Note: Descriptions are shown in the official language in which they were submitted.
WO 2021/220284
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E PROTEIN CHANNEL BLOCKERS AND ORF3 INHIBITORS AS ANTI-COVID-
19 AGENTS
CROSS REFERENCE TO RELATED APPLICATIONS
[001] The present application claims the benefit of priority of U.S.
Provisional Patent
Application No. 63/018,598, titled "E PROTEIN CHANNEL BLOCKERS AS ANTI-
COVID-19 AGENTS", filed May 1, 2020, and of U.S. Provisional Patent
Application No.
63/117,619, titled "E PROTEIN CHANNEL BLOCKERS AND ORF3 INHIBITORS AS
ANTI-COVID-19 AGENTS", filed November 24, 2020, the contents of both are
incorporated herein by reference in their entirety.
FIELD OF INVENTION
[002] The present invention is in the field of anti-viral therapy.
BACKGROUND
[003] Coronaviruses are positive-sense, single-stranded RNA viruses that are
often
associated with mild respiratory tract infections in humans. However, three
members of the
family have received notoriety due to their abnormal virulence: SARS-CoV-1 was
the
etiological agent of the SARS epidemic in the winter of 2002/3 that caused 774
deaths
amongst 8,098 cases; MERS-CoV was responsible for the MERS epidemic that
started from
2012 with 862 deaths from 2506 infections; Finally, SARS-CoV-2 is responsible
for the
ongoing COVID-2019 pandemic resulting in 1.31 million deaths out of 54,068,330
cases (as
of Sun Nov 15, 2020.
[004] Genomic analyses have indicated that SARS-CoV-1 and SARS-CoV-2 are very
similar to one another (ca. 80%) but are distinct from most other
Coronaviridae members
that infect humans. Both viruses have been placed in subgroup B in the
Betacoronavinis
genus within the Orthocoronavirinae subfamily of the Coronaviridae.
[005] Of all coronavirus' structural proteins, E is the least understood in
terms of
mechanism of action and structure. Functionally, the E protein has been
implicated in viral
assembly, release, and pathogenesis. Yet crucially, coronavirus E proteins are
important for
viral pathogenesis, and attenuated viruses lacking the protein have even been
suggested to
serve as vaccine candidates.
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[006] SARS-CoV-2 3a protein, also known as open reading frame 3a (ORF3a), is
implicated in assembly of homotetrameric potassium sensitive ion channels
(viroporin) and
may modulate virus release. Additionally, it is implicated in pathogenesis,
including up-
regulation of expression of fibrinogen subunits FGA. FGB and FGG in host lung
epithelial
cells, inducement of apoptosis in cell culture.
SUMMARY
[007] According to a first aspect, there is provided a method for treating or
preventing
SARS-CoV-2 virulence in a subject in need thereof, comprising administering to
the subject
a therapeutically effective amount of any one of: SARS-CoV-2 E protein channel
blocker
and a SARS-CoV-2 3a protein inhibitor, thereby treating or preventing SARS-CoV-
2
virulence in the subject.
[008] According to another aspect, there is provided a pharmaceutical
composition
comprising a S ARS-CoV-2 E protein channel blocker and/or SARS-CoV-2 3a
protein
inhibitor for use in the treatment or prevention of SARS-CoV-2 virulence in a
subject in
need thereof.
[009] In some embodiments, preventing comprises preventing any one of: SARS-
CoV-2
entry to a cell of the subject, uncoating of the SARS-CoV-2 in a cell of the
subject, release
of the SARS-CoV-2 from a cell of the subject, and any combination thereof.
[010] In some embodiments, the subject is infected or suspected of being
infected by
SARS-CoV-2.
[011] In some embodiments, the SARS-CoV-2 E protein channel blocker is at
least one
molecule selected from the group consisting of: 5-Azacytidine, Memantine,
Gliclazide,
Mavorixafor, Saroglitazar Magnesium, Mebrofenin, Cyclen, Kasugamycin,
Plerixafor, and
any salt thereof.
[012] In some embodiments, the SARS-CoV-2 E protein channel blocker is for use
at a
daily dose of 0.01 to 500 mg/kg.
[013] In some embodiments, the SARS-CoV-2 E protein channel blocker is
Ginsenoside.
[014] In some embodiments, the SARS-CoV-2 E protein channel blocker is
Memantine.
[015] In some embodiments, the SARS-CoV-2 3a protein inhibitor is at least one
molecule
selected from the group consisting of: Capreomycin, Pentamidine,
Spectinomycin,
Kasugamycin, Plerixafor, Flumatinib, Litronesib, Darapladib, Floxuridine, and
Fludarabine.
[016] In some embodiments, the SARS-CoV-2 3a protein inhibitor is Capreomycin.
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[017] In some embodiments, the prevention comprises prevention of any one of:
SARS-
CoV-2 entry to a cell of the subject, uncoating of the SARS-CoV-2, release of
the SARS-
CoV-2 from a cell of the subject, and any combination thereof.
[018] Further embodiments and the full scope of applicability of the present
invention will
become apparent from the detailed description given hereinafter. However, it
should be
understood that the detailed description and specific examples, while
indicating preferred
embodiments of the invention, are given by way of illustration only, since
various changes
and modifications within the spirit and scope of the invention will become
apparent to those
skilled in the art from this detailed description.
BRIEF DESCRIPTION OF THE AURES
[019] Figures 1A-1B include graphs showing membrane permeabilization assay.
Growth
curves (n=2) of bacteria as a function of SARS-CoV-2 E protein expression (1A,
right) or
as a function of SARS-CoV-2 3a protein expression (1B). Bacteria that express
the maltose
binding protein without a conjugated viral ion channel are shown in the left
panel as a
negative control. Bacteria that express the influenza M2 viroporin, as a
positive control, are
shown at the centre. Induction at different IPTG concentrations (as noted),
takes place when
the bacteria density reaches an 0.D.600 nm of 0.2. Growth 0.D.600 nm values
were
collected every 15 min. Fig. 1B shows growth curves of bacteria as a function
of SARS-
CoV-3a protein expression. Negative control (no channel; NC); no drug (ND).
[020] Figures 2A-2B include graphs showing I( conductivity assay. Impact of
viral
protein SARS-CoV-2 E protein on the growth of Ktuptake deficient bacteria
(left panel,
2A). Different protein expression levels are achieved by varying the
concentration of the
IPTG inducer, as noted. Bacterial growth rate as a function of [K+] is plotted
in the right
panel (2A). (2B) depicts the impact of SARS CoV-2 3a protein on the growth of
Ktuptake
deficient bacteria, using varying concentration of the IPTG inducer.
[021] Figures 3A-3B include graphs showing fluorescence-based fr conductivity
assay.
The fluorescence of bacteria that harbor pHluorin, a pH-sensitive GFP22, was
examined as
a function of SARS CoV-2 E protein expression (3A) or SARS CoV-2 3a protein
expression
(3B). Protein levels were governed by the level of the inducer (IPTG) as
indicated. The
results are an average of two independent experiments, with standard
deviations depicted as
error bars.
[022] Figures 4A-4B include graphs showing compound screening results using
the
positive and negative genetic tests. Impact of different drugs, as noted. and
E protein
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expression on the growth rates of bacteria. (4A) Negative genetic test in
which SARS-CoV-
2 E protein is expressed at an elevated level (40 [IM [IPTG]) and is therefore
deleterious to
bacteria. In this instance inhibitory drugs enhance bacterial growth. (4B)
Positive genetic
test in which SARS-CoV-2 E protein is expressed at low level (10 uM [IPTG]) in
Ktuptake
deficient bacteria. In this instance inhibitory drugs reduce bacterial growth.
In both panels
the impact on growth in comparison to growth without any drug is listed.
[023] Figures 5A-5C include graphs showing Mavorixafor screening results using
the
negative assay: Viral channel harmful to bacteria, wherein blocker increases
growth (5A),
positive assay: Viral channel essential to bacteria, wherein blocker decreases
growth (5B),
and fluorescence assay: Viral channel alters Fluorescence, wherein blocker
decreases
fluorescence change (5C) of SARS-CoV-2 E protein expressing bacteria.
[024] Figures 6A-6C include graphs showing Saroglitazar magnesium screening
results
using the negative assay: Viral channel harmful to bacteria, wherein blocker
increases
growth (6A), positive assay: Viral channel essential to bacteria, wherein
blocker decreases
growth (6B), and fluorescence assay: Viral channel alters Fluorescence,
wherein blocker
decreases fluorescence change (6C) of SARS-CoV-2 E protein expressing
bacteria.
[025] Figures 7A-7C include graphs showing Mebrofenin screening results using
the
negative assay: Viral channel harmful to bacteria, wherein blocker increases
growth (7A),
positive assay: Viral channel essential to bacteria, wherein blocker decreases
growth (7B),
and fluorescence assay: Viral channel alters Fluorescence, wherein blocker
decreases
fluorescence change (7C) of SARS-CoV-2 E protein expressing bacteria.
[026] Figures 8A-8C include graphs showing Cyclen screening results using the
negative
assay: Viral channel harmful to bacteria, wherein blocker increases growth
(8A), positive
assay: Viral channel essential to bacteria, wherein blocker decreases growth
(8B), and
fluorescence assay: Viral channel alters Fluorescence, wherein blocker
decreases
fluorescence change (8C) of SARS-CoV-2 E protein expressing bacteria.
[027] Figures 9A-9C include graphs showing Kasugamycin screening results using
the
negative assay: Viral channel harmful to bacteria, wherein blocker increases
growth (9A),
positive assay: Viral channel essential to bacteria, wherein blocker decreases
growth (9B),
and fluorescence assay: Viral channel alters Fluorescence, wherein blocker
decreases
fluorescence change (9C) of SARS-CoV-2 E protein expressing bacteria.
[028] Figures 10A-10C include graphs showing 5-Azacytidine screening results
using the
negative assay: Viral channel harmful to bacteria, wherein blocker increases
growth (10A),
positive assay: Viral channel essential to bacteria, wherein blocker decreases
growth (10B),
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and fluorescence assay: Viral channel alters Fluorescence, wherein blocker
decreases
fluorescence change (10C) of SARS-CoV-2 E protein expressing bacteria.
[029] Figures 11A-11C include graphs showing Plerixafor (octahydrochloride)
screening
results using the negative assay: Viral channel harmful to bacteria, wherein
blocker increases
growth (11A), positive assay: Viral channel essential to bacteria, wherein
blocker decreases
growth (11B), and fluorescence assay: Viral channel alters Fluorescence,
wherein blocker
decreases fluorescence change (11C) of SARS-CoV-2 E protein expressing
bacteria.
[030] Figures 12A-12C include graphs showing Plerixafor screening results
using the
negative assay: Viral channel harmful to bacteria, wherein blocker increases
growth (12A),
positive assay: Viral channel essential to bacteria, wherein blocker decreases
growth (12B),
and fluorescence assay: Viral channel alters Fluorescence, wherein blocker
decreases
fluorescence change (12C) of SARS-CoV-2 E protein expressing bacteria.
[031] Figures 13A-13C include graphs showing Capreomycin (sulfate) screening
results
using the negative assay: Viral channel harmful to bacteria, wherein blocker
increases
growth (13A), positive assay: Viral channel essential to bacteria, wherein
blocker decreases
growth (13B), and fluorescence assay: Viral channel alters Fluorescence,
wherein blocker
decreases fluorescence change (13C) of SARS-CoV-2 E protein expressing
bacteria.
[032] Figures 14A-14C include graphs showing Pentamidine (isethionate)
screening
results using the negative assay: Viral channel harmful to bacteria, wherein
blocker increases
growth (14A), positive assay: Viral channel essential to bacteria, wherein
blocker decreases
growth (14B), and fluorescence assay: Viral channel alters Fluorescence,
wherein blocker
decreases fluorescence change (14C) of SARS-CoV-2 3a protein expressing
bacteria.
[033] Figures 15A-15C include graphs showing Spectinomycin (dihydrochloride)
screening results using the negative assay: Viral channel harmful to bacteria,
wherein
blocker increases growth (15A), positive assay: Viral channel essential to
bacteria, wherein
blocker decreases growth (15B), and fluorescence assay: Viral channel alters
Fluorescence,
wherein blocker decreases fluorescence change (15C) of SARS-CoV-2 3a protein
expressing bacteria.
[034] Figures 16A-16C include graphs showing Kasugamycin (hydrochloride
hydrate)
screening results using the negative assay: Viral channel harmful to bacteria,
wherein
blocker increases growth (16A), positive assay: Viral channel essential to
bacteria, wherein
blocker decreases growth (16B), and fluorescence assay: Viral channel alters
Fluorescence,
wherein blocker decreases fluorescence change (16C) of SARS-CoV-2 3a protein
expressing bacteria.
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[035] Figures 17A-17C include graphs showing Plerixafor screening results
using the
negative assay: Viral channel harmful to bacteria, wherein blocker increases
growth (17A),
positive assay: Viral channel essential to bacteria, wherein blocker decreases
growth (17B),
and fluorescence assay: Viral channel alters Fluorescence, wherein blocker
decreases
fluorescence change (17C) of SARS-CoV-2 3a protein expressing bacteria.
[036] Figures 18A-18C include graphs showing Flumatinib screening results
using the
negative assay: Viral channel harmful to bacteria, wherein blocker increases
growth (18A),
positive assay: Viral channel essential to bacteria, wherein blocker decreases
growth (18B),
and fluorescence assay: Viral channel alters Fluorescence, wherein blocker
decreases
fluorescence change (18C) of SARS-CoV-2 3a protein expressing bacteria.
[037] Figures 19A-19C include graphs showing Litronesib screening results
using the
negative assay: Viral channel harmful to bacteria, wherein blocker increases
growth (19A),
positive assay: Viral channel essential to bacteria, wherein blocker decreases
growth (19B),
and fluorescence assay: Viral channel alters Fluorescence, wherein blocker
decreases
fluorescence change (19C) of SARS-CoV-2 3a protein expressing bacteria.
[038] Figures 20A-20C include graphs showing Darapladib screening results
using the
negative assay: Viral channel harmful to bacteria, wherein blocker increases
growth (20A),
positive assay: Viral channel essential to bacteria, wherein blocker decreases
growth (20B),
and fluorescence assay: Viral channel alters Fluorescence, wherein blocker
decreases
fluorescence change (20C) of SARS-CoV-2 3a protein expressing bacteria.
[039] Figures 21A-21C include graphs showing Floxuridine screening results
using the
negative assay: Viral channel harmful to bacteria, wherein blocker increases
growth (21A),
positive assay: Viral channel essential to bacteria, wherein blocker decreases
growth (21B),
and fluorescence assay: Viral channel alters Fluorescence, wherein blocker
decreases
fluorescence change (21C) of SARS-CoV-2 3a protein expressing bacteria.
[040] Figures 22A-22C include graphs showing Fludarabine screening results
using the
negative assay: Viral channel harmful to bacteria, wherein blocker increases
growth (22A),
positive assay: Viral channel essential to bacteria, wherein blocker decreases
growth (22B),
and fluorescence assay: Viral channel alters Fluorescence, wherein blocker
decreases
fluorescence change (22C) of SARS-CoV-2 3a protein expressing bacteria.
[041] Figure 23 includes a vertical bar graph showing the effect of various
tested drugs on
the viability of Vero-E6 cells which were infected with SARS-CoV-2 at a
multiplicity of
infection (MOI) of 0.01.
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DETAILED DESCRIPTION
[042] The present invention, in some embodiments, provides compositions
comprising a
SARS-CoV-2 E protein channel blocker and/or a SARS-CoV-2 3a protein inhibitor
for
treating or preventing SARS-CoV-2 virulence in a subject. The present
invention, in some
embodiments, provides compositions comprising a SARS-CoV-2 E protein channel
blocker
and/or a SARS-CoV-2 3a protein inhibitor, for preventing SARS-CoV-2 2 cell
entry,
uncoating and/or release from a cell.
SARS-CoV-2 E protein channel blockers
[043] The invention is based, at least in part, on the finding using three
bacteria-based
assays, that SARS-CoV-2 E protein is an ion channel. The invention is further
based, at least
in part, on a finding that Gliclazide, Memantine, Mavorixafor, Saroglitazar
Magnesium,
Mebrofenin, Cyclen, Kasugamycin, Azacytidine, and Plerixafor, inhibit SARS-CoV-
2 E
protein and therefore can be used to treat and prevent SARS-CoV-2 virulence.
[044] SARS-CoV-2 E protein is known to one skilled in the art and has a
GenBank
Accession no: QIH45055.1. According to some embodiments, the SARS-CoV-2 E
protein
comprises the amino acid sequence as set forth in SEQ ID NO I:
MYS FVS EETGTLIVNS VLLFLAFVVFLLVTLAILTALRLC AYCCNIVNVS LVKPS FY
VYSRVKNLNSSRVPDLLV. According to some embodiments, the SARS-CoV-2 E protein
comprises an analog of SEQ Ill NO: 1, such as an analog having at least 85%,
at least 90%,
at least 95% identity to SEQ ID NO: 1.
[045] According to some embodiments, the invention provides a method of
treating or
preventing SARS-CoV-2 virulence in a subject in need thereof, comprising
administering to
the subject a therapeutically effective amount of a SARS-CoV-2 E protein
channel blocker,
thereby treating or preventing SARS-CoV-2 virulence in the subject.
[046] In some embodiments, the invention provides a method of treating or
preventing
Coronavirus disease 2019 (COVID-19) in a subject in need thereof, comprising
administering to the subject a therapeutically effective amount of a SARS-CoV-
2 E protein
channel blocker, thereby treating or preventing COVID-19.
[047] According to some embodiments, the invention provides a method of
preventing
SARS-CoV-2 release from a cell. In some embodiments, the method comprises
contacting
a cell with a SARS-CoV-2 E protein channel blocker, thereby preventing SARS-
CoV-2
release from the cell.
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[048] According to some embodiments, the invention provides a method of
preventing
SARS-CoV-2 cell entry. In some embodiments, the method comprises contacting a
cell with
a SARS-CoV-2 E protein channel blocker, thereby preventing SARS-Co V -2 cell
entry.
[049] According to some embodiments, the invention provides a method of
preventing
SARS-CoV-2 uncoating. In some embodiments, the method comprises contacting a
cell with
a SARS-CoV-2 E protein channel blocker, thereby preventing SARS-CoV-2
uncoating.
[050] According to some embodiments, a cell is a cell of a subject. According
to some
embodiments, contacting comprises administering to the subject. According to
some
embodiments, the subject is a subject infected or suspected as being infected
by S ARS-CoV-
2.
[051] According to some embodiments, there is provided a method for treating
or
preventing SARS-CoV-2 virulence in a subject in need thereof, comprising
administering to
the subject a therapeutically effective amount of 5-Azacytidine, thereby
treating or
preventing SARS-CoV-2 virulence in said subject.
[052] According to some embodiments, there is provided a pharmaceutical
composition
comprising 5-Azacytidine, for use in the treatment and/or prevention of SARS-
CoV-2
virulence in a subject in need thereof.
[053] According to some embodiments, the SARS-CoV-2 E protein channel blocker
is at
least one molecule selected from: Memantine, Gliclazide, Mavorixafor,
Saroglitazar
Magnesium, Mebrofenin, Cyclen, Kasugamycin, Azacytidine, Plerixafor, or any
salt thereof.
[054] According to some embodiments, the invention provides a SARS-CoV-2 E
protein
channel blocker for use in treating or preventing SARS-CoV-2 virulence in a
subject in need
thereof.
[055] According to some embodiments, the invention provides a SARS-CoV-2 E
protein
channel blocker for use in preventing SARS-CoV-2 release from a cell.
[056] According to some embodiments, the SARS-CoV-2 E protein channel blocker
is
within a pharmaceutical composition. In some embodiments, the pharmaceutical
composition further comprises a pharmaceutically acceptable carrier.
[057] According to some embodiments, the invention provides a pharmaceutical
composition comprising Azacytidine, an analog or a salt thereof, for treating
a viral infection.
[058] According to some embodiments, the SARS-CoV-2 E protein channel blocker
is
Azacytidine, an analog or a salt thereof. According to some embodiments, the
SARS -CoV-
2 E protein channel blocker is 5-Azacytidine.
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[059] Azacytidine, as used herein, includes Azacytidine (CAS: 320-67-2; 4-
Amino-1-13-D-
ribofuranosyl-s-triazin-2(1H)-one), as well as pharmaceutically acceptable
salts, solvates,
hydrates, or mixtures thereof. Azacytidine is described, for example in
W02012135405A1.
The terms "5-Azacytidine" and "Azacytidine" are used herein interchangeably.
[060] According to some embodiments, the invention provides a pharmaceutical
composition comprising Memantine, an analog or a salt thereof, for use in the
treatment of
a viral infection. In some embodiments, the viral infection comprises a
coronaviruses
infection. In some embodiments, the viral infection comprises an infection by
virus having
an E protein being an ion channel. In some embodiments, the viral infection is
a
coronaviruses infection.
[061] According to some embodiments, the SARS-CoV-2 E protein channel blocker
is
Memantine, an analog or a salt thereof. According to some embodiments, the
SARS-CoV-2
E protein channel blocker is memantine hydrochloride.
[062] Memantine, as used herein, includes memantine (CAS: 19982-08-2; 1-amino-
3.5-
dimethyladamantane), as well as pharmaceutically acceptable salts, solvates,
hydrates, or
mixtures thereof. Memantine is described, for example, in U.S. Patents
3,391,142,
5,891,885, 5,919,826, and 6,187,338.
[063] According to some embodiments, the invention provides a pharmaceutical
composition comprising Gliclazide, an analog or a salt thereof, for treating a
viral infection.
In some embodiments, the viral infection is an infection by virus having an E
protein being
an ion channel.
[064] According to some embodiments, the SARS-CoV-2 E protein channel blocker
is
Gliclazide an analog or a salt thereof.
[065] Gliclazide, as used herein, includes gliclazide (CAS: 21187-98-4; 1-(3-
azabicyclo(3.3.0)oct-3-y1)-3-(p-tolylsulfonyl)urea) as well as
pharmaceutically acceptable
salts, solvates, hydrates, or mixtures thereof.
[066] According to some embodiments, the SARS-CoV-2 E protein channel blocker
is
selected from a group including Ginsenoside.
[067] According to some embodiments, the invention provides a pharmaceutical
composition comprising Mavorixafor, an analog or a salt thereof, for treating
a viral
infection.
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[068] According to some embodiments, the SARS-CoV-2 E protein channel blocker
is
Mavorixafor, an analog or a salt thereof. According to some embodiments, the
SARS-CoV-
2 h protein channel blocker is Mavorixafor.
[069] Mavorixafor, as used herein, includes Mavorixafor (CAS: 558447-26-0; N-
(1H-
benzimidazol-2-ylmethyl)-N- [(8S)-5,6,7,8-tetrahydroquinolin-8-yl]butane-1,4-
diamine), as
well as pharmaceutically acceptable salts, solvates, hydrates, or mixtures
thereof.
Mavorixafor is described, for example, in U.S. Patent US7332605, and as
compound 89 from
a series of 169 analogues in W02003055876.
[070] According to some embodiments, the invention provides a pharmaceutical
composition comprising Saroglitazar, an analog or a salt thereof, for treating
a viral infection.
[071] According to some embodiments, the SARS-CoV-2 E protein channel blocker
is
Saroglitazar, an analog or a salt thereof. According to some embodiments, the
SARS -CoV-
2 E protein channel blocker is Saroglitazar Magnesium.
[072] Saroglitazar, as used herein, includes Saroglitazar (CAS: 495-N9-09-2;
(aS)-a-
Ethox y -44242- meth y1-5 - [4-(mc thy lthio)phen yl] -1H-pyrrol-1-
yl]ethoxy]benzencpropanoic
Acid), as well as pharmaceutically acceptable salts, solvates, hydrates, or
mixtures thereof.
Saroglitazar is described, for example, in W02016181409.
[073] According to some embodiments, the invention provides a pharmaceutical
composition comprising Mebrofenin, an analog or a salt thereof, for treating a
viral infection.
[074] According to some embodiments, the SARS-CoV-2 E protein channel blocker
is
Mebrofenin, an analog or a salt thereof. According to some embodiments, the
SARS-CoV-
2 E protein channel blocker is Mebrofenin.
[075] Mebrofenin, as used herein, includes Mebrofenin (CAS: 78266-06-5; 21[243-
bromo-2,4,6-trimethylanilino)-2-oxoethy1]-(carboxymethyl)aminolacetic acid),
as well as
pharmaceutically acceptable salts, solvates, hydrates, or mixtures thereof.
Mebrofenin is
described, for example, in US9,878,984.
[076] According to some embodiments, the invention provides a pharmaceutical
composition comprising Cyclen, an analog or a salt thereof, for treating a
viral infection.
[077] According to some embodiments, the SARS-CoV-2 E protein channel blocker
is
Cyclen, an analog or a salt thereof. According to some embodiments, the SARS -
CoV-2 E
protein channel blocker is Cyclen.
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[078] Cyclen, as used herein, includes Cyclen (CAS: 294-90-6; 1,4,7,10-
Tetraazacyclododecane), as well as pharmaceutically acceptable salts,
solvates, hydrates, or
mixtures thereof. Cyclen is described, for example in US9421223132.
[079] According to some embodiments, the invention provides a pharmaceutical
composition comprising Kasugamycin, an analog or a salt thereof, for treating
a viral
infection.
[080] According to some embodiments, the SARS-CoV-2 E protein channel blocker
is
Kasugamycin, an analog or a salt thereof. According to some embodiments, the
SARS-CoV-
2 E protein channel blocker is Kasugamycin hydrochloride hydrate (CAS: 19408-
46-9).
[081] Kasugamycin, as used herein, includes Kasugamycin (CAS: 6980-18-3; 2-
amino-2-
[(2R,3S,5S,6R)-5-amino-2-methy1-6-[(2R,3S,5S,6S)-2,3,4,5,6-
pentahydroxycyclohexyl]oxyoxan-3-yl]iminoacetic acid), as well as
pharmaceutically
acceptable salts, solvates, hydrates, or mixtures thereof. Kasugamycin is
described, for
example in US3358001A.
[082] According to some embodiments, the invention provides a pharmaceutical
composition comprising Plerixafor, an analog or a salt thereof, for treating a
viral infection.
[083] According to some embodiments, the SARS-CoV-2 E protein channel blocker
is
Plerixafor, an analog or a salt thereof. According to some embodiments, the
SARS-CoV-2
E protein channel blocker is Plerixafor octahydrochloride.
[084] Plerixafor, as used herein, includes Plerixafor (CAS: 155148-31-5; 14[4-
(1,4,8,11-
tetrazacyclotetradec- 1-ylmethyl)phenyl] methyl] - 1,4,8,11-
tetrazacyclotetradecane), as well
as pharmaceutically acceptable salts, solvates, hydrates, or mixtures thereof.
Plerixafor is
described, for example in W02014125499A1.
SARS-CoV-2 3a protein inhibitor
[085] The invention is based, at least in part, on the finding using three
bacteria-based
assays, that SARS-CoV-2 3a protein inhibitors can serve as effective agents
for treating and
preventing S ARS -CoV-2 virulence.
[086] SARS-CoV-2 3a protein, also known as open reading frame 3a (ORF3a), is
known
to one skilled in the art and has a UniProt Accession no: PODTC3.
[087] The terms "3a protein" and "ORF3a" are used herein interchangeably.
[088] According to some embodiments, the SARS-CoV-2 3a protein comprises the
amino
acid sequence as set forth in SEQ ID NO
2:
MDLFMRIFTIGTVTLKQGEIKDATPSDFVRATATIPIQASLPFGWLIVGVALLAVFQ
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SASKIITLKKRWQLALSKGVHFVCNLLLLFVTVYSHLLLVAAGLEAPFLYLYALVY
FLQSINFVRIIMRLWLCWKCRSKNPLLYDANYFLCWHTNCYDYCIPYNSVTSSIVIT
SUDCITTSPISEHDYQIGGYTEKWESGVKDCV VLHSYFTSDY YQLYSTQLSTDTGVE
HVTFFIYNKIVDEPEEHVQ1HT1DGSSGVVNPVMEPIYDEPTTTTSVPL According to
some embodiments, the SARS-CoV-2 3a protein_comprises an analog of SEQ ID NO:
2,
such as an analog having at least 85%, at least 90%, at least 95% identity to
SEQ ID NO: 2.
[089] According to some embodiments, the invention provides a method of
treating or
preventing SARS-CoV-2 virulence in a subject in need thereof, comprising
administering to
the subject a therapeutically effective amount of a SARS -CoV-2 3a protein
inhibitor, thereby
treating or preventing SARS-CoV-2 virulence in the subject.
[090] In some embodiments, the invention provides a method of treating or
preventing
Coronavirus disease 2019 (COVID-19) in a subject in need thereof, comprising
administering to the subject a therapeutically effective amount of a SARS-CoV-
2 3a protein
inhibitor, thereby treating or preventing COVID-19.
[091] According to some embodiments, the invention provides a method of
preventing
SARS-CoV-2 release from a cell, the method comprising contacting the cell with
a SARS-
CoV-2 3a protein inhibitor, thereby preventing SARS-CoV-2 release from the
cell.
[092] According to some embodiments, the method comprising contacting the cell
with a
SARS-CoV-2 3a protein inhibitor, thereby preventing SARS-CoV-2 cell entry.
[093] In some embodiments, the method comprising contacting the cell with a
SARS -Co V-
2 3a protein inhibitor, thereby preventing SARS-CoV-2 uncoating.
[094] According to some embodiments, the subject is a subject infected or
suspected as
being infected by SARS-CoV-2.
[095] According to some embodiments, the SARS-CoV-2 3a protein inhibitor is at
least
one molecule selected from: Capreomycin, Pentamidine, Spectinomycin,
Kasugamycin,
Plerixafor, Flumatinib, Litronesib, Darapladib, Floxuridine, Fludarabine, or
salts thereof.
[096] According to some embodiments, the invention provides a SARS-CoV-2 3a
protein
inhibitor for use in the treatment or prevention of SARS-CoV-2 virulence, in a
subject in
need thereof.
[097] According to some embodiments, the invention provides a SARS-CoV-2 3a
protein
inhibitor for use in the prevention of SARS-CoV-2 release from a cell.
[098] According to some embodiments, the SARS-CoV-2 3a protein inhibitor is
within a
pharmaceutical composition.
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[099] In some embodiments, the viral infection is an infection by virus having
a 3a protein.
[0100] According to some embodiments, the invention provides a pharmaceutical
composition comprising Capreomycin, an analog or a salt thereof, for treating
a viral
infection.
[0101] According to some embodiments, the SARS-CoV-2 3a protein inhibitor is
Capreomycin, an analog or a salt thereof. According to some embodiments, the
SARS-CoV-
2 3a protein inhibitor is Capreomycin sulfate.
[0102] Capreomycin, as used herein, includes Capreomycin (CAS: 11003-38-6;
IUPAC:
(3S)-3,6-diamino-N-R(2S,5S,8E,11S,15S)-15-amino-11-[(4R)-2-amino-3,4,5,6-
tetrahydropyrimidin-4-y1]-8-[(carbamoylamino)methylidene] -2-(hydroxym ethyl )-
3 ,6,9,12,16-pentaoxo-1,4 ,7 , 10,13 -pentazacyclohexadec-5 -yl]
methyl]hexanamide; (3 S)-3 ,6-
diamino-N- [ [(2S ,5S.8E.11S ,15S)-15- amino -11- [(4R)-2- amino-3 ,4,5.6-
tetrahydropyrimidin-4-yl] -8- [(c arb amoylamino)methylidene] -2-methyl-3 ,6
,9,12,16-
pentaoxo-1,4.7 , 10,13 -pentazacyclohexadec-5 -yl] methyl]hexanamide), as
well as
pharmaceutically acceptable salts, solvates, hydrates, or mixtures thereof.
[0103] According to some embodiments, the invention provides a pharmaceutical
composition comprising Pentamidine, an analog or a salt thereof, for treating
a viral
infection.
[0104] According to some embodiments, the SARS-CoV-2 E protein channel blocker
is
Pentamidine, an analog or a salt thereof. According to some embodiments, the
SARS-CoV-
2 3a protein inhibitor is Pentamidine isethionate.
[0105] Pentamidine, as used herein, includes Pentamidine (CAS: 100-33-4;
IUPAC: 4,4'-
[pentane-1,5 -diylbi s(oxy)] dibenzenec arboximidamide), as well as
pharmaceutically
acceptable salts, solvates, hydrates, or mixtures thereof.
[0106] According to some embodiments, the invention provides a pharmaceutical
composition comprising Spectinomycin, an analog or a salt thereof, for
treating a viral
infection.
[0107] According to some embodiments, the SARS-CoV-2 E protein channel blocker
is
Spectinomycin, an analog or a salt thereof. According to some embodiments, the
SARS-
CoV-2 3a protein inhibitor is Spectinomycin dihydrochloride.
[0108] Spectinomycin, as used herein, includes Spectinomycin (CAS: 1695-77-8;
IUPAC:
1R.3S.5R.8R.10S ,11S,12S ,13R,14S)-8,12,14-trihydroxy-5-methyl-11,13-
13
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bis(methylamino)-2,4.9-trioxatricyclo [8.4 Ø03 ,8] tetradec an-7-one), as
well as
pharmaceutically acceptable salts, solvates, hydrates, or mixtures thereof.
[0109] According to some embodiments, the invention provides a pharmaceutical
composition comprising Kasugamycin, an analog or a salt thereof, for treating
a viral
infection.
[0110] According to some embodiments, the SARS-CoV-2 3a protein inhibitor is
Kasugamycin, an analog or a salt thereof. According to some embodiments, the S
ARS-CoV-
2 3a protein inhibitor is Kasugamycin hydrochloride hydrate.
[0111] Kasugamycin, as used herein, includes Kasugamycin (CAS:6980-18-3;
IUPAC: 2-
amino-2- [(2R,3S,5S ,6R)-5-amino-2-methy1-6- [(2R ,3S .5S ,6S)-2,3,4,5,6-
pentahydroxycyclohexyl]oxyoxan-3-yl]iminoacetic acid), as well as
pharmaceutically
acceptable salts, solvates, hydrates, or mixtures thereof.
[0112] According to some embodiments, the invention provides a pharmaceutical
composition comprising Plerixafor, an analog or a salt thereof, for treating a
viral infection.
[0113] According to some embodiments, the SARS-CoV-2 3a protein inhibitor is
Plerixafor,
an analog or a salt thereof. According to some embodiments, the SARS-CoV-2 3a
protein
inhibitor is Plerixafor.
[0114] Plerixafor, as used herein, includes Plerixafor (CAS: 155148-31-5;
IUPAC: 1,1'-
(1,4-phenylenebismethylene)bis(1,4,8,11- tetraazacyclotetradecane)), as well
as
pharmaceutically acceptable salts, solvates, hydrates, or mixtures thereof.
[0115] According to some embodiments, the invention provides a pharmaceutical
composition comprising Flumatinib, an analog or a salt thereof, for treating a
viral infection.
[0116] According to some embodiments, the SARS-CoV-2 3a protein inhibitor is
Flumatinib, an analog or a salt thereof. According to some embodiments, the
SARS-CoV-2
3a protein inhibitor is Flumatinib.
[0117] Flumatinib, as used herein, includes Flumatinib (CAS: 895519-90-1;
TUPAC: 4-[(4-
methylpiperazin- 1 -yl)methyl] -N-[6-methyl-5- [(4-pyridin-3-ylpyrimidin-2-
yl)amino]pyridin-3-yl] -3 -(trifluoromethyl)benzamide), as well as
pharmaceutically
acceptable salts, solvates, hydrates, or mixtures thereof.
[0118] According to some embodiments, the invention provides a pharmaceutical
composition comprising Litronesib, an analog or a salt thereof, for treating a
viral infection.
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[0119] According to some embodiments, the SARS-CoV-2 3a protein inhibitor is
Litronesib, an analog or a salt thereof. According to some embodiments, the
SARS-CoV-2
3a protein inhibitor is Litronesib.
[0120] Litronesib, as used herein, includes Litronesib (CAS: 910634-41-2;
IUPAC: N-
[(5R)-4-(2,2-dimethylpropanoy1)-5-[[2-(ethylamino)ethylsulfonylamino] methyl] -
5-phenyl-
1,3,4-thiadiazol-2-y1]-2,2-dimethylpropanamide), as well as pharmaceutically
acceptable
salts, solvates, hydrates, or mixtures thereof.
[0121] According to some embodiments, the invention provides a pharmaceutical
composition comprising Darapladib, an analog or a salt thereof, for treating a
viral infection.
[0122] According to some embodiments, the SARS-CoV-2 3a protein inhibitor is
Darapladib, an analog or a salt thereof. According to some embodiments, the
SARS-CoV-2
3a protein inhibitor is Darapladib.
[0123] Darapladib, as used herein, includes Darapladib (CAS: 356057-34-6;
IUPAC: N-(2-
Diethylaminoethyl)-2- [2- [(4-flu orophenyl)methylsu lfanyl] -4-o xo -6,7-d
ihy d ro -5H-
cyclopenta [d]pyrimidin- 1-yl] -N- [[444-(trifluoromethyl)phenyl]phenyl]
methyl] acetamide),
as well as pharmaceutically acceptable salts, solvates, hydrates, or mixtures
thereof.
[0124] According to some embodiments, the invention provides a pharmaceutical
composition comprising Floxuridine, an analog or a salt thereof, for treating
a viral infection.
[0125] According to some embodiments, the SARS-CoV-2 3a protein inhibitor is
Floxuridine, an analog or a salt thereof. According to some embodiments, the
SARS-CoV-
2 3a protein inhibitor is Floxuridine.
[0126] Floxuridine, as used herein, includes Floxuridine (CAS: 50-91-9; IUPAC:
5-Fluoro-
1-[4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl] -1H-pyrimidine-2,4-dione),
as well
as pharmaceutically acceptable salts, solvates, hydrates, Or mixtures thereof.
[0127] According to some embodiments, the invention provides a pharmaceutical
composition comprising Fludarabine, an analog or a salt thereof, for treating
a viral infection.
[0128] According to some embodiments, the SARS-CoV-2 3a protein inhibitor is
Fludarabine, an analog or a salt thereof. According to some embodiments, the
SARS-CoV-
2 3a protein inhibitor is Fludarabine.
[0129] Fludarabine, as used herein, includes Fludarabine (CAS: 21679-14-1;
IUPAC:
[(2R,3S ,4S ,5R)-5 -(6- amino-2-fluoro-purin-9-y1)-
3 ,4-dihydroxy-oxolan-2-
yl]methoxyphosphonic acid), as well as pharmaceutically acceptable salts,
solvates,
hydrates, or mixtures thereof.
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Pharmaceutical compositions
[0130] As used herein, the terms "treatment" or "treating" of a disease,
disorder, or condition
encompasses alleviation of at least one symptom thereof, a reduction in the
severity thereof,
or inhibition of the progression thereof. Treatment need not mean that the
disease, disorder,
or condition is totally cured. To be an effective treatment, a useful
composition herein needs
only to reduce the severity of a disease, disorder, or condition, reduce the
severity of
symptoms associated therewith, or provide improvement to a patient or
subject's quality of
life.
[0131] As used herein, the term "prevention" of a disease, disorder, or
condition
encompasses the delay, prevention, suppression, or inhibition of the onset of
a disease,
disorder, or condition. As used in accordance with the presently described
subject matter,
the term "prevention" relates to a process of prophylaxis in which a subject
is exposed to the
presently described compositions or formulations prior to the induction or
onset of the
disease/disorder process. The term "suppression" is used to describe a
condition wherein the
disease/disorder process has already begun but obvious symptoms of the
condition have yet
to be realized. Thus, the cells of an individual may have the
disease/disorder, but no outside
signs of the disease/disorder have yet been clinically recognized. In either
case, the term
prophylaxis can be applied to encompass both prevention and suppression.
Conversely, the
term "treatment" refers to the clinical application of active agents to combat
an already
existing condition whose clinical presentation has already been realized in a
patient.
[0132] In some embodiments, preventing comprises reducing the disease
severity, delaying
the disease onset, reducing the disease cumulative incidence, or any
combination thereof.
[0133] As used herein, the terms "administering," "administration," and like
terms refer to
any method which, in sound medical practice, delivers a composition containing
an active
agent to a subject in such a manner as to provide a therapeutic effect.
[0134] As used herein, the terms "subject" or "individual" or "animal" or
"patient" or
"mammal," refers to any subject, particularly a mammalian subject, for whom
therapy is
desired, for example, a human.
[0135] In some embodiments, a therapeutically effective dose of the
composition of the
invention is administered. The term "therapeutically effective amount" refers
to an amount
of a drug effective to treat a disease or disorder in a mammal. The term "a
therapeutically
effective amount" refers to an amount effective, at dosages and for periods of
time necessary,
to achieve the desired therapeutic or prophylactic result. The exact dosage
form and regimen
would be determined by the physician according to the patient's condition.
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[0136] The dosage administered will be dependent upon the age, health, and
weight of the
recipient, kind of concurrent treatment, if any, frequency of treatment, and
the nature of the
effect desired. The route of administration of the pharmaceutical compositions
will depend
on the disease or condition to be treated. Suitable routes of administration
include, but are
not limited to, parenteral injections, e.g., intradermal, intravenous,
intramuscular,
intralesional, subcutaneous, intrathecal, and any other mode of injection as
known in the art.
Although the bioavailability of peptides administered by other routes can be
lower than when
administered via parenteral injection, by using appropriate compositions it is
envisaged that
it will be possible to administer the compositions of the invention via
transdermal, oral,
rectal, vaginal, topical, nasal, inhalation and ocular modes of treatment. In
addition, it may
be desirable to introduce the pharmaceutical compositions of the invention by
any suitable
route, including intraventricular and intrathecal injection; intraventricular
injection may be
facilitated by an intraventricular catheter, for example, attached to a
reservoir.
[0137] In some embodiments, the composition of the invention is delivered
orally. In some
embodiments, the composition of the invention is an oral composition. In some
embodiments, the composition of the invention further comprises orally
acceptable carrier,
excipient, or a diluent.
[0138] According to some embodiments, the active agents of the invention
(e.g., SARS-
CoV-2 E protein channel blocker or protein 3a inhibiter) is for use at a daily
dose of 0.01 to
500 mg/kg.
[0139] According to some embodiments, the SARS-CoV-2 E protein channel blocker
is
Memantine or a salt thereof and is for use at a daily dose of between about 1
mg/day and
about 50 mg/day, about 1 mg/day and 45 mg/day, and 5 mg/day and 3 5mg/day.
[0140] According to some embodiments, the SARS-CoV-2 E protein channel blocker
is
Gliclazide or a salt thereof and is for use at a daily dose of between about 1
mg/day and 350
mg/day, 10 mg/day and 350 mg/day, 50 mg/day and 350 mg/day, 1 mg/day and 300
mg/day,
mg/day and 300 mg/day, and 50 mg/day and 250 mg/day.
[0141] According to some embodiments, the SARS-CoV-2 E protein channel blocker
is
Mavorixafor or a salt thereof and is for use at a daily dose of between about
50 mg/day and
about 100 mg/day, about 50 mg/day and 200 mg/day, and 50 mg/day and 400
mg/day.
[0142] According to some embodiments, the SARS-CoV-2 E protein channel blocker
is
Saroglitazar or a salt thereof and is for use at a daily dose of between about
0.1 mg/day and
about 5 mg/day, about 1 mg/day and 4 mg/day, and 1.5 mg/day and 4.5 mg/day.
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[0143] According to some embodiments, the SARS-CoV-2 E protein channel blocker
is
Mebrofenin or a salt thereof and is for use at a daily dose of between about 1
mg/day and
about 50 mg/day, about 1 mg/day and 45 mg/day, and 5 mg/day and 35 mg/day.
[0144] According to some embodiments, the SARS-CoV-2 E protein channel blocker
is
Cyclen or a salt thereof and is for use at a daily dose of between about 0.01
mg/day and
about 0.5 mg/day, about 0.1 mg/day and 0.5mg/day, and 0.05 mg/day and 0.3
mg/day.
[0145] According to some embodiments, the SARS-CoV-2 E protein channel
blocker, the
SARS-CoV-2 3a protein inhibitor, or both is Kasugamycin or a salt thereof and
is for use at
a daily dose of between about 1 mg/day and about 500 mg/day, about 5 mg/day
and
250mg/day, and 10 mg/day and 350 mg/day.
[0146] According to some embodiments, the SARS-CoV-2 E protein channel blocker
is
Azacytidine or a salt thereof and is for use at a daily dose of between about
1 mg/day and
about 100 mg/day, about 1 mg/day and 200 mg/day, and 1 mg/day and 300 mg/day.
[0147] According to some embodiments, the SARS-CoV-2 E protein channel
blocker, the
SARS-CoV-2 3a protein inhibitor, or both is Plerixafor or a salt thereof and
is for use at a
daily dose of between about 1 mg/day and about 50 mg/day, about 1 mg/day and
45 mg/day,
and 5 mg/day and 35 mg/day.
[0148] According to some embodiments, the SARS-CoV-2 3a protein inhibitor is
Capreomycin or a salt thereof and is for use at a daily dose of between about
50 mg/day and
about 1,000 mg/day, about 10 mg/day and 700 mg/day, and 20 mg/day and 800
mg/day.
[0149] According to some embodiments, the SARS-CoV-2 3a protein inhibitor is
Pentamidine or a salt thereof and is for use at a daily dose of between about
50 mg/day and
about 500 mg/day, about 30 mg/day and 400 mg/day, and 100 mg/day and 300
mg/day.
[0150] According to some embodiments, the SARS-CoV-2 3a protein inhibitor is
Spectinomycin or a salt thereof and is for use at a daily dose of between
about 500 mg/day
and about 5,000 mg/day, about 250 mg/day and 2,500 mg/day, and 100 mg/day and
4,500
mg/day.
[0151] According to some embodiments, the SARS-CoV-2 3a protein inhibitor is
Flumatinib or a salt thereof and is for use at a daily dose of between about
50 mg/day and
about 1,000 mg/day, about 100 mg/day and 1,500 mg/day, and 50 mg/day and 5,000
mg/day.
[0152] According to some embodiments, the SARS-CoV-2 3a protein inhibitor is
Litronesib
or a salt thereof and is for use at a daily dose of between about 10 mg/day
and about 3,000
mg/day, about 50 mg/day and 2,500 mg/day, and 20 mg/day and 2,000 mg/day.
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[0153] According to some embodiments, the SARS-CoV-2 3a protein inhibitor is
Darapladib or a salt thereof and is for use at a daily dose of between about
10 mg/day and
about 1,000 mg/day, about 50 mg/day and 500 mg/day, and 100 mg/day and 800
mg/day.
[0154] According to some embodiments, the SARS-CoV-2 3a protein inhibitor is
Floxuridine or a salt thereof and is for use at a daily dose of between about
1 mg/day and
about 100 mg/day, about 5 mg/day and 80 mg/day, and 10 mg/day and 100 mg/day.
[0155] According to some embodiments, the SARS-CoV-2 3a protein inhibitor is
Fludarabine or a salt thereof and is for use at a daily dose of between about
1 mg/day and
about 100 mg/day, about 2 mg/day and 80 mg/day, and 5 mg/day and 60 mg/day.
[0156] In some embodiments, the pharmaceutical composition comprises a
pharmaceutically acceptable carrier, adjuvant or excipient.
[0157] As used herein, the term "carrier," "adjuvant" or "excipient" refers to
any component
of a pharmaceutical composition that is not the active agent. As used herein,
the term
"pharmaceutically acceptable carrier" refers to non-toxic, inert solid, semi-
solid liquid filler,
diluent, encapsulating material, formulation auxiliary of any type, or simply
a sterile aqueous
medium, such as saline. Some examples of the materials that can serve as
pharmaceutically
acceptable carriers are sugars, such as lactose, glucose and sucrose, starches
such as corn
starch and potato starch, cellulose and its derivatives such as sodium
carboxymethyl
cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt,
gelatin, talc;
excipients such as cocoa butter and suppository waxes; oils such as peanut
oil, cottonseed
oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols,
such as propylene
glycol, polyols such as glycerin, sorbitol, mannitol and polyethylene glycol;
esters such as
ethyl oleate and ethyl laurate, agar; buffering agents such as magnesium
hydroxide and
aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline,
Ringer's solution;
ethyl alcohol and phosphate buffer solutions, as well as other non-toxic
compatible
substances used in pharmaceutical formulations. Some non-limiting examples of
substances
which can serve as a carrier herein include sugar, starch, cellulose and its
derivatives,
powered tragacanth, malt, gelatin, talc, stearic acid, magnesium stearate,
calcium sulfate,
vegetable oils, polyols, alginic acid, pyrogen-frec water, isotonic saline,
phosphate buffer
solutions, cocoa butter (suppository base), emulsifier as well as other non-
toxic
pharmaceutically compatible substances used in other pharmaceutical
formulations. Wetting
agents and lubricants such as sodium lauryl sulfate, as well as coloring
agents, flavoring
agents, excipients, stabilizers, antioxidants, and preservatives may also be
present. Any non-
toxic, inert, and effective carrier may be used to formulate the compositions
contemplated
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herein. Suitable pharmaceutically acceptable carriers, excipients, and
diluents in this regard
are well known to those of skill in the art, such as those described in The
Merck Index,
Thirteenth Edition. Budavari et al.. Eds., Merck & Co., Inc., Rahway, N.J.
(2001); the CTFA
(Cosmetic, Toiletry, and Fragrance Association) International Cosmetic
Ingredient
Dictionary and Handbook, Tenth Edition (2004); and the "Inactive Ingredient
Guide," U.S.
Food and Drug Administration (FDA) Center for Drug Evaluation and Research
(CDER)
Office of Management, the contents of all of which are hereby incorporated by
reference in
their entirety. Examples of pharmaceutically acceptable excipients, carriers
and diluents
useful in the present compositions include distilled water, physiological
saline, Ringer's
solution, dextrose solution, Hank's solution, and DMSO. These additional
inactive
components, as well as effective formulations and administration procedures,
are well
known in the art and are described in standard textbooks, such as Goodman and
Gillman' s.
The Pharmacological Bases of Therapeutics, 8th Ed., Gilman et al. Eds.
Pergamon Press
(1990); Remington's Pharmaceutical Sciences, 18th Ed., Mack Publishing Co.,
Easton, Pa.
(1990); and Remington: The Science and Practice of Pharmacy, 21st Ed.,
Lippincott
Williams & Wilkins, Philadelphia, Pa., (2005), each of which is incorporated
by reference
herein in its entirety. The presently described composition may also be
contained in
artificially created structures such as liposomes, ISCOMS, slow-releasing
particles, and
other vehicles which increase the half-life of the peptides or polypeptides in
serum.
Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid
crystals,
phospholipid dispersions, lamellar layers and the like. Liposomes for use with
the presently
described peptides are formed from standard vesicle-forming lipids which
generally include
neutral and negatively charged phospholipids and a sterol, such as
cholesterol. The selection
of lipids is generally determined by considerations such as lipo some size and
stability in the
blood. A variety of methods are available for preparing liposomes as reviewed,
for example,
by Coligan, J. E. et al, Current Protocols in Protein Science, 1999, John
Wiley & Sons, Inc.,
New York, and see also U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and
5,019,369.
[0158] The carrier may comprise, in total, from about 0.1% to about 99.99999%
by weight
of the pharmaceutical compositions presented herein.
Screening assays
[0159] According to some embodiments, there is provided a method of screening
effectiveness of an agent in treating or preventing a coronavirus infection,
the method
comprising providing a cell comprising a membrane permeabilized comnavirus E
or 3a
protein, contacting the cell with the agent, and determining effect of the
agent on growth of
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the cell, wherein a substantial effect of the agent on cellular growth is
indicative of the agent
as being effective for treating or preventing a coronavirus infection, thereby
screening
effectiveness of an agent in treating or preventing a coronavirus infection.
[0160] In some embodiments, the method is a negative assay. In some
embodiments, the cell
is charecterized by growth retardation due to the membrane permeabilized E
protein or 3a
protein. In some emobodemts, an agent that alleviates growth retardation is
indicative as
being effective for treating or preventing a coronavirus infection.
[0161] In some embodiments, the method is a positive assay. In some
embodiments, the cell
is a K+-uptake deficient cell grown in low [K+] media experience growth, due
to the channel
formed by the E protein or 3a protein. In some emobodemts, an agent that
induces growth
retardation is indicative as being effective for treating or preventing a
coronavirus infection.
[0162] In some embodiments, the coronavirus is SARS-CoV. In some embodiments,
SARS-
CoV is any one of SARS-CoV-1 and SARS-CoV-2. In some embodiments, the
coronavirus
E protein or 3a protein is a SARS-CoV-1 E protein or 3a protein, respectively.
In some
embodiments, the coronavirus E protein or 3a protein is a SARS-CoV-2 E
protein, or 3a
protein, respectively.
[0163] In some embodiments, the method comprises performing both the negative
assay and
the positive assay.
[0164] In some embodiments, the cell is a bacterial cell. In some embodiments,
the cell is
devoid of endogenous potassium uptake, besides an exogenously provided (e.g.,
expressed)
membrane permeabilized SARS-CoV E protein or by the 3a protein.
[0165] Non-limiting examples for growing a bacterial cell applicable for the
screening
methods provided herein, include: Astrahan, P. et al., Acta 1808, 394-8
(2011); Santner, P.
et al. Biochemistry 57, 5949-5956 (2018), and Taube, R., Alhadeff, R., Assa,
D., Krugliak,
M. & Arkin, I. T. PLoS One 9, e105387 (2014).
[0166] In some embodiment, the assay is for determining susceptibility of the
virus to
develop resistance against the agent.
[0167] As used herein, the term "about" when combined with a value refers to
plus and
minus 10% of the reference value. For example, a length of about 1,000
nanometers (nm)
refers to a length of 1000 nm 100 nm.
[0168] It is noted that as used herein and in the appended claims, the
singular forms "a,"
"an," and "the" include plural referents unless the context clearly dictates
otherwise. Thus.
for example, reference to "a polynucicotide" includes a plurality of such
polynucleotides and
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reference to "the polypeptide" includes reference to one or more polypeptides
and
equivalents thereof known to those skilled in the art, and so forth. It is
further noted that the
claims may be drafted to exclude any optional element. As such, this statement
is intended
to serve as antecedent basis for use of such exclusive terminology as
"solely." "only" and the
like in connection with the recitation of claim elements or use of a
"negative" limitation.
[0169] In those instances where a convention analogous to "at least one of A,
B, and C, etc."
is used, in general such a construction is intended in the sense one having
skill in the art
would understand the convention (e.g., "a system having at least one of A, B,
and C" would
include but not be limited to systems that have A alone, B alone, C alone, A
and B together,
A and C together, B and C together, and/or A, B, and C together, etc.). It
will be further
understood by those within the art that virtually any disjunctive word and/or
phrase
presenting two or more alternative terms, whether in the description, claims,
or drawings,
should be understood to contemplate the possibilities of including one of the
terms, either of
the terms, or both terms. For example, the phrase "A or B" will be understood
to include the
possibilities of "A" or "B" or "A and B."
[0170] It is appreciated that certain features of the invention, which are,
for clarity, described
in the context of separate embodiments, may also be provided in combination in
a single
embodiment. Conversely, various features of the invention, which are, for
brevity, described
in the context of a single embodiment, may also be provided separately or in
any suitable
sub-combination. All combinations of the embodiments pertaining to the
invention are
specifically embraced by the present invention and are disclosed herein just
as if each and
every combination was individually and explicitly disclosed. In addition, all
sub-
combinations of the various embodiments and elements thereof are also
specifically
embraced by the present invention and are disclosed herein just as if each and
every such
sub-combination was individually and explicitly disclosed herein.
[0171] Additional objects, advantages, and novel features of the present
invention will
become apparent to one ordinarily skilled in the art upon examination of the
following
examples, which are not intended to be limiting. Additionally, each of the
various
embodiments and aspects of the present invention as delineated hereinabove and
as claimed
in the claims section below finds experimental support in the following
examples.
[0172] Various embodiments and aspects of the present invention as delineated
hereinabove
and as claimed in the claims section below find experimental support in the
following
examples.
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EXAMPLES
[0173] Generally, the nomenclature used herein, and the laboratory procedures
utilized in
the present invention include molecular, biochemical, microbiological and
recombinant
DNA techniques. Such techniques are thoroughly explained in the literature.
See, for
example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989);
"Current
Protocols in Molecular Biology" Volumes I-III Ausubel, R. M., ed. (1994);
Ausubel et al.,
"Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore,
Maryland
(1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons,
New York
(1988); Watson et al., "Recombinant DNA", Scientific American Books, New York;
Birren
et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold
Spring Harbor
Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat.
Nos. 4,666,828;
4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory
Handbook",
Volumes I-III Cellis, J. E., ed. (1994); "Culture of Animal Cells - A Manual
of Basic
Technique" by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; "Current
Protocols in
Immunology" Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds),
"Basic and Clinical
Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and
Shiigi
(eds), "Strategies for Protein Purification and Characterization - A
Laboratory Course
Manual" CSHL Press (1996); all of which are incorporated by reference. Other
general
references are provided throughout this document.
Materials and Methods
Bacterial strains
[0174] Three strains of K12 Escherichia call were used in the current study:
DH10B,
LB650, and LR1. DH1OB cells were purchased from Invitrogen (Carlsbad, CA).
LB650
bacteria (AtrkG, AtrkH, and AkdpABC5 system) contain deletions in genes
connected to
potassium uptake (Stumpe, S. & Bakker, E. P. Arch Microbiol 167, 126-36
(1997)). LR1
bacteria contained a chromosomal copy of a pH sensitive green fluorescence
protein (GFP)
called pHluorin (Miesenbock, G and De Angelis, D A and Rothman, J E. Nature
394, 192-
(1998)).
Plasmids
[0175] The SARS-CoV-2 E protein, 3a protein, and the influenza M2 channel were
expressed as fusion proteins to the maltose binding protein using the pMAL-p2X
plasmid
(New England Biolabs, Ipswich, MA). Genes for the viral proteins have been
added with a
nucleotide sequence coding for linker of seven amino-acids, six histidines,
and a stop codon
at the 3' end. EcoRI and XbaI restriction sites were located at the 5' and 3'
ends, respectively.
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The sequences were synthesized by GenScript (Piscataway, NJ). Protein
expression was
achieved by adding isopropyl fl-D-1-thiogalactopyranoside (IPTG) to the growth
media, as
indicated.
Chemicals
[0176] IPTG was purchased from Biochemika-Fluka (Buchs, Switzerland). All
other
chemicals were purchased from Sigma-Aldrich laboratories (Rehovot, Israel).
Growth media
[0177] Lysogeny Broth (LB) was used for all bacterial growth (Bertani, G. J
Bacteriol 62,
293-300 (1951)) unless noted otherwise. LBK was similar to LB expect that KC1
replaces
NaC1 at 10 gr/L. All media contained ampicillin at 50 lag /ml.
Bacterial growth
[0178] Escherichia coli DH10B bacteria bearing or lacking (as a reference) the
viral chimera
were grown overnight in LB at 37 C. Thereafter, the growth culture was
diluted and the
bacteria were grown until their 0.D.600 reached 0.2. Fifty (50) 1 of
bacterial culture were
subsequently dispensed into 96-well flat-bottomed plates (Nunc, Roskilde,
Denmark)
containing 50 1 of the different treatments. Unless stated otherwise, IPTG
was added to the
cells to final concentrations ranging from 0 to 100 M. D-glucose was added to
a
concentration of 1%. Ninty six (96)-well plates were incubated for 16 hours at
37 C in an
Infinite 200 from the Tecan Group (Mannedorf, Switzerland) at a constant high
shaking rate.
0.D.600 readings were recorded every 15 min. For every measurement duplicates
or
triplicates were conducted.
[0179] For the Escherichia coli LB 650 bacteria, the same protocol was used,
except that
growth was done in LBK overnight. Thereafter, the growth medium was replaced
with LB
and the bacteria were diluted and grown until their 0.D.600 reached 0.2, and
diluted twofold
with the various treatments in each well. Unless stated otherwise IPTG was
added to the
LB650 bacteria to a final concentration of 10 M.
pHlux assay
[0180] Transformed LR1 cells were cultured overnight in LB media containing 1%
glucose
and 50 M ampicillin. Secondary cultures were prepared by diluting the primary
culture by
1:500 in LB media and allowing it to grow to an 0.D.600 of 0.6-0.8. Protein
synthesis was
induced by the addition of 50 M IPTG for two hours. Cultures without IPTG
induction
were used as control. Following two hours of induction, the 0.D.600 of all
cells were
measured, and after pelleting at 3,500 g for 10 min, the bacteria were
resuspended in
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McIlvaine buffer (200 mM Na 2HPO4, 0.9% NaC1 adjusted to pH 7.6 with 0.1 M
citric acid,
0.9% Nacl) to an optical density of 0.25 at 600 nm. 200 I of cell suspension
were
subsequently transferred with 30 I of McIlvaine buffer to 96 well plate. The
plate includes
a row with only assay buffer and cultures without induction. The fluorescence
measurements
were carried out in a microplate reader (Infinite F200 pro, Tecan) with the
emission set at
520 nm, while excitation shifted between 390 nm and 466 nm.
[0181] A liquid handling system (Tecan) was used to add 70 ill of 300 mM
citric acid with
0.9% NaCl to the bacteria. The fluorescence emission of each well after
addition of acid was
measured by alternate read out of the two filter pairs for 50 seconds. The
ratio for the two
differently excited emissions, F = F390 nm/F466 run was calculated and
translated into proton
concentration using the following equation:
[H+] = 0.132 = F-1.75'F .51
[0182] Vero-E6 cells were pretreated for 20 h with tested compounds (drugs
including their
concentration are described in Fig. 23) and were infected with SARS-CoV-2 at a
multiplicity
of infection (MOI) of 0.001 and 0.01 in the presence of the compounds as
indicated. The
medium with the same DMSO concentration was used as the no-drug control. Drug
efficacies in control of toxicity were assessed at 24 hours post infection by
MTT assay. All
infection experiments were performed in a BSL-3 facility.
Results
[0183] Three, recently developed bacteria-based assays (A strah an, P. et al.
Biochim Biophys
Acta 1808, 394-8 (2011); Santner, P. et al. Biochemistry 57, 5949-5956 (2018);
Taube, R.,
Alhadeff, R., Assa, D., Krugliak, M. & Arkin, I. T. PLoS One 9, e105387
(2014); Tomar, P.
P. S., Oren, R., Krugliak, M. & Arkin, I. T. Viruses 11(2019)) were tailord to
examine if
SARS-CoV-2 E protein is an ion channel.
[0184] These assays are quantitative, easy to implement rapidly, and are
amenable to high-
throughput screening for inhibitor identification. Moreover, each of these
there assays was
used on known viroporins and were shown to distinguish non-conducting
transmembrane
domains (Tomar, P. P. S., Oren, R., Krugliak, M. & Arkin, I. T., Viruses 11
(2019)). Finally,
one of the assays can also be used to predict, prior to clinical use, the
options that the virus
has to develop resistance against any particular inhibitor of the channel
(Assa, D., Alhadeff,
R., Krugliak, M. & Arkin, 1. T., J. Mol Biol 428, 4209-4217 (2016)).
[0185] In order to ensure proper membrane incorporation, the inventors made
use of of the
pMAL protein fusion and purification system (New England Biolabs). In this
construct
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which has been used successfully with multiple viroporins, SARS-CoV-2 E
protein or 3a
protein are fused to the carboxyl terminus of the periplasmic maltose binding
protein. As a
positive control, the inventors compared the activity of the proteins to the
M2 channel from
the influenza A virus, the archetypical viroporin that can be inhibited by
aminoadamantanes
(Pinto, L. H., Holsinger, L. J. & Lamb, R. A. Cell 69, 517-28 (1992)).
[0186] The first test that was undertaken was to examine if the SARS-CoV-2 E
and 3a
proteins' channel activity can lead to membrane permeabilization and thereby
negatively
impact bacterial growth (negative genetic test). As seen in numerous other
viroporins, when
expressed at increasing levels, channel activity hampers growth due to its
deleterious impact
on the proton motive force. Subsequently, channel-blocking drugs may be
identified readily
due to their ability to alleviate growth retardation. The data in Fig. 1 show
that expression
of the SARS-CoV-2 E or 3a protein causes significant bacterial growth
retardation
proportional to the protein's expression levels. This behaviour is similar to
that of a known
proton channel, the M2 influenza A protein.
[0187] The inventors recognized that growth retardation is not an uncommon
consequence
of heterologous protein expression in bacteria. In other words, spurious
factors could lead to
bacterial death in addition to channel activity. The inventors thus
demonstrate that bacterial
death is due to protein channel activity in the following three ways: (i) The
inventors identify
E and 3a protein channel blockers and show that they can revive bacterial
growth; (ii) The
inventors developed a complementary bacterial growth assay, where channel
activity is
essential for growth (positive genetic test); and (iii) The inventors show
that protein
expression increases H+ conductivity in an assay involving a pH sensitive GFP
(Santner, P.
et al. Biochemistry 57, 5949-5956 (2018)).
[0188] The second experimental test that the inventors have performed examines
K+
conductivity. Specifically, Ktuptake deficient bacteria (Stumpe, S. & Bakker,
E. P. Arch
Microbiol 167, 126-36 (1997)) are incapable of growth, unless the media is
supplemented
by Kt However. when a channel capable of K+ transport is heterologously
expressed, the
bacteria can thrive even under low K+ media (Taube, R. et al. PLoS One 9,
e105387 (2014);
Tomar, P. P. S., Oren, R., Krugliak, M. & Arkin, I. T. Viruses 11 (2019)).
Hence, in this
instance the viral channel is essential to bacterial growth (positive genetic
test). Finally,
results shown in Fig. 2 indicate that expression of SARS-CoV-2 E or 3a
proteins are able to
increase the growth rate of Ktuptake deficient bacteria in otherwise limiting
conditions (i.e.,
low [K+]).
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[0189] The final test to examine channel activity, was based on detecting
protein-mediated
Fr flux in bacteria that express a pH-sensitive green fluorescent protein (S
antner, P. et al.
Biochemistry 57, 5949-5956 (2018)). The addition of an acidic solution to the
media will
result in cytoplasmic acidification if the bacteria express a channel capable
of H transport.
Consequently, as seen in Fig. 3, expression of the E or 3a proteins from SARS-
CoV-2 results
in appreciable cytoplasmic acidification, indicative of its ability to
transport protons. Similar
acidification was detected in other viroporins, such as the influenza A M2
channel.
[0190] Considering that all three bacterial assays indicated that the SARS-CoV-
2 E and 3a
proteins are a potential viroporins, the inventors set forth to screen a small
data set of known
channel blockers. First, a library of 372 compounds from MedChemExpress (NJ,
USA) in
the area of "Membrane Transporter/Ion Channel". Each of these chemicals was
tested in the
positive and negative genetic tests detailed above against the E protein.
[0191] In the negative assay, bacteria experience appreciable growth
retardation due to the
expression of the SARS-CoV-2 E protein at elevated levels (Fig. 1). Therefore,
channel
blockers can be readily identified since they alleviate this growth
retardation. Note that this
screen inherently reduces potential toxicity since it selects for chemicals
that are not toxic to
the bacteria. Specifically, each of the chemicals in the pilot library was
added to the media,
followed by growth recording and comparison to bacteria that did not receive
any treatment.
Out of the 372 compound drug library, several chemicals relieved the growth
inhibition that
the bacteria experienced due to the SARS-CoV-2 E protein activity. Of
particular notice are
Gliclazide and Memantine that enhance bacterial growth, as shown in Fig. 4A.
[0192] In the positive assay screening, a reciprocal picture is obtained.
Ktuptake deficient
bacteria grown in low [K+1 media experience growth enhancement due to the (low
level)
expression of the SARS-CoV-2 E protein (Fig. 2). Therefore, channel blockers
can be
identified since they result in growth retardation. In a manner similar to the
negative assay,
each of the chemicals in the pilot library was added to the media followed by
growth
recording. Once again, Gliclazide and Memantine scored positively in the test,
in that they
both inhibited growth (Fig. 4B).
[0193] The above results are encouraging since the same chemicals scored
positively in
reciprocal assays. Scoring positively in both assays rules out any spurious
factors. When the
E protein is detrimental to bacteria, the chemicals enhanced growth. However,
when the E
protein is essential to bacteria, the same compounds were deleterious to
growth.
[0194] Our results demonstrate that the SARS-CoV-2 E protein is an ion
channel. Since
coronavirus E proteins are essential to virulence, it represents an attractive
drug target. Our
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screening efforts identified two inhibitors that block E protein channel
activity. Since both
drugs are approved for human use for other indications, they represent
candidates for swift
mitigation the CO VID-19 crisis.
[0195] Further, the screen was broadend and 3000 moleclues were screened
against the
SARS-CoV-2 E protein, and 3,000 moleclues were screened against SARS-CoV-2 3a
protein.
[0196] The resluts indicate that any one of: Memantine, Gliclazide,
Mavorixafor,
Saroglitazar Magnesium, Mebrofenin, Cyclen, Kasugamycin, Azacytidine, and
Plerixafor
can be used as SARS-CoV-2 E protein channel blacker, and accordingly be used
as attractive
COVID-19 drugs.
[0197] The results further indicate that any one of Capreomycin, Pentamidine,
Spectinomycin, Kasugamycin, Plerixafor, Flumatinib, Litronesib, Darapladib,
Floxuridine,
and Fludarabine, can be used as SARS-CoV-2 3a protein inhibitor, and
accordingly be used
as attractive COVID-19 drugs.
[0198] Further, the inventors showed the effect of the compounds tested herein
on the
viability of cells infected with SARS-CoV-2 virus. Specifically, the results
show there was
-60% reduction in the cell viability of the Vero-E6 cells when infected with
SARS-CoV-2
at a multiplicity of infection (MOI) of 0.01, whereas the cells pretreated
with the drugs
showed -10-50% reduction of the cell viability after infected with the virus
(Fig. 23).
[0199] Although the invention has been described in conjunction with specific
embodiments
thereof, it is evident that many alternatives, modifications and variations
will be apparent to
those skilled in the art. Accordingly, it is intended to embrace all such
alternatives,
modifications and variations that fall within the spirit and broad scope of
the appended
claims.
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