WO2006017346A1 - Treatment of viral infections by means o proteasome inhibitors - Google Patents

Abstract

The invention relates to treatment of cells or humans carrying or infected with a virus capable of causing an immunodeficiency disease with particular compounds, including proteasome inhibitors.

Description

TREATMENT OF HIV INFECTIONS BY MEANS O PROTEASOME INHIBITORS

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S Provisional Patent Application No.

60/587,810, filed July 13, 2004, the entire teachings of which are hereby incorporated by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Research supporting this application was carried out by the United States of America as represented by the Secretary, Department of Health and Human Services.

BACKGROUND OF THE INVENTION

The human immunodeficiency virus type 1 (HIV-I, also referred to as HTLV-III LAV or HTLV-III/LAV) and, to a lesser extent, human immunodeficiency virus type 2 (HIV- 2) is the etiological agent of the acquired immune deficiency syndrome (AIDS) and related disorders. Barre-Sinoussi, et al., Science, 220:868-871 (1983); Gallo, et al, Science,

224:500-503 (1984); Levy, et al., Science. 225:840-842 (1984); Popovic, et al., Science. 224:497-500 (1984); Sarngadharan, et al., Science. 224:506-508 (1984); Siegal, et al., N₁ Engl. J. Med.. 305:1439-1444 (1981); Clavel, F., AIDS, 1:135-140. This disease is characterized by a long asymptomatic period followed by the progressive degeneration of the immune system and the central nervous system. Studies of the virus indicate that replication is highly regulated, and both latent and lytic infection of the CD4 positive helper subset of T- lymphocytes occur in tissue culture. Zagurv, et al.. Science, 231 :850-853 (1986). The expression of the virus in infected patients also appears to be regulated as the titer of infectious virus remains low throughout the course of the disease. Both HIV-I and 2 share a similar structural and function genomic organization, having regulatory genes such as tat, rev, nef, in addition to structural genes such as env, gag and pol. While AIDS, itself, does not necessarily cause death, in many individuals the immune system is so severely depressed that various other diseases (secondary infections or unusual tumors) such as herpes, cytomegalovirus, Kaposi's sarcoma and Epstein-Barr virus related lymphomas among others occur, which ultimately results in death. These secondary infections may be treated using other medications. However, such treatment can be adversely affected by the weakened immune system. Some humans infected with the AIDS virus seem to live many years with little or no symptoms, but appear to have persistent infections. Another group of humans suffers mild immune system depression with various symptoms such as weight loss, malaise, fever and swollen lymph nodes. These syndromes have been called persistent generalized lymphadenopathy syndrome (PGL) and AIDS related complex (ARC) and may or may not develop into AIDS. In all cases, those infected with the HIV are believed to be persistently infective to others.

The activation of the latent HIV provirus from the asymptomatic period has been reported to be governed by a long terminal repeat (LTR) in the viral DNA. See, e.g. Ranki, A., et al., Lancet ii: 589-593 (1987); Fauci, A.S., et al., Science. 239:617-622 (1988); Zagury, D., et al., Science. 23J_:850-853 (1985); Mosca, J.D., Nature (London), 325:67-70 (1987). The activity of HIV-I is determined by the complex interaction of positive and negative transcriptional regulators that bind to specific sequences within the LTR. Cullen, B. R., et al., Cell, 58:423-426 (1989). Changes in the quantity or quality of these factors may underlie the activation of transcription of HIV-I and HIV-2 latent provirus by a myriad of stimuli. See, e.g. Fauci, A.S., Science. 239:617-622 (1988); Griffin, G.E., et al., Nature (London), 339:70- 73 (1989); Nabel, G., et al., Science. 239: 1299-1302 (1988). Specifically, phorbol 12- myristate-13-acetate (PMA) and Tumor Necrosis Factor-a (TNFa) are believed to be potent activators. In particular, TNFa is present in markedly enhanced levels in HIV infected individuals, suggesting that the cytokine plays an important role in the pathogenesis of AIDS. Lahdevirta, J., Am. J. Med.. 85:289-291 (1988).

Most known methods for treating individuals infected by HIV have focused on preventing integration of the provirus into the host cell's chromosome. One overlooked area of interest has been drugs that affect latently infected cells so that virus may be cleared from the infected individual completely. Many of the proposed therapeutic methods, however, have not proven clinically effective. While current antiretroviral agents can control HIV replication, they have not eliminated latently infected cells. Since viral reactivation is necessary for targeting by antiviral drugs (Brooks, et al. 2003. Molecular characterization, reactivation, and depletion of latent HIV. Immunity 19:413-23), identification of new approaches that eject HIV from latency when used together with effective antiviral therapy can lead to the reduction of latent HIV reservoirs in an infected patient.

It thus would be desirable to have new compounds and methods that can treat cells latently infected with HIV. It would be particularly desirable to have a new therapy that can be used to treat cells already infected, by means other than by preventing integration of the virus.

SUMMARY OF THE INVENTION

We have now discovered that certain compounds, including proteasome inhibitors, and compounds of the following formulae herein, are useful for methods of treating cells infected by immunodeficiency viruses and methods of preventing cells from becoming infected by immunodeficiency viruses, preferably human immunodeficiency viruses such as HIV.

In one aspect, the compounds are those of formula I or formula II:

formula (I) wherein R, is independently -C(O)NR²R ³³,, - -CC((OO))SSRR²²,, or -C(O)OR², wherein R² and R³ are each independently alkyl, cycloalkyl, aryl, heterocyclyl, heteroaryl, aralkyl, or heteroarylalkyl, each being optionally substituted with 1-3 substituents, and R⁴ and R⁵ are each independently alkyl; or

formula (II) wherein R⁴ and R⁵ are each independently alkyl.

Further aspects are those wherein the compounds of formulae (I) or (II) are:

wherein Ri is as defined above; and

Further aspects of the invention are methods of treating or preventing HIV infection in a subject comprising administration of an effective amount of a proteasome inhibitor, including those having the structure of the formulae herein. The proteasome inhibitor can have a pyrrolidin-2-one ring in its structure or a β-lactone ring in its structure.

In certain aspects, compounds (e.g., compounds of the formulae herein) are used for purposes of inducing lytic replication in cells latently infected by immunodeficiency viruses, preferably human immunodeficiency viruses such as HIV.

The compounds of the present invention can induce lytic replication in cells latently infected with an immunodeficiency virus such as HIV by targeting product(s) of a gene or genes, which is(are) differentially expressed in latently infected cells and/or lytic replicating cells.

In one embodiment, the compounds of the present invention can treat cells infected acutely and chronically by immunodeficiency viruses, for example, HIV, preferably HIV-I, and thus can be used to treat humans infected by HIV. For example, treatment of those diagnosed as having AIDS as well as those having ARC, PGL and those not yet exhibiting such conditions.

Other aspects are a method of inhibiting HIV replication in a cell comprising administration to the cell of an effective amount of a proteasome inhibitor; a method of treating latently HIV-infected cells in a subject comprising administration to the subject of an effective amount of a proteasome inhibitor; and a method of modulating lytic replication in an HIV-infected cell in a subject comprising administration to the subject of an effective amount of a proteasome inhibitor. In the methods, the cell is preferably a lymphocytic cell or a monocytic cell. In certain embodiments, the cell is a human cell.

In one aspect, the invention provides a method of treating HIV infection in a subject. The method comprises identifying a subject as in need of reactivation of a replication process in latent HIV-infected cells; administering to the subject an effective amount of a proteasome inhibitor to reactivate the replication process; and administering to the subject one or more HIV antiviral agents to inhibit induced lytic replication in the cells.

In another aspect, the invention provides a method of treating HIV infection in a subject. The method comprises the steps of identifying a subject or cell as in need of reactivation of a replication process in latent HIV-infected cells; administering to the subject or cell an effective amount of a proteasome inhibitor having a pyrrolidin-2-one ring in its structure to reactivate the replication process; and administering to the subject or cell one or more HIV antiviral agents to inhibit induced lytic viral replication in the subject and/or the cells. The method provides a means for decreasing or eliminating the pool of latently infected cells and also decreasing the viral population.

In another aspect, the invention provides a method of treating HIV infection in a subject. The method comprises the steps of identifying a subject or cell as in need of reactivation of a replication process in latent HIV-infected cells; administering to the subject or cell an effective amount of a proteasome inhibitor having a β-lactone ring in its structure to reactivate the replication process; and administering to the subject or cell one or more HIV antiviral agents to inhibit the induced lytic viral replication in the subject and/or the cells. The method provides a means for decreasing or eliminating the pool of latently infected cells and also decreasing the viral population.

In another aspect, the invention provides a method of treating HIV infection in a subject or cell. The method comprises the steps of identifying a subject or cell as in need of reactivation of a replication process in latent HIV-infected cells; administering to the subject or cell an effective amount of a proteasome inhibitor having the structure of formula (I) or (II):

formula (I)

wherein Ri is independently -C(O)NR²R³, -C(O)SR², or -C(O)OR², wherein R² and R³ are each independently alkyl, cycloalkyl, aryl, heterocyclyl, heteroaryl, aralkyl, or heteroarylalkyl, each being optionally substituted with 1 -3 substituents, and R⁴ and R⁵ are each independently alkyl; or

formula (II)

wherein R⁴ and R⁵ are each independently alkyl, to reactivate the replication process; and administering to the subject or cell one or more HIV antiviral agents to inhibit the induced lytic replication in the subject and/or the cells. The method provides a means for decreasing or eliminating the pool of latently infected cells and also decreasing the viral population.. In a preferred embodiment, the proteasome inhibitor is c/αsto-lactacystin β-lactone. In preferred embodiments, the additional agent(s) are a reverse transcriptase inhibitor, a protease inhibitor, or combination thereof, more preferably a combination of a reverse transcriptase inhibitor and a protease inhibitor.

In another aspect, the invention provides a method of modulating lytic replication in an HIV-infected cell in a subject identified as in need of lytic replication modulation. The method comprises administration to the subject of an effective amount of a proteasome inhibitor.

In preferred embodiments of any of the methods described above, the cell is a lymphocytic cell or a monocytic cell. In preferred embodiments, the cell is a human cell, i.e., any human cell capable of sustaining a latent provirus.

In another aspect, the invention provides a method of reducing latent HIV reservoirs in an HIV-infected subject. The method comprises the steps of identifying the subject as in need of reactivation of replication processes in latent HIV-infected cells; and administration to the subject of an effective amount of a proteasome inhibitor for reactivation of viral replication processes in latent HIV-infected cells, e.g., such that latent HIV reservoirs in an infected subject or infected cells are reduced.

In another aspect, the invention provides a method of increasing expression of p24 in a latently HIV-infected cell. The method comprises administration to the cell of an effective amount of a proteasome inhibitor, preferably a proteasome inhibitor of formula I or II herein.

In another aspect, the invention provides a method of activating latent HIV-provirus in a cell in a subject. The method comprises identifying the subject as in need of reactivation of replication processes in latent HIV-infected cells; and administration to the subject of an effective amount of a proteasome inhibitor for reactivation of replication processes in latent HIV-infected cells. In preferred embodiments, the proteasome inhibitor affects one or more catalytic activities of the proteasome, more preferably chymotryptic, tryptic, or peptidyl glutamyl catalytic activities, more preferably two or three of these activities.

In any of the methods described above, the method preferably comprises administration (to the cell or subject) of one or more additional anti-HIV therapeutic agents, more preferably one or more reverse transcriptase inhibitors, one or more protease inhibitors, or a combination thereof. In preferred embodiments of the above methods, the proteasome inhibitor is c/αsto-lactacystin β-lactone.

In still another aspect, the invention provides a method of screening for a compound capable of activating latent HIV-infected cells, the method comprising contacting a latent HIV-infected cell (e.g., an ACH-2 cell or Jl.1 cell or Ul cell) with a test compound and determining the level of p24 expression in the cell. In preferred embodiments, increased expression of p24 in cells treated with a test compound relative to non-treated cells indicates that the test compound is capable of activating latent HIV-infected cells. In preferred embodiments, the test compound comprises a pyrrolidin-2-one ring or a β-lactone ring structural moiety.

Other aspects are a method of reducing latent HIV reservoirs in an HIV-infected subject comprising administration to the subject of an effective amount of a proteasome inhibitor; a method of increasing (e.g., 10-15 fold relative to cells treated with AZT) expression of p24 in a latently HIV-infected cell comprising administration to the cell of an effective amount of a proteasome inhibitor (e.g., a compound of the formulae herein); a method of activating latent HIV-provirus in a cell in a subject comprising administration to the subject of an effective amount of a proteasome inhibitor (e.g., a compound of the formulae herein); a method of activating latent HIV-provirus in a cell in a subject comprising administration to the subject of an effective amount of a proteasome inhibitor (e.g., a compound of the formulae herein), wherein the proteasome inhibitor affects one or more catalytic activities of the proteasome. The one or more catalytic activities can be chymotryptic, tryptic, or peptidyl glutamyl. As such, in various embodiments the proteasome inhibitor(s) used in the methods herein are those that affect one, two or all three activities, in any combination. In one aspect, a broader range of proteasomal inhibition may be desired for viral reactivation. Clastolactacystin-beta-lactone, which causes viral reactivation, affects all three catalytic activities of the proteasome.

The methods delineated herein can further include administration of one or more additional anti-HIV therapeutic agents. The additional agent(s) can be one or more reverse transcriptase inhibitors, one or more protease inhibitors, or combination thereof. In one aspect the proteasome inhibitor in the methods herein is c/αsfo-lactacystin β-lactone.

Another aspect is a method of screening for a compound capable of activating latent HIV-infected cells comprising contacting an ACH-2 cell or Jl .1 cell or Ul cell with a test compound and determining the level of p24 expression; the method can be wherein increased expression of p24 in cells treated with a test compound relative to non-treated cells indicates a compound capable of activating or reactivating latent HIV-infected cells. The test compound can include those of any of the formulae delineated herein, including those having a pyrrolidin-2-one ring or a β-lactone ring structural moiety.

Another aspect is a method of reducing latent HIV-reservoirs in a subject by controlled activation of viral replication including administration of an effective amount of a proteasome inhibitor. Controlled activation is that activation initiated by administration of an effective amount of proteasome inhibitor such that the proteasome inhibitor reactivates (directly or indirectly) replication processes in latent HIV-infected cells. The latent HIV- reservoirs are collections of latent HIV-infected cells, that is cells in which the HIV- replication is considered to be in a latent state. The method can further include administration with one or more antiviral agents, thus both depleting the latent cell reservoir and inhibiting induced viral lytic replication, whereupon the cells in that state are subjected to and susceptible to the antiretroviral therapy, which controls viral proliferation.

The methods delineated herein include administering to a subject (e.g., a human or an animal) in need thereof an effective amount of one or more proteasome inhibitors, e.g., compounds as delineated herein. The methods can also include the step of identifying that the subject is in need of treatment of diseases or disorders described herein, e.g., identifying that the subject is in need of reactivation of a replication process or processes in latent HIV- infected cells. The identification can be in the judgment of a subject or a health professional and can be subjective (e.g., opinion) or objective (e.g., measurable by a test or a diagnostic method). Tests for HIV infection are known in the art and include polymerase chain reaction-based (PCR-based) amplification and detection of viral RNA; Western blot detection of anti-HIV antibodies; agglutination assays for anti-HIV antibodies; ELISA-based detection of HIV-specific antigens (e.g., p24); line immunoassay (LIA); and other methods known to one of ordinary skill in the art. In each of these methods, a sample of biological material, such as blood, plasma, semen, or saliva, is obtained from the subject to be tested. Thus, the methods of the invention can include the step of obtaining a sample of biological material (such as a bodily fluid) from a subject; testing the sample to determine the presence or absence of detectable HIV infection, HIV particles, or HIV nucleic acids; and determining whether the subject is in need of treatment according to the invention, i.e., identifying whether the subject is in need of reactivation of a replication process or processes in latent HIV-infected cells.

The methods delineated herein can further include the step of assessing or identifying the effectiveness of the treatment or prevention regimen in the subject by assessing the presence, absence, increase, or decrease of a marker, including a marker or diagnostic measure of HIV infection, HIV replication, viral load, or expression of an HIV infection marker; preferably this assessment is made relative to a measurement made prior to beginning the therapy. Such assessment methodologies are known in the art and can be performed by commercial diagnostic or medical organizations, laboratories, clinics, hospitals and the like. As described above, the methods can further include the step of taking a sample from the subject and analyzing that sample. The sample can be a sampling of cells, genetic material, tissue, or fluid (e.g., blood, plasma, sputum, etc.) sample. The methods can further include the step of reporting the results of such analyzing to the subject or other health care professional. The method can further include additional steps wherein (such that) the subject is treated for the indicated disease or disease symptom.

As used herein, the terms "subject" and "patient" are used interchangeably. As used herein, the terms "subject" and "subjects" refer to an animal, preferably a mammal including a non-primate (e.g., a cow, pig, horse, cat, dog, rat, and mouse) and a primate (e.g., a monkey, ape, monkey, or human), and more preferably a human. In one embodiment, the subject is an immunocompromised or immunosuppressed mammal, preferably a human (e.g., an HIV infected patient). In another embodiment, the subject is a farm animal (e.g., a horse, a cow, a pig, etc.) or a pet (e.g., a dog or a cat). In a preferred embodiment, the subject is a human.

The invention also provides pharmaceutical compositions comprising a compound of any of the formulae herein and a suitable carrier therefore for use in the methods and conditions referred to above.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the flow cytometric analysis of chronically infected ACH-2 cells before and after induction. Uninduced cells and cells from serial time points were fixed and permeabilized for intracellular p24 labeling. As an isotype control, cell samples were labeled with mouse IgGl . For each sample, 100,000 events were collected. In the figure, each sample histogram labeled for p24 (red) is overlaid with the control histogram labeled for the isotype control (green). (A) Uninduced ACH-2 cells, showing minimal p24 accumulation with 8.2% of cells infected, (B) ACH-2 cells at 0.5 hours post induction (p.i.) with 7.4% of cells positive for p24, (C) ACH-2 cells at 6 hours p.i, with 61.6% cells infected, (D), (E), and (F) ACH-2 cells at 12, 18, and 24 hours p.i., respectively, showing complete infection. Flow cytometric analysis was performed on all batches of cells to ensure active replication of HIV following induction with PMA. Data from one induction experiment is shown. Data indicate that viral replication occurs in an ordered manner post induction, and complete infection of cells is achieved within 12 hours post induction of chronically infected ACH-2 cells.

Figure 2 shows the levels of expression of multiply spliced (MS HIV-I) and unspliced (US HIV-I) mRNA prior and post induction of chronically infected ACH-2 cells. Real time RT-PCR reactions were carried out using Taqman probes specific for early (multiply spliced) and late (unspliced) transcripts of HIV-I , tagged with FAM and TAMRA fluorescent dyes at the 5' and 3' ends respectively. Reactions were performed in triplicate for each time point as described in the Methods section and average values are shown. Maximal fold change in mRNA levels for early transcripts (MS HIV-I) was observed 8 hours post induction. Fold change for late transcripts (US HIV-I) showed maximal increase 18 hours post induction. Figure 3 shows the hierarchical clustering of differentially expressed cellular genes before and after induction of chronically infected ACH-2 cells. The figure shows the hierarchical clustering of the cellular genes that showed significant differential expression (p < 0.001) across the time course (before induction up to 96 hours post induction), following reactivation of chronically infected ACH-2 cells as per the criteria described in the Methods. Genes that are shown in red showed up regulation, those in green were down regulated, while those that did not show any change with respect to normalized matched control are shown in black. The gray areas indicate missing data for the given gene and time point. The magnified panels indicate selected kinetic profiles that are seen before and following induction into active viral replication. (A) Up regulation of selected genes observed before induction; (B) Up regulation of genes immediately following induction; (C) Genes that are up regulated prior to induction and down regulated 12-24 hours post induction; (D) Genes that are up regulated in the early stage following reactivation, but are down regulated in the intermediate stage; (E) Genes that are down regulated before induction but are up regulated in the intermediate stage followed by down regulation in the late stage (48-96 hours p.i.).

Figure 4 shows the trends seen in pathways that show differential expression before and after induction of chronically infected ACH-2 cells. Pathway profiles observed prior to induction and following reactivation of ACH-2 cells with PMA over a period of 96 hours. The figure shows the number of genes in each pathway that were differentially expressed in a particular pathway, (A) indicates the pathways that were maximally altered prior to induction. (B) includes the pathways that showed maximum change during the early phase of the lytic cycle, (0.5-8 hours p.i.). (C) represents the pathways that showed maximal change during the period of 12-24 hours post induction. Most pathways did not show any change during the period of 48-96 hours post induction. The groups above are a selected representation of the various pathways that changed differentially prior to induction and/or over the time course studied. Classification of the altered genes into various pathways was performed using the CGAP pathway databases.

Figure 5 shows the hierarchical clustering of genes that show differential expression across three chronically infected cell lines prior to induction. Hierarchical clustering of differentially expressed genes that show a significant change in expression (p < 0.001), in the chronically infected cell lines ACH-2, Ul and Jl .1. Genes shown in red are up regulated, those in green exhibit down regulation, while black indicates normal expression. Gray areas indicate missing values. Many genes are altered similarly across the cell lines. Each cell line also shows some unique patterns of cellular expression. Data are the average of values from eight independent samples per cell line. The magnified portions of the cluster highlight some of the patterns of gene expression across the cell lines. (A) shows genes that are up regulated in all three cell lines; (B) shows genes that are down regulated in all three cell lines; (C) indicates the genes that are up regulated in ACH-2 and Jl .1 and down regulated in Ul ; (D) indicates genes which show no significant similarity in their expression in the three cell lines.

Figure 6 shows the effects of specific agents on HIV p24 production in chronically infected ACH-2 cells. Different concentrations of (A) the proteasome inhibitor, clastolactacystin-beta-lactone or (B) Resveratrol, an Egrl activator or (C) Trichostatin, a HDAC inhibitor were tested in chronically infected ACH-2 cells treated with 250 nM AZT. Samples were collected 24 hours after addition of agent and p24 concentrations were determined by ELISA. p24 production from cells treated by TNF-alpha was used as a positive control in ACH-2 cells compared to control (AZT treated cells). p24 production from untreated cells (No AZT) was also determined. Experiments were performed in triplicate and are representative of three independent experiments. Microarray data for the specific genes targeted are shown for each agent tested.

Figure 7 shows the effect of clastolactacystin-beta-lactone (CLBL), a proteasome inhibitor, on HIV latently infected Jurkat clones. Several different HIV latently infected cloned cell lines, derived from Jurkat lymphocytic cells, showed different maximal response to reactivation of latent HIV by 10 μM of CLBL. The effect of CLBL ranged from 50-500% of the effect caused by TNF-alpha on the same latently infected Jurkat clones.

Figure 8 shows the effect of a proteasome inhibitor on viral reactivation from aviremic patient samples. Aliquots of the aviremic patient sample were tested with different concentrations of CLBL and assayed for p24 expression 5 days post drug addition. p24 levels are expressed as a percentage of p24 expression caused by TNF-alpha (a known reactivating agent) on an aliquot of the aviremic patient sample. A dose dependent increase in p24 expression was observed with CLBL ranging from 130-160% of TNF alpha induced p24 expression levels.

Figure 9 shows the effect of proteasome inhibitor treatment on reactivation of aviremic patient samples. Table 1 : Functionally related genes that were differentially expressed prior to induction in chronically infected ACH-2 cells. List of selected classes of genes based on known function that are differentially expressed in latently infected ACH-2 cells, relative to uninfected parental cell line, A3.01. A number of genes involved in similar cellular functions previously not associated with presence of pro viral HIV were altered coordinately even during the latent non-replicative stage.

Table 1: Functionally related genes that were differentially expressed prior to induction in chronically infected ACH-2 cells.

DETAILED DESCRIPTION OF THE INVENTION It has been discovered that proteasome inhibitors, e.g., compounds of the formulae herein can be used to reactivate replication processes in cells infected by an immunodeficiency virus, preferably human cells infected with HIV and thus can be used for treatment in HIV-infected individuals.

As used herein, the term "alkyl" refers to a straight-chained or branched hydrocarbon group containing 1 to 12 carbon atoms. The term "lower alkyl" refers to a C1-C6 alkyl chain. The terms "cycloalkyl" or "cyclyl" refer to a hydrocarbon monocyclic or bicyclic ring system having at least one non-aromatic ring wherein 0, 1, 2, 3, or 4 atoms of each ring may be substituted by a substituent. Examples of cycloalkyl groups include cyclopentyl, cyclohexyl, cyclohexenyl, bicyclo[2.2.1]hept-2-enyl, dihydronaphthalenyl, and the like.

The term "aryl" refers to a hydrocarbon monocyclic, bicyclic or tricyclic aromatic ring system wherein 0, 1, 2, 3, or 4 atoms of each ring may be substituted by a substituent. Examples of aryl groups include phenyl, naphthyl and the like. The term "arylalkyl" or the term "aralkyl" refers to alkyl substituted with an aryl. The term "arylalkoxy" refers to an alkoxy substituted with aryl.

The term "heteroaryl" refers to an aromatic 5-8 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ring system having 1-3 ring heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selected from O, N, or S, and the remainder ring atoms being carbon (with appropriate hydrogen atoms unless otherwise indicated) wherein 0, 1, 2, 3, or 4 atoms of each ring may be substituted by a substituent. Examples of heteroaryl groups include pyridyl, furyl or furanyl, pyrrolyl, oxazolyl, pyrimidinyl, quinazolinyl, imidazolyl, benzimidazolyl, thienyl, indolyl, thiazolyl, and the like. The term "heteroarylalkyl" or the term "heteroaralkyl" refers to an alkyl substituted with a heteroaryl. The term "heteroarylalkoxy" refers to an alkoxy substituted with heteroaryl.

The term "heterocyclyl" or "heterocycloalkyl" refers to a nonaromatic 5-8 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ring system comprising 1- 3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selected from O, N, S, B, P or Si, wherein 0, 1, 2, 3, or 4 atoms of each ring may be substituted by a substituent. Examples of heterocyclyl groups include tetrahydrofuryl, piperidinyl, piperazinyl, pyrrolidinyl, morpholinyl, thiomorpholinyl, dihydrothiophenyl, 1,4- oxazepanyl, lH-pyridin-2-onyl and the like. The term "heterocycloalkylalkyl" or the term "heterocyclylalkyl" refers to an alkyl substituted with a heterocyclyl. The term "heteroarylalkoxy" refers to an alkoxy substituted with heterocyclyl. The term "substituted" refers to one or more substituents (which may be the same or different), each replacing a hydrogen atom. Examples of substituents include, but are not limited to, halogen (F, Cl, Br, or I), hydroxyl, amino, alkylamino, arylamino, dialkylamino, diarylamino, cyano, nitro, mercapto, oxo (i.e., carbonyl), thio, imino, formyl, carbamido, carbamyl, carboxyl, ester, N-alkyl-substituted amido, alkoxycarbonyl, alkylcarbonyl, alkyl, alkenyl, or alkyloxy.

The term "alkoxy" refers to an -O-alkyl radical. The term "ester" refers to a C(O)O- alkyl or C(O)O-aryl, heteroaryl, heterocyclyl, or cyclyl group. An "amido" is an C(O)NH₂, an "N-alkyl-substituted amido" is of the formula C(O)N(H)(alkyl).

While compounds having the precise structure of the formulae herein are preferred, as the terms are defined herein compounds coming within the formulae herein include structurally related compounds, including those compounds that are pharmaceutically acceptable salts, solvates, hydrates, and prodrugs of compounds delineated herein. Examples of prodrugs include esters and other pharmaceutically acceptable derivatives, which, upon administration to a subject, are capable of providing the parent compounds described herein (see Goodman and Gilman's, The Pharmacological basis of Therapeutics, 8^th ed., McGraw- Hill, Int. Ed. 1992, "Biotransformation of Drugs").

In another embodiment, the invention includes use of related compounds such as lactacystin or derivatives thereof, e.g., clasto-lactacystin-β-lactone , clasto-lactacystin (9R)-B- lactone, clasto-lactacystin dihydroxy acid, lactacystin amide, and descarboxylactacystin. See, e.g., Fenteany et al, Proc. Natl. Acad. Sci. USA, vol. 91, pp.3358-3362 (1994).

In certain embodiments, other proteasome inhibitors can be employed. For example, in certain embodiments, proteasome inhibitors such as MGl 32, epoxomicin, bortezomib, proteasome inhibitor I (Z-Ile-Glu(OtBu)-Ala-Leu-aldehyde), proteasome inhibitor II (Z-Leu- Leu-Phe-aldehyde), proteasome inhibitor III (Z-Leu-Leu-Leu-B(OH)₂, also referred to as MG232), natural ester bond-containing green tea polyphenols (GTPs), such as ( )- epigallocatechin-3-gallate [( )-EGCG] and ( )-gallocatechin-3-gallate [( )-GCG] (see, e.g., D.M. Smith et al, MoI. Med. 8(7), 382-392 (2002)), and 4-hydroxy-5-iodo-3- nitrophenylacetyl-Leu-Leu-leucinal-vinyl sulfone, and the like, can also be used in the methods and compositions of the invention. In certain embodiments, a boronic acid compound may be employed as a proteasome inhibitor (see, e.g, U.S. Patent No. 6,297,217). However, as described infra, tests to determine the ability of bortezomib to reactivate latent HIV were inconclusive due to the cytotoxicity of bortezomib. In certain preferred embodiments, the proteasome inhibitor for use according to the invention is not bortezomib.

Compounds of the present invention can readily be made. See, Merck Index, 417 (1 lth ed., 1989); Fenteany et al., Proc. Natl. Acad. Sci. USA, vol. 91, pp.3358-3362 (1994). Other compounds of the formulae herein can be prepared by procedures well known to those skilled in the synthesis art.

One compound, c/αsto-lactacystin β-lactone, can be used in the methods delineated herein, or can be formed in vivo from administration in its corresponding β-lactone ring opened forms (e.g., lactacystin), or other prodrug form.

It is believed that the compounds of the present invention can provide effective therapy of latently infected cells (i.e. cells infected by a virus which is an immunodeficiency virus such as FIV, SIV, HIV, etc.) as evidenced by the induction of lytic replication in latently infected cells.

Hence, in one embodiment the present invention can be used in treating those diagnosed as having AIDS as well as those having ARC, PGL and those seropositive but asymptomatic patients. For example, as a preventative, an effective amount of a compound can be used prophylactically as a preventative for high risk individuals.

Compounds of the present invention can be used to treat cells, especially mammalian cells and in particular human cells, infected by an immunodeficiency virus such as HIV. As a result of treatment with compounds of the present invention the number of latently infected cells can be significantly reduced. P24, a major structural protein (product of gag), has been widely used for monitoring HIV-I replication in cells and vireamia in individuals. Use of present compounds such as proteasome inhibitors (e.g., c/αsto-lactacystin β-lactone) at concentrations that do not adversely affect cells, can dramatically reduce the number of cells latently infected with HIV, e.g. preferably a reduction of HIV-I latently infected cells as shown by an increase (e.g., X- fold increase, where x is 2, 3, 4, 5, 10, 15, 20, 30, 40, or any integer between about 2 and about 50) in P24 levels in cells treated with test compound (e.g., proteasome inhibitors) relative to levels in untreated latent HIV-infected cells. In another aspect the increase can be determined relative to untreated uninfected cells.

The effective amount of a compound of the present invention used to obtain such a result can be at micromolar concentrations. Furthermore, the administration of the compounds of the present invention at effective concentrations, which inhibit HIV expression, has not been found to adversely affect treated cells.

The compounds of the present invention can be administered to HIV infected individuals or to individuals at high risk for HIV infection, for example, those having sexual relations with an HIV infected partner, intravenous drug users, etc. Because of its effect of inducing lytic replication, the compounds of the present invention and a pharmaceutical compositions comprising one or more compounds of the formulae herein can be used prophylactically as a method of prevention for such individuals to minimize their risk of cells becoming latently infected. The compound is administered at an effective amount as set forth below by methodology such as described herein.

As demonstrated in the examples which follow, compounds of the present invention induce lytic replication of HIV-I LTR and HIV-I in latently infected cells. In particular, it has been found that compounds of the present invention in a dose-dependent fashion cause latently infected cells to lytically replicate. Moreover, such induction is provided with essentially no adverse effects on cell survival or cellular mRNA or total cellular RNA synthesis. Thus, it is believed compounds of the present invention will have utility in clearing latent infections of an HIV infection and other retroviral infections in cells and in a human, and (in preferred embodiments) to ultimately entirely clear virus from an infected subject.

Preferably, for inducing lytic replication, one or more compounds of the invention is administered in an amount sufficient to activate lytic replication in at least about 25 percent of infected cells, more preferably an amount sufficient to induce lytic replication in at least about 50 percent of the infected cells and still more preferably induce lytic replication in at least about 75 percent of latently infected cells.

In general for the treatment of immunodeficiency viral infections, for example an HIV infection, a preferred effective dose of one or more compounds of the present invention, e.g., compounds of the formulae herein, will be in the range 0.1 mg to 5g per kilogram body weight of recipient per day, more preferably in the range of 0.1 mg to 1,000 mg per kilogram body weight per day, and still more preferably in the range of 1 to 600 mg per kilogram of body weight per day. A preferred effective dose of one or more therapeutic compounds can be readily determined based on known factors such as efficacy of the particular therapeutic agent used, age, weight and gender of the patient, and the like. See dosage guidelines as set forth e.g. in Remington, The Science and Practice of Pharmacy, 20^th Edition. The desired dose is suitably administered once or several more sub-doses administered at appropriate intervals throughout the day, or other appropriate schedule. These sub-doses may be administered as unit dosage forms, for example, containing 100 to 4,000 mg, preferably 100 to 2,000 mg.

Preferably a compound (e.g., of the formulae herein) is used in accordance with the present invention in an isolated form distinct as it may be naturally found and in a comparatively pure form, e.g., at least 85% by weight pure, more preferably at least 95% pure. For some treatments in accordance with the present invention, it may be desirable that the administered compound of the formulae herein be at least 98% or even greater than 99% pure. Such a material would be considered sterile for pharmaceutical purposes. Potential contaminants include side products that may result upon synthesis of a compound of the invention or materials that may be otherwise associated with the compound prior to its isolation and purification. The present compounds should preferably be sterile and pyrogen free. Purification techniques known in the art may be employed, for example chromatography.

Administration of the compounds of the invention may be by any suitable route including oral, rectal, nasal, topical (including buccal and sublingual), vaginal and parenteral (including subcutaneous, intramuscular, intravenous and intradermal) with oral or parenteral being preferred. It will be appreciated that the preferred route may vary with, for example, the condition and age of the recipient.

The administered ingredients may be used in therapy in conjunction with other medicaments such as reverse transcriptase inhibitors such as dideoxynucleosides, e.g. zidovudine (AZT), 2',3'-dideoxyinosine (ddl) and 2',3'-dideoxycytidine (ddC), lamivudine (3TC), stavudine (d4T), and TRIZIVIR (abacavir + zidovudine + lamivudine), nonnucleosides, e.g., efavirenz (DMP-266, DuPont Pharmaceuticals/Bristol Myers Squibb), nevirapine (Boehringer Ingleheim), and delaviridine (Pharmacia-Upjohn), TAT antagonists such as Ro 3-3335 and Ro 24-7429, protease inhibitors, e.g., indinavir (Merck), ritonavir (Abbott), saquinavir (Hoffmann LaRoche), nelfinavir (Agouron Pharmaceuticals), 141 W94 (Glaxo-Wellcome), atazanavir (Bristol Myers Squibb), amprenavir (GlaxoSmithKline), fosamprenavir (GlaxoSmithKline), tipranavir (Boehringer Ingleheim), KALETRA (lopinavir + ritonavir, Abbott), and other agents such as 9-(2-hydroxyethoxymethyl)guanine (acyclovir), interferon, e.g., alpha-interferon, interleukin II, and phosphonoformate (Foscarnet), or entry inhibitors, e.g., T20 (enfuvirtide, Roche/Trimeris) or UK-427,857 (Pfizer), or in conjunction with other immune modulation agents or treatments including bone marrow or lymphocyte transplants or other medications such as levamisol or thymosin which would increase lymphocyte numbers and/or function as is appropriate. Because many of these drugs are directed to different targets, e.g., viral integration, it is anticipated that an additive or synergistic result will be obtained by use of combinations. Further, proteasome inhibitors can be combined with the use of other lytic replication activators, for example, farnesyl transferase inhibitors or Egrl activators, as disclosed in U.S. provisional patent application Nos. 60/587,771 and 60/588,301, respectively, both filed on July 13, 2004 and incorporated herein by reference. In one embodiment, one or more compounds of the formulae herein are used in conjunction with one or more therapeutic agents useful for treatment or prevention of HIV, a symptom associated with HIV infection, or other disease or disease symptom such as a secondary infection or unusual tumor such as herpes, cytomegalovirus, Kaposi's sarcoma and Epstein-Barr virus-related lymphomas among others, that can result in HIV immuno¬ compromised subjects.

In certain embodiments of the invention, one or more compounds of the formulae herein are used in conjunction with a Standard HIV antiviral treatment regimen (e.g., HAART). This combination is advantageous in that the compound(s) of the formulae herein can activate latent HIV infected cells to replicate by stimulating lytic viral replication, thus making them susceptible to the co-administered standard HIV antiviral treatment regimens. In this manner, the latent or secondary reservoirs of HIV-infected cells are depleted through "controlled" activation (rather then serendipitous or uncontrolled activation), resulting in more complete elimination of virus, while controlling the spread of viral infection.

In other embodiments, the treatment methods herein include administration of a so- called HIV-drug "cocktail" or combination therapy, wherein a combination of reverse transcriptase inhibitor(s) and HIV protease inhibitor(s) is co-administered. In a preferred embodiment, a highly active anti-retroviral therapy (HAART) treatment regime is combined with treatment with a proteasome inhibitor according to the invention.

In yet other embodiments, a combination therapy according to the invention includes administration of an proteasome inhibitor together with an abl kinase inhibitor such as imatinib (use of imatinib for HIV treatment is described more fully in co-pending U.S. Provisional Patent Application No. 60/588,015, filed June 13, 2004).

In another aspect, the methods involve modulation of any gene that exhibits altered expression in chronically HIV-infected cells compared to uninfected parental cells, prior to induction into lytic replication. The methods herein can involve, or target, any of the genes listed in the tables herein. This modulation can be direct or indirect, that is, it can be by direct control of expression or binding activity of the target, or by indirect control of the expression or binding activity of the target. In any case, another aspect is modulation of replication activity of latent HIV-infected cells. In another aspect, the methods involve modulation of lyn, cdc42, MNDA, CEBP alpha oτMeisl by administration of the compounds of the formulae herein.

It appears that in chronically infected ACH-2 cells, the higher expression of proteasomes can lead to increased degradation of HIV mRNA. This effect, along with insufficient levels of full length Tat and Nef proteins would tend to inhibit HIV lytic replication. In such a case, addition of a proteasome inhibitor can release the suppressive effect of proteasomal degradation, potentiate the function of Tat by binding to the active sites on the proteasome, and thus allow for accumulation of sufficient early viral proteins to trigger viral reactivation. In an aspect of the invention, the methods delineated herein modulate one or a combination of Tat levels, Nef levels, or increase proteasome expression in latent HIV- infected cells. In any case, a resulting activation, or reactivation, or HIV lytic replication is achieved by administration (or contact) of the proteasome inhibitor (in an effective amount) with the latent infected cell, which is part of a treatment or prevention regimen.

The present invention includes use of both racemic mixtures and optically active stereoisomers of compounds of the formulae herein.

Compositions of compounds of the invention (e.g., of the formulae herein) used in combination with other compounds (e.g., reverse transcriptase inhibitors, protease inhibitors, and the like) may be employed alone or in combination with acceptable carriers such as those described below. For the treatment of immunodeficiency viral infections, for example an HIV infection, a suitable effective dose of a compound in such a composition will be in the range of 1 to 5,000 mg per kilogram body weight of recipient per day, preferably in the range of 10 to 4,000 mg per kilogram body weight of recipient per day. When multiple compounds having complementary activity are administered together it is expected one can use the lower portion of these ranges (or even less).

One or more compounds of the formulae herein may be administered alone, or as part of a pharmaceutical composition, comprising at least one proteasome inhibitor (e.g., a compound of the formulae herein) together with one or more acceptable carriers thereof and optionally other therapeutic ingredients, including those therapeutic agents discussed supra. The carrier(s) must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.

Combinations of substituents and variables envisioned by this invention are only those that result in the formation of stable compounds. The term "stable", as used herein, refers to compounds which possess stability sufficient to allow manufacture and which maintains the integrity of the compound for a sufficient period of time to be useful for the purposes detailed herein (e.g., formulation into therapeutic products, intermediates for use in production of therapeutic compounds, isolatable or storable intermediate compounds). The compounds delineated herein are commercially available or readily synthesized by one of ordinary skill in the art using methodology known in the art.

Some of the compounds of this invention have one or more double bonds, or one or more asymmetric centers. Such compounds can occur as racemates, racemic mixtures, single enantiomers, individual diastereomers, diastereomeric mixtures, and cis- or trans- or E- or Z- double isomeric forms. All such isomeric forms of these compounds are expressly included in the present invention. The compounds of this invention may also be represented in multiple tautomeric forms, in such instances, the invention expressly includes all tautomeric forms of the compounds described herein (e.g., alkylation of a ring system may result in alkylation at multiple sites, the invention expressly includes all such reaction products). All such isomeric forms of such compounds are expressly included in the present invention. All crystal forms of the compounds described herein are expressly included in the present invention.

The compositions include those suitable for oral, rectal, nasal, topical (including buccal and sublingual), vaginal or parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration. The formulations may conveniently be presented in unit dosage form, e.g., tablets and sustained release capsules, and in liposomes, and may be prepared by any methods well known in the art of pharmacy. Such methods include the step of bringing into association the to be administered ingredients with the carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers, liposomes or finely divided solid carriers or both, and then if necessary shaping the product.

Compositions of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous liquid or a non-aqueous liquid; or as an oil-in- water liquid emulsion or a water-in-oil liquid emulsion, or packed in liposomes and as a bolus, etc.

A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, preservative, surface-active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein.

Compositions suitable for topical administration include lozenges comprising the ingredients in a flavored basis, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia; and mouthwashes comprising the ingredient to be administered in a suitable liquid carrier.

Compositions suitable for topical administration to the skin may be presented as ointments, creams, gels and pastes comprising one or more compounds of the present invention and a pharmaceutically acceptable carrier. A suitable topical delivery system is a transdermal patch containing the ingredient to be administered. Compositions suitable for rectal administration may be presented as a suppository with a suitable base comprising, for example, cocoa butter or a salicylate.

Compositions suitable for nasal administration wherein the carrier is a solid include a coarse powder having a particle size, for example, in the range 20 to 500 microns which is administered in the manner in which snuff is taken, i.e., by rapid inhalation through the nasal passage from a container of the powder held close up to the nose. Suitable formulations wherein the carrier is a liquid, for administration, as for example, a nasal spray or as nasal drops, include aqueous or oily solutions of the active ingredient.

Compositions suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations containing in addition to the active ingredient such carriers as are known in the art to be appropriate.

Compositions suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampules and vials, and may be stored in a freeze dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.

It should be understood that in addition to the ingredients particularly mentioned above the formulations of this invention may include other agents conventional in the art having regard to the type of formulation in question, for example, those suitable for oral administration may include flavoring agents.

The use of the term "or" is unless otherwise indicated, to be construed as being inclusive. That is, the recitation of A, B or C is meant include A, B or C each alone, or in any combination (e.g., A and B, B and C, A and C, and A and B and C) thereof. AIl documents mentioned herein (including patents, patent applications, and other references) are incorporated herein by reference.

The present invention is further illustrated by the following examples. These ^x examples are provided to aid in the understanding of the invention and are not to be construed as limitations thereof.

EXAMPLE 1 MATERIALS AND METHODS

Cells: ACH-2, A3.01, Jl.1, and Ul cells (11, 15, 24, 25, 48) were obtained through the NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH. U-937 cells were obtained from American Type Culture Collection (Manassas, VA). ACH-2, Jl .1 and Ul are chronically infected cell lines harboring HIV-I LAV strain, while A3.01, Jurkat, and U-937 are the corresponding parental uninfected cell lines. Cells were grown in RPMI-1640 (Invitrogen, San Diego, CA) with 10% fetal bovine serum (FBS, Invitrogen), 5% penicillin-streptomycin (Invitrogen), and 2mM glutamine (Invitrogen). Cells were maintained at a concentration of 1x10 cells/ml in T- 175 flasks. Cell concentrations and cell viability were monitored throughout the experiment at all time points studied. Cells were induced by addition of 20 ng/mL of phorbol myristyl acetate (PMA or TPA, Sigma, St Louis, MO) for one hour, after which the cells were washed with phosphate buffered saline (PBS). Cells were harvested by centrifugation at 1000 rpm for 10 minutes, at the following times after induction, 0.5, 3, 6, 8, 12, 18, 24, 48, 72, and 96 hour(s). HIV-infected and uninfected cells maintained and harvested in parallel with the PMA-treated cells but not induced with PMA were also harvested. For the time course experiment, 3'-azido- 3'-deoxythymidine (AZT, Sigma) was not added to the ACH-2 or A3.01 cells, in order to keep conditions as close to an acute infection as allowed by the experimental model. Harvested cells were washed thrice with ice-cold PBS to remove media; cell pellets were snap frozen using ethanol-dry ice mixture and stored at -8O⁰C for subsequent RNA extraction. Three independent time course experiments (biological replicates) were performed using the protocols described above to ensure reproducibility. To compare expression profiles of chronically infected cell lines, ACH-2, Ul, and J 1.1 and their uninfected parental cell lines, A3.01, U-937 and Jurkat cells respectively, were grown under identical conditions but in presence of AZT (250 nM) in growth media and cells were harvested as described above. In these studies, no inducing agent was used in either chronically infected or uninfected parental cell lines so as to study changes in cellular gene expression cells latently infected with HIV and uninfected cells.

Flow Cytometry: To confirm viral replication following PMA induction, we measured the accumulation of intracellular p24 over a period of 48 hours by measuring cell populations labeled with anti-p24 FITC-labeled antibody by flow cytometry. Cells (ACH-2 and A3.01) were washed twice with ice-cold PBS and suspended in 50 μL ice-cold permeabilization buffer (BD Biosciences, San Jose, CA), and incubated at 4⁰C in the dark for

30 minutes. The cells were fixed using the CytoFix/CytoPerm kit (BD Biosciences) and 5 μL KC57-FITC-labeled p24 antibody (Beckman Coulter), was added to detect intracellular p24.

Labeling A3.01 samples with FITC-labeled p24 antibody served as controls to ensure that the parental cell line did not show any p24 accumulation over samples labeled with FITC-labeled mouse IgGl (Immunotech, Hialeah, FL), which was used as isotype control. Following incubation on ice for 30 minutes in the dark, the cells were washed thrice with permeabilization buffer, and resuspended in 300 μL of permeabilization buffer and analyzed using a Becton Dickinson FACSCAN instrument (BD Biosciences) in conjunction with

CellQuest software (BD Biosciences) for flow cytometric analysis.

Total RNA Extraction: Total RNA was extracted using RNEasy Midiprep Kits per manufacturer's protocol (Qiagen, Valencia, CA). RNA concentrations and purity were measured by spectrophotometry, RNA quality (absence of RNA degradation) was assessed by gel electrophoresis. RNA concentration was adjusted to the levels required for subsequent microarray experiment protocols by concentration in a SpeedVac (Savant Instruments, Holbrook, CA). RNA samples (6-7 μg/μL) were stored in 100 μL TE buffer at -8O⁰C.

Real Time RT-PCR Quantitation of Viral RNA and Cellular RNA: Quantitation of HIV viral mRNA was carried out by real-time PCR using an ABI 7000 instrument (Applied Biosystems (ABI), Foster City, CA). A housekeeping gene, glyceraldehyde phosphate dehydrogenase (GAPDH) whose expression was unchanged in ACH-2 cells before and after PMA, was used as a normalization control. RNA from the samples was subjected to DNase treatment to remove contaminating DNA, and the DNAse was inactivated using the DNase Free kit (Amersham Biosciences, Piscataway, NJ) according to manufacturer's protocols. 2 μg of RNA was reverse transcribed using the Taqman RT kit from ABI per manufacturer's specifications. Briefly, the reaction mixture (50 μl) was incubated at 65⁰C for 5 minutes followed by 37⁰C for 45 minutes, 94⁰C for 5 minutes, and then cooled on ice. 1/40 th aliquots of the corresponding samples were then used in a real-time PCR reaction using Taqman probes labeled with FAM and TAMRA at the 5' and 3' ends respectively. Primer probe pairs were designed using PrimerExpress (ABI). The reactions were carried out in triplicate for each time point and the fold changes observed were normalized to GAPDH, for each time point. Melting curve analysis and an assessment of the efficiency of the PCR reactions over a given concentration range were performed to determine if any errors occurred during the reactions, for example, primer-dimer formation or poor priming effects. Real time PCR reactions were set up as described above using primers and probes specific for early (multiply spliced mRNA, MS HIV-I) and late (unspliced mRNA, US HIV-I) respectively. The sequences for 5' and 3' primers to quantitate early mRNA were 5 'CGAAGAGCTCATCAGAACAGTCAS' and 5 'TTGGGAGGTGGGTCTGCTTS'. The sequence for the labeled probe was 5'CTTCTCTATCAAAGCAGACCCACCTCCS ' which overlapped with the splice site of HIV-I Rev sequence. The sequence detection primers for unspliced or late RNA were SK38 and SK39 from the HIV-I Gene Amplimer kit (ABI). A TAMRA labeled probe with sequence identical to SKl 9 (ABI) was used for Real time PCR quantitation of the late viral RNA species. Standards from the kit were diluted to calculate copy number of virus based on gag mRNA concentrations.

Real time RT-PCR analysis was also carried out for selected cellular genes using gene specific primer probe pairs and Taqman detection primers. Fold differences in mRNA expression in uninduced ACH-2 samples and the corresponding A3.01 samples, was determined using the protocol described for quantitation of viral mRNA. Real time RT-PCR quantitation was performed for genes PSMC5, p44slθ (proteasome subunits) Egrl (early growth response 1), HDACl (histone deacetylase 1), NK4, EIF4, SFRS3, to confirm that these genes were differentially expressed in the latently infected ACH-2 cells compared to the uninfected A3.01 parental cells. Primer-probe pairs specific for each gene were designed using PrimerExpress (ABI). The sequences for the detection primers and probes for each gene are available as supplemental data, Table Sl.

Microarray Studies: Total RNA obtained from induced chronically infected and corresponding uninfected parental cells were used for microarray experiments. For each time point, RNA from the induced chronically infected ACH-2 cells and RNA from the corresponding induced, uninfected A3.01 cells were compared to minimize effects due to PMA induction. Microarrays were obtained from the National Cancer Institute Microarray Facility, Advanced Technology Center (Gaithersburg, MD). The microarrays (Hs. UniGem2) contained 10,395 cDNA spots on each glass slide. The cDNAs were selected for spotting on the slides based on their known or probable involvement in oncogenesis, signal transduction, apoptosis, immune function, inflammatory pathways, cellular transport, transcription, protein translation and other important cellular functions. A number of expressed sequence tags (ESTs) from unknown genes homologous to known genes and cDNAs encoding housekeeping genes were also included in these gene sets. For each time point, 50 μg of total RNA from PMA induced ACH-2 cells and 70 μg of total RNA from PMA induced A3.01 cells was labeled with Cy-3-dUTP and Cy-5-dUTP respectively as previously described (34, 60). Higher amounts of RNA were used for Cy-5 labeling to minimize the disparities in dye incorporation. Each sample of RNA from PMA-induced, infected cells from a particular time point was compared with RNA from the corresponding PMA-induced, uninfected cells from the same time point for subsequent hybridization to the same array to ensure accurate comparisons and to eliminate inter-array variability. The labeled cDNAs were then combined and purified using MicroCon YM-30 (Millipore, Bedford, MA) spin column filters, to remove any unincorporated nucleotides. 8-10 μg each of Cot-1 DNA, (Boehringer Mannheim, Indianapolis, IN), yeast tRNA (Sigma) and polyA (Amersham Biosciences) were added to the reaction mixture and heated at 100⁰C for 1 minute. Hybridization of the labeled cDNA to the microarray was carried out at 65⁰C overnight, followed by washes with IX SSC, 0.2X SSC and 0.05X SSC respectively. The slides were dried by centrifugation at 1000 rpm for 3 minutes and then scanned as described below. RNA samples from three identical but independently conducted time course experiments were tested. Microarray experiments were performed at least twice for each time point (technical replicates) of each experiment. We also compared AZT-treated ACH-2 cells to untreated ACH-2 cells to determine whether any differences in gene expression might be solely due to AZT.

Microarray experiments for each chronically infected cell line (J 1.1, Ul and ACH-2) were also conducted as per the protocol described above. Samples from eight independent experiments per cell line were used for studying the gene expression patterns in chronically infected cell lines. To compensate for dye labeling bias that might be due to differences in

Cy5 and Cy3 labeling efficiency and preferential dye incorporation by some mRNA species,

RNA from the same samples labeled with Cy5 (70 μg RNA) and Cy3 (50 μg RNA) were co- hybridized to the same array, scanned, and data were analyzed for all the cell lines studied, using identical filtering and statistical tests, and genes showing dye incorporation bias were eliminated from further analysis as described below.

Microarray Scanning and Data Analysis: The slides were scanned using an Axon GenePix 4000 scanner (Axon Instruments, Union City, CA). The photomultiplier tube values (PMT) were adjusted to obtain equivalent intensities at both wavelengths used, 635 nm and 532 nm for the Cy5 and Cy3 channels respectively. Image analysis was performed using GenePix analysis software (Axon Instruments) and data analysis was performed using the microArray Database (mAdb) system hosted by the Center for Information Technology and Center for Cancer Research at NIH (http://nciarray.nci.nih.gov). Each array was normalized using Lowess normalization (71). Normalization across arrays over the time course was not feasible since no particular time point could be established as a median for cellular gene expression data, since viral gene expression and associated host cell gene expression was different for different time points. For each time point, there were at least 6 data sets (2 technical replicates X 3 biological replicates). For each time period (0.5-8 hours, 12-24 hours, 48-96 hours post induction.), there were at least 18 datasets (at least 3 time points per time period). Only arrays that passed initial spot size and intensity criteria, and whose normalization ratio (between the two signal intensities) was close to one (0.85-1.15), were analyzed. Filtering criteria were as follows: a) For each spot, signal intensity must be at least twice that of the background intensity; b) Each gene must have values in at least 70% of the arrays; c). Each array must have values for at least 70% of the gene spots. Genes that showed dye labeling bias in a particular cell line after normalization were excluded from that gene set prior to further analysis. This was determined using a one sample t-test on mean log ratios for replicate arrays with the same sample labeled with both Cy3 and Cy 5.

Statistical Analysis: Comparison of expression profiles of infected versus uninfected (both induced by PMA) cell lines at a given time point was performed using univariate parametric and multivariate permutation tests based on the one sample random variance t- statistic in BRB-ArrayTools (http://linus.nci.nih.gov/BRB-ArrayTools^') (62). Since RNA for infected and uninfected cell lines corresponding to the same time point were paired and co- hybridized on the same array, inter-array sources of variation were minimized and differential expression could be detected by a statistically significant non-zero mean log-ratio in biologically independent replicates. All biological replicates that passed the filtering criteria described above were used in the analyses. Technical replicates were averaged. The random variance model enabled variance information to be shared across genes without assuming that all genes have the same variance (69). For comparison of expression for latently infected versus uninfected cell lines, significance was based on p < 0.001 for a parametric one-sample random variance t-test. For evaluation of differential expression between infected and uninfected cell lines at fixed times after induction, a multivariate permutation test based on the one-sample random variance t-statistic was used in which the proportion of false discoveries was limited to 0.10 with 90% confidence (36, 62). Hierarchical clustering analyses on the resulting data sets were done using the mAdb system as well as Cluster and TreeView software programs (Stanford University, CA). Since statistically significant gene classes need not necessarily indicate the biological relevance of genes in a particular class, pathway analysis of the various genes that showed significant differential expression was performed by utilizing analysis tools provided by the NIH mAdb database (http://nciarray.nci.nih.gov) and querying the database of the Cancer Genome Anatomy Project (CGAP), (http://cgap.nci.nih.goW) with pathway information provided by KEGG (www.genome.ad.jp/kegg/) and Biocarta (www.biocarta.com) pathway databases. Gene ontology summary analyses, which allow for grouping of genes based on their molecular function were also performed to ensure that changes observed in our studies were not due to general dysregulation of all genes studied. Where possible, observed/expected ratio (OfE) for each functional class of genes was determined to ensure that observed changes in number of genes differentially expressed within a gene class were greater than that expected by chance (O/E > 1). This provided stringent thresholds for pathway and functional classification of the differentially expressed genes.

Latency Reactivation Studies: Cells (ACH-2, Jl.1 and Ul) were seeded at a concentration of 2x10 cells/mL in 24 well plates in a volume of 1 mL. Clastolactacystin- beta-lactone (CLBL) a proteasome inhibitor, resveratrol an Egrl activator, or trichostatin, a histone deacetylase inhibitor (Biomol Research Laboratories, Plymouth Meeting, PA) was dissolved in sterile dimethylsulfoxide (DMSO) and further diluted with media to obtain the desired final concentrations. The final concentration of DMSO in contact with the cells was never greater than 0.001% at any dose tested. AZT, (250 nM) was added to the chronically infected cells in order to inhibit p24 production that may be caused due to low levels of actively replicating virus present along with the chronically infected cells and to ensure that any increases in p24 expression would be attributable to activation of latent provirus and not due to subsequent amplification via additional rounds of viral replication. Cells were incubated with different concentrations of either CLBL, resveratrol, or trichostatin at 37⁰C. 200 μL samples of cell supernatant were collected at 24 hours after treatment. Cells incubated with tumor necrosis factor alpha (TNF-alpha, 0.5 μg/mL), also in the presence of AZT, served as a positive control. Cells treated with AZT alone served as a negative control. Cells not treated with AZT were also examined. Samples were mixed with lysing buffer (10% Triton-X-100, Sigma) to inactivate virus and diluted 5-fold with sample diluent (1% bovine serum albumin, 0.2% Tween-20 in RPMI-1640). p24 expression was assayed by ELISA using HIV-I p24 antigen capture kits (AIDS Vaccine Program, Frederick, MD) per manufacturer's specifications. Briefly, plates were washed with plate wash buffer and samples were added in duplicate wells. The samples (100 μL) were incubated for 2 hours at 37⁰C. The plates were washed, and rabbit anti-HIV p24 antibody was added at 1 :400 dilution. Following incubation for one hour, the plates were washed and goat anti-rabbit IgG peroxidase labeled antibody at 1 :300 dilution was added. The plates were incubated for one hour at 37⁰C, followed by washing and addition of a two-component substrate. Substrate solution consisted of equal volumes of TMB peroxidase substrate and peroxidase solution B (Kirkegaard and Perry Laboratories, Gaithersburg, MD). Samples were incubated for 30 minutes at room temperature and reactions were stopped by addition of IN hydrochloric acid solution. The absorbance was measured at 450 nm using a SpectraMax250 spectrophotometer (Molecular Devices Coφoration, Sunnyvale, CA). The samples were assayed in duplicate and experiments were performed at least thrice using independent cell samples.

EXAMPLE 2

We treated the ACH-2 chronically infected cell line with phorbol myristyl acetate (PMA or TPA) to trigger the initiation and completion of the lytic replication cycle. Virus production was confirmed by flow cytometry for the HIV late protein p24 (Figure 1). p24 production was low in the absence of PMA (8.2%) in ACH-2 cells not treated with AZT. A3.01, the parental uninfected cell line, did not show any p24 specific staining over isotype control (data not shown). At 6 hours post induction (p.i), 62% of the ACH-2 cells showed p24 production, and from 12 hours p.i. up to 96 hours p.i., nearly all cells were positive for p24 production, indicating that a lytic HIV infection was underway in essentially all the cells in the culture (Figure. 1). By 48 hours post induction, flow cytometry analysis showed high levels of p24 production, but with increased cell death due to cytopathic effects (data not shown).

Cell viability before and after induction of lytic replication was carefully monitored so as to ensure that changes in gene expression could be associated with the process of lytic replication and not due to excessive cell death due to HIV replication. From the start of PMA induction to up to 24 hours p.i., cell viability of induced ACH-2 cells remained at similar levels to that of uninfected, induced parental A3.01 cells and also to uninduced ACH-2 cells, indicating that cell death due to HIV lytic replication was not a significant factor in changes in gene expression for that period. Beyond 24 hours p.i., (48- 96 hr p.i.) cytopathic effects and increased cell death were observed in the induced ACH-2 cells (also confirmed by flow cytometry), indicating that changes in cellular gene expression during the 48- 96 hr p.i. may be attributed to a combination of viral replication as well as cellular mechanisms involved in cell death. RNA yields decreased for this time period, however RNA quality and purity were similar to the previous time points, suggesting that changes in cellular gene expression were not due to RNA degradation in these cells. Since nearly all cells undergoing lytic replication were positive for p24 antigen by 24 hours p.i. when cellular viability was unaffected, the changes in cellular gene expression from 0.5 hour p.i. up to 24 hours p.i. are most likely associated with the process of lytic replication in ACH-2 cells.

EXAMPLE 3

Viral mRNA Expression by Real Time RT-PCR

To confirm that production of early and late viral mRNA was underway in lytically induced cells, and to determine relative proportions of early (multiply spliced; tat, rev and nef) and late (unspliced; gag, pol, env) HIV-I mRNA over the time course, we used real time RT-PCR to quantitate the message classes. In our system, the multiply spliced mRNA expression increased after lytic induction and showed a maximum of 40 fold increase over uninduced ACH-2 cells at 8 hours post induction. Unspliced mRNA concentrations showed a gradual increase with a 148-fold increase at 18 hours post induction (Figure 2). The results indicate that viral RNA expression in our lytically induced cells followed known kinetic expression patterns (6, 35). There was a clear distinction in the peak expression of the early and late viral mRNA indicating that viral RNA expression followed a discrete temporal pattern in these cells.

EXAMPLE 4

Effects on ACH-2 Cellular Gene Expression Before and After Induction

For the cellular gene expression studies, we compared the cellular gene expression pattern of ACH-2 with that of its uninfected parental line, A3.01. Both the infected and uninfected parental lines were subjected to identical PMA treatments and sampling procedures over a 96 hour time course, and RNA samples from three separate time course experiments were each tested in duplicate. In all, 66 arrays were examined. The filtering criteria yielded 9122 analyzable gene spots out of the 10395 gene spots printed on each microarray. The expression of most cellular genes was similar in the uninfected and chronically infected cells and did not change during activation into a lytic infection cycle. We also performed experiments to detect any gene spots that may be false positive due to bias in dye labeling under our experimental conditions. A small set of genes that showed dye bias (43 genes), in our dye-labeling bias experiments were excluded from the data set. Data from technical and biological replicates for each time point, were normalized for each array as described in the Methods section. Statistical analyses to identify genes that were significantly differentially expressed were performed using univariate and multivariate random variance one sample t-test, using all the replicates that passed the filtering criteria. Based on the statistical analyses of the genes showing altered expression, 131 genes showed altered expression even prior to induction. 1740 of all the analyzable spots showed statistically significant (p < 0.001) altered gene expression at some point, either before induction or over the entire period of the lytic replication cycle (Figure 3).

EXAMPLE 5

Cellular Gene Expression Profiles Before Induction and During Lytic Replication

The changes observed during lytic replication following induction occurred in an orderly, time dependent manner. To better appreciate the time-dependent changes in cellular gene expression that accompany the portion of the lytic replication cycle occurring after activation of the chronically infected cells by PMA, we grouped our observations into three time periods after induction, a) Early (0.5-8 hours p.i), b) Intermediate (12-24 hours p.i) and c) Late (48-96 hours p.i), roughly corresponding to the times during lytic replication when the early and the late viral mRNA peak, and the end of the lytic cycle. Statistical analyses and hierarchical clustering of the data also grouped the various cellular genes into these time periods (data not shown). The changes in gene expression observed during the late period, (48-96 hours p.i.) cannot be solely associated with the process of lytic replication, since during this time period cells showed cytopathic effects. However, the data is presented in order to provide the most complete possible view of the cellular environment following lytic replication. Some genes show differential expression only during the early stages of lytic replication and return to levels observed in the uninfected cells, while certain other genes are initially unaltered and show differential expression at later time points. Certain groups of genes show similar patterns of regulation following induction (Figure 3). In the early time period 1334 genes were differentially expressed. In the intermediate time period, 756 genes were differentially expressed. The late time period (48-96 hours p.i) showed the least change with 566 genes exhibiting significant altered expression (p < 0.001). Many of the genes that were differentially expressed in the early time period also showed either similar or the opposite trend in their expression patterns during the other time periods, hence some genes were included in the analysis of both the time periods. A number of discrete patterns of gene regulation were observed. Several cellular genes showed distinct temporal expression patterns during the lytic replication cycle, an expected finding, but more interestingly, a smaller number of genes appeared to be differentially expressed in the latently infected ACH- 2 cells compared to their parental, uninfected cells, even before induction of the lytic cycle. A total of 131 genes already showed significant change (p < 0.001) in their expression prior to induction. They included genes encoding transcription factors, components of proteasomes, factors that control immune function, apoptosis and other functional classes. For gene classes that were annotated in the gene ontology database (GO database, www.geneontoloay.org) (5), observed/expected ratio for the number of genes within a functional class that were differentially expressed was set at greater than one (O/E > 1 ), so as .. to eliminate functional classes where the number of genes differentially expressed was not greater than that randomly expected. However, not all genes that were significantly differentially expressed are annotated in the database. Extensive literature studies for functional significance and classification were conducted in such cases to ensure that important classes of genes were still included in the analyses. An abbreviated listing of the genes grouped according to known functions that were differentially expressed before induction is given in Table 1.

EXAMPLE 6

Genes and Pathways Affected Prior to Induction

While a large number of pathways are altered during lytic replication, a fewer number of pathways are also altered prior to induction of a lytic replication cycle. This observation was interesting and is important for understanding the mechanisms involved in latency maintenance, hence we conducted further analyses on this data set. Pathways involved in cell-cell signaling, signal transduction, inhibition of T-cell receptor signaling, protein translation, and cell cycle transition/regulation show altered expression prior to induction. Most notably, a number of genes encoding different subunits of proteasomes, including those that constitute the 20S/26S core complex (PSMB4, PSMA6, PSMA5) as well as regulatory subunits like PSMD 13 (1 IS regulatory subunit) were up regulated prior to induction. PSMB4 has peptidase activity, which is inhibited by Tat during viral replication. Tat competes with the 11 S regulatory subunit, for binding to the 2OS core complex due to presence of a common binding site in Tat and the HS regulator alpha subunit (32, 59). Proteasomes are also involved in processing certain regions of HIV-I Nef preferentially, which leads to production of Nef-specific CTLs (cytotoxic T-lymphocytes) (44). Many other classes of genes encoding immune response modulators, integrins, cell cycle modulators (such as Egrl), nuclear import factors, and G-protein signaling molecules were also differentially expressed. A listing of genes that were differentially expressed prior to induction, based on their functional classification is given (Table 1). A list of pathways that were affected in the uninduced, chronically infected cells is given (supplemental data, Table S2) .

Real time RT PCR analysis was performed on a selected set of cellular genes using gene specific sequence detection primers and probes to determine if microarray results correlated with the actual normalized fold differences in the corresponding ACH-2 and A3.01 samples, prior to induction. For genes that were found to be statistically and/or biologically significant, the fold difference in expression levels was confirmed by their RT-PCR quantitation (supplemental data, Table S3).

EXAMPLE 7

Trends Seen in Pathway Profiles During Lytic Replication

While a number of genes that had altered expression during lytic replication could be classified based on their functionality, an alternate method was also used to determine if the genes that showed differential expression grouped into known cellular pathways. Genes encoding components of several distinct pathways were regulated in a coordinated fashion during lytic viral replication. Of the 1740 genes that were differentially expressed over the lytic infection cycle in ACH-2 cells, 697 genes were assigned to various known pathways using the CGAP pathway databases, used in conjunction with the microArray database (mAdb) data analysis tools (http://nciarray.nci.nih.gov). Based on the information in the database, these genes were found to lie in a total of 385 known pathways. The remainder of the genes could not be classified into any known pathways listed in the CGAP database. The principal pathways affected over the time course and classified based on the number of genes involved in a particular pathway that are differentially expressed are shown in Figure 4 (and in supplemental data, Table S2). We observed that a number of pathways involved in signaling, cell cycle, and transcription showed maximum changes even prior to induction. During the early stage post induction (0.5- 8 hours p.i.), a number of metabolic pathways and signaling pathways show similar patterns. The intermediate stage (12-24 hours p.i) showed maximal changes in pathways involved in immune response modulation and cell survival. These patterns correlate well with the levels of early (multiply spliced) and late (unspliced) HIV protein gene expression and known viral replication effects on the host cell(32, 44, 59).

EXAMPLE 8

Microarray Analysis of Chronically Infected Cell Lines

Our initial experiments with the ACH-2 cell line were aimed at studying the changes in cellular host gene expression profiles during a lytic infection following activation. However, our results showed that even prior to induction, many cellular genes were differentially expressed. This led us to study cellular gene expression in other chronically infected cell lines, in order to assess whether other chronically infected cell lines also showed alterations in cellular gene expression in the absence of active viral replication and if so, whether different cell lines showed similar patterns of altered gene expression. Different chronically infected HIV cell lines including Jl.1, a chronically infected T-lymphocytic cell line derived from Jurkat cells and Ul, a promonocytic chronically infected cell line derived from U937 cells, were studied using microarrays to determine the similarities and differences in their expression profiles. The p24 expression in all the latently infected cell lines was below lng/mL (0.2-0.8 ng/mL) indicating that the cells were not lyrically active at the time of harvesting the cells. Experiments were performed on eight independent cell samples for each cell line and similar parameters were applied for filter criteria, gene selection and statistical analysis as with the ACH-2 cell line, as described in the Methods section. In these sets of experiments, 24 arrays were analyzed and 8902 of the 10395 gene spots passed the selection criteria. Statistical analysis of expression ratios of AZT treated ACH-2 cells and untreated ACH-2 cells over their respective A3.01 controls did not show any significant differences in gene expression profiles, indicating that changes in gene expression were not due to low levels of actively replicating viral population (data not shown). Genes that had shown differential dye incorporation in our dye labeling bias experiments were excluded from each cell line data (43 genes in ACH-2, 18 genes in Jurkat, and 22 genes in U937 cell lines). Upon analyzing the resulting datasets, we found that 131 genes were differentially expressed in ACH-2, 65 genes were differentially expressed in J 1.1 and 155 genes were differentially expressed in Ul cells compared to their respective uninfected AZT treated parental cell lines. While stringent statistical thresholds were used for genes that were differentially expressed in each cell line, we reasoned that if a gene showed up as significantly differentially expressed (p < 0.001) in at least one cell line, then the expression data for those genes in the other cell lines may be important, even if not found statistically significant for that cell line. We found that ACH-2 and J 1.1 were more similar in their profiles, which may be due to their common T-cell lineage as compared to Ul, which is a promonocytic cell line (Figure 5).

EXAMPLE 9

Gene and Pathway Profile Analysis of Chronically Infected Cell Lines

An analysis of the gene expression profiles showed a limited number of genes that changed similarly across the three cell lines tested (Figure 5). These genes include cdc42, Lyn, MNDA, CEBPalpha, Meisl and others, which were down regulated in all cell lines. Genes that showed up regulation, include those encoding BTGl, BTG3, CDTl, pinin, and many others. Cdc42 is critical for activation of Nef associated kinase (PAK2), while Lyn is required for binding to the PXXP motif of HIV-Nef (43), (57). MNDA, CEBPalpha and Meisl are all tightly clustered. The proteins encoded by these genes are known to be critical in the progress of certain leukemias (18, 54, 65), but have not been hitherto related to HIV latency. Certain genes show similar differential expression in ACH-2 and Jl .1 but not in Ul cells. Also, some genes show opposite trends in the cell lines tested. For example, proteasome subunits are up regulated in ACH-2, while they are down regulated in Ul . A list of common pathways affected and some pathways that change selectively is given (supplemental data, Table S4). The list of differentially expressed genes common to all three cell lines is given along with their expression ratios (supplemental data, Table S5).

EXAMPLE 10

Reactivation of Chronically Infected Cells by Targeting Specific Gene Classes

We reasoned that the differential expression of certain cellular genes in latently infected cells might be involved in maintaining the virus in latency, and so hypothesized that treating latently infected cells with agents targeting the products of differentially expressed genes may force virus out of latency. This may be important because reactivation of proviral HIV into lytic infection by targeting cellular factors, notably including cellular factors differentially expressed in latent, chronically infected cell lines, may provide new approaches to reduce or eliminate latent viral reservoirs. To test our hypothesis, we focused on the proteasome class of genes and Egrl, due to the availability of specific agents. We also found other differentially expressed genes such as the histone deacetylases, HDACl and HDAC2, which may represent good targets for reactivation of viral replication. Treatment of latently infected cells with agents that alter histone acetylations already been shown to trigger lytic replication in latently infected cells (21 , 40, 67).

We observed that the proteasome class of genes showed increased expression in ACH-2 cells even prior to induction, but not so in JLl and Ul cell lines. Hence, we sought to determine whether inhibition of proteasomes would induce latent provirus into lytic replication. We studied the effects of different concentrations of a proteasome inhibitor, clastolactacystin-beta-lactone (19), on p24 protein levels in chronically infected ACH-2 cells. A previous study has shown that proteasome inhibitors are capable of increasing the efficiency of an acute HIV infection (58). We therefore studied the effect of the proteasome inhibitor in the presence of 250 nM of AZT (ICs₀= 50 nM), in order to ensure that any increases in p24 levels were not due to increased efficiency of infection by actively replicating virus present in chronically infected cells or to subsequent rounds of viral replication. ACH-2 cells treated with AZT alone showed slightly lower p24 amounts than cells that were not treated with AZT, consistent with the observation that the chronically infected cells support a low, but detectable amount of ongoing viral replication. TNF-a, a known activator of latently infected cells caused a 20-24 fold increase in p24 expression in ACH-2 cells treated with AZT. 5 μM of clastolactacystin-beta-lactone caused a 10-15 fold increase in p24 expression compared to AZT-treated ACH-2 cells, indicating that clastolactacystin-beta-lactone activated latent provirus into lytic replication. The increases in p24 expression observed in cells treated with clastolactacystin-beta-lactone were dose- dependent, indicating a drug related response (Figure. 6A). This effect was not observed in similarly treated J 1.1 and Ul cells (data not shown), which is consistent with the finding that the expression of a large number of proteasomal subunits was not up regulated in these chronically infected cells. We also tested PS-341, a highly specific proteasome inhibitor that binds to the Beta-5 subunit of the proteasome and is approved for clinical treatment of multiple myeloma (1, 3). However, due to the extreme cytotoxicity of PS-341 even at low concentration (100 nM) we were unable to observe any changes in p24 expression under our experimental conditions (1-1000 nM) in latently infected cells (data not shown).

In our microarray studies, the gene Egrl (early growth response 1) was down regulated in ACH-2 cells prior to induction and more importantly, up regulated during lytic replication. Egrl is involved in cell cycle regulation and cell differentiation, in response to a number of different growth factors (56, 64). Egrl activity is suppressed in many cancers including breast cancer and brain tumors, indicating that its activity is essential in cell cycle regulation (42),(38). It has recently been shown that resveratrol, an anti-oxidant stilbene (3, 5, 4'-trihydroxystilbene), can activate Egrl, thereby modulating p21cip expression and thus exert an effect on cell cycle regulation. Egrl expression also causes growth arrest (Gl-S, G2- M phases) (52). We therefore tested the ability of resveratrol to activate lytic replication. Treatment of ACH-2 cells with resveratrol caused a dose-dependent increase in p24 expression, indicative of viral reactivation (Figure 6B). At concentrations greater than 50 μM, an increase in cell death was observed, which lead to decreased p24 levels at higher concentrations.

Considerable prior work has been done on the influence of chromatin structure and histone acetylation status on HIV gene expression (21, 40, 67). These studies postulated that agents that can alter histone acetylation state of the host cell would activate lytic HIV replication and this was indeed found to be the case. However there has been no prior indication that the cellular machinery that maintains histones in the deacetylated state might be up regulated in cells latently infected with HIV. In our expression data, we observe that HDACl and HDAC2 are up regulated greater than two- fold prior to induction. Also, the gene encoding YYl, known to repress HIV-I LTR through HDAC recruitment was also significantly up regulated prior to induction in ACH-2 cells (17). We confirmed that treating cells latently infected with HIV with trichostatin, an agent that targets this class of differentially expressed gene products, activates lytic HIV replication (Figure 6C).

Table Sl: List of sequence detection primers and probe pairs for real time RT-PCR quantitation of selected genes.

Table S 1 : List of sequence detection primers and probe pairs for real time RT-PCR quantitation of selected genes.

Sequence detection primers and labeled probes were designed by PrimerExpress software from Applied Biosystems (ABI). The primer and probe names indicate the position in the cDNA sequence. F= forward, R= reverse, T= Taqman probe.

Table S2: Pathways that changed significantly prior and post induction of ACΗ-2 cells.

Table S2: Pathways that changed significantly prior and post induction of ACH-2 cells.

List of pathways that show altered expression based upon the number of genes involved in the pathways that showed significant differential expression during the four stages of the time course studied: Uninduced, 0.5-8 hours post induction (p.i.), 12-24 hour p.i., and 48-96 hours p.i. The values highlighted in each column indicate the pathways that were maximally altered during that time period. The pathway profiles that are shown in Figure 3 are included here, along with the respective pathway descriptions. During 48-96 hours p.i, not many pathways changed appreciably in relation to the other time periods, hence data for those pathways is not shown.

Table S3: Verification of differentially expressed gene expression levels by real time

RT-PCR quantitation.

Table S3: Verification of differentially expressed gene expression levels by real time RT-PCR quantitation.

Comparison of fold change in gene expression in latently infected ACH-2 cells prior to PMA induction, as compared to gene expression in uninfected parental A3.01 cells prior to PMA induction, by microarray and real time RT-PCR. RT-PCR data for the selected genes was normalized to data for GAPDH for each cell line, before assessing fold change in ACH-2 cells with respect to A3.01 cells. Differential expression of the selected genes was confirmed by RT-PCR quantitation.

Table S4: Pathways that change significantly in three chronically infected cell lines.

Table S4: Pathways that changed significantly in three chronically infected cell lines.

List of common cellular pathways differentially expressed in three chronically infected cell lines, ACH-2, Ul, and Jl.1. Classification was performed using the CGAP pathway database. The pathways are classified on the basis of the number of genes that showed significant differential expression in the chronically infected cell line over the corresponding uninfected parental cell line. Bold italicized values indicate similar number of genes affected in the pathways across all the three cell lines. Values in bold indicate pathways which are affected in ACH-2 and Jl .1 but not appreciably in Ul . Values in italics indicate pathways affected in ACH-2 and Ul , but not in Jl .1. Values in normal text indicate pathways that show some change in Jl .1 and Ul but not in ACH-2 cells.

Table S5: Genes that showed similar differential expression in all three chronically infected cell lines.

Table S5: Genes that showed similar differential expression in all three chronically infected cell lines.

Genes that were up regulated or down regulated in all three latently infected cell lines.

Normalized gene expression ratios that passed the various filter criteria and were shown to be significant by univariate (p < 0.001) and multivariate T-test for each cell line are listed.

Example 11

Effect of Proteasome Inhibitor on Latently Infected Jurkat clones We found that CLBL (l OμM) causes reactivation of latent provirus in these latently infected cell lines (developed in our laboratory) indicating that CLBL may be able to broadly activate HIV latently infected cells, although with differing efficiency depending on the cell line (Figure 7). Depending on the cell line, the effect observed was 50-500% of the reactivation caused by TNF-alpha in these cell lines.

Example 12

Effect of proteasome Inhibitor on aviremic patient sample #1

Enriched CD4+ T-cells were obtained from HIV-infected patients (see, e.g., Chun TW, Finzi D, Margolick J, Chadwick K, Schwartz D, Siliciano RF. In vivo fate of HIV-I- infected T cells: quantitative analysis of the transition to stable latency.Nat Med. 1995 Dec; 1(12): 1284-90) who were being treated with Highly Active Antiretroviral Therapy (HAART, combination therapy with several different antiretroviral agents) who had had viral loads below the limits of detection for some time. Although these patients had no detectable circulating HIV, they still had a population of latently infected cells. Enriched CD4 cells were aliquoted at IxIO⁶ cells/well and different concentrations of CLBL were tested. Cells were pre-treated with AZT (25OnM) to ensure that any p24 expression changes were due to reactivation of provirus and not from actively replicating virus that may be present at undetectably low levels in aviremic patients. Upon addition of CLBL in vitro, a dose dependent increase in p24 expression was observed in patient samples. For patient sample #1, the effect ranged from 130% - 160% of TNF-alpha's effect on reactivating latent virus, indicating that CLBL has a strong and potent effect on viral reactivation (Figure 8). Also, the dose at which reactivation was observed in the patient sample was about 5-10 fold lower than that observed in cell lines, indicating that a lower concentration range of reactivating agents may be sufficient for reactivating latent viral reservoirs from aviremic patients.

Example 13 Effect of Clastolactacystin-beta-lactone ( 1 uM) on Reactivation of Aviremic Patient Samples

As seen in Figure 9, different aviremic HIV-infected patient samples showed different levels of viral reactivation upon treatment with the proteasome inhibitor CLBL. Figure 9 shows the relative effect of lμM of Clastolactacystin-beta-lactone on two aviremic patient samples (samples #3 ( 8 year aviremic), and #4 ( 4-year aviremic)). The data indicate that aviremic patients do have existing latent viral reservoirs that respond to clastolactacystin- beta-lactone, but that different patients can have differing responses to treatment with clastolactacystin-beta-lactone. Without wishing to be bound by any theory, the degree of response may be dependent on a number of factors such as frequency of latent virus present, sensitivity to Clastolactacystin-beta-lactone and other factors.

In summary, CLBL caused reactivation of latent provirus from latently infected cell lines and from aviremic patient samples

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See also V. Krishnan and S.L. Zeichner, J. Virology, 78(17): 9458-9473 (2004).

All documents (including patents, patent applications and literature references) mentioned herein are incorporated herein by reference.

All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.

From the above description, one skilled in the art can easily ascertain the essential characteristics of the present invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, other embodiments are within the appended claims.

Claims

What is claimed is:

1. A method of treating HIV infection in a subject comprising identifying a subject as in need of reactivation of a replication process in latent HIV-infected cells; administering to the subject an effective amount of a proteasome inhibitor to reactivate the replication process; and administering to the subject one or more HIV antiviral agents to inhibit induced lytic replication in the cells.

2. A method of treating HIV infection in a subject comprising identifying a subject as in need of reactivation of a replication process in latent HIV-infected cells; administering to the subject an effective amount of a proteasome inhibitor having a pyrrolidin-2-one ring in its structure to reactivate the replication process; and administering to the subject one or more HIV antiviral agents to inhibit induced lytic replication in the cells.

3. A method of treating HIV infection in a subject comprising identifying a subject as in need of reactivation of a replication process in latent HIV-infected cells; administering to the subject an effective amount of a proteasome inhibitor having a β-lactone ring in its structure to reactivate the replication process; and administering to the subject one or more HIV antiviral agents to inhibit induced lytic replication in the cells.

4. A method of treating HIV infection in a subject comprising identifying a subject as in need of reactivation of a replication process in latent HIV-infected cells; administering to the subject an effective amount of a proteasome inhibitor having the structure of formula (I) or

(II):

formula (I) wherein Ri is independently -C(O)NR²R³, -C(O)SR², or -C(O)OR², wherein R² and R³ are each independently alkyl, cycloalkyl, aryl, heterocyclyl, heteroaryl, aralkyl, or heteroarylalkyl, each being optionally substituted with 1-3 substituents, and R⁴ and R⁵ are each independently alkyl; or

formula (II)

wherein R⁴ and R⁵ are each independently alkyl, to reactivate the replication process; and administering to the subject one or more HIV antiviral agents to inhibit induced lytic replication in the cells.

5. The method of claim 4, wherein the proteasome inhibitor is c/αsto-lactacystin β-lactone.

6. The method of claim 5, wherein the additional agent(s) are a reverse transcriptase inhibitor, a protease inhibitor, or combination thereof.

7. The method of claim 6, wherein the additional agent(s) are a combination of a reverse transcriptase inhibitor and a protease inhibitor.

8. A method of modulating lytic replication in an HIV-infected cell in a subject identified as in need of lytic replication modulation comprising administration to the subject of an effective amount of a proteasome inhibitor.

9. The method of any of claims 1-8, wherein the cell is a lymphocytic cell.

10. The method of any of claims 1-8, wherein the cell is a monocytic cell.

11. A method of reducing latent HIV reservoirs in an HIV-infected subject comprising identifying the subject as in need of reactivation of replication processes in latent HIV- infected cells; and administration to the subject of an effective amount of a proteasome inhibitor for reactivation of replication processes in latent HIV-infected cells.

12. A method of increasing expression of p24 in a latently HIV-infected cell comprising administration to the cell of an effective amount of a proteasome inhibitor.

13. A method of activating latent HIV-provirus in a cell in a subject comprising identifying the subject as in need of reactivation of replication processes in latent HIV-infected cells; and administration to the subject of an effective amount of a proteasome inhibitor for reactivation of replication processes in latent HIV-infected cells.

14. The method of claim 13, wherein the proteasome inhibitor affects one or more catalytic activities of the proteasome.

15. The method of claim 14, wherein one or more catalytic activities are chymotryptic, tryptic, or peptidyl glutamyl catalytic activities.

16. The method of any of claims 8-15, further comprising administration of one or more additional anti-HIV therapeutic agents.

17. The method of claim 16, wherein the additional agent(s) are a reverse transcriptase inhibitor, a protease inhibitor, or combination thereof.

18. The method of any of claims 8-17 wherein the proteasome inhibitor is c/αsto-lactacystin β-lactone.

19. A method of screening for a compound capable of activating latent HIV-infected cells comprising contacting a test compound with a latent HIV-infected cell (e.g., an ACH-2 cell or Jl .1 cell or Ul cell) and determining the level of p24 expression.

20. The method of claim 19, wherein increased expression of p24 in cells treated with a test compound relative to non-treated cells indicates that the test compound is capable of activating replication processes in latent HIV-infected cells.

21. The method of claim 20, wherein the test compound comprises a pyrrolidin-2-one ring or a β-lactone ring structural moiety.

22. A method of preventing HIV infection in a subject comprising identifying a subject as in need of reactivation of a replication process in latent HIV-infected cells; administering to the subject an effective amount of a proteasome inhibitor to reactivate the replication process; and administering to the subject one or more HIV antiviral agents to inhibit induced lytic replication in the cells.