U.S. patent application number 14/479952 was filed with the patent office on 2014-12-25 for inhibition of filovirus entry into cells and uses thereof.
This patent application is currently assigned to Board of Regents, University of Texas System. The applicant listed for this patent is Robert A. Davey, Andrey A. Kolokoltsov, Mohammad F. Saeed. Invention is credited to Robert A. Davey, Andrey A. Kolokoltsov, Mohammad F. Saeed.
Application Number | 20140378435 14/479952 |
Document ID | / |
Family ID | 52111410 |
Filed Date | 2014-12-25 |
United States Patent
Application |
20140378435 |
Kind Code |
A1 |
Davey; Robert A. ; et
al. |
December 25, 2014 |
Inhibition of Filovirus Entry Into Cells and Uses Thereof
Abstract
The present invention discloses method to treat infections
caused by filovirus. Such a method comprises blocking the PI3
kinase pathway or the calcium-associated pathway at the gene or
protein level. Also disclosed herein are the compounds useful in
the treatment of filoviral infection.
Inventors: |
Davey; Robert A.;
(Galveston, TX) ; Kolokoltsov; Andrey A.;
(Galveston, TX) ; Saeed; Mohammad F.; (League
City, TX) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Davey; Robert A.
Kolokoltsov; Andrey A.
Saeed; Mohammad F. |
Galveston
Galveston
League City |
TX
TX
TX |
US
US
US |
|
|
Assignee: |
Board of Regents, University of
Texas System
Austin
TX
|
Family ID: |
52111410 |
Appl. No.: |
14/479952 |
Filed: |
September 8, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12653916 |
Dec 21, 2009 |
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14479952 |
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Current U.S.
Class: |
514/211.07 ;
435/375; 514/279; 514/356; 514/604 |
Current CPC
Class: |
A61K 31/713 20130101;
A61K 31/366 20130101; A61K 31/4741 20130101; A61K 31/4745 20130101;
A61K 33/00 20130101; A61K 31/554 20130101; A61K 31/435 20130101;
A61K 31/18 20130101; A61K 31/277 20130101; A61K 31/5377
20130101 |
Class at
Publication: |
514/211.07 ;
514/279; 514/604; 514/356; 435/375 |
International
Class: |
A61K 31/554 20060101
A61K031/554; A61K 31/145 20060101 A61K031/145; A61K 31/435 20060101
A61K031/435; A61K 31/4745 20060101 A61K031/4745 |
Claims
1. A method of preventing or treating filovirus infection of a
cell, comprising: contacting the cell with a sole active agent that
is a calcium channel blocker, wherein the contacting inhibits the
entry of the filovirus into the cell.
2. The method of claim 1, wherein the calcium channel blocker is
Tetrandrine.
3. The method of claim 1, wherein the calcium channel blocker is
Diltiazem.
4. The method of claim 1, wherein the calcium channel blocker is
KN-93.
5. The method of claim 1, wherein the calcium channel blocker is a
dihydropyridine.
6. The method of claim 1, wherein the calcium channel blocker is
Nimodipine.
7. The method of claim 1, wherein the filovirus is an Ebola virus
or a Marburg virus.
8. A method of treating filovirus infection in an individual
infected with a filovirus consisting essentially of: administering
a pharmacologically effective amount of a calcium channel blocker,
thereby treating the individual infected with the filovirus.
9. The method of claim 8, wherein the calcium channel blocker is
Tetrandrine.
10. The method of claim 8, wherein the calcium channel blocker is
Diltiazem.
11. The method of claim 8, wherein the calcium channel blocker is
Nimodipine.
12. The method of claim 8, wherein the calcium channel blocker is
KN-93.
13. The method of claim 8, wherein the calcium channel blocker is a
dihydropyridine.
14. The method of claim 8, wherein said filovirus is an Ebola virus
or a Marburg virus.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims benefit of priority under 35 U.S.C.
.sctn.120 of international application PCT/US2008/007747, filed
Jun. 20, 2008, which claims benefit of priority under 35 U.S.C.
.sctn.119(e) of provisional U.S. Ser. No. 60/936,426, filed Jun.
20, 2007, now abandoned, the entirety of both of which are hereby
incorporated by reference.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates to the field of virology. In
general, the present invention discloses method(s) of treating
infection by members of the filovirus family of viruses by directly
inhibiting virus entry into the cells or by preventing envelope
protein induced toxicity. More specifically, the present invention
discloses methods of treating infection by filovirus such as Ebola
virus, Marburg virus and related viruses by blocking the PI3 kinase
signaling pathway or the calcium-associated signaling pathway.
[0004] 2. Description of the Related Art
[0005] Ebola virus is a member of the family Filoviridae and causes
severe hemorrhagic fevers in humans and nonhuman primates. It is an
emerging virus and outbreaks have been reported periodically at
2-10 year intervals since its initial identification in 1976. It is
a NIH category A agent and is one of the most widely publicized
human viruses. Most outbreaks have occurred in isolated communities
in Africa and so have been effectively contained. Release into a
large city community would likely have severe consequences not only
for those infected but as a result of mass panic and major economic
disruption. For these reasons Ebola virus remains one of the most
highly effective terrorist bioweapon threats.
[0006] In the wild, infections initiate from contact of people with
dead or dying virus-infected forest animals such as chimpanzees,
forest antelope and porcupines that have been regularly found on
the rainforest floor in affected areas (World Health Organization,
2004). However, these are dead-end hosts and the primary animal
reservoir remains unknown.
[0007] The Ebola virus genus includes four subtypes: Zaire, Sudan,
Ivory Coast and Reston. The Zaire strain is the most often
associated with outbreaks with very high mortality rates, on
average between 80 to 90% (Sadek et al., 1995; Peters, 1996, Peters
and LeDuc, 1999; Feldman et al., 1993; Baize et al., 1999;
Fisher-Hoch and McCormick, 1999). Virus is spread by contact with
blood or body fluids from infected individuals or animals and is
highly infectious. The final stages of Ebola virus infection are
characterized by fever, hemorrhage, hypotensive shock with an
apparent dependence on the reticuloendothelial and mononuclear
phagocytic cell systems (Peters and LeDuc, 1999; Baskerville et
al., 1978; Baskerville et al., 1985; Feldmann et al., 1996;
Schnittler and Feldmann, 1999).
[0008] Apart from palliative treatment, there is no effective
treatment for an Ebola virus infection. Some success has been found
using monoclonal antibodies against envelope proteins and
nucleoproteins or using passive transfer of immune serum from
convalescent patients (Mupapa et al., 1999; Xu et al., 1998) but
this is not practical in the situation of a virus outbreak.
Patients that survive typically have more rapid Ebola
virus-specific humoral and cellular responses than those that die.
Therefore delaying virus spread would give a greater opportunity
for the immune system to mount an effective anti-viral response.
Then drugs that can prevent or slow an ongoing infection would
likely be effective in treatment.
[0009] Previous research has suggested that mononuclear phagocytic
cells and endothelial cells are sites of Ebola virus replication
early in infection although evidence of replication has been
observed in many tissues including the liver, spleen and lymph
nodes (Connolly et al., 1999; Geisbert et al., 2000; Nabel, 1999;
Schnittler et al., 1993; Yang et al., 1998). The pantropic nature
of Ebola virus infection suggests a role for monocytes in
disseminating the virus to distant sites (Schnittler and Feldmann,
1999; Schnittler and Feldmann, 1998; Stroher et al., 2001). It has
been hypothesized that cytokines released from infected mononuclear
cells contribute highly to the hypotensive shock and cell damage
seen during the later course of infection (Schnittler and Feldmann,
1999; Stroher et al., 2001). Apoptosis is also seen in endothelial
cells from fatally infected patients (Baize et al., 1999). There
are probably numerous factors involved in the effects seen during
Ebola virus infections including virus induced cytokine production
that alters vascular permeability, as well as other yet to be
identified factors. Identification of these factors is important
for the discovery of drugs that can prevent and treat infection and
is a major goal of this proposal.
[0010] The Ebola virus envelope glycoprotein (GP) determines the
cell binding and entry properties of the virus. Ebola virus
glycoprotein contains both the receptor-binding domain and fusion
mechanism of the virus and is the primary target of a neutralizing
antibody response. It is the first virus protein that makes contact
with cells and may induce cell-signaling pathways that allow
establishment of infection. Ebola virus glycoprotein has also been
identified as a major viral determinant of vascular cell
cytotoxicity, permeability and injury (Yang et al., 2000). When
Ebola virus glycoprotein was expressed at levels comparable to
those seen in an in vivo infection, cell death resulted in a
variety of cell types. Treatment of cells with Ebola virus envelope
protein also resulted in rounding and detachment in culture
(Simmons et al., 2002). In rapid entry assays, it was observed that
Ebola virus takes a long time (4 h) to penetrate cells compared to
other viruses like Murine leukemia virus (20 min) or Vesicular
stomatitis virus. These observations suggest that Ebola virus
primes or triggers the cell for entry to take place. While it is
unlikely that Ebola virus glycoprotein cytotoxicity is solely
responsible for the pantropic destruction seen in Ebola
virus-induced hemorrhagic fever, this protein is still a major and
accessible virulence factor that requires further study.
[0011] Thus, prior art is deficient in methods to treat individuals
infected with filoviruses. The present invention fulfills this
long-standing need and desire in the art.
SUMMARY OF THE INVENTION
[0012] In one embodiment of the present invention, there is
provided a method of inhibiting entry of a filovirus into a cell.
This method comprises contacting the cell with an agent that blocks
activity of PI3 kinase signaling pathway protein(s) or
calcium-associated signaling pathway protein(s), thereby inhibiting
the entry of the filovirus into the cell.
[0013] In another embodiment of the present invention, there is
provided a method of inhibiting entry of a filovirus into a cell.
Such a method comprises contacting the cell with an agent that
downregulates expression of a gene(s) encoding PI3 kinase signaling
pathway protein(s) or of a gene(s) encoding calcium-associated
signaling pathway protein(s), thereby inhibiting the entry of the
filovirus into the cell.
[0014] In yet another embodiment of the present invention, there is
provided a method of treating an individual infected with a
filovirus. This method comprises administering pharmacologically
effective amounts of an agent that inhibits activity of the PI3
kinase signaling protein(s) or calcium associated signaling pathway
protein(s), thereby treating the individual infected with the
filovirus.
[0015] In still yet another embodiment of the present invention,
there is provided a method of treating an individual infected with
a filovirus. Such a method comprises administering
pharmacologically effective amounts of an agent that downregulates
the expression of gene(s) encoding PI3 kinase signaling pathway
protein(s) or calcium-associated signaling pathway protein(s),
where the downregulation of the gene inhibits entry of the
filovirus into the cells of the individual, thereby treating the
individual infected with the filovirus.
[0016] In another embodiment of the present invention, there is
provided a method of identifying compounds useful in the treatment
of infection caused by a filovirus. This method comprises
contacting a cell infected with the filovirus with the compound and
determining the levels of PI3 kinase signaling pathway protein(s)
or calcium associated signaling pathway protein(s), gene(s)
encoding said proteins or a combination thereof in the presence and
absence of said compound. The levels of the protein(s), the gene(s)
encoding the protein(s), or a combination thereof in the presence
of the compound are compared with the levels of the protein(s), the
gene(s) encoding the protein(s) or a combination thereof in the
absence of the compound, where a decrease in the levels of the
protein(s), gene(s) encoding the protein(s) or a combination
thereof in the presence of the compound is indicative that the
compound is useful in the treatment of infection caused by the
filovirus.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] FIGS. 1A-1C show validation of luciferase virus entry assay.
FIG. 1A shows that entry of Ebola virus GP pseudotype occurs with
slow kinetics. Luc-containing Ebola virus (open circle) and VSV
(solid circle) GP pseudotypes were incubated with HEK293 cells at
room temperature. After 10 mins, cells were washed to remove
unbound virus and incubated at 37.degree. C. At indicated time
intervals, aliquots of cells were withdrawn and luciferase entry
assay performed. For each virus, luciferase activity at different
time points was normalized to the maximum luciferase activity for
that virus. Each data point represents mean.+-.range of 2
independent experiments. FIG. 1B shows that anti-Ebola virus
neutralizing antibody specifically inhibits entry of Ebola virus GP
pseudotype. Luciferase entry assay was performed using Ebola virus
(open bars) or VSV (solid bars) GP pseudotype and HEK293 cells in
the presence of anti-Ebola virus neutralizing antibody (KZ52) or a
non-specific antibody (control). Data were normalized to luciferase
activity in cells incubated with respective untreated virus. Each
data point represents mean.+-.s.d. of 3 independent experiments.
FIG. 1C shows that entry of Ebola GP pseudotype is sensitive to
inhibitors of endosomal acidification. Luciferase entry assay was
performed using Ebola virus, VSV or Fr-MLV GP pseudotype and target
cells (HEK293 for VSV and Ebola virus or 293-CAT-1 for Fr-MLV) in
the presence of either 20 mM ammonium chloride (open bars) or 50 nM
bafilomycin A1 (solid bars). Data were normalized to luciferase
activity for untreated cells. Each data point represents
mean.+-.s.d. of 3 independent experiments.
[0018] FIG. 2A-2B shows the distribution of effects of siRNA on
infection of EBOV pseudotyped viruses. FIG. 2A shows the impact of
each single siRNA was plotted for the 720 targeted genes as a
function of fold-increase in standard deviation over that seen for
the negative control non-targeting siRNA (neg 1 and neg 2).
Positive controls targeting kif11 and firefly luciferase (fluc) are
also indicated. The fluc siRNA suppressed the infection reporter
gene encoded by the pseudotyped virus. 15 replicates of each
control are shown. For the siRNA that were part of the screen,
circles, squares and diamonds indicate distinct siRNA targeting
each gene. The cut-off of significance is indicated by the dashed
line and corresponds to three times the standard deviation of the
control signals. FIG. 2B shows RSA ranking of siRNA hits. All siRNA
that suppressed or elevated infection of EBOV envelope protein
pseudotyped virus were evaluated using the RSA algorithm that is
specifically designed to analyze siRNA screening data from screens
using multiple siRNA targeting each gene. Probability scores
assigned for each gene are shown. Scores of 10 indicate the gene
product was not classified as a potential hit. The threshold used
for the network analysis is indicated by the dashed line. All
scores below this line were used.
[0019] FIG. 3A-3C identified gene product networks impacting
infection by the EBOV pseudotyped virus. Three distinct kinase
network types were identified using the Ingenuity Pathway Analysis
software. These were MAP kinase (MAPK, FIG. 3A), PI3 kinase (PI3K,
FIG. 3B) and calcium/calmodulin dependent kinase (Ca.sup.2+/CALM,
FIG. 3C) dependent networks. Genes or gene products are represented
by inverted triangles and circles. Lines connecting symbols
represent known interactions in the Ingenuity database. Shaded
symbols indicate siRNA that were indicated as hits using the RSA
algorithm. Numbers below each symbol indicate assigned RSA
probability values.
[0020] FIGS. 4A-4D show that PI3K-Akt-1 pathway plays a critical
role in Ebola virus entry. FIG. 4A shows that wortmannin, but not
U0126 inhibits entry of Ebola virus. U0216 is an inhibitor of a
PI3K independent pathway, thus showing that PI3K inhibition is
specific. Entry assays were performed using VSV (solid bars) or
Ebola virus (open bars) GP pseudotype and HEK293 cells treated with
wortmannin (0.10) or U0126 (15 .mu.M). Data were normalized to
luciferase activity in DMSO (vehicle)-treated cells. Each data
point represents mean.+-.s.d. of 3 independent experiments. FIG. 4B
shows that PI3K inhibitor LY294002 and Akt-1 inhibitor inhibit
Ebola virus entry. Entry assays were performed using VSV (solid
bars) or Ebola virus (open bars) GP pseudotype and HEK293 cells
were treated with LY294002 (500) or Akt-1 inhibitor (1.00). Data
were normalized to luciferase activity in DMSO (vehicle)-treated
cells. Each data point represents mean.+-.s.d. of 3 independent
experiments. FIG. 4C shows that dominant negative mutant of the
regulatory subunit of PI3k reduces Ebola virus entry. HEK293 cells
were transfected with either pcDNA3 (empty vector) or a pcDNA3
encoding .DELTA.p85.alpha.. Entry assays were performed 36 h after
transfection using VSV (solid bars) or Ebola (open bars) GP
pseudotypes. Data were normalized to luciferase activity in
untransfected cells. Each data point represents mean.+-.s.d. of 2
independent experiments. FIG. 4D shows that inhibitors of
PI3K-Akt-1 pathway do not affect binding of Ebola virus to the
target cells. HEK293 cells were pre-treated with LY294002 (50
.mu.M) or Akt-1 inhibitor (1.0 .mu.M) for 1 h at 37.degree. C.,
followed by incubation with Ebola env pseudotyped virus for 10 min
at room temperature. Cells were then washed to remove unbound
virus, resuspended in luciferase assay buffer containing triton
X-100 detergent and luciferase activity measured. Data were
normalized to luciferase activity in vehicle-treated samples. Each
data point represents mean.+-.s.d. of 2 independent
experiments.
[0021] FIG. 5 shows that Ebola GP pseudotyped virus promotes Akt-1
phosphorylation. Serum starved HEK293 cells were incubated with
serum-free medium, Ebola GP pseudotyped virus or medium containing
10% fetal bovine serum, as indicated. After 1 h, cells were lysed
and phosphorylated Akt-1 was detected by Western blot (p-Akt-1,
upper panel). Subsequently, the same membrane was stripped and
reprobed for total Akt-1 (lower panel).
[0022] FIGS. 6A-6C show that Rac1 plays a role in Ebola virus entry
and serves as a downstream effector of Akt-1 signaling. FIG. 6A
shows that inhibitor of Rac1 inhibits Ebola virus entry. Cells were
treated with Rac1 inhibitor (100 .mu.M) and entry assays were
performed using VSV or Ebola GP virus pseudotypes. Each data point
represents mean.+-.s.d. of 3 independent experiments. FIG. 6B shows
expression of dominant negative Rac1 results in inhibition of Ebola
virus entry. HEK293 cells were transfected with either pcDNA3 (open
bars) or pcDNA3 encoding dominant-negative Rac1 (Rac1-T17N, solid
bars). Entry assays were performed 36 h later using VSV or Ebola GP
pseudotypes. Results are expressed as percent of luciferase
activity in untransfected cells. Each data point represents
mean.+-.range of 2 independent experiments. FIG. 6C shows that
expression of constitutively active Rac1 overcomes the inhibitory
effect of Akt-1 inhibitor on Ebola virus entry. HEK293 cells were
transfected with either pcDNA3 (open bars) or pcDNA3 encoding
constitutively active Rac1 (Rac1-G12V, solid bars). Entry assays
were performed 36 h later, in the presence of Akt-1 inhibitor (1.0
.mu.M) using VSV or Ebola virus GP pseudotypes. Results are
expressed as percent of luciferase activity in vehicle-treated
sample of each representative virus. Each data point represents
mean.+-.range of 2 independent experiments.
[0023] FIG. 7 is a model depicting role of PI3K in Ebola virus
entry. Attachment of Ebola virus to the cell surface receptor (step
1) activates PI3K (step 2) indirectly leading to phosphorylation
and activation of Akt-1 (step 3). Activated Akt-1 causes activation
of Rac 1, involving GDP to GTP exchange (step 4), which then
promotes assembly of the actin cytoskeleton, possibly at the site
of viral attachment (step 5), facilitating membrane trafficking,
virus endocytosis and/or fusions (steps 6 and 7).
[0024] FIG. 8 shows specific inhibition of Ebola entry by siRNA
targeting AK7, CAMK2 and DMPK. Cells were transfected with specific
siRNA by reverse transfection. After 2 days, (siRNA inhibition
peak) cells were challenged with Ebola envelope-pseudotyped viruses
carrying a Firefly Luciferase reporter gene. Cells were
simultaneously infected with a ecotropic murine leukemia virus
envelope (MLV)-pseudotype carrying a Renilla Luciferase reporter
gene (both luciferases are measured independently). The MOI for
each was less than 0.05 (fewer than 1/20 cells infected by each,
fewer than 1/400 cells by both). This internally controls for
adverse effects of siRNA challenge for each siRNA and allows rapid
identification of siRNA that specifically inhibit Ebola virus. The
bars indicate change in virus infectivity relative to negative
controls.
[0025] FIG. 9 shows that CAMK2 plays a role in Ebola virus
infection by treatment with the specific CAMK2 inhibitor KN-93.
Left: 293 cells were treated with KN-93 or vehicle control (DMSO)
and challenged with retrovirus pseudotypes bearing Ebola virus or
eMLV (Fr-MLV) envs. Luciferase activity was used to measure
infection. Right: 293 cells were treated as described with KN-93 or
its inactive homolog KN-92 (both at 10 .mu.M). Cells were infected
with either Ebola virus or Venezuelan equine encephalitis virus
(VEEV) pseudotyped VSV particles. KN-93 specifically blocked
infection with Ebola virus. Infection relative to untreated cells
is shown.
[0026] FIG. 10 shows cells treated with Tetrandine or Diltiazem at
5 or 10 .mu.M, respectively. They were then infected with
virus-like particles bearing Ebola virus (solid bars) or Venezuelan
equine encephalitis virus (open bars) envs. Infection was measured
by counting GFP-expressing cells.
[0027] FIGS. 11A-11B validate usage of drugs for treatment of
infection of the live virus. FIG. 11A shows effect of indicated
calmodulin inhibitor (Phenoxybenzamine hydrochloride (Pheno)) and
L-channel blocking drugs (Verapamil (Verap), Nimodopine (Nimod) and
Methoxyverapamil (MethV)) on Ebola virus entry into cells. Drugs
were tested on virus-like particles bearing Ebola (Eb) or
Venezuelan equine encephalitis virus envelope proteins. FIG. 11B
shows inhibition of infection of live Ebola Zaire virus by
inhibitors of PI3K (LY294002) and the calcium blocker
(methoxy-verampil). Vero E6 cells were infected with either live
Vesicular stomatitis virus (Indiana strain; solid bars) or live
Ebola virus (Zaire strain; open bars) after being treated either at
50 and 100 uM, respectively. After 6 hours, the drugs were removed
and new medium added. Cells were cultivated for 2 or 10 days for
Vesicular Stomatits virus or Ebola virus, respectively and plaques
were counted. Plaque numbers were normalized to DMSO (drug vehicle)
treated controls.
[0028] FIG. 12 shows CREB activation pathway of transcription. It
is proposed that Ebola virus binding to its (as yet unidentified)
receptor activates a calcium influx into the cell which in turn
activates calmodulin (CALM) and CAMK2 which trigger transcriptional
activation through CREB. This is key for Ebola virus infection.
[0029] FIG. 13A-13B shows the effects of drug inhibitors of PI3
Kinase and CAMK2 proteins on EBOV pseudotyped virus infection.
Cells were treated with LY294002, a specific inhibitor of the PI3K
catalytic subunit or KN-93, an inhibitor of CAMK2 or its inactive
derivative KN-92. FIG. 13A shows EBOV pseudotyped lentivirus was
added in the presence of each drug and then incubated with virus
for 6 h. Both drug and unbound virus were then removed and cells
were assayed for infection 40 h later by measuring expression of a
reporter gene (firefly luciferase). The average of 4
replicates.+-.st. dev. is shown. FIG. 130B demonstrates that drug
activity was not against the lentivirus core, a vesicular
stomatitis virus core was used to make additional pseudotyped
viruses. Aside from EBOV pseudotyped particles (open bars), an
additional pseudotype was made, using the envelope proteins of VEEV
(solid bars). Each drug was tested at 100 mM. The average of two
replicates.+-.st. dev. is shown.
[0030] FIG. 14 shows that verapamil protected cells against Ebola
virus induced cytopathic effect (CPE). Cells were infected with
wild type Ebola virus, Zaire strain, at an MOI of 1, without
treatment or in the presence of Verapamil (100 mM). After 7 days
images were taken. In the left panel a marked CPE is apparent with
many cells rounded up and floating or dead, whereas the cell
monolayer remained intact and confluent with treatment.
[0031] FIG. 15 shows the test for a synergistic inhibitory effect
of L-type calcium channel inhibitor (Verapamil) and calmodulin
antagonist (Trifluoperazine) on EBOV pseudotyped virus infection.
Cells were pretreated with indicated concentrations of Verapamil
and Trifluoperazine for 30 minutes and then EBOV pseudotyped virus
was added for 6 h, after which medium was replaced with fresh
medium. Luciferase activity was measured next day. The extent of
syngergism was calculated by the MacSynergy II software package (M.
Pritchard, University of Michigan). The graph shows the isobologram
for all concentrations tested and peaks indicate strong
synergy.
DETAILED DESCRIPTION OF THE INVENTION
[0032] The present invention is drawn to treatment of individuals
infected with filoviruses, which include but is not limited to
Ebola virus and Marburg virus. Specifically, the present invention
is drawn to treating such infections by directly inhibiting virus
entry into cells or by preventing envelope protein induced
toxicity. The envelope protein (env or GP) define the majority of
cell tropism for enveloped viruses by determining receptor
specificity and thereby, entry route into the cells. Virus GP
pseudotypes are virus-like particles that bear the GP proteins of
one virus over the core of another. They function as good proxies
to study virus entry in relative safety. Additionally, they are
accepted by most in the field to adopt the entry mechanism and
pathway. FIG. 11 demonstrates that the information gained using
such pseudotypes corresponds to that attained with live virus.
[0033] Briefly, it is demonstrated herein that inhibition of
protein activities in the PI3 kinase pathway (PI3K, Akt or Rac-1)
effectively blocked entry of Ebola envelope protein (GP) bearing
virus particles. PI3K and Akt make up a key cell signaling pathway
that become dysfunctional in many cancers. Also demonstrated herein
is that inhibition of calmodulin, CAMK2 enzyme or inhibition of
Ca.sup.2+ channels, effectively blocked entry of Ebola envelope
protein (envs) bearing virus particles. Each of these proteins
represents a link in the chain of a major cellular
calcium-associated signaling pathway. Calcium signaling has been
shown to be key in triggering numerous responses in cells including
gene transcription. It is a key cell signaling pathway that becomes
dysfunctional in many chronic diseases including high blood
pressure and Alzheimer's disease.
[0034] A rapid entry assay was used herein to determine the effect
of inhibition of PI3 kinase pathway on the entry of the filovirus
such as Ebola virus. The PI3 kinase pathway was inhibited using
siRNA, LY294002, Wortmannin or Akt-1 inhibitor. Such inhibition
resulted in inhibition of entry of Ebola virus in the cells but not
the binding of the Ebola virus to the target cells. Thus, it is
concluded herein that inhibition of PI3K, Akt or Rac-1 blocks the
Ebola virus infection at the point of virus entry. This is because
Ebola virus entry likely requires cytoskeleton rearrangement in the
cell that is stimulated by activation of PI3K and Akt. This pathway
has been shown to be involved in cancer formation. Hence, drugs
that treat cancer by suppressing the PI3 kinase pathway should
function as effective cures for infections caused by filovirus such
as Ebola virus. It is also contemplated that most filoviruses will
be affected by these drugs in the same manner as the Ebola virus
since they use the same pathway for infection. Additionally, since
this treatment does not work for murine leukemia viruses or VSV, it
is presumed that such a treatment is limited to filoviruses.
[0035] Furthermore, inhibition of CAMK2 using siRNA targeting CAMK2
and a CAMK2 inhibitor such as KN-93 blocked entry of Ebola virus
into cells. Additionally, since calmodulin acts to recruit and
trigger CAMK2 by binding the released calcium, calmodulin
inhibiting drugs such as phenoxybenzamine, W7 and trifluorperazine
were used to treat cells infected with Ebola virus. It was observed
that these drugs inhibited Ebola virus infection. Further,
treatment of cells with calcium channel blocking drugs such as
tetrandine and diltiazem specifically blocked Ebola virus
infection. Of the numerous calcium channel drugs specific for 4
classes of channels (L, N, P/Q and R) that were screened, L-channel
blocking drugs such as Verapamil and its derivative
methoxyverapamil, Nimodopine and other dihydropyridines were found
to be effective against Ebola virus entry. Since transient
treatment with these drugs is sufficient to irreversibly block
Ebola virus entry, it is contemplated that a short term usage of
these drugs should be effective in preventing Ebola virus infection
and spread.
[0036] Thus, it is concluded that inhibition of Ca.sup.2+ channels,
calmodulin or CaMK2 blocks Ebola virus infection at the point of
virus entry. This is because Ebola virus entry likely requires
transcriptional activation of the cell and synthesis of some
factor, possibly through activation of the nuclear transcription
factor, CREB. This pathway has been shown to be involved in
transmitting calcium-based signals to the cell nucleus by hormones
and other stimuli. Aside from the drugs tested herein, it is
contemplated that any calcium L-channel blocking drug will work
similarly. Additionally, most filoviruses will be affected by these
drugs as they use the same pathway for infection. Since the
treatment does not work for eMLV or VEEV, it is presumed that the
treatment is specific for filoviruses. It is also likely that the
calcium influx induced by Ebola virus particles is responsible for
the increase in vascular permeability syndrome (profuse internal
vascular bleeding and leakage of blood fluids from blood vessels,
etc) seen during infection that ultimately kills patients. If so,
then treatment with calcium channel blockers would also reduce this
cytotoxicity, treat Ebola virus associated disease symptoms and
could potentially cure the patient.
[0037] The present invention is directed to a method of inhibiting
entry of a filovirus into a cell, comprising: contacting the cell
with an agent that blocks activity of PI3 kinase signaling pathway
proteins or calcium-associated signaling pathway protein, thereby
inhibiting the entry of the filovirus into the cell. Representative
examples of the PI3 kinase signaling pathway protein may include
but is not limited to PI3 kinase, Akt, Rac-1 or a combination
thereof. Representative examples of such an agent may include but
is not limited to Wortmannin, LY294002, an Akt-1 inhibitor, a Rac-1
inhibitor or a dominant negative Rac-1. Alternatively,
representative examples of the calcium-associated pathway protein
may include but is not limited to calmodulin, CAMK2 enzyme or
Ca.sup.2+ channels. Representative examples of such an agent may
include but is not limited to KN-93, Tetrandine, Diltiazem or a
L-type channel blocker. Further, representative examples of the
L-type channel blocker may include but is not limited to verapamil,
methoxyverapamil or other dihydropyridine. Furthermore,
representative examples of the filovirus may include but is not
limited to an Ebola virus or a Marburg virus.
[0038] The present invention is also directed to a method of
inhibiting entry of a filovirus into a cell, comprising: contacting
the cell with an agent that down-regulates expression of a gene(s)
encoding the PI3 kinase signaling pathway protein(s) or a gene(s)
encoding calcium-associated signaling pathway protein(s), thereby
inhibiting the entry of the filovirus into the cell. Representative
examples of gene(s) downregulated by such an agent may include but
is not limited to the gene(s) that encodes PI3 kinase, Akt, Rac-1
or CAMK2. Additionally, representative examples of the filovirus
may include but is not limited to an Ebola virus or a Marburg
virus. Furthermore, examples of the agent that can be used in such
a method may include but is not limited to a siRNA, a peptide
nucleic acid, an inorganic chemical compound, an organic chemical
compound or a polypeptide.
[0039] The present invention is further directed to a method of
treating an individual infected with a filovirus, comprising:
administering pharmacologically effective amounts of an agent that
inhibits the activity of the PI3 kinase pathway proteins or
calcium-associated signaling pathway thereby treating the
individual infected with the filovirus. Such an inhibition may
block the entry of the filovirus into cells of the individual.
Further, representative examples of the PI3 kinase signaling
pathway proteins may include but is not limited to PI3 kinase, Akt,
Rac-1 or a combination thereof and those of the calcium-associated
signaling pathway may include but is not limited to calmodulin,
CAMK2 enzyme or Ca.sup.2+ channels. Representative examples of such
agents may include but is not limited to Wortmannin, LY294002, an
Akt-1 inhibitor, a Rac-1 inhibitor, a dominant negative Rac-1,
KN-93, Tetrandine, Diltiazem, dihydropyridine or a L-type channel
blocker. Additionally, representative examples of the L-type
channel blocker may include but is not limited to Verapamil or
methoxyverapamil. Representative examples of the filovirus may
include but is not limited to an Ebola virus or a Marburg
virus.
[0040] The present invention is still further directed to a method
of treating an individual infected with a filovirus, comprising:
administering pharmacologically effective amounts of an agent that
downregulates the expression of gene(s) encoding PI3 kinase
signaling pathway protein(s) or calcium-associated signaling
pathway protein(s), where the downregulation of the expression of
the gene(s) inhibits the entry of the filovirus into cells of the
individual, thereby treating the individual infected with the
filovirus. Examples of the agent useful in such a method may
include but is not limited to a siRNA, a peptide nucleic acid, an
inorganic chemical compound, an organic chemical compound or a
polypeptide. Additionally, examples of the genes whose expression
is downregulated by the agent may include but is not limited to
genes encoding PI3 kinase, Akt, Rac-1 calmodulin, CAMK2 or L-type
calcium channels. Examples of the filovirus may include but is not
limited to an Ebola virus or a Marburg virus.
[0041] The present invention is also directed to a method of
identifying compounds useful in treatment of infection caused by a
filovirus, comprising: contacting a cell infected with the
filovirus with the compound; determining the levels of PI3 kinase
signaling pathway protein(s) or calcium associated signaling
pathway protein(s), gene(s) encoding the proteins or a combination
thereof in the presence and absence of the compound; and comparing
the levels of the protein(s), the gene(s) encoding the protein(s),
or a combination thereof in the presence of the compound with the
levels of the protein(s), the gene(s) encoding the protein(s) or a
combination thereof in the absence of the compound, where a
decrease in the levels of the protein(s), the gene(s) encoding the
protein(s) or a combination thereof in the presence of the compound
is indicative that the compound is useful in the treatment of
infection caused by the filovirus. This method may further
comprise: performing viral entry assay to determine the ability of
the compound to inhibit entry of the filovirus in the cell.
Examples of the PI3 kinase signaling pathway protein may include
but are not limited to PI3 kinase, Akt or Rac-1 and those of the
calcium-associated signaling pathway protein may include but is not
limited to calmodulin, CAMK2 enzyme or Ca.sup.2+ channels.
Representative examples of the filovirus may include but are not
limited to an Ebola virus or a Marburg virus.
[0042] As used herein, the term, "a" or "an" may mean one or more.
As used herein in the claim(s), when used in conjunction with the
word "comprising", the words "a" or "an" may mean one or more than
one. As used herein "another" or "other" may mean at least a second
or more of the same or different claim element or components
thereof. As used herein, the term "PI3K" means PI3 Kinase. Whereas
the term "PI3 kinase signaling pathway" means those proteins that
form part of a signaling cascade including PI3 kinase. As used
herein, the term "GP" means the envelope glycoprotein of a virus.
As used herein, the term "h" means hour.
[0043] As used herein, the term "contacting" refers to any suitable
method of bringing the agent described herein into contact with a
virally infected cell. In vitro or ex vivo this is achieved by
exposing the infected cell to the agent in a suitable medium. For
in vivo applications, any known method of administration is
suitable as described herein.
[0044] The agents described herein can be administered
independently, either systemically or locally, by any method
standard in the art, for example, subcutaneously, intravenously,
parenterally, intraperitoneally, intradermally, intramuscularly,
topically, enterally, rectally, nasally, buccally, vaginally or by
inhalation spray, by drug pump or contained within transdermal
patch or an implant. Dosage formulations of the composition
described herein may comprise conventional non-toxic,
physiologically or pharmaceutically acceptable carriers or vehicles
suitable for the method of administration.
[0045] The agents described herein may be administered
independently one or more times to achieve, maintain or improve
upon a therapeutic effect. It is well within the skill of an
artisan to determine dosage or whether a suitable dosage of the
agents described herein comprises a single administered dose or
multiple administered doses. An appropriate dosage depends on the
subject's health, inhibition of the PI3 kinase or
calcium-associated signaling pathways and blockage of viral entry
into the cells, the route of administration and the formulation
used.
[0046] The following examples are given for the purpose of
illustrating various embodiments of the invention and are not meant
to limit the present invention in any fashion. One skilled in the
art will appreciate readily that the present invention is well
adapted to carry out the objects and obtain the ends and advantages
mentioned, as well as those objects, ends and advantages inherent
herein. Changes therein and other uses which are encompassed within
the spirit of the invention as defined by the scope of the claims
will occur to those skilled in the art.
Example 1
Materials and Methods
[0047] All chemicals were Ultragrade from Sigma (St. Louis, Mo.)
unless stated otherwise. Dharmafect Cell Culture Reagent (DCCR) and
DharmaFECT 1 transfection reagent used for siRNA transfection were
from Dharmacon (Lafayette, Colo.).
[0048] Cells were maintained in a humidified air-5% CO.sub.2
atmosphere incubator at 37.degree. C. 293 FT cells (Invitrogen,
Carlsblad, Calif.) were used to express envelope proteins by
plasmid transfection. Screens were performed using HEK293 cells and
were grown in DMEM (Invitrogen, Carlsblad, Calif.) supplemented
with 10% fetal bovine serum (Gemini Bioproducts, West Sacramento,
Calif.). Vero-E6 cells were used for verification of drug
activities on wild type virus and were cultured as for HEK293
cells.
Cultivation of Wild Type EBOV and Determination of Virus Titer
[0049] EBOV, Zaire strain, was cultivated on Vero-E6 cells by
infection at an MOI of 0.1. Culture supernatants were collected
after 10 d and clarified by centrifugation at 2000.times.g for 15
min. Virus concentration was determined by titration on Vero-E6
cells. Cells were incubated with virus for 1 h and then overlaid
with 0.8% tragacanth gum in DMEM/4% FBS. 10 d post-infection cells
were fixed with 2% formalin and stained with crystal violet to
count plaques. All experiments with EBOV were performed under
biosafety level 4 conditions in the Robert E. Shope BSL-4
Laboratory, UTMB.
Production of Envelope Protein Pseudotyped Lentiviruses
[0050] Pseudotyped lentiviruses were produced following published
methods [Kolokoltsov et al. 2005]. Virus envelope protein
pseudotyped lentiviruses (LV) were prepared for use in siRNA
screens and as controls in drug treatment experiments. Pseudotypes
were made bearing the envelope EBOV Zaire strain. For screening
purposes, the LV pseudotype encoded firefly luciferase as the
reporter of infection. Pseudotyped virus was assembled by
transfecting 293 FT cells (Invitrogen) with plasmids encoding HIV
LV core structural proteins (pLP1), HIV Rev (pLP2) together with
plasmids encoding the firefly luciferase reporter gene
(pLENTI6-fluc) and the envelope protein of the Zaire strain of EBOV
(pEBOVenv). The EBOV envelope expression construct was supplied by
Dr. Sanchez (CDC). The firefly luciferase reporter construct,
pLENTI6-fluc, was made by inserting a codon optimized firefly
luciferase gene between BamHI and XhoI sites of the pLENTI6 plasmid
(Invitrogen). Transfection was done by calcium phosphate [Chen and
Okayama 1987]. After 2 d, culture supernatants were collected,
filtered through a 0.45 mm filter to remove cell debris, and the
filtered virus was used immediately or stored frozen at -80.degree.
C.
[0051] The titer of the pseudotyped virus was determined by
limiting dilution on HEK293 cells. The concentration of virus was
then optimized to yield 20-50,000 cps per 10 ml of virus culture
supernatant added per well of a 96-well plate. The expression of
luciferase was detected 36 h post-infection using a Veritas 96-well
plate reading luminometer. Assays were performed in white walled
96-well Costar tissue culture plates (Corning, Lowell, Mass.). From
previous work, approximately 100 cps corresponded to 1 colony
forming units of virus. This set the MOI for infection to 0.05 per
well.
Production of Envelope Protein Pseudotyped Vesicular Stomatitis
Viruses
[0052] Vesicular stomatitis virus core pseudotyped virus was made
according to described methods [Matsuura et al. 2001]. HEK293 cells
were transfected with plasmids encoding EBOV (described above) or
VEEV envelope protein [Kolokoltsov et al. 2005]. After 24 h, the
cells were infected with a GFP-encoding VSV parent stock virus that
was defective for VSV-G expression (provided by Dr. M. Whitt,
University of Tennessee, Memphis) and progeny pseudotyped virus
harvested after a further 24 h. The titer of the pseudotyped virus
was determined by counting GFP-expressing infected cells, 18 h post
infection, using an epifluorescence microscope.
siRNA Library Screening and Analysis
[0053] The siRNA library used was a subset of the druggable genome
library (Ambion, Austin, Tex.) comprising siRNA targeting kinase
and phosphorylase genes. A total of 720 distinct genes were
targeted using 3 independent siRNA for each gene. Each siRNA was
tested independently in separate wells.
[0054] The method used was described [Kolokoltsov et al. 2007]. All
transfections were performed in 96-well format. The siRNA was
dissolved in 25 ml of 1.6% (v/v) stock of DharmaFECT 1 transfection
reagent in DCCR (Dharmacon, Calif.) and incubated for 30 min. Then
0.5.times.10.sup.4 HEK293 cells in 100 ml medium were added and
incubated for 24 h. The final concentration of pooled siRNA per
well was 40 nM. This amount was sufficient to reduce the expression
of recombinant firefly luciferase by 95% [Kolokoltsov et al. 2007].
Also, this amount was shown to effectively reduce expression of
numerous endogenous genes by at least 5-fold, as determined by
Western blot [Kolokoltsov et al. 2007].
[0055] For screening purposes, 48 h after transfection with siRNA,
pseudotyped virus was added to HEK293 cells at an MOI of 0.05.
Firefly luciferase expression was measured at 36 h post-infection
using Dual-glo reagent (Promega, Madison, Wis.) in a Veritas plate
reader (Turner Biosystems, CA). Controls used in the assays were
use of transfection reagent alone, and two non-targeting siRNA
(Ambion, Tex.). Two other siRNA served as positive controls and
indicators of transfection efficacy and knockdown efficacy. One was
against firefly luciferase (Ambion, Tex.) which suppressed
expression of pseudotyped virus-encoded firefly luciferase. The
second was against kif11 (Ambion, Tex.) which when suppressed, is
cytotoxic, suppressing productive infection. In all cases, cell
viability was checked by visual inspection using phase contrast
microscopy.
[0056] Analysis of screening results was performed in two phases.
The first phase used a recently reported algorithm, redundant siRNA
activity analysis (RSA), that was specifically designed to analyze
data from siRNA screens [Konig et al. 2007]. The algorithm takes
into account the fact that siRNA targeting the same gene may not be
equally effective at inducing a knockdown effect. The importance of
a gene in assay outcome is determined by assigning probability
values that define the statistical significance of the impact of a
set of siRNA targeting one gene that yielded a significant effect
on the assay being used. This approach has been shown to be more
effective at identifying active siRNA, with higher confirmation
rates, in high throughput screens than standard ranking methods
[Konig et al. 2007]. For this, the activity of the luciferase
infection reporter, in cells transfected with library siRNA, was
normalized to the mean of the signals obtained for the negative
control siRNA. The negative control siRNA had been performed at
least 39 times each for the assay and were distributed throughout
the sets of plates. The normalized data was then analyzed using the
RSA algorithm implemented in the Perl programming language. Two
sets of data were generated. One set analyzed reductions in
activity of the infection reporter and the other analyzed increases
in the infection signal. Values less than or equal to 0.58, or more
than or equal to 1.72 times the mean of the control siRNA were used
as the cut-off thresholds respectively. This corresponded to 3
times the average standard deviation of the signals from the
control sets.
[0057] In the second phase of analysis, the assigned probability
values generated by the RSA algorithm and associated gene
identifiers were submitted for analysis using the Ingenuity
Pathways Analysis software package (Ingenuity Systems,
www.ingenuity.com). Each gene identifier was mapped to known
networks in the Ingenuity Pathways Knowledge Base. A cutoff value
of less than or equal to a probability score of 0.2 was set as the
threshold for selection of genes of interest. For canonical
pathways, p-values were calculated by the Ingenuity software using
Fischer's exact test, which is a measure of the probability that
the selected genes are associated with a pathway by chance
alone.
Drug Treatments
[0058] To confirm involvement of siRNA identified signaling
proteins in EBOV entry and infection, cells were challenged with
virus in the presence of specific inhibitor drugs. KN-93 inhibits
calmodulin kinase 2 (CAMK2). KN-92 is a derivative of KN-93 that
has weak affinity for CAMK2 and served as a negative control for
off-target activities. LY294002 was used to target
phosphatidylinositol-3-kinase (PI3K) and is widely accepted to be
specific for PI3K with no appreciable off-target effects at the
concentration used. In each case the drugs were dissolved in DMSO
to give a 100.times. concentrated stock solution and were diluted
in culture medium immediately before use.
[0059] For testing of drugs, both pseudotyped virus and wild type
EBOV were used. Cells were prepared similarly for each assay. Cells
were seeded to 50% confluency, which is approximately 10.sup.4
cells for each well of a 96-well plate or 5.times.10.sup.5 cells
for each well of a 6-well plate. The plate was incubated at
37.degree. C. for 4 h to allow for cell attachment. For pseudotyped
virus, HEK293 cells were used in 96-well plates while Vero cells in
6-well plates were used for wild type EBOV. Each drug was added to
the cells 1 h before addition of virus. The medium was then
replaced with fresh medium containing drug and virus. Typical
infection dosage was at an MOI of 0.005 for wild type EBOV or 0.05
for pseudotyped virus.
[0060] For the pseudotyped lentivirus, assays were read after 40 h.
The medium was removed and Dual-glo luciferase substrate added
(Promega, Madison, Wis.), following the manufacturer's
recommendations. The plate was then read using a Veritas
luminometer (Turner Biosystems, Sunnyvale, Calif.). For wild type
EBOV, cell monolayers were treated as described for determination
of virus titer and plaques were counted. For the pseudotyped VSV
particles, assays were read at 18 h post infection by counting
GFP-expressing cells using an epifluorescence microscope.
Statistical analysis, curve fitting and calculation of IC50 values
was performed using Graphpad Prism software (GraphPad Prism version
4.00 for Windows, GraphPad Software, San Diego Calif. USA,
www.graphpad.com). Data were compared by one way ANOVA with the
Tukey-Kramer post test.
Example 2
Inhibition of Filovirus Entry into Cells by Blocking of the PI3
Kinase Pathway
[0061] A rapid entry assay developed for Ebola virus pseudotypes
was used herein. This is potentially a powerful diagnostic tool
(FIG. 1). Briefly, human-derived 293 HEK cells which are generally
accepted as good models for study of virus infection of cells in
the human body were used herein. Initial work identified the
potential role of PI3 Kinase isoforms in Ebola virus infection by
performing an siRNA screen by treating cells with siRNA targeting
cell kinase genes. The activity of genes important for infection by
each virus was identified by comparing cells infected with either
Ebola Zaire or ecotropic murine leukemia virus (eMLV) GP
pseudotyped viruses. Since eMLV and Ebola virus envs have different
receptor specificities and functions, differences between the
infection activity of each virus indicated specific genes important
for the respective virus. The screen was set up to give readout of
infection by two independent viruses. Firefly luciferase (fLuc) and
Renilla luciferase (RLuc) were used as reporters of infection for
Ebola virus and eMLV pseudotypes respectively. Through this screen
PI3Kinase was identified as important for Ebola virus infection but
did not influence eMLV.
[0062] To identify new potential leads for developing antiviral
therapies against EBOV an siRNA library targeting cell kinases and
phosphatases, often referred to as the kinome was screened. Both
sets of enzymes are important drug targets, as they are active in
many aspects of cell function that could impact virus infection.
These include receptor-ligand mediated signaling pathways, gene
regulation and cell cycle control. They are also often found
dysregulated in many chronic diseases such as cancers.
[0063] An EBOV envelope protein pseudotyped virus was used for
screening. A viral pseudotype is a virus particle that bears the
envelope proteins of a virus of interest over the core of another
virus, in this case a lentivirus. The pseudotyped virus particle
typically adopts the receptor specificity, cell tropism and entry
characteristics dictated by the foreign envelope protein.
Retroviruses are often used to make pseudotypes as they readily
adopt new envelope proteins which can function to give infection by
the retrovirus core. Retroviral pseudotypes are also advantageous
as they can be made in the absence of the natural envelope proteins
and with replication incompetent genomes that are modified to
express marker proteins upon infection. Such pseudotyped viruses
recapitulate the entry characteristics of the envelope protein
donor and EBOV retrovirus pseudotypes have been used to study EBOV
infection mechanisms. Most importantly, pseudotyped viruses offer
the ability to study the features of a highly pathogenic virus at
lower biocontainment and are more amenable to high-throughput
screening platforms. Nonetheless, it is still important to validate
all findings attained with pseudotyped viruses by testing live
virus as well.
[0064] Since three distinct siRNA were screened per gene target, it
was important to rank the importance of targets based on the
overall impact of each set of siRNA on infection. This is relevant
because not all siRNA perform equally well. Simple ranking of siRNA
effectiveness based on a single strong effect of one siRNA or the
average effect of each does not take this into account. For this,
redundant siRNA activity (RSA) analysis was used. This algorithm
assigns a probability score (p-value) that indicates the
statistical relevance for each gene being important for screening
outcome, based on the extent of impact on the assay and the
performance of each siRNA targeting the same gene [Konig et al.
2007]. The cut-off threshold used in this analysis was set to 3
times the standard deviation in signals seen in the negative and
positive controls (one standard deviation was 14%). Therefore a
change of or less than or equal to 0.58 or greater than or equal to
1.72 times the average infection rate seen for controls was used as
the threshold for significance. One quarter (26.2%) of the siRNA
were defined as potential hits, having a significant impact on
pseudotyped virus infection. These siRNA not only inhibited
infection but many (45% of the active siRNA) actually increased the
infection signal (FIG. 2). The latter may indicate that these siRNA
inhibited expression of cell proteins that misdirect virus into
non-productive pathways or are part of anti-viral host defense
mechanisms.
[0065] To gain further insight into potential roles that these
genes and gene products played in infection, all hits were analyzed
using Ingenuity Pathway Analysis software. While this software is
primarily designed to identify changes in gene expression patterns
using genechips, it was recently used to identify related genes
important for intracellular growth of Salmonella bacteria. Instead
of supplying infection assay data, the probability scores from the
RSA analysis were used to prioritize network assignments. This
provides an additional filter to remove false positive signals and
permit detection for genes that would otherwise be missed using a
simple ranking system. A total of 9 networks containing more than
15 genes each were indicated as important for infection by the EBOV
pseudotyped virus. Two of the top four networks were related and
shared MAP kinases. These gene products are key signaling proteins
involved in control of cell growth and proliferation. The remaining
networks did not share more than one gene in common. Of the
remaining networks, one containing PI3 kinases and another
containing calcium/calmodulin dependent kinases were ranked in the
top four (FIG. 3). PI3 kinases modulate signaling from membrane
bound receptors to promote cell growth, survival and movement.
Calcium/calmodulin kinases are key for nerve function and memory
but also play important roles in signal transduction to the nucleus
through CREB in non-neuronal cells.
[0066] A set of canonical, (well characterized) signaling pathways
were also indicated as important (Table 1). These contained each of
the gene products from the networks discussed above, except for the
calcium/calmodulin kinase-containing network. For each of the
canonical pathways identified, either MAP kinase or PI3 kinase
related gene products were prominent components. Fisher's exact
test yielded scores showing a non-random association of each set of
hits from the screen with the indicated pathways (Table 1).
TABLE-US-00001 TABLE 1 List of canonical pathways and gene product
networks identified important for infection by pseudotyped EBOV
particles. Number of siRNA targeting/ total genes in network
*p-value Canonical pathway SAPK/JNK Signaling 32/147 4.1 .times.
10.sup.-25 PI3K/AKT Signaling 30/176 2.0 .times. 10.sup.-21
Inositol Phosphate 26/173 6.7 .times. 10.sup.-20 metabolism Ephrin
Receptor 32/232 9.1 .times. 10.sup.-20 Signaling ERK/MAPK 30/226
1.6 .times. 10.sup.-17 Signaling Other networks Calcium/calmodulin
24/34 *N.A. signaling *p-values were calculated by the Ingenuity
Pathways Analysis package for canonical pathways using Fisher's
exact test as described in the methods.
[0067] To confirm the role of PI3K in Ebola virus infection, cells
were treated with the specific PI3K inhibitor LY294002 which was
expected to give a similar effect to that seen with siRNA. Cells
were plated 1 day prior to each experiment, then they were
preincubated with the indicated drug for 1 hr at the indicated
dosage before adding the virus. After 1 h preincubation virus was
added for another 3 h and then excess drug and virus was removed
and virus entry were measured (FIG. 4). The retrovirus pseudotypes
were used and luciferase activity used to give a measure of virus
entry. To rule out a virus core specific effect, similar studies
were performed with vesicular stomatitis virus core to make the
pseudotypes using the rapid entry assay. No effect was seen for any
of the drugs used. Therefore the impact of PI3k inhibition was
specific for Ebola virus.
[0068] PI3K is an initial signaling molecule in a cascade that
leads to stimulation of gene transcription. One the effectors in
this pathway is Akt. It was expected then that inhibition of Akt
would also lead to inhibition of Ebola virus entry and this was the
case (FIG. 4). Akt-1 was also phosphorylated after treatment with
Ebola virus particles (FIG. 5). Akt can influence a number of
downstream proteins. One of these is Rac-1 which is important for
cell cytoskeleton rearrangement. To test that Rac1 was the
downstream target of PI3K-Akt activation both a Rac-1 and a
dominant negative Rac1 protein were used. Cell treated with drug or
expressing the dominant negative protein gave lower entry signals
for Ebola virus. VSV entry remained unaffected (FIG. 6).
Example 2
Inhibition of Filovirus Entry into Cells by Blocking a
Calcium-Associated Signaling Pathway
[0069] Human-derived 293 HEK, Hela cells and African green
monkey-derived Vero cells, which are generally accepted as good
models for study of virus infection of cells in the human body were
used herein. Initial work identified the potential role of CAMK2
isoforms in Ebola virus infection by performing an siRNA screen. In
this experiment, cells were treated with siRNA targeting cell
kinase genes. The activity of genes important for infection by each
virus was identified by comparing cells infected with either Ebola
Zaire or ecotropic murine leukemia virus (eMLV) env pseudotyped
viruses. Since ecotropic murine leukemia virus and Ebola envs have
different receptor specificities and functions, differences between
the infection activity of each virus indicated specific genes
important for the respective virus. The screen was set up to give
readout of infection by two independent viruses. Firefly luciferase
(fLuc) and Renilla luciferase (RLuc) were used as reporters of
infection for Ebola virus and ecotropic murine leukemia virus
pseudotypes, respectively. Through this screen CAMK2D was
identified as important for Ebola virus infection but did not
influence eMLV (FIG. 8).
[0070] To confirm the role of CAMK2 in Ebola virus infection, cells
were treated with the specific CAMK2 inhibitor KN-93 which was
expected to give a similar effect to that seen with CAMK2D siRNA.
Cells were plated 1 day prior to each experiment, then they were
preincubated with the indicated drug for 1 hr at the indicated
dosage before adding the virus. After 1 h preincubation, virus was
added for another 1 h and then excess drug and virus was removed by
replacing the culture medium. Cells were incubated with drug for
another 5 h and then media was replaced to normal and infection was
determined by counting infected cells 30 hr later (FIG. 9). Again,
the retrovirus pseudotypes were used and luciferase activity used
to give a measure of virus infection. To rule out a virus core
specific effect similar studies were performed with vesicular
stomatitis virus core to make the pseudotypes. This core-encoded
green fluorescent protein as the marker of infection. In this case
KN-93 activity was compared to the inactive homolog KN-92 (FIG. 9,
right). In both cases a strong effect of KN-93 was demonstrated for
blocking only the Ebola infection. CAMK2 is an intermediary in
transcriptional activation through CREB.
[0071] CAMK2 is typically triggered by calcium influx into the
cytoplasm either from internal storage vesicles or from the
exterior of the cell. Calmodulin (Calm) acts to recruit and trigger
CAMK2 by binding the released calcium. To demonstrate this, the
Calmodulin inhibiting drugs, Phenoxybenzamine, W7 and
Trifluorperazine were used to treat cells infected with Ebola virus
or Venezuelan equine encephalitis virus-like particles. In each
case the drugs inhibited Ebola virus but not Venezuelan equine
encephalitis virus infection (FIGS. 11A-9B). For the activation of
this pathway calcium flow through calcium channels embedded in the
cell membrane is an important trigger.
[0072] If true, it was expected that calcium channel blocking drugs
should specifically inhibit entry of Ebola virus. Calcium channel
blocking drugs are one type of commonly prescribed blood pressure
medication used by many in the US and other countries. These drugs
are collectively termed as dihydropyridines and represent a class
of FDA approved drugs that can be taken orally with limited side
effects. Initially, two calcium channel blocking drugs, Tetrandine
and Diltiazem, were used to test this concept. Cells were treated
as for the CAMK2 inhibitor, KN-93 and then infected with VSV core
pseudotypes. Venezuelan equine encephalitis virus and Ebola virus
envelope particles were compared. As anticipated both drugs proved
to be potent and specific blockers of Ebola infection (FIG.
10).
[0073] Numerous calcium channel drugs that are specific for each of
the 4 classes of channel (L, N, P/Q and R) were also screened. It
was observed that that L-type channels elicit the effect seen.
Commercially available and FDA approved L-channel blocking drugs
were then carefully tested for potency. From this, Verapamil and
its derivative Methoxyverapamil were identified as being highly
effective against Ebola virus entry. However, Nimodopine and other
dihydropyridines were also effective. It is known that transient
treatment with each drug is sufficient to irreversibly block Ebola
virus entry into cells. This suggests that even a short term usage
of such drugs should be effective at preventing Ebola virus
infection and spread.
[0074] The siRNA profiling coupled with RSA and network analysis
predicted the involvement of signaling pathways in EBOV infection.
The usefulness of this prediction was tested by treatment of cells
with inhibitors of components for two of the identified networks.
Drugs that target the catalytic subunit of
phosphatidylinositol-3-kinase (PI3K) and calcium/calmodulin kinase
2 (CAMK2) were used. LY294002 inhibits the kinase activity of PI3K
and KN-93 prevents association of CAMK2 with calmodulin which is
required for its kinase activity. These targets were chosen as they
are integral parts of two distinct networks and blocking either of
these was predicted to inhibit the function of the entire network
for which they are part. Each drug is also highly specific for its
substrate, is cell membrane permeable and share low micromolar
affinities for their enzyme targets.
[0075] Each drug was tested for inhibition of infection of the
pseudotyped virus using a range of dosages and dose response curves
were fitted to the data by non-linear regression (FIG. 13A).
LY294002 was most active with a calculated IC50 value for infection
of 6.5.+-.1.1 mM (R.sup.2=0.94), while KN-93 had an IC50 of
21.4.+-.1.1 mM (R.sup.2=0.94). The KN-93 derivative, KN-92, which
has much weaker activity against CAMK2, was also tested and was
found to be less effective at blocking infection by the pseudotyped
virus, only approaching 50% reduction in infection at the highest
dose tested (100 mM). These findings suggested that the siRNA
profiling had successfully identified two important cell signaling
pathways for which activity is required for infection by the
pseudotyped virus. However, since a pseudotyped virus was used, the
siRNA and drugs may have impacted the expression of the infection
marker that was controlled by the lentivirus core.
[0076] To demonstrate that the drugs were specific for the function
of the EBOV envelope proteins and not the lentivirus core, an
additional pseudotyped virus was generated and tested. The
lentivirus core was substituted for that of vesicular stomatitis
virus (VSV). Again, the infection of the VSV core pseudotyped virus
was significantly impacted (P<0.001) by either LY294002 or KN-93
but not KN-92 (P>0.05). Since each pseudotype shared a common
envelope protein, the activity of the drugs most likely act at an
EBOV envelope protein-dependent step in infection. As a final test,
the EBOV envelope protein was replaced with that of Venezuelan
equine encephalitis virus (VEEV). In contrast to what was seen with
the EBOV pseudotyped virus, the VEEV pseudotyped particles were not
significantly affected by any of the drugs (P>0.05, FIG. 13B).
This finding confirmed that the drugs were specifically affecting
the function of the EBOV envelope proteins and not the activity of
the pseudotyped virus core.
[0077] FIG. 14 shows that verapamil protected cells against Ebola
virus induced cytopathic effect. Cells were infected with wild type
Ebola virus, Zaire strain, at an MOI of 1, without treatment or in
the presence of Verapamil (100 mM). After 7 days images were taken.
In the left panel a marked cytopathic effect is apparent with many
cells rounded up and floating or dead, whereas the cell monolayer
remained intact and confluent with treatment. Thus, treatment with
verapamil reduced or significantly prevented the Ebola virus
induced cytopathic effect.
[0078] FIG. 15 illustrates the synergistic inhibitory effect of the
L-type calcium channel inhibitor (Verapamil) and the calmodulin
antagonist (Trifluoperazine) on EBOV pseudotyped virus infection.
The graph shows the isobologram for all concentrations tested and
peaks indicate strong synergy.
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[0103] Any patents or publications mentioned in this specification
are indicative of the levels of those skilled in the art to which
the invention pertains. Further, these patents and publications are
incorporated by reference herein to the same extent as if each
individual publication was specifically and individually indicated
to be incorporated by reference.
* * * * *
References