WO2012118599A1

WO2012118599A1 - C-abl tyrosine kinase inhibitors useful for inhibiting filovirus replication

Info

Publication number: WO2012118599A1
Application number: PCT/US2012/023953
Authority: WO
Inventors: Daniel Kalman; Gary J. Nabel; Mayra GARCIA; Arik Arie COOPER
Original assignee: Emory University; US Department of Health and Human Services
Current assignee: Emory University; US Department of Health and Human Services
Priority date: 2011-02-28
Filing date: 2012-02-06
Publication date: 2012-09-07
Anticipated expiration: 2013-08-28

Abstract

This disclosure provides method of treating a Filoviridae viral infection, such as an Ebola virus infection or a Marburg virus infection, comprising providing an effective amount of a c-Abl tyrosine kinase inhibitor to a patient in need thereof. The c-Abl tyrosine kinase inhibitor may be a biological inhibitor that decreases expression of the c-Abl tyrosine kinase, such as a c-Abl tyrosine kinase specific siRNA. However it is preferred that the c-Abl tyrosine kinase inhibitor is a small molecule c-Abl tyrosine kinase antagonist. Suitable c-Abl tyrosine kinase antagonists include dasatinib, imatinib, and nilotinib and the pharmaceutically acceptable salts thereof.

Description

C-ABL TYROSINE KINASE INHIBITORS USEFUL FOR INHIBITING FILO VIRUS

REPLICATION

CROSS REFERENCE TO RELATED APPLICATION

This application claims priority from US Provisional Application No. 61/447,298, filed February 28, 2011, which is hereby incorporated by reference in its entirety.

STATEMENT OF GOVERNMENT SUPPORT

[0001] This invention was made in part with government support from the National Institutes of Health. The government has certain rights in this invention.

FIELD OF THE DISCLOSURE

[0002] c-Abl tyrosine kinase antagonists inhibit virus growth through their effect on VP-40 induced budding, and c-Abl tyrosine kinase antagonists inhibit virus growth. c-Abl specific siRNAs and c-Abl tyrosine kinase inhibitors are provided herein as Ebola and Marburg viral replication inhibitors. Compositions and methods for treating and preventing viral infection, including Ebola and Marburg viral infections, are also provided by this disclosure.

BACKROUND

[0003] The highly lethal Ebola virus is well known for its fulminant replication that ultimately overwhelms the ability of the human immune system to contain it.

[0004] Ebolavirus (EBOV) and Marburg virus (MARV) represent filoviruses that cause severe, often fatal, hemorrhagic disease in humans and non-human primates. Because of their high mortality and human transmissibility, they remain biological threats for which effective prophylactic and therapeutic interventions are needed.

[0005] Among the seven gene products of Ebola virus, NP, VP35 and VP24 are necessary and sufficient for nucleocapsid assembly, and additional expression of a fourth gene, VP40, permits release of virions. Viruses exploit a variety of cellular components to complete their life cycle. Among them, the c-Abl and related tyrosine kinases (TKs) have been implicated in actin motility and release of poxviruses and in the lifecycles of other bacterial and viral pathogens. Because similar host factors might affect the budding and release of the highly filamentous Ebola virus, inventors tested the hypothesis that c-Abl may regulate Ebola virus replication and delineated the mechanism by which it achieves this effect.

[0006] The present disclosure provides inhibitors of filovirus c-Abl tyrosine kinase pathway useful for inhibiting viral replication. The disclosure fulfills the need for additional prophylactic and therapeutic treatments for Ebola virus and Marburg virus and provides additional advantages discussed below.

SUMMARY

[0007] c-Abl specific siRNAs and c-Abl tyrosine kinase inhibitors are provided herein as Ebola and Marburg viral replication inhibitors.

[0008] A method of treating a Filoviridae viral infection, such as an Ebola virus infection or a Marburg virus infection, comprising providing an effective amount of a c-Abl tyrosine kinase inhibitor to a patient in need thereof is provided by the disclosure. The c-Abl tyrosine kinase inhibitor may be a biological inhibitor that decreases expression of the c-Abl tyrosine kinase, such as a c-Abl tyrosine kinase specific siRNA. However, it is preferred that the c-Abl tyrosine kinase inhibitor is a small molecule c-Abl tyrosine kinase antagonist. Suitable c-Abl tyrosine kinase antagonists include dasatinib, imatinib, tivozanib, ponatinib, bafetinib, saracatinib, fingolimod, AT 9283, KW 2449, danusertib, and nilotinib and the pharmaceutically acceptable salts thereof. In certain embodiments the c-Abl tyrosine kinase antagonist is dasatinib, imatinib, or nilotinib or a pharmaceutically acceptable salt.

[0009] The disclosure includes methods of treating a Filoviridae viral infection in which the c-Abl tyrosine kinase inhibitor is provided together with an additional active agent. Suitable additional active agents include ribavirin and interferon, such as interferon alpha- 2b.

[0010] Methods of treatment include both therapeutic methods in which the patient is known to be infected with a Filoviridae virus, such as Ebola virus or Marburg virus, and prophylactic methods. Because Ebola virus and Marburg virus are highly contagious, treating health care workers who will be exposed to these viruses prophylactically to reduce their chance of becoming infected is particularly included as an aspect of this disclosure.

[0011] The c-Abl tyrosine kinase inhibitor may be provided by any pharmaceutically acceptable method, such as administration as an oral dosage form or intravenously. In certain embodiments the c-Abl tyrosine kinase inhibitor is provided to an infected patient together with supportive treatment such as intravenous fluids given to prevent or reverse dehydration and/ or transfusions of platelets. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] FIGURE 1. Transient transfection of NP, VP40, VP35, VP24 and GP gives rise to Ebola VLPs in 293T cells. (A) Western blot analysis of EBOV VLP

immunoprecipitates from 293T cells transfected with empty vector control or the indicated combinations of VP24, VP35, VP40, NP and GP plasmids. Antibodies to GP were used for immunoprecipitation. (B) Electron microscope pictures of cells transfected with control plasmid or Ebola plasmids. Control cells are shown on the left. Morphology of Ebola virus nucleocapsids (middle) or VLPs (right) in 293T cells transfected with the five EBOV plasmids seen intracellularly (middle, arrows) and in supernatant fluids (right, arrows) by electron microscopy. Size standards are shown. The virions emerging from the cell surface contained nucleocapsids (right, arrows).

[0013] FIGURE 2. Effect of c-Abll knockdown and kinase inhibition on Ebola VLP release in transfected 293T cells. (A) Knockdown of c-Abll (lanes 1-3) or c-Abl2 (lanes 4-6) using non-targeting siRNA control or siRNA targeting c-Abll or c-Abl2 confirmed by Western blotting in cell lysates with antibodies specific to either c-Abll (lanes 1-3) or c-Abl2 (lanes 4-6). β-actin was used as a loading control. The results are representative of 5 independent experiments. 293T cells were transfected with plasmids encoding VP24, VP35, VP40, NP, and GP. In all cases, EBOV VLPs were analyzed by immunoprecipitation with GP followed by Western blotting for NP and VP40 (lanes 10-12). Cell lysates are shown in lanes 7-9. Data represent the mean + SEM of individual measures with cells from 4 independent experiments. (B) Knockdown of c-Abll using a non-targeted siRNA control (lane 13) or three individual siRNAs (S9, S10, Sll) targeting c-Abll (lanes 14-16) was analyzed by Western blotting in cell lysates, with eIF4E as a loading control. NP and VP40 present in cell lysates were determined (lanes 17-20), and EBOV VLPs in supernatants were measured as in (A) after knockdown with c-Abll individual siRNAs (lanes 21-24). Data represent the mean + SEM of individual measures with cells from 3 independent experiments. For A and B, quantitation of NP and VP40 protein bands is expressed as a percentage of the intensity of the siRNA control band (lower panels). (C) Western blot analysis of EBOV VLP release for NP and VP40 content with imatinib (lanes 25-27) or nilotinib (lanes 28-30).

Water was used as control for imatinib and DMSO for nilotinib. Quantitation of NP and VP40 was performed relative to the intensity of the solution control band. Data are presented as mean + SEM of individual measures with cells from 8 (for NP) and 3 (for VP40) independent experiments. Significant differences (P < 0.05) by paired Student' s t test between test and control are indicated (*).

[0014] FIGURE 3. Toxicity analysis, morphology of Ebola intracellular

nucleocapsids, and nucleocapsid formation. (A) 293T cells viability analysis. Toxicity of 10 or 20 μΜ imatinib or nilotinib or vehicle control was measured by 7-AAD exclusion in untransfected cells 36-48 hours after the drug was added. Data are presented as mean + SEM of 3 independent experiments. (B) Morphology of Ebola virus nucleocapsids in 293T cells transfected with empty vector (left panel) or the five EBOV plasmids in combination with water or DMSO controls (middle panels) or drug treatments at 20 μΜ (right panels) seen intracellularly (arrows) by electron microscopy. Size standards are shown. (C) Nucleocapsid formation. Effect of nilotinib (lane 4) in nucleocapsid formation on cells transfected with VP24, VP35, and NP. Anti-NP antibody was used for immunoprecipitation of nucleocapsids. Quantitation of NP and VP35 was performed relative to the intensity of the DMSO control band (lane 3). Input amount of protein is shown in lanes 1 and 2. Data are presented as mean + SEM of individual measures with cells from 3 independent experiments.

[0015] FIGURE 4. GP levels in 293T cell lysates after drug treatment. Western blotting analysis of GP from 293T cells transfected with VP24, VP35, VP40, NP and GP plasmids in the presence of imatinib (A) or nilotinib (B). Water was used as the solution control for imatinib and DMSO for nilotinib. Quantitation of GP was performed relative to the intensity of the solution control band. For nilotinib, intracellular GP levels were normalized to loading control. Data are presented as mean + SEM of individual measures with cells from 3 independent experiments.

[0016] FIGURE 5. c-Abll- specific siRNA and nilotinib effects on VP40 VLP egress. 293T cells were transfected with plasmid encoding VP40. VP40 VLPs were analyzed by immunoprecipitation with monoclonal antibody anti-FLAG followed by Western blotting for VP40 after transfection with a pool (A, lanes 4-6) or individual (B, lanes 11-14) siRNAs specific to cAbll . Cell lysates are shown in lanes 1-3 and 7-10. VP40 levels in VLPs were normalized to inputs. Quantitation of VP40 protein bands is expressed as a percentage of the intensity of the siRNA control band. Data represent the mean + SEM of individual measures with cells from 3 independent experiments for A. (C) Western blotting analysis of VP40 VLP release after immunoprecipitation with monoclonal antibody anti-FLAG (lanes 17 and 18). Cells were incubated with 20 μΜ nilotinib (lanes 16 and 18). DMSO was used as the solution control (lanes 15 and 17). Cell lysates are shown in lanes 15 and 16. Quantitation of VP40 was performed relative to the intensity of the solution control band. Data are presented as mean + SEM of individual measures with cells from 3 independent experiments.

Significant differences (P < 0.05) by paired Student's t test between test and control are indicated (*).

[0017] FIGURE 6. Tyrosine phosphorylation of c-Abll and VP40 after expression of c-Abll in 293T cells. (A) Western blotting analysis of c-Abll activation in 293T cells transfected with empty vector control (lanes 1 and 4) or wild type c-Abll expression vector (lanes 2, 3, 5 and 63) 24 hours before EBOV plasmid transfection. Autophosphorylation on Tyr 412 and 245 was measured by Western blotting for phospho tyro sine in cell lysates after c-Abll immunoprecipitation (lanes 2 and 5). 20 μΜ imatinib (lane 3) or 20 μΜ nilotinib (lane 6) was used as a control to confirm its specificity (lane 3). The blot was re-probed for c- Abll expression (middle panel), β-actin was used as a loading control. The results are representative of two three independent experiments. (B) Western blot analysis of phosphotyrosine content in lysates (lanes 7-9) or immunoprecipitates (lane 10 - 12) of cells transfected with wild type c-Abll and independent EBOV plasmids. (C) Western blot analysis of phosphotyrosine content in cells transfected with empty vector control (lanes 7 and 10, 13, 16, 19), wild type c-Abll (lanes 8 and 11, 14, 17, 20) or kinase-dead c-Abll (lanes 12, 15, 18, 219 and 12) in the presence of VP40. The blot was re-probed for VP40 and c-Abll expression (middle panels). eIF4E was used as a loading control (bottom panel). The results are representative of 2 individual experiments.

[0018] FIGURE 7. Effect of c-Abll expression on VP40 phosphorylation and sensitivity to tyrosine kinase antagonist inhibition and siRNA. (A) Western blotting analysis of transfected cell lysates (lanes 1-4) or VP40 immunoprecipitates (lanes 5-8) of cells transfected with empty vector control (lanes 1, 3, 5, and 7) or wild type c-Abll (lanes 2, 4, 6 and 8) in the presence of VP40 expression vector. DMSO control (lanes 1, 2 and 5, 6) or 20 μΜ of nilotinib (lanes 3, 4 and 7, 8) was added 12-18 hours after transfection. Kinase activity on VP40 was measured by Western blotting of inputs using an anti-phosphotyrosine antibody (upper panel). Western blotting was also performed for c-Abll and flag-tagged VP40 (middle panels). eIF4E was used as a loading control (lower panel). Analyses are representative of 3 independent experiments. (B) Western blotting analysis of input cellular lysates (lanes 9-11) or VP40 immunoprecipitates (lanes 12-14) in cells transfected with non- targeting siRNA control (lanes 9, 10, 12, and 13) or a smart pool of siRNA targeting c-Abll (lanes 11 and 14) in the absence (lanes 9, 11, 12, and 14) or presence (lanes 10, 13) of 20 μΜ of imatinib. Kinase activity on VP40 was measured by Western blotting of inputs using an anti-phosphotyrosine antibody (upper panel). Western blotting was also performed for c- Abll and flag-tagged VP40, middle panels, β-actin was used as a loading control (lower panel). Analyses are representative of 3 independent experiments. (C) Western blotting analysis of phosphotyrosine and c-Abll immunoprecipitates. Cells were transfected with EBOV VP40 and then treated with DMSO vehicle (lanes 15, 16, 18, 19, 21 and 22) or 20 μΜ nilotinib (lanes 17, 20 and 23). Cell lysates (lanes 15-17) were immunoprecipitated with an anti-pTyr antibody, PY20 (pTyr, lanes 18-20) or anti-c-Abll antibody, 8E9 (c-Abll, lanes 21- 23) and Western blotting performed for flag-tagged VP40 and c-Abll. β-actin was used as a loading control. The results are representative of 2 independent experiments. (D) Western blotting analysis of phosphotyrosine immunoprecipitates. Cells were transfected with non- targeting siRNA control (lanes 24, 27) or siRNA targeting c-Abll (lanes 25, 28) or c-Abl2 (lanes 26, 29) for a day before EBOV VP40 transfection. Cell lysates (lanes 24-26) were immunoprecipitated with an anti-pTyr antibody, PY20 (pTyr, lanes 27-29) and Western blotting performed for flag-tagged VP40. The results are representative of 3 independent experiments. (E) Western blotting analysis of cell lysates and extracellular VLPs with an anti-pTyr antibody, PY20, and anti- flag-tagged VP40 upon expression of empty vector (lanes 31 and 34) or WT VP40 (lanes 32 and 35) in the five plasmid mixture in 293T cells. Lanes 30 and 33 show no transfection. Inputs are shown in lanes 30-32 with β-actin as a loading control. VLPs were purified by sucrose density sedimentation gradients. The results are representative of 2 independent experiments. (F) Co-immunoprecipitation of NP and VP40. Cells were cotransfected with NP and VP40 and then treated with DMSO (lanes 39 and 44) or 20 μΜ nilotinib (lanes 40 and 45). NP and flag-tagged VP40 in cell lysates (lanes 36-40) and VP40 immunoprecipitates (lanes 41-45) were detected by Western blotting using rabbit anti-NP antiserum and anti-FLAG monoclonal antibody, β-actin was used as a loading control. NP levels in VLPs were normalized to NP levels in lysates and then expressed as a ratio of the DMSO control sample (lower panel). Data are presented as mean + SEM from 4 independent experiments and significance analyzed by paired Student's t test.

[0019] FIGURE 8. Localization of VP40 c-Abll -mediated tyrosine phosphorylation and effect of Tyr mutation on VLP release. (A) Expanded region of the matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) spectrum of the VP40 (amino acids 3-21) acquired for the non-phosphorylated (left) vs. the phosphorylated (right) peptides from 293T cells co-transfected with VP40 and c-Abll. Analysis was performed on a gel slice of a flag-tagged VP40 immunoprecipitate. Arrow indicates site of Y13 phosphorylation of VP40. (B) Western blotting analysis of VP40 phosphorylation using WT or VP40 mutants created by site-directed mutagenesis on Y13. Cells were transfected with empty vector control (lanes 1, 3, 5, and 7) or wild type c-Abll vector (lanes 2, 4, 6, and 8) in the absence (lanes 1, 2, 5, and 6) or presence (lanes 3, 4, 7 and 8) of Y13A VP40 labeled with a flag tag. WT VP40 was used as a reference in both cases. Phosphorylation on Tyr was measured by Western blotting (upper panel) in cell lysates (lanes 1-4) or in VP40 immunoprecipitates (lanes 5-8). Western blotting analysis was also carried out for VP40 by flag labeling (middle panel), β-actin was used as a loading control (lower panel). The results are representative of 4 independent experiments. (C) Western blotting analysis of cell lysates and extracellular VLPs upon expression of empty vector (lanes 9 and 12), WT VP40 (lanes 10 and 13) or Y13A VP40 (lanes 11 and 14) EBOV VP40 in the five plasmid mixture in 293T cells. Inputs are shown in lanes 9-11 with eIF4E as a loading control. VLPs were purified by sucrose density sedimentation gradients. The results are representative of 3 independent experiments. (D) Quantitation of the ratio of NP in VLPs relative to cell lysate NP based on panel C (lanes 9-11 and 12-14). Data are presented as mean + SEM of individual measures with cells from 3 independent experiments and significance analyzed using paired Student's t test.

[0020] FIGURE 9. Mass spectrometry analysis of VP40 modification in the absence of c-Abll tyrosine kinase expression vectors in transfected 293T cells. (A) Expanded region of the MALDI-TOF MS spectrum of the VP40 (amino acids 292-326) acquired for the non- phosphorylated (left) vs. the phosphorylated (right) peptides from 293T cells co-transfected with VP40 and c-Abll . Arrow indicates site of Y292 phosphorylation of VP40. (B) Expanded region of the MALDI-TOF MS spectrum of the VP40 (amino acids 4-21) acquired for the non-phosphorylated (left) vs. the phosphorylated (right) peptides is shown. Results are presented in the absence of c-Abll tyrosine kinase expression vectors. The increase in m/z values by 40 from 1030.8 to 1071. lm/z (indicated by arrows) is consistent with the molecular weight and charge of a phosphorylation. (C) Western blotting analysis of VP40 phosphorylation using WT or VP40 mutants created by site-directed mutagenesis on Y13, Y292, or Y13+Y292. Cells were transfected with empty vector control (lanes 1, 3, 5, 7, 9, 11, 13, and 15) or wild type c-Abll vector (lanes 2, 4, 6, 8, 10, 12, 14, and 16) in the presence of WT (lanes 1, 2, 9, and 10) or Y13A (lanes 3, 4, 11, and 12) or Y292A (lanes 5, 6, 13, and 14) or Y13A+Y292A (lanes 7, 8, 15, and 16) VP40 labeled with a flag tag. WT VP40 was used as a reference in all cases. Phosphorylation on Tyr was measured by Western blotting (upper panel) in cell lysates (lanes 1-8) or in VP40 immunoprecipitates (lanes 9-16). Western blotting analysis was also carried for VP40 by flag labeling (middle panel), β-actin was used as a loading control (lower panel). The results are representative of 3 independent experiments.

[0021] FIGURE 10. Nedd4 and c-Abll interaction with VP40. (A) Co- immunoprecipitation of Nedd4 (top panel) with VP40 in the absence (lane 3) or presence (lane 4) of 20 μΜ nilotinib. Inputs are shown on lanes 1 and 2. Western blotting analysis was also carried out for VP40 by flag labeling (middle panel). eIF4E was used as a loading control (lower panel). Data are presented as mean + SEM of 3 independent experiments. (B) Co-immunoprecipitation of Nedd4 (top panel, left) with VP40 (lane 7) or Y13A mutant (lane 8), or c-Abll (top panel, right) with VP40 (lane 11) or Y13A mutant (lane 12). Inputs are shown on lanes 5-6 and 9-10. Western blotting analysis was also carried out for VP40 by flag labeling (middle panel). eIF4E or β-actin were used as loading controls (lower panels). Data are presented as mean + SEM of 3 independent experiments. Significant differences (P < 0.05) by paired Student's t test between WT and mutant VP40 are indicated (*).

[0022] FIGURE 11. Effect of siRNA knockdown and c-Abll tyrosine kinase inhibition on EBOV replication. (A) Effect of a non-targeted siRNA control or individual siRNAs targeting c-Abll (S9, S10, SI 1) on Zaire EBOV release from Vero cells on day 7 after infection. Cells were infected at multiplicity of infection of 1. Background viral load for day 1 was subtracted. Data are presented as mean + SEM of individual measures with cells from 2 independent experiments. (B) Viral load was measured in supernatant fluids of Vero cells infected with Zaire EBOV and treated with nilotinib (20 μΜ) and measured by TCID₅₀ on days 0, 1, 2, 7, and 8 after infection compared to DMSO vehicle control (right panel). Background viral load for day 0 was subtracted. Data are presented as mean + SEM of 4 individual measures and significance analyzed by paired Student's t test. p.i. = postinfection.

[0023] FIGURE 12. Infection assays. (A) Vero cells viability analysis. Toxicity of 20 μΜ nilotinib or DMSO compared to untreated cells was measured by 7-AAD exclusion in uninfected Vero cells on day 1, 2, 3, and 7 after the drug was added. Data are presented as mean + SEM of 3 independent experiments. (B) Infection viability analysis. Toxicity of 20 μΜ nilotinib or DMSO was measured by an approximation of the number of floating cells in Zaire EBOV-infected Vero cells on days 0, 1, 2, 5, 7 and 8 after the drug was added. The results are representative of 2 independent experiments. (C) Adenovirus infection assay. Effect of 20 μΜ nilotinib on adenovirus 5 infection of HEK 293T cells. Data are presented as mean + SEM of triplicate wells from 2 independent experiments. (D) Pseudo virus infection assay. Effect of_10 or 20 μΜ nilotinib on EBOV-luciferase pseudotyped virus infection of 293 A cells. DMSO was used as control. Data are presented as mean + SEM of single wells from 4 independent experiments. (E) Knockdown of c-Abll in Vero cells using a non-targeted siRNA control (lane 1) or three individual siRNAs (S9, S10, SI 1) targeting c- Abll (lanes 2-4) was analyzed by Western blotting in cell lysates, with β-actin as a loading control. The results are representative of 2 independent experiments. RLU = relative light units; cps = counts per second.

DETAILED DESCRIPTION

TERMINOLOGY

[0024] Prior to setting forth the disclosure in detail, it may be helpful to provide definitions of certain terms to be used herein. Compounds are described using standard nomenclature. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which this disclosure belongs.

[0025] The use of the terms "a", "an", and "the" and similar referents in the context of the disclosure (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising", "having", "including", and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to") unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the disclosure unless otherwise claimed. "About" indicates an approximate amount, including the quantity it modifies. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention. [0026] Isotopes include those atoms having the same atomic number but different mass numbers. By way of general example, and without limitation, isotopes of hydrogen include tritium and deuterium and isotopes of carbon include ^UC, ¹³C, and ¹⁴C.

[0027] An "active agent" is any compound, element, or mixture, that when administered to a patient alone or in combination with one or more other agents confers a therapeutic benefit on the patient. When the active agent is a compound, solvates (including hydrates) of the free compound or salt, crystalline and non-crystalline forms, as well as various polymorphs or the compound are included. For example, an active agent can include optical isomers of the compound and pharmaceutically acceptable salts thereof either alone or in combination.

[0028] The term "dosage form" denotes a form of a pharmaceutical composition that contains an amount sufficient to achieve a therapeutic effect with a single administration. The term "oral dosage form" is meant to include a unit dosage form prescribed or intended for oral administration. An oral dosage form may or may not comprise a plurality of subunits such as, for example, microcapsules or microtablets, packaged for administration in a single dose.

[0029] The term "effective amount" means an amount effective, when administered to a human or non-human patient, to provide any therapeutic benefit such as an amelioration of symptoms, e.g., an amount effective to decrease the symptoms of viral infection, and preferably an amount sufficient to decrease the symptoms of Ebola virus or Marburg virus infection. An "effective amount" may also be an amount sufficient to decrease viral load or viral antibodies in the patient's blood tissues and symptoms. In certain circumstances a patient suffering from a virus may not present symptoms of being affected. Thus a therapeutically effective amount of a compound is also an amount sufficient to provide a positive effect on any indicia of disease, e.g. an amount sufficient to prevent a significant increase or to significantly reduce the detectable level of markers for viral infection in the patient's blood, serum, or tissues. A significant increase or reduction in the detectable level of viral infection markers is any detectable change that is statistically significant in a standard parametric test of statistical significance such as Student's T-test, where p < 0.05.

Lymphocytic leukemia markers are discussed in more detail in the "method of treatment" section.

[0030] A "patient" is any human or non-human animal in need of medical treatment. In preferred embodiments the patient is a human patient determined to have a filovirus infection, such as an Ebola virus infection or a Marburg virus infection. Medical treatment can include treatment of an existing condition, such as a disease or disorder, or prophylactic or preventative treatment. For example treatment can be treatment of a patient infected with a filovirus, such as an Ebola virus or a Marburg virus. In another embodiment treatment can be prophylactic treatment of a patient, such as a health care worker, who does not yet show symptoms of infection, wherein the treatment additionally comprises determining the patient has been in contact with a human or non-human animal infected with Ebola virus or Marburg virus with the past 0 to 14 days or will be in contact with a human or non-human animal infected with Ebola virus or Marburg virus in the following 0 to 14 days. Preferably prophylactic treatment will be continued for 1 to 10 days after the patient is no longer in contact with a human or non-human animal infected with Ebola virus or Marburg virus.

[0031] "Pharmaceutically acceptable salts" includes derivatives of the disclosed compounds, wherein the parent compound is modified by making non-toxic acid or base addition salts thereof, and further refers to pharmaceutically acceptable solvates, including hydrates, of such compounds and such salts. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid addition salts of basic residues such as amines; alkali or organic addition salts of acidic residues such as carboxylic acids; and the like, and combinations comprising one or more of the foregoing salts. The pharmaceutically acceptable salts include non-toxic salts and the quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. For example, non-toxic acid salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like; other acceptable inorganic salts include metal salts such as sodium salt, potassium salt, cesium salt, and the like; and alkaline earth metal salts, such as calcium salt, magnesium salt, and the like, and

combinations comprising one or more of the foregoing salts.

[0032] Pharmaceutically acceptable organic salts include salts prepared from organic acids such as acetic, trifluoroacetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, mesylic, esylic, besylic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic, HOOC-(CH₂)_n-COOH where n is 0-4, and the like; organic amine salts such as triethylamine salt, pyridine salt, picoline salt, ethanolamine salt, triethanolamine salt, dicyclohexylamine salt,

Ν,Ν'-dibenzylethylenediamine salt, and the like; and amino acid salts such as arginate, asparginate, glutamate, and the like, and combinations comprising one or more of the foregoing salts. CHEMICAL DESCRIPTION

[0033] c-Abl tyrosine kinase inhibitors include both biological molecules and small molecules that reduce the activity of c-Abl tyrosine kinase whether by reducing the levels of c-Abl protein as in the case of c-Abl tyrosine kinase specific siRNAs or by interacting directly with the c-Abl tyrosine kinase protein and thereby reducing its enzymatic activity (c- Abl tyrosine kinase antagonists).

[0034] Effective c-Abl tyrosine kinase antagonists for use in this method include dasatinib (CAS Reg. No. 302962-49-8), imatinib (CAS Reg. No. 152459-95-5), imatinib mesylate (CAS Reg. No. 220127-57-1), and nilotinib (CAS Reg. No. 641571-10-0). These molecules have the following chemical structures:

dasatinib

imatinib

nilotinib

[0035] Analogues of these molecules, which are also useful in the methods of treatment described herein, have been described. US Patent No. 6,596,746, is hereby incorporated by reference at columns 31-270 for its teachings regarding dasatinib and its analogues. US Patent No. 5,521,184 is hereby incorporated by reference at columns 21-29 for its teachings regards imatinib and its analogues. US Patent No. 7,169,791 is hereby incorporated by reference at columns 14- 17 and 26-52 for its teachings regards nilotinib and its analogues.

[0036] Other c-Abl tyrosine kinase inhibitors useful in the methods of treatment described herein include tivozanib (CAS Reg. No. 475108-18-0), ponatinib (CAS Reg. No. 1114544-31-8), bafetinib (CAS Reg. No. 887650-05-7), saracatinib (CAS Reg. No. 379231- 04-6), fingolimod (CAS Reg. No. 162359-55-9), AT9283 (CAS Reg. No. 896466-04-9), KW-2449 (CAS Reg. No. 1000669-72-6), and danusertib (CAS Reg. No. 827318-97-8).

BIOLOGICAL DESCRIPTION

[0037] Inventors have discovered that Ebola virus replication is regulated by the c- Abl tyrosine kinase. Release of Ebola virus-like particles (VLPs) in a cell culture co- transfection system was inhibited by c-Abl- specific siRNA or kinase inhibitors. This effect is mediated by the Ebola matrix protein, VP40. Phosphorylation of tyrosine (Y) 13 on VP40 is stimulated by c-Abl expression, and a VP40 Y13A mutation inhibited release of Ebola VLPs. Productive replication of the highly pathogenic Ebola virus Zaire strain is inhibited by c-Abl siRNAs and by nilotinib, a c-Abl kinase inhibitor. These findings identify a previously unrecognized mechanism of filovirus regulation useful in antiviral therapy.

[0038] Inventors have also identified an unexpected modification by c-Abl of an essential Ebola virus gene product, VP40. EBOV VLP egress is inhibited by c-Abl siRNAs.

[0039] Inventors examined the effect of c-Abl on Ebola virus assembly and release initially by using a cell transfection system previously shown to support virus-like particle (VLP) generation in vitro. Transfection of expression vectors encoding VP24, VP35, VP40, NP, and GP into the 293 human renal epithelial cell line induced VLPs (1-3) detectable by both immunoprecipitation and electron microscopy (FIG. 1). To determine whether c-Abll affected VLP release, Inventors knocked doTwn c-Abll or the related c-Abl2 with specific siRNAs (FIG. 2A, lanes 1-6). c-Abl2 siRNA had no effect on c-Abll levels (FIG. 2A, lane 1 vs. 3) or vice versa (FIG. 2A, lane 4 vs. 5). Notably, transfection of c-Abll siRNA decreased the quantity of VLPs by ~5-fold as measured by NP, and by ~2.5-fold as measured by VP40 protein levels following immunoprecipitation with GP (FIG. 2A, lane 11). No effect was observed on intracellular levels of Ebola NP or VP40 proteins (FIG. 2A, lanes 7-9). The effect was specific; similar effects were evident with three individual siRNAs for c-Abll (FIG. 2B, lanes 14-16 and 22-24), whereas c-Abl2 siRNA or a control siRNA had no effect (FIG. 2A, lanes 10 and 12), and c-Abll siRNAs did not alter expression of an unrelated control protein eIF4E (FIG 2B, compare lane 13 with 14-16). Moreover, c-Abll siRNAs had no effect on intracellular levels of Ebola NP or VP40 proteins (FIG. 2B, lanes 17-20). At the same time, they decreased VLP release (FIG 2B, lanes 21-24) as measured by NP and VP40 protein levels, suggesting that they affected egress of preassembled VLPs from the cell. Release of EBOV VLPs is decreased by c-Abll tyrosine kinase inhibitors

[0040] Inventors next determined the effect of specific Abl-family TK inhibitors on VLP production. Imatinib and nilotinib inhibit c-Abll kinase activity and are used clinically for treatment of chronic myelogenous leukemia; a disease caused by translocations or mutations that dysregulate c-Abll. Incubation of 293T cells with imatinib (FIG. 2C, left panel) or nilotinib (FIG. 2C, right panel) 12-18 hours after transfection of EBOV plasmids resulted in a dose-dependent reduction in VLP release measured by NP and VP40 protein levels. No toxicity was observed at the selected concentrations (FIG. 3A). Neither drug reduced the formation of intracellular nucleocapsids as detected by transmission electron microscopy (Fig. 3B) or Western blotting analysis (FIG. 3C), or affected intracellular levels of NP, VP40, and VP35 (Fig. 2C, upper panels and FIG. 3C, lanes 1 and 2), suggesting a specific effect of the drugs on trafficking or egress. On the other hand, levels of GP, reported to enhance release of VLPs, were not affected by the drug treatment (FIG. 4A and B).

Release of VP40 VLPs is also decreased by c-Abll tyrosine kinase inhibitors

[0041] In addition to nucleocapsid-containing VLPs, Inventors assessed the effect of c-Abll on VP40-induced VLPs lacking nucleocapsid. VP40 VLPs were reduced by c-Abll - specific siRNAs at levels similar to those observed in complete VLPs (FIG. 5A and B).

Furthermore, release of VP40 VLPs was similarly reduced by nilotinib (FIG. 5C).

c-Abll tyrosine kinase phosphorylates Ebola proteins

[0042] To define potential viral targets of c-Abll, Inventors investigated the presence of Tyr kinase phosphorylation sites in EBOV proteins. Notably, VP40 contains a sequence surrounding Y18 that is homologous to a sequence in the Tir protein of enteropathogenic E. coli, which serves as an Abl/Tec phosphorylation site and an SH2 binding site. To corroborate this observation, Inventors transfected 293T cells with a c-Abll expression vector in conjunction with an EBOV VP40 plasmid. Transfected c-Abll was catalytically active as evidenced by autophosphorylation on Y412 (FIG. 6A, lanes 2 and 5), an effect inhibited by imatinib (FIG. 6A, lane 3) and nilotinib (FIG. 6A, lane 6). Upon immunoprecipitation, VP40 was likewise phosphorylated and also associated with c-Abll (FIG. 7A, lane 6, upper and middle panels). The effect appeared specific; both phosphorylation of VP40 and its association with c-Abl were antagonized by nilotinib (FIG. 7A, lanes 6 vs. 8). Moreover, kinase dead c-Abll did not induce either detectable phosphorylation of VP40 or association with c-Abl upon immunoprecipitation (FIG. 6B, lane 11 vs. 12). Finally, co-transfection of c-Abll siRNAs with the VP40 and c-Abll expression vectors inhibited tyrosine

phosphorylation of VP40 and c-Abll association (FIG. 7B, lanes 12 vs. 14) similar to the drug (FIG. 7B, lanes 12 vs. 13). Activation of c-Abl by overexpression, or its inhibition by drug treatment, increases VP40 expression with a fold change of 1.75 + 0.19, a statistically significant difference (p= 0.02). This observation could be the result of either more transcription or less turnover of VP40. The basis for such changes in VP40 levels is unclear. However, these changes are independent of the effects on Ebola replication, a phenomenon that is only observed when c-Abll kinase activity is inhibited.

Endogenous c-Abll also phosphorylates VLP-associated VP40

[0043] Without co-transfection, immunoprecipitation with a phosphotyrosine mAb or c-Abll mAb followed by Western analysis with anti-VP40 confirmed that endogenous tyrosine phosphorylation of VP40 (FIG. 7C, lanes 19 vs. 20) and its association with c-Abll (FIG. 7C, lanes 22 vs. 23) were also sensitive to nilotinib. Endogenous phosphorylation of VP40 by c-Abll was confirmed after c-Abll siRNA treatment followed by

immunoprecipitation with a phosphotyrosine mAb and Western analysis with anti-VP40 (FIG. 7D, lanes 27 vs. 28). Using available antibodies, Inventors could not resolve endogenous phospho-c-Abl upon immunoprecipitation, either because the levels were too low, or because the protein cycles into a dephosphorylated state. Incorporation of phosphorylated VP40 into VLPs was observed by Western blotting analysis of isolated VLPs through a sucrose cushion (FIG. 7E, lane 35). Finally, the association of VP40 with NP was reduced by nilotinib (FIG. 4F, lanes 44 vs. 45) to background levels (FIG. 7F, lane 42), indicating a reduced NP-VP40 interaction. Together, these data provide evidence that c-Abll associates with VP40 and mediates its phosphorylation. Additionally, phosphorylated VP40 is incorporated into VLPs.

Y13 is a site of phosphorylation in VP40 by c-Abll

[0044] To assess the role of VP40 phosphorylation in Ebola VLP release, Inventors determined the sites of tyrosine phosphorylation in VP40 by mass spectrometry after cotransfection of VP40 and c-Abll. Tryptic peptides containing phosphorylated Y residues were predicted to increase the m/z ratio by 40 compared to unphosphorylated peptides, and matrix-assisted laser desorption ionization (MALDI) analysis detected several peptides with such m/z deviations, including Y13 (FIG. 8A, right vs. left) and Y292 (FIG. 9A, right vs. left panels). A similar modification of VP40 Y13 was evident with endogenous c-Abll, in the absence of co-transfected c-Abll (FIG. 9B, right vs. left panels).

[0045] Inventors then asked whether c-Abll mediated phosphorylation of a mutated VP40 containing a substitution of A for Y at position 13 (VP40^Y13A). No phosphorylation of immunoprecipitated VP40^Y13A was detectable by Western blot analysis (FIG. 8B, lanes 6 vs. 8), even upon co-transfection with c-Abll. In contrast, VP40 remained phosphorylated (FIG. 9C, lanes 10 vs. 14) while the double mutant (FIG. 8C, lanes 10 vs. 16) did not, suggesting that Y13 and not Y292 was a primary target of c-Abll.

Phosphorylation of Y13 by c-Abl is required for EBOV VLP egress

[0046] To determine whether phosphorylation of Y13 was involved in EBOV VLP egress, Inventors co-transfected wild type VP40 or the VP40^Y13A mutant with the other EBOV plasmids and assessed VLP release. No difference in intracellular VP40 or NP levels was evident (FIG. 8C, left panel, lanes 10 vs. 11). However, with the Y13A mutant, levels of released VLP-associated NP were reduced by 84% (FIG. 8C, lanes 13 vs. 14, and FIG. 8D). Cotransfection of VP40 and Nedd4, a protein known to interact at position Y13 of the matrix protein, did not show effects on the interaction with Nedd4 in the presence of nilotinib (FIG. 10A, bar graph) yet disrupted interactions with c-Abll (FIG. 7A). In addition, Y13A disrupted Nedd4 interaction with VP40 (FIG. 10B, left graph) while the interaction with c- Abl was retained (FIG. 10B, right graph). Together, these data suggest that phosphorylation of VP40 is necessary for the egress of VLPs. This effect is mediated by phosphorylation of Y13 by c-Abll, and independent of Nedd4. Accordingly, nilotinib abrogated VP40 phosphorylation and VLP egress.

c-Abll tyrosine kinase inhibitors reduce productive replication of wild type Ebola virus.

[0047] Inventors next determined whether c-Abll regulates productive replication of EBOV Zaire, a strain that is lethal in humans and NHPs, which was identified after an outbreak in 1976 in the Ebola River valley of Zaire, now the Democratic Republic of the Congo. Treatment of Vero E6 cells with the S9 or S10 c-Abll siRNAs reduced Zaire EBOV production by 8- to 10-fold seven days after infection (FIG. 11 A), measured by relative levels that generated 50 percent of a tissue culture infectious dose (TCID₅₀). After incubation of Vero cells with nilotinib, EBOV infectious virus production decreased by up to ~4 logs eight days after infection (FIG. 11B). Notably, there was no detectable cytopathic effect of the drug alone (FIG. 12A and B), and nilotinib had no effect on infection by an unrelated virus, adenovirus 5 (FIG. 12C), confirming the specificity of this inhibition. To discriminate an effect of the drug on entry rather than on egress, Inventors used a luciferase-pseudotyped HIV virus with EBOV envelope to infect 293A cells. EBOV GP-mediated entry was not affected by nilotinib in concentrations up to 20 μΜ (FIG. 12D). The greater magnitude of drug inhibition (FIG.1 IB), compared to c-Abll siRNA treatment (FIG. 11 A), was likely due to the ability of these small molecules to diffuse into cells, compared to the lower transfection efficiency of siRNA delivery in Vero cells (FIG. 12E). Collectively, these data demonstrate that c-Abll regulates Ebola Zaire growth in vitro and that c-Abll tyrosine kinase antagonists inhibit productive replication.

METHODS OF TREATMENT

[0048] c-Abll tyrosine kinase antagonists disclosed herein are useful for treating viral infections in patients, including filovirus infections, such as Ebola virus infection or Marburg virus infection.

[0049] This disclosure provides methods of treating viral infections, including filovirus infections, such as Ebola virus infections and Marburg virus infections, by providing a therapeutically effective amount of a c-Abll tyrosine kinase antagonist or pharmaceutically acceptable salt of thereof to patient infected with a virus. The c-Abll tyrosine kinase antagonist or salt thereof may be provided as the only active agent or may be provided together with one or more additional active agents. In certain embodiments the c-Abll tyrosine kinase antagonist is administered together with interferon alpha 2b or ribavirin. [0050] An effective amount of a c-Abll tyrosine kinase antagonist may be an amount sufficient to (a) inhibit the progression of the viral infection (b) cause a regression of the viral infection; or (c) cure of a viral infection such that the virus or viral antibodies can no longer be detected in a previously infected patient's blood or plasma. An amount of a c-Abll tyrosine kinase antagonist effective to inhibit the progress or cause a regression of viral infection includes an amount effective to stop the worsening of symptoms of viral infection or reduce the symptoms experienced by a patient infected with the virus. Alternatively a halt in progression or regression of viral infection may be indicated by any of several markers for the disease. For example, a lack of increase or reduction in the viral load or a lack of increase or reduction in the number of circulating viral antibodies in a patient' s blood are markers of a halt in progression or regression of viral infection. Other filovirus markers include elevated TNF-a levels, elevated interleukin levels, including IL-6, -10, -11, and -1β. Disease regression is usually marked by the return of interleukin and TNF levels to the normal range.

[0051] Symptoms of filovirus infection that may be affected by an effective amount of a c-Abll tyrosine kinase antagonist include fever, sore throat, weakness, severe headache, joint and muscle aches, diarrhea, vomiting, dehydration, cough, stomach pain, internal and external bleeding, and rash.

[0052] Included herein are methods of treatment in which a c-Abll tyrosine kinase antagonist is provided together with one or more additional active agents. Suitable additional active agents include interferon alpha- 2b and ribavirin. Certain drugs increase the amount of c-Abll tyrosine kinase antagonists in the bloodstream and may increase the efficacy of c- Abll tyrosine kinase antagonists. Methods of treatment include administering on or more of these compounds in combination with a c-Abll tyrosine kinase antagonist. For example a c- Abll tyrosine kinase antagonist may be administered together with another antiviral compound such as but not limited to ritonavir, atazanavir sulfate, indinavir, nelfinavir, saquinavir; an antibiotic such as but not limited to telithromycin, erythromycin, or clarithromycin; an antifungal such as but not limited to ketoconazole or itraconazole; or nefadar.

[0053] The compound c-Abll tyrosine kinase antagonist and additional active agent may be: (1) co-formulated and administered or delivered simultaneously in a combined formulation; (2) delivered by alternation or in parallel as separate formulations; or (3) by any other combination therapy regimen known in the art. When delivered in alternation therapy, the methods of the invention may comprise administering or delivering the c-Abll tyrosine kinase antagonist and an additional active agent sequentially, e.g., in separate solution, emulsion, suspension, tablets, pills or capsules, or by different injections in separate syringes. In general, during alternation therapy, an effective dosage of each active ingredient is administered sequentially, i.e., serially, whereas in simultaneous therapy, effective dosages of two or more active ingredients are administered together. Various sequences of intermittent combination therapy may also be used.

[0054] Methods of treatment include providing certain dosage amounts of a c-Abll tyrosine kinase antagonist to a patient. Dosage levels of each active agent of from about 0.1 mg to about 140 mg per kilogram of body weight per day are useful in the treatment of the above-indicated conditions (about 0.5 mg to about 7 g per patient per day). The amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the patient treated and the particular mode of administration. Dosage unit a compound of the invention. In certain embodiments 25 mg to 500 mg, or 25 mg to 200 mg of a compound of the invention are provided daily to a patient. Frequency of dosage may also vary depending on the compound used and the particular disease treated. However, for treatment of most infectious disorders, a dosage regimen of 4 times daily or less is preferred and a dosage regimen of 1 or 2 times daily is particularly preferred. Preferred dosages include dasatinib, about 10 to about 250 mg per day, or about 50 to 150 mg per day, or about 100 mg per day or about 140 mg per day; nilotinib, about 20 to 1500 mg per day, or about 150 to 1000 mg per day, or about 400 to 800 mg per day, or about 150 mg per day, or about 800 mg per day, or about 400 mg administerd twice per day; imatinib, about 50 to 500 mg per day, or about 100 to about 400 mg per day; tivozanib, about 0.1 to 20 mg per day, or about 0.5 to 10 mg per day, or about 1.5 mg per day; ponatinib, about 10 to 150 mg per day, or about 20 to 100 mg per day or about 45 mg per day; bafetinib, about 100 to 1200 mg per day, or about 200 to 500 mg per day, or about 480 mg per day; saracatinib, about 20 to 200 mg per day, or about 70 to 150 mg per day, or about 125 mg per day; fingolimod, about 0.1 to about 10 mg per day, or about 0.1 to 2 mg per day, or about 0.5 mg per day; AT9283 about 0.5 to about 40 mg per day, or about 1 to 5 mg per day; or danusertib, about 600 to 1500 mg per day.

[0055] It will be understood, however, that the specific dose level for any particular patient will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, and rate of excretion, drug combination and the severity of the particular disease in the patient undergoing therapy. EXAMPLES

ABBREVIATIONS

[0056] The following abbreviations may be helpful to understanding of the detailed examples set forth below.

NP, VP35, VP24 Ebola Viral Protein necessary for nucleocapsid assembly

VP40 Ebola Viral Protein responsible for virion release, matrix protein

TK Tyrosine kinase

EBOV Ebola Virus

MARV Marburg Virus

VLP Virus Like Particle

GP An EbolaViral Protein, envelope

MATERIALS AND METHODS

[0057] Cells. Human embryonic kidney 293T, 293A cells, and Vero E6 cells were obtained from the American Type Culture Collection (ATCC, Manassas VA). Cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS) and 100 U/mL penicillin and 100μg/mL streptomycin (P/S).

[0058] Virus. Ebola Zaire was kindly provided by Peter Jahrling. The filovirus stock was grown in Vero E6 cells within 150 mm tissue culture flasks with DMEM (Invitrogen) containing 4,500 mg/L d-glucose, 1-glutamine and supplemented with 2% heat-inactivated FBS (DMEM-2) at 37°C, 5% C0₂, and 85% humidity. At 8 days post-infection, supernatant was collected and aliquots of the virus were stored at -80°C.

[0059] Drugs. Imatinib mesylate and nilotinib were synthesized by Bill Bornmann at MD Anderson Cancer Center.

[0060] Plasmids. Expression vectors pGP(Z), pNP, pVP24, pVP35, and

pFLAG_VP40 (altogether pEBOV x5) contain a cytomegalovirus enhancer and promoter as previously described. Plasmid DNAs were purified using double cesium chloride

sedimentation gradients. ρΔ8.2 and luciferase were used in a pseudovirus infection assay in combination with pGP(Z) envelope. VP40 mutants were created by site-directed mutagenesis (Stratagene, La Jolla, CA). c-Abll WT and kinase dead plasmid were kindly provided by Dr. Y. Shaul, Weizmann Institute of Science, Israel. Nedd4 WT plasmid was obtained from Addgene (Cambridge, MA). [0061] Transient transfections. For the egress assay and electron microscopy, 2.5 x 10⁵ human embryonic kidney 293T cells were seeded in 6-well plates in DMEM + 10% FBS + P/S. On the following day the cells were transfected using 0.5 μg of pNP, pVP24, pVP35, WT, or mutant pFLAG_VP40 and 0.00625 μg of pGP(Z) with Lipofectamine™ 2000 (Invitrogen) transfection reagents according to the protocol supplied by the manufacturer. The backbone plasmid was used as a negative control. For VP40 VLPs the cells were transfected only with 0.5 μg of pFLAG VP40. For phosphorylation studies, 0.5 μg of WT FLAG_VP40 or FLAG_VP40 mutants were transfected alone or in combination with 2 μg of c-Abll WT with the exception of the experiment when 0.5 μg of pFLAG_VP40 was co- transfected with 1 μg of WT c-Abll or kinase dead c-Abll or when 1 μg of c-Abll was transfected alone. For endogenous phosphorylation of VP40 cells were transfected with 5μg of pFLAG_VP40. For siRNA experiments in the presence of VP40 and c-Abll, 0.5 μg of FLAG_VP40 was transfected in combination with 1 μg of c-Abll WT. For the interaction of VP40 with NP or Nedd5, cells were co-transfected with 1 μg of each plasmid. For the interaction of VP40 with c-Abll, 0.5 μg of WT FLAG_VP40 or FLAG_VP40 mutants were transfected alone or in combination with 2 μg of c-Abl WT. For nucleocapsid formation, the cells were cotransfected using 0.5 μg of pNP, pVP24, and pVP35. For endogenous c-Abll association or phosphorylation of VP40, 2.5 x 10⁶ HEK 293T cells seeded in 10 cm plates were transfected with 5 μg of pFLAG_VP40. For cushion analysis of particle formation, 2.5 x 10⁶ human embryonic kidney 293T cells were seeded in 10 cm plates in DMEM + 10% FBS + P/S. On the following day the cells were transfected using 2 μg of pNP, pVP24, pVP35, WT or mutant pFLAG_VP40 and 0.025 μg of pGP(Z) with Lipofectamine™ 2000 (Invitrogen). For ESI-LC-MS/MS studies, 2.5 x 10⁶ human embryonic kidney 293T cells were seeded in 10 cm plates in DMEM + 10% FBS + P/S. The following day the cells were transfected using 5 μg of pFLAG_VP40 and 5 μg of c-Abll or plasmid control with

Lipofectamine™ 2000 (Invitrogen). When used, drugs were added 12-18 hours after transfection. Cells were harvested 36-48 hours later. Supernatant fluids were kept at 4°C and cell pellets frozen at

-80°C.

[0062] siRNA. For siRNA experiments, 1.25 x 10⁵ 293T or Vero E6 cells per well were seeded in 6-well plates in DMEM + 10% FBS + P/S. On the following morning, a total of 100 pmol of smart pools of siRNA for c-Abll (ON-TARGETplus SMARTpool L-003100- 00-0005, Human ABL1, NM_005157) or c-Abl2 (ON-TARGETplus SMARTpool L-003101- 00-0005, Human ABL2, NM_005158) (Dharmacon, Lafayette, CO) were transfected using Lipofectamine 2000 (Invitrogen). Alternatively, three individual siRNAs against c-Abll (ON-TARGETplus J-003100-09, called S9-, ON-TARGETplus J-003100-10, called S10-, and ON-TARGETplus J-003100-11, called S11-, Human ABLl, NM_007313; NM_005157) (Dharmacon) were used at the same concentration. The following day, 293T cells were transfected for a second time with pGP(Z), pNP, pVP24, pVP35, and pFLAG_VP40. 36-48 hours later, cells were harvested. Supernatant fluids were kept at 4°C and cell pellets frozen at -80°C before immunoprecipitation. For Vero E6 cells, the transfection mixture was removed and the cells were infected at a multiplicity of infection of 1 with Zaire EBOV for two hours. The inoculum was then removed, fresh media added and supernatant fluids were collected on day 7 after infection. A non-targeting control siRNA was used as a reference.

[0063] Production of pseudotyped lentiviral viruses. The pseudotyped lentiviral vectors expressing a luciferase reporter gene were produced by cotransfecting 293T cells with expression vectors ρΔ8.2, luciferase, and pGP(Z) by Ca as previously reported. After 24 hours, the transfection mixture was removed and fresh media added. 72 hours later, supernatant fluid was collected and filtered.

[0064] 293A infection. 100 of pseudotyped lentiviruses were added to cells which had been pre-incubated for 2 hours with 20 μΜ nilotinib or dimethyl sulfoxide (DMSO) in a final volume of 200 per well. Luciferase activity was measured after 48 hours as previously described (56) using mammalian cell lysis buffer and luciferase assay reagent (Promega, Madison, WI).

[0065] Cell lysis and immunoprecipitation. Cell extracts for Western blotting and immunoprecipitations were resuspended in 450 mL cell lysis buffer (Cell Signaling

Technology, Danvers, MA) supplemented with a cocktail of protease (Roche, Indianapolis, IN) and phosphatase inhibitors (Sigma, St. Louis, MO) for 30 min. Cells were pelleted for 5 min at 13,200 rpm. 30 aliquots of the soluble fraction were kept as loading control and the remaining lysate was immunoprecipitated overnight at 4°C. For coimmunoprecipitation of VP40 and NP or Nedd4 or c-Abll, the lysate was subjected to immunoprecipitation with anti-FLAG coated beads (Sigma). For phosphorylation assays, lysates were

immunoprecipitated with the same beads, or with anti-P-Tyr (PY20) sc-508 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or anti-c-Abll (8E9) (BD Biosciences, San Diego, CA) followed by Protein G beads (Invitrogen) for an additional 3 hours at 4°C the next morning. For nucleocapsid formation, lysates were immunoprecipitated with polyclonal antibody anti- NP followed by Protein G beads (Invitrogen). Supernatant fluids were immunoprecipitated overnight at 4°C with a polyclonal antibody anti-EBOV GP obtained from immunized rabbits followed by Protein G beads (Invitrogen). For VP40 VLPs, supernatant fluids were immunoprecipitated overnight at 4°C with monoclonal antibody anti-FLAG (Sigma). In all the above cases, beads were pelleted, washed three times with cold IX PBS (Gibco, Carlsbad, CA) and resuspended in 40 μΐ_^ of lx loading buffer (Invitrogen). After 5 minutes of boiling, samples were loaded onto the gels. For ESI-LC-MS/MS, cells were lysed and

immunoprecipitated with Protein G beads for 2 hours at 4°C for pre-clearance. After centrifugation, soluble lysates were incubated with beads covered with monoclonal antibody anti-FLAG for an additional 3 hours at 4°C. Beads were then pelleted, washed three times with cold IX PBS (Gibco, Carlsbad, CA) and resuspended in 30 μΐ_^ of lx loading buffer (Invitrogen). After 5 minutes of boiling, samples were loaded onto 4-15% polyacrylamide Criterion™ precast gels (Bio-Rad, Hercules, CA) under denaturing conditions. After silver staining, bands corresponding to VP40 were excised and sent for MS/MS analysis.

[0066] Sucrose gradient sedimentation. Culture supernatants were clarified by low- speed centrifugation and 0.45 μπι filter and layered onto a 20% sucrose cushion. VLPs were pelleted through the cushion at 100,000g for 3 hours. Viral proteins in cell lysates and VLPs were analyzed by Western blotting.

[0067] Immunoblot assays. Protein lysates were run under denaturing conditions, transferred to PVDF membranes and blocked for one hour at room temperature in lx TBS + 0.1% Tween-20 + 5% weight/volume nonfat dry milk. Blots were incubated with primary antibodies at appropriate dilutions in of the above buffer for an hour at room temperature or in lx TBS + 0.1% Tween-20 + 5% weight/volume BSA with gentle agitation overnight at 4°C according to the manufacturer's suggestion. Membranes were probed with 1:1,000 dilution of primary antibodies against c-Abll (Bethyl, Montgomery, TX), 1 :1000 antibody against phospho-c-Abl (Tyr412) or anti-Nedd4 (Cell Signaling Technology), 2.5 g/mL dilution of c-Abl2 (Abeam, Cambridge, MA), 1:5,000 antibody against EBOV-NP or EBOV- GP from DNA-immunized mice or rabbits, 1:3,000 antibody against EBOV-VP35 from DNA-immunized guinea pigs, 1:100 of anti-P-Tyr (PY20) sc-508 (Santa Cruz Biotechnology, Inc.) or 4G10 (Millipore, Temecula, CA), or O.lng/ L of VP40 (IBT Bioservices,

Gaithersburg, MD), or 1:10,000 of anti-FLAG and 1:5,000 dilution of -actin (Sigma), or 1:1000 anti-eIF4E (Cell Signaling Technology) as loading controls; secondary antibodies were alkaline phosphatase-conjugated anti-mouse or anti-rabbit IgG (Santa Cruz Biotechnology), or anti-guinea pig IgG (Jackson ImmunoResearch Laboratories Inc., West Grove, PA). Secondary antibodies at a dilution of 1:5,000 were added to 10 mL of lx TBS + 0.1% Tween-20 + 2.5% weight/volume nonfat dry milk with gentle agitation for 1 hour at room temperature. Blots were washed three times with lx TBS + 0.1% Tween-20 at room temperature for 10 min and developed in 12 mL of ECL detection reagent (GE Healthcare, Piscataway, NJ) for 1 min. Semi-quantitative analysis was performed using ImageQuant TL 7.0 image analysis software (GE Healthcare).

[0068] Infectivity assay. Vero cells cultured in DMEM-2(Invitrogen) containing 4,500 mg/L d-glucose, 1-glutamine and supplemented with 2% heat-inactivated FBS

(DMEM-2) were incubated for two hours with 20 μΜ of nilotinib prior to infection with Ebola Zaire for two additional hours at a multiplicity of infection of 1. The inoculum was then removed and fresh media with the drug at the same concentration was added. DMSO vehicle was used as a negative control. Aliquots of supernatant fluids were taken at days 0, 1, 2, 5, 7 or 8 after infection for TCID₅₀ determination. For adenovirus 5 infection, we used the Adeno-XTM rapid titer kit and followed the protocol according to the manufacturer's indications (Clontech, Mountain View, CA).

[0069] TCID 0 assay. The quantity of Ebola virus in samples was estimated by calculating the 50% Tissue Culture Infectious Dose (TCID₅₀) as originally described by Reed and Muench (Am. J. Hygiene (1938) 27: 493-497.). Briefly, a 96-well tissue culture plate was seeded with Vero E6 cells and then incubated at 37 °C and 5% CO₂ for 24 hours. Serial 1-log dilutions of the virus in DMEM-2 were added to five wells per dilution of a 96-well tissue culture plate (BD, Franklin Lakes, NJ). After 10 days, the monolayers were observed with phase contrast microscopy to identify CPE. Cells were then stained by adding 0.2 ml of a solution of crystal violet (PML Microbiologicals, WilsonviUe, OR) to each well. Wells that remained unstained were identified as having CPE and thus counted as infected.

[0070] Electron microscopy. 48 hours after transfection, 293T cells were lifted from plates by resuspending them with PBS and then pelleted in a 15 ml conical tube by

centrifugation at 1,000 rpm for 5 min. The supernatant was removed, and 2 ml of fixing solution was added (3% glutaraldehyde and 3% formaldehyde in 0.1 M sodium cacodylate buffer (pH 7.3); (Tousimis Research Corp., Rockville, MD). The specimens were mixed gently and analyzed in the EM facility at National Cancer Institute-Frederick.

[0071] Reduction, alkylation and washing of gel bands for MS/MS. Silver- stained gel bands were destained and washed with several changes of water until no yellow color was visible. Gels were then reduced, alkylated, and prepared for digestion by washing twice with 200 μΐ 0.05 M Tris, pH 8.5 + 30% acetonitrile for 20 minutes on a mechanical shaker, and once with 100 μΐ acetonitrile for several minutes until the gel was opaque white. After removing the acetonitrile, the gel pieces were dried for 20-30 min in a Speed- Vac

concentrator.

[0072] Enzymatic digestion. Gels were digested by adding 0.10 μg modified trypsin (sequencing grade, Roche Molecular Biochemicals, Indianapolis, IN) in 50 μΐ 0.025 M Tris, pH 8.5, or enough volume to completely hydrate the gel. The tubes were placed in a heating block at 32°C and left overnight. Peptides were extracted with 2x 50 μΐ 50% acetonitrile + 2% TFA and the combined extracts were reduced in volume in a Speed- Vac and brought up to 20 μΐ_^ with 0.1% formic acid. 5 μΐ_^ aliquots were analyzed by LC-MS/MS.

[0073] ESI-LC-MS/MS. LC-MS/MS analysis was done on a Waters Ultima Q-Tof hybrid quadrupole/time-of-flight mass spectrometer with a nanoelectro spray source.

Capillary voltage was set at 1.8 kV and cone voltage 32 V; collision energy was set according to mass and charge of the ion, from 18 eV to 50 eV. Chromatography was performed on an LC Packings HPLC with a CI 8 PepMap column using a two-hour linear acetonitrile gradient with flow rate of 200 nl/ min. Raw data files were processed using the MassLynx

ProteinLynx software and .pkl files were submitted for searching at www.matrixscience.com using the Mascot algorithm.

[0074] Cell viability and cytopathic effect. Vero cells were harvested 1, 2, 3 and 7 days and 293T cells 36-48 hours after the addition of 20 μΜ nilotinib or DMSO control. Cell viability was determined by 7-AAD exclusion in a BD LSR cell analyzer (BD Biosciences) using FlowJo version 9.1 (Tri Star Inc., Ashland, OR). Cytopathic effect was estimated by visual examination of Vero E6 cell monolayers infected with Ebola virus several days after infection.

[0075] Statistical analysis. Data are expressed as means + SEM. Paired Student's t test was used either between control and test drugs or test siRNAs, or WT versus mutant proteins. For statistical analysis, GraphPad Prism software was used. For all tests, a value of P < 0.05 was accepted as statistically significant.

Claims

CLAIMS What is claimed is:

1. A method of therapeutically treating a filovirus infection in a patient or prophylactically treating a filovirus infection in a patient comprising providing an effective amount of a c-Abl tyrosine kinase inhibitor to the patient.

2. The method of Claim 1 wherein the c-Abl tyrosine kinase inhibitor is a c-Abl tyrosine kinase antagonist.

3. The method of Claim 1, wherein the filovirus is Ebola virus or Marburg virus.

4. The method of Claim 1, wherein the c-Abl tyrosine kinase antagonist is selected from dasatinib, imatinib, tivozanib, ponatinib, fingolimid, AT9283, danusertib, bafetinib, saracatinib and nilotinib and the pharmaceutically acceptable salts thereof.

5. The method of Claim 4, wherein the c-Abl tyrosine kinase antagonist is selected from dastinib, imatinib, and nilotinib and the pharmaceutically acceptable salts thereof

6. The method of any one of Claims 1 to 5, wherein the c-Abl tyrosine kinase inhibitor is provided together with an additional active agent.

7. The method of Claim 6, wherein the additional active agent is ribavirin or interferon alpha- 2b.

8. The method of any one of Claims 1 to 7, wherein the patient is a health care worker exposed to Ebola virus or Marburg virus, who does not yet show symptoms of Ebola virus or Marburg virus infection.

9. The method of any one of Claim 1 to 7 of prophylactically treating a filovirus infection in a patient who does not yet show symptoms of infection, wherein the additionally comprising determining the patient has been in contact with a human or non-human animal infected with Ebola virus or Marburg virus with the past 0 to 14 days or will be in contact with a human or non-human animal infected with Ebola virus or Marburg virus in the following 0 to 14 days.

10. The method of Claim 9, wherein prophylactically treating the patient includes administering the c-Abll tyrosine kinase antagonist to the patient at least once daily from the day it is determined the patient has been in contact or will be in contact with a human or non- human animal infected with Ebola virus or Marburg virus until at least one day after the contact has ended.

11. The method of any one of Claims 1 to 10, wherein the c-Abl tyrosine kinase inhibitor is provided to the patient as oral dosage form.

12. The method of any one of Claims 1 to 10, wherein the c-Abl tyrosine kinase inhibitor is provided to the patient by intravenous administration.

13. The method of any one of Claims 1 to 7, or 11 to 12 wherein the c-Abl tyrosine kinase inhibitor is provided together with intravenous fluids and/ or transfusions of platelets.