WO2007002587A2 - Inhibitors of epstein barr virus nuclear antigen 1 - Google Patents

Abstract

The invention relates to a high throughput assay and methods for detecting inhibitors. The invention also relates to the inhibitors identified by the assay methods, and methods of using the inhibitors to treat and prevent disease.

Description

INHIBITORS QF EPSTEIN BARR VIRUS NUCLEAR ANTIGEN 1

Related Application

This application claims the benefit under 35 U.S. C. § 119(e) of U.S. provisional application serial number 60/693685, filed June 24, 2005, the disclosure of which is incorporated by reference herein.

Field of the Invention

The invention relates to a high throughput assay and methods for detecting inhibitors. The invention also relates to the inhibitors identified by the assay methods, and methods of using the inhibitors to treat and prevent disease.

Background of the Invention

Epstein Barr Virus (EBV) infection is prevalent in all human populations. EBV is also an important cause of lymphoma, particularly in immune compromised individuals. The EBV disease burden is largest in people with HIV infection, with transplanted tissues, or with other genetic or acquired immune deficiency states (Guterman et al. (1996) Clin Neuropathol 15:79-86; Kieff et al. (2001). p. 1185-1248. In Knipe, PM (ed.), Fundamental Virology, 4 ed. Lippincott Williams & Wilkins, Philadelphia; Wutzler et al. (1995). Arch Virol 140:1979-95). In such people EBV much more commonly causes lymphoproliferative diseases including Post Transplant type Lymphoproliferative Disease (PTLD), various B and T cell Lymphomas (L) and Hodgkin's Disease (HD). EBV is also the cause of anaplastic nasopharyngeal cancer (NPC), a common epithelial cell malignancy in people of Southern Chinese descent, in North Africans and other near Eastern people, and in northern Native American populations (Kieff et al. (2001). p. 1185-1248. In Knipe, PM (ed.), Fundamental Virology, 4 ed. Lippincott Williams & Wilkins, Philadelphia; Sam et al. (1993) Int J Cancer 53:957-62. Shibata et al. (1993) 5/oo</ 81:2102-9).

EBV replicates in oropharyngeal epithelial cells and establishes long term latency in B lymphocytes, from which EBV reactivates to initiate foci of lytic infection in the oropharyngeal epithelium. In B cells EBV can establish 3 types of latency. Latency III is characterized by expression of 6 EBV Nuclear antigens (EBNAs), two integral membrane proteins (LMPl and 2), EBER small RNAs, and BamHl A rightward transcripts (BARTs) (Kieff et al. (2001). p. 1185-1248. In Knipe, PM (ed.), Fundamental Virology, 4 ed. Lippincott Williams & Wilkins, Philadelphia). In latency III, EBV causes infected B lymphocyte proliferation, which is indistinguishable from early onset PTLD. Normally, latency III cells proliferate and disseminate in primary EBV infection, are recognized by the developing T lymphocyte immune response, and are readily eliminated by specifically committed CD4 and CD8 T lymphocytes. In some infected B cells, in vivo, EBV persists in latency II, wherein only EBNAl, LMPl and LMP2, EBERS and BARTs are expressed. EBV gene expression in HD and NPC is characteristically latency II. Alternatively, some latently infected B lymphocytes characteristically express only EBNAl, EBERs and BARTs, defined as latency I. EBV infection in Burkitt's lymphoma cells is usually latency I. While some studies have been consistent with the notion that EBV episomes persist in some stationary cells without EBNAl expression, these studies are in part confounded by the limits of resolution of the analyses and the slightly lower sensitivity of EBNAl detection assays (Babcock et al. (1998) Immunity 9:395-404; Babcock et al. (2000) Immunity 13:497-506; Babcock et al. (2000) Pr oc Natl Acad Sd USA 97:12250-5; 34). The EBV genome is almost always an episome in EBV infected cells including those of associated malignancies (Kieff et al. (2001). p. 1185-1248. In Knipe, PM (ed.), Fundamental Virology, 4 ed. Lippincott Williams & Wilkins, Philadelphia).

Endogenously produced EBNAl is not processed by proteasome pathways (Dantuma et al. (2002) Curr Top Microbiol Immunol 269:23-36; Khanna et al. (1997) Int Immunol 9: 1537-43 ; Leonchikset al. (2002) FEBS Lett 522:93-8., Levitskaya et al. (1997) Proc Natl AcadSci USA 94:12616-21., Sharipo et al. (2001) FEBS Lett 499:137-42). Therefore, EBV infected cells that express EBNAl can escape recognition by EBNAl specific CD8 cytotoxic T cells (Khanna et al. (1995) Microbiol Rev 59:387-405; Khanna et al. (1995) Virology 214:633-7).

EBNAl is the only EBV protein required for EBV DNA persistence as an episome (Lee et al. (1999) J Virol 73:2974-82., Sugden et al. (1985) MoI Cell Biol 5:410-3., Yates et al. (1985) Nature 313:812-5). EBNAl dimerizes and binds as a dimer to multiple potentially hairpin, dimeric EBNAl binding sequences in the cis-acting element, oriP, which is also essential for EBV episome replication, maintenance, and enhanced transcription (Frappier et al. (1992) J Virol 66:1786-90., Hsieh et al. (1993) Embo J 12:4933-44, Middleton et al. (1992) J Virol 66:489-95, Rawlins et al. (1985) Cell 42:859-68, Wysokenski et al. (1989) J Virol 63:2657-66). OriP alone is a potential origin of plasmid replication in human cells and its efficiency is substantially augmented EBNAl. OriP consists of 20 copies of a 30 base pair dimeric EBNAl binding site, which EBNAl engages to enhance transcription, and two tandem repeats and a pair of inverted repeats of a dimeric EBNAl binding site, which can be an origin for DNA replication, particularly in the presence of EBNAl; the enhancer and origin components of oriP are separated by about lkb of DNA, which also contributes to replication (Ambinder et al. (1991) J Virol 65:1466-78, Ambinder et al. (1990) J Virol 64:2369-79., Chen et al. (1993) J Virol 67:4875-85., Puglielli et al. (1996). J Virol 70:5758- 68., Rawlins et al. (1985) Cell 42:859-68., Reismanet al. (1985) MoI Cell Biol 5:1822-32., Sugden et al. (1985) MoI Cell Biol 5:410-3., Yates et al. (1985) Nature 313:812-5)

EBNA 1 is composed of 641 amino acids and has five major functional domains; two chromosome Binding and Transcription enhancing arginine rich domains (amino acids 1-89, and 323-386), a glycine alanine repeat and cis-acting proteasome protective domain (amino acids 90-322), an NLS (amino acids 379-385), and a DNA binding/ dimerization/ multimerization DNA replication domain (amino acids 386-641), which is also required for transcription enhancement (Bochkarev et al. (1996) Cell 84:791-800, Ceccarelli et al. (2000) J Virol 74:4939-48, Frappier et al. (1994) J Biol Chem 269:1057-62, Frappier et al. (1992) J Virol 66:1786-90, Frappier et al. (1991) J Biol Chem 266:7819-26, Goldsmith et al. (1993) J Virol 67:3418-26, Hung et al. (2001) Proc Natl Acad Sci USA 98:1865-70, 28).

EBNAl is not required for interaction with EBNAl cognate sequences in EBV transformed lymphoblastoid cell lines in which EBV has integrated into cell DNA. Furthermore, EBNAl has little effect on the oriP enhancer when that element is integrated into almost all sites in chromosomal DNA. These observations make it unlikely that EBNAl alters cell gene transcription or contributes to EBV effects on cell growth and survival. Indeed, EBV genomes that lack EBNAl can fully transform cells, albeit with reduced efficiency. However, integrated EBV genomes cannot enter the lytic infection cycle and cannot give rise to new infectious EBV.

No drugs currently exist for prevention or treatment of EBV-associated diseases. Drugs exist that can inhibit EBV replication in human cells, but unfortunately the genome persists in lymphoid cells in a latent state and cannot be eliminated. Therefore, inhibition or replication does not work and virus rapidly emerges from latency as soon as replication inhibitors are stopped. As latency itself is the cause of most EBV-associated diseases, there exists a need for treatments that can reduce or eradicate latent EBV infection. Summarv of the Invention

EBNAl interactions with cellular proteins have been described and these could have effects independent of EBNAl interaction with its cognate DNA. Since EBNAl is required for EBV episome persistence in dividing cells and enhances latent infection associated EBV gene expression from episomes (Gahn et al. (1995) J Virol 69:2633-6, Jones et al. (1989) J Virol 63:101-10, Reisman et al. (1986) MoI Cell Biol 6:3838-46, Summers et al. (1996) J Virol 70: 1228-31), compounds that inhibit EBNAl binding to cognate sites on the episome would interfere with EBNAl -mediated transcriptional enhancement and also block latent episomal EBV genome replication and persistence in lymphocytes. Thus, inhibiting EBNAl dimerization and DNA binding would be useful for preventing or treating EBV associated malignancies and may also eliminate EBV episomes from humans. We have therefore developed screening assays so as to undertake screens to identify compounds (e.g.small molecules) that inhibit EBNAl function.

The invention provides in some aspects a novel cell-based assay for drug screening for identifying inhibitors of Epstein Barr virus nuclear antigen 1 (EBNAl) function in maintaining an EBNAl -dependent episome. The assay has been developed in a high through-put format to allow rapid screening for compounds, including small molecules, that reduce the EBNAl -dependent maintenance of the episome in cells, and therefore that can reduce or eliminate latent EBV infection. The format of this assay allows for the rapid screening of large libraries of compounds in an automated setting. The assay is cell-based, and therefore provides a reasonable representation of the in vivo environment, increasing the probability that hit compounds will show efficacy in animal models and in treatment.

One aspect of the invention is a method for identifying compounds that selectively inhibit Epstein Barr virus nuclear antigen 1 (EBNAl) function in maintaining an EBNAl- dependent episome. The method provides at least one cell that expresses a first reporter sequence operably linked to an EBNAl -dependent promoter sequence and a second reporter sequence operably linked to a promoter sequence that is not EBNAl -dependent, contacting the at least one cell with a compound, and determining expression of the first reporter sequence and the second reporter sequence. In this aspect of the invention a compound that decreases expression of the first reporter sequence in a greater amount than the second reporter sequence is a compound that selectively inhibits Epstein Barr virus nuclear antigen 1 (EBNAl) function in maintaining an EBNAl -dependent episome. In one embodiment, the compound decreases expression of the first reporter sequence but not the second reporter sequence. In another embodiment, the at least one cell is at least one human cell. In another embodiment, the first reporter sequence is contained in a first cell and the second reporter sequence is contained in a second cell. In still another embodiment, the first and second reporter sequences are contained in the same cell. In yet another embodiment, the first reporter sequence is contained in a first construct. In one embodiment, the first construct is a plasmid. In another embodiment, the second reporter sequence is contained in a second construct. In one embodiment, the second construct is a plasmid. In yet another embodiment, the first reporter and the second reporter are, or encode, similar types of molecules.

In one embodiment the molecules are luciferase proteins, enzymes or fluorescent proteins, In another embodiment, the expression of the first reporter and the second reporter are assayed using enzyme assays, fluorescence assays, immunoassays, luminescence assays, or nucleic acid expression assays. In one embodiment the assays are similar types of assays. In another embodiment, the at least one cell recombinantly expresses EBNAl.

In one embodiment, the method provides for testing the compound that selectively inhibits for its effect on cell viability of EBV-infected and/or non-infected cells. In another embodiment, the compound is tested on cells in which the EBV genome is maintained as an episome or in which the EBV genome is integrated into a chromosome. In another embodiment, the method provides for testing the compound that selectively inhibits for its effect on binding of EBNAl protein to EBNAl -responsive promoter sequence or a chromosome. In another embodiment, the testing is performed using a gel shift assay. In still another embodiment, the method provides for testing the compound that selectively inhibits for inhibition of plasmid replication or transcription. In another embodiment, the method provides for testing the compound that selectively inhibits for reduction of latent EBV infection of a cell. In one embodiment, the infection is in a lymphocyte. In still another embodiment the method provides for testing the compound that selectively inhibits for inhibition of dimerization of EBNAl . In one embodiment, the method provides for testing the compound that selectively inhibits for an increase in EBNAl degradation. In another embodiment the at least one cell is a B lymphocyte.

Another aspect of the invention provides a method for identifying compounds that selectively inhibit Epstein Barr virus nuclear antigen 1 (EBNAl) function in maintaining an EBNAl -dependent episome by providing at least one cell that expresses a reporter sequence operably linked to an EBNAl -dependent promoter sequence, contacting the at least one cell with a compound, and determining expression of the reporter sequence The compound that decreases expression of the reporter sequence is a compound that selectively inhibits Epstein Barr virus nuclear antigen 1 (EBNAl) function in maintaining an EBNAl -dependent episome. One embodiment additionally includes determining toxicity of the compound. In one embodiment, determining toxicity comprises measuring ATP level or other measure of cell viability (e.g. the ability to exclude dye, or ³H thymidine incorporation) in the at least one cell that has been contacted with the compound. In another embodiment, the at least one cell is at least one human cell. In another embodiment, the expression of the reporter sequence is determined in a first cell and the toxicity is determined in a second cell. In one embodiment, the expression of the reporter sequence and the toxicity are determined in the same cell. In another embodiment, the reporter sequence is contained in a construct.

In still another embodiment, the construct is a plasmid. In another embodiment, the reporter sequence encodes a luciferase protein, enzyme or fluorescent protein. In yet another embodiment, the expression of the reporter sequence is assayed using an enzyme assay, fluorescence assay, immunoassay, luminescence assay, or nucleic acid expression assay. In yet another embodiment, the at least one cell recombinantly expresses EBNAl.

Another embodiment provides further testing of the compound that selectively inhibits for its effect on cell viability of EBV-infected and/or non-infected cells. In another embodiment the compound is tested on cells in which the EBV genome is maintained as an episome or in which the EBV genome is integrated into a chromosome. Another embodiment provides further testing of the compound that selectively inhibits for its effect on binding of EBNAl protein to EBNAl -responsive promoter sequence or a chromosome. In one embodiment, the testing is performed using a gel shift assay. Another embodiment provides further testing of the compound that selectively inhibits for inhibition of plasmid replication or transcription. Another embodiment provides further testing of the compound that selectively inhibits for reduction of latent EBV infection of a cell. In one embodiment, the infection is in a lymphocyte. Another embodiment provides further testing of the compound that selectively inhibits for inhibition of dimerization of EBNAl . Another embodiment provides further testing of the compound that selectively inhibits for an increase in EBNAl degradation. In another embodiment, the at least one cell is a B lymphocyte.

In another aspect, the invention provides methods for treating or preventing an Epstein Barr virus related disorder. This method involves administering to a subject in need of such treatment an effective amount of a compound that selectively inhibits Epstein Barr virus nuclear antigen 1 (EBNAl) function in maintaining an EBNAl -dependent episome, wherein the compound is a (4-oxo-2-thioxo-l,3-thiazolidin-3-yl)propanoate or propanoic acid, a [((methyl)phenoxy)methyl]benzoic acid, 2-[[3-[bis(2-hydroxyethyl)amino]-5,10- bis(l-ρiperidyl)-2,4,7,9-tetrazabicyclo[4.4.0]deca-2,4,7,9,ll-ρentaen-8-yl]-(2- hydroxyethyl)amino]ethanol (Dipyridamole), 6',7',10,11-Tetramethoxymethane 2HCl (Ementine), Emodine, 2-p-methoxyphenylmethyl-3-acetoxy-4-hydroxypyrrolidine (Anisomycin), ethyl 3-{[(4-methoxyphenoxy)acetyl]amino}benzoate (Compound G), N-I- adamantyl-4-[5-(4-methoxybenzylidene)-4-oxo-2-thioxo-l,3-thiazolidin-3-yl]butanamide (Compound H) or 2-(lH-benzimidazol-2-yl)-4-metliylphthalazin-l(2H)-one (Compound Q). In one embodiment, the (4-oxo-2-thioxo-l,3-thiazolidin-3-yl)propanoate or propanoic acid is 2-(dimethylamino)ethyl 3-[5-(4-methylbenzylidene)-4-oxo-2-thioxo-l,3-thiazolidin-3- yl]propanoate hydrochloride (Compound I), or 3-[5-(4-ethylbenzylidene)-4-oxo-2-thioxo-l,3- thiazolidin-3-yl]propanoic acid (Compound J), (Z)-5-(3,4-dihydroxybenzylidene)-3-methyl- 2-thioxothiazolidin-4-one (Compound H20), (Z)-5-(4-ethoxybenzylidene)-3-methyl-2- thioxothiazolidin-4-one (Compound H25), roscovitine (Compound 101), shikonin (Compound 102), or a salt, solvate, or analog thereof.

In certain embodiments, the [((methyl)phenoxy)methyl]benzoic acid is 3-[(4-{[l-(2- methylphenyl)-4,6-dioxo-2-thioxotetrahydro-5(2H) pyrimidinylidene]methyl}phenoxy)methyl]benzoic acid (Compound K), 3-[(4-{[l-(4- methylphenyl)-2,4,6-trioxotetrahydro-5(2H)- pyrimidinylidene]methyl}phenoxy)methyl]benzoic acid (Compound L), 3-[(4-{[l-(3- methylphenyl)-2,4,6-trioxotetrahydro-5(2H)- pyrimidinylidene]methyl}phenoxy)methyl]benzoic acid (Compound M), 3-[(4-{[l-(3- methylphenyl)-4,6-dioxo-2-thioxotetrahydro-5(2H)- pyrimidinylidene]methyl}phenoxy)methyl]benzoic acid (Compound N), 3-({4-[(2,4,6- trioxotetrahydro-5(2H)-pyrimidinylidene)methyl]phenoxy}methyl)benzoic acid (Compound O), or a salt, solvate, or analog thereof.

In other embodiments, the compound is compound is N-l-adamantyl-4-[5-(4- methoxybenzylidene)-4-oxo-2-thioxo-l,3-thiazolidin-3-yl]butanamide, 3-[(4-{[l-(2- methylphenyl)-4,6-dioxo-2-thioxotetrahydro-5(2H)- pyrimidinylidene]methyl}phenoxy)methyl]benzoic acid or 3-[(4-{[l-(3-methylphenyl)-4,6- dioxo-2-thioxotetrahydro-5(2H)-pyrimidinylidene]methyl}phenoxy)methyl]benzoic acid or a salt, solvate, or analog thereof. In a particular embodiment, the compound is compound N-I- adamantyl-4- [5-(4-methoxybenzylidene)-4-oxo-2-thioxo- 1 ,3 -thiazolidin-3 -yljbutanamide or a salt, solvate, or analog thereof.

Another aspect of the invention provides a method for treating or preventing an Epstein Barr virus related disorder. This aspect involves administering to a subject in need of such treatment an effective amount of a compound that selectively inhibits Epstein Barr virus nuclear antigen 1 (EBNAl) function in maintaining an EBNAl -dependent episome, where the compound comprises at least one of the following chemical structures:

A further aspect of the invention provides a method for treating or preventing an Epstein Barr virus related disorder. This method includes administering to a subject in need of such treatment an effective amount of a compound that selectively inhibits Epstein Barr virus nuclear antigen 1 (EBNAl) function in maintaining an EBNAl -dependent episome, where the compound is at least one of the following chemical structures:

In some embodiments, the Epstein Barr virus related disorder is cancer or lymphoproliferative disease. In certain embodiments, the cancer is lymphoma or carcinoma. In specific embodiments, the lymphoma is Hodgkin's Disease, Non-Hodgkin's Lymphoma or Burkitt's Lymphoma. In another embodiment, the carcinoma is nasopharyngeal cancer. In another embodiment, the lymphoproliferative disease is post transplantation lymphoproliferative disease (PTLD).

In still other embodiments, the Epstein Barr virus related disorder is infectious mononucleosis. In another embodiment, the subject is immunocompromised. In another embodiment, the immunocompromised subject has malaria, is infected with HIV, is a subject receiving a tissue or organ transplant, or is receiving immunosuppressive therapy. In another embodiment, the compound is administered concurrently with a cancer therapy.

According to another aspect of the invention, polypeptides including SEQ ID NOs: 1 and/or 5 are provided. The polypeptides are not full-length EBNAl and do not have EBNAl function, e.g., are not effective in maintaining episomes. To the contrary, the polypeptides of the invention are inhibitors of EBNAl function. Preferably the polypeptides include SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4.

In some embodiments, the polypeptides are less than about 180 amino acids in length, preferably less than about 100 amino acids in length, more preferably less than about 75 amino acids in length, more preferably less than about 50 amino acids in length, more preferably less than about 40 amino acids in length, still more preferably less than about 30 amino acids in length, and most preferably less than about 20 amino acids in length.

In certain embodiments, the polypeptide is a fragment of EBNAl . In some embodiments, the polypeptide is non-hydrolyzable. Another aspect of the invention provides methods for treating or preventing an Epstein Barr virus related disorder, as described herein. The methods include administering to a subject in need of such treatment an effective amount of the foregoing polypeptides to selectively inhibit Epstein Barr virus nuclear antigen 1 (EBNAl) function in maintaining an EBNAl -dependent episome.

The use of the compounds, molecules and polypeptides disclosed herein, in the preparation of medicaments, particularly for the treatment of Epstein Barr virus related disorders also is provided.

These and other aspects of the invention are described in further detail below in connection with the detailed description of the invention.

Brief Description of the Drawings

Figure 1 is a chart detailing different types of latent Epstein Barr virus (EBV) conditions.

Figure 2 is a drawing showing domain structure of Epstein Barr Nuclear Antigen 1 (EBNAl) protein.

Figure 3 is a schematic drawing of the procedure for screening assays 1 and 2.

Figure 4 is a schematic diagram detailing the steps of the primary high-throughput screen.

Figure 5 is a schematic diagram detailing the potential modes of action of EBNAl inhibitors identified by the screening assay.

Figure 6 shows chemical structure drawings of four compounds identified by the screening methods of the invention (G, H, I, and J).

Figure 7 shows chemical structure drawings of four compounds identified by the screening methods of the invention (K, L, M, and N).

Figure 8 shows chemical structure drawings of two compounds identified by the screening methods of the invention (O and Q).

Figure 9 is an image of a gel-shift assay illustrating inhibition of EBNAl binding to DNA by compounds H, K, N, and R. Compounds were added at 5, 10, 25, 50 or lOOμM. Controls include: addition of BSA, competition with 10Ox wild-type or mutant unlabeled DNA, supershift with EBNAl antibody, addition of IgG (no supershift), addition of DMSO.

Figure 10 is a chart showing cell viability, FL, and GFP positive cell number in control (BJAB) and transiently-transfected BJAB-FElcells treated with Compound H. Figure 11 shows the effect of Compound H on the viability of an EBV transformed human cord blood derived B lymphocyte cell line (LCL 1022). Figure 1 Ia is a chart showing cell viability. The left column represents control cells and the right column represents cells treated by Compound H. Figure 1 Ib is a chart tabulating cell numbers of either IB4 cells (with an integrated EBV genome) or LCL 1022 cells (episomal EBV genome) treated with compound H or control (DMSO).

Figure 12 shows photographs of either IB4 cells (with an integrated EBV genome) or LCL 1022 cells (episomal EBV genome) treated with compound H or control.

Figure 13 shows that certain synthetic small molecules and the natural compound Shikonin inhibit EBNAl specific DNA binding in vitro. Compounds were added at lOμM or 50μM. Controls include addition of BSA, competition with wild-type or mutant unlabeled DNA, supershift with EBNAl antibody, addition of IgG (no supershift), and addition of DMSO.

Figure 14 shows that a peptide in β sheet 3 of the EBNAl DNA binding domain (amino acids 560-574) abrogates EBNAl DNA binding in vitro. Two other peptides, EBNA amino acids 564-578 and 568-582, did not have similar activity at 0.6μM.

Detailed Description of the Invention

Compounds that functionally inhibit the ability of EBNAl to maintain an EBNAl- dependent episome could be used for the elimination of Epstein Barr virus (EBV) episomes and for halting the growth of latency III infected lymphocytes in culture and in experimental models. This would provide a compelling model and lead compounds for approaches to the therapy or prevention of EBV-related disorders such as EBV-associated Post Transplant type Lymphoproliferative Disease (PTLD), AIDS-associated lymphoma, Hodgkin's Lymphoma or nasopharyngeal cancer (NPC). This could also be the first direct step toward the elimination of a herpes virus from its sites of latent infection in humans. As used herein, the term "episome" refers to a extrachromosomal genetic element which replicates within a cell independently of the chromosome and is able, in some circumstances, to integrate into the host chromosome.

The invention provides in part a human cell-based assay system to identify inhibitors of Epstein Barr Nuclear Antigen 1 (EBNAl) function in maintaining an EBNAl -dependent episome with EBNAl -dependent gene expression using an EBNAl -dependent promoter preferably linked to a suitable reporter. As used herein, an "EBNAl -dependent promoter sequence" is a promoter sequence that requires functional (i.e. non-inhibited) EBNAl for expression. Included in the assay system is a suitable control promoter that is not EBNAl- dependent (i.e. EBNAl -independent) and has a compatible reporter. The system is designed so that the activity of a compound in inhibiting expression from the EBNAl -dependent promoter can be directly compared to the (non-) effect on the EBNAl -independent promoter/reporter in the same cell. Preferably, the reporters have compatible assays of similar type so as to provide the most meaningful control for an EBNAl -specific effect.¹

Generally, the assay methods involve assaying for compounds which modulate (up- or down-regulate) the level of expression of a detectable marker as an indicator of inhibition of EBNAl -dependent maintenance of the episome. As used herein, a compound is said to "decrease expression" if expression of a detectable marker is down-regulated. A compound that "selectively inhibits" EBNAl -dependent maintenance of the episome is a compound that selectively affects EBNAl -dependent expression or expression of an EBNAl -dependent reporter. It is understood that any mechanism of action described herein for the inhibitor compounds is not intended to be limiting, and the scope of the invention is not bound by any such mechanistic descriptions provided herein.

One preferred method is a primary high throughput screen (HTS) that utilizes a luciferase reporter system. This cell-based assay makes use of human EBV-negative Burkitt's Lymphoma cells (BJAB). Briefly, the cells are transfected with an episome including an EBNAl -dependent enhancer and promoter upstream of a Secreted Alkaline Phosphatase (SEAP) reporter gene. SEAP expression is a measure of EBNAl activity. A cell viability assay, such as CellTiter-Glo®, which measures total ATP level in live cells, therefore serves as a measure of toxicity of the compound.

As detailed in the Examples, this HTS has been used to successfully identify nontoxic compounds which reduce or eliminate SEAP expression, and are therefore inhibitors of EBNAl -dependent expression of episomal genes (Example 1). These inhibitors can be formulated as pharmaceutical compositions for the purpose of treating a subject in need of such inhibitor treatment, i.e. a subject having a latent EBV infection.

A second preferred assay utilizes the same type of cells as described above, which uses Firefly Luciferase as a measure of EBNAl activity, along with Renilla Luciferase as a measure of chemical toxicity or generalized transcription, processing or translational effects, and untransfected cells as a baseline control. Use of this second HTS as thus far yielded ten non-toxic compounds that reduce or eliminate Firefly Luciferase activity without a corresponding reduction of Renilla Luciferase activity (see Examples).

As used herein, the term "inhibitor of Epstein Barr virus nuclear antigen 1 (EBNAl) function" means a compound (e.g. a small molecule) that inhibits or reduces the normal function of EBNAl in maintaining an EBNAl -dependent epsiome. In some aspects of the invention, the inhibitor prevents binding of EBNAl to DNA. In some aspects of the invention, the inhibitor prevents binding of EBNAl to a cellular protein or protein complex. In some aspects of the invention, the inhibitor prevents initial plasmid (episomal) replication or recruitment of proteins to initiation sites.

The inhibitor compounds of the invention may include small molecules, chemicals, polypeptides (for example, competitive ligands and antibodies, or antigen-binding fragments thereof), and may also include nucleic acids. The inhibitor compounds are identified using the assays provided herein, including those in the Examples section. For example, an inhibitor compound may be tested for its ability to inhibit EBNAl function in maintaining an EBNAl -dependent episome by the methods of the invention.

A candidate inhibitor compound that produces a decrease in EBNAl function(s), or a surrogate marker of EBNAl function (e.g. Firefly Luciferase or SEAP) is considered an inhibitor of EBNAl function. The decrease in the detectable marker is at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99%. In preferred embodiments, a decrease of about 35% or more is detected. In particularly preferred embodiments, a decrease of about 50% or more is detected.

Candidate inhibitor molecules useful in accordance with the invention encompass numerous chemical classes, although typically they are organic compounds. Typically, the candidate inhibitor molecules are small organic compounds, i.e., those having a molecular weight of more than 50 Daltons (Da.) yet less than about 2500 Da., preferably less than about 1000 Da. and, more preferably, less than about 500 Da. Candidate inhibitor molecules comprise functional chemical groups necessary for structural interactions with proteins and/or nucleic acid moleculess, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups and more preferably at least three of the functional chemical groups. The candidate inhibitor molecules can comprise cyclic carbon or heterocyclic structure and/or aromatic or polyaromatic structures substituted with one or more of the above-identified functional groups. Candidate inhibitor molecules also can be biomolecules such as peptides, saccharides, fatty acids, sterols, isoprenoids, purines, pyrimidines, derivatives or structural analogs of the above, or combinations thereof and the like.

Candidate inhibitors identified thus far include 2-[[3-[bis(2-hydroxyethyl)amino]- 5,10-bis(l -ρiperidyl)-2,4,7,9-tetra2abicyclo[4.4.0]deca-2,4.7,9, 11 -pentaen-8-yl]-(2- hydroxyethyl)amino]ethanol (Dipyridamole), 6',7',10,11-Tetramethoxymethane 2HCl (Ementine), Emodine, 2-p-methoxyphenylmethyl-3 -acetoxy-4-hydroxypyrrolidine (Anisomycin), ethyl 3-{[(4-methoxyphenoxy)acetyl]amino}benzoate (Compound G, Figure 6), N-l-adamantyl-4-[5-(4-methoxybenzylidene)-4-oxo-2-thioxo-l,3-thiazolidin-3- yljbutanamide (Compound H, Figure 6), (4-oxo-2-thioxo-l,3-thiazolidin-3-yl)propanoate or propanoic acid is 2-(dimethylamino)ethyl 3-[5-(4-methylbenzylidene)-4-oxo-2-thioxo-l,3- thiazolidin-3-yl]propanoate hydrochloride (Compound I, Figure 6), or 3-[5-(4- ethylbenzylidene)-4-oxo-2-thioxo-l,3-thiazolidin-3-yl]propanoic acid (Compound J, Figure 6), 3-[(4-{[l-(2-methylphenyl)-4,6-dioxo-2-thioxotetrahydro-5(2H) pyrimidinylidene]methyl}phenoxy)methyl]benzoic acid (Compound K, Figure 7), 3-[(4-{[l- (4-methylphenyl)-2,4,6-trioxotetrahydro-5(2H)- pyrimidinylidene]methyl}phenoxy)methyl]benzoic acid (Compound L, Figure 7), 3-[(4-{[l- (3-methylphenyl)-2,4,6-trioxotetrahydro-5(2H)- pyrimidinylidene]methyl}phenoxy)methyl]benzoic acid (Compound M, Figure 7), 3-[(4-{[l- (3-methylphenyl)-4,6-dioxo-2-thioxotetrahydro-5(2H)- pyrimidinylidene]methyl}phenoxy)methyl]benzoic acid (Compound N, Figure 7), 3-({4- [(2,4,6-trioxotetrahydro-5(2H)-pyrimidinylidene)methyl]phenoxy}methyl)benzoic acid (Compound O, Figure 8), 2-(lH-benzimidazol-2-yl)-4-methylphthalazin-l(2H)-one (Compound Q, Figure 8), (Z)-5-(3,4-dihydroxybenzylidene)-3-methyl-2-thioxothiazolidin-4- one (Compound H20), (Z)-5-(4-ethoxybenzylidene)-3-methyl-2-thioxothiazolidin-4-one (Compound H25), roscovitine (Compound 101), and shikonin (Compound 102). These compounds encompass a variety of structural features, including but not limited to:

or

In some aspects of the invention, the inhibitor compounds are isolated nucleic acid molecules that are useful for practicing the invention. In other embodiments, the compositions include isolated polypeptides that are encoded by the above-described nucleic acid molecules. The polypeptides used in the methods of the invention embrace polypeptides as well as polypeptide fragments. The inhibitor polypeptides of the invention include fragments, (i.e. pieces) of inhibitor compounds. These fragments are shorter than the full- length inhibitor polypeptides. The inhibitor polypeptides and fragments of the invention can be screened for inhibition of EBNAl function using the same type of assays as described herein (e.g. in the Examples section). Using such assays, the inhibitor compounds that have the best inhibitory activity can be identified. Where the inhibitor compound is a nucleic acid molecule, the molecule typically is a DNA or RNA molecule, although modified nucleic acid molecules as defined herein are also contemplated. Polypeptide inhibitor compounds have been identified. These include peptides, i.e., short polypeptides. In particular, it has been discovered that polypeptides that include CYFMVFL (SEQ ID NO: 1) are effective in reducing EBNAl DNA binding and dimerization. Three overlapping 15 amino acid peptides (SEQ ID NO:2, SEQ ID NO: 3 and SEQ ID NO:4) that include SEQ ID NO:1 were shown to have this activity. A further 15 amino acid peptide (SEQ ID NO: 5) that does not contain SEQ ID NO:1 also was shown to have this activity Polypeptides including SEQ ID NOs: 1 and/or 5 thus are provided as inhibitors of EBNAl function. Such polypeptides may be of varying size and composition, as long as they contain SEQ ID NOs: 1 and/or 5. In preferred embodiments, the polypeptides are fragments of EBNAl that do not have EBNAl function. These fragments can include other amino acid sequences (i.e., non-EBNAl sequences), for example, at the amino- or carboxy-terminal ends. However, in other embodiments, the polypeptide does not contain EBNAl sequences other than SEQ ID NOs: 1, 2, 3, 4 and/or 5. Preferably the polypeptides are less than about 180 amino acids in length, more preferably less than about 100 amino acids in length, more preferably less than about 75 amino acids in length, more preferably less than about 50 amino acids in length, more preferably less than about 40 amino acids in length, still more preferably less than about 30 amino acids in length, and most preferably less than about 20 amino acids in length. The invention also includes polypeptides of all lengths intermediate to the lengths mentioned herein.

Changes to the structure of an EBNAl inhibitor compound to form variants or analogs of such a compound can be made according to established principles in the art. Such changes can be made to increase the therapeutic efficacy of the compound, reduce side effects of the compound, increase or decrease the hydrophobicity or hydrophilicity, and the like. Changes to the structure include the addition of additional functional groups, such as for targeting the compound to a particular organ or cell type of a subject, and substitution of one or more portions of the compound. In general, substitutions involve conservative substitutions of particular moieties or subunits of the compound. For example, when preparing variants of a compound which is a polypeptide, one of ordinary skill in the art will recognize that conservative amino acid substitutions will be preferred, i.e., substitutions which retain a property of the original amino acid such as charge, hydrophobicity, hydrophobicity, etc. Examples of conservative substitutions of amino acids include substitutions made amongst amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q₅ N; and (g) E, D. Preferred substitutions include substitutions amongst β -branched amino acids.

Preferably, polypeptide-based compounds are non-hydrolyzable. To provide such peptide compounds, one may select polypeptides from a library of non-hydrolyzable polypeptides, such as polypeptides containing one or more D-amino acids or polypeptides containing one or more non-hydrolyzable peptide bonds linking amino acids. Alternatively, one can select polypeptides which are optimal for disrupting EBNAl function (e.g., binding to either DNA or protein, including multimerization), and then modify such polypeptides as necessary to reduce the potential for hydrolysis by proteases. For example, to determine the susceptibility to proteolytic cleavage, polypeptides may be labeled and incubated with cell extracts or purified proteases and then isolated to determine which peptide bonds are susceptible to proteolysis, e.g., by sequencing polypeptides and proteolytic fragments. Alternatively, potentially susceptible peptide bonds can be identified by comparing the amino acid sequence of a polypeptide with the known cleavage site specificity of a panel of proteases. Based on the results of such assays, individual peptide bonds which are susceptible to proteolysis can be replaced with non-hydrolyzable peptide bonds by in vitro synthesis of the polypeptide.

Many non-hydrolyzable peptide bonds are known in the art, along with procedures for synthesis of polypeptides containing such bonds. Non-hydrolyzable bonds include - PSi[CH₂NH]- reduced amide peptide bonds, -psi[COCH₂]- ketomethylene peptide bonds, - psi[CH(CN)NH]- (cyanomethylene)amino peptide bonds, -psi [CH₂CH(OH)]- hydroxyethylene peptide bonds, -PSi[CH₂O]- peptide bonds, and -PSi[CH₂S]- thiomethylene peptide bonds.

Polypeptides preferably are short enough to be synthesized and isolated readily, yet long enough to effectively disrupt EBNAl binding to either DNA or protein. Preferred polypeptides thus are between seven and twenty amino acids in length, e.g., 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 amino acids. More preferably, polypeptides are between 7 and 15 amino acids in length. Those skilled in the art are well- versed in methods for preparing and isolating such polypeptides, such as synthetic chemistry or even recombinant biological methods.

Polypeptides useful in the invention can be linear, or maybe circular or cyclized by natural or synthetic means. For example, disulfide bonds between cysteine residues may cyclize a polypeptide sequence. Bifunctional reagents can be used to provide a linkage between two or more amino acids of a polypeptide. Other methods for cyclization of polypeptides, such as those described by Anwer et al. (Int. J. Pep. Protein Res. 36:392-399, 1990) and Rivera - Baeza et al. (Neuropeptides 30:327-333, 1996) are also known to those of skill in the art.

Nonpeptide analogs of polypeptides, e.g., those which provide a stabilized structure or lessened biodegradation, are also contemplated. Preferably, the nonpeptide moieties permit the peptide to retain its natural confirmation, or stabilize a preferred, e.g., bioactive, confirmation. One example of methods for preparation of nonpeptide mimetic analogs from polypeptides is described inNachman et al., Reguϊ. Pept. 57:359-370 (1995). "Polypeptide" as used herein embraces all of the foregoing.

The polypeptide sequence can also be delivered into cells by providing a recombinant protein fused with peptide carrier molecules. These carrier molecules, which are also referred to herein as protein transduction domains (PTDs), and methods for their use, are known in the art. Examples of PTDs, though not intended to be limiting, are tat, antennapedia, and synthetic poly-arginine; nuclear localization domains also can be included in the polypeptide molecules for targeting to the nucleus of cells. These delivery methods are known to those of skill in the art and are described in US patent 6,080,724, and US patent 5,783,662, the entire contents of which are hereby incorporated by reference.

Candidate inhibitor compounds 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, synthetic organic combinatorial libraries, phage display libraries of random peptides, and the like. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or ^■ readily produced. Additionally, natural and synthetically produced libraries and compounds can be readily modified through conventional chemical, physical, and biochemical means. Further, known pharmacological inhibitor compounds may be subjected to directed or random chemical modifications such as acylation, alkylation, esterification, amidification, etc. to produce structural analogs of the inhibitor compounds. Examples of libraries are those provided by ChemDiv, Inc. (San Diego, CA), The ChemBridge Corporation Library (ChemBridge Corporation, San Diego, CA), the National Institute of Neurological Disorders and stroke (NINDS), the Natural Product Library (TimTec, Inc., Newark, DE ) and The Prestwick Chemical Library (Prestwick Chemical, Inc., Washington, DC). ChemDiv, Inc. provides custom libraries for discovery or hit profiling. The small molecule discovery collection includes 750,000 individual, highly purified and re-suppliable compounds split onto International Diversity (acquired compounds) and CombiLab (compounds synthesized in-house) collections. The custom-made target-specific libraries are compiled into protein-class focused libraries and disease area/pathway focused libraries. Focused libraries already available include protein-class libraries focused on GPCRs, kinases, phosphatases, ion channels, nuclear receptors, and proteases, and pathway/disease libraries focused on signal transduction, diabetes II, antiinflammatories, and anti-infectives. The National Institute of Neurological Disorders and stroke (NINDS) offers a collection of over 1,040 FDA approved drugs and bioactive compounds known to have a neurological effect. The ChemBridge Corp. library (30,000 compounds) consists of Diverse and CNS sets. The Diverse set contains compounds that were selected based on 3D pharmacophore analysis to cover the broadest part of biologically relevant pharmacophore diversity space. The CNS set is a therapeutic area focused collection of pre-designed, drug-like, small molecule compounds. Computational methods are applied to select compounds with increased probability of oral bioavailability and possibly blood brain barrier penetration. The natural product library contains 2000 natural/semi-natural products with a broad diversity of acceptable chemical structures purified to a 95% screening grade or chemically re- synthesized. Compounds that may be problematic to a cell based assay system, e.g. very high molecular weight species and simple sugars, are filtered out. The Prestwick Chemical Library is a collection of 1120 high-purity chemical compounds (all off patent) carefully selected for: structural diversity, broad spectrum covering several therapeutic areas (from neuropsychiatry to cardiology, immunology, anti-inflammatory, analgesia and more) and known safety and bioavailability in humans.

The high throughput screen (HTS) of the invention is an assay that allows the screening of one or more inhibitor compounds at a time. Generally the screening method involves assaying for inhibitor compounds that inhibit EBNAl function in maintaining an EBNAl -dependent episome. The assay mixture comprises at least one candidate inhibitor compound. A plurality of assay mixtures can be run in parallel with different inhibitor compound concentrations to obtain a different response to the various concentrations. In some methods one of these concentrations can serve as a negative control, i.e., at zero concentration of candidate inhibitor compound or at a concentration of candidate inhibitor compound below the limits of assay detection. The high throughput screening assay may be based in a multiwell plate. In one aspect, a cell is contacted with at least one candidate inhibitor compound in a multiwell plate. The level of transcription is determined using a detectable marker, e.g. a luciferase reporter, operably linked to an EBNAl -dependent promoter sequence. Optionally, a second reporter sequence operably linked to a promoter sequence that is not EBNAl -dependent is used.

An exemplary transcription assay is described herein, which may be used to identify candidate inhibitor compounds that can inhibit EBNAl function in maintaining an EBNAl- dependent episome. In general, the mixture of the foregoing assay materials is incubated under conditions whereby, in the presence of the candidate inhibitor compounds, EBNAl- dependent reporter activity in EBNAl -transformed lymphoblasts is reduced or eliminated. The order of addition of components, incubation temperature, time of incubation, and other parameters of the assay may be readily determined. Such experimentation merely involves optimization of the assay parameters, not the fundamental composition of the assay. Incubation temperatures typically are between 4°C and 40⁰C. Incubation times preferably are minimized to facilitate rapid, high throughput screening, and typically are between 0.1 and 72 hours. After incubation, the reporter activity is detected by any convenient method available to the user. It is contemplated that cell-based assays as described herein can be performed using cell samples and/or cultured cells. Biopsy cells and tissues as well as cell lines grown in culture are useful in the methods of the invention.

A variety of methods may be used to detect the expression of a polypeptide-encoding sequence. Detection may be effected in any convenient way for cell-based assays such as use of a detectable marker. For cell-based and cell-free assays, one of the components usually comprises, or is coupled to, a detectable marker. In one method, expression is detected using the expression of a detectable marker. In some embodiments the detectable marker is Firefly Luciferase or Renilla Luciferase. Other markers may be used to detect the expression level of a polypeptide-encoding sequence, e.g. green fluorescent protein (GFP) or the chloramphenicol acetyl transferase (CAT) gene. The method of detection of the marker may depend on the nature of the marker and other assay components. A wide variety of markers can be used, for example, the marker may be directly detected through optical or electron density, radioactive emissions, nonradiative energy transfers, luminescence, fluorescence (for example green fluorescent protein (GFP), etc.), or indirectly detected with antibody conjugates, strepavidin-biotin conjugates, epitope tags such as the FLAG epitope, assays of enzyme activity such as horse-radish peroxidase, etc. Methods for detecting and using markers are well known in the art.

As will be understood by the person of ordinary skill in the art, it is desirable to measure the amount of EBNAl function in a linear range of the assay system, such that small but significant decreases in detectable marker relative to control well may be observed. It is well within the skill of the art to determine a volume and concentration of a candidate inhibitor compound which causes a suitable response in cells so that EBNAl function may be reliably detected.

An expression vector is one into which a desired DNA sequence may be inserted by restriction and ligation such that it is operably linked to regulatory sequences and may be expressed as an RNA transcript. Expression vectors can be, for example, plasmids or episomes. The expression vector is one which is able to replicate in a host cell or be replicated after its integration into the genome of a host cell, and which is further characterized by one or more endonuclease restriction sites at which the vector may be cut in a determinable fashion and into which a desired DNA sequence may be ligated such that the new recombinant vector retains its ability to replicate in the host cell. In the case of plasmids, replication of the desired sequence may occur many times as the plasmid increases in copy number within the host bacterium or just a single time per host before the host reproduces by mitosis. In the case of phage, replication may occur actively during a lytic phase or passively during a lysogenic phase.

Vectors may further contain one or more marker sequences suitable for use in the identification of cells which have or have not been transformed or transfected with the vector. Markers include, for example, genes encoding proteins which increase or decrease either resistance or sensitivity to antibiotics or other compounds, genes which encode enzymes whose activities are detectable by standard assays known in the art (e.g., β-galactosidase, luciferase or alkaline phosphatase), and genes which visibly affect the phenotype of transformed or transfected cells, hosts, colonies or plaques (e.g., green fluorescent protein).

A selection marker should be such that it allows - i.e. under appropriate selection conditions - host cells and/or host organisms that have been (successfully) transformed with the nucleotide sequence of the invention to be distinguished from host cells/organisms that have not been (successfully) transformed. Some preferred but non-limiting examples of such markers are genes that provide resistance against antibiotics (such as puromycin or blasticidin), genes that provide for temperature resistance, or genes that allow the host cell or host organism to be maintained in the absence of certain factors, compounds and/or (food) components in the medium that are essential for survival of the non-transformed cells or organisms.

As used herein, a coding sequence and regulatory sequences are said to be "operably linked" when they are covalently linked in such a way as to place the expression or transcription of the coding sequence under the influence or control of the regulatory sequences. If it is desired that the coding sequences be translated into a functional protein, two DNA sequences are said to be operably linked if induction of a promoter in the 5 ' regulatory sequences results in the transcription of the coding sequence and if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the promoter region to direct the transcription of the coding sequences, or (3) interfere with the ability of the corresponding RNA transcript to be translated into a protein. Thus, a promoter region is operably linked to a coding sequence if the promoter region is capable of effecting transcription of that DNA sequence such that the resulting transcript can be translated into the desired protein or polypeptide. Such a promoter will be an EBNAl -dependent promoter for assays of compound activity, and a non-EBNAl -dependent promoter (or EBNAl -independent) for control assays.

A polypeptide-encoding nucleotide sequence may include the entire coding sequence of a protein, or regions of a coding sequence that encode fragments of a protein. Regions of a protein include but are not limited to domains, such as the N-domain or the C-domain, and subunits of proteins. In one embodiment, a nucleotide sequence encoding a small interfering RNA molecule (siRNA) or other detectable RNA is inserted in place of the polypeptide- encoding nucleotide sequence.

The precise nature of the regulatory sequences needed for gene expression may vary between species or cell types, but shall in general include, as necessary, 5' non-transcribed and 5' non-translated sequences involved with the initiation of transcription and translation respectively, such as a TATA box, capping sequence, CAAT sequence, and the like. Especially, such 5' non-transcribed regulatory sequences will include an EBNAl -dependent or independent promoter region which includes a promoter sequence for transcriptional control of the operably linked gene. Regulatory sequences may also include enhancer sequences or upstream activator sequences as desired. The vectors of the invention may optionally include 5' leader or signal sequences. A leader sequence should be such that - in the intended host cell or host organism - it allows for the desired post-translational modifications and/or such that it directs the transcribed mRNA to a desired part or organelle of a cell. A leader sequence may also allow for secretion of the expression product from said cell. As such, the leader sequence may be any pro-, pre-, or prepro-sequence operable in the host cell or host organism. In some instances, the nucleotide sequences inserted into the expression vector of the invention may include sequences that encode signal peptides. The signal sequence may refer to a region coding for a portion of a protein that is later cleaved off. The invention embraces each of these sequences with, or without, the portion of the sequence that encodes a signal peptide.

For some further non-limiting examples of the selection markers, leader sequences, expression markers and further elements that may be present/used in the genetic constructs of the invention - such as terminators, transcriptional and/or translational enhancers and/or integration factors - reference is made to the general handbooks such as J. Sambrook, et al., eds, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, F.M. Ausubel, et al., eds, Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York, W.B. Wood et al., "The nematode Caenorhabditis elegans", Cold Spring Harbor Laboratory Press (1988) and D.L. Riddle et al., "C ELEGANSH", Cold Spring Harbor Laboratory Press (1997), as well as to the examples that are given in WO 95/07463, WO 96/23810, WO 95/07463, WO 95/21191, WO 97/11094, WO 97/42320, WO 98/06737, WO 98/21355, US-A-6,207,410, US-A- 5,693,492 and EP 1 085 089. Other examples will be clear to the skilled person.

Inhibitors of EBNAl function identified by the methods of the invention have in vitro and in vivo diagnostic and therapeutic utilities. For example, these compounds can be administered to cells in culture, e.g. in vitro or ex vivo, or in a subject, e.g., in vivo, to treat, prevent or diagnose a variety of disorders. As used herein, the term "subject" is intended to include humans and non-human animals. Preferred subjects include a human patient having an Epstein Barr virus related disorder. Other preferred subjects include subjects that are treatable with the compositions of the invention.

One aspect the invention provides a method for treating or preventing an Epstein Barr virus related disorder. An "Epstein Barr virus related disorder" is one which is caused by EBV infection or latent infection. Such disorders include, but are not limited to, those incurred in an subject with a genetic or acquired immune deficiency states. Examples of such disorders include diseases such as Post Transplant type Lymphoproliferative Disease (PTLD), various B and T cell Lymphomas (L), Hodgkin's Disease (HD), anaplastic nasopharyngeal cancer (NPC), a lymphoma, a carcinoma, infectious mononucleosis, or malaria. Inhibitors of EBNAl function can be administered to a subject who is at risk of becoming immune suppressed, such as a subject about to undergo chemotherapy or a subject about to receive a tissue or organ transplant. In some aspects of the invention, the subject is a subject of Southern Chinese descent, a subject who is of North African or other near Eastern descent, or northern Native American descent.

The compounds of the present invention may include or be diluted into a pharmaceutically-acceptable carrier. As used herein, "pharmaceutically acceptable carrier" or "physiologically acceptable carrier" means one or more compatible solid or liquid fillers, diluents or encapsulating substances which are suitable for administration to a subject, including a human or other mammal such as a primate, dog, cat, horse, cow, sheep, or goat. Such carriers include any and all salts, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. The term "carrier" denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application. The carriers are capable of being commingled with the preparations of the present invention, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficacy or stability. Preferably, the carrier is suitable for oral, intranasal, intravenous, intramuscular, subcutaneous, parenteral, spinal, intradermal or epidermal administration (e.g., by injection or infusion). Suitable carriers can be found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa. Depending on the route of administration, the active compound, i.e., EBNAl inhibitor may be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

When administered, the pharmaceutical preparations of the invention are applied in pharmaceutically-acceptable amounts and in pharmaceutically-acceptable compositions. The term "pharmaceutically acceptable" means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredients. The components of the pharmaceutical compositions also are capable of being co-mingled with the molecules of the present invention, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficacy. Such preparations may routinely contain salts, buffering agents, preservatives, compatible carriers, and optionally other therapeutic agents, such as supplementary immune potentiating agents including adjuvants, chemokines and cytokines. When used in medicine, the salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically-acceptable salts thereof and are not excluded from the scope of the invention.

A salt retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects (see e.g., Berge, S.M., et al. (1977) J Pharm. Sci. 66: 1-19). Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from nontoxic inorganic acids, such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous and the like, as well as from nontoxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl substituted alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids and the like. Base addition salts include those derived from alkaline earth metals, such as sodium, potassium, magnesium, calcium and the like, as well as from nontoxic organic amines, such as N,N'-dibenzylethylenediamine, N-methylglucamine, chioroprocaine, choline, diethanolamine, ethylenediamine, procaine and the like.

The pharmaceutical preparations of the invention also may include isotonicity agents. This term is used in the art interchangeably with iso-osmotic agent, and is known as a compound which is added to the pharmaceutical preparation to increase the osmotic pressure to that of 0.9% sodium chloride solution, which is iso-osmotic with human extracellular fluids, such as plasma. Preferred isotonicity agents are sodium chloride, mannitol, sorbitol, lactose, dextrose and glycerol.

Optionally, the pharmaceutical preparations of the invention may further comprise a preservative, such as benzalkonium chloride. Suitable preservatives also include but are not limited to: chlorobutanol (0.3 - 0.9% W/V), parabens (0.01 - 5.0%), thimerosal (0.004 - 0.2%), benzyl alcohol (0.5 - 5%), phenol (0.1 - 1.0%), and the like.

The formulations provided herein also include those that are sterile. Sterilization processes or techniques as used herein include aseptic techniques such as one or more filtration (0.45 or 0.22 micron filters) steps.

The pharmaceutical compositions may contain suitable buffering agents, including: acetic acid in a salt; citric acid in a salt; boric acid in a salt; and phosphoric acid in a salt. The pharmaceutical compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well-known in the art of pharmacy. All methods include the step of bringing the active agent into association with a carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing the active compound into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product.

The administration of inhibitor(s) identified by the methods of the invention can be combined with other therapeutic agents. The inhibitor and other therapeutic agent may be administered simultaneously or sequentially. When the other therapeutic agents are administered simultaneously they can be administered in the same or separate formulations, but are administered at the same time. The other therapeutic agents are administered sequentially with one another and with the inhibitor , when the administration of the other therapeutic agents and the inhibitor is temporally separated. The separation in time between the administration of these compounds may be a matter of minutes or it may be longer. Other therapeutic agents include but are not limited to chemotherapeutics, therapeutics administered to a subject with HIV, antimalarial drugs, etc.

Compositions suitable for parenteral administration conveniently comprise a sterile aqueous or non-aqueous preparation of EBNA inhibitor compounds, which is preferably isotonic with the blood of the recipient. This preparation may be formulated according to known methods using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation also may be a sterile injectable solution or suspension in a nontoxic parenterally-acceptable diluent or solvent, for example, as a solution in 1,3 -butane diol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono-or di-glycerides. In addition, fatty acids such as oleic acid may be used in the preparation of injectables. Carrier formulations suitable for oral, subcutaneous, intravenous, intramuscular, etc. administration can be found in Remington 's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA.

The inhibitor compounds can be prepared with carriers that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are patented or generally known to those skilled in the art. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

The therapeutics of the invention can be administered by any conventional route, including injection or by gradual infusion over time. The administration may, for example, be oral, subcutaneous, intravenous, intraperitoneal, intramuscular, intracavity, intratumor, or transdermal. In some embodiments subcutaneous or intramuscular administration is preferred. When antibodies are used therapeutically, preferred routes of administration include intravenous and by pulmonary aerosol. Techniques for preparing aerosol delivery systems containing antibodies are well known to those of skill in the art. Generally, such systems should utilize components which will not significantly impair the biological properties of the antibodies, such as the paratope binding capacity (see, for example, Sciarra and Cutie, "Aerosols," in Remington's Pharmaceutical Sciences, 18th edition, 1990, pp. 1694-1712; incorporated by reference). Those of skill in the art can readily determine the various parameters and conditions for producing antibody aerosols without resorting to undue experimentation.

The pharmaceutical preparations of the invention, when used in alone or in cocktails, are administered in therapeutically effective amounts. Effective amounts are well known to those of ordinary skill in the art and are described in the literature. A therapeutically effective amount will be determined by the parameters discussed below; but, in any event, is that amount which establishes a level of the drug(s) effective for treating a subject , such as a human subject, having one of the conditions described herein. An effective amount means that amount alone or with multiple doses, necessary to delay the onset of, inhibit completely or lessen the progression of or halt altogether the onset or progression of the condition being treated. When administered to a subject, effective amounts will depend, of course, on the particular condition being treated; the severity of the condition; individual patient parameters including age, physical condition, size and weight; concurrent treatment; frequency of treatment; and the mode of administration. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. It is preferred generally that a maximum dose be used, that is, the highest safe dose according to sound medical judgment. An "effective amount" is that amount of an inhibitor of EBNAl function that alone, or together with further doses, produces the desired response, e.g. , reduces EBV latent infection or treats a lymphoproliferative disorder in a subject. The term is also meant to encompass the amount of an EBNAl inhibitor that in combination with a chemotherapeutic agent produces the desired response. This may involve only slowing the progression of the disease temporarily, although more preferably, it involves halting the progression of the disease permanently. This can be monitored by routine methods. The desired response to treatment of the disease or condition also can be delaying the onset or even preventing the onset of the disease or condition.

Such amounts will depend, of course, on the particular condition being treated, the severity of the condition, the individual patient parameters including age, physical condition, size and weight, the duration of the treatment, the nature of concurrent therapy (if any), the specific route of administration and like factors within the knowledge and expertise of the health practitioner. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. It is generally preferred that a maximum dose of the individual components or combinations thereof be used, that is, the highest safe dose according to sound medical judgment. It will be understood by those of ordinary skill in the art, however, that a patient may insist upon a lower dose or tolerable dose for medical reasons, psychological reasons or for virtually any other reasons.

The pharmaceutical compositions used in the foregoing methods preferably are sterile and contain an effective amount of an inhibitor of EBNAl function for producing the desired response in a unit of weight or volume suitable for administration to a patient. The response can, for example, be measured by determining the physiological effects of the ENBAl inhibitor, such as reduction of EBV latent infection or a decrease of disease symptoms. Other assays will be known to one of ordinary skill in the art and can be employed for measuring the level of the response.

The doses of inhibitor compounds administered to a subject can be chosen in accordance with different parameters, in particular in accordance with the mode of administration used and the state of the subject. Other factors include the desired period of treatment. In the event that a response in a subject is insufficient at the initial doses applied, higher doses (or effectively higher doses by a different, more localized delivery route) may be employed to the extent that patient tolerance permits. In general, doses can range from about 10 μg/kg to about 100,000 μg/kg. In one embodiment, the dose is about 50 mg. In another embodiment, the dose is about 250 mg. In still another embodiment, the dose is about 500 mg, 1000 mg or greater. Based upon the composition, the dose can be delivered once, continuously, such as by continuous pump, or at periodic intervals. The periodic interval may be weekly, bi-weekly, or monthly. The dosing can occur over the period of one month, two months, three months or more to elicit an appropriate humoral and/or cellular immune response. Desired time intervals of multiple doses of a particular composition can be determined without undue experimentation by one skilled in the art. Other protocols for the administration of EBNAl inhibitors will be known to one of ordinary skill in the art, in which the dose amount, schedule of administration, sites of administration, mode of administration and the like vary from the foregoing.

Administration of inhibitor compounds to mammals other than humans, e.g. for testing purposes or veterinary therapeutic purposes, is carried out under substantially the same conditions as described above.

The present invention is further illustrated by the following Examples, which in no way should be construed as further limiting. The entire contents of all of the references (including literature references, issued patents, published patent applications, and co-pending patent applications) cited throughout this application are hereby expressly incorporated by reference.

Examples

Materials and methods

Reporter constructs

EBNAl dependent oriP-Cp-FL reporter was constructed by cloning a Klenow blunt- ended 4.34 kilo base pair (kbp) EcoRl/Eco72i DNA fragment from the EBV Bam C plasmid encompassing the 1.8 kbp oriP DNA that included 20 copies of 30 base pair (bp) family of repeats, 4 copies of a dyad symmetry of 2 EBNAl binding sites and 2.6 kbp downstream DNA including the EBV Cp promoter into a Klenow blunted Xhol site of promoter-less pGL2 basic plasmid with Firefly Luciferase (FL) (pFL-oriPCp). The 1.055 kbp EcoRI/Clal pSV40 Puro DNA from pBABE PURO was blunt ended and ligated into the blunt ended BamHI site of pFL-oriPCp to yield poriP-Cp-FL-Puro. Small molecule library screening

Small molecule screening used the Harvard Institute of Chemistry and Cell Biology (ICCB) facilities and 44,000 compounds including Bioactive NINDS, ChemDiv, Chembridge, and NCI open libraries. Raw and analyzed data of primary HTS screening results were deposited at ICCB database where structure and vendor information are available.

Example 1: High-throughput screens using luciferase reporter and cell viability assays (Assay 1)

Aliquots of BJAB human lymphoblast cells expressing EBNAl were transfected with the full natural 4 kbp, EBNAl dependent, EBV OriP to Cp enhancer and promoter upstream of a Secreted Alkaline Phosphatase (SEAP) gene on an episome. The reporter activity was 100-fold EBNAl -enhanced as was evident by transfection of the reporter into BJAB cells that expressed or did not express EBNAl. Non-specific toxicity was assessed by Glo-type luminescence which homogeneously measures total ATP level in metabolically live cells (CellTiter-Glo^® Luminescent Cell Viability Assay, Promega) (Figure 3, Assay 1). After 6-8 days of selection and expansion in Puromycin, cells were aliquoted into 384 well plates with 10⁴ cells/well. The initial screen of a diverse ChemDiv (San Diego, CA) combilab 28,864 component chemical library required approximately 200 plates for duplicate screening. Stock concentration of 10 mM in 100% DMSO was formatted for 384 well plate assays. This and other chemical libraries were provided from the Harvard ICCB collection (iccb.med.harvard.edu/screening/compound_libraries).

Four compounds, Dipyridamole, Ementine, Emodine and Anisomycin, inhibited EBNAl dependent SEAP reporter activity 50% and consistently retained >70% GIo luminescence. The conditions for the HTS assay were 4500 cells/well in 75 μl of RPMI 1640 with 5% serum and 160 nl compound/well (10 μM final concentration). Plates were incubated for two days and 8 μl was assayed for SEAP activity. GIo assay was done on 20 μl of the residual culture. These conditions were highly reproducible in identifying the same four compounds, which had reproducible low level toxicity. Example 2: High-throughput screens using luciferase reporter based assays (Assay 2)

A second cell-based assay used the same BJAB EBNAl -expressing cells as in the first assay. The same 4 kbp EBV DNA oriP enhancer and Cp promoter episomal plasmid was also used. Firefly Luciferase was used as the EBNAl dependent reporter in the oriP based episome (oriP-CpFL). An integrated SV40 enhancer and promoter driving Renilla luciferase was used as the EBNAl -independent control for nonspecific toxic or generalized transcription, processing or translational effects (see Figure 3, Assay 2).

The High Throughput Screening system used Dual Glo™ Luciferase Assay System (Promega, Madison, WI). A library of 44,000 small molecules was screened for effect on the EBNAl -dependent promoter and lack of effect on the control SV40 promoter. This cell- based assay system was optimized and pre-validated using 960 bioactive compounds at 10 μM in a 384-well plate format.

The cell line used for the Assay 2 HTS was E10F5 cells, which are EBV-negative BJAB Burkitt's Lymphoma cells that stably express three elements: FLAG-EBNAl (amino acids 8-641) as an effector protein, SV40 promoter-driven Renilla Luciferase (RL) as EBNAl -independent reporter for cytotoxicity measurement and oriP-CP-FL-Puro as an EBNAl-dependent reporter. The cell line for HTS was derived from BJAB-FLAG-EBNA1 (BJAB-FEl) (1) by transfection with linearized SV40-RL plasmid (Promega) and linearized pcDNA6HisA (Invitrogen, Carlsbad, CA) followed by selection with Blastcidin (10 μg/ml). The cells then were transfected with oriP-Cp-FL-Puro reporter followed by Puromycin selection (1 μg/ml) for 1 week. Briefly, ten million BJAB-FEl cells were transfected with 5 μg of pSV40-RL and 0.5 μg pcDNA6-HisA. The following day, 2 ml cells were put in selection media with 20 μg/ml Blastcidin. After 7 days, cells were plated at 30, 100, or 300 cells per well in 96 wells containing 150 μl selective media. Outgrowing cells were tested for RL using Dual Glo™ Luciferase Assay System (Promega). One clone, Cl, had the highest RL activity and was cloned in 96 well plates at 1 or 3 cells per well in selective medium. In 4 weeks, clones were retested for RL activity. Clones Bl and ElO had highest activity, were mycoplasma-free by PCR, and were used for transfection with poriP-Cp-FL reporter. Two sets of 2OxIO⁶ ElO cells expressing EBNAl with integrated SV40p-hRL reporter were transfected with 20 μg oriP-Cp-FL-Puro and selected in RPMI 1640/10% new gem serum plus μg/ml Puromycin for 8 days (E10F5). Background FL and RL from BJAB or BJABFEl cells was only about 200 and <500, respectively. RL activity from HTS cell line (E10F5) was approximately 5-fold higher than FL activity, indicating that residual FL activity post- quenching does not affect RL activity. Moreover, FL signal to noise (EBNAl vs. BJAB cells) from 2 million cells was 480-fold (680,000 vs. 1400), 2 days post transfection.

This assay was also designed in part to determine the gross biochemical effect of the chemical at effective concentrations on EBV DNA and RNA persistence in the oriP-Cp plasmid BJAB cells used in the chemical screen. For these experiments, the effect of drug was determined over a five day period in comparison with control media. Plasmid DNA and FL and RL RNA content was measured by real-time PCR. Decreased plasmid DNA and FL RNA with stable persistence of RL RNA was the expected phenotype of an inhibitor of EBNAl that affected the ability of EBNAl to dimerize and bind to DNA. These two characteristics have been closely linked in previous EBNAl genetic analyses, but may be unlinked by a putative chemical that does not inhibit dimerization but does inhibit DNA binding.

The assay of Firefly Luciferase (FL) as EBNAl -dependent reporter and Renilla Luciferase (RL) as an EBNAl -independent reporter used the Dual Glo™ Luciferase Assay System (Promega) to measure FL and RL sequentially in culture plate. Using the cells described above, 10,000 cells/well were incubated with 10 μM chemical compound in 35 μl media for two days at 37⁰C. Two days post plating, 35 μl of FL buffer was added. After incubation for 10 min. at room temperature, plates were read in a Wallac Victor 2™ multi- label counter for 0.1 sec/ well. RL buffer was then added, incubated for 10 min. at room temperature and read in the Wallac Victor 2™ multi-label counter. Screening was done in duplicate to minimize experimental error (Figure 4). Data points were analyzed using ID Business Solutions software and stored in Oracle data base server of Harvard ICCB.

In addition to the compounds identified in the initial assay (Assay 1), this second assay initially identified 128 hits. Ten compounds survived re-screening and testing for reproducible inhibition of EBNAl dependent FL more that 75% with less than 25% inhibition of RL at 30 μM final concentration (see Table 1 and Figures 6-8). Table 1. Ten small molecules that inhibit EBNA1 activity

H 90 7δ 66 ~1 128 91 87 + + C₂#⁾3!^2θ# 2

I 77 53 41 3-10 88 76 63 -

J 39 74 65 <3 133 79 95 +• C1 ^1 ^IO ^2

K 94 86 70 «3 105 79 90 O2&2&&Φ

L 93 87 76 <3 96 BS 85 +

M 93 88 74 «3 84 85 81 C2^2{M2θs

H 89 68 41 10~3 10.1 80 81

O 93 80 58 «3 108 85 90

Q 96 91 70 «3 148 98 90 i; ID marked on Kfβff Lab note

2; inhibition assay on stably maintained EBNAI-depβndnentoriP reporter at indicated concentrations of compounds for 3 days in duplicate of 3Θ4 wed piate and a 24 well plate from triplicate experiments 3; effective concentration at which EBNA1 dependent oriP reporter activity was Inhibited by 50% 4; inhibition assay on transiently transfectβd EBNA1 dependent oriP reporter for 2 days in 24 well plate 6; electraphoredic mobility gel shift assay of EBNAI (306-641) using 1 copy FR, EBNA1 binding site

Once primary hits were identified by high throughput screening, dose-dependency was established using the same efficacy/toxicity cell based assay with 2 fold increments and decrements of chemical concentration. Compounds that appeared to have activity at concentrations that were at least 4-fold lower than toxicity were taken through secondary screens to further define the mechanism of action.

Example 3: Secondary assay for determining mechanism of action

A secondary assay was used to determine the underlying biochemical basis for the chemical effect on EBNAl mediated episome maintenance and/or transcription (Figure 5). These experiments involved more detailed assessments of the effect of the chemical on EBNAl dimerization and interaction with DNA. Further investigation of such effects on dimerization or DNA binding were facilitated by a range of EBNAl C-terminus fragments (amino acids 379-481) expressed in E. coll (Shah et al (1992) J. Virol. 66: 3355-3362; Ambinder et al (1991) J. Virol. 65:1466-1478)

The secondary screening also served to establish whether the chemical had the expected effect on EBV-transformed lymphoblastoid cell growth. An EBNAl inhibitor would preferentially inhibit the growth of EBV-transformed cell lines with EBV episome and have much less effect on the growth of lymphocyte cell lines (LCLs) such as the IB4 cell line with integrated EBV DNA (Kang et al. (2001) Proc Natl Acad Sci USA 98:15233-8). In contrast, we have many early passage EBV episome-positive LCLs which are completely dependent on EBNAl for their growth.

Recent data has indicated that EBNAl residues 379-641 can alone mediate initial plasmid replication in B lymphoblasts (Chaudhuri et al. (2001) Proc Natl Acad Sci USA 98:10085-9, Dhar et al. (2001) Cell 106:287-96, Hirai et al. (2001) Curr Top Microbiol Immunol 258:13-33, Schepers et al. (2001) Embo J20:4588-602, Shirakata et al. Virology 263:42-54). In order to determine whether a possible effect through EBNAl amino acids 379-641 was in initial plasmid replication or in putative recruitment of ORC and mem complexes to initiation sites, DNA replication assays were performed following transfection of EBNAl positive BJAB cells with oriP plasmid in the presence or absence of compound. After 2 days post transfection, the circular plasmid DNA was isolated from cells and digested by Dpnl and BamHI. To determine whether the inhibition of the EBNAl N-terminus association with chromosomes or mediation of transcriptional activation, EBNAl N-terminus interaction with chromosomes were assessed using EBNAl N-terminus-EGFP fusion proteins that have been previously described (Hung et al. (2001) Proc Natl Acad Sci USA 98:1865-70).

Compounds were tested for inhibition of E. co/z-expressed ΕBNA1 binding, in vitro, to cognate DNA (FR). Twenty ng of ΕBNA1 amino acids 386-641, which include the DNA binding and dimerization domain, were incubated with compound at various concentrations prior to addition of ³²P-labeled FR probe and separated on 5% polyacrylamide gels. Compounds H, K, and N exhibited inhibitory activity at 100 μM and compound H showed best inhibition at 10 μM (Figure 9).

Compound H activity was further tested in BJAB or BJAB-FΕ1/SV40-RL (ElO) cells transiently transfected with a oriP-FL-CMV-GFP plasmid. After 3 days, cell viability, FL, and GFP-positive cell number were assayed in DMSO or DMSO with 5 μM or 10 μM compound H. FL activity was 149-fold in DMSO versus 39- or 23-fold in 5 or 10 μM compound H. The number of GFP positive cells decreased in parallel from 12,4 % in DMSO versus 5.2 or 3.8% in 5 μM or 10 μM compound H treated cells, respectively, as further evidence of loss of oriP dependent plasmid. Cell viability was unaffected (Figure 10).

The effect of compound H on the viability of an EBV transformed human cord blood derived B lymphocyte cell line (LCL 1022) was then tested, using IB4 cells as a control. IB4 cells have an integrated EBV genome that is unresponsive to EBNAl inhibition. 1000 IB4 cells per well or 10,000 LCL 1022 cells were plated, grown for 10 days in triplicate 96 well plates in presence of DMSO control or 5 μM compound H. Half of the media was replaced with fresh media containing DMSO or compound H every 3-4 days. At day 10, the number of wells with outgrowth was counted and the relative cell growth was determined from 18 wells each by (CellTiter-Glo^® Luminescent Cell Viability Assay, Promega), which measures ATP released from metabolically live cells (Figure 11). Compound H had a strong negative effect on LCL 1022 and a slight positive effect on IB4 cells (Figure 12).

Example 4: Peptide sequences that inhibit EBNAl interaction with cognate DNA sequence as measured by EBNAl labeled DNA probe gel shift assays.

Peptides were screened using gel shift assays to identify inhibitors of EBNAl function. Successive EBNAl 15 mer peptides overlapping by 11 amino acids, beginning at EBNAl aa376 and ending at aa641, were screened for their ability to inhibit the gel shift of EBNAl aa387-641 or EBNAl aa459-641 with p32-labeled EBNAl cognate DNA sequence. This is the same gel shift inhibition test that was used to assay the activity of the chemical compounds as described above.

EBNAl aa387-641 or EBNAl aa459-641 were incubated with each of the 15 mers before the addition of labeled EBNAl cognate DNA sequence. The mixtures were then assayed on polyacrylamide gel. A Phosphorimager was used to quantify the amount of gel shifted labeled DNA in controls and in response to each of the 62 15 mer peptides. Most EBNAl 15 mer peptides had no inhibitory activity at the initial screening level of 1500 fold excess peptide over EBNAl aa387-641 or EBNAl aa 459-641 protein. The only peptides that had reproducible inhibitory activity at levels 1500 fold excess peptide or less were the peptide shown in the following table.

Table 2. EBNAl peptides that inhibit EBNAl/cognate DNA gel shift.

We conclude that the sequence CYFMVFL (SEQ ID NO: 1) is inhibitory of the EBNAl gel shift in the absence of further sequence at its amino or carboxy terminus. The sequence EGTWVAGVFVYGGSK (SEQ ID NO:5) is also specifically inhibitory.

Example 5: Preclinical testins of inhibitor compounds in (LCD mice

Compounds that have specific effects on EBNAl function in the foregoing assays are tested in mouse models, for example using the ability of LCLs to establish Post Transplant type Lymphoproliferative Disease-like or EBV-associated lymphoma in SCID mice after intraperitoneal inoculation (Brink et al. (1997) J Clin Pathol 50:911-8). Established LCL mice will be treated with chemicals at sub-toxic concentrations to determine whether the compounds affect tumor regression.

Example 6: Activity of additional compounds

The assays described in the foregoing Examples were repeated using several additional compounds. The compounds and results are provided in Table 3, summarized as follows.

- Compound H20 inhibited EBNAl -dependent transcription at 5μM.

- Compound 101 (roscovitine) inhibited EBNAl -dependent transcription at 30μM.

- Compound 102 (shikonin) inhibited EBNAl -dependent transcription at 0.2μM and inhibited in vitro EBNAl -dependent DNA gel shift activity at 5μM (Fig. 13).

- Compound H25 inhibited EBNAl -dependent DNA gel shift at lOμM (Fig. 13). Inhibition of EBNAl -dependent DNA gel shift using Compound H was demonstrated above, at lOμM. Compound K was retested using the same conditions with freshly purchased compound. It reproducibly exhibited inhibition of DNA binding in vitro at lOμM (Fig. 13).

- As shown in Fig. 14, EBNAl peptide CYFMVFLQTHIFAEV (SEQ ID NO:4) inhibited EBNAl -dependent DNA gel shift at Q.6μM.

Table 3 is shown below: TABLE 3

ID Specific action sites Name FW(g) Structure vendor catalogue #

Inhibit EBNA1 DNA N-1-adamantyl-4-[5-(4-methoxybenzylidene)-4- 470.65 Chembridge 5551021 binding activity in oxo-2-thioxo-1,3-thiazolidin-3-yl]butanamide vitro at 1OuM

Inhibit EBNA1 DNA Chembridge 6190924 binding activity in vitro at 1OuM

OJ

H20 Chembridge 5889410

H25 Inhibit EBNA1 DNA (Z)-5-(4-ethoxybenzylidene)-3-methyl-2- 279 Chembridge 5554765 binding activity in thioxothiazolidin-4-one vitro at 1OuM

101 Inhibit EBNA1 roscovitine 354.45 Biomol CC205 transactivation

102 Biomol CT115

EBNA1 Inhibit EBNA1 DNA CYFMVFLQTHIFAEV 1848.21 hydrophobic amino acid in beta synthesized synthesized peptide binding activity in sheet strand in EBNA1 crystal a.a.560- vitro at 0.6uM structure 574

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

All references disclosed herein are incorporated by reference in their entirety.

We claim:

Claims

CIaims

1. A method for treating or preventing an Epstein Ban^* virus related disorder, comprising administering to a subject in need of such treatment an effective amount of a compound that selectively inhibits Epstein Barr virus nuclear antigen 1 (EBNAl) function in maintaining an EBNAl -dependent episome, wherein the compound is a (4-oxo-2-thioxo-l,3- thiazolidin-3-yl)propanoate or propanoic acid, a [((methyl)phenoxy)methyl]benzoic acid, 2- [[3-[bis(2-hydroxyethyl)amino]-5,10-bis(l-piperidyl)-2,4,7,9-tetrazabicyclo[4.4.0]deca- 2,4,7,9, 11 -pentaen-8-yl]-(2-hydroxyethyl)amino]ethanol, &,T, 10, 11 -Tetramethoxymethane, Emodine, 2-p-methoxyphenylmethyl-3-acetoxy-4-hydroxypyrrolidine, ethyl 3-{[(4- methoxyphenoxy)acetyl]amino}benzoate, N-l-adamantyl-4-[5-(4-methoxybenzylidene)-4- oxo-2-thioxo- 1 ,3 -thiazolidin-3-yl]butanamide, 2-( 1 H-benzimidazol-2-yl)-4- methylphthalazin-l(2H)-one, (Z)-5-(3,4-dihydroxybenzylidene)-3-methyl-2- thioxothiazolidin-4-one, (Z)-5-(4-ethoxybenzylidene)-3-methyl-2-thioxothiazolidin-4-one, roscovitine, shikonin, or a salt, solvate, or analog thereof.

2. The method of claim 1, wherein the (4-oxo-2-thioxo-l,3-thiazolidin-3-yl)propanoate or propanoic acid is 2-(dimethylamino)ethyl 3-[5-(4-methylbenzylidene)-4-oxo-2-thioxo-l,3- thiazolidin-3-yl]propanoate hydrochloride, or 3-[5-(4-ethylbenzylidene)-4-oxo-2-thioxo-l,3- thiazolidin-3-yl]propanoic acid, or a salt, solvate, or analog thereof.

3. The method of claim 1, wherein the [((methyl)phenoxy)methyl]benzoic acid is 3-[(4- { [1 -(2-methylρhenyl)-4,6-dioxo-2-thioxotetrahydro-5(2H)- pyrimidinylidene]methyl}phenoxy)methyl]benzoic acid, 3-[(4-{ [1 -(4-methylphenyl)-2,4,6- trioxotetrahydro-5(2H)-pyrimidinylidene]methyl}phenoxy)methyl]benzoic acid, 3-[(4-{[l-(3- methylphenyl)-2,4,6-trioxotetrahydro-5(2H)- pyrimidinylidene]methyl}phenoxy)methyl]benzoic acid, 3-[(4-{ [1 -(3-methylphenyl)-4,6- dioxo-2-thioxotetrahydro-5(2H)-pyrimidinylidene]methyl}phenoxy)methyl]benzoic acid, or 3-({4-[(2,4,6-trioxotetrahydro-5(2H)-pyrimidinylidene)methyl]phenoxy}methyl)benzoic acid, or a salt, solvate, or analog thereof.

4. The method of claim 1 , wherein the compound is compound is N- 1 -adamantyl-4- [5 - (4-methoxybenzylidene)-4-oxo-2-thioxo-l,3-thiazolidin-3-yl]butanamide, 3-[(4-{[l-(2- methylphenyl)-4,6-dioxo-2-thioxotetrahydro-5(2H)- pyrimidinylidene]methyl}phenoxy)methyl]benzoic acid or 3-[(4-{[l-(3-methylphenyl)-4,6- dioxo-2-thioxotetrahydro-5(2H)-pyrimidinylidene]methyl}phenoxy)methyl]benzoic acid or a salt, solvate, or analog thereof.

5. The method of claim 4, wherein the compound is compound N-l-adamantyl-4-[5-(4- methoxybenzylidene)-4-oxo-2-thioxo-l,3-thiazolidin-3-yl]butanamide or a salt, solvate, or analog thereof.

6. A method for treating or preventing an Epstein Barr virus related disorder, comprising: administering to a subject in need of such treatment an effective amount of a compound that selectively inhibits Epstein Barr virus nuclear antigen 1 (EBNAl) function in maintaining an EBNAl -dependent episome, wherein the compound comprises at least one of the chemical structures:

7. A method for treating or preventing an Epstein Barr virus related disorder, comprising: administering to a subject in need of such treatment an effective amount of a compound that selectively inhibits Epstein Barr virus nuclear antigen 1 (EBNAl) function in maintaining an EBNAl -dependent episome, wherein the compound comprises at least one of the chemical structures:

8. A polypeptide comprising SEQ ID NO: 1 and/or SEQ ID NO:5, wherein the polypeptide is not EBNAl .

9. The polypeptide of claim 8, comprising SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4.

10. The polypeptide of claim 8, wherein the polypeptide is less than about 180 amino acids in length.

11. The polypeptide of claim 8, wherein the polypeptide is less than about 100 amino acids in length.

12. The polypeptide of claim 8, wherein the polypeptide is less than about 75 amino acids in length.

13. The polypeptide of claim 8, wherein the polypeptide is less than about 50 amino acids in length.

14. The polypeptide of claim 8, wherein the polypeptide is less than about 40 amino acids in length.

15. The polypeptide of claim 8, wherein the polypeptide is less than about 30 amino acids in length.

16. The polypeptide of claim 8, wherein the polypeptide is less than about 20 amino acids in length.

17. The polypeptide of any of claims 8-16, wherein the polypeptide is a fragment of EBNAl.

18. The polypeptide of any of claims 8-16, wherein the polypeptide is non-hydrolyzable.

19. A method for treating or preventing an Epstein Barr virus related disorder, comprising: administering to a subject in need of such treatment an effective amount of a polypeptide of any of claims 8-18 to selectively inhibit Epstein Barr virus nuclear antigen 1 (EBNAl) function in maintaining an EBNAl -dependent episome.

20. The method of any of claims 1-7 or 19, wherein the Epstein Barr virus related disorder is cancer or lymphoproliferative disease.

21. The method of claim 20, wherein the cancer is lymphoma or carcinoma.

22. The method of claim 21 , wherein the lymphoma is Hodgkin' s Disease, Non- Hodgkin's Lymphoma or Burkitt Lymphoma.

23. The method of claim 21 , wherein the carcinoma is nasopharyngeal cancer.

24. The method of claim 20, wherein the lymphoproliferative disease is post transplantation lymphoproliferative disease (PTLD).

25. The method of claim any of claims 1-7 or 19, wherein the Epstein Barr virus related disorder is infectious mononucleosis.

26. The method of claim any of claims 1-7 or 19, wherein the subject is immunocompromised.

27. The method of claim 26, wherein the immunocompromised subject has malaria, is infected with HIV, is a subject receiving a tissue or organ transplant, or is receiving immunosuppressive therapy.

28. The method of claim 26, wherein the compound is administered concurrently with a cancer therapy.

29. A method for identifying compounds that selectively inhibit Epstein Barr virus nuclear antigen 1 (EBNAl) function in maintaining an EBNAl -dependent episome, comprising providing at least one cell that expresses a first reporter sequence operably linked to an EBNAl -dependent promoter sequence and a second reporter sequence operably linked to a promoter sequence that is not EBNAl -dependent, contacting the at least one cell with a compound, and determining expression of the first reporter sequence and the second reporter sequence, wherein a compound that decreases expression of the first reporter sequence in a greater amount than the second reporter sequence is a compound that selectively inhibits Epstein Barr virus nuclear antigen 1 (EBNAl) function in maintaining an EBNAl -dependent episome.

30. The method of claim 29, wherein the compound decreases expression of the first reporter sequence but not the second reporter sequence.

31. The method of claim 29, wherein the at least one cell is at least one human cell.

32. The method of claim 29, wherein the first reporter sequence is contained in a first cell and the second reporter sequence is contained in a second cell.

33. The method of claim 29, wherein the first and second reporter sequences are contained in the same cell.

34. The method of claim 29, wherein the first reporter sequence is contained in a first construct.

35. The method of claim 34, wherein the first construct is a plasmid.

36. The method of claim 29, wherein the second reporter sequence is contained in a second construct.

37. The method of claim 36, wherein the second construct is a plasmid.

38. The method of claim 29, wherein the first reporter and the second reporter are, or encode, similar types of compounds.

39. The method of claim 38, wherein the compounds are luciferase proteins, enzymes or fluorescent proteins.

40. The method of claim 29, wherein the expression of the first reporter and the second reporter are assayed using enzyme assays, fluorescence assays, immunoassays, luminescence assays, or nucleic acid expression assays.

41. The method of claim 40, wherein the assays are similar types of assays.

42. The method of claim 29, wherein the at least one cell recombinantly expresses EBNAl.

43. The method of claim 29, further comprising testing the compound that selectively inhibits for its effect on cell viability of EBV-infected and/or non-infected cells.

44. The method of claim 43, wherein the compound is tested on cells in which the EBV genome is maintained as an episome or in which the EBV genome is integrated into a chromosome.

45. The method of claim 29, further comprising testing the compound that selectively inhibits for its effect on binding of EBNAl protein to EBNAl -responsive promoter sequence or a chromosome.

46. The method of claim 45, wherein the testing is performed using a gel shift assay.

47. The method of claim 29, further comprising testing the compound that selectively inhibits for inhibition of plasmid replication or transcription.

48. The method of claim 29, further comprising testing the compound that selectively inhibits for reduction of latent EBV infection of a cell.

49. The method of claim 48, wherein the infection is in a lymphocyte.

50. The method of claim 29, further comprising testing the compound that selectively inhibits for inhibition of dimerization of EBNAl.

51. The method of claim 29, further comprising testing the compound that selectively inhibits for an increase in EBNAl degradation.

52. The method of claims 29-51 , wherein the at least one cell is a B lymphocyte.

53. A method for identifying compounds that selectively inhibit Epstein Barr virus nuclear antigen 1 (EBNAl) function in maintaining an EBNAl -dependent episome, comprising providing at least one cell that expresses a reporter sequence operably linked to an EBNAl -dependent promoter sequence, contacting the at least one cell with a compound, and determining expression of the reporter sequence, wherein a compound that decreases expression of the reporter sequence is a compound that selectively inhibits Epstein Barr virus nuclear antigen 1 (EBNAl) function in maintaining an EBNAl -dependent episome.

54. The method of claim 53, further comprising determining toxicity of the compound.

55. The method of claim 54, wherein determining toxicity comprises measuring ATP level in the at least one cell that has been contacted with the compound.

56. The method of claim 53, wherein the at least one cell is at least one human cell.

57. The method of claim 53, wherein the expression of the reporter sequence is determined in a first cell and the toxicity is determined in a second cell.

58. The method of claim 53, wherein the expression of the reporter sequence and the toxicity are determined in the same cell.

59. The method of claim 53, wherein the reporter sequence is contained in a construct.

60. The method of claim 59, wherein the construct is a plasmid.

61. The method of claim 53, wherein the reporter sequence encodes a luciferase protein, enzyme or fluorescent protein.

62. The method of claim 53, wherein the expression of the reporter sequence is assayed using an enzyme assay, fluorescence assay, immunoassay, luminescence assay, or nucleic acid expression assay.

63. The method of claim 53 , wherein the at least one cell recombinantly expresses EBNAl.

64. The method of claim 53, further comprising testing the compound that selectively inhibits for its effect on cell viability of EBV-infected and/or non-infected cells.

65. The method of claim 64, wherein the compound is tested on cells in which the EBV genome is maintained as an episome or in which the EBV genome is integrated into a chromosome.

66. The method of claim 53, further comprising testing the compound that selectively inhibits for its effect on binding of EBNAl protein to EBNAl -responsive promoter sequence or a chromosome.

67. The method of claim 66, wherein the testing is performed using a gel shift assay.

68. The method of claim 53, further comprising testing the compound that selectively inhibits for inhibition of plasmid replication or transcription.

69. The method of claim 53, further comprising testing the compound that selectively inhibits for reduction of latent EBV infection of a cell.

70. The method of claim 69, wherein the infection is in a lymphocyte.

71. The method of claim 53, further comprising testing the compound that selectively inhibits for inhibition of dimerization of EBNAl.

72. The method of claim 53, further comprising testing the compound that selectively inhibits for an increase in EBNAl degradation.

73. The method of claims 53-72, wherein the at least one cell is a B lymphocyte.