held at various holding potentials ranging between +100 to ⁇ 100 mV. Currents were recorded using a Warner model BD-525D amplifier, filtered at 1 kHz, sampling at 5 kHz and digitally recorded on the hard disk of a PC using software developed in house.
  • Drugs to be tested for their ability to inhibit SARS E protein ion channel activity were made up at 50 mM is a solution of 50% DMSO: 50% methanol.
  • ion channel activity 100 ⁇ M to 400 ⁇ M of compound was added to the CIS chamber while stirring for 30 seconds. Bilayar currents were recorded before channel activity, during channel activity and after the addition of the drug.
  • cinnamoylguanidine (Bit036), a compound which was shown in earlier experiments to be antiviral and to inhibit ion channel proteins from other viruses.
  • E protein was dissolved to 1 mg/ml, 5 mg/ml and 10 mg/ml in, 6 M Urea, 10% Glycerol, 5% SDS, 500 mM DTT, 0.002% Bromophenol Blue, 62.5 mM Tris HCl (pH 8.3), Peptides in solutions were heated at 100° C. for 20 minutes before 30 ⁇ L samples were run on stacking gel 4-20% (Gradipora). SeeBlue® pre-stained standard (Invitrogen) was used for molecular weight markers.
  • the purified synthetic peptide was reconstituted into planar lipid bilayers (21). Typically, 3 ⁇ g of SARS full-length E protein was added to the CIS chamber, while stirring. This CIS chamber contained 500 mM NaCl and the TRANS chamber contained 50 mM NaCl. In 60 experiments, ion currents due to SARS E protein ion channel activity were observed after about 5-15 minutes of stirring. Activity was detected more rapidly and reliably with a holding potential of approximately ⁇ 100 mV across the bilayer. Currents recorded at ⁇ 100 mV, (A) and at ⁇ 60 mV (B) in one of these experiments are shown in FIG. 6 .
  • FIG. 7 a shows typical current traces recorded oyer a range of potentials in NaCl solutions.
  • the IV curve shows that at the lower voltages the average current flow across the bilayer is small but at higher potentials there is an increase in average current across the bilayer, resulting in a non-linear IV relationship.
  • the average reversal potential was +48.3 ⁇ 23 mV (mean ⁇ 1SEM), indicating that the channels were about 37 times more permeable to Na+ ion than to Cl ⁇ ions.
  • the reversal potential is close to the Na+ equilibrium potential (+53 mV), therefore the channel is selective for Na+ ions.
  • the channel conductance varied between 95-164 pS; the average conductance was 130 ⁇ 13 pS.
  • FIG. 8 b shows recording of currents in KCl solutions at a range of potentials. In this experiment the currents reversed at ⁇ 31 mV. In seven similar experiments E protein ion channel average reversal potential was +34.5 ⁇ 2.5 mV. Therefore the SARS E protein ion channel is about 7.2 times more permeable to K + ions than Cl ⁇ ions. In seven experiments, the channel conductance varied ranging between 24-166 pS, the average conductance was 83.4 ⁇ 26 pS.
  • SARS E protein N-terminal peptide also formed ion channels in KCl solution that were similarly selective for K+ ions compared to the full-length E protein.
  • the average channel reversal potential was +39.5 ⁇ 3.6 mV, therefore the channel is about 11 times more permeable to K + ions than Cl ⁇ ions.
  • SDS-PAGE of the purified full-length E protein peptide showed bands corresponding to the full-length E protein (Data not shown). Larger bands of varying size up to about 20 kDa were detected, suggesting that SARS E protein may form homo-oligomers.
  • SARS E Portein Ion Channel is Blocked by Cimmamoylguamidine and Other Compounds
  • the average current across the bilayer was reduced to baseline by 100 ⁇ M cinnamoylguanidine.
  • 100 to 200 ⁇ M cinnamoulguanidine reduced the average current across the bilayer about 4 fold.
  • 100 to 200 ⁇ M cinnamoylguanidine blocked channels formed by full-length E protein in KCl solutions.
  • SARS E protein can form ion channels in lipid bilayer membranes.
  • the first 40 amino acids of the N-terminal which contains thg hydrophobic domain of the SARS virus E protein is sufficient for the formation of ion channels on planar lipid bilayers.
  • the N-terminal E protein ion channel has the same selectivity and conductance as the full-length E protein ion channel.
  • the SARS virus full length E protein ion channel activity and N-terminal domain E protein ion channel activity on planar lipid biiayers in NaCl and KCl solutions was inhibited by addition of between 100 ⁇ M to 200 ⁇ M cinnamoylguanidine to the CIS chamber. Inhibition or partial inhibition of the E protein ion channel activity by cinnamoylguanidine has been observed in seven independent experiments in NaCl solution and four independent experiments in KCl solution.
  • coronaviruses encode an E protein with a hydrophobic N-terminus transmembrane domain therefore all coronaviruses E proteins could form ion channels on planar lipid bilayers. This indicates that the E protein could be a suitable target for antiviral drugs and potentially stop the spread of coronavirus from infected host cells. Drugs that block the E protein ion channel could be effective antiviral therapy for the treatment of several significant human and veterinary coronavirus diseases including SARS and the common cold.
  • a bio-assay of SARS-CoV E protein function in bacterial cells was developed.
  • a synthetic cDNA fragment encoding SARS-CoV E protein was cloned into the expression plasmid pPL451, creating a vector in which E protein expression is temperature inducible, as described in Example 4.
  • Inhibition of the growth of E. coli cells expressing E protein at 37° C. was observed as an indicator of p7 ion channel function dissipating the normal Na+ gradient maintained by the bacterial cells.
  • Table 7 lists the scores for inhibition of SARS-CoV E protein in the bacterial bio-assay.
  • SARS-CoV SARS Antiviral Assay for Testing Compounds Against Replication of SARS Coronavirus
  • Monolayers of Vero cells grown in 25 cm 2 flasks were infected at a multiplicity of 1:50 and treated immediately post infection with compounds at two concentrations, 10 uM and 2 uM. A control infected monolayer remained untreated.
  • Samples of culture media were taken at 48 hours post infection. Two aliquots from each of the samples (titrations 1 and 2) were serially log diluted and 12 replicates of log dilutions ⁇ 4 to ⁇ 7 added to cells is microtitre plates. Four days later, wells in the microtitre plates were scored for cytopathic effect (CPE) and the titration values calculated based on the number of CPE positive wells at the 4 dilutions. Control titres were 4.8 and 5.9 TCDD 50 ⁇ 10 6 (average 5.35 ⁇ 10 6 )
  • trans-3-(1-napthyl)acryloylguanidine and cinnamoylguanidine a decrease in virus titre of approximately 80% was observed at a concentration of 10 uM and a reduction of approximately 50% was seen to persist at 2 ⁇ M trans-3-(1-naphthyl)acsyloylguanidine.
  • Table 8 provides Virus titration data presented as % of a control (SARS CoV grown for 48 hours in the absence of compounds).
  • a peptide corresponding to the full-length 229E-E protein was synthesized manually using FMOC chemistry and solid phase peptide synthesis. The synsthesis was done at the Biomolecular Resource Facility (John Curtin School of Medical Research, ANU, Australia) using a Symphony R Peptide Synthesiser from Protein Technologies Inc. (Woburn, Miss.
  • the crude synthetic peptide was purified using the ProteoPlusTM kit (Qbiogene inc. CA), following manufactures instructions. Briefly, the peptides were diluted in loading buffer (60 mM Tris-HCl pH 8.3, 6M urea, 5% SDS, 10% glycerol, 0.2% Bromophenol blue, ⁇ 100 mM ⁇ -mercaptoethanol) and run on 4-20% gradient polyacrylamide gels (Gradipore, NSW, Australia) in tris-glycine electrophoresis buffer (25 mM Triss 250 mM glycine, 0.1% SDS). The peptides were stained with gel code blue (Promega, NSW) and the bands corresponding to the full-length peptide were excised out of the gel.
  • loading buffer 60 mM Tris-HCl pH 8.3, 6M urea, 5% SDS, 10% glycerol, 0.2% Bromophenol blue, ⁇ 100 mM ⁇ -mercaptoethanol
  • the gel slice was transferred to the ProteoPLUSTM tube and filled with tris-glycine electrophoresis buffer.
  • the tubes were emerged in tris-glycine electrophoresis buffer and subjected to 100 volts for approximately 1 hour.
  • the polarity of the electric current was reversed for 1 minute to increase the amount of protein recovered.
  • the peptides were harvested and centrifuged at 13,000 rpm for 1 minute.
  • the purified peptides were dried in a Speedvac and the weight of the final product was used to calculate the yield.
  • Lipid bilayer studies were performed as described elsewhere (Sumtrom, 1996; Miller, 1986).
  • a lipid mixture of palmitoyl-oleoyl-phosphatidylethanolamine, palmitoyl-oleoyl-phosphatidylserine and palmitoyl-oleoyl-phosphatidylcholine (5:3:2) (Avanti Polar Lipids, Alabaster, Ala.) was used.
  • the lipid mixture was painted onto an aperture of 150-200 ⁇ m in the wall of a 1 ml delrin cup. The aperture separates two chambers, cis and trans, both containing salt solutions at different concentrations.
  • the cis chamber was connected to ground and the trans chamber to the input of an Axopatch 200 amplifier. Normally the cis chamber contained either 500 mM NaCl or 500 mM KCl and the trans 50 mM NaCl or 50 mM KCl.
  • the bilayer formation was monitored electrically by the amplitude of the current pulse generated by a current ramp. The potentials were measured in the trans chamber with respect to the cis.
  • the synthetic peptide was added to the cis chamber and stirred until channel activity was seen. The currents were filtered at 1000 Hz, digitized at 5000 Hz and stored on magnetic disk.
  • the 229E E synthetic peptide was dissolved in 2,2,2-trifluorethanol (TFE) at 0.05 mg/ml to 1 mg/ml. 10 ⁇ l of this was added to the cis chamber (1 ml aqueous volume) of the bilayer apparatus, which was stirred via a magnetic “flea”. Ionic currents, indicating channel activity in the bilayer, were typically detected within 15-30 min. After channels were detected the holding potential across the bilayer was varied between ⁇ 100 mV and +100 mV to characterise the size and polarity of current flow and enable the reversal potential to be determined.
  • TFE 2,2,2-trifluorethanol
  • FIG. 9 shows examples of raw current data for the 229E E ion channel at various holding potentials (cis relative to trans) in asymmetrical KCl solutions (500/50 mM).
  • the graph is a representative plot of average bilayer current (pA; y-axis) versus holding potential (mV; x-axis).
  • Compound stock solutions were typically prepared at 500 mM in DMSO. This solution was further diluted to 50 mM, or lower concentration in 50% DMSO/50% methanol and 2 ⁇ l of the appropriately diluted compound was added to the cis and/or trans chambers to yield the desired final concentration.
  • a bio-asssy of 229E-CoV E-protein function in bacterial cells was developed.
  • a synthetic eDNA fragment encoding 229E-CoV E-protein was cloned into the expression plasmid pPL451, creating a vector in which E protein expression is temperature inducible, as described in Example 4.
  • Inhibition of the growth of E. coli cells expressing E protein at 37° C. was observed as an indicator of p7 ion channel function dissipating the normal Na+ gradient maintained by the bacterial cells.
  • Table 9 list the scores for inhibition of 229E-CoV E-protein in the bacterial bio-assay.
  • the infective inoculum was removed and replaced with fresh medium (DMEM supplemented with 10% fetal calf serum) containing various test concentrations of compounds or the appropriate level of solvent used for the compounds (control). Plates were subsequently incubated at 35° C. (in 5% CO 2 ) for 3-5 days post infection, after which time culture supernatant was removed and the cells were stained with 0.1% crystal violet solution in 20% ethanol for 10 minutes. Plaques were counted in all wells and the percentage reduction in plaque number compared to solvent control was calculated. Measurements were performed in duplicate to quadruplicate wells.
  • an ELISA assay was developed measuring the release of the viral N-protein into culture supernatants from monolayers of OC43-infected MRC-5 cells (human lung fibroblasts; ATCC CCL-171): First, a virus working stock was prepared by amplification in MRC-5 cells. This was then used to infect confluent monolayers of MRC-5 cells grown in 6-well tissue culture plates by exposure to the virus at an MOI of approx. 0.01 pfu/cell for 1 hour at 35° C. in 5% CO 2 .
  • the infective inoculum was removed and replaced with fresh medium (DMEM supplemented with 10% fetal calf serum) containing various test concentrations of compounds or the appropriate level of solvent used for the compounds (control). Plates were subsequently incubated at 35° C. (in 5% CO 2 ) for 5 days post infection, after which time cuitare supernatant was harvested and cellular debris removed by centrifugation at 5000 ⁇ g for 10 minutes. For N-antigen deteetion, 100 ⁇ l samples of clarified culture supernatant were added to duplicate wells of a 96-well Maxi-Sorb plate; 100 ⁇ l of RIPA baffer was added per well with mixing and the plate was covered and incubated at 4° C.
  • DMEM fetal calf serum
  • a peptide eonresponding to the full-length MHV-A59 E protein (sequence: MFNLFLTDTVWYVGQIIFIFAVCLMVTIIVVAFLASIKLCIQLCGLCNTL VLSPSIYLYDRSKQLYKYYNEEMRLPLLEVDDI; accession number NP — 068673) was synthesized manually using FMOC chemistry and solid phase peptide synthesis The synthesis was done at the Biomolecular Resource Facility (John Curtin School of Medical Research, ANU, Australia) using a Symphony R Peptide Synthesiser from Protein Technologies Inc.
  • the crude synthetic peptide was purified using the ProteoPlusTM kit (Qbiogene inc. CA), following manufactures instructions. Briefly, the peptides were diluted in loading buffer (60 mM Tris-HCl pH 8.3, 6M urea, 5% SDS, 10% glycerol, 0.2% Bromophenol blue, ⁇ 100 mM, ⁇ -mercaptoethanol) and run on 4-20 % gradient polyacrylamide gels (Gradipore, NSW, Australia) in tris-glycine electrophoresis buffer (25 mM Tris, 250 mM glycine, 0.1 % SDS). The peptides were stained with gel code blue (Promega, NSW) and the bands corresponding to the full-length peptide were excised out of the gel.
  • loading buffer 60 mM Tris-HCl pH 8.3, 6M urea, 5% SDS, 10% glycerol, 0.2% Bromophenol blue, ⁇ 100 mM, ⁇ -mercapto
  • the gel slice was transferred to the ProteoPLUSTM tube and filled with tris-glycine electrophoresis buffer.
  • the tubes were emerged in tris-glycine electrophoresis buffer and subjected to 100 volts for approximately 1 hour.
  • the polarity of the electric current was reversed for 1 minute to increase the amount of protein recovered.
  • the peptides were harvested and centrifuged at 13,000 rpm for 1 minute.
  • the purified peptides were dried in a Speedvac and the weight of the final product was used to calculate the yield.
  • Lipid bilayer studies were performed as described elsewhere (Sunstrom, 1996; Miller, 1936).
  • a lipid mixture of palmitoyl-oleoyl-phosphatidylethanolamine, palmitoyl-oleoyl-phosphatidylserine and palmitoyl-oleoyl-phosphatidylcholine (5:3:2) (Avanti Polar Lipids, Alabaster, Ala.) was used.
  • the lipid mixture was painted onto an aperture of 150-200 ⁇ m in the wall of a 1 ml delrin cup. The aperture separates two chambers, cis and trans, both containing salt solutions at different concentrations.
  • the cis chamber was connected to ground and the trans chamber to the input of an Axopatch 200 amplifier. Normally the cis chamber contained either 500 mM MaCl or 500 mM KCl and the trans 50 mM NaCl or 50 mM KCl.
  • the bilayer formation was monitored electrically by the amplitude of the current pulse generated by a current ramp. The potentials were measured in the trans chamber with respect to the cis.
  • the synthetic peptide was added to the cis chamber and stirred until channel activity was seen. The currents were filtered at 1000 Hz, digitized at 5000 Hz and stored on magnetic disk.
  • the MHV E synthetic peptide was dissolved in 2,2,2-trifluoroethanol (TFE) at 0.05 mg/ml to 1 mg/ml. 10 ⁇ l of this was added to the cis chamber (1 ml aqueous volume) of the bilayer apparatus, which was stirred via a magnetic “flea”. Ionic currents, indicating channel activity in the bilayer, were typically detected within 15-30 min. After channels were detected the holding potential across the bilayer was varied between ⁇ 100 mV and +100 mV to characterise the size and polarity of current flow and enable the reversal potential to be determined.
  • TFE 2,2,2-trifluoroethanol
  • FIG. 11 shows examples of raw current data for the MHV E ion channel at various holding potentials (cis relative to trans) in asymmetrical NaCl solutions (500/50 mM).
  • the graph is a representative plot of average bilayer current (pA; y-axis) versus holding potential (mV; x-axis).
  • Compound stock solutions were typically prepared at 500 mM in DMSO. This solution was further dilated to 50 mM, or lower ooncenrration in 50% DMSO/50% methanol and 2 ⁇ l of the appropriately diluted compound was added to the cis and/or trans chambers to yield the desired final concentration.
  • a bio-assay of MHV E-protein function in bacterial cells was developed.
  • a synthetic oDNA fragment encoding MHV E-protein was cloned into the expression plasmid pPL451, creating a vector in which E protein expression is temperature inducible, as described in Example 4.
  • Inhibition of the growth of E. coli cells expressing E protein at 37° C. was observed as as indicator of p7 ion channel function dissipating the normal Na+ gradient maintained by the bacterial cells.
  • Table 12 lists the scores for inhibition of MHV E protein in the bacterial bio-assay.
  • the infective inoculum was removed and replaced with fresh medium (DMEM supplemented with 10% horse serum) containing various test coneentration of compounds or the appropriate level of solvent used for the compounds (control). Plates were subsequently incubated at 37° C. (in 5% CO 2 ) for 16-24 hours post infection, after which time culture snpematant was removed and the cells were stained with 0.1% crystal violet solution in 20% ethanol for 10 minutes. Plaques were counted in all wells and the percentage reduction in plaque number compared to solvent control was calculated. Measurements were performed in duplicate to quadruplicate wells.
  • an assay measuring reduction in the number of plaques formed in monolayers of PRCV infected ST cells was developed: Confluent ST cells in 6 well plates were infected with a quaternary passage of porcine respiratory virus (PRCV) strain AR310 at three dilations 10 ⁇ 1 , 50 ⁇ 1 and 10 ⁇ 2 in PBS to provide a range of plaques numbers to coount. 100 ⁇ l of diluted virus was added per well in a volume of 1 ml of media.
  • PRCV porcine respiratory virus
  • the viral supernatant was removed and 2 ml/well of overlay containing 1% Sealiaque agarose in 1 ⁇ MEM, 5 % FCS was added to each well.
  • Compounds to be tested were added to the overlay mixture by diluting the compounds ftom a 0.5M frozen stock to a concentration so that the same volume of compound/solvent would be added to the overlay for each concentration of compound.
  • the volume of componnd/solvent never exceeded 0.07% of the volume of the overlay.
  • the solvent used to dissolve compounds was DMSO and methanol mixed in equal proportions.
  • Compounds were tested for anti-plaque forming activity at four concentrations, 0.1 uM, 1 uM, 10 uM and 20 uM. Quadruplicates were performed at each concentration.
  • Controls were performed where the same volume of solvent was added to the overlay.
  • the overlay was allowed to set at room temp for 20 mins.
  • the plates were then incubated at 37° C. for 7 days.
  • the monolayers were then fixed and stained by adding 1 ml/well of 0.5% methylene blue, 4% formaldehyde.
  • HCV hepatitis C virus
  • Lipid bilayer studies were performed as described elsewhere (Miller, 1986).
  • a lipid mixture of palmitoyl-oleoyl-phosphatidylethanolamine, palmitoyl-oleoyl-phosphatidylserine and palmitoyl-oleoyl-phoshatidylcholine (5:3:2) (Avanti Polar Lipids, Alabaster, Ala.) was used.
  • the lipid mixture was painted onto an aperture of 150-200 um in the wall of a 1 ml delrin cup.
  • the aperture separates two chambers, cis and trans, both containing salt solutions at different concentrations.
  • the cis chamber was connected to ground and the trans chamber to the input of an Axopatch 200 amplifier.
  • the cis chamber contained 500 mM KCl and the trans 50 mM KCl.
  • the bilayer formation was monitored electrically by the amplitude of the current pulse generated by a current ramp. The potentials were measured in the trans chamber with respect to the cis.
  • the protein was added to the cis chamber and stirred until channel activity was seen. The currents were filtered at 1000 Hz, digitized at 2000 Hz and stored on magnetic disk.
  • the P7 peptide was dissolved in 2,2,2-trifluorethanol (TFE) at 10 mg/ml. 10 ul of this was added to the cis chamber of the bilayer which was stirred. Channel activity was seen within 15-20 min.
  • TFE 2,2,2-trifluorethanol
  • the channels formed by the P7 peptide were blocked by 5-(N,N-hexamethylene) amiloride (HMA).
  • cDp7.coli cDp7.coli
  • cDp7.mam codons for expression in E. coli
  • cDp7.mam codons ware biased for expression in mammalian cell lines.
  • cDp7.coli was cloned into the plasmid pPL451 as a BamHI/EcoRI fragment for expression in E. coli .
  • cDp7.mam was cltoned into vectors (for example, pcDNA3. vaccinia virus, pfastBac-1) for expression of p7 in mammalian cell lines.
  • VLP virus-like particles
  • pcDNAGag HIV-1 Gag protein expressed under control of the T7 promoter
  • Example 33 The two methods of detecting p7 ion channel functional activity, described in Examples 33-35, were employed to assay the ability of compounds to inhibit the p7 channel.
  • compounds were tested for their ability to inhibit p7 channel activity in planar lipid bilayers.
  • compounds were tested for their ability to reduce the number of VLPs released from cells expressing both p7 and HIV-1 Gag.
  • HCV p7 Ion Channel Inhibits Bacterial Cell Growth
  • a bio-assay of p7 function in bacterial cells was developed.
  • the p7-encoding synthetic cDNA fragment cDp7.coli was cloned into the expression plasmid pPL451, creating the vector pPLp7, in which p7 expression is temperature inducible, as described in Example 4.
  • Inhibition of the growth of E. coli cells expressing p7 at 37° C. was observed as an indicator of p7 ion channel function dissipating the normal Na+ gradient maintained by the bacterial cells.
  • Table 16 lists the scores for inhibition of HCV p7 protein in the bacterial bio-assay.
  • Equine Arteritis Virus EAV
  • the infective inoculum was removed and nd the cells were overlayed with a 1% sea plaque overlay (Cambrex Bio Science) in MEM containing 10% FCS containing and 10, 5 or 1 ⁇ M of compounds to be tested or the appropriate level of solvent used for the compounds (control). Plates were subsequently incubated at 37° C. (in 5% CO 2 ) for 3 days post infection, after which time culture supernatant was removed and the cells were stained with 0.1% crystal violet solution in 20% ethanol for 10 minutes. Plaques were counted in all wells and the percentage reduction in plaque number compared to solvent control was calculated. Measurements were performed in duplicate to quadruplicate wells.
  • the C-terminal 40 amino acids of the M protein of the Dengue virus type 1 strain Singapore S275/90 (Fu et al 1992) (ALRHPGFTVIALFLAHAIGTSITOKGIIFILLMLVTPSMA) was synthesised using the Fmoc method. The synthesis was done on a Symphony Peptide Synthesiser form Protein Technologies Inc (Tucson, Ariz.) as used to give C-terminal amides, the coupling was done with HBTU and hydroxybenzotriazole in N-methylpyrolidone. Each of the synthesis cycle used double coupling and a 4-fold excess of the amino acids. Temporary ⁇ -N Fmoc-protecting groups were removed using 20% piperidine in DMF.
  • Lipid bilayer studies were performed as described elsewhere (Sunstrom, 1996; Miller, 1986).
  • a lipid mixture of palmiutoyl-oleoyl-phosphatidylethanolamine, palmitoyl-oleoyl-phosphatidylscrine and palmitoyl-oleoyl-phosphatidylcholine (5:3:2) (Avanti Polar Lipids, Alabaster, Ala.) was used.
  • the lipid mixture was painted onto an aperture of 150-200 ⁇ m in the wall of a 1 ml delrin cup. The aperture separates two chambers, sis and trans, both containing salt solutions at different concentrations.
  • the cis chamber was connected to ground and the trans chamber to the input of an Axopatch 200 amplifier. Normally the cis chamber contained 500 mM KCl and the trans 50 mM KCl. The bilayer formation was monitored electrically by the amplitude of the current pulse generated by a current ramp. The potentials were measured in the trans chamber with respect to the cis. The protein was added to the cis chamber and stirred until channel activity was seen. The currents were filtered at 1000 Hz, digitized at 5000 Hz and stored on magnetic disk.
  • the dengue virus M protein C-terminal peptide (DMVC) was dissolved in 2,2,2-trifluorethanol (TFE) at 0.05 mg/ml to 1 mg/ml. 10 ⁇ l of this was added to the cis chamber of the bilayer which was stirred. Channel activity was seen within 15-30 min.
  • HMA Hexamethylene Amiloride
  • Solutions of 50 mM HMA were prepared by first making a 500 mM solution in DMSO. This solution was further diluted to 50 mM HMA using 0.1 M HCl. 2 ⁇ l of the 50 mM HMA was added to the cis chamber after channel activity was seen. The cis chamber contained 1 ml of solution making the final concentration of HMA 100 ⁇ M.
  • the cultures were allowed to grow for 7 days and then Alamar Blue, a fluorescent dye that measures the metabolism of the cultures (red/ox), was added to each culture and the fluorescence value for each culture was measured.
  • the negative control without experimental compound or virus was fixed at 100%.
  • the positive controls and the cultures with compound were scored by calculating their average fluorescence as a percentage of the negative control. At least six replicate wells were measured for each experimental condition.
  • the p24-antigen data for twelve compounds representing various substituted acyl-guanidines was compared with the activity scores obtained for those compounds in the Vpu bacterial assay.
  • the data from each assay was initially rank ordered for effectiveness.
  • the rank order for the Vpu bacterial assay was determined from all activity scores, the highest score indicating the greatest effectiveness.
  • the rank order for the anti-HTV-1 assay was determined based on the overall average value of p24 antigen measured in culture supernatants at all of the drug concentration tested, with the lowest score indicating the greatest effectiveness.
  • the two rank orders generated were then compared statistically by generating the Spearman's Rank correlation coefficient.
  • the bacterial assay may therefore be a useful tool in screening for compounds that exhibit anti-viral activity.
  • Bacterial assay p24 Compound rank order rank order di ⁇ circumflex over ( ) ⁇ 2 (3-bromocinnamoyl)guanidine 1 1 0 3-(trifluoromethyl)cinnamoylguanidine 2 2 0 3-methylcinnamoylguanidine 3 3 0 cinnamoylguanidine 4 4 0 trans-3-(1-napthyl)acryloylguanidine 5.5 7 2.25 6-methoxy-2-naphthoylguanidine 5.5 5 0.25 4-phenylbenzoylguanidine 7 11 16 (5-phenyl-penta-2,4-dienoyl)guanidine 8 9 1 N-(3-phenylpropanoyl)-N′- 9 12 9 phenylguanidine Hexamethylene am
  • MHV plaque reduction activity data for 96 compounds screened were sorted from greatest to least percent plaque reduction and rank orders were assigned to the list of compounds. This was performed for the data generated by exposure to both 10 ⁇ M and 1 ⁇ M concentrations of the compounds, giving rise to two rank order lists. Similarly, a rank order list was generated for the MHVE bacterial bioassay scores for the same 96 compounds. Where one or more compounds had the same score, the rank values for that group were averaged.
  • This table summarises the Rs and P values generated as a result of the indicated pairwise comparisons between rank orders.
  • the rank order comparison of 96 compounds assayed in the bacterial bio-assay and the antiviral assay show that MHVE bacterial assay rank order for the compounds tested is significantly positively correlated with the rank orders generated by the MHV plaque reduction assay.
  • the significant correlation between the assays is highly indicative that either assay may be utilised to identify compounds that may be useful.
  • the bacterial assay may thereby be a useful tool in screening for compounds that exhibit anti-viral activity.
  • 229E plaque reduction activity data for 97 compounds screened against 2.5 ⁇ M compound concentration were sorted from greatest to least percent plaque reduction and rank orders were assigned to the list of compounds.
  • a rank order list was generated for the 229E E bacterial bioassay scores for the same 97 compounds. Where one or more compounds had the same scores the rank values for that group were averaged.
  • This table summarises the Rs and P values generated as a result of the indicated pairwise comparisons between rank orders.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • Medicinal Chemistry (AREA)
  • Epidemiology (AREA)
  • Virology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Oncology (AREA)
  • Communicable Diseases (AREA)
  • Molecular Biology (AREA)
  • Pulmonology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • AIDS & HIV (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
  • Compositions Of Macromolecular Compounds (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Furan Compounds (AREA)
  • Agricultural Chemicals And Associated Chemicals (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Heterocyclic Compounds That Contain Two Or More Ring Oxygen Atoms (AREA)
  • Quinoline Compounds (AREA)
  • Pyridine Compounds (AREA)
  • Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
US13/553,239 2003-06-26 2012-07-19 Antiviral compounds and methods Abandoned US20130035328A1 (en)

Priority Applications (4)

Application Number Priority Date Filing Date Title
US13/553,239 US20130035328A1 (en) 2003-06-26 2012-07-19 Antiviral compounds and methods
US14/615,616 US20150313909A1 (en) 2003-06-26 2015-02-06 Antiviral compounds and methods
US15/602,526 US10472332B2 (en) 2003-06-26 2017-05-23 Antiviral compounds and methods
US16/554,990 US11192863B2 (en) 2003-06-26 2019-08-29 Antiviral compounds and methods

Applications Claiming Priority (11)

Application Number Priority Date Filing Date Title
AU2003903251A AU2003903251A0 (en) 2003-06-26 2003-06-26 Antiviral compounds and methods
AU2003903251 2003-06-26
AU2003903850 2003-07-25
AU2003903850A AU2003903850A0 (en) 2003-07-25 2003-07-25 Anti-coronavirus compounds and methods
AU2003904692 2003-08-29
AU2003904692A AU2003904692A0 (en) 2003-08-29 Anti-flavivirus compounds and methods
AU2004902902 2004-05-31
AU2004902902A AU2004902902A0 (en) 2004-05-31 Anti-flavivirus compounds and methods
PCT/AU2004/000866 WO2004112687A2 (fr) 2003-06-26 2004-06-26 Compositions et methodes antivirales
US56229605A 2005-12-22 2005-12-22
US13/553,239 US20130035328A1 (en) 2003-06-26 2012-07-19 Antiviral compounds and methods

Related Parent Applications (3)

Application Number Title Priority Date Filing Date
US10/562,296 Continuation US20070099968A1 (en) 2004-06-26 2004-06-24 Antiviral compounds and methods
PCT/AU2004/000866 Continuation WO2004112687A2 (fr) 2003-06-26 2004-06-26 Compositions et methodes antivirales
US56229605A Continuation 2003-06-26 2005-12-22

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US14/615,616 Continuation US20150313909A1 (en) 2003-06-26 2015-02-06 Antiviral compounds and methods

Publications (1)

Publication Number Publication Date
US20130035328A1 true US20130035328A1 (en) 2013-02-07

Family

ID=33545379

Family Applications (4)

Application Number Title Priority Date Filing Date
US13/553,239 Abandoned US20130035328A1 (en) 2003-06-26 2012-07-19 Antiviral compounds and methods
US14/615,616 Abandoned US20150313909A1 (en) 2003-06-26 2015-02-06 Antiviral compounds and methods
US15/602,526 Expired - Lifetime US10472332B2 (en) 2003-06-26 2017-05-23 Antiviral compounds and methods
US16/554,990 Expired - Lifetime US11192863B2 (en) 2003-06-26 2019-08-29 Antiviral compounds and methods

Family Applications After (3)

Application Number Title Priority Date Filing Date
US14/615,616 Abandoned US20150313909A1 (en) 2003-06-26 2015-02-06 Antiviral compounds and methods
US15/602,526 Expired - Lifetime US10472332B2 (en) 2003-06-26 2017-05-23 Antiviral compounds and methods
US16/554,990 Expired - Lifetime US11192863B2 (en) 2003-06-26 2019-08-29 Antiviral compounds and methods

Country Status (8)

Country Link
US (4) US20130035328A1 (fr)
EP (2) EP2617709B8 (fr)
JP (1) JP5030587B2 (fr)
KR (1) KR101153254B1 (fr)
BR (1) BRPI0411900B8 (fr)
CA (1) CA2529949C (fr)
NZ (1) NZ544671A (fr)
WO (1) WO2004112687A2 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9440926B2 (en) 2005-06-24 2016-09-13 Biotron Limited Antiviral compounds and methods

Families Citing this family (45)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2617709B8 (fr) 2003-06-26 2022-12-21 Biotron Limited Dérivés de guanidine comme agents antiviraux
US7745442B2 (en) * 2003-08-20 2010-06-29 Parion Sciences, Inc. Methods of reducing risk of infection from pathogens
AU2013205388B2 (en) * 2005-06-24 2016-05-05 Biotron Limited Antiviral compounds and methods
GB0526244D0 (en) 2005-12-22 2006-02-01 Novartis Ag Organic compounds
GB0526240D0 (en) * 2005-12-22 2006-02-01 Novartis Ag Organic compounds
AU2013219202B2 (en) * 2007-08-03 2016-12-08 Biotron Limited Hepatitis C antiviral compositions and methods
AU2008286240B2 (en) * 2007-08-03 2013-05-23 Biotron Limited Hepatitis C antiviral compositions and methods
US20110217265A1 (en) * 2008-09-23 2011-09-08 Glenn Jeffrey S Screening for Inhibitors of HCV Amphipathic Helix (AH) Function
UA111163C2 (uk) 2010-07-22 2016-04-11 Гайлід Сайєнсіз, Інк. Способи й сполуки для лікування вірусних інфекцій paramyxoviridae
TWI767201B (zh) 2014-10-29 2022-06-11 美商基利科學股份有限公司 絲狀病毒科病毒感染之治療
MA52371A (fr) 2015-09-16 2021-09-22 Gilead Sciences Inc Méthodes de traitement d'infections dues aux coronaviridae
WO2018081863A1 (fr) * 2016-11-04 2018-05-11 University Of Wollongong Dérivés à substitution en position 6 de l'hexaméthylène amiloride en tant qu'inhibiteurs de upa et leurs utilisations
CA3056072C (fr) 2017-03-14 2022-08-23 Gilead Sciences, Inc. Methodes de traitement d'infections par le coronavirus felin
JP2020518578A (ja) 2017-05-01 2020-06-25 ギリアード サイエンシーズ， インコーポレイテッド （Ｓ）−２−エチルブチル２−（（（Ｓ）−（（（２Ｒ，３Ｓ，４Ｒ，５Ｒ）−５−（４−アミノピロロ［２，１−ｆ］［１，２，４］トリアジン−７−イル）−５−シアノ−３，４−ジヒドロキシテトラヒドロフラン−２−イル）メトキシ）（フェノキシ）ホスホリル）アミノ）プロパノエートの結晶形態
ES3000461T3 (en) 2017-07-11 2025-02-28 Gilead Sciences Inc Compositions comprising an rna polymerase inhibitor and cyclodextrin for treating viral infections
JP2023512656A (ja) 2020-01-27 2023-03-28 ギリアード サイエンシーズ， インコーポレイテッド ＳＡＲＳ ＣｏＶ－２感染を治療するための方法
JP2021138619A (ja) * 2020-03-02 2021-09-16 フマキラー株式会社 抗コロナウイルス剤
TWI890963B (zh) 2020-03-12 2025-07-21 美商基利科學股份有限公司 1'-氰基核苷之製備方法
US20230127965A1 (en) * 2020-03-30 2023-04-27 Thomas Jefferson University Methods for treating, ameliorating, or preventing viral infections
AU2021251689B2 (en) 2020-04-06 2024-06-13 Gilead Sciences, Inc. Inhalation formulations of 1'-cyano substituted carbanucleoside analogs
TW202532084A (zh) 2020-05-29 2025-08-16 美商基利科學股份有限公司 瑞德西韋之治療方法
IL299202A (en) 2020-06-24 2023-02-01 Gilead Sciences Inc 1'-cyano nucleoside analogs and uses thereof
WO2022040321A1 (fr) * 2020-08-19 2022-02-24 Natreon, Inc. Protection contre une infection à coronavirus par des extraits et des composants d'extrait
US11773122B2 (en) 2020-08-24 2023-10-03 Gilead Sciences. Inc. Phospholipid compounds and uses thereof
EP3960165A1 (fr) * 2020-08-25 2022-03-02 Fondation EspeRare Inhibiteurs de nhe-1 pour le traitement des infections à coronavirus
PE20231983A1 (es) 2020-08-27 2023-12-12 Gilead Sciences Inc Compuestos y metodos para el tratamiento de infecciones virales
TWI811812B (zh) 2020-10-16 2023-08-11 美商基利科學股份有限公司 磷脂化合物及其用途
EP4346772A1 (fr) 2021-05-26 2024-04-10 Gilead Sciences, Inc. Formulations phospholipidiques d'analogues de carba-nucléoside 1'-cyano-substitué
JP2024528237A (ja) 2021-08-06 2024-07-26 インターベット インターナショナル ベー． フェー． 獣医学的ウイルス性疾患の治療方法
EP4387977A1 (fr) 2021-08-18 2024-06-26 Gilead Sciences, Inc. Composés phospholipidiques et leurs procédés de production et d'utilisation
TWI878811B (zh) 2022-03-02 2025-04-01 美商基利科學股份有限公司 用於治療病毒感染的化合物及方法
CA3244278A1 (fr) 2022-03-02 2023-09-07 Gilead Sciences Inc Composés et méthodes pour le traitement d’infections virales
US20230382940A1 (en) 2022-03-03 2023-11-30 Gilead Sciences, Inc. Antiviral compounds and methods of making and using the same
CA3252494A1 (fr) 2022-03-03 2023-09-07 Gilead Sciences Inc Composés antiviraux et leurs procédés de fabrication et d'utilisation
JPWO2023167055A1 (fr) * 2022-03-03 2023-09-07
EP4536223A1 (fr) 2022-06-06 2025-04-16 Gilead Sciences, Inc. Méthodes de traitement d'infections virales y compris le sars-cov-2
US20240051962A1 (en) 2022-06-29 2024-02-15 Gilead Sciences, Inc. Solid forms of a nucleoside analogue and uses thereof
EP4547665A1 (fr) 2022-06-30 2025-05-07 Gilead Sciences, Inc. Formes solides d'un analogue nucléosidique et leurs utilisations
CN120659609A (zh) * 2022-11-21 2025-09-16 西奥多·亨德森 使用抗病毒化合物和螺内酯进行治疗
US20240309028A1 (en) 2023-02-16 2024-09-19 Gilead Sciences, Inc. Phospholipid compounds and methods of making and using the same
WO2025049699A1 (fr) 2023-08-31 2025-03-06 Gilead Sciences, Inc. Composés antiviraux et leurs procédés de fabrication et d'utilisation
US20250084088A1 (en) 2023-08-31 2025-03-13 Gilead Sciences, Inc. Antiviral compounds and methods of making and using the same
WO2025054278A1 (fr) 2023-09-06 2025-03-13 Gilead Sciences, Inc. Formes solides d'un analogue de nucléoside et leurs utilisations
TW202517647A (zh) 2023-09-28 2025-05-01 美商基利科學股份有限公司 用於治療病毒感染的化合物及方法
US12357577B1 (en) 2024-02-02 2025-07-15 Gilead Sciences, Inc. Pharmaceutical formulations and uses thereof

Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE2531343A1 (de) * 1975-07-14 1977-02-10 Henkel & Cie Gmbh Antimikrobielle mittel
US4496573A (en) * 1982-08-24 1985-01-29 William H. Rorer, Inc. 1-Pyridylmethyl-3-acyl guanidines
DE4301739A1 (de) * 1993-01-22 1994-07-28 Sauer Professor Dr Gerhard Guanidin-Derivate zur Behandlung von Primär-Tumoren und viralen Erkrankungen
US5567734A (en) * 1994-01-25 1996-10-22 Hoechst Aktiengesellschaft Phenyl-substituted alkylcarboguanidides carrying perfluoroalkyl groups, a process for their preparation, their use as a medicament or a diagnostic agent, and a medicament containing them
US5719169A (en) * 1993-07-31 1998-02-17 Hoechst Aktiengesellschaft Substituted benzoylguanidines, their use as a medicament or diagnostic, and medicament containing them
US5733934A (en) * 1995-04-18 1998-03-31 Hoechst Aktiengesellschaft Antiarrythmic and cardioprotective substituted indenoylguanidines
US6011059A (en) * 1997-12-24 2000-01-04 Bristol-Myers Squibb Company Acyl guanidine sodium/proton exchange inhibitors and method
WO2000021538A1 (fr) * 1998-10-12 2000-04-20 The Australian National University Procede de modulation de l'activite fonctionnelle du canal ionique
US7041702B1 (en) * 1997-10-21 2006-05-09 Scion Pharmaceuticals, Inc. Pharmaceutically active compounds and methods of use

Family Cites Families (30)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US2734904A (en) * 1956-02-14 Xcxnhxc-nh
BE639386A (fr) 1962-10-30
FR1435379A (fr) * 1964-07-30 1966-04-15 American Cyanamid Co Procédé pour stabiliser le chlorure de polyvinyle rigide contre les effets de la lumière
US3527758A (en) * 1967-04-13 1970-09-08 Merck & Co Inc Process for the preparation of pyrazinoylguanidines from a pyrazinoic azide and a guanidine
DE2006186A1 (de) 1970-02-11 1971-08-19 Huebner Vamag Verfahren zum Einsetzen eines Sitz ringes fur den Absperrkorper einer Armatur
DD200618A1 (de) * 1981-10-05 1983-05-25 Manfred Augustin Verfahren zur herstellung von neuen n-aroyl-und hetaroylimiden
US4894376A (en) * 1988-02-26 1990-01-16 Trustees Of The University Of Pennsylvania Methods of treating diseases characterized by hyperexcitability of neurons
JPH0789859A (ja) * 1990-04-05 1995-04-04 Celtrix Pharmaceut Inc ピラジン誘導体を含む薬剤学的組成物と眼の新血管新生を阻止する治療法
IN177137B (fr) * 1992-12-11 1996-11-16 Hoechst India
JPH0725768A (ja) * 1993-07-09 1995-01-27 Mitsubishi Chem Corp 血管内膜肥厚抑制剤
DE4415873A1 (de) * 1994-05-05 1995-11-09 Hoechst Ag Substituierte bizyklische Heteroaroylguanidine, Verfahren zu ihrer Herstellung, ihre Verwendung als Medikament oder Diagnostikum sowie sie enthaltendes Medikament
DE4421536A1 (de) * 1994-06-20 1995-12-21 Hoechst Ag Perfluoralkylgruppen tragende phenylsubstituierte Alkenylcarbonsäure-guanidine, Verfahren zu ihrer Herstellung, ihre Verwendung als Medikament oder Diagnostikum sowie sie enthaltendes Medikament
JPH08225513A (ja) * 1994-12-21 1996-09-03 Kanebo Ltd ナフトイルグアニジン誘導体
AT408330B (de) 1995-01-27 2001-10-25 Colop Stempelerzeugung Skopek Selbstfärbestempel
DE19518796A1 (de) * 1995-05-22 1996-11-28 Hoechst Ag Fluorphenylsubstituierte Alkenylcarbonsäure-guanidine, Verfahren zu ihrer Herstellung, ihre Verwendung als Medikament oder Diagnostikum sowie sie enthaltendes Medikament
DE19527305A1 (de) * 1995-07-26 1997-01-30 Hoechst Ag Substituierte Zimtsäureguanidide, Verfahren zu ihrer Herstellung, ihre Verwendung als Medikament oder Diagnostikum sowie sie enthaltendes Medikament
US5628984A (en) * 1995-07-31 1997-05-13 University Of North Carolina At Chapel Hill Method of detecting lung disease
JPH0967332A (ja) * 1995-08-29 1997-03-11 Yamanouchi Pharmaceut Co Ltd Ｎ−（２−プロペノイル）グアニジン誘導体
DE19621482A1 (de) * 1996-05-29 1997-12-04 Hoechst Ag Substituierte 1-Naphthoylguanidine, Verfahren zu ihrer Herstellung, ihre Verwendung als Medikament oder Diagnostikum sowie sie enthaltendes Medikament
DE19622222A1 (de) * 1996-06-03 1997-12-04 Hoechst Ag Verwendung von Inhibitoren des zellulären Na·+·/H·+·-Exchangers (NHE) zur Herstellung eines Medikament zur Normalisierung der Serumlipide
AUPO545297A0 (en) * 1997-03-04 1997-03-27 Fujisawa Pharmaceutical Co., Ltd. Guanidine derivatives
IL126276A0 (en) * 1997-09-24 1999-05-09 Hoechst Marion Roussel De Gmbh The use of an inhibitor of the na+/H+ exchanger for the production of a medicament for the treatment or prophylaxis of disorders of the central nervous system
DK1032556T3 (da) * 1997-10-21 2008-03-25 Wyeth Corp Farmaceutisk aktive forbindelser og anvendelsesfremgangsmåder
CN1285001A (zh) * 1997-12-18 2001-02-21 塞普拉科有限公司 同时鉴定新型生物靶和用于药物开发的引导结构的方法
DE19849722A1 (de) * 1998-10-28 2000-05-04 Aventis Pharma Gmbh Substituierte Phenyl-alkenoylguanidine, Verfahren zu ihrer Herstellung, ihre Verwendung als Medikament oder Diagnostikum sowie sie enthaltendes Medikament
DE19859727A1 (de) * 1998-12-23 2000-06-29 Aventis Pharma Gmbh Die Verwendung von Hemmern des Natrium-Wasserstoff-Austauschers zur Herstellung eines Medikaments zur Verhinderung von altersbedingten Organ-Dysfunktionen, altersbedingten Erkrankungen zur Lebensverlängerung
AUPQ223999A0 (en) * 1999-08-16 1999-09-09 University Of Sydney, The Intracellular feedback controls in the diagnosis and treatment of human disease
AUPS022802A0 (en) * 2002-01-31 2002-02-21 Macfarlane Burnet Institute For Medical Research And Public Health Limited, The Anti-viral compounds
EP2617709B8 (fr) 2003-06-26 2022-12-21 Biotron Limited Dérivés de guanidine comme agents antiviraux
NZ610715A (en) * 2005-06-24 2014-07-25 Biotron Ltd Antiviral compounds and methods

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE2531343A1 (de) * 1975-07-14 1977-02-10 Henkel & Cie Gmbh Antimikrobielle mittel
US4496573A (en) * 1982-08-24 1985-01-29 William H. Rorer, Inc. 1-Pyridylmethyl-3-acyl guanidines
DE4301739A1 (de) * 1993-01-22 1994-07-28 Sauer Professor Dr Gerhard Guanidin-Derivate zur Behandlung von Primär-Tumoren und viralen Erkrankungen
US5719169A (en) * 1993-07-31 1998-02-17 Hoechst Aktiengesellschaft Substituted benzoylguanidines, their use as a medicament or diagnostic, and medicament containing them
US5567734A (en) * 1994-01-25 1996-10-22 Hoechst Aktiengesellschaft Phenyl-substituted alkylcarboguanidides carrying perfluoroalkyl groups, a process for their preparation, their use as a medicament or a diagnostic agent, and a medicament containing them
US5733934A (en) * 1995-04-18 1998-03-31 Hoechst Aktiengesellschaft Antiarrythmic and cardioprotective substituted indenoylguanidines
US7041702B1 (en) * 1997-10-21 2006-05-09 Scion Pharmaceuticals, Inc. Pharmaceutically active compounds and methods of use
US6011059A (en) * 1997-12-24 2000-01-04 Bristol-Myers Squibb Company Acyl guanidine sodium/proton exchange inhibitors and method
WO2000021538A1 (fr) * 1998-10-12 2000-04-20 The Australian National University Procede de modulation de l'activite fonctionnelle du canal ionique

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
Iwanowicz et al. Bioorganic & Medicinal Chemistry Letters 12 (2002) 2931-2934. *
Lamb et al. Virology (1997), 229(1), 1-11 *
Lamb et al. Virology (1997), 229(1), 1-11. *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9440926B2 (en) 2005-06-24 2016-09-13 Biotron Limited Antiviral compounds and methods
US10683263B2 (en) 2005-06-24 2020-06-16 Biotron Limited Antiviral compounds and methods

Also Published As

Publication number Publication date
EP1646371A4 (fr) 2010-09-22
US20170260147A1 (en) 2017-09-14
CA2529949A1 (fr) 2004-12-29
CA2529949C (fr) 2013-08-06
EP1646371A2 (fr) 2006-04-19
KR20060103086A (ko) 2006-09-28
KR101153254B1 (ko) 2012-07-02
WO2004112687A2 (fr) 2004-12-29
US11192863B2 (en) 2021-12-07
JP2007508234A (ja) 2007-04-05
EP2617709B8 (fr) 2022-12-21
US20150313909A1 (en) 2015-11-05
NZ544671A (en) 2009-02-28
US10472332B2 (en) 2019-11-12
BRPI0411900A (pt) 2006-09-19
JP5030587B2 (ja) 2012-09-19
BRPI0411900B8 (pt) 2021-05-25
BRPI0411900B1 (pt) 2018-11-21
US20190389816A1 (en) 2019-12-26
EP2617709A1 (fr) 2013-07-24
WO2004112687A3 (fr) 2007-07-12
EP2617709B1 (fr) 2022-11-16

Similar Documents

Publication Publication Date Title
US11192863B2 (en) Antiviral compounds and methods
US7256005B2 (en) Methods for identifying iminosugar derivatives that inhibit HCV p7 ion channel activity
JP2010202674A (ja) 病原体感染を処置するためのチロシンキナーゼインヒビターの組成物および使用
WO1997028802A1 (fr) Inhibition de la replication virale
US20120064062A1 (en) Inhibitors of bacterial plasminogen activators
KR20230063347A (ko) 항바이러스성 펩토이드 조성물
US20210060002A1 (en) Methods of preventing or treating flavivirus virus infections and methods of inhibiting the entry of flvivirus, enterovirus or lentivirus into host cells
US20070099968A1 (en) Antiviral compounds and methods
AU2004248859C1 (en) Antiviral acylguanidine compounds and methods
CN101111475A (zh) 抗病毒化合物及方法
HK1187600A (en) Guanidine derivatives as antiviral agents
US11376306B2 (en) Peptides and uses therefor as antiviral agents
JP4624562B2 (ja) イオンチャネル機能活性の調節方法
KR20050092103A (ko) 이온 채널 활성을 억제하는 이미노슈가 유도체의 용도
US20230190769A1 (en) E protein channel blockers and orf3 inhibitors as anti-covid-19 agents
US20230390235A1 (en) Therapeutic agent for covid-19
EP0684818B1 (fr) Procede d&#39;inversion de la resistance de souches d&#39;hiv-1 a la zidovudine
WO2011108785A1 (fr) Composé peptidique possédant une activité inhibant l&#39;infection ou la prolifération d&#39;orientia tsutsugamushi, et composition pharmaceutique utilisant ce composé pour soulager le typhus des broussailles
WO2018189566A1 (fr) Utilisation d&#39;un composé chimique en tant qu&#39;agent thérapeutique
EP0879055A1 (fr) Inhibition de la replication virale

Legal Events

Date Code Title Description
STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION