WO2019118803A1 - Chemical entities for lytic activation of kshv and therapeutic targeting of viral enzymes/proteins - Google Patents

Abstract

A method for activating and killing Kaposi's sarcoma herpesvirus (KSHV) includes (i) contacting latent KSHV with an amount of a compound according to Formula I, or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof, effective to activate at least some of the latent KSHV, and (ii) contacting the activated KSHV with an amount of an α-hydroxytropolone effective to kill at least some of the activated KSHV. With respect to Formula I, R1 is -N(R2)-C(0)-(CH2)x-R3 where R2 is hydrogen or C1-C3 alkyl, R3 is a substituted amino group or a substituted or unsubstituted piperazinyl or piperidinyl group, and x is an integer from 0-10.

Description

CHEMICAL ENTITIES FOR LYTIC ACTIVATION OF KSHV AND THERAPEUTIC TARGETING OF VIRAL ENZYMES/PROTEINS

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of the earlier filing date of U.S. Provisional Application No. 62/598,807, filed December 14, 2017, which is incorporated by reference in its entirety herein.

FIELD

This disclosure concerns a method for activating and then killing Kaposi’s sarcoma herpesvirus (KSHV), wherein one agent activates the KSHV and a second agent kills the activated vims.

ACKNOWLEDGMENT OF GOVERNMENT SUPPORT

This invention was made with government support under NIH Z01 Project # ZIA BC 010493 and SC1GM111158 awarded by the National Institutes of Health. The government has certain rights in the invention.

PARTIES TO JOINT RESEARCH AGREEMENT

The United States Government and Research Foundation of The City University of New York on behalf of Brooklyn College.

BACKGROUND

Kaposi’s sarcoma-associated herpesvirus (KSHV), also known as HHV-8, is classified as an oncogenic human herpesvirus and is one of the most frequent causes of cancer in human immunodeficiency virus (HlV)-infected patients. KSHV is the most common cause of cancer in Sub-Saharan Africa, and Kaposi’s sarcoma is the second most frequent tumor in HIV-infected patients. KSHV is also the etiologic agent of primary effusion lymphoma and the B-cell hyperplasia known as multicentric Castleman’s disease. With -45,000 new cases and 27,000 deaths worldwide in 2012, KSHV-associated cancer presents a significant global health burden (Global Cancer Observatory, http://globocan.iarc.fr/). Like HIV, a major problem in the treatment of KSHV is viral latency which modulates expression of cellular genes and allows the vims to subvert host immunity. SUMMARY

Embodiments of a method for activating and killing Kaposi’s sarcoma herpesvirus (KSHV) are disclosed. The method includes (i) contacting latent KSHV with an amount of a compound according to Formula I, or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof, effective to activate at least some of the latent KSHV, and (ii) contacting the activated KSHV with an amount of an a-hydroxytropolone effective to kill at least some of the activated KSHV. With respect to Formula I, R¹ is -N(R²)-C(0)-(CH₂)_X-R³ where R² is hydrogen or C1-C3 alkyl, R³ is a substituted amino group or a substituted or unsubstituted piperazinyl or piperidinyl group, and x is an integer from 0-10.

Formula I

In some embodiments, the compound according to Formula I is

, or any combination thereof.

In any or all of the above embodiments, the a-hydroxytropolone may have a chemical structure according to Formula II, or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof:

Formula II where R⁴ is a hydrogen or C₁-C₅ alkyl, R⁵ is hydrogen, hydroxy, alkoxy, alkoxycarbonyl, aryl, or halo, and R⁶ is hydrogen, an ester, a ketone, sulfonyl, or aryl, or R⁴ and R⁶ together with the atoms to which they are attached form a substituted or unsubstituted heteroaliphatic ring. In some embodiments, the a-hydroxytropolone is

or any combination thereof.

In any or all of the above embodiments, contacting the latent KSHV with the compound according to Formula I may include administering the effective amount of the compound according to Formula I to a subject infected, or suspected of being infected, with KSHV; and contacting the activated KSHV with the a-hydroxytropolone may include administering the effective amount of the a-hydroxytropolone to the subject. Administration may be performed simultaneously or sequentially. In some embodiments, the compound according to Formula 1 is administered prior to administering the a-hydroxytropolone. The a-hydroxytropolone may be administered to the subject within a therapeutic time window of the compound according to Formula I. In any or all of the above embodiments, the compound according to Formula I and/or the a-hydroxytropolone may be administered simultaneously as a single pharmaceutical composition or administered

simultaneously or sequentially by the same or different routes as two pharmaceutical compositions.

The foregoing and other objects, features, and advantages of the invention will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

FIG. 1 shows a 40-nucleotide RNA duplex including the expression and nuclear retention element of (ENE, left) and the ENE to which an (A)₉ oligonucleotide was hybridized to generate the ENE triple helix (right).

FIG. 2 is a color photograph showing fluorescence obtained when an activation compound as disclosed herein binds to the ENE triple helix.

FIG. 3 shows the structure of a KSHV episome including a recombinant virus rKSHV.2l9.

FIG. 4 is a series of color photographs showing results of incubating iSLK-2l9 cells with 1% DMSO as a control (FIG. 3 A), 2.5 mM sodium butyrate (FIG. 3B), 100 mM Compound 15, 100 mM Compound 15 and 2.5 mM sodium butyrate, 100 mM Compound 18 and 100 mM Compound 18 and 2.5 mM sodium butyrate. The left panels are phase micrographs, the center panels show GFP fluorescence, and the right panels show RFP fluorescence.

FIG. 5 is photographs of stained agarose gels showing inhibition of KSHV pORF29C nuclease activity by certain a-hydroxytropolones (aHTs).

FIGS. 6 A and 6B show a duplex DNA sequence (6 A) and photographs of stained agarose gels showing inhibition of KSHV pORF29C nuclease activity by an aHT as disclosed herein (6B).

FIGS. 7A and &B show the effects of an aHT as disclosed herein on KSHV lytic replication. KSHV viral copies were determined by a quantitative real-time assay in cells (7 A) and DNase I-treated supernatants (7B) after 24 hours (black bars) and 72 hours (gray bars).

SEQUENCE LISTING

The nucleic and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and three letter code for amino acids, as defined in 37 C.F.R. § 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand. The Sequence Listing is submitted as an ASCII text file, created on December 9, 2018, 0.9 kB, which is incorporated by reference herein.

SEQ ID NO: 1 is the nucleotide sequence ATGTATTTAGGATTGGAACTTCTTGAG

SEQ ID NO: 2 is the nucleotide sequence CTCAAGAAGTTCCAATCCTAAATACATA

DETAILED DESCRIPTION

Embodiments of compounds and methods for activating and killing Kaposi’s sarcoma herpesvirus (KSHV) are disclosed. The method is a“kick and kill” strategy in which a first agent lytically activates latent KSHV and a second agent kills the activated virus e.g., by inhibiting replication.

Like HIV, a major problem in the treatment of KSHV is viral latency which modulates expression of cellular genes and allows the virus to subvert host immunity. The latent to lytic switch is marked by production of a highly abundant long noncoding transcript (lncRNA) designated polyadenylated nuclear (PAN) that interacts with cell- and virus-encoded factors to regulate the immune response gene expression. A challenge to developing an effective antiviral strategy for KSHV is that lytic activation is necessary before antiviral agents targeting virus- associated proteins and enzymes can be employed. Some embodiments of the disclosed method employ two classes of molecules used in combination to (i) drive KSHV out of latency, and (ii) target nuclease activity of the KSHV terminase molecular motor, thereby inhibiting replication and killing the KSHV.

I. Definitions and Abbreviations

The following explanations of terms and abbreviations are provided to better describe the present disclosure and to guide those of ordinary skill in the art in the practice of the present disclosure. As used herein,“comprising” means“including” and the singular forms“a” or“an” or “the” include plural references unless the context clearly dictates otherwise. The term“or” refers to a single element of stated alternative elements or a combination of two or more elements, unless the context clearly indicates otherwise.

Unless explained otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. The materials, methods, and examples are illustrative only and not intended to be limiting. Other features of the disclosure are apparent from the following detailed description and the claims.

Unless otherwise indicated, all numbers expressing quantities of components, temperatures, times, and so forth, as used in the specification or claims are to be understood as being modified by the term“about.” As used herein, the term“about” or the symbol

refers to a ±10% variation from the nominal value unless otherwise indicated or inferred. Accordingly, unless otherwise implicitly or explicitly indicated, or unless the context is properly understood by a person of ordinary skill in the art to have a more definitive construction, the numerical parameters set forth are approximations that may depend on the desired properties sought and/or limits of detection under standard test conditions/methods as known to those of ordinary skill in the art. When directly and explicitly distinguishing embodiments from discussed prior art, the embodiment numbers are not approximates unless the word“about” is recited.

At various places in the present specification, substituents are disclosed in groups or in ranges. It is specifically intended that the description include each and every individual subcombination of the members of such groups and ranges. For example, the term“C1-C5 alkyl” is specifically intended to individually disclose Cl, C2, C3, C4, C5, C1-C5, C1-C4, C1-C3, C1-C2, C2-C5, C2-C4, C2-C3, C3-C5, C3-C4, and C4-C5 alkyl.

Activated virus: As used herein, the term“activated virus” refers to a virus that is proliferating, also known as the lytic part of the viral life cycle.

Alkoxy: A radical (or substituent) having the structure -OR, where R is a substituted or unsubstituted alkyl. Methoxy (-OCH₃) is an exemplary alkoxy group. In a substituted alkoxy, R is alkyl substituted with a non-interfering substituent.“Thioalkoxy” refers to -S-R, where R is substituted or unsubstituted alkyl. “Haloalkyloxy” means a radical -OR where R is a haloalkyl.

Alkoxycarbonyl (ester): A chemical functional group -C(0)OR where R is substituted or unsubstituted alkyl.

Alkyl: A hydrocarbon group having a saturated carbon chain. The chain may be cyclic, branched or unbranched. Examples, without limitation, of alkyl groups include methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl and decyl. Unless otherwise specified, an alkyl group may be substituted or unsubstituted.

Aryl: A monovalent aromatic carbocyclic group of, unless specified otherwise, from 6 to 15 carbon atoms having a single ring (e.g., phenyl) or multiple condensed rings in which at least one ring is aromatic (e.g., quinoline, indole, benzodioxole, and the like), provided that the point of attachment is through an atom of an aromatic portion of the aryl group and the aromatic portion at the point of attachment contains only carbons in the aromatic ring. If any aromatic ring portion contains a heteroatom, the group is a heteroaryl and not an aryl.

Arylalkyl: An aryl group (such as a phenyl group) appended to an alkyl radical including, but not limited to, benzyl, ethylbenzene, propylbenzene, butylbenzene, pentylbenzene,

methylindole, ethylindole, and the like. Arylalkyl groups, such as benzyl groups, may be unsubstituted or substituted with one, two or three substituents, with substituent(s) independently selected from alkyl, heteroalkyl, aliphatic, heteroaliphatic, thioalkoxy, haloalkyl (such as -CF₃), halo, nitro, cyano, -OR (where R is hydrogen or alkyl), -N(R)R’ (where R and R’ are independently of each other hydrogen or alkyl), -COOR (where R is hydrogen or alkyl) or -C(0)N(R’)R” (where R’ and R” are independently selected from hydrogen or alkyl).

Sulfonyl: A functional group with the general formula:

where R and R' independently are selected from various groups, including by way of example aliphatic, substituted aliphatic, cyclic aliphatic, substituted cyclic aliphatic, aryl, substituted aryl, heteroaryl, and substituted heteroaryl.

Effective amount (or dose): An amount sufficient to effect a change, such as a change in replication activity of KSHV.

a-Hydroxytropolone: An organic compound having the formula C₇H₄(0H)₂0:

Latent virus: A virus in its latent stage is dormant within the cells of a subject, also known as the lysogenic part of the viral life cycle. Latency is a phase in which proliferation of vims particles ceases, but the viral genome is not eradicated. A latent virus may later reactivate and proliferate.

Pharmaceutically acceptable: A substance that can be taken into a subject without significant adverse toxicological effects on the subject. The term "pharmaceutically acceptable form" means any pharmaceutically acceptable derivative or variation, such as stereoisomers, stereoisomer mixtures, enantiomers, solvates, hydrates, isomorphs, polymorphs, pseudomorphs, neutral forms, salt forms, and prodrug agents.

Pharmaceutically acceptable carrier: The pharmaceutically acceptable carriers (vehicles) useful in this disclosure are conventional. Remington: The Science and Practice of Pharmacy, The University of the Sciences in Philadelphia, Editor, Lippincott, Williams, & Wilkins, Philadelphia, PA, 2 I^st Edition (2005), describes compositions and formulations suitable for pharmaceutical delivery of one or more therapeutic compositions and additional pharmaceutical agents. In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. In some examples, the pharmaceutically acceptable carrier may be sterile to be suitable for administration to a subject (for example, by parenteral, intramuscular, or subcutaneous injection). In addition to biologically-neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate. In some examples, the pharmaceutically acceptable carrier is non-natural or synthetic. The carrier also can be formulated in a unit-dosage form that carries a preselected therapeutic dosage of the active agent, for example in a pill, vial, bottle, or syringe.

Pharmaceutically acceptable salt: A biologically compatible salt of a compound that can be used as a drug, which salts are derived from a variety of organic and inorganic counter ions well known in the art and include, by way of example only, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, and the like; and when the molecule contains a basic

functionality, salts of organic or inorganic acids, such as hydrochloride, hydrobromide, tartrate, mesylate, acetate, maleate, oxalate, and the like. Pharmaceutically acceptable acid addition salts are those salts that retain the biological effectiveness of the free bases while formed by acid partners that are not biologically or otherwise undesirable, e.g., inorganic acids such as

hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like, as well as organic acids such as acetic acid, trifluoroacetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, benzene sulfonic acid (besylate), cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the like. Pharmaceutically acceptable base addition salts include those derived from inorganic bases such as sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Exemplary salts are the ammonium, potassium, sodium, calcium, and magnesium salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2- dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine,

methylglucamine, theobromine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins, and the like. Exemplary organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine. (See, for example, S. M. Berge, et al., “Pharmaceutical Salts,” J. Pharm. Sci., 1977; 66: 1-19, which is incorporated herein by reference.)

Stereoisomers: Compounds described herein can contain an asymmetric atom (also referred as a chiral center) and some of the compounds can contain two or more asymmetric atoms or centers, which can thus give rise to stereoisomers. Stereoisomers have the same molecular formula and sequence of bonded atoms, but differ only in the three-dimensional orientation of the atoms in space. Stereoisomers that are not mirror images of one another are termed

“diastereomers” and those that are non- superimpos able mirror images of each other are termed “enantiomers.” When a compound has an asymmetric center, for example, if a carbon atom is bonded to four different groups, a pair of enantiomers is possible. An enantiomer can be characterized by the absolute configuration of its asymmetric center and is described by the R- and S-sequencing rules of Cahn and Prelog, or by the manner in which the molecule rotates the plane of polarized light and designated as dextrorotatory or levorotatory (/._<?., as (+) or (-) isomers respectively). A chiral compound can exist as either individual enantiomer or as a mixture thereof. A mixture containing equal proportions of the enantiomers is called a“racemic mixture.” In some embodiments, optical isomers can be obtained in enantiomerically enriched or pure form by standard procedures known to those skilled in the art, which include, for example, chiral separation, diastereomeric salt formation, kinetic resolution, and asymmetric synthesis. £ZZ isomers are isomers that differ in the stereochemistry of a double bond. An E isomer (from entgegen, the German word for "opposite") has a trans- configuration at the double bond, in which the two groups of highest priority are on opposite sides of the double bond. A Z isomer (from zusammen, the German word for "together") has a cA-configuration at the double bond, in which the two groups of highest priority are on the same side of the double bond. It also should be understood that the compounds of the present teachings encompass all possible regioisomers in pure form and mixtures thereof. In some embodiments, the preparation of the present compounds can include separating such isomers using standard separation procedures known to those skilled in the art, for example, by using column chromatography, thin-layer chromatography, simulated moving-bed

chromatography, and high-performance liquid chromatography, either alone or in combination. However, mixtures of regioisomers can be used similarly to the uses of each individual regioisomer of the present teachings as described herein and/or known by a skilled artisan. It is specifically contemplated that the depiction of one regioisomer includes any other regioisomers and any regioisomeric mixtures unless specifically stated otherwise.

Substituent: An atom or group of atoms that replaces another atom in a molecule as the result of a reaction. The term "substituent" typically refers to an atom or group of atoms that replaces a hydrogen atom, or two hydrogen atoms if the substituent is attached via a double bond, on a parent hydrocarbon chain or ring. The term“substituent” may also cover groups of atoms having multiple points of attachment to the molecule, e.g., the substituent replaces two or more hydrogen atoms on a parent hydrocarbon chain or ring. In such instances, the substituent, unless otherwise specified, may be attached in any spatial orientation to the parent hydrocarbon chain or ring. Exemplary substituents include, for instance, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, acyl, aldehyde, amido, amino, aminoalkyl, aryl, arylalkyl, arylamino, carbonate, carboxyl, cyano, cycloalkyl, dialkylamino, halo, haloaliphatic (e.g., haloalkyl), haloalkoxy, heteroaliphatic, heteroaryl, heterocycloaliphatic, hydroxyl, oxo, sulfonamide, sulfhydryl, thio, and thioalkoxy groups.

Substituted: A fundamental compound, such as an aryl or aliphatic compound, or a radical thereof, having coupled thereto one or more substituents, each substituent typically replacing a hydrogen atom on the fundamental compound. Solely by way of example and without limitation, a substituted aryl compound may have an aliphatic group coupled to the closed ring of the aryl base, such as with toluene. Again, solely by way of example and without limitation, a long-chain hydrocarbon may have a hydroxyl group bonded thereto.

Tautomers: Constitutional isomers of organic compounds that differ only in the position of the protons and electrons, and are interconvertible by migration of a hydrogen atom. Tautomers ordinarily exist together in equilibrium.

Therapeutic agent: An agent that provides a beneficial, or therapeutic, effect to a subject or a given percentage of subjects.

Therapeutically effective amount (or dose): An amount sufficient to provide a beneficial, or therapeutic, effect to a subject or a given percentage of subjects.

Therapeutic time window: The length of time during which an effective, or therapeutic dose, of a compound remains therapeutically effective in vivo.

Treating or treatment: With respect to disease, either term includes (1) preventing the disease, e.g., causing the clinical symptoms of the disease not to develop in an animal that may be exposed to or predisposed to the disease but does not yet experience or display symptoms of the disease, (2) inhibiting the disease, e.g., arresting the development of the disease or its clinical symptoms, or (3) relieving the disease, e.g., causing regression of the disease or its clinical symptoms.

II. Compounds for Activating and Killing KSHV

A combination of two compounds is used in a“kick and kill” strategy for activating and killing KSHV. KSHV expresses a highly abundant long noncoding transcript (lncRNA) designated polyadenylated nuclear (PAN) that interacts with cell- and virus-encoded factors to regulate the immune response gene expression. At its 3' terminus, PAN exhibits a unique structure - the expression and nuclear retention element (ENE) - which assumes a triple helix configuration to sequester and shield the poly(A) tail from exonucleases. While this motif imparts distinctive structural properties on PAN, the biological significance of the ENE triple helix is unclear.

Nonetheless, the inventors have discovered that certain compounds which bind to the ENE triple helix can promote lytic induction of KSHV. In some embodiments, the activating compound that promotes lytic induction of KSHV has a structure according to Formula I, or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof:

Formula I

With respect to Formula I, R¹ is -N(R²)-C(0)-(CH₂)_X-R³ where R² is hydrogen or C1-C3 alkyl, R³ is a substituted amino group or a substituted or unsubstituted piperazinyl or piperidinyl group, and x is an integer from 0-10. In some embodiments, R² is hydrogen. In any of the foregoing

embodiments, x may be 1, 2, 3, 4, or 5. In some embodiments, x is 1, 2, or 3. In certain examples, x is 1.

In any or all of the above embodiments, R³ is a substituted amino group or a substituted or unsubstituted piperazinyl or piperidinyl group. The substituted amino group may be -N(R)R' where R is hydrogen or C1-C3 alkyl and R' is alkyl or arylalkyl. In some embodiments, R is hydrogen and R' is alkyl or arylalkyl. In one embodiment, R is hydrogen and R' is a branched or unbranched alkyl, such as a C1-C10 alkyl. In an independent embodiment, R and R' independently are C1-C3 alkyl. In another independent embodiment, R is hydrogen and R' is arylalkyl. In still another independent embodiment, R³ is a substituted or unsubstituted piperazinyl group attached to the remainder of R¹ via a nitrogen atom in the ring.

Certain exemplary compounds according to Formula I are shown in Table 1. Table 1

The killing compound is an a-hydroxytropolone that exhibits antiviral activity against KSHV. Some a-hydroxytropolones are potent small molecular inhibitors of herpesvirus nucleotidyltransferases. The a-hydroxytropolone may be a naturally occurring compound or a synthetic a-hydroxytropolone. In some embodiments, the a-hydroxytropolone has a chemical structure according to Formula II:

Formula II

With respect to Formula II, R⁴ is hydrogen or C1-C5 alkyl, R⁵ is hydrogen, hydroxy, alkoxy, alkoxycarbonyl, aryl, or halo, and R⁶ is hydrogen, an ester, a ketone, sulfonyl, or aryl, or R⁴ and R⁶ together with the atoms to which they are attached form a substituted or unsubstituted

heteroaliphatic ring. In some embodiments, R⁴ is hydrogen or C1-C5 alkyl, R⁵ is hydrogen, hydroxy, alkoxy, alkoxycarbonyl, or aryl, and R⁶ is hydrogen, an ester, a ketone, sulfonyl, or aryl.

In some embodiments, R⁴ is methyl, ethyl, or propyl. In certain examples, R⁴ is methyl. In any or all of the above embodiments, R⁵ is hydrogen, hydroxy, alkoxy, alkoxycarbonyl, aryl, or halo. In some embodiments, R⁵ is hydrogen, hydroxy, alkoxy, alkoxycarbonyl, or halo. In certain embodiments, R⁵ is hydrogen, hydroxy, methoxycarbonyl, or bromo.

In any or all of the above embodiments, R⁶ may be -C(0)-(CH₂)o-₄CH₃, -C(0)-0-(CH₂)o- 4CH3, hydroxy, or aryl. In some embodiments, R⁶ is -C(0)-(CH₂)o-₄CH₃, -C(0)-0-(CH₂)o-₄CH₃, or aryl, such as an aryl ketone (i.e., -C(O)-aryl). In certain examples, R⁶ is -C(0)CH₃, -C(0)0CH₃, - C(0)0CH₂CH₃, -C(0)-C₆H₅, -C(0)-C₆H₄-C₆H₅ wherein the biphenyl group is attached to the carbonyl via Cl or C2 (structure below), or H, or C⁶ and C⁴ together with the atoms to which they

are bound form

(biphenyl)

Some exemplary a-hydroxytropolones according to Formula II are shown in Table 2.

Table 2

In some embodiments, the a-hydroxytropolone is aHTl, aHT2, aHT3, aHT6, aHT7, aHTlO, or any combination thereof. In certain embodiments, the a-hydroxytropolone is aHTl, aHT2, aHT3, or any combination thereof.

III. Pharmaceutical Compositions

In one embodiment, a pharmaceutical composition includes a compound according to Formula I, or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable additive such as pharmaceutically acceptable carriers, thickeners, diluents, buffers, preservatives, surface active agents and the like in addition to the molecule of choice. In another embodiment, a pharmaceutical composition includes an a-hydroxytropolone as disclosed herein, or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable additive. In an independent embodiment, a pharmaceutical composition includes (i) a compound according to Formula I, or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof, (ii) an a-hydroxytropolone as disclosed herein, or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof, and (iii) at least one pharmaceutically acceptable additive. In any or all of the above embodiments, the pharmaceutical compositions can also include one or more additional active ingredients such as antiviral agents, antimicrobial agents, anti-cancer agents, anti-inflammatory agents, anesthetics, and the like. The pharmaceutically acceptable carriers useful for these formulations are conventional. Remington 's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, PA, 19* Edition (1995), for example, describes compositions and formulations suitable for pharmaceutical delivery of the compounds herein disclosed.

The pharmaceutical compositions may be in a dosage unit form such as an injectable fluid, an oral delivery fluid (e.g., a solution or suspension), a nasal delivery fluid (e.g., for delivery as an aerosol or vapor), a semisolid form (e.g., a topical cream), or a solid form such as powder, pill, tablet, or capsule forms.

In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually contain injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions (for example, powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically-neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.

The compounds according to Formula I and the a-hydroxytropolones (hereinafter referred to as“the agents”) disclosed herein can be administered to subjects by a variety of routes, including by intramuscular, subcutaneous, intravenous, intra-arterial, intra-articular, intraperitoneal, intrathecal, intracerebroventricular, parenteral, oral, rectal, intranasal, intrapulmonary, transdermal, or topical routes. In other alternative embodiments, the agents can be administered ex vivo by direct exposure to cells, tissues or organs originating from a subject.

To formulate the pharmaceutical compositions, the agents can be combined with various pharmaceutically acceptable additives, as well as a base or vehicle for dispersion of the compound. Desired additives include, but are not limited to, pH control agents, such as arginine, sodium hydroxide, glycine, hydrochloric acid, citric acid, and the like. In addition, isotonizing agents (for example, sodium chloride, mannitol, sorbitol), adsorption inhibitors (for example, Tween^® 80 polyethylene sorbitol ester or Miglyol^® 812 triglycerides), solubility enhancing agents (for example, cyclodextrins and derivatives thereof), stabilizers (for example, serum albumin), and reducing agents (for example, glutathione) can be included. Adjuvants, such as aluminum hydroxide (for example, Amphogel, Wyeth Laboratories, Madison, NJ), Freund’s adjuvant, MPL™ (3-O-deacylated monophosphoryl lipid A; Corixa, Hamilton, IN) and IL-12 (Genetics Institute, Cambridge, MA), among many other suitable adjuvants well known in the art, can be included in the compositions. When the composition is a liquid, the tonicity of the formulation, as measured with reference to the tonicity of 0.9% (w/v) physiological saline solution taken as unity, is typically adjusted to a value at which no substantial, irreversible tissue damage will be induced at the site of administration· Generally, the tonicity of the solution is adjusted to a value of about 0.3 to about 3.0, such as about 0.5 to about 2.0, or about 0.8 to about 1.7.

The agents can be dispersed in a base or vehicle, which can include a hydrophilic compound having a capacity to disperse the compound, and any desired additives. The base can be selected from a wide range of suitable compounds, including but not limited to, copolymers of

polycarboxylic acids or salts thereof, carboxylic anhydrides (for example, maleic anhydride) with other monomers (for example, methyl (meth)acrylate, acrylic acid and the like), hydrophilic vinyl polymers, such as polyvinyl acetate, polyvinyl alcohol, polyvinylpyrrolidone, cellulose derivatives, such as hydroxymethylcellulose, hydroxypropylcellulose and the like, and natural polymers, such as chitosan, collagen, sodium alginate, gelatin, hyaluronic acid, and nontoxic metal salts thereof. Often, a biodegradable polymer is selected as a base or vehicle, for example, polylactic acid, poly(lactic acid-glycolic acid) copolymer, polyhydroxybutyric acid, poly(hydroxybutyric acid- glycolic acid) copolymer and mixtures thereof. Alternatively or additionally, synthetic fatty acid esters such as polyglycerin fatty acid esters, sucrose fatty acid esters and the like can be employed as vehicles. Hydrophilic polymers and other vehicles can be used alone or in combination, and enhanced structural integrity can be imparted to the vehicle by partial crystallization, ionic bonding, cross-linking and the like. The vehicle can be provided in a variety of forms, including fluid or viscous solutions, gels, pastes, powders, microspheres and films for direct application to a mucosal surface.

The agents can be combined with the base or vehicle according to a variety of methods, and release of the agents can be by diffusion, disintegration of the vehicle, or associated formation of water channels. In some circumstances, the agent is dispersed in microcapsules (microspheres) or nanocapsules (nanospheres) prepared from a suitable polymer, for example, isobutyl 2- cyanoacrylate (see, for example, Michael et al.., J. Pharmacy Pharmacol. 43:1-5, 1991), and dispersed in a biocompatible dispersing medium, which yields sustained delivery and biological activity over a protracted time.

The compositions of the disclosure can alternatively contain, as pharmaceutically acceptable vehicles, substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, and triethanolamine oleate. For solid compositions, conventional nontoxic pharmaceutically acceptable vehicles can be used which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like.

Pharmaceutical compositions for administering the agents can also be formulated as a solution, microemulsion, or other ordered structure suitable for high concentration of active ingredients. The vehicle can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity for solutions can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of a desired particle size in the case of dispersible formulations, and by the use of surfactants. In many cases, it will be desirable to include isotonic agents, for example, sugars, polyalcohols, such as mannitol and sorbitol, or sodium chloride in the composition. Prolonged absorption of the compound can be brought about by including in the composition an agent which delays absorption, for example, monostearate salts and gelatin.

In certain embodiments, the agents can be administered in a time release formulation, for example in a composition which includes a slow release polymer. These compositions can be prepared with vehicles that will protect against rapid release, for example a controlled release vehicle such as a polymer, microencapsulated delivery system or bioadhesive gel. Prolonged delivery in various compositions of the disclosure can be brought about by including in the composition agents that delay absorption, for example, aluminum monostearate hydrogels and gelatin. When controlled release formulations are desired, controlled release binders suitable for use in accordance with the disclosure include any biocompatible controlled release material which is inert to the active agent and which is capable of incorporating the compound and/or other biologically active agent. Numerous such materials are known in the art. Useful controlled-release binders are materials that are metabolized slowly under physiological conditions following their delivery (for example, at a mucosal surface, or in the presence of bodily fluids). Appropriate binders include, but are not limited to, biocompatible polymers and copolymers well known in the art for use in sustained release formulations. Such biocompatible compounds are non-toxic and inert to surrounding tissues, and do not trigger significant adverse side effects, such as nasal irritation, immune response, inflammation, or the like. They are metabolized into metabolic products that are also biocompatible and easily eliminated from the body.

Exemplary polymeric materials for use in the present disclosure include, but are not limited to, polymeric matrices derived from copolymeric and homopolymeric polyesters having hydrolyzable ester linkages. A number of these are known in the art to be biodegradable and to lead to degradation products having no or low toxicity. Exemplary polymers include polyglycolic acids and polylactic acids, poly(DL-lactic acid-co-glycolic acid), poly(D-lactic acid-co-glycolic acid), and poly(L-lactic acid-co-glycolic acid). Other useful biodegradable or bioerodable polymers include, but are not limited to, such polymers as poly(epsilon-caprolactone),

poly(epsilon-caprolactone-CO-lactic acid), poly(epsilon.-caprolactone-CO-glycolic acid), poly(beta-hydroxy butyric acid), poly(alkyl-2-cyanoacrilate), hydrogels, such as poly(hydroxyethyl methacrylate), polyamides, poly(amino acids) (for example, L-leucine, glutamic acid, L-aspartic acid and the like), poly(ester urea), poly(2-hydroxyethyl DL-aspartamide), polyacetal polymers, polyorthoesters, polycarbonate, polymaleamides, polysaccharides, and copolymers thereof. Many methods for preparing such formulations are well known to those skilled in the art (see, for example, Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978). Other useful formulations include controlled-release

microcapsules (U.S. Patent Nos. 4,652,441 and 4,917,893), lactic acid-glycolic acid copolymers useful in making microcapsules and other formulations (U.S. Patent Nos. 4,677,191 and 4,728,721) and sustained-release compositions for water-soluble peptides (U.S. Patent No. 4,675,189).

The pharmaceutical compositions of the disclosure typically are sterile and stable under conditions of manufacture, storage and use. Sterile solutions can be prepared by incorporating the compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated herein, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the compound and/or other biologically active agent into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated herein. In the case of sterile powders, methods of preparation include vacuum drying and freeze-drying which yields a powder of the compound plus any additional desired ingredient from a previously sterile-filtered solution thereof. The prevention of the action of microorganisms can be accomplished by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.

In accordance with the various treatment methods of the disclosure, the agents can be delivered to a subject in a manner consistent with conventional methodologies associated with management of the disorder for which treatment or prevention is sought. In accordance with the disclosure herein, a prophylactically or therapeutically effective amount of each of the agents (compound according to Formula I and a-hydroxytropolone) is administered to a subject in need of such treatment for a time and under conditions sufficient to prevent, inhibit, and/or ameliorate a KSHV infection or one or more symptom(s) thereof.

The agents can be administered to the subject by the oral route or in a single bolus delivery, via continuous delivery (for example, continuous transdermal, mucosal or intravenous delivery) over an extended time period, or in a repeated administration protocol (for example, by an hourly, daily or weekly, repeated administration protocol). The therapeutically effective dosages of the agents can be provided as repeated doses within a prolonged prophylaxis or treatment regimen that will yield clinically significant results to alleviate one or more symptoms or detectable conditions associated with a targeted condition as set forth herein. Determination of effective dosages in this context is typically based on animal model studies followed up by human clinical trials and is guided by administration protocols that significantly reduce the occurrence or severity of targeted disease symptoms or conditions in the subject. Suitable models in this regard include, for example, murine, rat, avian, porcine, feline, non-human primate, and other accepted animal model subjects known in the art. Alternatively, effective dosages can be determined using in vitro models. Using such models, only ordinary calculations and adjustments are required to determine an appropriate concentration and dose to administer a therapeutically effective amount of the compound (for example, amounts that are effective to elicit a desired immune response or alleviate one or more symptoms of a targeted disease). In alternative embodiments, an effective amount or effective dose of the agents may simply inhibit or enhance one or more selected biological activities correlated with a disease or condition, as set forth herein, for either therapeutic or diagnostic purposes.

The actual dosages of the agents will vary according to factors such as the disease indication and particular status of the subject (for example, the subject’s age, size, fitness, extent of symptoms, susceptibility factors, and the like), time and route of administration, other drugs or treatments being administered concurrently, as well as the specific pharmacology of the agent for eliciting the desired activity or biological response in the subject. Dosage regimens can be adjusted to provide an optimum prophylactic or therapeutic response. A therapeutically effective amount is also one in which any toxic or detrimental side effects of the agent is outweighed in clinical terms by therapeutically beneficial effects. A non-limiting range for a therapeutically effective amount of a compound according to Formula I or an a-hydroxytropolone within the methods and formulations of the disclosure is 0.001 mg/kg body weight to 100 mg/kg body weight, such as 0.01 mg/kg body weight to 20 mg/kg body weight, 0.01 mg/kg body weight to 10 mg/kg body weight 0.05 mg/kg to 5 mg/kg body weight, or 0.1 mg/kg to 2 mg/kg body weight. Dosage can be varied by the attending clinician to maintain a desired concentration at a target site (for example, systemic circulation). Higher or lower concentrations can be selected based on the mode of delivery, for example, trans- epidermal or oral delivery versus intravenous or subcutaneous delivery. Dosage can also be adjusted based on the release rate of the administered formulation, for example, of sustained release oral versus injected particulate or transdermal delivery formulations, and so forth.

IV. Methods of Activating and Killing KSHV

A method for activating and killing KSHV includes (i) contacting latent KSHV with an amount of a compound according to Formula I, or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof, as disclosed herein, effective to activate at least some of the latent KSHV to provide activated KSHV ; and (ii) contacting the activated KSHV with an amount of an a- hydroxytropolone effective to kill at least some of the activated KSHV. The method may be performed in vitro, ex vivo, or in vivo. In some embodiments, contacting the latent KSHV with the compound according to Formula I comprises administering the effective amount of the compound according to Formula I to a subject infected, or suspected of being infected, with KSHV, and contacting the activated KSHV with the a-hydroxytropolone comprises administering the effective amount of the a- hydroxytropolone to the subject. The compound according to Formula I and the a- hydroxytropolone may be administered simultaneously or sequentially in any order to the subject.

A subject may be identified through any suitable means as being infected with KSHV or suspected of being infected with KSHV. KSHV infection may be determined by serologic tests for both latent and/or lytic viral antigens. For example, latency-associated nuclear antigen (LANA) antibodies may be measured by immunoblotting and indirect immunofluorescence assays. KSHV lytic antigen enzyme-linked immunosorbent assays (ELISA), such as ELIS As against open reading frame 65 (ORF65) and/or K8.1 antigens, may also be used. A KSHV infection may also be diagnosed by clinical presentation such as presence of skin lesions and/or internal tumors characteristic of Kaposi sarcoma. Internal tumors may be detected by computerized tomography, X-rays, bronchoscopy, and/or gastrointestinal endoscopy.

In some embodiments, the compound according to Formula I and the a-hydroxytropolone are administered simultaneously to the subject. In other embodiments, the compound according to Formula I and the a-hydroxytropolone are administered sequentially to the subject.

Advantageously, when administered sequentially, the compound according to Formula I and the a-hydroxytropolone are administered within a timeframe such that the therapeutic time windows of both agents overlap, thereby activating latent KSHV and exposing the activated KSHV to the a-hydroxytropolone. In some embodiments, the compound according to Formula I is administered first to the subject, and the a-hydroxytropolone is administered to the subject after a period of time has elapsed. The a-hydroxytropolone is administered to the subject within a therapeutic time window of the compound according to Formula I so that KSHV is in an activated state when the a-hydroxytropolone is administered. The a-hydroxytropolone may be administered within a timeframe of up to several days after administration of the compound according to Formula I, such as within a timeframe of zero seconds to two weeks, zero seconds to one week, zero seconds to 96 hours, 1-72 hours, 1-48 hours, 4-48 hours, 6-48 hours, 12-48 hours, 12-24 hours, or 24-48 hours after administration of the compound according to Formula I. For example, the compound according to Formula I may be administered daily at a first time and the a- hydroxytropolone may be administered daily at a second time. In another example, the compound according to Formula I may be administered every other day, and the a-hydroxytropolone may be administered on the intervening days. In an independent embodiment, the a-hydroxytropolone is administered first to the subject, and the compound according to Formula I is administered after a period of time has elapsed. The compound according to Formula I is administered to the subject within a therapeutic time window of the a-hydroxytropolone so that the a-hydroxytropolone remains effective when the KSHV is activated by the compound according to Formula I.

If administered simultaneously, the compound according to Formula I and the a- hydroxytropolone may be administered in separate pharmaceutical compositions or in a single pharmaceutical composition including both agents. A therapeutically effective amount of the pharmaceutical composition(s) is administered. A therapeutically effective amount of a pharmaceutical composition comprising the compound according to Formula I is an amount sufficient to activate at least some latent KSHV, if present, within the subject. A therapeutically effective amount of a pharmaceutical composition comprising the a-hydroxytropolone is an amount sufficient to kill at least some activated KSHV in the subject. The therapeutically effective amount of the pharmaceutical composition(s) may be administered in a single dosage or in divided dosages over a period of time. If administered in separate compositions, the agents may be administered by the same or different routes. For instance, as one nonlimiting example, one agent may be administered intravenously while the other agent is administered orally.

In any or all of the above embodiments, the compound according to Formula I may be a compound shown in Table 1 and/or the a-hydroxytropolone may be a compound shown in Table 2. In some embodiments, the compound according to Formula I is:

In one embodiment, a single dose of the compound according to Formula I and a single dose of an a-hydroxytropolone administered simultaneously or sequentially may be sufficient to activate and kill KSHV, thereby reducing or eliminating a KSHV infection in a subject. In other embodiments, doses of the compound according to Formula I and the a-hydroxytropolone are administered simultaneously or sequentially at intervals for a period of time effective to reduce or eliminate the KSHV infection in the subject. The intervals may range from a few hours to a few days, such as from four hours to fourteen days, from four hours to seven days, from six hours to seven days, from eight hours to seven days, or from twelve hours to two days. The effective period of time may be from one day to several weeks or months, such as from one day to six months. In any or all of the above embodiments, the method may further include administering an additional therapeutic agent to the subject. In some embodiments, the additional therapeutic agent comprises imiquimod, thalidomide, lenalidomide, pomalidomide, bortezomib, imatinib, sorafenib, maraviroc, bleomycin, vinblastine, vincristine, ali tretinoin, daunorubicin, doxorubicin, ganciclovir, penciclovir, foscarnet, rapamycin, paclitaxel, an anti-angiogenic agent, a matrix metalloproteinase inhibitor, radiotherapy, or any combination thereof.

Advantageously, embodiments of the disclosed method may provide a more potent therapy with fewer unwanted side effects than conventional KSHV therapies. For example, current viral activators, e.g., sodium butyrate, target cellular proteins such as histone deacetylase inhibitors, leading to extensive stress and damage to the host cell. In contrast, some embodiments of the compounds according to Formula I, e.g., Compound 15, target a RNA triple helix of a virus- specific long non-coding RNA and show no cytotoxic effects towards the host cell. Thus, both the lytic activators and the a-hydroxytropolones target viral functions and do not harm the host cell.

V. Examples

Example 1

Identification of KSHV PAN Binding Ligands

Two variants of the KSHV PAN ENE element were subjected to small molecular microarray (SMM) screening, namely a 40-nt RNA duplex (ENE) and the same duplex to which a fluorescently labeled (A)₉ oligonucleotide was hybridized in trans, generating the ENE triple helix (ENE/(A)_n core) as shown in FIG. 1. The RNA motif of interest was prepared by in vitro transcription and its structure in isolation was verified by chemical and enzymatic footprinting.

A library of 20,000 commercially available primary alcohols, and primary amines (both aromatic and aliphatic) was assembled and biased to resemble“drug like” chemical space, with physical properties that increase the likelihood of cell permeability. The library was arrayed and printed in duplicate onto isocyanate-functionalized glass slides (4,000/slide) along with appropriate controls to generate the array. The SMMs were incubated with a Cy5-labeled test and control RNAs to rule out promiscuous binders. The RNAs included duplex and triplex versions of the KSHV ENE encoded by the 3' terminus of the PAN lncRNA. Binding was recorded as a fluorescence signal.

FIG. 2 shows that positive compounds reacted only with the PAN triplex and did not react with the buffer or PAN duplex. Array data were analyzed using Axon GenePix^® software

(Molecular Devices, LLC, Sunnyvale, CA) and JMP^® statistical software (SAS Institute Inc., Cary, NC) to generate a composite Z score for each molecule in the library in order to represent the increase in fluorescence of a given array spot upon incubation with RNA. Hit molecules were defined as having a Z score three standard deviations from the mean of all compounds in the library as well as the ability to bind to the test RNA and not the control. The screening identified 26 chemotypes specific for the ENE triplex that were further characterized by NMR spectroscopy and FT-ICR mass spectrometry. It is noted that SMM screening does not identify the ligand binding site, but only the association of the ligand with the target RNA.

A luminescence-based cytotoxicity test using the specialized iSLK-2l9 Kaposi sarcoma- derived cell line reduced the number of ENE binders for further biological testing. A recombinant vims, rKSHV.2l9, was constructed using iSLK-2l9 cells that express the red fluorescent protein (RFP) from the KSHV lytic PAN promoter, the green fluorescent protein (GFP) from the EF-la promoter, and a puromycin resistance gene as a selectable marker (FIG. 3). iSLK-2l9 cells were maintained at 10 pg/ml puromycin and exposed to 1% DMSO (control), 2.5 mM sodium butyrate (a positive control that inhibits histone deacetylase), 100 mM identified ENE triplex binders, and a combination of the identified compounds and 2.5 mM sodium butyrate. Images were obtained on day four after seeding the cells - 48 h after addition of sodium butyrate, 66 h after treatment with the identified compound. The results with DMSO, sodium butyrate (NaB), and Compounds 15 and 18 are shown in FIG. 4. In FIG. 4, the left panels are phase micrographs, the center panels show GFP fluorescence, and the right panels show RFP fluorescence. The KSHV.219 virus

constitutively expresses puromycin N-acetyl-transferase and GFP, while RFP fluorescence is indicative of lytic activation (Vieira and O-Hearn, Virology 2004, 325(2):225-240). Among the tested ENE triplex binders, Compound 15 promoted lytic induction of KSHV in this viral indicator system. Compound 15 also was found to not be cytotoxic.

p (Compound 18)

Several of the compounds identified in the SMM screening were not effective lytic activators of KSHV. For example, all of the compounds shown in Table 3, some of which share apparent structural similarities with Compound 15, were biologically inactive. Table 3 - Biologically Inactive Compounds

Example 2

Identification of a-Hydroxytropolones with Anti-KSHV Activity Herpesviruses have a linear dsDNA genome encased in an icosahedral capsid. Nascent viral DNA in host cells exists as branched concatemers comprising multiple copies of unit-length viral genome. Concatemers are resolved into genome-length units and inserted into a capsid by the virus-coded terminase through a mechanism similar to that of tailed dsDNA bacteriophages. In the prototypic herpes simplex virus type 1 (HSV-l), the terminase comprises three subunits - pULl5, pUL28, and pUL33. The three subunits interact in vitro and in infected cells, and both pUL28 and pULl5 interact with the portal protein pUL6. pULl5, pUL28, and pUL33 form a complex in the host cytoplasm that is imported into the nucleus via the nuclear localization signal on pULl5.

Their essential role in virus replication suggests herpesvirus terminase proteins as promising antiviral targets.

Since KSHV terminase proteins had not previously been characterized, the inventors characterized the corresponding C-terminal nuclease domain of KSHV, designated ORF29 (ORF29C). Nuclease activity assays, fluorescence-based thermal shift analysis, and structure modeling demonstrated that ORF29C is an RNase H-like nuclease and a component of the KSHV terminase molecular motor.

Several a-hydroxytropolones were found to inhibit KSHV ORF29C nuclease activity.

Without wishing to be bound by a particular theory, the a-hydroxytropolones target the two-metal- ion catalytic mechanisms common to HIV integrase and the reverse transcriptase RNase H domain. Inhibition was evaluated using a supercoiled DNA substrate. ORF29C-derived nuclease products were analyzed by 1% agarose electrophoresis and SYBR^® Gold nucleic acid gel staining. Each compound was evaluated at final concentrations of 20.0, 4.0, 0.80, 0.16, 0.032, and 0.006 mM.

FIG. 5 shows the results of inhibition with a-HT1-a-HT9 (see Table 2 for structures). The migration positions of covalently closed circular, open circular, and linear plasmid DNA are designated ccc, oc, and 1, respectively. Increasing the a-hydroxytropolone concentration led to gradual accumulation of ccc DNA. Visual inspection showed that aHTl, aHT2, and aHT3 demonstrated dose-dependent inhibition of pORF29C nuclease activity. aHTl and aHT2 most significantly affected the nuclease activity. At 0.8 pM, aHTl exhibited modest inhibition (FIG. 5, lane 3), and at 4.0 pM, most of the DNA substrate was refractory to cleavage (lane 2). aHT2 displayed a similar inhibitory effect. Based on recovery of covalently closed circular DNA at an inhibitor concentration of 4.0 pM, aHT3, aHT6, and aHT7 were slightly less active, while minimal closed circular DNA accumulated with the remaining compounds.

Differential scanning fluorimetry was used to determine effects of the a-hydroxytropolones on thermal stability. Visual inspection of nuclease activity in FIG. 5 indicated that aHTl and aHT2 most significantly indicated nuclease activity, and Table 3 indicates that these compounds induced the most significant change in pORF29C thermal stability relative to a control (absence of a- hydroxy tropolone) .

Table 3 - AT_m for WT pORF29C in

presence of aHT inhibitors

The ability of aHTIO to inhibit cleavage of duplex DNA by WT pORF29C was evaluated. FIG. 6A shows the sequence of the 27-nt/28-nt DNA oligonucleotide used as a substrate. The duplex DNA was fluorescein labeled on the 5' terminus of the upper strand, as indicated by the asterisk. FIG. 6B shows the pORF29C cleavage profiles in the absence and presence of aHTIO. Lanes C - control input DNA, lanes O - incubation of duplex DNA with purified pORF29C; lanes D - incubation of duplex DNA with DNasel. All incubations were for 15 minutes at 37 °C.

The sensitivity of KSHV to inhibition by aHT4 was examined in the inducible TREx BCBL-lTra cell line. TREx BCBL-l-Rta cells contain a doxycycline (DOX)-inducible replication and transcription activator (Rta) that activates viral lytic replication. TREx BCBL-l-Rta cells (provided by Joseph Zielgelbauer, NCI) were grown and maintained in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum, penicillin (100 U/ml), and

streptomycin (100 pg/ml; all from Gibco) at 37 °C under 5% CO2. Selection for the ORF50 gene (Rta) was maintained by addition of hygromycin B (100 pg/ml; Thermo Fisher Scientific). To induce KSHV lytic replication, cells were seeded at 10⁶ cells/ml and treated with doxycycline (1 pg/ml; Sigma-Aldrich) or both DOX and sodium butyrate (1 mM; Sigma-Aldrich). Where indicated, cells were also treated with phosphonoacetic acid (500 pM; PAA; Sigma-Aldrich), a known inhibitor of herpesvirus DNA polymerase or aHT4 (5 or 20 pM). At 24 or 72 h, cells and supernatants were collected for the detection of viral genome copies. All experiments were performed as biological triplicates. Cell-associated DNA was extracted using a Qiagen body fluids minikit by following the manufacturer’s instructions. Supernatants were treated with DNase I (Zymo) to remove any unencapsidated viral DNA. Extraction was performed using the QIAamp^® MinElute^® vims spin kit (Qiagen) according to the manufacturer’s protocol. KSHV viral load (VL) was assessed by quantitative real-time PCR. Supernatant DNA was tested using 10 pL of the elution in triplicate reaction mixtures. The average KSHV copy was adjusted to copies per milliliter based upon the fraction of material tested. The cellular DNA was tested in triplicate reactions by TaqMan™ quantitative PCR (Thermo Fisher) to both KSHV, specifically targeting the K6 gene region, and also the human endogenous retrovirus 3 gene (ERV-3), which is used as a cell quantitation assay. KSHV copies were normalized to one million cellular estimates as previously described.

Treatment of TREx-BCBL-l-RTa cells with DOX, either alone or in combination with the histone deacetylase inhibitor sodium butyrate (NaB) leads to reactivation, which is significantly reduced in the presence of the DNA polymerase inhibitor phosphonoacetic acid (PAA) at a final concentration of 500 pM after 24 and 72 h. For both DOX and the DOX-NaB combination, aHT4 at a final concentration of 5 pM (FIG. 7) or 20 pM (not shown) provided equally significant suppression of vims replication in both cell pellets (FIG. 7A) and DNase I-treated supernatants (FIG. 7B). Statistical significant was determined by t test: *, P < 0.05, **, P < 0.01, ***, P < 0.001, ns, not significant. The effect of aHT4 on viral replication was not due to cytotoxicity as this compound induced no cell death at 5 mM and only moderate effects at 20 mM.

Example 3

Treatment of a KSHV Infection

A subject having a KSHV infection, or suspected of having a KSHV infection, is identified and selected. The subject may be identified and selected on the basis of serological testing, clinical presentation, and/or imaging methods. The subject is administered a compound according to Formula I or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof as disclosed herein at amounts determined by a clinician to be therapeutically effective. The subject is also administered an a-hydroxytropolone or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof as disclosed herein at amounts determined by a clinician to be therapeutically effective. The compound according to Formula I and the a-hydroxytropolone may be administered simultaneously or sequentially. The compound according to Formula I and the a-hydroxytropolone may be administered in a single pharmaceutical composition (if administered simultaneously) or as two separate pharmaceutical compositions administered by the same or different routes. The subject may be administered repeated doses of the compound according to Formula I and/or the a- hydroxytropolone at intervals for a period of time sufficient to reduce or eliminate the KSHV infection. In some instances, the progress of the treatment may be monitored, e.g., by serologic tests, to determine when to cease treatment. In some instances, the compound according to Formula I is a compound shown in Table 1 and/or the a-hydroxytropolone is a compound shown in Table 2.

The subject may be administered with an additional therapeutic agent. For example, the subject may further be administered imiquimod, thalidomide, lenalidomide, pomalidomide, bortezomib, imatinib, sorafenib, maraviroc, bleomycin, vinblastine, vincristine, alitretinoin, daunorubicin, doxorubicin, ganciclovir, penciclovir, foscamet, rapamycin, paclitaxel, an anti- angiogenic agent, a matrix metalloproteinase inhibitor, radiotherapy, or any combination thereof.

Representative Embodiments

Certain representative embodiments of the disclosed methods are set forth in the numbered clauses below.

1. A method for activating and killing Kaposi’s sarcoma herpesvirus (KSHV), comprising: contacting latent KSHV with an amount of a compound according to Formula I, or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof, wherein R¹ is -N(R²)-C(0)- (CH₂)_X-R³ where R² is hydrogen or C1-C3 alkyl, R³ is a substituted amino group or a substituted or unsubstituted piperazinyl or piperidinyl group, and x is an integer from 0-10, effective to activate at least some of the latent KSHV to provide activated KSHV

Formula I; and

contacting the activated KSHV with an amount of an a-hydroxytropolone effective to kill at least some of the activated KSHV.

2. The method of clause 1, wherein R² is hydrogen.

3. The method of clause 1 or clause 2, wherein R³ is -N(R)R' where R is hydrogen or

C1-C3 alkyl and R' is alkyl or arylalkyl.

4. The method of clause 1, wherein R³ is a substituted piperazinyl group.

5. The method of clause 1, wherein the compound according to Formula I is

, or any combination thereof.

6. The method of clause 1, wherein the compound according to Formula I is

7. The method of any one of clauses 1-6, wherein the a-hydroxtropolone has a chemical structure according to Formula II, or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof:

Formula II

where R⁴ is hydrogen or C1-C5 alkyl, R⁵ is hydrogen, hydroxy, alkoxy, alkoxycarbonyl, or aryl, and R⁶ is hydrogen, carboxylate, ester, ketone, sulfonyl or aryl.

8. The method of clause 7, wherein R⁴ is methyl.

9. The method of clause 7 or clause 8, wherein R⁵ is hydrogen, hydroxy, or methoxycarbonyl.

10. The method of any one of clauses 7-9, wherein R⁶ is -C(0)CH₃, -C(0)0CH₃, -C(O)- CeH₅, or -C(0)-C₆H₄-C₆H₅.

11. The method of any one of clauses 1-6, wherein the a-hydroxytropolone is

or any combination thereof.

12. The method of any one of clauses 1-11, wherein: contacting the latent KSHV with the compound according to Formula I comprises administering the effective amount of the compound according to Formula I to a subject infected, or suspected of being infected, with KSHV; and contacting the activated KSHV with the a-hydroxytropolone comprises administering the effective amount of the a-hydroxytropolone to the subject.

13. The method of clause 12, wherein the compound according to Formula I is administered to the subject prior to administering the a-hydroxytropolone to the subject. 14. The method of clause 13, wherein the a-hydroxytropolone is administered to the subject within a therapeutic time window of the compound according to Formula I.

15. The method of clause 12 or clause 13, wherein the compound according to Formula I and the a-hydroxytropolone are administered simultaneously.

16. The method of any one of clauses 12-15, wherein administering the compound according to Formula I to the subject comprises administering a therapeutically effective amount of a pharmaceutical composition comprising the compound according to Formula I to the subject.

17. The method of any one of clauses 12-16, wherein administering the a- hydroxytropolone to the subject comprises administering a therapeutically effective amount of a pharmaceutical composition comprising the a-hydroxytropolone to the subject.

18. The method of any one of clauses 12-15 wherein administering the compound according to Formula I and the a-hydroxytropolone to the subject comprises administering a therapeutically effective amount of a pharmaceutical composition comprising the compound according to Formula I and the a-hydroxytropolone to the subject.

19. The method according to any one of clauses 12-18, further comprising administering an additional therapeutic agent to the subject.

20. The method according to clause 19, wherein the additional therapeutic agent comprises imiquimod, thalidomide, lenalidomide, pomalidomide, bortezomib, imatinib, sorafenib, maraviroc, bleomycin, vinblastine, vincristine, alitretinoin, daunorubicin, doxorubicin, ganciclovir, penciclovir, foscarnet, rapamycin, paclitaxel, an anti-angiogenic agent, a matrix metalloproteinase inhibitor, radiotherapy, or any combination thereof.

In view of the many possible embodiments to which the principles of the disclosed invention may be applied, it should be recognized that the illustrated embodiments are only preferred examples of the invention and should not be taken as limiting the scope of the invention. Rather, the scope of the invention is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.

Claims

We claim:

1. A method for activating and killing Kaposi’s sarcoma herpesvirus (KSHV), comprising:

contacting latent KSHV with an amount of a compound according to Formula I, or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof, wherein R¹ is -N(R²)-C(0)- (CH₂)_X-R³ where R² is hydrogen or C1-C3 alkyl, R³ is a substituted amino group or a substituted or unsubstituted piperazinyl or piperidinyl group, and x is an integer from 0-10, effective to activate at least some of the latent KSHV to provide activated KSHV

Formula I; and

contacting the activated KSHV with an amount of an a-hydroxytropolone effective to kill at least some of the activated KSHV.

2. The method of claim 1 , wherein R² is hydrogen.

3. The method of claim 1 or claim 2, wherein R³ is -N(R)R' where R is hydrogen or C1-C3 alkyl and R' is alkyl or arylalkyl.

4. The method of claim 1 , wherein R³ is a substituted piperazinyl group.

5. The method of claim 1, wherein the compound according to Formula I is

, or any combination thereof.

6. The method of claim 1 , wherein the compound according to Formula I is

7. The method of any one of claims 1-6, wherein the a-hydroxtropolone has a chemical structure according to Formula II, or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof:

Formula II

where R⁴ is hydrogen or C1-C5 alkyl, R⁵ is hydrogen, hydroxy, alkoxy, alkoxycarbonyl, aryl, or halo, and R⁶ is hydrogen, ester, ketone, sulfonyl or aryl, or R⁴ and R⁶ together with the atoms to which they are attached form a substituted or unsubstituted heteroaliphatic ring.

8. The method of claim 7, wherein R⁴ is methyl.

9. The method of claim 7 or claim 8, wherein R⁵ is hydrogen, hydroxy,

methoxycarbonyl, or bromo.

10. The method of any one of claims 7-9, wherein R⁶ is -C(0)CH₃, -C(0)0CH₃, -C(0)0CH₂CH₃, -OOl-CeHs, -C(0)-C₆H₄-C₆H₅, or C⁶ and C⁴ together with the atoms to which / Ύ°

they are bound form '— O

11. The method of any one of claims 1-6, wherein the a-hydroxytropolone is

or any combination thereof.

12. The method of any one of claims 1-11, wherein:

contacting the latent KSHV with the compound according to Formula I comprises administering the effective amount of the compound according to Formula I to a subject infected, or suspected of being infected, with KSHV ; and

contacting the activated KSHV with the a-hydroxytropolone comprises administering the effective amount of the a-hydroxytropolone to the subject.

13. The method of claim 12, wherein the compound according to Formula I is administered to the subject prior to administering the a-hydroxytropolone to the subject.

14. The method of claim 13, wherein the a-hydroxytropolone is administered to the subject within a therapeutic time window of the compound according to Formula I.

15. The method of claim 12 or claim 13, wherein the compound according to Formula I and the a-hydroxytropolone are administered simultaneously.

16. The method of any one of claims 12-15, wherein administering the compound according to Formula I to the subject comprises administering a therapeutically effective amount of a pharmaceutical composition comprising the compound according to Formula I to the subject.

17. The method of any one of claims 12-16, wherein administering the a- hydroxytropolone to the subject comprises administering a therapeutically effective amount of a pharmaceutical composition comprising the a-hydroxytropolone to the subject.

18. The method of any one of claims 12-15 wherein administering the compound according to Formula I and the a-hydroxytropolone to the subject comprises administering a therapeutically effective amount of a pharmaceutical composition comprising the compound according to Formula I and the a-hydroxytropolone to the subject.

19. The method according to any one of claims 12-18, further comprising administering an additional therapeutic agent to the subject.

20. The method according to claim 19, wherein the additional therapeutic agent comprises imiquimod, thalidomide, lenalidomide, pomalidomide, bortezomib, imatinib, sorafenib, maraviroc, bleomycin, vinblastine, vincristine, alitretinoin, daunorubicin, doxorubicin, ganciclovir, penciclovir, foscarnet, rapamycin, paclitaxel, an anti-angiogenic agent, a matrix metalloproteinase inhibitor, radiotherapy, or any combination thereof.