WO2011109217A2 - Méthodes de traitement prophylactique ou thérapeutique de troubles viraux dépendants de l'arn polymérase par administration d'inhibiteurs de la jak2 kinase - Google Patents

Méthodes de traitement prophylactique ou thérapeutique de troubles viraux dépendants de l'arn polymérase par administration d'inhibiteurs de la jak2 kinase Download PDF

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WO2011109217A2
WO2011109217A2 PCT/US2011/026049 US2011026049W WO2011109217A2 WO 2011109217 A2 WO2011109217 A2 WO 2011109217A2 US 2011026049 W US2011026049 W US 2011026049W WO 2011109217 A2 WO2011109217 A2 WO 2011109217A2
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jak2
agent
subject
hiv
heximl
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PCT/US2011/026049
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WO2011109217A3 (fr
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Jawahar Raina
Eduardo Javier Mascareno
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Immunodiagnostics, Inc.
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Publication of WO2011109217A2 publication Critical patent/WO2011109217A2/fr
Publication of WO2011109217A3 publication Critical patent/WO2011109217A3/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/444—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a six-membered ring with nitrogen as a ring heteroatom, e.g. amrinone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/517—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with carbocyclic ring systems, e.g. quinazoline, perimidine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention is directed to the use of methods of treating or preventing viral disorders using JAK2 kinase inhibitors and agents that inhibit HEXIM1 tyrosine phosphorylation.
  • HIV human immunodeficiency virus
  • CDC Centers for Disease Control
  • nucleoside reverse transcriptase inhibitors such as AZT, ddl, ddC, d4T, 3TC, and abacavir
  • nucleotide reverse transcriptase inhibitors such as tenofovir
  • non-nucleoside reverse transcriptase inhibitors such as nevirapine, efavirenz and delavirdine
  • protease inhibitors such as saquinavir, ritonavir, indinavir, nelfmavir, amprinavir, lopinavir and atazanavir
  • fusion inhibitors such as enfuvirtide.
  • the present invention is based, at least in part, on the elucidation of the role of JAK2 kinase and the tyrosine phosphorylation of HEXIM1 in the replication of RNA polymerase dependent viruses. Indeed, the present invention is predicated, in part, on the finding that JAK2 kinase plays an important role in phosphorylating HEXIM1, and that upon such phosphorylation, RNA polymerase II is activated so as to promote viral gene expression and viral replication after infection of a cell.
  • RNA polymerase dependent virus based disorders such as Human Immunodeficiency Virus (HIV) (HIV-1 and HIV-2), Influenza (Influenzavirus A such as the H1N1 virus, Influenzavirus B and Influenzavirus C), Hepatitis (Hepatitis virus A, Hepatitis virus C, Hepatitis virus D and Hepatitis virus E), and the West Nile Virus.
  • HIV Human Immunodeficiency Virus
  • Influenza Influenzavirus A such as the H1N1 virus, Influenzavirus B and Influenzavirus C
  • Hepatitis virus A, Hepatitis virus C, Hepatitis virus D and Hepatitis virus E the West Nile Virus.
  • the present invention provides a method for treating or preventing a viral disorder in a subject having or at risk of having said viral disorder, wherein said virus is RNA polymerase dependent, by administering to the subject an effective amount of a composition including a JAK2 kinase inhibitor or an agent that inhibits tyrosine phosphorylation of HEXIM1, thereby treating or preventing the viral disorder in said subject.
  • the viral disorder is selected from the group consisting of HIV, influenzavirus, influenzavirus A, influenzavirus B, influenzavirus C, H1N1, hepatitis A, hepatitis C, hepatitis D, hepatitis E, and west nile virus.
  • the present invention provides a method for inhibiting the replication of a virus in a subject, by administering to the subject infected with said virus, wherein said virus is dependent on RNA polymerase for replication, an effective amount of a composition including a JAK2 kinase inhibitor or an agent that inhibits tyrosine phosphorylation of HEXIM1, thereby inhibiting viral replication in the subject.
  • the virus is selected from the group consisting of HIV, influenzavirus, influenzavirus A, influenzavirus B, influenzavirus C, H1N1, hepatitis A, hepatitis C, hepatitis D, hepatitis E, and west nile virus.
  • the RNA polymerase is RNA polymerase II.
  • the invention is directed to a method for treating or preventing HIV infection in a subject having or at risk of HIV infection, by administering to the subject an effective amount of a composition including a JAK2 kinase inhibitor or an agent that inhibits tyrosine phosphorylation of HEXIM1, thereby treating or preventing HIV in said subject.
  • the invention is directed to a method for inhibiting the
  • B3841854.1 replication of HIV in a subject having or at risk of HIV infection, by administering to the subject an effective amount of a composition including a JAK2 kinase inhibitor or an agent that inhibits tyrosine phosphorylation of HEXIMl , thereby inhibiting HIV replication in the subject.
  • the method includes administering to the subject an effective amount of a JAK2 kinase inhibitor, for example, one that inhibits tyrosine kinase activity.
  • the JAK2 inhibitor is selected from the group consisting of AG490, tyrphostin, lestaurtinib (CEP701), AZD1480, member of CIS/SOCS/Jab family, synthetic component AG490, 2-methyl-l-phenyl-4-pyridin-2-yl-2-(2-pyridin-2- ylethyl)butan-l-one, dimethoxyquinazoline compound, 4-(phenyl)-amino-6,7- dimethoxyquinazoline, 4-(4'-hydroxyphenyl)-amino-6,7-dimethoxyquinazoline, 4-(3'- bromo-4'-hydroxylphenyl)-amino-6,7-dimethoxyquinazoline, and 4-(3
  • the JAK2 kinase inhibitor is AG490 or 2-methyl-l-phenyl-4-pyridin-2-yl-2- (2-pyridin-2-ylethyl)butan-l-one.
  • the JAK2 kinase inhibitor is selected from the group consisting of low molecular weight inhibitors, antibodies or antibody fragments, peptide or R A aptamers, antisense constructs, small inhibitory R As and ribozymes.
  • the JAK2 kinase inhibitor is selected from the group consisting of an RNA interfering agent, an siRNA, an shRNA and dsRNA.
  • the methods include administering to the subject an effective amount of an agent that inhibits tyrosine phosphorylation of HEXIMl .
  • the agent inhibits tyrosine phosphorylation of HEXIMl at the YLEL domain of HEXIMl .
  • the JAK2 kinase inhibitor or the agent that inhibits tyrosine phosphorylation of HEXIMl is administered orally.
  • the methods include monitoring the effectiveness of treatment, for example, monitoring the activity of JAK2 kinase in the subject or monitoring the phosphorylation of HEXIMl at the YLEL domain.
  • the methods of the present invention include the treatment or prevention of HIV infection in a subject having or at risk of HIV infection, by
  • the invention is directed to a method for treating or preventing HIV infection in a subject having or at risk of HIV
  • B3841854.1 infection by administering to the subject an effective amount of an agent that inhibits the release of HEXIM1 from a cyclin Tl-cdk9 complex, thereby treating or preventing HIV in said subject.
  • the agent that inhibits tyrosine phosphorylation of HEXIM1 may be selected from the group consisting of AG490, tyrphostin, lestaurtinib (CEP701), AZD1480, member of CIS/SOCS/Jab family, synthetic component AG490, 2-methyl-l-phenyl-4- pyridin-2-yl-2-(2-pyridin-2-ylethyl)butan-l-one, dimethoxyquinazoline compound, 4- (phenyl)-amino-6,7-dimethoxyquinazoline, 4-(4'-hydroxyphenyl)-amino-6,7- dimethoxyquinazoline, 4-(3'-bromo-4'-hydroxylphenyl)-amino-6,7-dimethoxyquinazoline, and 4-(3',5'-dibromo-4'-hydroxylphenyl)-amino-6,7-dimethoxyquinazoline,
  • the agent that inhibits tyrosine phosphorylation of HEXIM1 is a tyrphostin, such as AG490, or 2-methyl-l-phenyl-4-pyridin-2-yl-2-(2-pyridin-2-ylethyl)butan-l-one.
  • the agent is an antisense nucleic acid molecule selected from the group consisting of an R A interfering agent, an siR A, an shR A and dsRNA.
  • the agent is administered orally.
  • the invention is directed to a method for identifying a therapeutic for treatment of HIV, by exposing a candidate substance to a composition including a JAK2 kinase or HEXIM1, monitoring the activity of the JAK2 kinase or HEXIM1, and identifying the candidate substance as a therapeutic for treatment of HIV if the candidate substance reduces the activity of the JAK2 kinase or the phosphorylation of HEXIM1.
  • the method involves monitoring the tyrosine phosphorylation of HEXIM1, wherein a reduction in the phosphorylation of HEXIM1 identifies the candidate substance as a therapeutic for treatment of HIV, and/or monitoring the JAK2 mediated phosphorylation of HEXIM1 at the YLEL motif of HEXIM1, wherein a reduction in the phosphorylation of HEXIM1 at the YXXL motif identifies the candidate substance as a therapeutic for treatment of HIV.
  • the method includes monitoring the release of HEXIM1 from a cyclin Tl-cdk9 complex.
  • Fig. 1A and Fig. IB depict the results of the HIV-1 p24 antigen determination by Lateral Flow p24 antigen test on day 22 after exposure to Z3: Left to right, H9 cells (C), cells with inhibitor (Z3), Cells infected with HIV-1 (D-nef) and HIV-1 + Z3 (D-nef,Z3).
  • the present invention is based, at least in part, on the elucidation of the role of JAK2 kinase and the tyrosine phosphorylation of HEXIM1 in the replication of R A polymerase dependent viruses. Indeed, the present invention is predicated, in part, on the finding that JAK2 kinase plays an important role in phosphorylating HEXIM1, and that upon such phosphorylation, RNA polymerase II is activated so as to promote viral gene expression and viral replication after infection of a cell.
  • RNA polymerase dependent virus based disorders including, but not limited to lentivirus based disorders, such as Human Immunodeficiency Virus (HIV) (HIV-1 and HIV-2), Influenza
  • Influenzavirus A such as the HlNl virus, Influenzavirus B and Influenzavirus C
  • Hepatitis virus A Hepatitis virus C
  • Hepatitis virus D Hepatitis virus D
  • Hepatitis virus E Hepatitis virus E
  • the present invention is predicated upon the finding that JAK2 kinase serves to phosphorylate HEXIM1, specifically, at the YLEL epitope present on HEXIM1, so as to trigger the release of HEXIM1 from the transcription elongation factor P-TEFb complex (cyclin Tl-cdk9).
  • a viral replicating factor for example, TAT (an HIV replicating factor)
  • TAT an HIV replicating factor
  • the P-TEFb complex Upon the interaction of the viral replicating factor with the P-TEFb complex, the P-TEFb complex, particularly, cdk9, triggers serine phosphorylation and transcription elongation of RNA Polymerase II so as to allow for transcription and replication of the viral genome.
  • RNA polymerase dependent virus based disorders such as Human Immunodeficiency Virus (HIV) (HIV-1 and HIV-2), Influenza (Influenzavirus A such as HlNl, Influenzavirus B and Influenzavirus C), Hepatitis (Hepatitis virus A, Hepatitis virus C, Hepatitis virus D and Hepatitis virus E), and the West Nile Virus, can be treated by use of JAK2 kinase inhibitors or agents that inhibit the tyrosine phosphorylation of HEXIM1.
  • HMV Human Immunodeficiency Virus
  • Influenza Influenzavirus A
  • Hepatitis virus A Hepatitis virus C
  • Hepatitis virus D and Hepatitis virus E Hepatitis virus E
  • West Nile Virus West Nile Virus
  • the present invention is directed to a method for treating or preventing a viral disorder in a subject having or at risk of having said viral disorder, wherein said virus is RNA polymerase dependent, by administering to the subject an effective amount of a composition including a JAK2 kinase inhibitor or an agent that inhibits the tyrosine phosphorylation of HEXIM1 , thereby treating or preventing the viral disorder (for example, HIV, influenzavirus, influenzavirus A such as HlNl, influenzavirus
  • the present invention is directed to a method for inhibiting the replication of a virus in a subject, by administering to the subject infected with said virus (for example, HIV, influenzavirus, influenzavirus A such as H1N1, influenzavirus B, influenzavirus C, hepatitis A, hepatitis C, hepatitis D, hepatitis E, and west nile virus), wherein said virus is dependent on RNA polymerase for replication, an effective amount of a composition including a JAK2 kinase inhibitor or an agent that inhibits the tyrosine phosphorylation of HEXIM1, thereby inhibiting viral replication in the subject.
  • said virus for example, HIV, influenzavirus, influenzavirus A such as H1N1, influenzavirus B, influenzavirus C, hepatitis A, hepatitis C, hepatitis D, hepatitis E, and west nile virus
  • the present invention is directed to a method for treating or preventing HIV infection in a subject having or at risk of HIV infection, by administering to the subject an effective amount of a composition including a JAK2 kinase inhibitor or an agent that inhibits the tyrosine phosphorylation of HEXIM1 , thereby treating or preventing HIV in said subject.
  • the present invention includes methods for inhibiting the replication of HIV in a subject having or at risk of HIV infection, by administering to the subject an effective amount of a composition including a JAK2 kinase inhibitor or an agent that inhibits the tyrosine phosphorylation of HEXIM1, thereby inhibiting HIV replication in the subject.
  • RNA polymerase refers to the art recognized enzyme that assists in cellular production of RNA during transcription. Specifically, RNA polymerase catalyzes the initiation and elongation of each type of RNA, i.e., mRNA, tRNA and rRNA, from a DNA template. In a particular embodiment, RNA polymerase refers specifically to RNA polymerase II, also referred to as RNA Pol II.
  • RNA polymerases often serve an important function in viral genome replication and/or viral genome transcription, depending on the nature of the virus.
  • DNA viruses often utilize host cell DNA dependent RNA polymerases for transcription of viral DNA, thereby producing viral mRNAs.
  • RNA viruses use RNA polymerases to effect viral genome transcription and replication.
  • plus (+) strand RNA viruses possess RNA genomes that serve as mRNAs. While these viruses do not require RNA polymerase to initiate viral genome expression given that the RNA genome serves as the mRNA, viral encoded RNA dependent RNA polymerase is required to produce (-) strand RNA which in turn allows for production of more (+) strand RNA.
  • These (+) strand RNA can serve as mRNAs, templates to make more (-) strand RNA or the genomes of progeny viruses.
  • minus (-) strand RNA viruses possess a viral genome which does not serve as mRNA. Accordingly, such viruses require the use of a viral RNA polymerase to transcribe the minus (-) strand RNA viruses to form (+) strands.
  • RNA polymerase II serves to catalyze the replication of HIV genomic material and, further, to generate viral mR A utilized to generate HIV proteins during translation.
  • the single stranded HIV RNA is converted to double-stranded HIV DNA which subsequently enters the host cell's nucleus and integrates within the host cell's DNA.
  • the integrated viral DNA is referred to as a provirus.
  • the host cell's endogenous RNA polymerase is hijacked by the provirus so as to catalyze the replication of HIV genomic material and, further, to generate viral mRNA.
  • TAT an HIV encoded transcription factor
  • TAR TAT -Responsive element
  • RNA polymerase dependent virus refers to a virus that depends on RNA polymerase, for example, RNA Polymerase II, for expression or replication of the viral genome or spread of the virus.
  • RNA polymerase dependent viruses include certain RNA viruses that require RNA polymerase for viral genome replication such as, but not limited to, Human Immunodeficiency Virus (HIV) (HIV-1 and HIV-2), Influenzaviruses (Influenzavirus A such as H1N1,
  • Influenzavirus B and Influenzavirus C Hepatitis viruses
  • Hepatitis viruses Hepatitis virus A, Hepatitis virus C, Hepatitis virus D and Hepatitis virus E
  • the West Nile Virus Hepatitis virus A, Hepatitis virus C, Hepatitis virus D and Hepatitis virus E
  • JAK2 Janus Kinase 2
  • JAK2 JAK2
  • JAK2 kinase refer to the art recognized Janus kinase 2 protein that is involved in signaling of certain pathways, for example, the type II cytokine receptor pathway, the GM-CSF pathway and the gpl30 receptor pathway. JAK2 is encoded by the JAK2 gene.
  • the NCBI accession number for the JAK2 protein is AAY22962.
  • JAK2 kinase inhibitor or "JAK2 inhibitor” refer to any JAK2 kinase inhibitor that is currently known in the art or that will be identified in the future, and includes any chemical entity that, upon administration to a subject or cell, results in inhibition of a biological activity associated with the activity of JAK2 kinase in the cell or in the patient, including any of the downstream biological effects otherwise resulting from the activity of JAK2.
  • Such JAK2 kinase inhibitors include any agent that can block JAK2 mediated phosphorylation or any of the downstream biological effects of JAK2 mediated phosphorylation that are relevant to treating viral disorders in a patient.
  • Such an inhibitor can act by binding directly to ATP so as to prevent the phosphorylation of JAK2, thereby inhibiting its subsequent kinase activity.
  • JAK2 kinase inhibitors include, but are not limited to, low molecular weight inhibitors, antibodies or antibody fragments, peptide or RNA aptamers, antisense constructs, small inhibitory RNAs (i.e., RNA
  • the JAK2 kinase inhibitor is selected from the group consisting of an R A interfering agent, an siRNA, an shRNA and dsRNA.
  • JAK2 kinase inhibitors can be selected from the group consisting of AG490, tyrphostin, lestaurtinib (CEP701), AZD1480, member of
  • the JAK2 kinase inhibitor is a tyrphostin such as AG490.
  • the JAK2 kinase inhibitor is the small molecule Z3.
  • HEXIM1 refers to the art recognized human
  • HEXIM1 hexamethylene bis-acetamide inducible 1
  • MAQ-1 hexamethylene bis-acetamide inducible 1
  • HEXIM1 interacts with the pTEFb complex in a manner that activates RNA Polymerase II.
  • NCBI accession number for the HEXIM1 protein is BAA36166.
  • an element means one element or more than one element.
  • the term "modulated" with respect to JAK2 kinase includes changing the expression, activity and/or function of JAK2 kinase in such a manner that it differs from the naturally-occurring expression, function and/or activity of JAK2 kinase under the same conditions.
  • the expression, function and/or activity can be greater or less than that of naturally occurring JAK2 kinase, e.g. , owing to a change in kinase activity, etc.
  • the various forms of the term "modulate” include stimulation (e.g. , increasing or upregulating a particular response or activity) and inhibition (e.g., decreasing or downregulating a particular response or activity).
  • the term "compound” includes any agent, e.g., nucleic acid molecules, antisense nucleic acid molecule, peptide, peptidomimetic, small molecule, or other drug, which binds to JAK2 or has a stimulatory or inhibitory effect on, for example, JAK2 expression or activity, kinase affinity or stability.
  • the compound may modulate transcription of JAK2 or kinase activity of JAK2.
  • inhibitor or “inhibitory agent” includes agents which decrease the expression and/or activity of JAK2 or, alternatively, agents which inhibit the tyrosine
  • inhibitory agents include, but are not limited to active protein and nucleic acid molecules, peptides and peptidomimetics of JAK2. Inhibitory agents also include naturally occurring inhibitors.
  • agents of the invention can directly or indirectly modulate, e.g., inhibit, the expression and/or activity of JAK2.
  • exemplary agents are described herein or can be identified using screening assays that select for such compounds, as described in detail below.
  • the "test compound” or “agent” screened includes molecules that are not known in the art to modulate, e.g., inhibit, JAK2 activity and/or expression as described herein.
  • a plurality of agents are tested using the instant methods.
  • library of test compounds is intended to refer to a panel comprising a multiplicity of test compounds.
  • the agent or test compound is a compound that directly interacts with JAK2 or directly interacts with a molecule with which JAK2 interacts (e.g., a compound that inhibits the interaction between JAK2 and a JAK2 target molecule, e.g. , HEXIM1).
  • the compound is one that indirectly modulates, e.g., inhibits, JAK2 expression and/or activity, e.g., by inhibiting the activity of a molecule that is upstream or downstream of JAK2 in a signal transduction pathway involving JAK2.
  • the agent or test compound can serve to inhibit the phosphorylation of JAK2 by interacting with ATP, thereby preventing subsequent JAK2 mediated
  • HEXIM 1 phosphorylation of downstream proteins
  • Such compounds can be identified using screening assays that select for such compounds, as described in detail below.
  • target molecule or "binding partner” is a molecule with which JAK2 binds or interacts in nature, and which interaction results in a biological response.
  • the target molecule can be a protein or a nucleic acid molecule.
  • Exemplary target molecules of the invention include proteins in the same signaling pathway as the JAK2 protein, e.g., proteins which may function upstream (including both stimulators and inhibitors of activity) or downstream of the JAK2 protein in a pathway involving for example, phosphorylation of JAK2 protein, phosphorylation of HEXIM 1, release of HEXIM 1 for the pTEFb complex, modulation of pTEFb complex activity, modulation of TAT interaction with RNA polymerase, modulation of CDK9 mediated phosphorylation of RNA polymerase, modulation of RNA polymerase activity, modulation of the transcription
  • proteins in the same signaling pathway as the JAK2 protein e.g., proteins which may function upstream (including both stimulators and inhibitors of activity) or downstream of the JAK2 protein in a pathway involving for example, phosphorylation of JAK2 protein, phosphorylation of HEXIM 1, release of HEXIM 1 for the pTEFb complex, modulation of pTEF
  • B3841854.1 of the viral genome modulation of the replication of the viral genome, and modulation of the spread of a virus.
  • interact as used herein is meant to include detectable interactions between molecules, such as can be detected using, for example, a yeast two hybrid assay or coimmunoprecipitation.
  • interact is also meant to include "binding" interactions between molecules. Interactions may be protein-protein or protein-nucleic acid in nature.
  • the term "contacting" i.e., contacting a cell e.g. infected with a virus such as HIV, with a compound
  • contacting is intended to include incubating the compound and the cell together in vitro (e.g., adding the compound to cells in culture) or administering the compound to a subject such that the compound and cells of the subject are contacted in vivo.
  • the term "indicator composition” refers to a composition that includes a protein of interest (e.g., JAK2 kinase), for example, a cell that naturally expresses the protein, a cell that has been engineered to express the protein by introducing an expression vector encoding the protein into the cell, or a cell free composition that contains the protein (e.g. , purified naturally-occurring protein or recombinantly-engineered protein).
  • a protein of interest e.g., JAK2 kinase
  • cell free composition refers to an isolated composition which does not contain intact cells.
  • cell free compositions include cell extracts and compositions containing isolated proteins.
  • an "agonist" of the JAK2 proteins can retain substantially the same, or a subset, of the biological activities of the naturally occurring form of a JAK2 protein.
  • An "antagonist" of a JAK2 protein can inhibit one or more of the activities of the naturally occurring form of the JAK2 protein by, for example, competitively modulating a cellular activity of a JAK2 protein.
  • an "antisense” nucleic acid comprises a nucleotide sequence which is complementary to a “sense” nucleic acid encoding a protein, e.g., complementary to the coding strand of a double-stranded cDNA molecule, complementary to an mRNA sequence or complementary to the coding strand of a gene. Accordingly, an antisense nucleic acid can hydrogen bond to a sense nucleic acid.
  • small molecule is a term of the art and includes molecules that are less than about 1000 molecular weight or less than about 500 molecular weight. In one embodiment, small molecules do not exclusively comprise peptide bonds. In another embodiment, small molecules are not oligomeric. Exemplary small molecule compounds which can be screened for activity include, but are not limited to, peptides,
  • the compounds are small, organic non-peptidic compounds.
  • a small molecule is not biosynthetic.
  • peptide includes relatively short chains of amino acids linked by peptide bonds.
  • peptidomimetic includes compounds containing non- peptidic structural elements that are capable of mimicking or antagonizing peptides.
  • treatment is defined as the application or administration of a therapeutic agent to a patient, or application or administration of a therapeutic agent to an isolated tissue or cell line from a patient, who has a disease, disorder, or infection, a symptom of a disease, disorder, or infection or a predisposition toward a disease, disorder, or infection, with the purpose of curing, healing, alleviating, relieving, altering, remedying, ameliorating, improving or affecting the disease, disorder, or infection, the symptoms of disease, disorder, or infection or the predisposition toward a disease, disorder, or infection.
  • a therapeutic agent includes, but is not limited to, nucleic acid molecules, small molecules, peptides, peptidomimetics, antibodies, ribozymes, and sense and antisense oligonucleotides described herein.
  • the present invention is directed to JAK2 kinase inhibitors and agents that inhibit tyrosine phosphorylation of HEXIMl, and their use in therapeutic and prophylactic methods for the treatment of RNA polymerase dependent viruses.
  • the JAK2 kinase inhibitor or agent inhibiting HEXIMl phosphorylation may be low molecular weight inhibitors, antibodies or antibody fragments, peptide or RNA aptamers, antisense constructs, small inhibitory RNAs ⁇ i.e., RNA interference by dsRNA, RNAi), and ribozymes.
  • the JAK2 kinase inhibitor or agent inhibiting may be low molecular weight inhibitors, antibodies or antibody fragments, peptide or RNA aptamers, antisense constructs, small inhibitory RNAs ⁇ i.e., RNA interference by dsRNA, RNAi), and ribozymes.
  • the JAK2 kinase inhibitor or agent inhibiting may be low molecular weight
  • HEXIMl tyrosine phosphorylation is selected from the group consisting of an RNA interfering agent, an siRNA, an shRNA and dsRNA.
  • exemplary JAK2 kinase inhibitors or agents inhibiting HEXIMl tyrosine phosphorylation further include such inhibitors identified by the methods described herein.
  • JAK2 kinase inhibitors or agents inhibiting HEXIMl tyrosine phosphorylation can be selected from the group consisting of tyrphostin, AG 490 ((E)-2-Cyano-3-(3,4-dihydrophenyl)-N-(phenylmethyl)-2-pr openamide), lestaurtinib (CEP701), AZD1480, member of CIS/SOCS/Jab family, 2-methyl-l-phenyl-4-pyridin-2-yl-
  • B3841854.1 2-(2-pyridin-2-ylethyl)butan-l-one (herein designated as Z3), dimethoxyquinazoline compound, 4-(phenyl)-amino-6,7-dimethoxyquinazoline, 4-(4'-hydroxyphenyl)-amino-6,7- dimethoxyquinazoline, 4-(3'-bromo-4'-hydroxylphenyl)-amino-6,7-dimethoxyquinazoline, and 4-(3',5'-dibromo-4'-hydroxylphenyl)-amino-6,7-dimethoxyquinazoline.
  • the JAK2 kinase inhibitor or agent inhibiting HEXIMl tyrosine phosphorylation is the small molecule Z3.
  • an inhibitory agent of the invention is a small molecule which interacts with JAK2 to thereby inhibit the activity of the JAK2 kinase or the tyrosine phosphorylation of HEXIMl .
  • the inhibitory agent of the invention is a small molecule which interacts with HEXIMl to thereby inhibit the tyrosine phosphorylation of HEXIMl .
  • Small molecule inhibitors of JAK2 or HEXIMl tyrosine phosphorylation can be identified using database searching programs capable of scanning a database of small molecules of known three-dimensional structure for candidates which fit into the target protein site known in the art.
  • Suitable software programs include, for example, CATALYST (Molecular Simulations Inc., San Diego, CA), UNITY (Tripos Inc., St Louis, MO), FLEXX (Rarey et al, J. Mol. Biol. 261 : 470-489 (1996)), CHEM-3DBS (Oxford Molecular Group, Oxford, UK), DOCK (Kuntz et al, J. Mol. Biol 161 : 269-288 (1982)), and MACCS-3D (MDL Information Systems Inc., San Leandro, CA).
  • the molecules found in the search may not necessarily be leads themselves, however, such candidates might act as the framework for further design, providing molecular skeletons to which appropriate atomic replacements can be made.
  • the scaffold, functional groups, linkers and/or monomers may be changed to maximize the electrostatic, hydrogen bonding, and hydrophobic interactions with the target protein.
  • phosphorylation can also be identified using computer-assisted molecular design methods
  • B3841854.1 comprising searching for fragments which fit into a binding region subsite and link to a predefined scaffold can be used.
  • the scaffold itself may be identified in such a manner.
  • Programs suitable for the searching of such functional groups and monomers include LUDI (Boehm, J Comp. Aid. Mol. Des. 6:61-78 (1992)), CAVEAT (Bartlett et al. in "Molecular Recognition in Chemical and Biological Problems", special publication of The Royal Chem. Soc, 78: 182-196 (1989)) and MCSS (Miranker et al. Proteins 11 : 29-34 (1991)).
  • Yet another computer-assisted molecular design method for identifying JAK2 or HEXIM1 phosphorylation small molecule inhibitors comprises the de novo synthesis of potential inhibitors by algorithmic connection of small molecular fragments that will exhibit the desired structural and electrostatic complementarity with the active binding or phosphorylation site of the JAK2 or HEXIM1 protein.
  • the methodology employs a large template set of small molecules with are iteratively pieced together in a model of the JAK2 or HEXIM1 binding or phosphorylation site. Programs suitable for this task include GROW (Moon et al. Proteins 11 :314-328 (1991)) and SPROUT (Gillet et al. J Comp. Aid. Mol. Des. 7:127 (1993)).
  • the suitability of small molecule inhibitor candidates can be determined using an empirical scoring function, which can rank the binding affinities for a set of inhibitors.
  • an empirical scoring function which can rank the binding affinities for a set of inhibitors.
  • Other modeling techniques can be used in accordance with this invention, for example, those described by Cohen et al. (J. Med. Chem. 33: 883- 894 (1994)); Navia et al. (Current Opinions in Structural Biology 2: 202-210 (1992));
  • Exemplary small molecule inhibitors for use in the present invention include, but are not limited to, tyrphostin, AG 490 ((E)-2-Cyano-3-(3,4-dihydrophenyl)-N- (phenylmethyl)-2-pr openamide), lestaurtinib (CEP701), AZD1480, member of
  • the small molecule inhibitor is a tyrphostin such as AG490. In another embodiment, the small molecule inhibitor is Z3.
  • An inhibitory agent of the invention can be, for example, an antisense nucleic acid molecule that is complementary to a gene encoding a JAK2 or to a portion of said gene, or a recombinant expression vector encoding said antisense nucleic acid molecule.
  • antisense nucleic acids to downregulate the expression of a particular polypeptide in a cell is well known in the art (see e.g., Weintraub, H. et al. , Antisense RNA as a molecular tool for genetic analysis, Reviews - Trends in Genetics, Vol. 1(1) 1986; Askari, F.K. and McDonnell, W.M. (1996) N. Eng. J. Med.
  • An antisense nucleic acid molecule comprises a nucleotide sequence that is complementary to the coding strand of another nucleic acid molecule ⁇ e.g. , an mRNA sequence) and accordingly is capable of hydrogen bonding to the coding strand of the other nucleic acid molecule.
  • Antisense sequences complementary to a sequence of an mRNA can be complementary to a sequence found in the coding region of the mRNA, the 5 ' or 3' untranslated region of the mRNA or a region bridging the coding region and an
  • an antisense nucleic acid can be complementary in sequence to a regulatory region of the gene encoding the mRNA, for instance a transcription initiation sequence or regulatory element.
  • an antisense nucleic acid of the invention is a compound that mediates RNAi.
  • RNA interfering agents include, but are not limited to, nucleic acid molecules including RNA molecules which are homologous to the target gene or genomic sequence, e.g., a JAK2 family member, or a fragment thereof, "short interfering RNA” (siRNA), "short hairpin” or “small hairpin RNA” (shRNA), and small molecules which interfere with or inhibit expression of a target gene by RNA interference (RNAi).
  • RNA interference is a post-transcriptional, targeted gene-silencing technique that uses double- stranded RNA (dsRNA) to degrade messenger RNA (mRNA) containing the same sequence as the dsRNA (Sharp, P.A. and Zamore, P.D. 287, 2431-2432 (2000); Zamore, P.D., et al. Cell 101, 25-33 (2000). Tuschl, T. et al. Genes Dev. 13, 3191-3197 (1999)).
  • the process occurs when an endogenous ribonuclease cleaves the longer dsRNA into shorter, 21- or 22-nucleotide-long RNAs, termed small interfering RNAs or siRNAs.
  • the smaller RNA segments then mediate the degradation of the target mRNA. Kits for synthesis of RNAi are commercially available from, e.g. New England Biolabs and
  • Antisense polynucleotides may be produced from a heterologous expression cassette in a transfectant cell or transgenic cell.
  • the antisense polynucleotides may comprise soluble oligonucleotides that are administered to the external milieu, either in the
  • B3841854.1 culture medium in vitro or in the circulatory system or in interstitial fluid in vivo. Soluble antisense polynucleotides present in the external milieu have been shown to gain access to the cytoplasm and inhibit translation of specific mR A species.
  • antisense nucleic acids of the invention can be designed according to the rules of Watson and Crick base pairing.
  • the antisense nucleic acid molecule can be complementary to the entire coding region of JAK2 mRNA, but more preferably is an oligonucleotide which is antisense to only a portion of the coding or noncoding region of JAK2 mRNA.
  • the antisense oligonucleotide can be complementary to the region surrounding the translation start site of JAK2 mRNA.
  • An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length.
  • an antisense nucleic acid is designed so as to be complementary to a region preceding or spanning the initiation codon on the coding strand or in the 3' untranslated region of an mRNA.
  • An antisense nucleic acid for inhibiting the expression of a JAK2 polypeptide in a cell can be designed based upon the nucleotide sequence encoding the a JAK2 polypeptide, constructed according to the rules of Watson and Crick base pairing.
  • an antisense nucleic acid can exist in a variety of different forms.
  • the antisense nucleic acid can be an oligonucleotide that is complementary to only a portion of a JAK2 gene.
  • one or more antisense oligonucleotides can be added to cells in culture media, typically at about 200 ⁇ g oligonucleotide/ml.
  • an antisense nucleic acid of the invention can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art.
  • an antisense nucleic acid ⁇ e.g., an antisense oligonucleotide
  • an antisense nucleic acid can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g. , phosphorothioate derivatives and acridine substituted nucleotides can be used.
  • modified nucleotides which can be used to generate the antisense nucleic acid include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine, 5- (carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5- carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6- isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2- methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7- methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D- mannosylqueosine, 5'-methoxycar
  • B3841854.1 isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2- thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5- oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino- 3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine.
  • the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).
  • an antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., nucleic acid transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest).
  • Regulatory sequences operatively linked to a nucleic acid cloned in the antisense orientation can be chosen which direct the expression of the antisense RNA molecule in a cell of interest, for instance promoters and/or enhancers or other regulatory sequences can be chosen which direct constitutive, tissue specific or inducible expression of antisense RNA.
  • an inducible eukaryotic regulatory system such as the Tet system (e.g. , as described in Gossen, M. and Bujard, H. (1992)
  • the antisense expression vector is prepared as described above for recombinant expression vectors, except that the cDNA (or portion thereof) is cloned into the vector in the antisense orientation.
  • the antisense expression vector can be in the form of, for example, a recombinant plasmid, phagemid or attenuated virus.
  • the antisense expression vector is introduced into cells using a standard transfection technique, as described above for recombinant expression vectors.
  • antisense nucleic acids of the invention can be designed according to the rules of Watson and Crick base pairing.
  • the antisense nucleic acid molecule can be complementary to the entire coding region of a JAK2 mRNA, but more preferably is an oligonucleotide which is antisense to only a portion of the coding or noncoding region of a JAK2 mRNA.
  • the antisense oligonucleotide can be complementary to the region surrounding the translation start site of a JAK2 mRNA.
  • An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length.
  • An antisense nucleic acid of the invention can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art.
  • an antisense nucleic acid e.g., an antisense oligonucleotide
  • an antisense nucleic acid can be chemically synthesized using naturally occurring
  • modified nucleotides which may be used to generate the antisense nucleic acid include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5- carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1- methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5- methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5 '-methoxy
  • the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation ⁇ i.e., R A transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).
  • the antisense nucleic acid molecules of the invention are typically administered to a subject or generated in situ such that they hybridize with or bind to cellular mR A and/or genomic DNA encoding a JAK2 protein to thereby inhibit expression of the protein, e.g., by inhibiting transcription and/or translation.
  • the hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule which binds to DNA duplexes, through specific
  • antisense nucleic acid molecules of the invention include direct injection at a tissue site.
  • antisense nucleic acid molecules can be modified to target selected cells and then administered systemically. For example, for systemic
  • antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecules to peptides or antibodies which bind to cell surface receptors or antigens.
  • the antisense nucleic acid molecules can also be delivered to cells using the vectors described herein.
  • vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are preferred.
  • the antisense nucleic acid molecule of the invention is an a-anomeric nucleic acid molecule.
  • An a-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual ⁇ -units, the strands run parallel to each other (Gaultier, et al. (1987) Nucleic Acids. Res. 15:6625- 6641).
  • the antisense nucleic acid molecule can also comprise a 2'-o-methylribonucleotide (Inoue, et al. (1987) Nucleic Acids Res. 15:6131-6148) or a chimeric R A-DNA analogue (Inoue, et al. (1987) FEBS Lett. 215:327-330).
  • an antisense nucleic acid of the invention is a ribozyme.
  • Ribozymes are catalytic RNA molecules with ribonuclease activity which are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region.
  • ribozymes ⁇ e.g., hammerhead ribozymes (described in Haselhoff and Gerlach (1988) Nature 334:585-591)) can be used to catalytically cleave JAK2 mRNA transcripts to thereby inhibit translation of JAK2 mRNA.
  • a ribozyme having specificity for a JAK2 -encoding nucleic acid can be designed based upon the nucleotide sequence of a JAK2 cDNA disclosed herein ⁇ see Figure 1).
  • a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in a JAK2 -encoding mRNA. See, e.g., Cech, et al. U.S. Patent No. 4,987,071; and Cech, et al. U.S. Patent No. 5,116,742.
  • JAK2 mRNA can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Bartel, D. and Szostak, J.W. (1993) Science 261 : 1411-1418.
  • JAK2 gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region of the JAK2 ⁇ e.g. , the JAK2 promoter and/or enhancers) to form triple helical structures that prevent transcription of the JAK2 gene in target cells.
  • nucleotide sequences complementary to the regulatory region of the JAK2 e.g. , the JAK2 promoter and/or enhancers
  • the JAK2 nucleic acid molecules of the present invention can be modified at the base moiety, sugar moiety or phosphate backbone to serve as antisense or antigene agents.
  • the deoxyribose phosphate backbone of the nucleic acid molecules can be modified to generate peptide nucleic acids (see Hyrup, B. et al. (1996) Bioorganic & Medicinal Chemistry 4 (1): 5-23).
  • the terms "peptide nucleic acids" or "PNAs” refer to nucleic acid mimics, e.g., DNA mimics, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained.
  • the neutral backbone of PNAs has been shown to allow for specific hybridization to DNA and RNA under conditions of low ionic strength.
  • the synthesis of PNA oligomers can be performed using standard solid phase
  • PNAs of JAK2 nucleic acid molecules can be used in therapeutic applications as described herein.
  • PNAs can be used as antisense or antigene agents for sequence-specific modulation of gene expression by, for example, inducing transcription or translation arrest or inhibiting replication.
  • JAK2 polypeptide can be used as an immunogen to generate antibodies that bind JAK2 using standard techniques for polyclonal and monoclonal antibody preparation.
  • a full-length Jak2 polypeptide can be used or, alternatively, the invention provides antigenic peptide fragments of JAK2 for use as immunogens.
  • the antigenic peptide of JAK2 comprises at least 8 amino acid residues of the amino acid sequence as shown in Figure 1 and encompasses an epitope of JAK2 such that an antibody raised against the peptide forms a specific immune complex with JAK2.
  • the antigenic peptide comprises at least 10 amino acid residues, more preferably at least 15 amino acid residues, even more preferably at least 20 amino acid residues, and most preferably at least 30 amino acid residues.
  • Preferred epitopes encompassed by the antigenic peptide are regions of JAK2 that are located on the surface of the polypeptide, e.g., hydrophilic regions, as well as regions with high antigenicity.
  • a JAK2 immunogen typically is used to prepare antibodies by immunizing a suitable subject, ⁇ e.g., rabbit, goat, mouse or other mammal) with the immunogen.
  • An appropriate immunogenic preparation can contain, for example, recombinantly expressed JAK2 polypeptide or a chemically synthesized JAK2 polypeptide.
  • the preparation can further include an adjuvant, such as Freund's complete or incomplete adjuvant, or similar immunostimulatory agent. Immunization of a suitable subject with an immunogenic JAK2 preparation induces a polyclonal anti- JAK2 antibody response.
  • antibody refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site which specifically binds (immunoreacts with) an antigen, such as JAK2.
  • immunologically active portions of immunoglobulin molecules include F(ab) and F(ab')2 fragments which can be generated by treating the antibody with an enzyme such as pepsin.
  • the invention provides polyclonal and monoclonal antibodies that bind JAK2.
  • B3841854.1 refers to a population of antibody molecules that contain only one species of an antigen binding site capable of immunoreacting with a particular epitope of JAK2.
  • a monoclonal antibody composition thus typically displays a single binding affinity for a particular JAK2 polypeptide with which it immunoreacts.
  • Polyclonal anti-JAK2 antibodies can be prepared as described above by immunizing a suitable subject with a JAK2 immunogen or a nucleic acid molecule encoding the same.
  • the anti- JAK2 antibody titer in the immunized subject can be monitored over time by standard techniques, such as with an enzyme linked immunosorbent assay (ELISA) using immobilized JAK2.
  • ELISA enzyme linked immunosorbent assay
  • the antibody molecules directed against JAK2 can be isolated from the mammal (e.g. , from the blood) and further purified by well known techniques, such as protein A chromatography to obtain the IgG fraction.
  • antibody- producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique originally described by Kohler and Milstein (1975) Nature 256:495-497) (see also, Brown, et al. (1981) J. Immunol.
  • an immortal cell line typically a myeloma
  • lymphocytes typically splenocytes
  • the culture supematants of the resulting hybridoma cells are screened to identify a hybridoma producing a monoclonal antibody that binds JAK2.
  • immortalized cell lines can be applied for the purpose of generating an anti- JAK2 monoclonal antibody (see, e.g., G. Galfre, et al. (1977) Nature 266:55052; Gefter, et al. Somatic Cell Genet., cited supra; Lerner, Yale J. Biol. Med., cited supra; Kenneth,
  • the immortal cell line e.g. , a myeloma cell line
  • murine hybridomas can be made by fusing lymphocytes from a mouse immunized with an immunogenic preparation of the
  • B3841854.1 present invention with an immortalized mouse cell line.
  • Preferred immortal cell lines are mouse myeloma cell lines that are sensitive to culture medium containing hypoxanthine, aminopterin and thymidine ("HAT medium").
  • HAT medium any of a number of myeloma cell lines can be used as a fusion partner according to standard techniques, e.g., the P3-NSl/l-Ag4-l, P3- x63-Ag8.653 or Sp2/0-Agl4 myeloma lines. These myeloma lines are available from ATCC.
  • HAT-sensitive mouse myeloma cells are fused to mouse splenocytes using polyethylene glycol ("PEG").
  • Hybridoma cells resulting from the fusion are then selected using HAT medium, which kills unfused and unproductively fused myeloma cells (unfused splenocytes die after several days because they are not transformed).
  • Hybridoma cells producing a monoclonal antibody of the invention are detected by screening the hybridoma culture supernatants for antibodies that bind JAK2, e.g., using a standard ELISA assay.
  • a monoclonal anti- JAK2 antibody can be identified and isolated by screening a recombinant
  • kits for generating and screening phage display libraries are commercially available ⁇ e.g. , the Pharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01; and the Stratagene
  • recombinant anti- JAK2 antibodies such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, are within the scope of the invention.
  • Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in Robinson, et al. International Application No. PCT/US86/02269; Akira, et al. European Patent Application 184,187; Taniguchi, M., European Patent Application 171,496; Morrison et al. European Patent Application 173,494; Neuberger, et al. PCT International Publication No. WO 86/01533; Cabilly, et al. U.S. Patent No. 4,816,567; Cabilly, et al. European Patent Application 125,023; Better, et al.
  • an anti- JAK2 antibody is a fully human antibody.
  • an inhibitory agent of the invention is an inhibitory form of human JAK2, also referred to herein as a dominant negative inhibitor.
  • Many proteins are known to homodimerize and to heterodimerize.
  • One means to inhibit the activity of molecules that form dimers is through the use of a dominant negative inhibitor that has the ability to dimerize with a functional molecule but that lacks the ability to perform its normal biological activity (see e.g., Petrak, D. et al. (1994) J. Immunol. 153:2046-2051). By dimerizing with JAK2, such dominant negative inhibitors can inhibit their functional activity.
  • an inhibitory agent of the invention can be a form of a JAK2 polypeptide that has the ability to dimerize with other proteins but that lacks the ability to perform its normal biological activity, such as phosphorylation of HEXIMl .
  • This dominant negative form of a JAK2 polypeptide may be, for example, a mutated form of JAK2 in which the conformational structure of the JAK2 has been altered to prevent
  • Such dominant negative human JAK2 proteins can be expressed in cells using a recombinant expression vector encoding the JAK2 polypeptide, which is introduced into the cell by standard transfection methods.
  • a mutant form of JAK2 lacking residues critical for phosphorylation for example, at the YXXL motif of the JAK2 kinase
  • nucleotide sequences encoding the corresponding domains of JAK2 are removed from the JAK2 coding sequences by standard recombinant DNA techniques.
  • the truncated DNA is inserted into a recombinant expression vector, which is then introduced
  • the JAK2 inhibitors or agents that inhibit HEXIM1 tyrosine phosphorylation described herein can be used primarily in therapeutic methods for the treatment or prevention of RNA polymerase dependent viral disorders, such as HIV, influenza, hepatitis and west nile virus.
  • JAK2 kinase inhibitors or agents that inhibit HEXIM1 tyrosine phosphorylation are involved in the modulation of the phosphorylation of HEXIM1, modulation of the release of HEXIM1 for the pTEFb complex, modulation of pTEFb complex activity, modulation of TAT interaction with RNA polymerase, modulation of CDK9 mediated phosphorylation of RNA polymerase, modulation of RNA polymerase activity, modulation of the transcription of the viral genome, modulation of the replication of the viral genome, and modulation of the spread of a RNA polymerase dependent virus.
  • phosphorylation of the present invention for example, nucleic acid molecules,
  • compositions suitable for administration can be incorporated into pharmaceutical compositions suitable for administration.
  • Such compositions typically comprise the nucleic acid molecule, or polypeptide and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.
  • the use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.
  • a pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration.
  • routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral ⁇ e.g., inhalation), transdermal
  • Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediammetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • a coating such as lecithin
  • surfactants for example, sodium bicarbonate
  • chlorobutanol phenol, ascorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions can be prepared by incorporating the JAK2 kinase inhibitor (e.g., AG490, small molecules such as Z3, antisense molecules and anti-JAK2 antibody) or agents that inhibit HEXIM1 tyrosine phosphorylation in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • JAK2 kinase inhibitor e.g., AG490, small molecules such as Z3, antisense molecules and anti-JAK2 antibody
  • agents that inhibit HEXIM1 tyrosine phosphorylation in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • sterile powders for the preparation of sterile injectable solutions, the preferred methods
  • B3841854.1 preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition.
  • the tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
  • a binder such as microcrystalline cellulose, gum tragacanth or gelatin
  • an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch
  • a lubricant such as magnesium stearate or Sterotes
  • a glidant such as colloidal silicon dioxide
  • the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
  • a suitable propellant e.g., a gas such as carbon dioxide, or a nebulizer.
  • Systemic administration can also be by transmucosal or transdermal means.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.
  • Transmucosal administration can be accomplished through the use of nasal sprays or suppositories.
  • the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
  • the compounds can also be prepared in the form of suppositories ⁇ e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery. Vaginal suppositories or foams for local mucosal delivery may also be prepared to block sexual transmission.
  • suppositories ⁇ e.g., with conventional suppository bases such as cocoa butter and other glycerides
  • retention enemas for rectal delivery.
  • Vaginal suppositories or foams for local mucosal delivery may also be prepared to block sexual transmission.
  • the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • a controlled release formulation including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art.
  • the materials can also be
  • Liposomal suspensions including liposomes targeted to infected cells with monoclonal antibodies to viral antigens and liposomes targeted to macrophages containing, for example,
  • phosphatidylserine can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811 and U.S. Patent No. 5,643,599, the entire contents of which are incorporated herein.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
  • Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD 50 (the dose lethal to 50% of the population) and the ED 50 (the dose therapeutically effective in 50% of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD 5 o/ED 5 o.
  • Compounds which exhibit large therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
  • the data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.
  • the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED 50 with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC 50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture.
  • IC 50 i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms
  • levels in plasma may be measured, for example, by high performance liquid chromatography.
  • a therapeutically effective amount of polypeptide ranges from about 0.001 to 30 mg/kg body weight, preferably about 0.01 to 25 mg/kg body weight, more preferably about 0.1 to 20 mg/kg body weight, and even more preferably about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight.
  • an effective dosage ranges from about 0.001 to 30 mg/kg body weight, preferably about 0.01 to 25 mg/kg body weight, more preferably about 0.1 to 20 mg/kg body weight, and even more preferably about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight.
  • an effective dosage ranges from about 0.001 to 30 mg/kg body weight, preferably about 0.01 to 25 mg/kg body weight, more preferably about 0.1 to 20 mg/kg body weight, and even more preferably about 1 to 10 mg/kg, 2 to 9 mg/kg,
  • treatment of a subject with a therapeutically effective amount of the JAK2 kinase inhibitor or agent that inhibits HEXIM1 tyrosine phosphorylation can include a single treatment or, preferably, can include a series of treatments.
  • a subject is treated with antibody or polypeptide in the range of between about 0.1 to 20 mg/kg body weight, one time per week for between about 1 to 10 weeks, preferably between 2 to 8 weeks, more preferably between about 3 to 7 weeks, and even more preferably for about 4, 5, or 6 weeks.
  • the effective dosage of the JAK2 kinase inhibitor or agent that inhibits HEXIM1 tyrosine phosphorylation used for treatment may increase or decrease over the course of a particular treatment. Changes in dosage may result and become apparent from the results of diagnostic assays as described herein.
  • the present invention encompasses JAK2 kinase inhibitors or agents that inhibit HEXIM1 tyrosine phosphorylation which modulate expression or activity.
  • a JAK2 kinase inhibitor or agent that inhibits HEXIM1 tyrosine phosphorylation may, for example, be a small molecule.
  • small molecules include, but are not limited to, peptides, peptidomimetics, amino acids, amino acid analogs,
  • the dose(s) of the small molecule will vary, for example, depending upon the identity, size, and condition of the subject or sample being treated, further depending upon the route by which the composition is to be administered, if applicable, and the effect which the practitioner desires the small molecule to have upon the nucleic acid or polypeptide of the invention.
  • Exemplary doses include milligram or microgram amounts of the JAK2 inhibitor or agent that inhibits HEXIM1 tyrosine phosphorylation, e.g., small molecule, per kilogram of subject or sample weight (e.g., about 1 microgram per kilogram to about 500 milligrams per kilogram, about 100 micrograms per kilogram to about 5 milligrams per kilogram, or about 1 microgram per kilogram to about 50 micrograms per kilogram. It is furthermore understood that appropriate doses of a small molecule depend upon the potency of the small molecule with respect to the expression or activity to be modulated. Such appropriate doses may be determined using the assays described herein. When one or more of these small molecules is to be administered to an animal (e.g.
  • a physician, veterinarian, or researcher may, for example, prescribe a relatively low dose at first, subsequently increasing the dose until an appropriate response is obtained.
  • the specific dose level for any particular animal subject will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, any drug combination, and the degree of expression or activity to be modulated.
  • an antibody may be conjugated to a therapeutic moiety such as a cytotoxin, a therapeutic agent or a radioactive metal ion.
  • a cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologues thereof.
  • Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thiogu
  • alkylating agents e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU)
  • BSNU carmustine
  • CCNU lomustine
  • cyclothosphamide busulfan
  • dibromomannitol dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin
  • anthracyclines e.g., daunorubicin (formerly daunomycin) and
  • doxorubicin doxorubicin
  • antibiotics e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)
  • anti-mitotic agents e.g., vincristine and vinblastine.
  • the drug moiety is not to be construed as limited to classical chemical therapeutic agents.
  • the drug moiety may be a protein or polypeptide possessing a desired biological activity.
  • proteins may include, for example, a toxin such as abrin, ricin A,
  • B3841854.1 pseudomonas exotoxin, or diphtheria toxin a protein such as tumor necrosis factor, alpha- interferon, beta-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator; or, biological response modifiers such as, for example,
  • lymphokines interleukin-1 ("IL-1”), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colony stimulating factor (“GM-CSF”), granulocyte colony stimulating factor (“G-CSF”), or other growth factors.
  • IL-1 interleukin-1
  • IL-2 interleukin-2
  • IL-6 interleukin-6
  • GM-CSF granulocyte macrophage colony stimulating factor
  • G-CSF granulocyte colony stimulating factor
  • an antibody can be conjugated to a second antibody to form an antibody heteroconjugate as described by Segal in U.S. Patent No. 4,676,980.
  • the nucleic acid molecules of the invention can be inserted into vectors and used as gene therapy vectors.
  • Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see U.S. Patent No. 5,328,470) or by stereotactic injection (see e.g., Chen, et al. (1994) Proc. Natl. Acad. Sci., USA 91 :3054- 3057).
  • the pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded.
  • the pharmaceutical preparation can include one or more cells which produce the gene delivery system.
  • compositions can be included in a container, pack, or dispenser together with instructions for administration.
  • the present invention is generally directed to a method for treating or preventing a viral disorder in a subject having or at risk of having said viral disorder, wherein said virus is R A polymerase dependent, by administering to the subject an effective amount of a composition including a JAK2 kinase inhibitor or agent that inhibits HEXIMl tyrosine phosphorylation, thereby treating or preventing the viral disorder (for example, HIV, influenzavirus, influenzavirus A such as HlNl, influenzavirus B, influenzavirus C, hepatitis A, hepatitis C, hepatitis D, hepatitis E, and west nile virus) in said subject.
  • a composition including a JAK2 kinase inhibitor or agent that inhibits HEXIMl tyrosine phosphorylation for example, HIV, influenzavirus, influenzavirus A such as HlNl, influenzavirus B, influenzavirus C, hepatitis A, hepatitis C, hepatitis D, hepatitis E
  • the present invention is directed to a method for inhibiting the replication of a virus in a subject, by administering to the subject infected with said virus, wherein said virus is dependent on RNA polymerase for replication (for example, HIV, influenzavirus, influenzavirus A such as HlNl, influenzavirus B, influenzavirus C, hepatitis A, hepatitis C, hepatitis D, hepatitis E, and west nile virus), an effective amount of a composition including a JAK2 kinase inhibitor or agent that inhibits HEXIMl tyrosine phosphorylation, thereby inhibiting viral replication in the subject.
  • RNA polymerase for replication for example, HIV, influenzavirus, influenzavirus A such as HlNl, influenzavirus B, influenzavirus C, hepatitis A, hepatitis C, hepatitis D, hepatitis E, and west nile virus
  • the methods of the present invention are based, at least in part, on the elucidation of the role of JAK2 kinase and the tyrosine phosphorylation of HEXIMl in the replication of RNA polymerase dependent viruses.
  • the inventors have identified that JAK2 kinase and the tyrosine phosphorylation of HEXIMl at its YLEL epitope play an important role in activating RNA polymerase II so as to promote viral gene expression and viral replication after infection of a cell.
  • RNA polymerase dependent virus based disorders such as HIV, Influenza, Hepatitis C and West Nile Virus.
  • JAK2 kinase serves to phosphorylate HEXIMl, specifically, at the YLEL epitope present on HEXIMl, so as to trigger the release of HEXIMl from the transcription elongation factor P-TEFb complex (cyclin Tl-cdk9).
  • a viral replicating factor for example, TAT (an HIV replicating factor)
  • TAT an HIV replicating factor
  • the P-TEFb complex specifically, cdk9, triggers phosphorylation and
  • RNA Polymerase II transcription elongation of RNA Polymerase II so as to allow for transcription and replication of the viral genome.
  • RNA polymerase dependent virus based disorders including lentivirus based disorders, such as Human Immunodeficiency Virus (HIV) (HIV-1 and HIV-2), Influenza
  • Hepatitis virus A Hepatitis virus C, Hepatitis virus D and Hepatitis virus E
  • the West Nile Virus can be treated by use of JAK2 kinase inhibitors or agents that inhibit HEXIM1 tyrosine phosphorylation at the YLEL motif.
  • the subject methods employ agents that inhibit JAK2 expression, processing, post-translational modification, or activity, or the expression, processing, post- translational modification, or activity of another molecule in a JAK2 signaling pathway, e.g., HEXIM1, such that JAK2 or the activity of a molecule in a JAK2 signal transduction pathway is modulated, for example, inhibited.
  • a JAK2 signaling pathway e.g., HEXIM1
  • the subject methods are useful in both clinical and non-clinical settings.
  • the instant methods can be performed in vitro. In another embodiment, the instant methods can be performed in a cell in vitro and then the treated cell can be administered to a subject.
  • subject is intended to include living organisms in which an immune response can be elicited.
  • Preferred subjects are mammals. Particularly preferred subjects are humans. Other examples of subjects include monkeys, dogs, cats, mice, rats, cows, horses, goats, sheep as well as other farm and companion animals.
  • Inhibition of JAK2 expression and/or activity or inhibition of HEXIM1 tyrosine phosphorylation, in humans as well as veterinary applications, provides a means to regulate RNA polymerase dependent viral disorders arising from JAK2 expression and/or activity or HEXIM1 tyrosine phosphorylation in various disease states and is encompassed by the present invention.
  • the inhibitory methods of the invention can operate by modulating the phosphorylation of JAK2 protein, the phosphorylation of HEXIM1, the release of HEXIM1 for the pTEFb complex, the activity of pTEFb complex, TAT interaction with RNA polymerase, CDK9 mediated phosphorylation of RNA polymerase, the activity of RNA polymerase activity and/or the transcription of the viral genome.
  • B3841854.1 with the disorder, either in the long term or short term (i.e., amelioration of the condition) or simply a transient beneficial effect to the subject.
  • the present invention is directed to a method for treating or preventing HIV infection in a subject having or at risk of HIV infection, by administering to the subject an effective amount of a composition including a JAK2 kinase inhibitor or an agent that inhibits HEXIMl tyrosine phosphorylation, thereby treating or preventing HIV in said subject.
  • the present invention includes methods for inhibiting the replication of HIV in a subject having or at risk of HIV infection, by administering to the subject an effective amount of a composition including a JAK2 kinase inhibitor or an agent that inhibits HEXIMl tyrosine phosphorylation, thereby inhibiting HIV replication in the subject.
  • the HIV may either be HIV-1 or HIV-2.
  • the present invention is based, at least in part, on the identification of the role that JAK2 kinase plays in activating RNA polymerase, thereby allowing for the expression, replication and spread of the HIV genome.
  • RNA polymerase II serves to catalyze the replication of HIV genomic material and, further, to generate viral mRNA utilized to generate HIV proteins during translation.
  • the single stranded HIV RNA is converted to double-stranded HIV DNA which subsequently enters the host cell's nucleus and integrates within the host cell's DNA.
  • the integrated viral DNA is referred to as a provirus.
  • the host cell's endogenous RNA polymerase is hijacked by the provirus so as to catalyze the replication of HIV genomic material and, further, to generate viral mRNA.
  • TAT an HIV encoded transcription factor
  • TAR TAT -Responsive element
  • JAK2 kinase serves to activate the RNA polymerase to catalyze the replication of HIV genomic material and, further, to generate viral mRNA utilized to generate HIV proteins during translation. Specifically, JAK2 kinase serves to
  • HEXIMl phosphorylate HEXIMl at the YLEL epitope present on HEXIMl, so as to trigger the release of HEXIMl from the transcription elongation factor P-TEFb complex (cyclin Tl- cdk9).
  • the c-termini of HEXIMl (KQELIKEYLELEKCLS) plays a key role in both the phosphorylation of HEXIMl by JAK2 kinase and the interaction of HEXIMl with cyclin Tl .
  • the post-translational modification of HEXIMl i.e., the tyrosine phosphorylation of the YLEL motif (amino acid residues 296-299) of HEXIMl by JAK2 kinase, facilitates the release of HEXIMl from cyclin Tl at the KEYL motif (amino acid residues 289-292)
  • HEXIMl from the p-TEFb complex and, in particular, from the cyclin Tl, enables TAT (an HIV replicating factor) to complex with the P-TEFb complex thereby hijacking the host cell's transcriptional machinery for purposes of promoting viral replication.
  • TAT an HIV replicating factor
  • the P-TEFb complex specifically, cdk9, triggers phosphorylation and transcription elongation of RNA Polymerase II so as to allow for transcription and replication of the HIV genome.
  • inhibition of JAK2 kinase activity or inhibition of the tyrosine phosphorylation of HEXIMl should serve to lock the access of tat to cyclinTl, thereby inhibiting HIV replication.
  • the present invention sets forth that the inhibition of JAK2 through administration of a JAK2 inhibitor or, alternatively, the inhibition of HEXIMl tyrosine phosphorylation through administration of an agent effecting such activity, can serve to treat and/or prevent HIV.
  • the JAK2 kinase inhibitor in eliciting the desired therapeutic or prophylactic effect, can operate by modulating the tyrosine phosphorylation of JAK2 protein, the tyrosine phosphorylation of HEXIMl, the release of HEXIMl for the pTEFb complex, the activity of pTEFb complex, TAT interaction with RNA polymerase, CDK9 mediated serine phosphorylation of RNA polymerase, the activity of RNA polymerase activity and/or the transcription of the viral genome.
  • the present invention is directed to a method for treating or preventing influenza in a subject having or at risk of infection by an infiuenzavirus, for example, any of influenza A ⁇ e.g., H1N1) , B or C, by administering to the subject an effective amount of a composition including a JAK2 kinase inhibitor or an agent that inhibits HEXIMl tyrosine phosphorylation, thereby treating or preventing influenza in said subject.
  • an infiuenzavirus for example, any of influenza A ⁇ e.g., H1N1) , B or C
  • a composition including a JAK2 kinase inhibitor or an agent that inhibits HEXIMl tyrosine phosphorylation thereby treating or preventing influenza in said subject.
  • the present invention includes methods for inhibiting the replication of the influenza virus, for example, influenza A ⁇ e.g., H1N1), B or C, in a subject having or at risk of infection by an infiuenzavirus, by administering to the subject an effective amount of a composition including a JAK2 kinase inhibitor or an agent that inhibits HEXIMl tyrosine phosphorylation, thereby inhibiting replication of the influenza virus in the subject.
  • influenza A ⁇ e.g., H1N1
  • B or C a subject having or at risk of infection by an infiuenzavirus
  • the present invention is based, at least in part, on the identification of the role of JAK2 kinase in the replication of influenzaviruses.
  • (-) strand influenza viral RNAs enter the nucleus, where they serve as templates for the synthesis of mRNA's for the synthesis of viral proteins in the cytoplasm.
  • the RNA-dependent RNA polymerase carried by the virus is critical for translating the (-) strand viral RNAs into protein, particularly in light of the fact that the host cells has no enzymes which can copy such long RNA molecules.
  • RNA polymerases specifically, RNA dependent RNA polymerase, trigger the production of additional (-) strand RNAs necessary for assembling new virions.
  • RNA polymerases carried by the infiuenzavirus, helps in producing a full length (+) strand, which, in turn, is copied to a full-length (-) strand RNA for use in assembling new virions.
  • RNA polymerase II Transcription by the influenza virus RNA-dependent RNA polymerase is dependent on cellular RNA processing activities that are known to be associated with cellular RNA polymerase II (Pol II) transcription, namely, capping and splicing.
  • the influenza virus RNA polymerase complex interacts with the large subunit of Pol II via its C-terminal domain.
  • the viral polymerase binds hyperphosphorylated forms of Pol II, indicating that it targets actively transcribing Pol II.
  • immunofluorescence analysis is consistent with a new model showing that influenza virus polymerase accumulates at Pol II transcription sites. Accordingly, regulation of RNA polymerase II activities, for example, by
  • JAK2 kinase inhibitors or agents that inhibit the tyrosine phosphorylation of HEXIMl can serve to modulate influenza virus replication, thereby treating influenza virus infection.
  • JAK2 kinase serves to activate the RNA polymerase to catalyze the replication of the influenza genomic material and, further, to generate viral mRNA utilized to generate HIV proteins during translation.
  • JAK2 kinase serves to phosphorylate HEXIMl, specifically, at the YLEL epitope present on HEXIMl, so as to trigger the release of HEXIMl from the transcription elongation factor P-TEFb complex (cyclin Tl-cdk9).
  • P-TEFb complex hijacks the host cell's transcriptional machinery for purposes of promoting replication of the infiuenzavirus.
  • inhibiting JAK2 through administration of a JAK2 inhibitor or, alternatively, inhibiting HEXIMl tyrosine phosphorylation through administration of an agent effecting such activity can serve to treat and/or prevent influenza.
  • the JAK2 kinase inhibitor or agent that inhibits tyrosine phosphorylation of HEXIM 1 can operate by modulating the phosphorylation of JAK2 protein, the phosphorylation of HEXIMl, the release of HEXIMl for the pTEFb complex, the activity of pTEFb complex, CDK9 mediated phosphorylation
  • RNA polymerase B3841854.1 of RNA polymerase, the activity of RNA polymerase activity and/or the transcription of the influenza virus genome.
  • the present invention is directed to a method for treating or preventing hepatitis in a subject having or at risk of infection by an influenza virus, for example, any of influenza A ⁇ e.g., H1N1), C, D or E, by administering to the subject an effective amount of a composition including a JAK2 kinase inhibitor or an agent that inhibits HEXIMl tyrosine phosphorylation, thereby treating or preventing hepatitis in said subject.
  • an influenza virus for example, any of influenza A ⁇ e.g., H1N1), C, D or E
  • the present invention includes methods for inhibiting the replication of the hepatitis virus, for example, hepatitis A, C, D or E, in a subject having or at risk of infection by hepatitis, by administering to the subject an effective amount of a composition including a JAK2 kinase inhibitor or an agent that inhibits HEXIMl tyrosine phosphorylation, thereby inhibiting replication of the hepatitis virus in the subject.
  • a composition including a JAK2 kinase inhibitor or an agent that inhibits HEXIMl tyrosine phosphorylation thereby inhibiting replication of the hepatitis virus in the subject.
  • JAK2 kinase plays an important role in the replication of various hepatitis viruses.
  • JAK2 kinase plays an important role in activating RNA polymerase to effect viral expression and replication, a role that is equally important with respect specifically to hepatitis viruses.
  • Hepatitis A and E utilize a virus encoded RNA-dependent RNA polymerase to replicate their respective genomes.
  • Hepatitis C additionally utilizes an RNA dependent RNA polymerase, for example, NS5B, to effect viral replication.
  • Hepatitis D utilizes RNA polymerase II to copy its genome, specifically replicating (-) strand viral RNA to a (+) strand RNA.
  • inhibiting JAK2 through administration of a JAK2 inhibitor or, alternatively, inhibiting HEXIMl tyrosine phosphorylation through administration of an agent effecting such activity can serve to treat and/or prevent hepatitis by inhibiting RNA polymerase activity.
  • the JAK2 kinase inhibitor or agent inhibiting HEXIMl tyrosine phosphorylation can operate by modulating the phosphorylation of JAK2 protein, the phosphorylation of HEXIMl, the release of HEXIMl for the pTEFb complex, the activity of pTEFb complex, CDK9 mediated phosphorylation of RNA polymerase, the activity of RNA polymerase activity and/or the transcription of the hepatitis virus genome.
  • JAK2 kinase inhibitors or agents that inhibits HEXIMl tyrosine phosphorylation
  • JAK2 kinase inhibitors or agents that inhibit HEXIMl tyrosine phosphorylation of the invention are administered to subjects in a biologically compatible form suitable for pharmaceutical administration in vivo to effect the desired therapeutic effect, for example, to treat HIV infection by inhibiting viral replication.
  • biologically compatible form suitable for administration in vivo is meant a form of the protein to be administered in which any toxic effects are outweighed by the therapeutic effects of the modulating agent.
  • subject is intended to include living organisms in which an immune response can be elicited, e.g., mammals. Examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof.
  • Administration of a JAK2 kinase inhibitor or agent that inhibits HEXIMl tyrosine phosphorylation as described herein can be in any combination of a JAK2 kinase inhibitor or agent that inhibits HEXIMl tyrosine phosphorylation as described herein can be in any
  • pharmacological form including a therapeutically active amount of an agent alone or in combination with a pharmaceutically acceptable carrier.
  • a therapeutically active amount of the therapeutic compositions of the present invention is defined as an amount effective, at dosages and for periods of time necessary to achieve the desired result.
  • a therapeutically active amount of a JAK2 kinase inhibitor or agent that inhibits HEXIMl tyrosine phosphorylation may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of peptide to elicit a desired response in the individual.
  • Dosage regimen may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.
  • compositions of the present invention can be administered by any suitable route known in the art including for example intravenous, subcutaneous, intramuscular, transdermal, intrathecal or intracerebral or administration to cells in ex vivo treatment protocols. Administration can be either rapid as by injection or over a period of time as by slow infusion or administration of slow release formulation.
  • a JAK2 kinase inhibitor can also be linked or conjugated with agents that provide desirable pharmaceutical or pharmacodynamic properties.
  • a JAK2 kinase inhibitor or agent that inhibits HEXIMl tyrosine phosphorylation can be coupled to any substance known in the art to promote infection of HIV infected cells such as an antibody specific for receptors on such infected cells, and administered by intravenous injection.
  • a JAK2 kinase inhibitor or agent that inhibits HEXIMl tyrosine phosphorylation can be stably linked to a polymer such as polyethylene glycol to obtain desirable properties of solubility, stability, half-life and other pharmaceutically
  • the a JAK2 kinase inhibitor or agent that inhibits HEXIM1 tyrosine phosphorylation can be in a composition which aids in delivery into the cytosol of a cell.
  • the agent may be conjugated with a carrier moiety such as a liposome that is capable of delivering the peptide into the cytosol of a cell.
  • a carrier moiety such as a liposome that is capable of delivering the peptide into the cytosol of a cell.
  • the a JAK2 kinase inhibitor or agent that inhibits HEXIM1 tyrosine phosphorylation can be modified to include specific transit peptides or fused to such transit peptides which are capable of delivering the JAK2 kinase inhibitor or agent that inhibits HEXIM1 tyrosine phosphorylation into a cell.
  • the agent can be delivered directly into a cell by microinjection.
  • compositions are usually employed in the form of pharmaceutical preparations. Such preparations are made in a manner well known in the pharmaceutical art.
  • One preferred preparation utilizes a vehicle of physiological saline solution, but it is
  • pharmaceutically acceptable carriers such as physiological concentrations of other non-toxic salts, five percent aqueous glucose solution, sterile water or the like may also be used.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the therapeutic compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions. It may also be desirable that a suitable buffer be present in the composition.
  • Such solutions can, if desired, be lyophilized and stored in a sterile ampoule ready for reconstitution by the addition of sterile water for ready injection.
  • the primary solvent can be aqueous or alternatively non-aqueous.
  • JAK2 kinase inhibitors or agents that inhibit HEXIM1 tyrosine phosphorylation can also be incorporated into a solid or semi-solid biologically compatible matrix which can be implanted into tissues requiring treatment.
  • the carrier can also contain other pharmaceutically-acceptable excipients for modifying or maintaining the pH, osmolality, viscosity, clarity, color, sterility, stability, rate of dissolution, or odor of the formulation.
  • the carrier may contain still other pharmaceutically-acceptable excipients for modifying or maintaining release or absorption or penetration across the blood-brain barrier.
  • excipients are those substances usually and customarily employed to formulate dosages for parenteral administration in either unit dosage or multi-dose form or for direct infusion by continuous or periodic infusion.
  • Dose administration can be repeated depending upon the pharmacokinetic parameters of the dosage formulation and the route of administration used. It is also
  • B3841854.1 provided that certain formulations containing the JAK2 kinase inhibitor or agent that inhibits HEXIMl tyrosine phosphorylation are to be administered orally. Such formulations are preferably encapsulated and formulated with suitable carriers in solid dosage forms.
  • Suitable carriers, excipients, and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, calcium silicate, microcrystalline cellulose, olyvinylpyrrolidone, cellulose, gelatin, syrup, methyl cellulose, methyl- and propylhydroxybenzoates, talc, magnesium, stearate, water, mineral oil, and the like.
  • the formulations can additionally include lubricating agents, wetting agents, emulsifying and suspending agents, preserving agents, sweetening agents or flavoring agents.
  • compositions may be formulated so as to provide rapid, sustained, or delayed release of the active ingredients after administration to the patient by employing procedures well known in the art.
  • the formulations can also contain substances that diminish proteolytic degradation and/or substances which promote absorption such as, for example, surface active agents.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.
  • the specific dose can be readily calculated by one of ordinary skill in the art, e.g., according to the approximate body weight or body surface area of the patient or the volume of body space to be occupied. The dose will also be calculated dependent upon the particular route of administration selected. Further refinement of the calculations necessary to determine the appropriate dosage for treatment is routinely made by those of ordinary skill in the art. Such calculations can be made without undue experimentation by one skilled in the art in light of the activity disclosed herein in assay preparations of target cells. Exact dosages are determined in conjunction with standard dose-response studies.
  • the amount of the composition actually administered will be determined by a practitioner, in the light of the relevant circumstances including the condition or conditions to be treated, the choice of composition to be administered, the age, weight, and response of the individual patient, the severity of the patient's symptoms, and the chosen route of administration.
  • nucleic acids including recombinant expression vectors encoding JAK2 inhibitors, antisense R A, or dominant negative inhibitors
  • the agents can be introduced into cells of the subject using methods known in the art for introducing nucleic acid (e.g., DNA) into cells in vivo. Examples of such methods encompass both non-viral and viral methods, including:
  • Naked DNA can be introduced into cells in vivo by directly injecting the DNA into the cells (see e.g., Acsadi et al. (1991) Nature 332:815-818; Wolff et al. (1990) Science 247: 1465-1468).
  • a delivery apparatus ⁇ e.g., a "gene gun" for injecting DNA into cells in vivo can be used.
  • Such an apparatus is commercially available ⁇ e.g., from BioRad).
  • Cationic Lipids Naked DNA can be introduced into cells in vivo by complexing the DNA with cationic lipids or encapsulating the DNA in cationic liposomes.
  • suitable cationic lipid formulations include N-[-l-(2,3-dioleoyloxy)propyl]N,N,N- triethylammonium chloride (DOTMA) and a 1 :1 molar ratio of l,2-dimyristyloxy-propyl-3- dimethylhydroxyethylammonium bromide (DMRIE) and dioleoyl
  • DOPE phosphatidylethanolamme
  • Naked DNA can also be introduced into cells in vivo by complexing the DNA to a cation, such as polylysine, which is coupled to a ligand for a cell-surface receptor (see for example Wu, G. and Wu, C.H. (1988) J. Biol. Chem. 263: 14621; Wilson et al. (1992) J. Biol. Chem. 267:963-967; and U.S. Patent No.
  • a cation such as polylysine
  • Binding of the DNA-ligand complex to the receptor facilitates uptake of the DNA by receptor-mediated endocytosis.
  • a DNA-ligand complex linked to adenovirus capsids which naturally disrupt endosomes, thereby releasing material into the cytoplasm can be used to avoid degradation of the complex by intracellular lysosomes (see for example Curiel et al. (1991) Proc. Natl. Acad. Sci. USA 88:8850; Cristiano et al. (1993) Proc. Natl. Acad. Sci. USA 90:2122-2126).
  • Retroviruses Defective retroviruses are well characterized for use in gene transfer for gene therapy purposes (for a review see Miller, A.D. (1990) Blood 76:271).
  • a recombinant retrovirus can be constructed having a nucleotide sequences of interest incorporated into the retroviral genome. Additionally, portions of the retroviral genome can be removed to render the retrovirus replication defective. The replication defective retrovirus is then packaged into virions which can be used to infect a target cell through the use of a helper virus by standard techniques. Protocols for producing recombinant retroviruses and for infecting cells in vitro or in vivo with such viruses can be found in
  • suitable retroviruses include pLJ, ZIP, pWE and pEM which are well known to those skilled in the art.
  • suitable packaging virus lines include ⁇ 2 and Retroviruses have been used to introduce a variety of genes into many different cell types, including epithelial cells, endothelial cells, lymphocytes, myoblasts, hepatocytes, bone marrow cells, in vitro and/or in vivo (see for example Eglitis, et al. (1985) Science 230: 1395-1398; Danos and Mulligan (1988) Proc. Natl. Acad. Sci. USA 85:6460-6464; Wilson et al. (1988) Proc. Natl. Acad. Sci.
  • Retroviral vectors require target cell division in order for the retroviral genome (and foreign nucleic acid inserted into it) to be integrated into the host genome to stably introduce nucleic acid into the cell. Thus, it may be necessary to stimulate replication of the target cell.
  • Adenoviruses The genome of an adenovirus can be manipulated such that it encodes and expresses a gene product of interest but is inactivated in terms of its ability to replicate in a normal lytic viral life cycle. See for example Berkner et al. (1988)
  • Suitable adenoviral vectors derived from the adenovirus strain Ad type 5 dl324 or other strains of adenovirus ⁇ e.g., Ad2, Ad3, Ad7 etc.) are well known to those skilled in the art. Recombinant adenoviruses are advantageous in that they do not require dividing cells to be effective gene delivery vehicles and can be used to infect a wide variety of cell types, including airway epithelium (Rosenfeld et al.
  • introduced adenoviral DNA (and foreign DNA contained therein) is not integrated into the genome of a host cell but remains episomal, thereby avoiding potential problems that can occur as a result of insertional mutagenesis in situations where introduced DNA becomes integrated into the host genome ⁇ e.g., retroviral DNA).
  • adenoviral genome for foreign DNA is large (up to 8 kilobases) relative to other gene delivery vectors (Berkner et al. cited supra; Haj-Ahmand and Graham (1986) J. Virol. 57:267).
  • Most replication-defective adenoviral vectors are most replication-defective adenoviral vectors.
  • B3841854.1 currently in use are deleted for all or parts of the viral El and E3 genes but retain as much as 80 % of the adenoviral genetic material.
  • Adeno-associated virus is a naturally occurring defective virus that requires another virus, such as an adenovirus or a herpes virus, as a helper virus for efficient replication and a productive life cycle. (For a review see
  • An AAV vector such as that described in Tratschin et al. (1985) Mol. Cell. Biol. 5:3251-3260 can be used to introduce DNA into cells.
  • a variety of nucleic acids have been introduced into different cell types using AAV vectors (see for example Hermonat et al. (1984) Proc. Natl. Acad. Sci. USA 81 :6466-6470; Tratschin et al. (1985) Mol. Cell. Biol. 4:2072-2081; Wondisford et al. (1988) Mol. Endocrinol. 2:32-39; Tratschin et al. (1984) J. Virol.
  • DNA introduced into a cell can be detected by a filter hybridization technique ⁇ e.g. , Southern blotting) and RNA produced by transcription of introduced DNA can be detected, for example, by Northern blotting, RNase protection or reverse transcriptase- polymerase chain reaction (RT-PCR).
  • RNA produced by transcription of introduced DNA can be detected, for example, by Northern blotting, RNase protection or reverse transcriptase- polymerase chain reaction (RT-PCR).
  • RT-PCR reverse transcriptase- polymerase chain reaction
  • the gene product can be detected by an appropriate assay, for example by immunological detection of a produced protein, such as with a specific antibody, or by a functional assay to detect a functional activity of the gene product.
  • a retroviral expression vector encoding a JAK2 kinase inhibitor or agent that inhibits HEXIM1 tyrosine phosphorylation is used to express the inhibitor in cells in vivo.
  • retroviral vectors can be prepared according to standard methods known in the art (discussed further above).
  • Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50.
  • Compounds which exhibit large therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such
  • B3841854.1 compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
  • the data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.
  • the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture.
  • IC50 i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms
  • levels in plasma may be measured, for example, by high performance liquid chromatography.
  • JAK2 kinase inhibitors or agents that inhibit HEXIM1 phosphorylation e.g., drugs or compounds
  • the effectiveness of an agent determined by a screening assay as described herein to decrease JAK2 gene expression, protein levels, or to downregulate JAK2 activity can be monitored in clinical trials of subjects having RNA polymerase viral disorders.
  • the expression or activity of a JAK2 gene, and preferably, other genes that have been implicated in a disorder can be used as a "read out" or markers of the phenotype of a particular cell.
  • genes including JAK2 encoding genes, that are modulated in cells by treatment with a JAK2 kinase inhibitor (e.g., compound, drug or small molecule) which modulates JAK2 activity (e.g., identified in a screening assay as described herein) can be identified.
  • a JAK2 kinase inhibitor e.g., compound, drug or small molecule
  • JAK2 activity e.g., identified in a screening assay as described herein
  • JAK2 kinase inhibitor e.g., compound, drug or small molecule
  • JAK2 activity e.g., identified in a screening assay as described herein
  • the levels of gene expression can be quantified by Northern blot analysis or RT-PCR, as described herein, or alternatively by measuring the amount of protein produced, by one of the methods as described herein, or by measuring the levels of activity of JAK2 or other genes.
  • the gene expression pattern can serve as a marker, indicative of the physiological response of the cells to the agent. Accordingly, this response state may be determined before, and at various points during treatment of the individual with the agent.
  • the present invention provides a method for monitoring the effectiveness of treatment of a subject with an agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate identified by the screening assays described herein) comprising the steps of (i) obtaining a pre-administration sample from a subject prior to administration of the agent; (ii) detecting the level of expression of a JAK2 protein, mR A, or genomic DNA in the pre- administration sample; (iii) obtaining one or more post-administration samples from the subject; (iv) detecting the level of expression or activity of the JAK2 protein, mRNA, or genomic DNA in the post-administration samples; (v) comparing the level of expression or activity of the JAK2 protein, mRNA, or genomic DNA in the pre-administration sample with the JAK2 protein, mRNA, or genomic DNA in the post administration sample or samples; and (vi) altering the administration of the agent
  • an agent e.g.,
  • the ability of a JAK2 inhibitor or agent that inhibits HEXIM1 tyrosine phosphorylation to modulate RNA polymerase dependent viral infection, for example, HIV, influenza, hepatitis and west nile virus, in a subject can be measured by detecting an improvement in the condition of the patient after the administration of the inhibitor. Such improvement can be readily measured by one of ordinary skill in the art using indicators appropriate for the specific condition of the patient. Monitoring the response of the patient by measuring changes in the condition of the patient is preferred in situations where the collection of biopsy materials would pose an increased risk and/or detriment to the patient.
  • tyrosine-phospho-HEXIMl antibodies could be used in an ELISA test to monitor viral replication by measuring the level of tyrosine phosphorylated YXXL-Heximl in subjects.
  • compositions containing a JAK2 inhibitor or agent that inhibits HEXIM1 tyrosine phosphorylation can be used.
  • the present invention also provides methods for detecting the presence of JAK2 in a sample from a patient.
  • the invention further provides methods (also referred to herein as “screening assays") for identifying further JAK2 kinase inhibitors or agents for preventing the tyrosine phosphorylation of HEXIM1, i.e., candidate or test compounds or agents (e.g.,
  • peptidomimetics small molecules or other drugs which modulate, for example one or more JAK2 activity or upstream or downstream activities thereof, e.g., the tyrosine
  • phosphorylation of JAK2 protein the tyrosine phosphorylation of HEXIM1, the release of HEXIM1 for the pTEFb complex (for example, at the YXXL motif), the modulation of pTEFb complex activity, the modulation of TAT interaction with RNA polymerase, the modulation of CDK9 mediated phosphorylation of RNA polymerase, the modulation of RNA polymerase activity, the modulation of the transcription of the viral genome, the modulation of the replication of the viral genome, and the modulation of the spread of a virus.
  • the assays can be used to identify agents that modulate, in particular, inhibit, the function of JAK2 and/or a JAK2 -binding molecule, such as, but not limited to HEXIM1.
  • agents may interact with JAK2 or the JAK2 -binding molecule (e.g., to inhibit their activity).
  • the function of JAK2 or the JAK2 -binding molecule can be affected at any level, including transcription, protein expression, protein localization, and/or cellular activity.
  • the subject assays can also be used to identify, e.g., agents that alter the interaction of JAK2 or the JAK2 -binding molecule with a binding partner, substrate, or cofactors, or modulate, e.g., decrease, the stability of such interaction.
  • the subject screening assays can measure the activity of JAK2 or a JAK2 -binding protein directly (e.g., phosphorylation or ubiquitination), or can measure a downstream event controlled by modulation of JAK2 or a JAK2 -binding protein (e.g., the
  • phosphorylation of JAK2 protein the phosphorylation of HEXIM1, the release of HEXIM1 for the pTEFb complex (for example, at the YXXL motif), the modulation of pTEFb complex activity, the modulation of TAT interaction with RNA polymerase, the modulation of CDK9 mediated phosphorylation of RNA polymerase, the modulation of RNA polymerase activity, the modulation of the transcription of the viral genome, the modulation of the replication of the viral genome, and the modulation of the spread of a virus).
  • the subject screening assays employ indicator compositions. These indicator compositions comprise the components required for performing an assay that detects and/or measures a particular event.
  • the indicator compositions of the invention provide a reference readout and changes in the readout can be monitored in the presence of one or more test compounds. A difference in the readout in the presence and the absence of the compound indicates that the test compound is a modulator of the molecule(s) present in the indicator composition.
  • the indicator composition used in the screening assay can be a cell that expresses a JAK2 or a JAK2 -binding molecule.
  • a cell that naturally expresses or, more preferably, a cell that has been engineered to express the protein by introducing into the cell an expression vector encoding the protein may be used.
  • a cell that is preferentially infected by the virus in question can be used.
  • the cell is a mammalian cell, e.g., a human cell.
  • the cell can be either a T lymphocyte or macrophage.
  • the indicator composition can be a cell-free composition that includes the protein ⁇ e.g., a cell extract or a composition that includes e.g., either purified natural or recombinant protein).
  • the indicator composition used in the screening assays of the invention can be a cell that expresses a JAK2 kinase and/or HEXIMl, for example, as set forth in Figure 1, or biologically active fragment thereof.
  • the indicator composition comprises more than one polypeptide.
  • the subject assays are performed in the presence of JAK2 kinase and/or at least one JAK2 -binding molecule, such as, but not limited to HEXIMl .
  • Compounds that modulate, for example, inhibit, the expression and/or activity of JAK2, identified using the assays described herein can be useful for treating a subject suffering from RNA polymerase dependent viral disorders that would benefit from the inhibition of JAK2 production.
  • secondary assays can be used to confirm that the inhibiting agent affects the JAK2 molecule or the HEXIMl in a specific manner.
  • compounds identified in a primary screening assay can be used in a secondary screening assay to determine whether the compound inhibits a JAK2 -related activity or HEXIMl tyrosine phosphorylation.
  • the invention pertains to a combination of two or more of the assays described herein.
  • an inhibitor can be identified using a cell-based or a cell-free assay, e.g., to detect binding, and the ability of the agent to modulate the activity of JAK2 can be confirmed using a biological read-out to measure, e.g., HEXIMl phosphorylation, in vitro or in vivo.
  • JAK2 kinase inhibitors or agents that inhibit HEXIMl tyrosine phosphorylation identified as described herein ⁇ e.g., a small molecule) may be used in an animal model to determine the efficacy, toxicity, or side effects of treatment with such an inhibitor.
  • an inhibitor identified as described herein may be used in an animal model to determine the mechanism of action of such inhibitor.
  • the screening assays of the invention are high throughput or ultra high throughput (e.g., Fernandes, P.B., Curr Opin Chem Biol. 1998 2:597; Sundberg, SA, Curr Opin Biotechnol. 2000, 11 :47).
  • test compound includes any reagent or test agent which is employed in the assays of the invention and assayed for its ability to influence either the production, expression and/or activity of JAK2 kinase or the tyrosine phosphorylation of HEXIM1. More than one compound, e.g., a plurality of compounds, can be tested at the same time for their ability to modulate cytokine production, expression and/or activity in a screening assay.
  • screening assay preferably refers to assays which test the ability of a plurality of compounds to influence the readout of choice rather than to tests which test the ability of one compound to influence a readout.
  • the subject assays identify compounds not previously known to have the effect that is being screened for.
  • high throughput screening may be used to assay for the activity of a compound.
  • the compounds to be tested can be derived from libraries (i.e., are members of a library of compounds). While the use of libraries of peptides is well established in the art, new techniques have been developed which have allowed the production of mixtures of other compounds, such as benzodiazepines (Bunin, et al. (1992). J. Am. Chem. Soc. 114: 10987; DeWitt et al. (1993). Proc. Natl. Acad. Sci., USA 90:6909) peptoids (Zuckermann. (1994). J. Med. Chem. 37:2678) oligocarbamates (Cho, et al.
  • the compounds of the present invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries, synthetic library methods requiring deconvolution, the One-bead one-compound' library method, and synthetic library methods using affinity chromatography selection.
  • biological libraries are limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam, K.S. (1997) Anticancer Drug Des. 12: 145).
  • Other exemplary methods for the synthesis of molecular libraries can be found in the art, for example in: Erb, et al. (1994). Proc. Natl. Acad. Sci.,
  • Exemplary compounds which can be screened for activity include, but are not limited to, peptides, nucleic acids, carbohydrates, small organic molecules, and natural product extract libraries.
  • Candidate/test compounds include, for example, 1) peptides such as soluble peptides, including Ig-tailed fusion peptides and members of random peptide libraries (see, e.g., Lam, K.S., et al. (1991) Nature 354:82-84; Houghten, R., et al. (1991) Nature 354:84-
  • Fab expression library fragments, and epitope-binding fragments of antibodies ); 4) small organic and inorganic molecules ⁇ e.g., molecules obtained from combinatorial and natural product libraries); 5) enzymes ⁇ e.g., endoribonucleases, hydrolases, nucleases, proteases, synthatases, isomerases, polymerases, kinases, phosphatases, oxido-reductases and
  • mutant forms of molecules ⁇ e.g., dominant negative mutant forms of JAK2 or a JAK2 -binding protein.
  • test compounds of the present invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including:
  • biological libraries spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the One-bead one-compound' library method; and synthetic library methods using affinity chromatography selection.
  • the biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam, K.S. (1997) Anticancer Drug Des. 12: 145).
  • Compounds identified in the subject screening assays may be used, e.g., in methods of modulating phosphorylation of JAK2 protein, phosphorylation of HEXIMl, release of HEXIMl for the pTEFb complex, pTEFb complex activity, TAT interaction with RNA polymerase, CDK9 mediated phosphorylation of RNA polymerase, RNA polymerase activity, modulation of the transcription of the viral genome, the replication of the viral genome, and the spread of a virus. It will be understood that it may be desirable to formulate such compound(s) as pharmaceutical compositions (described supra) prior to contacting them with cells.
  • test compound can then be further evaluated for its effect on cells, for example by contacting the compound of interest with cells either in vivo ⁇ e.g., by administering the compound of interest to a subject) or ex vivo ⁇ e.g., by isolating cells from the subject and contacting the isolated cells with the compound of interest or, alternatively, by contacting the compound of interest with a cell line) and determining the effect of the compound of interest on the cells, as compared to an appropriate control (such as untreated cells or cells treated with a control compound, or carrier, that does not modulate the biological response).
  • an appropriate control such as untreated cells or cells treated with a control compound, or carrier, that does not modulate the biological response.
  • the instant invention also pertains to compounds identified in the subject screening assays.
  • kits for carrying out the screening assays, modulatory methods or diagnostic assays of the invention can include an indicator composition comprising a JAK2 kinase and/or HEXIMl, means for measuring a readout ⁇ e.g., protein secretion) and instructions for using the kit to identify modulators of biological effects of JAK2 or HEXIMl tyrosine phoshorylation.
  • a kit for carrying out a screening assay of the invention can include cells deficient in JAK2 or HEXIMl, means for measuring the readout and instructions for using the kit to identify modulators of a biological effect of JAK2 or HEXIMl tyrosine phosphorylation.
  • the kit may further include a tyrosine phospho-HEXIMl antibody for use in an ELISA assay to measure the level of HEXIM1 phosphorylated at the tyrosine residue of the YLEL epitope.
  • the invention provides a kit for carrying out a modulatory method of the invention.
  • the kit can include, for example, a modulatory agent of the invention (e.g. , a JAK2 kinase inhibitor or an agent that inhibits HEXIM1) in a suitable carrier and packaged in a suitable container with instructions for use of the inhibitor to modulate a biological effect of JAK2 or HEXIM1 tyrosine
  • R A polymerase dependent viral disorder such as HIV, Influenza, Hepatitis or West Nile Virus.
  • kits for diagnosing a disorder associated with a biological activity of a JAK2 kinase or HEXIM1 in a subject can include a reagent for determining expression of JAK2 (e.g. , a nucleic acid probe for detecting JAK2 mRNA or an antibody for detection of JAK2 protein) or tyrosine phosphorylation of HEXIM1 (e.g., a tyrosine phospho-HEXIMl antibody), a control to which the results of the subject are compared, and instructions for using the kit for diagnostic purposes.
  • JAK2 e.g. , a nucleic acid probe for detecting JAK2 mRNA or an antibody for detection of JAK2 protein
  • tyrosine phosphorylation of HEXIM1 e.g., a tyrosine phospho-HEXIMl antibody
  • Example 1 Inhibition of HIV replication by JAK2 kinase inhibitor AG490 in cultured human Jurkat cells
  • DMEM cell culture medium supplemented with 10% Fetal Bovine Serum, heat inactivated, ImM Pyruvate- Jurkat cell (Invitrogen).
  • HIV-1 delta nef virus 200-bp nef deleted HIV-1 Eli virus - frozen at -80°C (IDI, Inc.)
  • HIV-1 p24 Elisa assay kit (IDI, Inc. product # 103)
  • HIV-1 p24 one-step cassette test kit (IDI, Inc. product # IT200)
  • a vial of HIV-1 delta nef Eli frozen virus was removed from the -75°C freezer.
  • a 100 micro-molar solution of AG490 inhibitor was prepared as follows. Initially a 1 mg/ml solution of AG490 in DMSO was prepared. Using a syringe and 22G needle, 0.2ml of the DMSO stock was removed and diluted with GMEM medium to 2 ml. The solution was filter sterilized by passage through a 0.2 micron disc filter (Millipore).
  • tissue culture plate from the incubator was placed under the sterile hood and surface swiped with 70%> ethanol.
  • Each well of the duplicate cultures was labeled as follows: (1) Blank, B; (2) delta nef virus, DNV; (3) AG490, AG; and (4) Delta nef virus AG490, VAG.
  • p24 antigen was assayed in culture supernatants on days 8, 10, 13, 15, 18 and 21 using a p24 Elisa kit (Product # 103 from IDI, Inc.) following the kit instructions as follows.
  • the culture plate was transferred to the safety hood and swiped carefully with 70% EtOH. With a micropippetor and sterile tip, ⁇ of culture supernatant was removed from each well set of Jurkat wells and transferred to an Eppendorf tube containing 20 ⁇ 1 of a 10X solution of PBST (PBS+ 0.05%) Tween 20).
  • the Eppendorf tubes were capped and transferred to a 55°C water bath for 10 minutes. Subsequently, the Eppendorf tubes were returned to the sterile hood and diluted with heat inactivated culture supernatants to a final volume of 220 ⁇ 1 with PBSTB
  • the p24 antigen assay was conducted on days 8, 10, 13, 15, 18 and 21 as described above in order to determine p24 antigen levels in the culture supernatants. Results were recorded using BioRad Gel Doc system.
  • the results reflect a clear inhibition of HIV replication upon administration of JAK2 kinase inhibitor AG490. Specifically, the results depict 92% inhibition of HIV- 1 replication in Jurkat cells, thereby demonstrating that AG490 can be used to inhibit HIV-1 replication. Finally, we note that the AG490 half-life may not be more than 48 hours.
  • Example 2 Inhibition of Endogenous HIV replication by JAK2 kinase inhibitor Z3 in cultured human Hut-78 (H9) cells
  • Confluent H9 cells were diluted in DMEM + 10% fetal calf serum (FCS) to a cell density of approximately lxl0 6 /ml, and 1ml cells were seeded per well in 6-well culture
  • FCS fetal calf serum
  • DMSO-vehicle, Z3 in DMSO and HIV-1 virus were added to wells as follows. DMSO-vehicle (C)), Z3 in DMSO, (Z3), DMSO-vehicle+ HIV-1 virus (D-nef ), and Z3 in DMSO + D-nef virus (D-nef-Z3).
  • the culture dishes were incubated at 37°C, 10% C0 2 , for 8 days. On day 8, culture supernatant p24 antigen was assayed by p24 Lateral Flow Antigen test. Residual p24 was detected in HIV-1 infected cells, without and with Z3.
  • Hut-78 (H9) cells ATCC No. HTB-176TM
  • DMEM cell culture medium supplemented with 10% Fetal Bovine Serum, heat inactivated - H9 cell (Invitrogen).
  • HIV-1 delta nef virus 200-bp nef deleted HIV-1 Eli virus - frozen at -80°C (IDI, Inc.).
  • HIV-1 p24 one-step cassette test kit (IDI, Inc. product # IT200).
  • H9 cells Confluent cultures of H9 cells at 2-3X106 cells per ml, 98%> viable were acquired. Cells were diluted to 1X10 6 cells per ml. H9 cells were appropriately diluted with media as defined above. 1 ml diluted H9 cells were transferred to 4 wells each of a 24 tissue culture plate. The plate was subsequently transferred to a precision 37°C incubator, 10%> C0 2 for 1 hour.
  • a vial of HIV-1 delta nef Eli frozen virus is removed from the -75°C freezer.
  • a 10 micro-molar solution of Z3 inhibitor was prepared as follows. Initially a 1 mg/ml solution Z3 in DMSO was prepared. The Z3 solution was filter-sterilized using a 0.2 micron disc filter (Millipore). The 10 micro-molar solution is stored at 4°C.
  • the H9 tissue culture plate from the incubator was placed under the sterile hood.
  • the plate was surface swiped with 70% ethanol.
  • Each well of the duplicate cultures was labeled as follow: (1) Blank, B; (2) delta nef virus, DNV; (3) Z3; and (4) Delta nef virus Z3, VZ3.
  • ⁇ of sterile Z3 was added to the Z3 and VZ3 wells (25 ⁇ ).
  • ⁇ of sterile DMSO was added to the B and DNV wells.
  • 0.1 ml delta nef virus was added to the DNV and VZ3 wells above.
  • Appropriate media was subsequently added to the wells in amounts to bring up the volume to 4ml per well.
  • p24 antigen was assayed in culture supernatants as follows: With a micropippetor and sterile tip, ⁇ of culture supernatant was removed from each set of H9 wells and transferred to an Eppendorf tube containing 20 ⁇ 1 of a 10X solution of PBST (PBS+0.05%) Tween 20). The Eppendorf tubes were capped and transferred to a 55°C water bath for 10 minutes. Subsequently, the Eppendorf tubes were returned to the sterile hood where p24 antigen levels were determined by the p24 Lateral Flow p24 antigen test in accordance with the manufacturer's product instructions. Results were recorded using BioRad Gel Doc System.
  • the H9 culture plate was transferred to the sterile hood and subsequently surface- wiped with70%> EtOH.
  • An appropriate number of 15 sterile culture tubes were collected under the sterile hood and labeled as indicated on the H9 culture wells.
  • cells were removed from the wells by repeatedly drawing cells into the pipette and transferring to the correspondingly labeled 15ml tube. Cells were pelleted by centrifugation at 1000 rpm for 5 minutes.
  • the tubes were returned to the sterile hood where medium was aspirated aseptically from the tubes.
  • culture medium as above, cells were transferred to a new a 6-well cluster dish.
  • DMSO-vehicle and Z3 inhibitor were added to appropriate wells as above. Medium was subsequently added to balance Z3 concentration at 25 ⁇ . Subsequently, the plate was returned to the 37°C incubator.
  • Figures 1 A and IB depict the results of the HIV-1 p24 antigen determination by Lateral Flow p24 antigen test on day 22 after exposure to Z3: Left to right, H9 cells (C), cells with inhibitor (Z3), Cells infected with HIV-1 (D-nef) and HIV-1 + Z3 (D-nef,Z3).
  • the results reflect a clear inhibition of HIV replication in H9 cells upon administration of JAK2 kinase inhibitor Z3. Specifically, the results depict 90% inhibition of HIV-1 replication in Jurkat cells, thereby demonstrating that Z3 can be used to inhibit HIV-1 replication.

Abstract

La présente invention concerne des méthodes de traitement prophylactique ou thérapeutique des troubles viraux dépendants de l'ARN polymérase à l'aide d'inhibiteurs de la JAK2 kinase ou d'agents inhibant la phosphorylation de la tyrosine HEXIM1. Dans un aspect particulier, la présente invention concerne le traitement prophylactique ou thérapeutique d'une infection par le VIH chez un sujet par administration d'inhibiteurs de JAK2 kinase ou d'agents inhibant la phosphorylation de la tyrosine HEXIM1.
PCT/US2011/026049 2010-03-02 2011-02-24 Méthodes de traitement prophylactique ou thérapeutique de troubles viraux dépendants de l'arn polymérase par administration d'inhibiteurs de la jak2 kinase WO2011109217A2 (fr)

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