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WO2009020559A2 - Agents inhibant les interactions du p-tefb et leurs procédés d'utilisation - Google Patents

Agents inhibant les interactions du p-tefb et leurs procédés d'utilisation Download PDF

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Publication number
WO2009020559A2
WO2009020559A2 PCT/US2008/009298 US2008009298W WO2009020559A2 WO 2009020559 A2 WO2009020559 A2 WO 2009020559A2 US 2008009298 W US2008009298 W US 2008009298W WO 2009020559 A2 WO2009020559 A2 WO 2009020559A2
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Prior art keywords
polypeptide
brd4
amino acid
acid sequence
tefb
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WO2009020559A3 (fr
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Eric M. Verdin
Dwayne A. Bisgrove
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J David Gladstone Institutes
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J David Gladstone Institutes
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/11—Protein-serine/threonine kinases (2.7.11)
    • C12Y207/11023—[RNA-polymerase-subunit] kinase (2.7.11.23)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • Bromodomains are conserved sequence elements identified in several protein families and constitute chromatin targeting modules that mediate attachment to acetylated histones.
  • Bromodomain-containing protein 4 (BRD4) is a member of the BET family of bromodomain proteins, which characteristically have two tandem N-terminal bromodomains followed by an extraterminal (ET) domain.
  • BRD4 has been identified as an interaction partner with the positive transcription elongation factor b (P-TEFb) complex.
  • P-TEFb is a heterodimer of cyclin-dependent kinase 9 (CDK9) and cyclin Tl , T2 or K.
  • the P-TEFb heterodimer has been shown to control the elongation phase of transcription via phosphorylation of the C-terminal domain of the large subunit (RPBl) of RNA polymerase II (RNAPII).
  • RBPl large subunit
  • RNAPII RNA polymerase II
  • the present invention provides methods of treating disorders associated with abnormal cellular growth and/or proliferation.
  • the methods generally involve administering an agent that reduces interaction of BRD4 with P-TEFb.
  • the present invention further provides methods of treating an immunodeficiency virus infection.
  • the methods generally involve administering an agent that reduces interaction of BRD4 with P-TEFb and/or a viral protein.
  • the present invention further provides methods of identifying an agent that reduces binding of BRD4 to P- TEFb.
  • the present invention further provides BRD4 polypeptides.
  • Figures IA and IB depict interaction of BRD4 with P-TEFb via a short C-terminal domain.
  • Figures 2 A-E depict the effect of a C-terminal peptide conserved among BRD4,
  • hBRD4 SEQ ID NO:4
  • mBRD4 SEQ ID NO:5
  • xBRD4 SEQ ID NO:6
  • hBRDT SEQ ID NO:7
  • mBRDT SEQ ID NO:8
  • fs(l)h SEQ ID NO:
  • Figure 3 depicts interaction of BRD4 with P-TEFb subunits CDK9 and cyclin Tl .
  • Figure 4 depicts results showing that the P-TEFb interacting domain of BRD4 is an autonomous transactivation domain.
  • Figures 5A and 5B depict inhibition of Tat transactivation of the HIV LTR by the P-
  • FIG. 6 depicts the effect of the P-TEFb-interacting domain of BRD4 on binding of
  • FIG. 7A depicts a protein transduction domain/BRD4 peptide ("PTD-BRD4"; SEQ
  • Figures 7B and 7C depict the effect of a BRD4 P-TEFb interacting domain peptide
  • FIG. 8 depicts the effect of PTD-BRD4 peptides on the viability of A2 cells in vitro.
  • abnormal cellular proliferation refers to division and/or growth occurring more rapidly or to a significantly greater extent than typically occurs in a normally functioning cell of the same type.
  • polypeptide refers to a polymeric form of amino acids of any length, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones.
  • fusion proteins including, but not limited to, fusion proteins with a heterologous amino acid sequence, fusions with heterologous and homologous leader sequences, with or without N-terminal methionine residues; immunologically tagged proteins; and the like.
  • NH 2 refers to the free amino group present at the amino terminus of a polypeptide.
  • COOH refers to the free carboxyl group present at the carboxyl terminus of a polypeptide.
  • a "substantially isolated” or “isolated” polypeptide or antibody is one that is substantially free of the materials with which it is associated in nature. By substantially free is meant at least 50%, at least 70%, at least 80%, or at least 90% free of the materials with which it is associated in nature.
  • an “isolated” peptide or polypeptide also refers to a peptide, polypeptide or fusion polypeptide, which, by virtue of origin or manipulation: (1) is not associated with all or a portion of a polypeptide with which it is associated in nature, (2) is linked to a polypeptide other than that to which it is linked in nature, or (3) does not occur in nature.
  • an isolated polypeptide e.g., a BRD4 polypeptide; a P-TEFb polypeptide
  • an isolated antibody is at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 99%, or more, pure.
  • treatment refers to obtaining a desired pharmacologic and/or physiologic effect.
  • the effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse affect attributable to the disease.
  • Treatment covers any treatment of a disease in a mammal, particularly in a human, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, e.g., causing regression of the disease, e.g., to completely or partially remove symptoms of the disease.
  • treatment encompasses prevention of establishment of a systemic infection following initial contact with the virus; and prophylactic treatment of an individual not yet infected with the virus.
  • an "effective amount” or “therapeutically effective amount” means a dosage sufficient to provide for treatment for the disease state being treated or to otherwise provide the desired effect (e.g., induction of an effective immune response, reduction in serum viral load, etc.).
  • the precise dosage will vary according to a variety of factors such as subject-dependent variables (e.g., age, immune system health, etc.), the disease (e.g., the particular immunodeficiency virus), and the treatment being effected.
  • an "effective amount” is that amount necessary to substantially improve the likelihood of treating the infection, in particular that amount which improves the likelihood of successfully preventing infection or eliminating infection when it has occurred.
  • the terms "individual,” “host,” “subject,” and “patient,” used interchangeably herein, refer to a mammal, including, but not limited to, murines, felines, simians, humans, mammalian farm animals, mammalian sport animals, and mammalian pets.
  • the term includes mammals that are susceptible to infection by an immunodeficiency virus.
  • a "biological sample” encompasses a variety of sample types obtained from an individual and can be used in a diagnostic or monitoring assay.
  • the definition encompasses blood and other liquid samples of biological origin, solid tissue samples such as a biopsy specimen or tissue cultures or cells derived therefrom and the progeny thereof.
  • the definition also includes samples that have been manipulated in any way after their procurement, such as by treatment with reagents; washed; or enrichment for certain cell populations, such as CD4 + T lymphocytes, glial cells, macrophages, tumor cells, peripheral blood mononuclear cells (PBMC), and the like.
  • the term "biological sample” encompasses a clinical sample, and also includes cells in culture, cell supernatants, tissue samples, organs, bone marrow, and the like.
  • immunodeficiency virus refers to human immunodeficiency virus- 1 (HIV-I); human immunodeficiency virus-2 (HIV-2); any of a variety of HIV subtypes and quasispecies; simian immunodeficiency virus (SIV); and feline immunodeficiency virus (FIV).
  • the present invention provides methods of treating disorders associated with abnormal cellular growth and/or proliferation.
  • the methods generally involve administering an agent that reduces interaction of BRD4 with P-TEFb.
  • the present invention further provides methods of treating an immunodeficiency virus infection.
  • the methods generally involve administering an agent that reduces interaction of BRD4 with P-TEFb and/or a viral protein.
  • the present invention further provides methods of identifying an agent that reduces binding of BRD4 with
  • the present invention further provides BRD4 peptides and recombinant BRD4 polypeptides.
  • the present invention provides methods of treating a disease state in a mammalian subject, where the disease state is characterized by abnormal or undesired cellular growth and/or abnormal or undesired cellular proliferation.
  • the methods generally involve administering to a mammalian subject in need thereof an effective amount of an agent that reduces interaction of P-TEFb with BRD4.
  • TEFb to BRD4 by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, or more, compared to the binding of P-TEFb to BRD4 in the absence of the agent.
  • an agent that reduces interaction between P-TEFb and BRD4 inhibits binding between a C-terminal portion of BRD4 and P-TEFb.
  • an agent that reduces interaction between P-TEFb and BRD4 inhibits binding between P-TEFb and a C-terminal portion of a polypeptide having a length of about 1300-1400 amino acids (e.g., about 1362 amino acids), and comprising an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100%, sequence identity to the full length BRD4 sequence set forth in SEQ ID NO: 1. '
  • the C-terminal portion of BRD4 comprises an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to amino acids 1329-1362, 1318-1362, 1315-1362, 1228-1362, 1209-1362, or 722- 1362 of SEQ ID NO: 1, or at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in one of SEQ ID NOs: 4-9.
  • the C- terminal portion of BRD4 can have a length of from about 30 amino acids to about 1000 amino acids, e.g., the C-terminal portion of BRD4 can have a length of from about 30 amino acids (aa) to about 35 aa (e.g., 30 aa, 31 aa, 32 aa, 33 aa, 34 aa, 35 aa), from about 35 aa to about 40 aa, from about 40 aa to about 45 aa, from about 45 aa to about 50 aa (e.g., 45 aa, 46 aa, 47 aa, 48 aa, 49 aa, or 50 aa), from about 50 aa to about 60 aa, from about 60 aa to about 75 aa, from about 75 aa to about 100 aa, from about 100 aa to about 150 aa (e.g., 100 aa, 120 aa, 125 aa, 130 aa, 135
  • an agent that reduces interaction of BRD4 with P-TEFb inhibits
  • RNAPII RNA polymerase II
  • an agent that reduces interaction of BRD4 with P-TEFb inhibits P-TEFb-mediated phosphorylation of the C-terminal domain of RPBl of RNAPII by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, or more, compared to the level of phosphorylation of the C-terminal domain of RPBl of RNAPII in the absence of the agent.
  • an agent that reduces interaction between P-TEFb and BRD4 specifically inhibits binding of P-TEFb to BRD4, e.g., the agent inhibits binding of P-TEFb to BRD4, but does not substantially inhibit binding of BRD4 to proteins other than P-TEFb.
  • P-TEFb includes a cyclin-dependent kinase-9 (CDK9) subunit and a cyclin subunit
  • an agent that reduces interaction between P-TEFb and BRD4 inhibits binding of BRD4 to the CDK9 subunit of P-TEFb. In other embodiments, an agent that reduces interaction between P-TEFb and BRD4 inhibits binding of BRD4 to the cyclin subunit of P-TEFb. In other embodiments, an agent that reduces interaction between P-TEFb and BRD4 inhibits binding of BRD4 to both the CDK9 and the cyclin subunit of P-TEFb.
  • the CDK9 subunit of P-TEFb comprises an amino acid sequence having at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to one of the amino acid sequences set forth in GenBank Accession Nos. AAF72183, NP_001252, and AAV38706, or one of the sequences set forth in SEQ ID NOs: 10, 11, and 12.
  • cyclin Tl amino acid sequences are found in, e.g., GenBank Accession Nos. EAW57998, NPJ301231, 060563, and AAC39638; and set forth in SEQ ID N0s:13, 14, 15, and 16, respectively.
  • a cyclin Tl subunit comprises an amino acid sequence having at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to one of the amino acid sequences set forth in GenBank Accession Nos. EAW57998, NP 001231, 060563, and AAC39638, or one of the sequences set forth in SEQ ID NOs: 13, 14, 15, and 16.
  • a cyclin T2 subunit comprises an amino acid sequence having at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to one of the amino acid sequences set forth in GenBank Accession Nos. AAI14367, AAW56073, NP_490595, NP 001232, EAXl 1647 and EAXl 1646; or one of the sequences set forth in SEQ ID NOs: 17, 18, 19, 20, 21, and 22.
  • Amino acid sequences of cyclin K are found in, e.g., GenBank Accession Nos.
  • a cyclin K subunit comprises an amino acid sequence having at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to one of the amino acid sequences set forth in GenBank Accession Nos.
  • Bromodomain containing-4 protein exists as a long isoform and a short isoform.
  • an agent that reduces interaction between P-TEFb and BRD4 inhibits binding of the long isoform of BRD4 with P-TEFb.
  • a BRD4 short isoform polypeptide comprises an amino acid sequence having at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity the amino acid sequence set forth in SEQ ID NO:36.
  • a BRD4 long isoform polypeptide comprises an amino acid sequence having at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 1.
  • a subject method of reducing undesired or abnormal cell growth or proliferation is useful for treating various disorders characterized by undesired or abnormal cell growth or proliferation.
  • disorders include, but are not limited to, cancer; cardiac hypertrophy (e.g., undesired growth of cardiac myocytes); prostate hypertrophy; pulmonary vascular obstructive disease; restenosis (e.g., restenosis following angioplasty or other vascular trauma); disorders resulting from vascular smooth muscle cell proliferation; psoriasis, a condition characterized by the cellular hyperproliferation of keratinocytes; and atherosclerosis.
  • cardiac hypertrophy e.g., undesired growth of cardiac myocytes
  • prostate hypertrophy e.g., pulmonary vascular obstructive disease
  • restenosis e.g., restenosis following angioplasty or other vascular trauma
  • disorders resulting from vascular smooth muscle cell proliferation psoriasis, a condition characterized by the cellular hyperpro
  • TEFb is useful for the treatment of cancer.
  • the methods generally involve administering to an individual in need thereof an effective amount of an agent that reduces the interaction of BRD4 with P-TEFb.
  • the agent is a subject BRD4 polypeptide.
  • a subject method is a method of treating breast cancer in a mammalian subject, wherein administration of a subject BRD4 polypeptide inhibits an interaction between estrogen receptor alpha (ERa) and a cyclin Tl subunit of P-TEFb thereby reducing ERa transcriptional activity.
  • ERa estrogen receptor alpha
  • P-TEFb cyclin Tl subunit of P-TEFb
  • BRD4 with P-TEFb is an amount that is effective to reduce a tumor load by at least about 5%, at least about 10%, at least about 20%, at least about 25%, at least about 50%, at least about 75%, at least about 85%, or at least about 90%, up to total eradication of the tumor, when compared to a suitable control.
  • an "effective amount" of an agent that reduces interaction of BRD4 with P-TEFb is an amount that is sufficient to reduce tumor load by at least about 5%, at least about 10%, at least about 20%, at least about 25%, at least about 50%, at least about 75%, at least about 85%, or at least about 90%, up to total eradication of the tumor, when compared to a suitable control.
  • a suitable control may be a genetically identical animal not treated with the agent.
  • a suitable control may be the tumor load present before administering the agent.
  • Other suitable controls may be a placebo control.
  • Whether a tumor load has been decreased can be determined using any known method, including, but not limited to, measuring solid tumor mass; counting the number of tumor cells using cytological assays; fluorescence-activated cell sorting (e.g., using antibody specific for a tumor-associated antigen) to determine the number of cells bearing a given tumor antigen; computed tomography scanning, magnetic resonance imaging, and/or x-ray imaging of the tumor to estimate and/or monitor tumor size; measuring the amount of tumor-associated antigen in a biological sample, e.g., blood or serum; and the like.
  • a biological sample e.g., blood or serum
  • the methods are effective to reduce the growth rate of a tumor by at least about 5%, at least about 10%, at least about 20%, at least about 25%, at least about 50%, at least about 75%, at least about 85%, or at least about 90%, up to total inhibition of growth of the tumor, when compared to a suitable control.
  • "effective amounts" of an agent that reduces interaction of BRD4 with P-TEFb are amounts that are sufficient to reduce tumor growth rate by at least about 5%, at least about 10%, at least about 20%, at least about 25%, at least about 50%, at least about 75%, at least about 85%, or at least about 90%, up to total inhibition of tumor growth, when compared to a suitable control.
  • a suitable control may be tumor growth rate in a genetically identical animal not treated with the agent.
  • a suitable control may be the tumor load or tumor growth rate present before administering the agent.
  • Other suitable controls may be a placebo control.
  • Whether growth of a tumor is inhibited can be determined using any known method, including, but not limited to, an in vivo assay for tumor growth; an in vitro proliferation assay; a 3 H-thymidine uptake assay; and the like.
  • the subject methods are useful for treating a wide variety of cancers, including carcinomas, sarcomas, leukemias, and lymphomas.
  • Carcinomas that can be treated using a subject method include, but are not limited to, esophageal carcinoma, hepatocellular carcinoma, basal cell carcinoma (a form of skin cancer), squamous cell carcinoma (various tissues), bladder carcinoma, including transitional cell carcinoma (a malignant neoplasm of the bladder), bronchogenic carcinoma, colon carcinoma, colorectal carcinoma, gastric carcinoma, lung carcinoma, including small cell carcinoma and non-small cell carcinoma of the lung, adrenocortical carcinoma, thyroid carcinoma, pancreatic carcinoma, breast carcinoma, ovarian carcinoma, prostate carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, renal cell carcinoma, ductal carcinoma in situ or bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, WiIm' s tumor, cervical carcinoma, uterine carcinoma, testicular carcinoma
  • Sarcomas that can be treated using a subject method include, but are not limited to, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, chordoma, osteogenic sarcoma, osteosarcoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's sarcoma, leiomyosarcoma, rhabdomyosarcoma, and other soft tissue sarcomas.
  • Other solid tumors that can be treated using a subject method include, but are not limited to, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, and retinoblastoma.
  • Leukemias that can be treated using a subject method include, but are not limited to, a) chronic myeloproliferative syndromes (neoplastic disorders of multipotential hematopoietic stem cells); b) acute myelogenous leukemias (neoplastic transformation of a multipotential hematopoietic stem cell or a hematopoietic cell of restricted lineage potential; c) chronic lymphocytic leukemias (CLL; clonal proliferation of immunologically immature and functionally incompetent small lymphocytes), including B-cell CLL, T-cell CLL prolymphocytic leukemia, and hairy cell leukemia; and d) acute lymphoblastic leukemias (characterized by accumulation of lymphoblasts).
  • Lymphomas that can be treated using a subject method include, but are not limited to, B-cell lymphomas (e.g., Burkitt's lymphoma); Hodgkin's lymphom
  • Individuals who are suitable for treatment with a subject method include individuals who have been diagnosed as having a cancer; individuals who have been treated for a cancer, and who failed such treatment; and individuals who have been treated for a cancer, who exhibited remission of the cancer, and in whom the cancer subsequently reappeared.
  • Combination therapies include individuals who have been diagnosed as having a cancer; individuals who have been treated for a cancer, and who failed such treatment; and individuals who have been treated for a cancer, who exhibited remission of the cancer, and in whom the cancer subsequently reappeared.
  • an agent that reduces interaction of BRD4 with P-TEFb is administered to an individual in combination (e.g., in the same formulation or in separate formulations) with one or more additional therapeutic agents or treatment methods ("combination therapy").
  • An agent that reduces interaction of BRD4 with P-TEFb can be administered in admixture with another therapeutic agent or can be administered in a separate formulation.
  • an agent that reduces interaction of BRD4 with P-TEFb and an additional therapeutic agent can be administered substantially simultaneously (e.g., within about 60 minutes, about 50 minutes, about 40 minutes, about 30 minutes, about 20 minutes, about 10 minutes, about 5 minutes, or about 1 minute of each other) or separated in time by about 1 hour, about 2 hours, about 4 hours, about 6 hours, about 10 hours, about 12 hours, about 24 hours, about 36 hours, or about 72 hours, or more.
  • Therapeutic agents and/or treatment methods that can be administered in combination with an agent that reduces interaction of BRD4 with P-TEFb include surgery (e.g., surgical removal of cancerous tissue), radiation therapy, bone marrow transplantation, chemotherapeutic treatment, biological response modifier treatment, and certain combinations of the foregoing.
  • Radiation therapy includes, but is not limited to, x-rays or gamma rays that are delivered from either an externally applied source such as a beam, or by implantation of small radioactive sources.
  • Chemotherapeutic agents are non-peptidic (i.e., non-proteinaceous) compounds that reduce proliferation of cancer cells, and encompass cytotoxic agents and cytostatic agents.
  • Non-limiting examples of chemotherapeutic agents include alkylating agents, nitrosoureas, antimetabolites, antitumor antibiotics, plant (vinca) alkaloids, and steroid hormones.
  • Agents that act to reduce cellular proliferation are known in the art and widely used.
  • Such agents include alkylating agents, such as nitrogen mustards, nitrosoureas, ethylenimine derivatives, alkyl sulfonates, and triazenes, including, but not limited to, mechlorethamine, cyclophosphamide (CytoxanTM), melphalan (L-sarcolysin), carmustine (BCNU), lomustine (CCNU), semustine (methyl-CCNU), streptozocin, chlorozotocin, uracil mustard, chlormethine, ifosfamide, chlorambucil, pipobroman, triethylenemelamine, triethylenethiophosphoramine, busulfan, dacarbazine, and temozolomide.
  • alkylating agents such as nitrogen mustards, nitrosoureas, ethylenimine derivatives, alkyl sulfonates, and triazenes, including, but not limited to, mechlorethamine, cyclopho
  • Antimetabolite agents include folic acid analogs, pyrimidine analogs, purine analogs, and adenosine deaminase inhibitors, including, but not limited to, cytarabine (CYTOSAR-U), cytosine arabinoside, fluorouracil (5-FU), floxuridine (FudR), 6-thioguanine, 6-mercaptopurine (6-MP), pentostatin, 5-fluorouracil (5-FU), methotrexate, 10-propargyl-5,8-dideazafolate (PDDF, CB3717), 5,8-dideazatetrahydrofolic acid (DDATHF), leucovorin, fludarabine phosphate, pentostatine, and gemcitabine.
  • CYTOSAR-U cytarabine
  • cytosine arabinoside including, but not limited to, fluorouracil (5-FU), floxuridine (FudR), 6-thioguanine, 6-mercap
  • Suitable natural products and their derivatives include, but are not limited to, Ara-C, paclitaxel (Taxol®), docetaxel (Taxotere®), deoxycoformycin, mitomycin-C, L- asparaginase, azathioprine; brequinar; alkaloids, e.g. vincristine, vinblastine, vinorelbine, vindesine, etc.; podophyllotoxins, e.g. etoposide, teniposide, etc; antibiotics, e.g.
  • anthracycline daunorubicin hydrochloride (daunomycin, rubidomycin, cerubidine), idarubicin, doxorubicin, epirubicin and morpholino derivatives, etc.; phenoxizone biscyclopeptides, e.g. dactinomycin; basic glycopeptides, e.g. bleomycin; anthraquinone glycosides, e.g. plicamycin (mithramycin); anthracenediones, e.g. mitoxantrone; azirinopyrrolo indolediones, e.g. mitomycin; macrocyclic immunosuppressants, e.g. cyclosporine, FK-506 (tacrolimus, prograf), rapamycin, etc. ; and the like.
  • phenoxizone biscyclopeptides e.g. dactinomycin
  • basic glycopeptides e.g.
  • anti -proliferative cytotoxic agents are navelbene, CPT-11, anastrazole, letrazole, capecitabine, reloxafine, cyclophosphamide, ifosamide, and droloxafine.
  • Microtubule affecting agents that have antiproliferative activity are also suitable for use and include, but are not limited to, allocolchicine (NSC 406042), Halichondrin B (NSC 609395), colchicine (NSC 757), colchicine derivatives (e.g., NSC 33410), dolstatin 10 (NSC 376128), maytansine (NSC 153858), rhizoxin (NSC 332598), paclitaxel (Taxol®), Taxol® derivatives, docetaxel (Taxotere®), thiocolchicine (NSC 361792), trityl cysterin, vinblastine sulfate, vincristine sulfate, natural and synthetic epothilones including but not limited to, eopthilone A, epothilone B, discodermolide; estramustine, nocodazole, and the like.
  • Hormone modulators and steroids that are suitable for use include, but are not limited to, adrenocorticosteroids, e.g. prednisone, dexamethasone, etc.; estrogens and pregestins, e.g. hydroxyprogesterone caproate, medroxyprogesterone acetate, megestrol acetate, estradiol, clomiphene, tamoxifen; etc.; and adrenocortical suppressants, e.g.
  • adrenocorticosteroids e.g. prednisone, dexamethasone, etc.
  • estrogens and pregestins e.g. hydroxyprogesterone caproate, medroxyprogesterone acetate, megestrol acetate, estradiol, clomiphene, tamoxifen; etc.
  • adrenocortical suppressants e.g.
  • estradiosteroids may inhibit T cell proliferation.
  • chemotherapeutic agents include metal complexes, e.g. cisplatin (cis-DDP), carboplatin, etc.; ureas, e.g. hydroxyurea; and hydrazines, e.g. N-methylhydrazine; epidophyllotoxin; a topoisomerase inhibitor; procarbazine; mitoxantrone; leucovorin; tegafur; etc.
  • metal complexes e.g. cisplatin (cis-DDP), carboplatin, etc.
  • ureas e.g. hydroxyurea
  • hydrazines e.g. N-methylhydrazine
  • epidophyllotoxin e.g. N-methylhydrazine
  • epidophyllotoxin e.g. N-methylhydrazine
  • epidophyllotoxin e.g. N-methylhydrazine
  • mycophenolic acid mycophenolic acid, thalidomide, desoxyspergualin, azasporine, leflunomide, mizoribine, azaspirane (SKF 105685); Iressa® (ZD 1839, 4-(3-chloro-4-fluorophenylamino)-7-methoxy-6- (3 -(4-morpholinyl)propoxy)quinazoline) ; etc.
  • Taxanes include paclitaxel, as well as any active taxane derivative or pro-drug.
  • “Paclitaxel” (which should be understood herein to include analogues, formulations, and derivatives such as, for example, docetaxel, TAXO LTM, TAXOTERETM (a formulation of docetaxel), 10-desacetyl analogs of paclitaxel and 3'N-desbenzoyl-3'N-t-butoxycarbonyl analogs of paclitaxel) may be readily prepared utilizing techniques known to those skilled in the art (see also WO 94/07882, WO 94/07881, WO 94/07880, WO 94/07876, WO 93/23555, WO 93/10076; U.S. Pat. Nos.
  • Paclitaxel should be understood to refer to not only the common chemically available form of paclitaxel, but analogs and derivatives (e.g., TaxotereTM docetaxel, as noted above) and paclitaxel conjugates (e.g., paclitaxel-PEG, paclitaxel-dextran, or paclitaxel-xylose).
  • analogs and derivatives e.g., TaxotereTM docetaxel, as noted above
  • paclitaxel conjugates e.g., paclitaxel-PEG, paclitaxel-dextran, or paclitaxel-xylose.
  • Taxane also included within the term "taxane” are a variety of known derivatives, including both hydrophilic derivatives, and hydrophobic derivatives.
  • Taxane derivatives include, but not limited to, galactose and mannose derivatives described in International Patent Application No. WO 99/18113; piperazino and other derivatives described in WO 99/14209; taxane derivatives described in WO 99/09021, WO 98/22451, and U.S. Patent No. 5,869,680; 6-thio derivatives described in WO 98/28288; sulfenamide derivatives described in U.S. Patent No. 5,821,263; and taxol derivative described in U.S. Patent No. 5,415,869. It further includes prodrugs of paclitaxel including, but not limited to, those described in WO 98/58927; WO 98/13059; and U.S. Patent No. 5,824,701.
  • Biological response modifiers suitable for use in connection with a subject method of cancer treatment include, but are not limited to, (1) inhibitors of tyrosine kinase (RTK) activity; (2) inhibitors of serine/threonine kinase activity; (3) tumor-associated antigen antagonists, such as antibodies that bind specifically to a tumor antigen; (4) apoptosis receptor agonists; (5) interleukin-2; (6) IFN- ⁇ ; (7) IFN- ⁇ ; (8) colony-stimulating factors; and (9) inhibitors of angiogenesis.
  • RTK tyrosine kinase
  • the present invention provides methods of treating an immunodeficiency virus infection in a mammalian subject, the methods generally involving administering to an individual in need thereof an effective amount of an agent that reduces interaction of BRD4 with P-TEFb and/or reduces the interaction between P-TEFb and a viral protein (e.g., Tat).
  • a viral protein e.g., Tat
  • BRD4 with P-TEFb and/or reduces the interaction between P-TEFb and a viral protein is an amount that, when administered in one or more doses, is effective to increase the number of CD4 + cells in the individual.
  • an "effective amount" of an agent that reduces interaction of BRD4 with P-TEFb and/or a viral protein is an amount that, when administered in one or more doses, is effective to increase the number of CD4 + T cells in the individual by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 50%, at least about 75%, at least about 2-fold, at least about 2.5-fold, at least about 5-fold, at least about 10-fold, at least about 25-fold, at least about 50- fold, or at least about 100-fold, or more, compared to the number of CD4 + T cells in the individual in the absence of treatment with the agent.
  • BRD4 with P-TEFb and/or reduces interaction between P-TEFb and a viral protein is an amount that, when administered in one or more doses, is effective to decrease viral load in the individual.
  • an "effective amount" of an agent that reduces interaction of BRD4 with P-TEFb and/or reduces interaction between P-TEFb and a viral protein is an amount that, when administered in one or more doses, is effective to decrease viral load in the individual by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, or more, compared to the viral load in the individual in the absence of treatment with the agent.
  • BRD4 with P-TEFb and/or reduces the interaction between P-TEFb and a viral protein is an amount that is effective to reduce the level of transcription of an immunodeficiency virus in an infected cell (e.g., in a cell infected with the immunodeficiency virus) in an individual by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or more, compared to the level of transcription of the immunodeficiency virus in an infected cell in the individual in the absence of treatment with the agent.
  • an agent that reduces interaction of BRD4 with P-TEFb reduces the interaction of P-TEFb with the viral transactivator protein (Tat).
  • an agent that reduces interaction of BRD4 with P-TEFb inhibits binding of P- TEFb to Tat by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or more, compared to the binding of P-TEFb to Tat in the absence of the agent.
  • the mammalian subject is a human infected with an immunodeficiency virus, and the immunodeficiency virus is a human immunodeficiency virus- 1 (HIV-I).
  • the mammalian subject is a non-human primate infected with an immunodeficiency virus, and the immunodeficiency virus is SIV.
  • Methods of determining whether the subject methods are effective in treating an immunodeficiency virus infection are any known test for indicia of immunodeficiency virus infection, including, but not limited to, measuring viral load, e.g., by measuring the amount of immunodeficiency virus in a biological sample, e.g., using a polymerase chain reaction (PCR) with primers specific for an immunodeficiency virus polynucleotide sequence; detecting and/or measuring a polypeptide encoded by an immunodeficiency virus, e.g., p24, gpl20, reverse transcriptase, using, e.g., an immunological assay with an antibody specific for the polypeptide; and measuring CD4 cell count in the individual.
  • measuring viral load e.g., by measuring the amount of immunodeficiency virus in a biological sample, e.g., using a polymerase chain reaction (PCR) with primers specific for an immunodeficiency virus polynucleotide sequence
  • an agent that reduces interaction of BRD4 with P-TEFb and/or reduces interaction between P-TEFb and a viral protein is administered to an individual in combination (e.g., in the same formulation or in separate formulations) with one or more additional therapeutic agents ("combination therapy").
  • An agent that reduces interaction of BRD4 with P-TEFb and/or reduces interaction between P-TEFb and a viral protein can be administered in admixture with another therapeutic agent or can be administered in a separate formulation.
  • an agent that reduces interaction of BRD4 with P-TEFb and/or reduces interaction between P-TEFb and a viral protein and an additional therapeutic agent can be administered substantially simultaneously (e.g., within about 60 minutes, about 50 minutes, about 40 minutes, about 30 minutes, about 20 minutes, about 10 minutes, about 5 minutes, or about 1 minute of each other) or separated in time by about 1 hour, about 2 hours, about 4 hours, about 6 hours, about 10 hours, about 12 hours, about 24 hours, about 36 hours, or about 72 hours, or more.
  • Therapeutic agents that can be administered in combination with an agent that reduces interaction of BRD4 with P-TEFb and/or reduces interaction between P-TEFb and a viral protein include agents that inhibit one or more immunodeficiency virus functions, which functions include, but are not limited to, viral replication; viral protease activity; viral reverse transcriptase activity; viral entry into a cell; viral integrase activity; activity of one or more of Rev, Tat, Nef, Vpr, Vpu, and Vif; and the like.
  • Therapeutic agents that can be administered in combination therapy with an agent that reduces interaction of BRD4 with P-TEFb and/or reduces interaction between P-TEFb and a viral protein include, but are not limited to, anti-inflammatory agents, anti-viral agents, antifungal agents, anti-mycobacterial agents, antibiotics, amoebicidal agents, trichomonocidal agents, analgesics, anti-neoplastic agents, anti-hypertensives, anti-microbial agents, or combinations of the foregoing.
  • patients with an immunodeficiency virus infection are treated with a combination of one or more agents that reduce interaction of BRD4 with P-TEFb and/or reduce interaction between P-TEFb and a viral protein, and with one or more of the following; beta-lactam antibiotics, tetracyclines, chloramphenicol, neomycin, gramicidin, bacitracin, sulfonamides, nitrofurazone, nalidixic acid, cortisone, hydrocortisone, betamethasone, dexamethasone, fluocortolone, prednisolone, triamcinolone, indomethacin, sulindac, acyclovir, amantadine, rimantadine, recombinant soluble CD4 (rsCD4), a fusion inhibitor (e.g., a T20 peptide, a T-1249 peptide; Trimeris); an anti-CD4
  • TEFb and/or reduces interaction between P-TEFb and a viral protein is administered in combination therapy with one or more nucleoside reverse transcriptase inhibitors (RTFs; where nucleoside reverse transcriptase inhibitors include AZT, ddl, 3TC, ddC, d4T, and abacavir); and/or one or more protease inhibitors (where protease inhibitors include indinavir, saquinavir, ritonavir, nelfinavir, amprevanir, and lopinavir); and/or one or more non-nucleoside reverse transcriptase inhibitors (where non-nucleoside reverse transcriptase inhibitors include nevirapine, delavirdine, emiravine, and efavirenz); and/or a fusion inhibitor (e.g., T20, T- 1249); and/or a CCR5 blocker (e.g., SCH-C, SCH
  • An agent that reduces interaction of BRD4 with P-TEFb and/or reduces interaction between P-TEFb and a viral protein can be administered to an individual in combination with any highly active antiretroviral therapy (HAART) or Structured Treatment Interruptions (STI) regimen currently in use.
  • HAART highly active antiretroviral therapy
  • STI Structured Treatment Interruptions
  • an agent that reduces interaction of BRD4 with P-TEFb and/or reduces interaction between P-TEFb and a viral protein is a BRD4 polypeptide, as described below.
  • a BRD4 polypeptide is administered in an amount of from about 20 mg to about 2000 mg per dosing event, e.g., from about 20 mg to about 200 mg, from about 200 mg to about 300 mg, from about 300 mg to about 400 mg, from about 400 mg to about 500 mg, from about 500 mg to about 600 mg, from about 600 mg to about 700 mg, from about 700 mg to about 800 mg, from about 800 mg to about 900 mg, from about 900 mg to about 1000 mg, from about 1000 mg to about 1200 mg, from about 1200 mg to about 1400 mg, from about 1400 mg to about 1600 mg, from about 1600 mg to about 1800 mg, or from about 1800 mg to about 2000 mg per dosing event.
  • the milligram quantities are based on the amount of BRD4 peptide or recomb
  • a single dose is administered.
  • multiple doses of an agent that reduces interaction of BRD4 with P-TEFb and/or reduces interaction between P-TEFb and a viral protein are administered.
  • the interval at which the doses are administered can vary, depending on a variety of factors, and the interval between any given dose and the subsequent dose is from about 1 day to about 1 month or more, e.g., from about 1 day to about 2 days, from about 2 days to about 7 days, from about 7 days to about 2 weeks, from about 2 weeks to about 4 weeks, or longer.
  • an agent that reduces interaction of BRD4 with P-TEFb and/or reduces interaction between P-TEFb and a viral protein is administered once per month, twice per month, three times per month, every other week (qow), once per week (qw), twice per week (biw), three times per week (tiw), four times per week, five times per week, six times per week, every other day (qod), daily (qd), twice a day (qid), or three times a day (tid) over a period of time ranging from about one day to about one week, from about two weeks to about four weeks, from about one month to about two months, from about two months to about four months, from about four months to about six months, from about six months to about eight months, from about eight months to about 1 year, from about 1 year to about 2 years, or from about 2 years to about 4 years, or for a period of more than 4 years.
  • dosing event refers to administration of an agent to a patient in need thereof, which event may encompass one or more releases of an agent from a drug dispensing device.
  • An agent that reduces interaction of BRD4 with P-TEFb is administered to an individual in need thereof in a subject treatment method.
  • An agent that reduces interaction of BRD4 with P-TEFb (referred generically below as an "active agent” or “drug") is formulated with one or more pharmaceutically acceptable excipients.
  • active agent or drug
  • pharmaceutically acceptable excipients are known in the art and need not be discussed in detail herein. Pharmaceutically acceptable excipients have been amply described in a variety of publications, including, for example, A. Gennaro (2000) "Remington: The Science and Practice of Pharmacy," 20th edition, Lippincott, Williams, & Wilkins; Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) H.C.
  • compositions such as vehicles, adjuvants, carriers or diluents
  • pharmaceutically acceptable auxiliary substances such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents and the like, are readily available to the public
  • an active agent may be administered to the host using any convenient means capable of resulting in the desired reduction in immunodeficiency disease or a disease state associated with abnormal cellular proliferation.
  • the active agent can be incorporated into a variety of formulations for therapeutic administration. More particularly, an active agent can be formulated into pharmaceutical compositions by combination with appropriate, pharmaceutically acceptable carriers or diluents, and may be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants and aerosols.
  • an active agent may be administered in the form of their pharmaceutically acceptable salts, or an active agent may be used alone or in appropriate association, as well as in combination, with other pharmaceutically active compounds.
  • the following methods and excipients are merely exemplary and are in no way limiting.
  • an active agent can be used alone or in combination with appropriate additives to make tablets, powders, granules or capsules, for example, with conventional additives, such as lactose, mannitol, corn starch or potato starch; with binders, such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins; with disintegrators, such as corn starch, potato starch or sodium carboxymethylcellulose; with lubricants, such as talc or magnesium stearate; and if desired, with diluents, buffering agents, moistening agents, preservatives and flavoring agents.
  • conventional additives such as lactose, mannitol, corn starch or potato starch
  • binders such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins
  • disintegrators such as corn starch, potato starch or sodium carboxymethylcellulose
  • lubricants such as talc or magnesium stearate
  • An active agent can be formulated into preparations for injection by dissolving, suspending or emulsifying them in an aqueous or nonaqueous solvent, such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol; and if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives.
  • an aqueous or nonaqueous solvent such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol.
  • An active agent can be utilized in aerosol formulation to be administered via inhalation.
  • An active agent can be formulated into pressurized acceptable propellants such as dichlorodifluoromethane, propane, nitrogen and the like.
  • an active agent can be made into suppositories by mixing with a variety of bases such as emulsifying bases or water-soluble bases.
  • An active agent can be administered rectally via a suppository.
  • the suppository can include vehicles such as cocoa butter, carbowaxes and polyethylene glycols, which melt at body temperature, yet are solidified at room temperature.
  • Unit dosage forms for oral or rectal administration such as syrups, elixirs, and suspensions may be provided wherein each dosage unit, for example, teaspoonful, tablespoonful, tablet or suppository, contains a predetermined amount of the composition containing one or more inhibitors.
  • unit dosage forms for injection or intravenous administration may comprise an active agent in a composition as a solution in sterile water, normal saline or another pharmaceutically acceptable carrier.
  • unit dosage form refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of an active agent calculated in an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier or vehicle.
  • the specifications for an active agent depend on the particular compound employed and the effect to be achieved, and the pharmacodynamics associated with each compound in the host.
  • An active agent can be administered as injectables.
  • injectable compositions are prepared as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared.
  • the preparation may also be emulsified or the active ingredient encapsulated in liposome vehicles.
  • an active agent is delivered by a continuous delivery system.
  • continuous delivery system is used interchangeably herein with “controlled delivery system” and encompasses continuous (e.g., controlled) delivery devices (e.g., pumps) in combination with catheters, injection devices, and the like, a wide variety of which are known in the art.
  • controlled delivery devices e.g., pumps
  • Mechanical or electromechanical infusion pumps can also be suitable for use with the present invention.
  • Examples of such devices include those described in, for example, U.S. Pat. Nos. 4,692,147; 4,360,019; 4,487,603; 4,360,019; 4,725,852; 5,820,589; 5,643,207; 6,198,966; and the like.
  • delivery of active agent can be accomplished using any of a variety of refillable, pump systems. Pumps provide consistent, controlled release over time.
  • the agent is in a liquid formulation in a drug-impermeable reservoir, and is delivered in a continuous fashion to the individual.
  • the drug delivery system is an at least partially implantable device.
  • the implantable device can be implanted at any suitable implantation site using methods and devices well known in the art.
  • An implantation site is a site within the body of a subject at which a drug delivery device is introduced and positioned.
  • Implantation sites include, but are not necessarily limited to a subdermal, subcutaneous, intramuscular, or other suitable site within a subject's body. Subcutaneous implantation sites are used in some embodiments because of convenience in implantation and removal of the drug delivery device.
  • Drug release devices suitable for use in the invention may be based on any of a variety of modes of operation.
  • the drug release device can be based upon a diffusive system, a convective system, or an erodible system (e.g., an erosion-based system).
  • the drug release device can be an electrochemical pump, osmotic pump, an electroosmotic pump, a vapor pressure pump, or osmotic bursting matrix, e.g., where the drug is incorporated into a polymer and the polymer provides for release of drug formulation concomitant with degradation of a drug-impregnated polymeric material (e.g., a biodegradable, drug-impregnated polymeric material).
  • the drug release device is based upon an electrodiffusion system, an electrolytic pump, an effervescent pump, a piezoelectric pump, a hydrolytic system, etc.
  • Drug release devices based upon a mechanical or electromechanical infusion pump can also be suitable for use with the present invention.
  • Examples of such devices include those described in, for example, U.S. Pat. Nos. 4,692,147; 4,360,019; 4,487,603; 4,360,019; 4,725,852, and the like.
  • a subject treatment method can be accomplished using any of a variety of refillable, non-exchangeable pump systems. Pumps and other convective systems can be used.
  • Osmotic pumps are used in some embodiments due to their combined advantages of more consistent controlled release and relatively small size (see, e.g., PCT published application no. WO 97/27840 and U.S. Pat. Nos.
  • Exemplary osmotically-driven devices suitable for use in the invention include, but are not necessarily limited to, those described in U.S. Pat. Nos. 3,760,984; 3,845,770; 3,916,899; 3,923,426; 3,987,790; 3,995,631 ; 3,916,899; 4,016,880; 4,036,228; 4,111,202; 4,111,203; 4,203,440; 4,203,442; 4,210,139; 4,327,725; 4,627,850; 4,865,845; 5,057,318; 5,059,423; 5,112,614; 5,137,727; 5,234,692; 5,234,693; 5,728,396; and the like.
  • the drug delivery device is an implantable device.
  • the drug delivery device can be implanted at any suitable implantation site using methods and devices well known in the art.
  • an implantation site is a site within the body of a subject at which a drug delivery device is introduced and positioned. Implantation sites include, but are not necessarily limited to a subdermal, subcutaneous, intramuscular, or other suitable site within a subject's body.
  • an active agent is delivered using an implantable drug delivery system, e.g., a system that is programmable to provide for administration of the agent.
  • implantable drug delivery system e.g., a system that is programmable to provide for administration of the agent.
  • exemplary programmable, implantable systems include implantable infusion pumps.
  • Exemplary implantable infusion pumps, or devices useful in connection with such pumps, are described in, for example, U.S. Pat. Nos. 4,350,155; 5,443,450; 5,814,019; 5,976,109; 6,017,328; 6,171,276; 6,241,704; 6,464,687; 6,475,180; and 6,512,954.
  • a further exemplary device that can be adapted for the present invention is the Synchromed infusion pump (Medtronic).
  • Suitable excipient vehicles are, for example, water, saline, dextrose, glycerol, ethanol, or the like, and combinations thereof.
  • the vehicle may contain minor amounts of auxiliary substances such as wetting or emulsifying agents or pH buffering agents.
  • auxiliary substances such as wetting or emulsifying agents or pH buffering agents.
  • Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in the art. See, e.g.. Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania, 17th edition, 1985.
  • the composition or formulation to be administered will, in any event, contain a quantity of the agent adequate to achieve the desired state in the subject being treated.
  • compositions such as vehicles, adjuvants, carriers or diluents, are readily available to the public.
  • pharmaceutically acceptable auxiliary substances such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents and the like, are readily available to the public.
  • an active agent is administered in an amount of from about 0.1 mg to about 10 mg per dose, e.g., from about 0.1 mg to about 0.25 mg, from about 0.25 mg to about 0.5 mg, from about 0.5 mg to about 1 mg, from about 1 mg to about 2.5 mg, from about 2.5 mg to about 5 mg, from about 5 mg to about 7.5 mg, or from about 7.5 mg to about 10 mg.
  • an active agent is administered in an amount of from about 10 mg to about 2000 mg per dose, e.g., from about 10 mg to about 20 mg, from about 20 mg to about 25 mg, from about 25 mg to about 50 mg, from about 50 mg to about 75 mg, from about 75 mg to about 100 mg, from about 100 mg to about 125 mg, from about 125 mg to about 150 mg, from about 150 mg to about 175 mg, from about 175 mg to about 200 mg, from about 200 mg to about 225 mg, from about 225 mg to about 250 mg, from about 250 mg to about 300 mg, from about 300 mg to about 350 mg, from about 350 mg to about 400 mg, from about 400 mg to about 450 mg, from about 450 mg to about 500 mg, from about 500 mg to about 750 mg, from about 750 mg to about 1000, from about lOOOmg to about 1250mg, from about 1250mg to about 1500mg, from about 1500mg to about 1750mg, or from about 1750mg to about 2000
  • the amount of an active agent per dose is determined on a per body weight basis.
  • an active agent is administered in an amount of from about 0.5 mg/kg to about 100 mg/kg, e.g., from about 0.5 mg/kg to about 1 mg/kg, from about 1 mg/kg to about 2 mg/kg, from about 2 mg/kg to about 3 mg/kg, from about 3 mg/kg to about 5 mg/kg, from about 5 mg/kg to about 7 mg/kg, from about 7 mg/kg to about 10 mg/kg, from about 10 mg/kg to about 15 mg/kg, from about 15 mg/kg to about 20 mg/kg, from about 20 mg/kg to about 25 mg/kg, from about 25 mg/kg to about 30 mg/kg, from about 30 mg/kg to about 40 mg/kg, from about 40 mg/kg to about 50 mg/kg per dose, from about 50 mg/kg to about 60 mg/kg, from about 60 mg/kg to about 70 mg/kg, from about 70 mg/kg
  • dose levels can vary as a function of the specific compound, the severity of the symptoms and the susceptibility of the subject to side effects. Preferred dosages for a given compound are readily determinable by those of skill in the art by a variety of means.
  • multiple doses of an active agent are administered.
  • the frequency of administration of an active agent can vary depending on any of a variety of factors, e.g., severity of the symptoms, etc.
  • an active agent is administered once per month, twice per month, three times per month, every other week (qow), once per week (qw), twice per week (biw), three times per week (tiw), four times per week, five times per week, six times per week, every other day (qod), daily (qd), twice a day (qid), or three times a day (tid).
  • an active agent is administered continuously.
  • the duration of administration of an active agent can vary, depending on any of a variety of factors, e.g., patient response, etc.
  • an active agent can be administered over a period of time ranging from about one day to about one week, from about two weeks to about four weeks, from about one month to about two months, from about two months to about four months, from about four months to about six months, from about six months to about eight months, from about eight months to about 1 year, from about 1 year to about 2 years, or from about 2 years to about 4 years, or more.
  • An active agent is administered to an individual using any available method and route suitable for drug delivery, including in vivo and ex vivo methods, as well as systemic and localized routes of administration.
  • routes of administration include intranasal, intramuscular, intratracheal, subcutaneous, intradermal, topical application, intravenous, rectal, nasal, oral, intratumoral, peritumoral, and other enteral and parenteral routes of administration. Routes of administration may be combined, if desired, or adjusted depending upon the agent and/or the desired effect. The compound can be administered in a single dose or in multiple doses.
  • An active agent can be administered to a host using any available conventional methods and routes suitable for delivery of conventional drugs, including systemic or localized routes.
  • routes of administration contemplated by the invention include, but are not necessarily limited to, enteral, parenteral, or inhalational routes.
  • Parenteral routes of administration other than inhalation administration include, but are not necessarily limited to, topical, transdermal, subcutaneous, intramuscular, intraorbital, intracapsular, intraspinal, intrasternal, and intravenous routes, i.e., any route of administration other than through the alimentary canal.
  • Parenteral routes of administration can also include intratumoral and peritumoral.
  • Parenteral administration can be carried to effect systemic or local delivery of the agent. Where systemic delivery is desired, administration typically involves invasive or systemically absorbed topical or mucosal administration of pharmaceutical preparations.
  • the agent can also be delivered to the subject by enteral administration.
  • Enteral routes of administration include, but are not necessarily limited to, oral and rectal (e.g., using a suppository) delivery.
  • Methods of administration of the agent through the skin or mucosa include, but are not necessarily limited to, topical application of a suitable pharmaceutical preparation, transdermal transmission, injection and epidermal administration.
  • a suitable pharmaceutical preparation for transdermal transmission, absorption promoters or iontophoresis are suitable methods.
  • Iontophoretic transmission may be accomplished using commercially available "patches" which deliver their product continuously via electric pulses through unbroken skin for periods of several days or more.
  • a colloidal dispersion system may be used for targeted delivery of the peptidic compound to specific tissue.
  • Colloidal dispersion systems include macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.
  • Liposomes are artificial membrane vesicles which are useful as delivery vehicles in vitro and in vivo. It has been shown that large unilamellar vesicles (LUV), which range in size from 20 400 nm can encapsulate a substantial percentage of an aqueous buffer comprising large macromolecules. RNA and DNA can be encapsulated within the aqueous interior and be delivered to cells in a biologically active form (Fraley, et ai, (1981) Trends Biochem. Sci., 6:77).
  • the composition of the liposome is usually a combination of phospholipids, particularly high-phase-transition-temperature phospholipids, usually in combination with steroids, especially cholesterol.
  • phospholipids or other lipids may also be used.
  • the physical characteristics of liposomes depend on pH, ionic strength, and the presence of divalent cations.
  • lipids useful in liposome production include phosphatidyl compounds, such as phosphatidylglycerol, phosphatidylcholine, phosphatidyl serine, phosphatidyl-ethanolamine, sphingolipids, cerebrosides, and gangliosides.
  • Particularly useful are diacylphosphatidyl glycerols, where the lipid moiety contains from 14-18 carbon atoms, particularly from 16-18 carbon atoms, and is saturated.
  • Illustrative phospholipids include egg phosphatidylcholine, dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine.
  • Exemplary liposome/therapeutic nucleic acid compositions suitable for use in a subject method are described in Louria-Hayon et al. (2002) Vaccine 20:3342.
  • targeting of liposomes can be classified based on anatomical and mechanistic factors.
  • Anatomical classification is based on the level of selectivity, for example, organ-specific, cell-specific, and organelle-specific.
  • Mechanistic targeting can be distinguished based upon whether it is passive or active. Passive targeting utilizes the natural tendency of liposomes to distribute to cells of the reticuloendothelial system (RES) in organs which contain sinusoidal capillaries.
  • RES reticuloendothelial system
  • Active targeting involves alteration of the liposome by coupling the liposome to a specific ligand such as a monoclonal antibody, sugar, glycolipid, or protein, or by changing the composition or size of the liposome in order to achieve targeting to organs and cell types other than the naturally occurring sites of localization.
  • a specific ligand such as a monoclonal antibody, sugar, glycolipid, or protein
  • the surface of the targeted delivery system may be modified in a variety of ways.
  • lipid groups can be incorporated into the lipid bilayer of the liposome in order to maintain the targeting ligand in stable association with the liposomal bilayer.
  • Various well known linking groups can be used for joining the lipid chains to the targeting ligand (see, e.g., Yanagawa, et al, (1988) Nuc. Acids Symp. Sen, 19:189; Grabarek, et al, (1990) Anal Biochem., 185:131; Staros, et al., (1986) Anal Biochem. 156:220 and Boujrad, et al, (1993) Proc.
  • Targeted delivery of a therapeutic peptidic formulation can also be achieved by conjugation of the therapeutic formulation to a surface of viral and non- viral recombinant expression vectors, to an antigen or other ligand, to a monoclonal antibody or to any molecule which has the desired binding specificity.
  • the present invention provides in vitro methods (e.g., in vitro cell-free methods; in vitro cell-based methods) of identifying an agent that inhibits binding between BRD4 and P- TEFb.
  • the methods generally involve: a) contacting a BRD4 polypeptide and P-TEFb polypeptide with a test agent, where the contacting is under conditions suitable for binding of the BRD4 polypeptide and the P-TEFb polypeptide; and b) determining the effect, if any, of the test agent on binding of the BRD4 polypeptide to the P-TEFb polypeptide.
  • Determining the effect (if any) of the test agent on binding of the BRD4 polypeptide to the P-TEFb polypeptide can involve measuring the binding of the two polypeptides to one another. Methods for determining binding of two polypeptides to one another are well known in the art; and any known method can be used. Suitable methods include, e.g., enzyme-linked immunosorbent assay (ELISA), bioluminescence resonance energy transfer (BRET), fluorescence resonance energy transfer (FRET), immunoprecipitation, radioimmunoassay, and the like.
  • ELISA enzyme-linked immunosorbent assay
  • BRET bioluminescence resonance energy transfer
  • FRET fluorescence resonance energy transfer
  • the BRD4 polypeptide will comprise a detectable label.
  • the BRD4 polypeptide will comprise a first detectable label
  • the P-TEFb polypeptide will comprise a second detectable label, where the first and the second detectable labels are distinguishable from one another.
  • a test agent that inhibits binding of a BRD4 polypeptide to a P-
  • TEFb polypeptide is a candidate agent for treating abnormal cellular proliferation.
  • a test agent that inhibits binding of a BRD4 polypeptide to a P-TEFb polypeptide is a candidate agent for treating an immunodeficiency virus infection.
  • Suitable BRD4 polypeptides include, but are not limited to, full-length BRD4 polypeptides, C-terminal BRD4 fragments, and BRD4 fusion proteins.
  • the BRD4 polypeptide is a full-length BRD4 polypeptide.
  • the BRD4 polypeptide is a C-terminal fragment of BRD4 that binds P-TEFb.
  • a suitable C-terminal fragment of BRD4 include a polypeptide that comprises an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to amino acids 1329-1362, 1318-1362, 1315-1362, 1228-1362, 1209- 1362, or 722-1362 of SEQ ID NO: 1.
  • the C-terminal portion of BRD4 can have a length of from about 30 amino acids to about 1000 amino acids, e.g., the C-terminal portion of BRD4 can have a length of from about 30 amino acids (aa) to about 35 aa (e.g., 30 aa, 31 aa, 32 aa, 33 aa, 34 aa, 35 aa), from about 35 aa to about 40 aa, from about 40 aa to about 45 aa, from about 45 aa to about 50 aa (e.g., 45 aa, 46 aa, 47 aa, 48 aa, 49 aa, or 50 aa), from about 50 aa to about 60 aa, from about 60 aa to about 75 aa, from about 75 aa to about 100 aa, from about 100 aa to about 150 aa (e.g., 100 aa, 120 aa, 125 aa, 130 aa, 1
  • a suitable BRD4 polypeptide includes a polypeptide comprising an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to a contiguous stretch of from about 1000 aa to about 1100 aa, from about 1100 aa to about 1200 aa, from about 1200 aa to about 1300 aa, or from about 1300 aa to about 1362 aa, of the amino acid sequence set forth in SEQ ID NO:1.
  • the BRD4 polypeptide is a fusion protein comprising a BRD4 polypeptide and a heterologous (non-BRD4) polypeptide.
  • the BRD4 polypeptide may be a native BRD4 polypeptide or a recombinant BRD4 polypeptide.
  • the BRD4 polypeptide is recombinant.
  • the BRD4 polypeptide is chemically synthesized.
  • the BRD4 polypeptide is purified.
  • Suitable P-TEFb polypeptides include, but are not limited to, a P-TEFb heterodimer; a
  • the P-TEFb polypeptide is a P- TEFb heterodimer.
  • the P-TEFb polypeptide is a subunit of P-TEFb, e.g., a CDK9 polypeptide, a cyclin polypeptide (e.g., cyclin Tl, cyclin T2, cyclin K).
  • the P-TEFb polypeptide is recombinant, hi some embodiments, the P-TEFb polypeptide is purified.
  • a complex of purified CDK9 and cyclin Tl e.g., a CDK9/cyclin Tl heterodimer
  • Suitable P-TEFb polypeptides include those described supra.
  • test agent inhibits binding of a BRD4 polypeptide and a P-TEFb polypeptide can be determined using any of a variety of methods, e.g., enzyme-linked immunosorbent assay (ELISA), BRET, FRET, immunoprecipitation, radioimmunoassay, and the like. Such assays are described in more detail below.
  • ELISA enzyme-linked immunosorbent assay
  • BRET BRET
  • FRET fluor FRET
  • immunoprecipitation e.g., radioimmunoassay
  • test agents of interest inhibit binding of a BRD4 polypeptide to a P-TEFb polypeptide by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90, or at least about 95%, compared to the level of binding in the absence of the test agent.
  • a test agent of interest is one that inhibits binding between a test agent of interest and a test agent of interest
  • the screening method is carried out in vitro, in a cell-free assay.
  • the screening method is carried out in vitro, in a cell-based assay, where the BRD4 polypeptide and the P-TEFb polypeptide are present in a cell.
  • the cell is contacted with a test agent; and the effect, if any, of the test agent on binding of the BRD4 polypeptide to the P-TEFb polypeptide is determined.
  • a FRET- or BRET-based assay may be used.
  • the cell is genetically modified with one or more nucleic acids comprising nucleotide sequences encoding the subject isolated peptide or recombinant polypeptide and the P-TEFb heterodimer or subunit thereof.
  • the nucleic acids can be expression constructs which provide for production of the subject isolated peptide or recombinant polypeptide and the P-TEFb heterodimer, or subunit thereof, in the cell.
  • test agent By “test agent,” “candidate agent,” and grammatical equivalents herein, which terms are used interchangeably herein, is meant any molecule (e.g. proteins (which herein includes proteins, polypeptides, and peptides), small (e.g., 5 Da-1000 Da, 5 Da to 500 Da, 100 Da-750 Da, 200 Da-500 Da, 1000 Da to 5000 Da, 5000 Da to 10,000 Da, less than 5000 Da, or less than 10,000 Da in size), or organic or inorganic molecules, polysaccharides, polynucleotides, etc.) which are to be tested for activity in inhibiting binding between a subject isolated peptide or recombinant polypeptide and a P-TEFb heterodimer, or subunit thereof.
  • proteins which herein includes proteins, polypeptides, and peptides
  • small e.g., 5 Da-1000 Da, 5 Da to 500 Da, 100 Da-750 Da, 200 Da-500 Da, 1000 Da to 5000 Da, 5000 Da to
  • test agents encompass numerous chemical classes, e.g., small organic compounds having a molecular weight of more than 50 daltons and less than about 10,000 daltons, less than about 5,000 daltons, or less than about 2,500 daltons.
  • Test agents can comprise functional groups necessary for structural interaction with proteins, e.g., hydrogen bonding, and can include at least an amine, carbonyl, hydroxyl or carboxyl group, or at least two of the functional chemical groups.
  • the test agents can comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups.
  • Test agents are also found among biomolecules including peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof.
  • Test agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides and oligopeptides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc. to produce structural analogs. Moreover, screening may be directed to known pharmacologically active compounds and chemical analogs thereof, or to new agents with unknown properties such as those created through rational drug design.
  • test agents are synthetic compounds.
  • a number of techniques are available for the random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides. See for example WO 94/24314, hereby expressly incorporated by reference, which discusses methods for generating new compounds, including random chemistry methods as well as enzymatic methods.
  • WO 94/24314 one of the advantages of the present method is that it is not necessary to characterize the candidate agent prior to the assay; only candidate agents that affect binding between a BRD4 polypeptide and a P-TEFb polypeptide need be identified.
  • test agents are provided as libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts that are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means. Known pharmacological agents may be subjected to directed or random chemical modifications, including enzymatic modifications, to produce structural analogs.
  • test agents include proteins (including antibodies, antibody fragments (i.e., a fragment containing an antigen-binding region, e.g., a FAb), single chain antibodies, and the like), nucleic acids, and chemical moieties.
  • the candidate modulators are naturally occurring proteins or fragments of naturally occurring proteins.
  • cellular extracts containing proteins, or random or directed digests of proteinaceous cellular extracts may be tested, as is more fully described below.
  • libraries of prokaryotic and eukaryotic proteins may be made for screening against any number of ubiquitin ligase compositions.
  • Other embodiments include libraries of bacterial, fungal, viral, and mammalian proteins, with the latter being preferred, and human proteins being especially preferred.
  • test agents are organic compounds.
  • test agents are synthesized from a series of substrates that can be chemically modified. "Chemically modified” herein includes traditional chemical reactions as well as enzymatic reactions.
  • These substrates generally include, but are not limited to, alkyl groups (including alkanes, alkenes, alkynes and heteroalkyl), aryl groups (including arenes and heteroaryl), alcohols, ethers, amines, aldehydes, ketones, acids, esters, amides, cyclic compounds, heterocyclic compounds (including purines, pyrimidines, benzodiazepine, beta-lactams, tetracylines, cephalosporins, and carbohydrates), steroids (including estrogens, androgens, cortisone, ecodysone, etc.), alkaloids (including ergots, vinca, curare, pyrollizdine, and mitomycines), organometallic compounds, hetero-atom bearing compounds, amino acids, and nucleosides. Chemical (including enzymatic) reactions may be done on the moieties to form new substrates or candidate agents which can then be tested using the present invention.
  • alkyl groups including alkanes,
  • determining refers to both quantitative and qualitative determinations and as such, the term “determining” is used interchangeably herein with “assaying,” “measuring,” and the like.
  • Determining the effect, if any, of a test agent on binding between a BRD4 polypeptide and a P-TEFb polypeptide can be carried out using any of a variety of assays, including, but not limited to, immunological assays (e.g., enzyme-linked immunosorbent assays; radioimmunoassay; and the like); fluorescence resonance energy transfer (FRET)-based assays; bioluminescence resonance energy transfer (BRET)-based assays; or any other assay that detects protein-protein binding.
  • immunological assays e.g., enzyme-linked immunosorbent assays; radioimmunoassay; and the like
  • FRET fluorescence resonance energy transfer
  • BRET bioluminescence resonance energy transfer
  • reagents like salts, neutral proteins, e.g. albumin, detergents, etc., including agents that are used to facilitate optimal enzyme activity and/or reduce non-specific or background activity.
  • Reagents that improve the efficiency of the assay such as protease inhibitors, anti-microbial agents, etc. may be used.
  • the components of the assay mixture are added in any order that provides for the requisite activity. Incubations are performed at any suitable temperature, typically between 4°C and 40°C. Incubation periods are selected for optimum activity, but may also be optimized to facilitate rapid high-throughput screening. Typically between 0.1 hour and 1 hour will be sufficient.
  • Assays of the invention include controls, where suitable controls include a sample (e.g., a sample comprising the BRD4 polypeptide and the P-TEFb polypeptide in the absence of the test agent). Generally a plurality of assay mixtures is run in parallel with different agent concentrations to obtain a differential response to the various concentrations. Typically, one of these concentrations serves as a negative control, i.e. at zero concentration or below the level of detection. Detectably labeled polypeptides
  • one or both of the BRD4 polypeptide and the P-TEFb polypeptide is detectably labeled ("tagged").
  • Peptides and polypeptides modified to comprises a tag and useful in the screening methods of the invention are specifically contemplated herein.
  • tag is meant an attached molecule or molecules useful for the identification or isolation of the attached molecule(s), which can be substrate binding molecules.
  • a tag can be an attachment tag or a label tag.
  • tag-X Components having a tag are referred to as "tag-X", wherein X is the component (e.g., a BRD4 polypeptide; a subject isolated BRD4 peptide; a subject recombinant BRD4 polypeptide; a P-TEFb heterodimer; or a subunit of a P-TEFb heterodimer, such as cyclin Tl, cyclin T2, cyclin K, or CDK9).
  • component e.g., a BRD4 polypeptide; a subject isolated BRD4 peptide; a subject recombinant BRD4 polypeptide; a P-TEFb heterodimer; or a subunit of a P-TEFb heterodimer, such as cyclin Tl, cyclin T2, cyclin K, or CDK9
  • tag refers to any suitable tag that can be directly (i.e., a primary label) or indirectly (i.e., a secondary label) detected; for example a label can be visualized and/or measured or otherwise identified so that its presence or absence can be known.
  • label include, but are not limited to, fluorescent labels (e.g. a green fluorescent protein, a red fluorescent protein, a yellow fluorescent protein, etc.) and label enzymes.
  • Exemplary tags include, but are not limited to, an optically-detectable label, a partner of a binding pair, and a surface substrate binding molecule (or attachment tag). As will be evident to the skilled artisan, many molecules may find use as more than one type of tag, depending upon how the tag is used. In one embodiment, the tag or label as described below is incorporated into the polypeptide as a fusion protein. [00151] As will be appreciated by those in the art, tag-components of the invention can be made in various ways, depending largely upon the form of the tag. A BRD4 polypeptide can be linked to a tag via a covalent bond. Examples of tags are described below. Exemplary tags
  • the tag is a polypeptide which is provided as a portion of a chimeric molecule comprising a first polypeptide fused to another, heterologous polypeptide or amino acid sequence, hi one embodiment, such a chimeric molecule comprises a fusion of a first polypeptide with a tag polypeptide.
  • the tag is generally placed at the amino-or carboxyl- terminus of the polypeptide.
  • the tag is usually a genetically encodable tag (e.g., fluorescent polypeptide, immunodetectable polypeptide, and the like).
  • the tag polypeptide can be, for example, an immunodetectable label (i.e., a polypeptide or other moiety which provides an epitope to which an anti-tag antibody can selectively bind), a polypeptide which serves as a ligand for binding to a receptor (e.g., to facilitate immobilization of the chimeric molecule on a substrate); an enzyme label (e.g., as described further below); or a fluorescent label (e.g., as described further below).
  • an immunodetectable label i.e., a polypeptide or other moiety which provides an epitope to which an anti-tag antibody can selectively bind
  • a polypeptide which serves as a ligand for binding to a receptor e.g., to facilitate immobilization of the chimeric molecule on a substrate
  • an enzyme label e.g., as described further below
  • a fluorescent label e.g., as described further below
  • Tag polypeptides provide for, for example, detection using an antibody against the tag polypeptide, and/or a ready means of isolating or purifying the tagged polypeptide (e.g., by affinity purification using an anti-tag antibody or another type of receptor-ligand matrix that binds to the tag).
  • a ready means of isolating or purifying the tagged polypeptide e.g., by affinity purification using an anti-tag antibody or another type of receptor-ligand matrix that binds to the tag.
  • the production of tag-polypeptides by recombinant means is within the knowledge and skill in the art.
  • optically detectable label includes labels that are detectably due to inherent properties (e.g., a fluorescent label), or which can be reacted with a substrate or act as a substrate to provide an optically detectable (e.g., colored) reaction product (e.g., HRP).
  • fluorescent label is meant any molecule that may be detected via its inherent fluorescent properties, which include fluorescence detectable upon excitation.
  • Suitable fluorescent labels include, but are not limited to, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosin, coumarin, methyl-coumarins, pyrene, Malacite green, stilbene, Lucifer Yellow, Cascade BlueTM, Texas Red, IAEDANS, EDANS, BODIPY FL, LC Red 640, Cy 5, Cy 5.5, LC Red 705 and Oregon green.
  • Suitable optical dyes are described in the 2002 Molecular Probes Handbook, 9th Ed., by Richard P. Haugland, hereby expressly incorporated by reference.
  • Suitable fluorescent labels include, but are not limited to, green fluorescent protein
  • GFP Greenhouse Protein
  • BFP blue fluorescent protein
  • enhanced yellow fluorescent protein EYFP; 1.
  • fluorescent labels are employed.
  • at least two fluorescent labels are used which are members of a fluorescence resonance energy transfer (FRET) pair.
  • FRET can be used to detect association/dissociation of for example, a subject isolated peptide (or recombinant polypeptide) and a P-TEFb heterodimer, or subunit thereof; and the like.
  • FRET pairs are used in in vitro assays.
  • FRET is phenomenon known in the art wherein excitation of one fluorescent dye is transferred to another without emission of a photon.
  • a FRET pair consists of a donor fluorophore and an acceptor fluorophore (where the acceptor fluorophore may be a quencher molecule).
  • the fluorescence emission spectrum of the donor and the fluorescence absorption spectrum of the acceptor must overlap, and the two molecules must be in close proximity.
  • the distance between donor and acceptor at which 50% of donors are deactivated (transfer energy to the acceptor) is defined by the Forster radius, which is typically 10-100 angstroms.
  • Changes in the fluorescence emission spectrum comprising FRET pairs can be detected, indicating changes in the number of that are in close proximity (i.e., within 100 angstroms of each other). This will typically result from the binding or dissociation of two molecules, one of which is labeled with a FRET donor and the other of which is labeled with a FRET acceptor, wherein such binding brings the FRET pair in close proximity.
  • FRET pairs useful in the invention include, but are not limited to, EDANS/fluorescein, IAEDANS/fluorescein, fluorescein/tetramethylrhodamine, fluorescein/Cy 5, IEDANS/D ABCYL, fluorescein/QSY-7, fluorescein/LC Red 640, fluorescein/Cy 5.5 and fluorescein/LC Red 705.
  • a fluorescent donor molecule and a nonfluorescent acceptor molecule may be employed.
  • fluorescent emission of the donor will increase when quencher is displaced from close proximity to the donor and fluorescent emission will decrease when the quencher is brought into close proximity to the donor.
  • Useful quenchers include, but are not limited to, DABCYL, QSY 7 and QSY 33.
  • Useful fluorescent donor/quencher pairs include, but are not limited to ED ANS/D ABCYL, Texas Red/D ABCYL, BODIPY/D ABCYL, Lucifer yellow/D ABCYL, coumarin/D ABCYL and fluorescein/QSY 7 dye.
  • detection involves bioluminescence resonance energy transfer
  • BRET is a protein-protein interaction assay based on energy transfer from a bioluminescent donor to a fluorescent acceptor protein.
  • the BRET signal is measured by the amount of light emitted by the acceptor to the amount of light emitted by the donor. The ratio of these two values increases as the two proteins are brought into proximity.
  • the BRET assay has been amply described in the literature. See, e.g., U.S. Patent Nos. 6,020,192; 5,968,750; and 5,874,304; and Xu et al. (1999) Proc. Natl. Acad. Sci. USA 96:151-156.
  • BRET assays may be performed by analyzing transfer between a bioluminescent donor protein and a fluorescent acceptor protein. Interaction between the donor and acceptor proteins can be monitored by a change in the ratio of light emitted by the bioluminescent and fluorescent proteins.
  • binding may be assayed by fluorescence anisotropy.
  • Fluorescence anisotropy assays are amply described in the literature. See, e.g., Jameson and Sawyer (1995) Methods Enzymol. 246:283-300.
  • label enzyme an enzyme which may be reacted in the presence of a label enzyme substrate which produces a detectable product.
  • Suitable label enzymes also include optically detectable labels (e.g., in the case of HRP).
  • Suitable label enzymes for use in the present invention include but are not limited to, horseradish peroxidase (HRP), alkaline phosphatase and glucose oxidase. Methods for the use of such substrates are well known in the art.
  • HRP horseradish peroxidase
  • alkaline phosphatase alkaline phosphatase
  • glucose oxidase glucose oxidase
  • Such products may be opaque, such as the reaction of horseradish peroxidase with tetramethyl benzedine, and may have a variety of colors.
  • Other label enzyme substrates such as Luminol (available from Pierce Chemical Co.), have been developed that produce fluorescent reaction products. Methods for identifying label enzymes with label enzyme substrates are well known in the art and many commercial kits are available. Examples and methods for the use of various label enzymes are described in Savage et al., Previews 247:6-9 (1998), Young, J. Virol. Methods 24:227-236 (1989), which are each hereby incorporated by reference in their entirety.
  • radioisotope any radioactive molecule. Suitable radioisotopes for use in the invention include, but are not limited to 14 C, 3 H, 32 P, 33 P, 35 S, 125 I, and 131 I. The use of radioisotopes as labels is well known in the art.
  • labels may be indirectly detected, that is, the tag is a partner of a binding pair.
  • partner of a binding pair is meant one of a first and a second moiety, wherein said first and said second moiety have a specific binding affinity for each other.
  • Suitable binding pairs for use in the invention include, but are not limited to, antigen/antibodies (for example, digoxigenin/anti-digoxigenin, dinitrophenyl (DNP)/anti-DNP, dansyl-X-anti-dansyl, fluorescein/anti-fluorescein, lucifer yellow/anti-lucifer yellow, and rhodamine anti-rhodamine), biotin/avidin (or biotin/streptavidin) and calmodulin binding protein (CBP)/calmodulin.
  • antigen/antibodies for example, digoxigenin/anti-digoxigenin, dinitrophenyl (DNP)/anti-DNP, dansyl-X-anti-dansyl, fluorescein/anti-fluorescein, lucifer yellow/anti-lucifer yellow, and rhodamine anti-rhodamine
  • biotin/avidin or biotin/streptavidin
  • CBP
  • binding pairs include polypeptides such as the FLAG-peptide (Hopp et al., BioTechnol, 6:1204-1210 (1988)); the KT3 epitope peptide (Martin et al., Science, 255:192- 194 (1992)); tubulin epitope peptide (Skinner et al., J. Biol. Chem., 266: 15 163- 15 166 (1991)); and the T7 gene 10 protein peptide tag (Lutz-Freyemuth et al., Proc. Natl. Acad. Sci. USA, a:6393-6397 (1990)) and the antibodies each thereto.
  • polypeptides such as the FLAG-peptide (Hopp et al., BioTechnol, 6:1204-1210 (1988)); the KT3 epitope peptide (Martin et al., Science, 255:192- 194 (1992)); tubulin epitope peptide (S
  • binding pair partners serves as the tag, as steric considerations in ubiquitin ligation may be important.
  • binding pair partners may be used in applications other than for labeling, such as immobilization of the protein on a substrate and other uses as described below.
  • a partner of one binding pair may also be a partner of another binding pair.
  • an antigen first moiety
  • first antibody second moiety
  • second antibody third moiety
  • a partner of a binding pair may comprise a label, as described above. It will further be appreciated that this allows for a tag to be indirectly labeled upon the binding of a binding partner comprising a label. Attaching a label to a tag which is a partner of a binding pair, as just described, is referred to herein as "indirect labeling.”
  • the tag is a surface substrate binding molecule.
  • surface substrate binding molecule and grammatical equivalents thereof is meant a molecule have binding affinity for a specific surface substrate, which substrate is generally a member of a binding pair applied, incorporated or otherwise attached to a surface.
  • Suitable surface substrate binding molecules and their surface substrates include, but are not limited to poly-histidine (poly-his) or poly-histidine-glycine (poly- his-gly) tags and Nickel substrate; the Glutathione-S Transferase tag and its antibody substrate (available from Pierce Chemical); the influenza hemagglutinin (HA) tag polypeptide and its antibody 12CA5 substrate (Field et al., MoI. Cell.
  • surface binding substrate molecules useful in the present invention include, but are not limited to, polyhistidine structures (His- tags) that bind nickel substrates, antigens that bind to surface substrates comprising antibody, haptens that bind to avidin substrate (e.g., biotin) and CBP that binds to surface substrate comprising calmodulin.
  • polyhistidine structures His- tags
  • antigens that bind to surface substrates comprising antibody
  • haptens that bind to avidin substrate (e.g., biotin)
  • CBP that binds to surface substrate comprising calmodulin.
  • Biotinylation of target molecules and substrates is well known, for example, a large number of biotinylation agents are known, including amine-reactive and thiol-reactive agents, for the biotinylation of proteins, nucleic acids, carbohydrates, carboxylic acids; see, e.g., chapter 4, Molecular Probes Catalog, Haugland, 6th Ed. 1996, hereby incorporated by reference.
  • a biotinylated substrate can be attached to a biotinylated component via avidin or streptavidin.
  • haptenylation reagents are also known. Methods for labeling of proteins with radioisotopes are known in the art. For example, such methods are found in Ohta et al., Molec. Cell 3:535-541 (1999), which is hereby incorporated by reference in its entirety.
  • the covalent attachment of the tag may be either direct or via a linker.
  • the linker is a relatively short coupling moiety that is used to attach the molecules.
  • a coupling moiety may be synthesized directly onto a component of the invention, ubiquitin for example, and contains at least one functional group to facilitate attachment of the tag.
  • the coupling moiety may have at least two functional groups, which are used to attach a functionalized component to a functionalized tag, for example.
  • the linker is a polymer. In this embodiment, covalent attachment is accomplished either directly, or through the use of coupling moieties from the component or tag to the polymer.
  • the covalent attachment is direct, that is, no linker is used.
  • the component can contain a functional group such as a carboxylic acid which is used for direct attachment to the functionalized tag.
  • a functional group such as a carboxylic acid which is used for direct attachment to the functionalized tag.
  • the component and tag may be attached in a variety of ways, including those listed above. What is important is that manner of attachment does not significantly alter the functionality of the component.
  • tag-BRD4 polypeptide the tag should be attached in such a manner as to allow binding between the BRD4 polypeptide and a P-TEFb polypeptide.
  • the tag is functionalized to facilitate covalent attachment, as is generally outlined above.
  • tags are commercially available which contain functional groups, including, but not limited to, isothiocyanate groups, amino groups, haloacetyl groups, maleimides, succinimidyl esters, and sulfonyl halides, all of which may be used to covalently attach the tag to a second molecule, as is described herein.
  • the choice of the functional group of the tag will depend on the site of attachment to either a linker, as outlined above or a component of the invention.
  • amino modified or hydrazine modified tags will be used for coupling via carbodimide chemistry, for example using 1 -ethyl-3-(3- dimethylaminopropyl)-carbodimide (EDAC) as is known in the art (see Set 9 and Set 11 of the Molecular Probes Catalog, supra; see also the Pierce 1994 Catalog and Handbook, pages T-155 to T-200, both of which are hereby incorporated by reference).
  • the carbodiimide is first attached to the tag, such as is commercially available for many of the tags described herein.
  • test agents are assessed for any cytotoxic activity (other than anti-growth and/or antiproliferative activity) it may exhibit toward a living eukaryotic cell, using well-known assays, such as trypan blue dye exclusion, an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2 H-tetrazolium bromide) assay, and the like. Agents that do not exhibit cytotoxic activity are considered candidate agents. Exemplary screening method
  • a C-terminal BRD4 peptide that binds P-TEFb includes a C-terminal BRD4 fragment as described herein, e.g., an isolated peptide comprising an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to amino acids 1329-1362, 1318-1362, 1315-1362, 1228-1362, 1209-1362, or 722-1362 of SEQ ID NO: l, or at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to the amino acid
  • a C-terminal BRD4 polypeptide can have a length of from about 30 amino acids to about 1000 amino acids, e.g., the C-terminal portion of BRD4 can have a length of from about 30 amino acids (aa) to about 35 aa (e.g., 30 aa, 31 aa, 32 aa, 33 aa, 34 aa, 35 aa), from about 35 aa to about 40 aa, from about 40 aa to about 45 aa, from about 45 aa to about 50 aa (e.g., 45 aa, 46 aa, 47 aa, 48 aa, 49 aa, or 50 aa), from about 50 aa to about 60 aa, from about 60 aa to about 75 aa, from about 75 aa to about 100 aa, from about 100 aa to about 150 aa (e.g., 100 aa, 120 aa, 125 aa, 130 aa,
  • Non-limiting examples of suitable BRD4 PID include the following peptides:
  • the BRD4 PID has the amino acid sequence of SEQ ID NO:4, or a P-TEFb-binding variant thereof.
  • the P-TEFb polypeptide can be recombinant.
  • epitope-tagged e.g.,
  • human recombinant cyclin Tl and epitope-tagged human recombinant CDK9 are expressed using recombinant baculovirus (e.g., BaculoGold; PharMingen).
  • baculovirus e.g., BaculoGold; PharMingen.
  • Spodoptera frugiperda cells Sf9 insect cells are infected with the recombinant baculovirus comprising nucleotide sequences encoding epitope-tagged cyclin Tl, or with recombinant baculovirus comprising nucleotide sequences encoding epitope-tagged CDK9.
  • the infected cells are incubated for 2-3 days and harvested by centrifugation.
  • Recombinant proteins are purified on Ni + -NTA-agarose columns and eluted using imidazole. Eluted proteins are pooled (forming a sample including both CDK9 and cyclin Tl) and loaded onto Mono S columns followed by elution with KCl. The peak corresponding to the CDK9-cyclin Tl heterodimer is collected.
  • BRD4 PID can be chemically synthesized.
  • the BRD4 PID is biotinylated.
  • Biotinylated BRD4 PID can be purified.
  • a test agent that inhibits BRD4/P-TEFb binding is identified using an enzyme-linked immunosorbent assay (ELISA).
  • ELISA enzyme-linked immunosorbent assay
  • Wells of a multi-well plastic plate e.g., a 96-well plate, a 192-well plate, a 384-well plate, etc.
  • Biotinylated BRD4 PID is immobilized onto the streptavidin-coated wells of the plate.
  • Immobilized BRD4 PID is contacted with purified CDK9/cyclin Tl heterodimer in the presence or absence of a test agent. Binding of the CDK9/cyclin Tl heterodimer to immobilized BRD4 PID is detected using antibody specific for the CDK9 subunit or the cyclin Tl subunit.
  • an antibody specific for cyclin Tl is used.
  • the anti-cyclin Tl antibody can be detectably labeled, either directly, or indirectly.
  • Suitable direct labels include radioisotopes, fluorescent proteins, and enzymes (e.g., alkaline phosphatase, horse radish peroxidase, luciferase, and the like) that yield a detectable product (e.g., a fluorescent product, a colored product, etc.).
  • An example of an indirect label is an enzyme-conjugated secondary antibody that binds the anti-cyclin Tl antibody.
  • Suitable enzymes include, e.g., alkaline phosphatase, horse radish peroxidase, luciferase, and the like.
  • a subject screening method is a method of identifying a candidate agent for treating a disease associated with abnormal cellular proliferation.
  • the method generally involves: (a) contacting a BRD4 polypeptide and a P-TEFb polypeptide with a test agent in vitro, where the contacting is under conditions suitable for binding of the BRD4 polypeptide to the P-TEFb polypeptide, and (b) determining the effect, if any, of the test agent on binding of the BRD4 polypeptide to the P-TEFb polypeptide, wherein a test agent that inhibits binding of the BRD4 polypeptide to the P-TEFb polypeptide is a candidate agent for inhibiting a disease associated with abnormal cellular proliferation.
  • a disease associated with abnormal cellular proliferation includes, but is not limited to, cancer; cardiac hypertrophy (e.g., undesired growth of cardiac myocytes); prostate hypertrophy; pulmonary vascular obstructive disease; restenosis (e.g., restenosis following angioplasty or other vascular trauma); disorders resulting from vascular smooth muscle cell proliferation; psoriasis, a condition characterized by the cellular hyperproliferation of keratinocytes; or atherosclerosis.
  • cardiac hypertrophy e.g., undesired growth of cardiac myocytes
  • prostate hypertrophy e.g., pulmonary vascular obstructive disease
  • restenosis e.g., restenosis following angioplasty or other vascular trauma
  • disorders resulting from vascular smooth muscle cell proliferation e.g., psoriasis, a condition characterized by the cellular hyperproliferation of keratinocytes; or atherosclerosis.
  • a test agent that inhibits binding between a BRD4 polypeptide and a P-TEFb polypeptide, and that is therefore considered a candidate agent is further tested for an effect on reducing proliferation of a cancerous cell.
  • Such a test is carried out using well- established methods of measuring cell proliferation. For example, a cancerous cell line is contacted with the candidate agent; and 3 H-thymidine incorporation into genomic DNA is measured as an indication of proliferation.
  • a candidate agent induces apoptosis in a cancerous cell.
  • Apoptosis can be assayed using any known method. Assays can be conducted on cell populations or an individual cell, and include morphological assays and biochemical assays.
  • Assays can be conducted on cell populations or an individual cell, and include morphological assays and biochemical assays.
  • a non-limiting example of a method of determining the level of apoptosis in a cell population is TUNEL (TdT-mediated dUTP nick-end labeling) labeling of the 3'-OH free end of DNA fragments produced during apoptosis (Gavrieli et al. (1992) J. Cell Biol. 119:493).
  • the TUNEL method consists of catalytically adding a nucleotide, which has been conjugated to a chromogen system or to a fluorescent tag, to the 3'-OH end of the 180-bp (base pair) oligomer DNA fragments in order to detect the fragments.
  • the presence of a DNA ladder of 180-bp oligomers is indicative of apoptosis.
  • Procedures to detect cell death based on the TUNEL method are available commercially, e.g., from Boehringer Mannheim (Cell Death Kit) and Oncor (Apoptag Plus). Another marker that is currently available is annexin, sold under the trademark APOPTESTTM.
  • This marker is used in the "Apoptosis Detection Kit,” which is also commercially available, e.g., from R&D Systems.
  • apoptosis a cell membrane's phospholipid asymmetry changes such that the phospholipids are exposed on the outer membrane.
  • Annexins are a homologous group of proteins that bind phospholipids in the presence of calcium.
  • a second reagent, propidium iodide (PI) is a DNA binding fluorochrome.
  • Subject screening methods include methods of identifying agents for treating infection by an immunodeficiency virus.
  • a subject method comprises a) contacting a BRD4 polypeptide and a P-TEFb polypeptide with a test agent in vitro, where the contacting is under conditions suitable for binding between the BRD4 polypeptide and the P-TEFb polypeptide; and b) determining the effect, if any, of the test agent on binding of the BRD4 polypeptide to the P-TEFb polypeptide.
  • a test agent that inhibits binding of the BRD4 polypeptide to the P-TEFb polypeptide, as compared with binding of the BRD4 polypeptide to the P-TEFb polypeptide and in the absence of the test agent, is a candidate agent for treating infection by an immunodeficiency virus.
  • a subject screening method is a method of identifying agents which inhibit Tat-mediated recruitment of a P-TEFb polypeptide to a transcriptional promoter of an immunodeficiency virus.
  • the method may comprise contacting a test agent with an BRD4 polypeptide as disclosed herein and a P-TEFb polypeptide; and determining the effect, if any, of the test agent on binding of the P-TEFb polypeptide to the BRD4 polypeptide, wherein a test agent that inhibits binding of the P-TEFb kinase to the isolated peptide or recombinant polypeptide is a candidate agent for inhibiting Tat-mediated recruitment of the P- TEFb polypeptide to a viral promoter of an immunodeficiency virus.
  • BRD4 POLYPEPTIDES BRD4 POLYPEPTIDES
  • the present invention provides isolated BRD4 polypeptides (including BRD4 polypeptide fragments and recombinant BRD4 polypeptides).
  • the BRD4 polypeptides are useful in subject treatment methods, as described above.
  • the BRD4 polypeptides are also useful in subject screening methods, as described above.
  • a subject isolated BRD4 polypeptide is capable of inhibiting an interaction between a P-TEFb polypeptide (e.g., a P-TEFb heterodimer, a P-TEFb CDK9 subunit, or a P-TEFb cyclin subunit), and an endogenous intracellular protein.
  • a subject isolated BRD4 polypeptide is capable of binding to a P-TEFb polypeptide, and inhibiting P-TEFb mediated phosphorylation of the C-terminal domain of the large subunit (RPBl) of RNA polymerase II (RNAPII).
  • the present disclosure provides isolated BRD4 peptides capable of inhibiting an interaction between estrogen receptor alpha (ERa) and a cyclin Tl subunit of P-TEFb.
  • the present disclosure also provides isolated BRD4 polypeptide that inhibit interaction between a viral transcriptional enhancer (e.g., Tat) and a P-TEFb polypeptide and compositions comprising the isolated peptides.
  • a viral transcriptional enhancer e.g., Tat
  • a subject isolated BRD4 polypeptide binds to P-TEFb, wherein
  • P-TEFb is a heterodimer of cyclin-dependent kinase 9 (CDK9) and cyclin Tl, T2 or K.
  • a subject isolated peptide binds to P-TEFb CDK9 subunit.
  • a subject isolated peptide binds to P-TEFb cyclin subunit (e.g., cyclin Tl, cyclin T2, or cyclin K).
  • a subject isolated peptide binds to both P-TEFb CDK9 subunit and to P-TEFb cyclin subunit (e.g., cyclin Tl, cyclin T2, or cyclin K).
  • the CDK9 subunit of P-TEFb is evolutionarily conserved as demonstrated by the significant amino acid sequence conservation between species as varied as human; fish, D. rerio; fly, D. melanogaster; sea urchin, S. purpuratus; and worm, C. elegans. Peterlin et al, (2006) Molecular Cell 23: 297-305. When compared with human protein sequences, fish, fly, sea urchin and worm were demonstrated to have 95, 84, 69 and 74% similarity, respectively, as determined by the number of identical and highly similar residues using ClustalW (a general purpose multiple sequence alignment program made available by the European Bioinformatics Institute). Peterlin et al, (2006) Molecular Cell 23: 297-305.
  • a subject isolated BRD4 polypeptide binds to P-TEFb, wherein the CDK9 subunit of P-TEFb comprises the amino acid sequence set forth in one of GenBank Accession Nos. AAF72183, NP 001252 and AAV38706.
  • a subject isolated peptide binds to P-TEFb, wherein the CDK9 subunit of P-TEFb comprises an amino acid sequence having at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to one of the amino acid sequences set forth in GenBank Accession Nos. AAF72183, NP 001252, and AAV38706.
  • GenBank Accession Nos. AAF72183, NP_001252, and AAV38706 are set forth in SEQ ID NOs: 10-12.
  • cyclin Tl subunit of P-TEFb is evolutionarily conserved as demonstrated by the significant amino acid sequence identity between mouse and human.
  • Murine cyclin Tl exhibits 90% amino acid sequence identity as compared with human cyclin Tl .
  • Bieniasz et al The EMBO Journal (1998) 17, 7056-7065.
  • a subject isolated peptide binds to P-TEFb, wherein the P-
  • TEFb comprises a cyclin Tl subunit and said cyclin Tl subunit comprises an amino acid sequence having at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to one of the amino acid sequences set forth in GenBank Accession Nos. EAW57998, NP OOl 231, 060563, and AAC39638, e.g., one of the amino acid sequences set forth in SEQ ID NOs:13-16.
  • a subject isolated peptide binds to P-TEFb, wherein the P-TEFb comprises a cyclin T2 subunit and said cyclin T2 subunit comprises an amino acid sequence having at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to one of the amino acid sequences set forth in GenBank Accession Nos. AAI14367, AAW56073, NP_490595, NP_001232, EAXl 1647 and EAXl 1646, e.g., one of the amino acid sequences set forth in SEQ ID NOs: 17- 22.
  • a subject isolated peptide binds to P-TEFb, wherein the P-
  • TEFb comprises a cyclin K subunit and said cyclin K subunit comprises an amino acid sequence having at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to one of the amino acid sequences set forth in GenBank Accession Nos.
  • a subject BRD4 polypeptide comprises an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to amino acids 1329-1362, 1318-1362, 1315-1362, 1228-1362, 1209-1362, or 722-1362 of SEQ ID NO: 1, or at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in one of SEQ ID NOs: 4-9.
  • a subject BRD4 polypeptide can have a length of from about 30 amino acids to about 1000 amino acids, e.g., the C-terminal portion of BRD4 can have a length of from about 30 amino acids (aa) to about 35 aa (e.g., 30 aa, 31 aa, 32 aa, 33 aa, 34 aa, 35 aa), from about 35 aa to about 40 aa, from about 40 aa to about 45 aa, from about 45 aa to about 50 aa (e.g., 45 aa, 46 aa, 47 aa, 48 aa, 49 aa, or 50 aa), from about 50 aa to about 60 aa, from about 60 aa to about 75 aa, from about 75 aa to about 100 aa, from about 100 aa to about 150 aa (e.g., 100 aa, 120 aa, 125 aa, 130 aa, 135
  • a subject isolated BRD4 polypeptide comprises amino acids
  • a subject isolated peptide comprises amino acids 1329-1362 of SEQ ID NO: 1 and has a length of 34 amino acids, hi other embodiments, a subject isolated peptide comprises an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or 100% amino acid amino acid sequence identity to amino acids 1329-1362 of SEQ ID NO: 1 ( Figure 12B).
  • a subject BRD4 polypeptide has a length of about 34 amino acids and comprises an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or 100% amino acid amino acid sequence identity to amino acids 1329-1362 of SEQ ID NO: 1.
  • a subject isolated BRD4 polypeptide comprises amino acids
  • a subject isolated BRD4 polypeptide comprises amino acids 1318-1362 of SEQ ID NO: 1, and has a length of 45 amino acids.
  • a subject isolated BRD4 polypeptide comprises an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or 100% amino acid amino acid sequence identity to amino acids 1318-1362 of SEQ ID NO: 1.
  • a subject isolated BRD4 polypeptide comprises an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or 100% amino acid amino acid sequence identity to amino acids 1318-1362 of SEQ ID NO: 1, and has a length of 45 amino acids.
  • a subject isolated BRD4 polypeptide comprises an amino acid sequence selected from the group of amino acid sequences shown in Figure 2, panel A (SEQ ID NOs: 4-9).
  • a subject isolated BRD4 polypeptide has an amino acid sequence length of 48 amino acids and comprises the amino acid sequence set forth in SEQ ID NO: 4. In another embodiment, a subject isolated BRD4 polypeptide has an amino acid sequence length of 48 amino acids and comprises the amino acid sequence set forth in SEQ ID NO: 5. In another embodiment, a subject isolated BRD4 polypeptide has an amino acid sequence length of 46 amino acids and comprises the amino acid sequence set forth in SEQ ID NO: 6. In another embodiment, a subject isolated BRD4 polypeptide has an amino acid sequence length of 47 amino acids and comprises the amino acid sequence set forth in SEQ ID NO: 7.
  • a subject isolated BRD4 polypeptide has an amino acid sequence length of 47 amino acids and comprises the amino acid sequence set forth in SEQ ID NO: 8. In another embodiment, a subject isolated BRD4 polypeptide has an amino acid sequence length of 48 amino acids and comprises the amino acid sequence set forth in SEQ ID NO: 9. In other embodiments, a subject isolated BRD4 polypeptide comprises an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or 100% amino acid sequence identity to an amino acid sequence set forth in one of SEQ ID NOs: 4-9.
  • a subject isolated BRD4 polypeptide comprises amino acids
  • a subject isolated BRD4 polypeptide comprises amino acids 1228-1362 of SEQ ID NO: 1, and has a length of 135 amino acids.
  • a subject isolated peptide comprises an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or 100% amino acid amino acid sequence identity to amino acids 1228-1362 of SEQ ID NO: 1.
  • a subject isolated BRD4 polypeptide comprises an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or 100% amino acid amino acid sequence identity to amino acids 1228-1362 of SEQ ID NO: 1, and has a length of 135 amino acids.
  • a subject isolated BRD4 polypeptide comprises amino acids
  • a subject isolated BRD4 polypeptide comprises amino acids 1209-1362 of SEQ ID NO: 1, and has a length of 154 amino acids.
  • a subject isolated BRD4 polypeptide comprises an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or 100% amino acid amino acid sequence identity to amino acids 1209-1362 of SEQ ID NO: 1.
  • a subject isolated BRD4 polypeptide comprises an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or 100% amino acid amino acid sequence identity to amino acids 1209-1362 of SEQ ID NO: 1, and has a length of 154 amino acids.
  • a subject isolated BRD4 polypeptide comprises amino acids
  • a subject isolated BRD4 polypeptide comprises amino acids 722-1362 of SEQ ID NO: 1, and has a length of 641 amino acids.
  • a subject isolated BRD4 polypeptide comprises an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or 100% amino acid amino acid sequence identity to amino acids 722-1362 of SEQ ID NO: 1.
  • a subject isolated BRD4 polypeptide comprises an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or 100% amino acid amino acid sequence identity to amino acids 722- 1362 of SEQ ID NO: 1, and has a length of 641 amino acids.
  • a subject isolated BRD4 polypeptide also lacks an N-terminal portion of SEQ ID NO: 1.
  • a subject isolated BRD4 polypeptide specifically lacks an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or 100% amino acid sequence identity to amino acids 1- 721, 1-1208, 1-1227, 1-1314, 1-1317 or 1-1328 of SEQ ID NO: 1.
  • a subject isolated BRD4 polypeptide which binds to P-TEFb may comprise one or more mutations or amino acid substitutions relative to the human BRD4 amino acid sequence (SEQ ID NO: 1).
  • a BRD4 polypeptide comprising a D1346N mutation or an S1351R mutation within the C-terminus of human BRD4 retains P-TEFb binding activity.
  • a subject isolated BRD4 polypeptide which binds to P-TEFb does not comprise certain mutations or amino acid substitutions or additions which would disrupt or eliminate binding to P-TEFb.
  • a subject BRD4 polypeptide which binds to P-TEFb does not comprise an R1337P mutation within the C- terminus of human BRD4 (amino acids 1209-1362 of SEQ ID NO: 1), as said mutation disrupts the BRD4-P-TEFb interaction.
  • an isolated BRD4 peptide which binds to P-TEFb does not comprise a mutation of amino acids F 1357 Ej 358 and E 1359 to A 1357 A 1358 and 1.
  • Whether a change in the amino acid sequence of a polypeptide results in a functional homolog may be readily determined by assessing the ability of the variant polypeptide to produce a response similar to that of the wild-type protein. Polypeptides in which more than one replacement has taken place may readily be tested in the same manner.
  • a subject BRD4 polypeptide comprises one or more post- translational or chemical modifications.
  • modifications include, for example, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent crosslinks, formation of cystine, formation of pyroglutamate, formylation, gamma carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino
  • the disclosure also contemplates a subject BRD4 polypeptide bound to a solid support.
  • Exemplary solid supports include the following: particles, strands, precipitates, gels, sheets, tubing, spheres, containers, capillaries, pads, slices, films, plates and slides.
  • subject BRD4 polypeptide is a fusion protein comprising a fusion partner which increases its solubility and/or facilitates its purification, identification, detection, and/or structural characterization.
  • Exemplary fusion partners include, for example, glutathione S-transferase (GST), protein A, protein G, calmodulin-binding peptide, thioredoxin, maltose binding protein, hemagglutinin (HA), myc, poly arginine, poly His (e.g., (His) 6 ), poly His- Asp, and a FLAG epitope.
  • GST glutathione S-transferase
  • protein A protein G
  • protein G calmodulin-binding peptide
  • thioredoxin maltose binding protein
  • HA hemagglutinin
  • myc hemagglutinin
  • poly arginine poly His
  • poly His e.g., (His) 6
  • poly His- Asp poly His- Asp
  • FLAG epitope e.g., FLAG epitope.
  • Additional exemplary domains include domains that alter protein localization in vivo, such as signal
  • Polypeptides may contain multiple copies of the same fusion domain or may contain fusions to two or more different domains.
  • the fusions may occur at the N-terminus of the polypeptide, at the C-terminus of the polypeptide, or at both the N- and C-terminus of the polypeptide.
  • linker sequences between a subject polypeptide and the fusion domain in order to facilitate construction of the fusion protein or to optimize protein expression or structural constraints of the fusion protein.
  • the polypeptide may be constructed so as to contain protease cleavage sites between the fusion polypeptide and polypeptide of the invention in order to remove the tag after protein expression or thereafter.
  • suitable endoproteases include, for example, Factor Xa and TEV proteases.
  • a subject BRD4 polypeptide can be detectably labeled.
  • Various labels include radioisotopes, fluorescers (e.g., fluorescent dyes), chemiluminescers, enzymes, a member of a specific binding pair, particles, e.g. magnetic particles, and the like.
  • Specific binding pairs include, but are not limited to, biotin and streptavidin; digoxin and antidigoxin; lectin and carbohydrate moieties; antibody and hapten; antibody and antigen; etc.
  • the disclosure also contemplates the generation of mimetics, e.g. peptide or non- peptide agents, which are able to mimic binding of the authentic protein to another cellular partner.
  • mimetics e.g. peptide or non- peptide agents
  • the critical residues of a protein which are involved in molecular recognition of a substrate protein may be determined and used to generate peptidomimetics that may bind to the substrate protein.
  • the peptidomimetic may then be used as an inhibitor of the wild-type protein by binding to the substrate and covering up the critical residues needed for interaction with the wild-type protein, thereby preventing interaction of the protein and the substrate.
  • peptidomimetic compounds may be generated which mimic those residues in binding to the substrate.
  • non- hydrolyzable peptide analogs of such residues may be generated using benzodiazepine (e.g., see Freidinger et al., in Peptides: Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988), azepine (e.g., see Huffman et al., in Peptides: Chemistry and Biology, G. R.
  • a peptide mimetic is a molecule that mimics the biological activity of a peptide but is no longer peptidic in chemical nature.
  • a peptidomimetic is a molecule that no longer contains any peptide bonds (that is, amide bonds between amino acids).
  • the term peptide mimetic is sometimes used to describe molecules that are no longer completely peptidic in nature, such as pseudo-peptides, semi-peptides and peptoids. Examples of some peptidomimetics by the broader definition (where part of a peptide is replaced by a structure lacking peptide bonds) are described below.
  • peptidomimetics contemplated by the disclosure provide a spatial arrangement of reactive chemical moieties that closely resembles the three-dimensional arrangement of active groups in the peptide on which the peptidomimetic is based. As a result of this similar active- site geometry, the peptidomimetic has effects on biological systems which are similar to the biological activity of the peptide.
  • the disclosure also contemplates peptidomimetic compositions which are analogs that mimic the activity of biologically active isolated peptides disclosed herein, i.e., the peptidomimetics are capable of binding to P-TEFb thereby inhibiting the binding of Tat to P- TEFb or the binding of BRD4 to P-TEFb.
  • the peptidomimetic contemplated by the disclosure may be substantially similar in three-dimensional shape and/or biological activity to the isolated peptides described herein.
  • the isolated peptides described above therefore have utility in the development of small chemical compounds with similar biological activities and therefore with similar therapeutic utilities.
  • the techniques of developing peptidomimetics are conventional. For example, peptide bonds can be replaced by non-peptide bonds that allow the peptidomimetic to adopt a similar structure, and therefore biological activity, to the original peptide. Further modifications can also be made by replacing chemical groups of the amino acids with other chemical groups of similar structure.
  • the development of peptidomimetics can be aided by determining the tertiary structure of the original peptide, either free or bound to a binding partner, by NMR spectroscopy, crystallography and/or computer-aided molecular modeling.
  • the present disclosure provides compounds exhibiting enhanced therapeutic activity in comparison to the isolated peptides described herein.
  • the peptidomimetic compounds obtained by the above methods having the biological activity of the isolated peptides described above and similar three dimensional structure, are contemplated by the present disclosure. It will be readily apparent to one skilled in the art that a peptidomimetic can be generated from any of the modified isolated peptides described above or from an isloated peptide bearing more than one of the modifications described above. It will furthermore be apparent that the peptidomimetics contemplated by the disclosure can be further used for the development of even more potent non-peptidic compounds, in addition to their utility as therapeutic compounds.
  • peptides with reduced isostere pseudopeptide bonds are contemplated. Substitution of peptide bonds by pseudopeptide bonds confers resistance to proteolysis. A number of pseudopeptide bonds have been described that in general do not affect peptide structure and biological activity.
  • the reduced isostere pseudopeptide bond is a suitable pseudopeptide bond that is known to enhance stability to enzymatic cleavage with no or little loss of biological activity (Couder, et al. (1993), Int. J. Peptide Protein Res., 41 : 181- 184).
  • amino acid sequences of these peptides may be identical to the sequences of the isolated peptides described herein except that one or more of the peptide bonds are replaced by an isostere pseudopeptide bond.
  • the most N-terminal peptide bond is substituted, since such a substitution would confer resistance to proteolysis by exopeptidases acting on the N-terminus.
  • the synthesis of peptides with one or more reduced isostere pseudopeptide bonds is known in the art (Couder, et al. (1993), ibid).
  • peptides with a retro-inverso pseudopeptide bond are contemplated.
  • Peptide bonds may also be substituted by retro-inverso pseudopeptide bonds to confer resistance to proteolysis (Dalpozzo, et al. (1993), Int. J. Peptide Protein Res., 41 :561- 566, incorporated herein by reference).
  • the amino acid sequences of the peptides may be identical to the sequences of the isolated peptides described herein except that one or more of the peptide bonds are replaced by a retro-inverso pseudopeptide bond.
  • N-terminal peptide bond is substituted, since such a substitution will confer resistance to proteolysis by exopeptidases acting on the N-terminus.
  • the synthesis of peptides with one or more reduced retro-inverso pseudopeptide bonds is known in the art (Dalpozzo, et al. (1993), ibid).
  • Peptoid derivatives of peptides represent another form of modified peptides that retain the important structural determinants for biological activity, yet eliminate the peptide bonds, thereby conferring resistance to proteolysis (Simon, et al., 1992, Proc. Natl. Acad. Sci. USA, 89:9367-9371).
  • Peptoids are oligomers of N-substituted glycines. A number of N-alkyl groups have been described, each corresponding to the side chain of a natural amino acid (Simon, et al. (1992), ibid).
  • all or a portion of the amino acids may be replaced with the corresponding N-substituted glycine.
  • the N-terminal residue may be the only one that is replaced, or a few amino acids may be replaced by the corresponding N-substituted glycines.
  • mimetopes of a subject BRD4 polypeptide can be provided.
  • Such peptidomimetics can have such attributes as being non- hydrolyzable (e.g., increased stability against proteases or other physiological conditions which degrade the corresponding peptide), increased specificity and/or potency for inhibition of PV replication, and increased cell permeability for intracellular localization of the peptidomimetic.
  • peptide analogs of the contemplated by the disclosure can be generated using, for example, benzodiazepines (e.g., see Freidinger et al. in Peptides: Chemistry and Biology, G. R.
  • peptidomimetics based on more substantial modifications of the backbone of the subject BRD4 peptides can be used.
  • Peptidomimetics which fall in this category include (i) retro-inverso analogs, and (ii) N-alkyl glycine analogs (so-called peptoids).
  • the peptidomimetic can be derived as a retro-inverso analog of the peptide.
  • retro-inverso analogs can be made according to the methods known in the art, such as that described by the Sisto et al. U.S. Pat. No. 4,522,752.
  • the illustrated retro-inverso analog can be generated as follows.
  • the geminal diamine corresponding to the N- terminal tryptophan is synthesized by treating a protected tryptophan analog with ammonia under HOBT-DCC coupling conditions to yield the N-Boc amide, and then effecting a Hofmann-type rearrangement with I,I-bis-(trifluoroacetoxy)iodobenzene (TIB), as described in Radhakrishna et al. (1979) J. Org. Chem.
  • TIB I,I-bis-(trifluoroacetoxy)iodobenzene
  • the product amine salt is then coupled to a side-chain protected (e.g., as the benzyl ester) N-Fmoc D-lys residue under standard conditions to yield the pseudodipeptide.
  • the Fmoc (fluorenylmethoxycarbonyl) group is removed with piperidine in dimethylformamide, and the resulting amine is trimethylsilylated with bistrimethylsilylacetamide (BSA) before condensation with suitably alkylated, side-chain protected derivative of Meldrum's acid, as described in U.S. Pat. No. 5,061,811 to Pinori et al., to yield the retro-inverso tripeptide analog WKH.
  • BSA bistrimethylsilylacetamide
  • the pseudotripeptide is then coupled with with an L-methionine analog under standard conditions to give the protected tetrapeptide analog.
  • the protecting groups are removed to release the product, and the steps repeated to enlogate the tetrapeptide to the full length peptidomimetic. It will be understood that a mixed peptide, e.g. including some normal peptide linkages, will be generated.
  • sites which are most susceptible to proteolysis are typically altered, with less susceptible amide linkages being optional for mimetic switching.
  • the final product, or intermediates thereof, can be purified by HPLC.
  • the peptidomimetic can be derived as a retro- enantio analog of a BRD4 peptide.
  • Retro-enantio analogs such as this can be synthesized from commercially available D-amino acids (or analogs thereof) and standard solid- or solution- phase peptide-synthesis techniques.
  • a suitably amino-protected (t-butyloxycarbonyl, Boc) D-trp residue (or analog thereof) is covalently bound to a solid support such as chloromethyl resin.
  • the resin is washed with dichloromethane (DCM), and the BOC protecting group removed by treatment with TFA in DCM.
  • DCM dichloromethane
  • the resin is washed and neutralized, and the next Boc-protected D-amino acid (D-lys) is introduced by coupling with diisopropylcarbodiimide.
  • the resin is again washed, and the cycle repeated for each of the remaining amino acids in turn (D-his, D-met, etc).
  • the protecting groups are removed and the peptide cleaved from the solid support by treatment with hydrofluoric acid/anisole/dimethyl sulfide/thioanisole.
  • the final product is purified by HPLC to yield the pure retro-enantio analog.
  • trans-olefin derivatives can be made for a subject BRD4 polypeptide.
  • the trans-olefin analog of a Brd4 peptide can be synthesized according to the method of Y. K. Shue et al. (1987) Tetrahedron Letters 28:3225. Referring to the illustrated example, Boc-amino L-IIe is converted to the corresponding ⁇ -amino aldehyde, which is treated with a vinylcuprate to yield a diastereomeric mixture of alcohols, which are carried on together.
  • the allylic alcohol is acetylated with acetic anhydride in pyridine, and the olefin is cleaved with osmium tetroxide/sodium periodate to yield the aldehyde, which is condensed with the Wittig reagent derived from a protected tyrosine precursor, to yield the allylic acetate.
  • the allylic acetate is selectively hydrolyzed with sodium carbonate in methanol, and the allylic alcohol is treated with triphenylphosphine and carbon tetrabromide to yield the allylic bromide.
  • This compound is reduced with zinc in acetic acid to give the transposed trans olefin as a mixture of diastereomers at the newly- formed center.
  • the diastereomers are separated and the pseudodipeptide is obtained by selective transfer hydrogenolysis to unveil the free carboxylic acid.
  • the pseudodipeptide is then coupled at the C-terminus, according to the above example, with a suitably protected tyrosine residue, and at the N-terminus with a protected alanine residue, by Standard techniques, to yield the protected tetrapeptide isostere.
  • the terapeptide is then further condensed with the olefinic tripeptide analog derived by similar means to build up the full peptide.
  • the protecting groups are then removed with strong acid to yield the desired peptide analog, which can be further purified by HPLC.
  • pseudodipeptides synthesized by the above method to other pseudodipeptides, to make peptide analogs with several olefinic functionalities in place of amide functionalities.
  • pseudodipeptides corresponding to Met-Arg or Tyr-Lys, etc. could be made and then coupled together by standard techniques to yield an analog of a subject BRD4 polypeptide which has alternating olefinic bonds between residues.
  • Still another class of peptidomimetic derivatives includes the phosphonate derivatives.
  • the BRD4 peptidomimetic may incorporate the l-azabicyclo[4.3.0]nonane surrogate (see Kim et al. (1997) J. Org. Chem. 62:2847), or an N-acyl piperazic acid (see Xi et al. (1998) J. Am. Chem. Soc. 120:80), or a 2-substituted piperazine moiety as a constrained amino acid analogue (see Williams et al. (1996) J. Med. Chem. 39:1345-1348).
  • certain amino acid residues can be replaced with aryl and bi-aryl moieties, e.g., monocyclic or bicyclic aromatic or heteroaromatic nucleus, or a biaromatic, aromatic-heteroaromatic, or biheteroaromatic nucleus.
  • the contemplated peptidomimetics can be optimized by, e.g., combinatorial synthesis techniques combined with such high throughput screening as described herein.
  • mimetopes include, but are not limited to, protein-based compounds, carbohydrate-based compounds, lipid-based compounds, nucleic acid-based compounds, natural organic compounds, synthetically derived organic compounds, anti-idiotypic antibodies and/or catalytic antibodies, or fragments thereof.
  • a mimetope can be obtained by, for example, screening libraries of natural and synthetic compounds for compounds capable of inhibiting an interaction between a BRD4 polypeptide and a P-TEFb kinase protein or a functional equivalent thereof.
  • a mimetope can also be obtained, for example, from libraries of natural and synthetic compounds, in particular, chemical or combinatorial libraries (i.e., libraries of compounds that differ in sequence or size but that have the same building blocks).
  • a mimetope can also be obtained by, for example, rational drug design.
  • the three-dimensional structure of a compound contemplated by the present disclosure can be analyzed by, for example, nuclear magnetic resonance (NMR) or x-ray crystallography.
  • the three-dimensional structure can then be used to predict structures of potential mimetopes by, for example, computer modeling.
  • the predicted mimetope structures can then be produced by, for example, chemical synthesis, recombinant DNA technology, or by isolating a mimetope from a natural source (e.g., plants, animals, bacteria and fungi).
  • a natural source e.g., plants, animals, bacteria and fungi.
  • a subject BRD4 polypeptide is a recombinant BRD4 polypeptide.
  • the present disclosure provides recombinant BRD4 polypeptides capable of inhibiting an interaction between a P-TEFb heterodimer, or subunit thereof, and an endogenous intracellular protein.
  • the subject recombinant polypeptides are capable of binding to a P-TEFb heterodimer, or subunit thereof, and thereby inhibiting P- TEFb mediated phosphorylation of the C-terminal domain of the large subunit (RPBl) of RNA polymerase II (RNAPII).
  • the present disclosure provides recombinant polypeptides capable of inhibiting an interaction between estrogen receptor alpha (ERa) and a cyclin Tl subunit of P-TEFb.
  • a subject recombinant BRD4 polypeptide comprises an inhibitor domain, wherein the inhibitor domain inhibits a binding interaction between a viral transcription enhancer and a P-TEFb polypeptide, and a protein transduction domain that provides for entry of the recombinant polypeptide into a mammalian cell.
  • the recombinant polypeptide comprises an inhibitor domain, wherein the inhibitor domain has a length of about 20 amino acids to about 150 amino acids, e.g., from about 20 aa to about 25 aa, from about 25 aa to about 50 aa, from about 50 aa to about 75 aa, from about 75 aa to about 100 aa, from about 100 aa to about 125 aa, or from about 125 aa to about 150 aa.
  • a subject recombinant BRD4 polypeptide comprises, in order from amino terminus to carboxyl terminus: a) an inhibitor domain; and b) a protein transduction domain. In other embodiments, a subject recombinant BRD4 polypeptide comprises, in order from amino terminus to carboxyl terminus: a) a protein transduction domain; and b) an inhibitor domain.
  • a subject recombinant BRD4 polypeptide comprises an inhibitor domain, wherein the inhibitor domain inhibits a binding interaction between a viral transcription enhancer and a P-TEFb polypeptide, and a protein transduction domain that provides for entry of the recombinant polypeptide into a mammalian cell, wherein the inhibitor domain comprises an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or 100% amino acid sequence identity to amino acids 1329-1362, 1318-1362, 1315-1362, 1228-1362, 1209-1362, or 722-1362 of SEQ ID NO: 1.
  • the inhibitor domain comprises an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to amino acids 1329-1362, 1318-1362, 1315-1362, 1228-1362, 1209-1362, or 722-1362 of SEQ ID NO: 1, or at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in one of SEQ ID NOs: 4-9; and the inhibitor domain has a length of from about 30 amino acids to about 1000 amino acids, e.g.,
  • the inhibitor domain has a length of 34 amino acids and comprises an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or 100% amino acid amino acid sequence identity to amino acids 1329-1362 of SEQ ID NO: 1.
  • the inhibitor domain has a length of 45 amino acids and comprises an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or 100% amino acid amino acid sequence identity to amino acids 1318-1362 of SEQ ID NO: 1.
  • the inhibitor domain has a length of 135 amino acids and comprises an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or 100% amino acid amino acid sequence identity to amino acids 1228-1362 of SEQ ID NO: 1.
  • the inhibitor domain has a length of has a length of 154 amino acids and comprises an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or 100% amino acid amino acid sequence identity to amino acids 1209-1362 of SEQ ID NO: 1.
  • the inhibitor domain has a length of 641 amino acids and comprises an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or 100% amino acid amino acid sequence identity to amino acids 722-1362 of SEQ ID NO: 1.
  • a subject recombinant BRD4 polypeptide comprises an inhibitor domain that inhibits a binding interaction of a viral transcription enhancer with a P- TEFb polypeptide, and a protein transduction domain that provides for entry of the recombinant polypeptide into a mammalian cell, wherein the inhibitor domain comprises an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or 100% amino acid sequence identity to one of the amino acid sequences shown in SEQ ID NOs: 4-9.
  • the inhibitor domain has a length of from about 45 amino acids to about 50 amino acids. In certain embodiments, where the inhibitor domain has an amino acid sequence as set forth in this paragraph, the inhibitor domain also has an amino acid sequence length equal to that set forth in one of SEQ ID NOs: 4-9.
  • a subject recombinant BRD4 polypeptide specifically lacks an N-terminal portion of the BRD4 amino acid sequence provided in SEQ ID NO: 1.
  • a subject recombinant polypeptide lacks amino acids 1-721, 1-1208, 1-1227, 1- 1314, 1-1317 or 1-1328 of the amino acid sequence provided in SEQ ID NO: 1.
  • a subject recombinant polypeptide comprises an inhibitor domain that inhibits a binding interaction of a viral transcription enhancer with a P-TEFb kinase and a protein transduction domain that provides for entry of the recombinant polypeptide into a mammalian cell, wherein the protein transduction domain (PTD) has a length of from about 10 amino acids to about 50 amino acids, e.g., the PTD has a length of from about 10 aa to about 15 aa, from about 15 aa to about 20 aa, from about 20 aa to about 25 aa, from about 25 aa to about 30 aa, from about 30 aa to about 35 aa, from about 35 aa to about 40 aa, from about 40 aa to about 45 aa, or from about 45 aa to about 50 aa.
  • PTD protein transduction domain
  • a subject recombinant BRD4 polypeptide comprises an inhibitor domain polypeptide that inhibits a binding interaction of a viral transcription enhancer with a P-TEFb kinase and a second peptide that provides for entry of the recombinant polypeptide into a mammalian cell.
  • the polypeptide may be fused to a second peptide which promotes "transcytosis," e.g., uptake of the peptide by cells.
  • the peptide may be a portion of the HIV transactivator (Tat) protein, such as the fragment corresponding to residues 37-62 or 48-60 of TAT, portions which have been observed to be rapidly taken up by a cell in vitro (Green and Loewenstein, (1989) Cell 55:1179-1188).
  • the internalizing peptide may be derived from the Drosophila antennapedia protein, or homologs thereof.
  • the 60 amino acid long homeodomain of the homeo-protein antennapedia has been demonstrated to translocate through biological membranes and can facilitate the translocation of heterologous polypeptides to which it is coupled.
  • polypeptides may be fused to a peptide consisting of about amino acids 42-58 of Drosophila antennapedia or shorter fragments for transcytosis (Derossi et al. (1996) J Biol Chem 271 :18188-18193; Derossi et al. (1994) J. Biol Chem 269:10444-10450; and Perez et al. (1992) J. Cell Sd. 102:717-722).
  • the transcytosis polypeptide may also be a non-naturally-occurring membrane-translocating sequence (MTS), such as the peptide sequences disclosed in U.S. Pat. No. 6,248,558.
  • MTS membrane-translocating sequence
  • a subject recombinant BRD4 polypeptide comprises an inhibitor domain that inhibits a binding interaction of a viral transcription enhancer with a P- TEFb kinase and a protein transduction domain that provides for entry of the recombinant polypeptide into a mammalian cell, wherein the protein transduction domain comprises an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or 100% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 2. (YGRKKRRQRRR).
  • a subject recombinant BRD4 polypeptide comprises the amino acid sequence
  • YGRKKRRORRROSMLDOORELARKREOERRRREAMAATIDMNFOSDLLSIFEENLF (SEQ ID NO:38), where the protein transduction domain is underlined, and where the inhibitor domain corresponds to amino acids 1318-1362 of SEQ ID NO:1.
  • a subject recombinant BRD4 polypeptide comprises an inhibitor domain that inhibits a binding interaction of a viral transcription enhancer with a P- TEFb kinase and a protein transduction domain that provides for entry of the recombinant polypeptide into a mammalian cell, wherein the viral transcription enhancer is an immunodeficiency virus Tat protein.
  • a subject recombinant BRD4 polypeptide can be detectably labeled.
  • Various labels include radioisotopes, fluorescers (e.g., fluorescent dyes), chemiluminescers, enzymes, a member of a specific binding pair, particles, e.g. magnetic particles, and the like.
  • Specific binding pairs include, but are not limited to, biotin and streptavidin; digoxin and antidigoxin; lectin and carbohydrate moieties; antibody and hapten; antibody and antigen; etc.
  • the present invention provides a nucleic acid comprising a nucleotide sequence that encodes a subject BRD4 polypeptide, where the nucleotide sequence does not encode the full length protein having the amino acid sequence set forth in SEQ ID NO: 1.
  • a subject nucleic acid can be isolated, recombinant, or synthetic.
  • a subject nucleic acid comprises a nucleotide sequence that encodes a subject BRD4 polypeptide, where the nucleotide sequence is operably linked to a transcriptional regulatory sequence.
  • the nucleic acids described herein can be included in a recombinant vector, such as, for example, an expression vector.
  • the present invention also provides recombinant vectors, including expression vectors, comprising a subject nucleic acid.
  • the present invention further provides genetically modified host cells, where the host cells are genetically modified with a subject nucleic acid.
  • a subject isolated nucleic acid comprises a nucleotide sequence which encodes amino acids 1329-1362, 1318-1362, 1315-1362, 1228-1362, 1209-1362, or 722-1362 of SEQ ID NO: 1.
  • a subject isolated nucleic acid comprises a nucleotide sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100%, nucleotide sequence identity with that portion of SEQ ID NO: 3 which encodes amino acids 1329-1362, 1318-1362, 1315-1362, 1228-1362, 1209-1362, or 722-1362 of SEQ ID NO: 1.
  • a subject isolated nucleic acid comprises a nucleotide sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100%, nucleotide sequence identity with that portion of SEQ ID NO: 3 which encodes amino acids 1329-1362 of SEQ ID NO: 1.
  • a subject isolated nucleic acid comprises a nucleotide sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100%, nucleotide sequence identity with nucleotides 3952- 4086 of SEQ ID NO: 3 which encodes amino acids 1318-1362 of SEQ ID NO:1.
  • a subject isolated nucleic acid comprises a nucleotide sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100%, nucleotide sequence identity with nucleotides 3943-4086 of SEQ ID NO: 3 which encodes amino acids 1315-1362 of SEQ ID NO:1.
  • a subject isolated nucleic acid comprises a nucleotide sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100%, nucleotide sequence identity with nucleotides3674-4086 of SEQ ID NO: 3 which encodes amino acids 1228-1362 of SEQ ID NO:1.
  • a subject isolated nucleic acid comprises a nucleotide sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100%, nucleotide sequence identity with nucleotides 3627-4086 of SEQ ID NO: 3 which encodes amino acids 1209-1362 of SEQ ID NO:1.
  • a subject isolated nucleic acid comprises a nucleotide sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100%, nucleotide sequence identity with nucleotides 2164-4086 of SEQ ID NO: 3 which encodes amino acids 722-1362 of SEQ ID NO: 1.
  • a subject isolated nucleic acid comprises a nucleic acid sequence which encodes an amino sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100%, amino acid sequence identity to an amino acid sequence set forth in one of SEQ ID NOs: 4-9.
  • the present invention provides recombinant vectors, including expression vectors, comprising a subject nucleic acid.
  • Suitable expression vectors include, but are not limited to, viral vectors (e.g. viral vectors based on vaccinia virus; poliovirus; adenovirus (see, e.g., Li et al., Invest Opthalmol Vis Sci 35:2543 2549, 1994; Borras et al., Gene Ther 6:515 524, 1999; Li and Davidson, PNAS 92:7700 7704, 1995; Sakamoto et al., H Gene Ther 5:1088 1097, 1999; WO 94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO 95/00655); adeno- associated virus (see, e.g., AIi et al., Hum Gene Ther 9:81 86, 1998, Flannery et al., PNAS 94:6916 6921, 1997; Bennett et al.,
  • viral vectors
  • SV40 herpes simplex virus
  • a lentivirus a human immunodeficiency virus
  • a retroviral vector e.g., Murine Leukemia Virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus
  • retroviral vector e.g., Murine Leukemia Virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus
  • Suitable expression vectors are known to those of skill in the art, and many are commercially available.
  • the following vectors are provided by way of example; for bacterial host cells: pQE vectors (Qiagen), pBluescript plasmids, pNH vectors, lambda-ZAP vectors (Stratagene); pTrc99a, pKK223-3, pDR540, and pRIT2T (Pharmacia); for eukaryotic host cells: pXTl, pSG5 (Stratagene), pSVK3, pBPV, pMSG, and pSVLSV40 (Pharmacia).
  • any other plasmid or other vector may be used so long as it is compatible with the host cell.
  • Suitable eukaryotic vectors include, for example, bovine papilloma virus-based vectors,
  • Epstein-Barr virus-based vectors Epstein-Barr virus-based vectors, vaccinia virus-based vectors, SV40, 2-micron circle, pcDNA3.1, pcDNA3.1/GS, pYES2/GS, pMT, p IND, pIND(Spl), pVgRXR (Invitrogen), and the like, or their derivatives.
  • Such vectors are well known in the art (Botstein et al., Miami Wntr. SyTnp. 19:265-274, 1982; Broach, In: "The Molecular Biology of the Yeast Saccharomyces: Life Cycle and Inheritance", Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y., p.
  • the recombinant vector can include one or more coding regions that encode a polypeptide (a "selectable marker") that allow for selection of the recombinant vector in a genetically modified host cell comprising the recombinant vector.
  • selectable markers include those providing antibiotic resistance; e.g., blasticidin resistance, neomycin resistance.
  • selectable marker genes that are useful include the hygromycin B resistance gene (encoding Aminoglycoside phosphotranferase (APH)) that allows selection in mammalian cells by conferring resistance to hygromycin; the neomycin phosphotranferase gene (encoding neomycin phosphotransferase) that allows selection in mammalian cells by conferring resistance to G418; and the like.
  • APH Aminoglycoside phosphotranferase
  • a BRD4 polypeptide-encoding nucleotide sequence can be operably linked to one or more transcriptional control elements, e.g., a promoter.
  • suitable eukaryotic promoters include cytomegalovirus (CMV) immediate early, herpes simplex virus (HSV) thymidine kinase, early and late SV40, long terminal repeats (LTRs) from retrovirus, and mouse metallothionein-I.
  • the promoter is a constitutive promoter.
  • constitutive promoters include: ubiquitin promoter, CMV promoter, JeT promoter (U.S. Pat.
  • the promoter is an inducible promoter.
  • inducible/repressible promoters include: Tet-On, Tet-Off, Rapamycin-inducible promoter, and MxI. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art.
  • Suitable promoters for use in prokaryotic host cells include, but are not limited to, a bacteriophage T7 RNA polymerase promoter; a trp promoter; a lac operon promoter; a hybrid promoter, e.g., a lac/tac hybrid promoter, a tac/trc hybrid promoter, a trp/lac promoter, a T7/lac promoter; a trc promoter; a tac promoter, and the like; an araBAD promoter; in vivo regulated promoters, such as an ⁇ aG promoter or a related promoter (see, e.g., U.S. Patent Publication No.
  • sigma70 promoter e.g., a consensus sigma70 promoter (see, e.g., GenBank Accession Nos. AX798980, AX798961, and AX798183); a stationary phase promoter, e.g., a dps promoter, an spv promoter, and the like; a promoter derived from the pathogenicity island SPI-2 (see, e.g., WO96/17951); an actA promoter (see, e.g., Shetron- Rama et al. (2002) Infect. Immun.
  • rpsM promoter see, e.g., Valdivia and Falkow (1996). MoI. Microbiol. 22:367-378
  • a tet promoter see, e.g., Hillen,W. and Wissmann,A. (1989) In Saenger,W. and Heinemann,U. (eds), Topics in Molecular and Structural Biology, Protein-Nucleic Acid Interaction. Macmillan, London, UK, Vol. 10, pp. 143-162
  • SP6 promoter see, e.g., Melton et al. (1984) Nucl. Acids Res. 12:7035-7056; and the like.
  • the present invention further provides genetically modified host cells, where a subject genetically modified host cell is genetically modified with a subject nucleic acid (e.g., a subject recombinant expression vector).
  • a subject nucleic acid e.g., a subject recombinant expression vector
  • Host cells are in many embodiments unicellular organisms, or are grown in culture as single cells.
  • the host cell is a eukaryotic cell.
  • Suitable eukaryotic host cells include, but are not limited to, yeast cells, insect cells, plant cells, fungal cells, and algal cells.
  • Suitable eukaryotic host cells include, but are not limited to, Pichiapastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichia sp., Saccharomyces cerevisiae, Saccharomyces sp., Hansenula polymorpha, Kluyveromyces sp., Kluyveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknowense, Fusarium sp., Fusarium gramineum
  • the host cell is a prokaryotic cell.
  • Suitable prokaryotic cells include, but are not limited to, any of a variety of laboratory strains of Escherichia coli, Lactobacillus sp., Salmonella sp., Shigella sp., and the like. See, e.g., Carrier et al. (1992) J. Immunol. 148:1176-1181 ; U.S. Patent No. 6,447,784; and Sizemore et al. (1995) Science 270:299-302.
  • suitable Salmonella strains include, but are not limited to, Salmonella typhi and S. typhimurium.
  • Suitable Shigella strains include, but are not limited to, Shigella ⁇ exneri, Shigella sonnei, and Shigella disenteriae.
  • the laboratory strain is one that is non-pathogenic.
  • suitable bacteria include, but are not limited to, Bacillus subtilis, Pseudomonas pudita, Pseudomonas aeruginosa, Pseudomonas mevalonii, Rhodobacter sphaeroides, Rhodobacter capsulatus, Rhodospirillum rubrum, Rhodococcus sp., and the like.
  • the host cell is Escherichia coli. COMPLEXES
  • the present invention provides an in vitro complex, where the complex comprises an a subject BRD4 polypeptide and a P-TEFb heterodimer, or subunit thereof sufficient for binding to the BRD4 polypeptide.
  • Standard abbreviations may be used, e.g., bp, base pair(s); kb, kilobase(s); pi, picoliter(s); s or sec, second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); kb, kilobase(s); bp, base pair(s); nt, nucleotide(s); i.m., intramuscular(ly); i.p., intraperitoneal(ly); s.c, subcutaneous(ly); and the like.
  • EXPERIMENTAL PROCEDURES e.g., bp, base pair(s); kb, kilobase(s); pi, picoliter(s); s or sec, second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); kb, kilobase(s); bp, base pair(s);
  • Open reading frames encoding the short (aa 1-722) and long (aa 1-1362) isoforms of human BRD4 were isolated by RT-PCR from leukocyte cDNA (Clontech, Palo Alto, CA) and Jurkat cDNA, respectively and inserted into pFLAG-CMV2 (Sigma- Aldrich, St Louis, MO). Deletion mutants were cloned by PCR. Site-directed mutagenesis was performed with the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). Drosophila female sterile homeotic (fs(l)h) and murine BRDT were cloned by RT-PCR from whole Drosophila cDNA and mouse testes cDNA, respectively.
  • Lysates were dialysed into dialysis buffer (20 mM HEPES, 20% glycerol, 150 mM NaCl, 0.2 mM EDTA, 0.5 mM DTT and 1 mM PMSF).
  • FLAG-tagged proteins were immunoprecipitated with anti-FLAG-agarose beads (Sigma-Aldrich, St. Louis, MO).
  • Co- immunoprecipitating proteins were detected by western blotting with rabbit anti-FLAG (Sigma-Aldrich, St. Louis, MO), anti-Cyclin Tl or anti-CDK9 (Santa Cruz Biotechnology, Santa Cruz, CA).
  • GST Pulldowns GST, GST-BRD4 1209-13 62, and GST-BRD4 1209- i 328 were over- expressed in BL-21 and purified on GST-Sepharose beads as recommended by the manufacturer (GE Healthcare).
  • Human cyclin Tl- and CDK9-encoding RNAs were prepared with the T7 Ribomax Express in vitro transcription kit (Promega, Madison, WI). Radiolabeled cyclin Tl and CDK9 were prepared by in vitro translation with 35 S-methionine and RNase- treated rabbit reticulocyte lysates.
  • GST pulldowns were performed by incubating washed beads coated with GST, GST-BRD4i 2 o 9-1362 or GST-BRD4 1209- i 328 with radiolabeled cyclin Tl or CDK9. After extensive washing, purified proteins were detected by Coomassie blue staining and fluorography (Amplify, GE Healthcare).
  • Luciferase Assays For the GAL4 transactivation experiments, HeLa cells were co- transfected with p5xGAL4-UAS-luciferase reporter plasmid and increasing amounts of GAL4- BRD4 1209-1362, 1209-1362FEE, or 1209-1328. Cell lysates and luciferase activity were prepared and analyzed using the dual-luciferase reporter assay system (Promega). Inhibition of HIV transcription was measured by transfecting HeLa cells with 25 ng HIV LTR-luciferase reporter, 5 ng pRSV-Tat, and 0-400 ng pFLAG-CMV2-BRD4 expression plasmids (Fugene 6, Roche). The total amount of DNA was kept constant with empty pFLAG-CMV2.
  • Latent HIV clone A2 contains a single latent HIV provirus that expresses Tat and GFP upon reactivation (10).
  • A2 cells were preincubated for 2 h with 2-10 ⁇ M Tat PTD-BRD ⁇ 209- I 362 , Tat PTD alone or DRB. TNF ⁇ was then added to the cells at 10 ng/ml, and reactivation of the latent HIV provirus detected 18 h later by FACS analysis (FACSCalibur, BD Biosciences).
  • BRD4-associated P-TEFb was detected by immunoblotting with antisera specific for cyclin Tl or CDK9.
  • P-TEFb was not detected in immunoprecipitates of the N- terminal half of BRD4 (amino acid residues 1-722), suggesting that the association occurs in the C-terminal half of BRD4 (Fig. IA).
  • Deletion of the bromodomains in the full length BRD4 had little effect on the interaction of P-TEFb and BRD4, suggesting that the association is not mediated by these domains.
  • the ET domain is a poorly defined domain thought to mediate protein-protein interactions (Florence et al, 2001 Front Biosci 6, Dl 008-1018).
  • the C- terminal half of BRD4 (amino acid residues 722-1362) was sufficient to result in immunoprecipitation of P-TEFb.
  • This region of BRD4 is missing in the short isoform and contains no known functional motifs (Fig. IA).
  • FIG. 1 shows a multi-alignment of the C-terminal domain of human, mouse, and Xenopus BRD4; human and mouse BRDT; and Drosophila melanogaster fs(l)h.
  • Xenopus BRD4 is 90% conserved
  • human and mouse BRDT are both 75% conserved
  • Drosophila fs(l)h is 69% conserved.
  • FLAG-tagged expression constructs for human BRD4, Drosophila fs(l)h and murine BRDT as well as C-terminal deletion mutants of each were introduced into 293T cells by transient transfection. Nuclear (BRD4 and fs(l)h) or whole- cell (BRDT) extracts were prepared and ⁇ -FLAG immunoprecipitations carried out.
  • Fig. IA and Fig. IB FLAG-tagged deletion mutants of BRD4 were overexpressed in 293T cells. Nuclear extracts (NE) and anti-FLAG immunoprecipitates were immunoblotted for CDK9 and cyclin Tl. Fig.
  • FLAG-tagged BRD4 constructs encoding amino acid residues 1209-1362, 1209-1362 ⁇ 1279-1304, 1209-1362 ⁇ 1306-1330, 1209- 1362 ⁇ 1329-1362, 1209-1362 ⁇ 1329-1345, and 1209-1362 ⁇ 1345-1362 were overexpressed in 293T cells.
  • Anti-FLAG immunoprecipitates were immunoblotted with anti-CDK9 or anti- cyclin Tl .
  • Figs. 2 A - E. Fig. 2 A Alignment of the C-termini of human, murine and Xenopus
  • BRD4 human and murine BRDT and Drosophila female sterile homeotic (fs(l)h). Identical residues are indicated in black, conserved residues in grey. The location of ⁇ -helices predicted by computer analysis are indicated below.
  • Fig. 2B Vectors encoding the C-termini of human BRD4 (amino acid residues 1209-1362), BRD4 ⁇ (1209-1344), fs(l)h (1885-2038), or fs(l)h ⁇ (1885-2020) were transfected into 293T cells. Anti-FLAG immunoprecipitates were immunoblotted with anti-FLAG, anti-CycTl and anti-CDK9. Fig.
  • FIG. 2C Expression constructs encoding the C-terminus of BRDT (amino acid residues 821-956) or a C-terminal deletion mutant (BRDT ⁇ 821-938) were transfected into 293T cells. Whole cell lysates were prepared and BRDT was immunoprecipitated with anti-FLAG beads.
  • Fig. 2D C-terminal residues conserved between BRD4, BRDT, and fs(l)h were mutated in FLAG-BRD4 (amino acid residues 1209-1362) (indicated by an asterisk in panel A.).
  • Fig. 2E Plasmids encoding FLAG tagged wild-type BRD4 (1-1362) or mutant (1-1362FEE) were each transfected into 293T cells. Anti-FLAG immunoprecipitates were blotted for anti-FLAG and anti-CDK9.
  • GST pull-down experiments were performed to determine which subunit of P-TEFb mediates the interaction with BRD4.
  • GST fusion proteins representing either GST alone, GST- BRD4 120 9 -136 2 or the deletion mutant GST-BRD4i 2 o9-i328 were overexpressed and purified from bacterial lysates. Washed beads coated with each GST fusion protein were incubated with either in vitro translated 35 S-radiolabeled human cyclin Tl or CDK9. After extensive washing, interacting proteins were eluted, visualized by Coomassie blue staining after SDS-PAGE and processed for autoradiography. Both in vitro translated cyclin Tl and CDK9 interacted with GST-BRD4i 209 -i 362 but not with GST-BRD4i 2 o 9- i328 (Fig. 3), suggesting that each subunit of P-
  • FIG. 3 GST pulldowns were performed by incubating - GST, GST-BRD4 (amino acid residues 1209-1632) or GST-BRD4 (amino acid residues 1209-1328) coated agarose beads with 35 S-radiolabelled human cyclin Tl or human CDK9. After extensive washing, proteins were eluted with Laemmli buffer, separated by SDS-PAGE, and visualized by Coomassie blue staining and autoradiography.
  • Fig. 4 Increasing amounts of a plasmid encoding the GAL4 DNA binding domain
  • BRD4 amino acid residues 1209-1362
  • BRD4 (1209-1328) or BRD4 amino acid residues 1209- 1362)(FEE 1357 AAA) were co-transfected with p5xGAL4xUAS-Luc into HeLa cells. After 36 h, lysates were prepared, and luciferase levels determined.
  • the viral transactivator Tat plays a critical role in HIV transcription via its ability to recruit the P-TEFb complex to the HIV promoter. Recruitment of P-TEFb by Tat markedly enhances the processivity of the HIV promoter-associated RNA polymerase II complex.
  • the effect of BRD4 expression on HFV transcription was determined by measuring the effect of BRD4 on an HIV transcriptional reporter in the presence of Tat. Overexpression of full-length BRD4 inhibited Tat-mediated HIV transcription, suggesting that it competes with Tat for P- TEFb (Fig. 5B). Overexpression of either the C-terminal 153 amino acids (1209-1362) or 102 amino acids (1260-1362) of BRD4 interfered with HIV transcription to an even greater extent than the full-length isoform.
  • Fig. 5 A and Fig. 5B Diagram showing the constructs used in this experiment: full-length BRD4, BRD4 (amino acid residues 1209-1362) and BRD4 (amino acid residues 1260-1362).
  • Fig. 5B HeLa cells were transfected with 25 ng HIV LTR-luciferase reporter and 5 ng Tat expression plasmid and increasing amount of a BRD4 expression plasmid (0—400 ng).
  • Example 5 BRD4 Suppresses the Tat/P-TEFb interaction.
  • cyclin Tl is recruited to the HIV LTR via Tat and the TAR element found at the 5' end of all viral transcripts. This association brings CDK9 to the LTR and promotes transcriptional elongation by enhancing phosphorylation of the C-terminal domain of RNA polymerase II.
  • in vitro competition experiments were performed to assess the effect of BRD4 on the Tat-P-TEFb interaction (Fig. 6).
  • Binding reactions consisting of a constant amount of biotinylated synthetic Tat and recombinant P-TEFb, as well as varying amounts of in vitro translated BRD4i 20 9.i 362 or BRIM 120 Q -1328 , were prepared.
  • the ability of Tat to bind to P-TEFb (CDK9) in the presence of BRD4 peptides was measured by western blotting ( ⁇ -CDK9) after binding of Tat to streptavidin-agarose and centrifugation.
  • Increasing amounts of BRD4i 209 .i 362 but not BRD4i 2 o 9 -i 328 decreased the amount of P-TEFb (CDK9) that interacted with Tat (Fig. 6). This result indicates that BRD4 expression interferes with Tat binding to P-TEFb and suggests that BRD4 could inhibit the Tat-mediated recruitment of P-TEFb to the viral LTR.
  • BRD4 peptides corresponding to either amino acid residues 1209-1362 or 1209-1328 obtained after in vitro transcription/translation.
  • Biotinylated Tat and associated factors were pulled down by streptavidin-agarose followed by western blotting using an antiserum specific for CDK9 or streptavidin-horse radish peroxidase (HRP) (to verify equal pulldown of Tat).
  • the BRD4 peptides are visualized by autoradiography after SDS-PAGE.
  • BRD4 was synthesized (Fig. 7A).
  • the PTD confers receptor- and energy-independent entry of fused proteins into cells.
  • Increasing amounts of either the PTD peptide alone, the PTD-BRD4 peptide or the chemical CDK9 inhibitor, 5,6-dichloro-l- ⁇ -D-ribofuranosylbenzimidazole (DRB) were added to HeLa cells transfected with an HIV LTR-luciferase reporter and a Tat expression plasmid. HIV transcription was inhibited six-fold by increasing amounts of PTD- BRD4 peptide but not the PTD peptide alone. Similar results were obtained with the DRB control.
  • Fig. 7B HeLa cells were transfected with 25 ng HIV LTR-luciferase with (+) or without (-) 5 ng Tat expression plasmid. The PTD alone (pale grey), PTD-BRD4 peptide (grey), or DRB (black) were added to the cell-culture supernatant at concentrations ranging from 0.01 to 10 ⁇ M. Luciferase activities were assayed 24 h after transfection.
  • Fig. 7C Clonal cell line A2 contains a single latent HIV provirus and expresses GFP following reactivation of HIV by TNF- ⁇ treatment.
  • A2 cells were pretreated for 2 h with either the PTD-BRD4 peptide (grey circles), PTD alone (white circles) or DRB (black triangles) at the indicated concentrations, followed by the addition of TNF ⁇ (10 ng/ml). After 18 h, levels of HIV proviral transcription were measured by FACS for GFP.

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Abstract

La présente invention a pour objet des procédés de traitement de troubles associés à une croissance et/ou une prolifération anormale de cellules. Les procédés impliquent généralement un agent qui réduit l'interaction de Brd4 avec le P-TEFb. La présente invention a en outre pour objet des procédés de traitement d'une infection par un virus induisant une immunodéficience. Les procédés impliquent généralement l'administration d'un agent qui réduit l'interaction de Brd4 avec le P-TEFb et/ou une protéine virale. La présente invention a en outre pour objet des procédés d'identification d'un agent qui réduit la liaison de Brd4 au P-TEFb. La présente invention concerne en outre des peptides de Brd4 et des polypeptides recombinants de Brd4.
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