WO2014186423A1

WO2014186423A1 - Combination therapy for treating hiv and aids

Info

Publication number: WO2014186423A1
Application number: PCT/US2014/037935
Authority: WO
Inventors: Harold C. Smith; Ryan P. BENNETT; Kimberly Prohaska
Original assignee: Oyagen, Inc.
Priority date: 2013-05-13
Filing date: 2014-05-13
Publication date: 2014-11-20

Abstract

The present invention relates to the use of a Vif inhibitor and an APOBEC3G (A3G) activator in combination to inhibit HIV infectivity in a host cell and to treat or prevent HIV infection or AIDS in a patient. In one embodiment, the present invention relates to a method for inhibiting infectivity of HIV in a host cell, which method comprises contacting a host cell comprising A3G host defense factor with a combination of antiviral-effective amounts of a Vif inhibitor and an A3G activator, thereby inhibiting infectivity of HIV in the host cell by simultaneously inhibiting Vif-dependent degradation of A3G and activating A3G deaminase activity in the host cell, where the Vif inhibitor inhibits Vif-dependent degradation of A3G by disrupting or inhibiting dimerization of Vif in the host cell, and where the A3G activator activates A3G deaminase activity by disrupting or inhibiting A3G:nucleic acid molecule interaction in the host cell.

Description

COMBINATION THERAPY FOR TREATING HIV AND AIDS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority benefit of U.S. Provisional Patent Application Serial No. 61/822,835, filed May 13, 2013, the disclosure of which is hereby incorporated by reference herein in its entirety.

GOVERNMENT RIGHTS STATEMENT

[0002] The present invention was made with U.S. Government support under National

Institutes of Health Grant No . R21NS067671-01. The U. S . Government has certain rights in the invention.

FIELD OF THE INVENTION

[0003] The present invention generally relates to, inter alia, the use of a Vif inhibitor and an

APOBEC3G (A3G) activator in combination to inhibit HIV infectivity in a host cell, to treat or prevent HIV infection or AIDS in a patient, and to design a personalized treatment regimen for treating or preventing HIV infection or AIDS in a patient.

BACKGROUND OF THE INVENTION

[0004] Human immunodeficiency virus type 1 (HIV-1) is a lentivirus and the causative agent of acquired immunodeficiency syndrome (AIDS) and presently infects approximately 34 million persons worldwide with approximately 1.9 million infected persons in North America alone. Recent studies have shown that HIV/AIDS has become a global epidemic that is not under control in developing nations. The rapid emergence of drug-resistant strains of HIV throughout the world has placed a priority on innovative approaches for the identification of novel drug targets that may lead to a new class of anti-retroviral therapies.

[0005] The virus contains a 10-kb single-stranded RNA genome that encodes three major classes of gene products that include: (i) structural proteins (Gag, Pol and Env); (ii) essential transacting proteins (Tat, Rev); and (iii) "auxiliary" proteins that are not required for efficient virus replication in permissive cells (Vpr, Vif, Vpu, Nef). There has been a heightened interest in Vif as an antiviral target because of the discovery that the primary function of Vif (Viral Infectivity Factor) is to overcome the action of a cellular antiviral protein known as APOBEC3G or A3G. [0006] In 1984, it was determined that HIV was the virus that causes AIDS and researchers declared that a vaccine would be available within two years. Nearly three decades later, there is still no vaccine available and the primary focus remains on developing therapeutics for those already infected. Currently, the primary HIV preventative is the combination treatment known as

STRIBILD™. This medication contains 3 components of the highly active anti-retroviral therapy (HAART) regimen (i.e., one integrase (IN) and two reverse transcriptase (RT) inhibitors). However, HIV strains with resistance to some or all of these components had already emerged prior to the availability of STRIBILD™, thus rendering it ineffective against such strains. Furthermore, not only has HIV developed resistance to STRIBILD™ components, but it also has developed resistance to all HAART medications to date, including inhibitors of all HIV enzymatic and viral entry targets. In fact, it is common to see drug resistance even among treatment -naive individuals worldwide, emphasizing that at least some of the current drugs have limited efficacy in a subset of untreated, infected individuals. The barrier to developing resistance to HIV drugs is low and often a single codon change in the targeted protein is sufficient to cause resistance to more than one inhibitor of the same class (i.e., M46I/L/V in the HIV protease confers resistance to 7 out of 8 inhibitors). The ever-present problem of drug resistance together with the lack of success in developing a vaccine accentuate the need for novel HIV prevention and treatment strategies that are unlikely to develop resistance.

[0007] The present invention is directed toward overcoming these and other deficiencies in the art.

SUMMARY OF THE INVENTION

[0008] The present invention generally relates to, inter alia, the use of a Vif inhibitor and an

[0009] In one aspect, the present invention relates to a method for inhibiting infectivity of HIV in a host cell. This method involves contacting a host cell comprising APOBEC3G (A3G) host defense factor with a combination of antiviral-effective amounts of a Vif inhibitor and an A3G activator, thereby inhibiting infectivity of HIV in the host cell by simultaneously inhibiting Vif- dependent degradation of A3G and activating A3G deaminase activity in the host cell. As provided in this method, the Vif inhibitor inhibits Vif-dependent degradation of A3G by disrupting or inhibiting dimerization of Vif in the host cell, and the A3G activator activates A3G deaminase activity by disrupting or inhibiting A3G:nucleic acid molecule interaction in the host cell. Suitable Vif inhibitors and A3G activators for use in this method are as described herein.

[0010] In another aspect, the present invention relates to a method for treating or preventing HIV infection or AIDS in a patient. This method involves administering to a patient in need of such treatment or prevention a combination of a therapeutically effective amount of a Vif inhibitor and an APOBEC3G (A3G) activator. In accordance with this method, the Vif inhibitor is administered at least once at a dosage effective to disrupt or inhibit multimerization of Vif in a cell of the patient, the A3G activator is administered at least once at a dosage effective to disrupt or inhibit A3G:nucleic acid molecule interaction in a cell of the patient, and the Vif inhibitor and the A3G activator are

administered in combination and up to the maximum tolerated dose of each agent. Suitable Vif inhibitors and A3G activators for use in this method are as described herein.

[0011] In another aspect, the present invention relates to a method of designing a personalized treatment regimen for treating or preventing HIV infection or AIDS in a patient. This method involves: determining the therapeutically effective dosage range of a Vif inhibitor in a patient; and determining the therapeutically effective dosage range of an APOBEC3G (A3G) activator. In accordance with this method, determining the therapeutically effective dosage ranges of the Vif inhibitor and the A3G activator is based on a combination treatment of these two agents. Suitable Vif inhibitors and A3G activators for use in this method are as described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012] For the purpose of illustrating the invention, there are depicted in the drawings certain embodiments of the invention. However, the invention is not limited to the precise arrangements and instrumentalities of the embodiments depicted in the drawings.

[0013] The patent or application file may contain at least one drawing executed in color.

Copies of this patent or patent application publication with color drawings, if any, will be provided by the U.S. Patent and Trademark Office upon request and payment of the necessary fee.

[0014] FIG. 1 is an illustration of the life cycle of an HIV virus in the presence of Vif dimers

(illustration on the left) and in the absence of Vif dimers (illustration on the right).

[0015] FIG. 2 is an illustration showing how HIV attacks host defense factor A3G at two stages, including an early blocking stage and a late blocking stage. [0016] FIG. 3 is an illustration showing how enabling of the A3G host-defense in a cell can inhibit HIV infection in the cell.

[0017] FIG. 4 illustrates certain camptothecin (CPT) derivatives for use as Vif inhibitors of the present disclosure, including deoxy-CPT, deoxy-lactam-CPT, thiol-CPT, and deoxy-thiol-CPT.

[0018] FIG. 5 illustrates certain camptothecin (CPT) derivatives for use as Vif inhibitors of the present disclosure, including 9-glycineamido-20(S)-CPT, 9-gly-lactam-CPT, 9-gly-deoxy-lactam-CPT, 9-gly-thiol-CPT, and 9-gly-deoxy-thiol-CPT.

[0019] FIG. 6 illustrates certain topotecan derivatives for use as Vif inhibitors of the present disclosure, including lactam-topotecan, deoxy-lactam- topotecan, thiol- topotecan, and deoxy-thiol- topotecan.

[0020] FIG. 7 are schematics of the synthetic pathways of various embodiments of the Vif inhibitor for use in the present invention, including deoxycamptothecin lactam (denoted as formula 2 in the figure) (corresponds to Formula (I-c)), 9-glycinamido camptothecin lactam (denoted as formula 5 in the figure) (corresponds to Formula (I-d)), 9-glycinamido deoxycamptothecin lactam (denoted as formula 7 in the figure) (corresponds to Formula (I-e)), topotecan lactam (denoted as formula 9 in the figure) (corresponds to Formula (I-f)), and deoxytopotecan lactam (denoted as formula 10 in the figure) (corresponds to Formula (I-g)).

[0021] FIG. 8 is a bar graph showing infectivity results for OYA002-16 (Vif inhibitor) combined with SMAA-2.0 (A3G activator).

[0022] FIG. 9 is a bar graph showing infectivity results for OYA002-16 (Vif inhibitor) combined with SMAA-2.6 (A3G activator).

[0023] FIG. 10 is a bar graph showing infectivity results for OYA004-06 (Vif inhibitor) combined with SMAA-2.6 (A3G activator). DETAILED DESCRIPTION OF THE INVENTION

[0024] The present invention is based, in part, on the discovery that combining a Viral

Infectivity Factor (Vif) inhibitor and an APOBEC3G (A3G) activator is effective as a combination therapy for treating or preventing HIV or AIDS. More particularly, the present invention generally relates to, inter alia, the use of a Vif inhibitor and an A3G activator in combination to inhibit HIV infectivity in a host cell, to treat or prevent HIV infection or AIDS in a patient, and to design a personalized treatment regimen for treating or preventing HIV infection or AIDS in a patient. As provided herein, in certain aspects, the present invention relates to the use of a Vif inhibitor and an A3G activator in combination to enable HIV restriction by the A3G host defense factor due to simultaneously preventing Vif-dependent degradation (through the use of a Vif dimerization antagonist) and activating the cellular potential for A3G deaminase activity (through the use of an antagonist of R A binding to A3G).

[0025] FIGS. 1-3 illustrate the relationship between Vif inhibition and A3G activation as it pertains to inhibiting HIV infection of a host cell and treating or preventing HIV and AIDS in a patient. FIG. 1 illustrates the life cycle of an HIV virus in the presence of Vif dimers (illustration on the left) and in the absence of Vif dimers (illustration on the right). As shown in FIG. 1, when Vif dimers are present, the HIV life cycle is not interrupted; when Vif dimers are not present, HIV DNA functionality is destroyed. FIG. 2 illustrates how HIV attacks host defense factor A3G at two stages, including an early blocking stage and a late blocking stage. In particular, as shown in FIG. 2, HIV suppresses A3G host defense activity in permissive cells as follows: (1) Early Block: HIV infection induces A3G inactivation through complex formation with host cell RNAs; and (2) Late Block: Vif is expressed and Vif dimers bind to A3G to direct its destruction before A3G can be packaged with virions. FIG. 3 illustrates how enabling of the A3G host-defense in a cell can inhibit HIV infection in the cell. As shown in FIG. 3, the HIV life cycle is enabled when A3G is bound to RNA (illustration on the left), and the A3G host-defense is enabled when A3G is not bound by RNA (illustration on the right).

Therefore, as provided herein, inhibiting HIV infectivity in a cell or treating or preventing HIV infection or AIDS in a patient can be achieved by combining a Vif inhibitor that inhibits Vif self- association and an A3G activator that activates the A3G host-defense in a cell.

[0026] Provided herein are suitable Vif inhibitors and A3G activators for use in the methods of the present invention. As described, the Vif inhibitors and A3G activators are compounds that exhibit the desired activity. Below are certain definitions relating to aspects of the compounds described herein.

[0027] As used herein, the following definitions shall apply unless otherwise indicated. For purposes of this invention, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th Ed. Additionally, general principles of organic chemistry are described in "Organic Chemistry", Thomas Sorrell, University Science Books, Sausalito: 1999, and "March's Advanced Organic Chemistry", 5th Ed., Ed.: Smith, M. B. and March, J., John Wiley & Sons, New York: 2001, which are herein incorporated by reference in their entirety.

[0028] Unless otherwise specified, alkyl is intended to include linear, branched, and cyclic hydrocarbon structures and combinations thereof. A combination would be, for example,

cyclopropylmethyl. Ci_6alkyl groups are those having one to six carbon atoms. Examples of Ci_6alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, s-and t-butyl and the like. Cycloalkyl (which includes cyclic hydrocarbon groups) is a subset of alkyl. Examples of cycloalkyl groups include c- propyl, c-butyl, c-pentyl, norbornyl and the like.

[0029] Alkoxy or alkoxyl refers to groups of from 1 to 8 carbon atoms of a straight, branched or cyclic configuration and combinations thereof attached to the parent structure through an oxygen. Examples include methoxy, ethoxy, propoxy, isopropoxy, cyclopropyloxy, cyclohexyloxy and the like. A particular subgroup of alkoxy is Ci_6alkoxy, which refers to alkoxy having 1, 2, 3, 4, 5, or 6 carbon atoms.

[0030] Aryl and heteroaryl ring systems mean (i) a phenyl group (or benzene) or a monocyclic 5- or 6-membered hetero aromatic ring containing 1 -4 heteroatoms selected from O, N, and S; (ii) a bicyclic 9- or 10-membered aromatic or heteroaromatic ring system containing 0-4 heteroatoms selected from O, N, and S; or (iii) a tricyclic 13- or 14-membered aromatic or heteroaromatic ring system containing 0-5 heteroatoms selected from O, N, and S. The aromatic 6- to 14-membered carbocyclic rings include, e.g., benzene, naphthalene, indane, tetralin, and fluorene and the 5- to 10- membered aromatic heterocyclic rings include, e.g., imidazole, pyridine, indole, thiophene,

benzopyranone, thiazole, furan, benzimidazole, quinoline, isoquinoline, quinoxaline, pyrimidine, pyrazine, tetrazole and pyrazole. As used herein aryl and heteroaryl refer to residues in which one or more rings are aromatic, but not all need be.

[0031] The term "halogen" (or "halo") means fluorine, chlorine, bromine or iodine. In one embodiment, halogen may be fluorine or chlorine.

[0032] The terms "haloalkyl" and "haloalkoxy" mean alkyl or alkoxy, respectively, substituted with one or more halogen atoms (e.g., -CF₃).

[0033] The term "heterocyclic group" includes within its scope aromatic, non-aromatic, unsaturated, partially saturated and fully saturated heterocyclic ring systems. In general, such groups may be monocyclic or bicyclic and may contain, for example, 3 to 12 ring members, more usually 5 to 10 ring members. Examples of monocyclic groups are groups containing 3, 4, 5, 6, 7, and 8 ring members, more usually 3 to 7, and preferably 5 or 6 ring members. A particular non-limiting example is a morpholinyl group.

[0034] Radicals and substituents (Rⁿ) are generally defined when introduced and retain that definition throughout the specification and in all independent claims.

[0035] The salt forms of the compounds of the present disclosure, including, without limitation, the compounds of formulas (I), (II), (III), (IV), and their analogs, are typically pharmaceutically acceptable salts, and examples of pharmaceutically acceptable salts are discussed in Berge et al. (1977) "Pharmaceutically Acceptable Salts," J. Pharm. Sci., Vol. 66, pp. 1-19. However, salts that are not pharmaceutically acceptable may also be prepared as intermediate forms which may then be converted into pharmaceutically acceptable salts.

[0036] The term "pharmaceutically acceptable salt" refers to salts prepared from

pharmaceutically acceptable non-toxic acids or bases including inorganic acids and bases and organic acids and bases. When the compounds used in the present invention are basic, salts may be prepared from pharmaceutically acceptable non-toxic acids including inorganic and organic acids. Suitable pharmaceutically acceptable acid addition salts for the compounds of the present invention include acetic, adipic, alginic, ascorbic, aspartic, benzenesulfonic (besylate), benzoic, boric, butyric, camphoric, camphorsulfonic, carbonic, citric, ethanedisulfonic, ethanesulfonic, ethylenediammetetraacetic, formic, fumaric, glucoheptonic, gluconic, glutamic, hydrobromic, hydrochloric, hydroiodic, hydroxynaphthoic, isethionic, lactic, lactobionic, laurylsulfonic, maleic, malic, mandelic, methanesulfonic, mucic, naphthylenesulfonic, nitric, oleic, pamoic, pantothenic, phosphoric, pivalic, polygalacturonic, salicylic, stearic, succinic, sulfuric, tannic, tartaric acid, teoclatic, p-toluenesulfonic, and the like. When the compounds contain an acidic side chain, suitable pharmaceutically acceptable base addition salts for compounds that may be used in the present invention include, but are not limited to, metallic salts made from aluminum, calcium, lithium, magnesium, potassium, sodium and zinc or organic salts made from lysine, arginine, Ν,Ν'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine) and procaine. Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium cations and carboxylate, sulfonate and

phosphonate anions attached to alkyl having from 1 to 20 carbon atoms.

[0037] Where the compounds of the present disclosure, including, without limitation, the compounds of formulas (I), (II), (III), (IV), and their analogs, contain an amine function, these may form quaternary ammonium salts, for example by reaction with an alkylating agent according to methods well known to persons having ordinary skill in the art. Such quaternary ammonium compounds are within the scope of formulas (I), (II), (III), (IV), and their analogs.

[0038] Compounds of the present disclosure, including, without limitation, the compounds of formulas (I), (II), (III), (IV), and their analogs, containing an amine function may also form N-oxides. A reference herein to a compound of the present disclosure, including, without limitation, the compounds of formulas (I), (II), (III), (IV), and their analogs, that contains an amine function also includes the N-oxide.

[0039] Where a compound contains several amine functions, one or more than one nitrogen atom may be oxidized to form an N-oxide. Particular examples of N-oxides are the N-oxides of a tertiary amine or a nitrogen atom of a nitrogen-containing heterocycle.

[0040] N-Oxides can be formed by treatment of the corresponding amine with an oxidizing agent such as hydrogen peroxide or a per-acid (e.g. a peroxycarboxylic acid). See, e.g., Advanced Organic Chemistry, by Jerry March, 4th Edition, Wiley Interscience. More particularly, N-oxides can be made by the procedure of L. W. Deady (Syn. Comm. (1977), 7, 509-514) in which the amine compound is reacted with m-chloroperoxybenzoic acid (MCPBA), for example, in an inert solvent such as dichloromethane.

Vif Inhibitors

[0041] Vif inhibitors for use in the methods of the present disclosure are as disclosed herein.

[0042] Vif binds to and induces the destruction of APOBEC3G (also referred to herein as "A3G"), which is a broad antiviral host-defense factor. Therefore, Vif is essential for HIV infection. Vif subunits interact to form multimers and this property has been shown to be necessary for HIV infectivity. The segment of Vif that mediates subunit interaction was previously determined to be pro line-pro line- leucine-proline (PPLP). In various embodiments, small molecule compounds or other agents that disrupt Vif self-association (also referred to herein as "Vif dimerization" or "Vif multimerization") are suitable as Vif inhibitors in accordance with the present invention.

[0043] In one embodiment, the Vif inhibitor of the present invention is effective to inhibit Vif dimerization by direct or indirect inhibition of binding of Vif dimers at the Vif dimerization domain, said Vif dimerization domain comprising the amino acid sequence of pro line -pro line- leucine-proline (PPLP). In another embodiment, the Vif inhibitor of the present invention is effective to inhibit Vif from binding to A3G. In another embodiment, the Vif inhibitor of the present invention is effective to inhibit Vif-dependent degradation of A3G. In another embodiment, the Vif inhibitor of the present invention is effective to inhibit Vif-dependent degradation of A3G by inhibiting interaction of Vif with one or more enzymes selected from the group consisting of Cullin 5, Elongin B, and Elongin C, thereby inhibiting ubiquitination of A3G.

[0044] In certain embodiments, the Vif inhibitor of the present invention can include, without limitation, camptotechin, topotecan, irinotecan, and analogs thereof and those having a related chemical scaffold (chemotype) thereof. Certain camptothecin derivatives for use as Vif inhibitors of the present disclosure are provided in FIG. 4 and FIG. 5 hereof. Certain topotecan derivatives for use as Vif inhibitors of the present disclosure are provided in FIG. 6.

[0045] In one aspect, the present invention provides small molecule compounds that are effective as inhibitors of Vif self-association. In some embodiments, a suitable compound that is effective as a Vif inhibitor includes a compound of formula (I):

(I) or a pharmaceutically acceptable salt thereof, wherein:

Q is selected from NH, O, and S;

R^20a and R^20b are individually selected from hydrogen, hydroxy, and Ci_₆alkyl; R²¹ is selected from hydrogen, -NHC(=0)(CH₂)_pNR²³R²⁴, and -(CH₂)_PNR²³R²⁴; p is 0, 1, 2, 3, or 4;

R²² is selected from hydrogen and hydroxyl;

R²³ and R²⁴ are individually selected from hydrogen and Ci_₆alkyl

R²⁵ and R²⁶ are individually selected from hydrogen and -N0₂. [0046] In one sub-group of compounds, Q is O.

[0047] In one sub-group of compounds, Q is selected from NH and S.

[0048] In a particular sub-group of compounds, Q is NH.

[0049] In another particular sub-group of compounds, Q is S.

[0050] In one sub-group of compounds, R^20a is Ci_6alkyl.

[0051] In one sub-group of compounds, R^20a and R^20b are individually selected from hydrogen, hydroxy, methyl, and ethyl.

[0052] In one sub-group of compounds R^20a is selected from hydrogen and hydroxy.

[0053] In a particular sub-group of compounds, R^20a is hydrogen.

[0054] In another particular sub-group of compounds, R^20a is hydroxy.

[0055] In one sub-group of compounds R^20b is Ci_₆alkyl.

[0056] In one sub-group of compounds R^20b is hydrogen.

[0057] In a particular sub-group of compounds, R^20b is ethyl.

[0058] In one sub-group of compounds, R^20a is hydrogen or hydroxy and R^20b is ethyl.

[0059] In a particular sub-group of compounds, R^20a is hydrogen and R^20b is ethyl.

[0060] In a particular sub-group of compounds, R^20a is hydroxy and R^20b is ethyl.

[0061] In one sub-group of compounds, R²¹ is hydrogen.

[0062] In another sub-group of compounds, R²¹ is -NHC(=0)(CH₂)_pNR²³R²⁴ (e.g., - NHC(=0)CH₂NR²³R²⁴, such as -NHC(=0)CH₂NH₂)

[0063] In another sub-group of compounds, R²¹ is -(CH₂)_PNR²³R²⁴ (e.g., -CH₂N(CH₃) ₂).

[0064] In one subgroup of compounds, p is 1, 2, 3, or 4. In a particular subgroup, p is 1 or 2. In a more particular subgroup of compounds, p is 1.

[0065] In one subgroup of compounds, R²² is hydrogen.

[0066] In one subgroup of compounds, R²² is hydroxyl.

[0067] In one subgroup, R²³ and R²⁴ are individually selected from hydrogen and methyl. [0068] In one subgroup of compounds, R and R are both hydrogen.

[0069] In one subgroup of compounds, R²⁵ and R²⁶ are both -N0₂.

[0070] In particular embodiments, a suitable Vif inhibitor of formula (I) is a compound selected from the group consisting of

(I-a) (I-b)

(I-c) (I-d)

(I-e) (I-f)

(i-g)

[0071] The compound of formula (I-a) is also referred to herein as "OYA-002-16." The compound of formula (I-b) is also referred to herein as "OYA-004-006." The compound of formula (I-c) is also referred to herein as deoxycamptothecin lactam (denoted as formula 2 in FIG. 7). The compound of formula (I-d) is also referred to herein as 9-glycinamido camptothecin lactam (denoted as formula 5 in FIG. 7). The compound of formula (I-e) is also referred to herein as 9-glycinamido deoxycamptothecin lactam (denoted as formula 7 in FIG. 7). The compound of formula (I-f) is also referred to herein as topotecan lactam (denoted as formula 9 in FIG. 7). The compound of formula (I-g) is also referred to herein as and deoxytopotecan lactam (denoted as formula 10 in FIG. 7).

[0072] The Vif inhibitor compounds for use in the methods of the present invention can include functional derivatives of any of the Vif inhibitor compounds disclosed herein, and pharmaceutically acceptable salts thereof.

A3G Activators

[0073] APOBEC3G (A3G) activators for use in the methods of the present disclosure are as disclosed herein.

[0074] A3G is a host defense factor. A3G was discovered as an HIV restriction factor during studies of the HIV Vif (viral infectivity factor) protein. In the absence of Vif, HIV cannot establish a spreading infection in non-permissive cells (i.e. CD4 cells, macrophages); however, its expression is not required for permissive cell infection (i.e. 293T, HeLa cells) [1, 2]. In 2002, Sheehy, et al.

identified A3G as the cellular defense factor [3]. In the absence of Vif, A3G is incorporated into HIV virions [4, 5] and binds to the viral core [6]. During viral replication, A3G extensively deaminates viral minus-strand DNA, converting dC to dU [7, 8]. This causes either: (1) degradation of viral DNA by DNA repair enzymes [9] or (2) mutated minus-strand DNA serving as a template for plus-strand synthesis, whereby aberrant dU residues lead to dG to dA transitions. This alters the viral open reading and introduces premature stop and missense codons [10-13]. Each case leads to the production of less infectious virions and decreased HIV infectivity.

[0075] In addition to A3G, A3B, A3C, A3DE, A3F, and some A3H haplotypes have been shown to be effective against HIV infection [4, 14-24], although to different extents. However, A3F and A3G have been the only proteins that have consistently demonstrated antiviral activity [25-29]. As provided herein, A3G is a therapeutic target due to the fact that its mR A is 3- to 10-fold more abundant than A3F in CD4⁺ T cells, the natural primary target for HIV infection [30-33], suggesting the potential of increased A3G protein abundance compared to A3F. Furthermore, it has demonstrated that A3G is more effective at restricting HIV infection than A3F [34], which indicates that A3G can be effective for neutralizing HIV.

[0076] Although HIV target cells express A3G, HIV is able to block its function both early and late in the viral life cycle. Upon HIV infection, cytokine signaling causes A3G to bind to cellular R A and become sequestered as large (megaDalton) ribonucleoprotein complexes (A3G-RNP), in which form it is inactive as an enzyme and antiviral [35]. RNase digestion of A3G-RNP in vitro reduced A3G to protein monomers and dimers and stimulated its deaminase activity [36]. Furthermore, newly synthesized A3G not yet in A3G-RNP was shown to be the source of active protein that is

encapsidated with virions and restricts infection through hypermutation of HIV ssDNA [36]. The latter function is dependent on removal of HIV genomic RNA by HIV RNase H [6, 37]. It has been shown that RNA alone binding to A3G is sufficient to inhibit its deaminase activity in vitro [38]. Thus, decreasing cellular A3G:RNA binding is expected to activate a sub-fraction of A3G to inhibit HIV when it enters target cells. This also makes more A3G available for packaging with nascent virions to prevent the establishment of a spreading infection.

[0077] In addition to causing A3G's inactivation, HIV expresses the Vif protein, which targets

A3G degradation via the proteasome [4, 5, 39, 40]. This occurs through Vif s ability to bind to the ubiquitination machinery. A consensus SOCS (suppressor of cytokine signaling) -box in the C-terminus of Vif binds to the Elongin C subunit of the E3 ubiquitin ligase complex that also contains Cullin 5 and Elongin B [41]. Vif also contains a zinc binding HCCH motif that confers an interaction with Cullin 5 [42]. Vif serves as a bridge for A3G to Elongin C and Cullin 5 in the E3 ubiquitin ligase complex, leading to polyubiquitination of both Vif and A3G [39, 41, 42]. Studies have shown that only polyubiquitination of Vif on one or more of its 16 lysine residues is required for proteosomal degradation of A3G and Vif [43]. Site-directed mutagenesis demonstrated that an aspartic acid at position 128 (D128) in A3G is required for Vif-mediated degradation [44-46]. In contrast, relatively large regions within the N-terminus of Vif are involved in its interaction with A3G [47-50]. Additional experiments using Vif deletion mutants revealed that residues 151-164 are critical for Vif

multimerization, a feature required for HIV infectivity of non-permissive cells [51]. Subsequent phage display revealed that peptides with a PXP motif bound to PPLP within Vif (residues 161 -164), which blocked Vif multimerization in vitro [52]. It has been demonstrated that linking a cell -transducing peptide to PPLP-containing Vif peptides facilitated their cellular transduction and inhibited HIV infectivity [52, 53]. It has further been demonstrated that A3G incorporation into viral particles was enhanced upon peptide treatment, resulting in marked suppression of HIV infectivity [53]. Mutating the PPLP motif to AAAP severely reduced the Vif-A3G interaction and enabled A3G antiviral activity [54]. Taken together, the data reveal that Vif dimerization is essential for both viral infectivity and its interaction with A3G. Moreover, the dimerization domain can be disrupted in vivo, demonstrating its potential as a drug target.

[0078] In one aspect, the present invention provides small molecule compounds that are effective as A3G activators. In particular embodiments, the A3G activators of the present disclosure are effective to disrupt or inhibit A3G:R A interaction in the cell.

[0079] In some embodiments, a suitable compound that is effective as an A3G activator includes a compound of formula (II):

or a pharmaceutically acceptable salt thereof, wherein: A, B, and C are each individually selected from CH and N;

W is absent or is selected from CH and N;

X is selected from CH and N;

Y is selected from CH, O, and S; the dotted line is either a single bond or a double bond;

Z is selected from C(=0) and CH₂;

R¹¹ is selected from phenyl and 5- or 6-membered heteroaryl, wherein said phenyl or 5- or 6- membered heteroaryl is substituted with 0, 1, 2, or 3 substituents individually selected from halo, Ci_ ₆alkyl, cyano, Ci_6alkoxy, COOR¹⁵, hydroxyCi_6alkyl, and 4- to 6-membered heterocyclic group; n is 0, 1, 2, or 3;

R¹² is selected from halo, C_halky!, cyano, Ci_6alkoxy, COOR¹⁵, and a 4- to 6-membered heterocyclic group; and

R¹⁵ is selected from hydrogen and Ci_₆ alkyl.

[0080] In one sub-group of compounds, A, B, and C are each CH, forming a phenyl ring.

[0081] In one sub-group of compounds, the A, B, C ring is pyridine (i.e., one of A, B and C is

N, and the remaining two are CH).

[0082] In one sub-group of compounds, the A, B, C, ring is selected from the following residues, where the dotted line represents the bond to the W, X, Y ring, where said residues are optionally substituted by 1, 2, or 3 R¹² groups:

[0083] In one sub-group of compounds, the A, B, C ring, together with its substituent(s), is selected from the following residues, where the dotted line represents the bond to the center (W, X, Y) ring:

[0084] In one sub-group of compounds, W is absent, such that the center ring of formula (II) is a 5-membered ring. For example, in some embodiments, the center (W, X, Y) ring is selected from oxazolyl, thienyl, and thiazolyl.

[0085] In one particular subgroup, W is absent, X is CH, and Y is S.

[0086] In one particular subgroup, W is absent, X is N, and Y is S.

[0087] In one particular subgroup, W is absent, X is N, and Y is O.

[0088] In one sub-group of compounds, W is CH or N, such that the center ring of formula (II) is a 6-membered ring.

[0089] In one particular subgroup, W is CH, X is N, and Y is CH.

[0090] In another particular subgroup, W is N, X is N, and Y is CH.

[0091] In one sub-group of compounds, Z is C(=0).

[0092] In one sub-group of compounds, Z is CH₂.

[0093] In one sub-group of compounds, R¹¹ is selected from unsubstituted phenyl and unsubstituted 5- or 6-membered heteroaryl.

[0094] In one sub-group of compounds, R¹¹ is selected from phenyl and 5- or 6-membered heteroaryl, each being substituted with 1, 2, or 3 substituents individually selected from halo, Ci_₆alkyl, cyano, Ci_6alkoxy, COOR¹⁵, hydroxyCi_6alkyl, and 4- to 6-membered heterocyclic group. [0095] In one subgroup of compounds, R¹¹ is optionally substituted phenyl, where substituents are as described above.

[0096] In one subgroup of compounds, R¹¹ is optionally substituted 5- or 6-membered heteroaryl (e.g., pyrazolyl, thienyl, thiazolyl, oxazolyl), where substituents are as described above.

[0097] In a particular embodiment, R¹¹ is substituted pyrazolyl.

[0098] In a more particular embodiment, R¹¹ is l ,3-dimethyl-lH-pyrazol-5-yl.

[0099] As indicated above, n is n is 0, 1 , 2, or 3. Where n is greater than 1 , each R¹² group is individually selected (i.e., present R¹² groups may be the same or different).

[00100] R¹⁵ is selected from hydrogen and Ci_₆ alkyl. In one subgroup, R¹⁵ is selected from hydrogen and methyl. In a more particular subgroup, R¹⁵ is hydrogen.

[00101] The A3G activator compounds for use in the methods of the present invention can include functional derivatives of any of the A3G activator compounds disclosed herein, and pharmaceutically acceptable salts thereof.

[00102] In particular embodiments, a suitable A3G activator of formula (II) is a compound of formula (Il-a) (also referred to herein as "SMAA2.0") or a compound of formula (II -b) (also referred to herein as "SMAA2.6" as follows:

(II-a) (Il-b) and analogs thereof and those having a related chemical scaffold (chemotype).

[00103] In other embodiments, a suitable A3G activator can be a compound of formula (III)

(also known as Altanserin) or formula (IV) (also referred to herein as "OYA-38") as provided below:

(III) (IV) and analogs thereof and those having a related chemical scaffold (chemotype).

[00104] The A3G activator compounds for use in the methods of the present invention include functional derivatives of any of the A3G activator compounds disclosed herein, and pharmaceutically acceptable salts thereof.

Methods of Using Combinations of Vif Inhibitors and A3G Activators

[00105] Both R A inhibition of A3G and Vif-mediated A3G degradation represent potential targets for HIV treatment. However, by targeting only Vif dimerization, A3G will remain minimally active due to its interaction with RNA. On the other hand, activation of A3 G by decreasing its association with RNA will still leave it vulnerable to degradation. The present invention provides for a combination of a Vif inhibitor and an A3G activator, as data has shown that both targets can be addressed simultaneously for enhanced HIV infectivity inhibtion. First, in addition to facilitating A3G degradation, Vif promotes the sequestration of A3G in large complexes that may involve RNA interactions [55]. Thus, although inhibiting Vif would lead to increased A3G abundance, it may still be predominantly inactive. In contrast, Vif also has been shown to preferentially degrade newly synthesized A3G that has not yet been sequestered as A3G-RNP [37], so shifting A3G to a lower molecular weight form may leave it more susceptible to degradation. Thus, considering that HIV virions contain little to no Vif protein [56, 57], activation of A3G in target cells will allow it to inhibit incoming virus, whereas antagonizing Vif dimerization will increase A3G abundance.

[00106] In one aspect, the present invention relates to a method for inhibiting infectivity of HIV in a host cell. The host cell can be from any human or animal source. This method involves contacting a host cell comprising APOBEC3G (A3G) host defense factor with a combination of antiviral-effective amounts of a Vif inhibitor and an A3G activator, thereby inhibiting infectivity of HIV in the host cell by simultaneously inhibiting Vif-dependent degradation of A3G and activating A3G deaminase activity in the host cell. As provided in this method, the Vif inhibitor inhibits Vif- dependent degradation of A3G by disrupting or inhibiting dimerization of Vif in the host cell, and the A3G activator activates A3G deaminase activity by disrupting or inhibiting A3G:nucleic acid molecule interaction in the host cell. Suitable Vif inhibitors and A3G activators for use in this method are as described herein.

[00107] In another aspect, the present invention relates to a method for treating or preventing HIV infection or AIDS in a patient. This method involves administering to a patient in need of such treatment or prevention a combination of a therapeutically effective amount of a Vif inhibitor and an APOBEC3G (A3G) activator. In accordance with this method, the Vif inhibitor is administered at least once at a dosage effective to disrupt or inhibit multimerization of Vif in a cell of the patient, the A3G activator is administered at least once at a dosage effective to disrupt or inhibit A3G:nucleic acid molecule interaction in a cell of the patient, and the Vif inhibitor and the A3G activator are

[00108] In one embodiment of this method, the Vif inhibitor and the A3G activator are administered simultaneously. In another embodiment, the A3G activator is administered prior to administration of the Vif inhibitor. In a further embodiment, the Vif inhibitor is administered prior to administration of the A3G activator. In another embodiment, the Vif inhibitor and the A3G activator are administered in multiple alternating doses.

[00109] In one embodiment, this method further involves administering a therapeutically effective amount of at least one other agent for treating HIV selected from the group consisting of HIV reverse transcriptase inhibitors, non-nucleoside HIV reverse transcriptase inhibitors, HIV protease inhibitors, HIV fusion inhibitors, HIV attachment inhibitors, CCR5 inhibitors, CXCR4 inhibitors, HIV budding or maturation inhibitors, and HIV integrase inhibitors.

[00110] In accordance with this method, the HIV infection is due to an HIV virus selected from the group consisting of HIV- 1 and HIV-2.

[00111] In another aspect, the present invention relates to a method of designing a personalized treatment regimen for treating or preventing HIV infection or AIDS in a patient. This method involves: determining the therapeutically effective dosage range of a Vif inhibitor in a patient; and determining the therapeutically effective dosage range of an APOBEC3G (A3G) activator. In accordance with this method, determining the therapeutically effective dosage ranges of the Vif inhibitor and the A3G activator is based on a combination treatment of these two agents. Suitable Vif inhibitors and A3G activators for use in this method are as described herein.

[00112] In one embodiment of this method, the combination treatment comprises

simultaneously administering the Vif inhibitor and the A3G activator. In another embodiment, the combination treatment comprises administering the A3G activator prior to the Vif inhibitor. In a further embodiment, the combination treatment comprises administering the Vif inhibitor prior to the A3G activator. In another embodiment, the combination treatment comprises administering the Vif inhibitor and the A3G activator in multiple alternating doses.

[00113] In one embodiment, the combination treatment of this method further involves administering a therapeutically effective amount of at least one other agent for treating HIV selected from the group consisting of HIV reverse transcriptase inhibitors, non-nucleoside HIV reverse transcriptase inhibitors, HIV protease inhibitors, HIV fusion inhibitors, HIV attachment inhibitors, CCR5 inhibitors, CXCR4 inhibitors, HIV budding or maturation inhibitors, and HIV integrase inhibitors.

[00114] In accordance with this method, the HIV infection is due to an HIV virus selected from the group consisting of HIV- 1 and HIV-2.

[00115] As provided herein, the invention involves the use of Vif inhibitor compounds and A3G activator compounds, which include pharmaceutical compositions. Pharmaceutically acceptable carriers that are useful include, but are not limited to, glycerol, water, saline, ethanol and other pharmaceutically acceptable salt solutions such as phosphates and salts of organic acids. Examples of these and other pharmaceutically acceptable carriers are described in Remington's Pharmaceutical Sciences (1991, Mack Publication Co., New Jersey), the disclosure of which is incorporated by reference as if set forth in its entirety herein.

[00116] The pharmaceutical compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. This suspension or solution may be formulated according to the known art, and may comprise, in addition to the active ingredient, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable formulations may be prepared using a non-toxic peritoneally-acceptable diluent or solvent, such as water or 1,3-butane diol, for example. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or di-glycerides.

[00117] Pharmaceutical compositions that are useful in the methods of the invention may be administered, prepared, packaged, and/or sold in formulations suitable for oral, rectal, vaginal, peritoneal, topical, pulmonary, intranasal, buccal, ophthalmic, or another route of administration.

Other contemplated formulations include projected nanoparticles, liposomal preparations, resealed erythrocytes containing the active ingredient, and immuno logically-based formulations.

[00118] The compositions of the invention may be administered via numerous routes, including, but not limited to, oral, rectal, vaginal, peritoneal, topical, pulmonary, intranasal, buccal, or ophthalmic administration routes. The route(s) of administration will be readily apparent to the skilled artisan and will depend upon any number of factors including the type and severity of the disease being treated, the type and age of the veterinary or human patient being treated, and the like.

[00119] As used herein, "peritoneal administration" of a pharmaceutical composition includes any route of administration characterized by physical breaching of a tissue of a subject and

administration of the pharmaceutical composition through the breach in the tissue. Peritoneal administration thus includes, but is not limited to, administration of a pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a tissue-penetrating non-surgical wound, and the like. In particular, peritoneal administration is contemplated to include, but is not limited to, subcutaneous, intraperitoneal, intramuscular, intrasternal injection, and kidney dialytic infusion techniques.

[00120] A pharmaceutical composition can consist of the active ingredient alone, in a form suitable for administration to a subject, or the pharmaceutical composition may comprise the active ingredient and one or more pharmaceutically acceptable carriers, one or more additional ingredients, or some combination of these. The active ingredient may be present in the pharmaceutical composition in the form of a physiologically acceptable ester or salt, such as in combination with a physiologically acceptable cation or anion, as is well known in the art.

[00121] The formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient into association with a carrier or one or more other accessory ingredients, and then, if necessary or desirable, shaping or packaging the product into a desired single- or multi-dose unit.

[00122] Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions that are suitable for ethical administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and perform such modification with merely ordinary, if any, experimentation. Subjects to which

administration of the pharmaceutical compositions of the invention is contemplated include, but are not limited to, humans and other primates, mammals including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, and dogs.

[00123] Controlled- or sustained-release formulations of a pharmaceutical composition of the invention may be made using conventional technology.

[00124] Formulations of a pharmaceutical composition suitable for peritoneal administration comprise the active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampules or in multi-dose containers containing a preservative. Formulations for peritoneal administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Such formulations may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents. In one embodiment of a formulation for peritoneal administration, the active ingredient is provided in dry (i.e., powder or granular) form for reconstitution with a suitable vehicle (e.g., sterile pyrogen- free water) prior to peritoneal administration of the reconstituted composition.

[00125] The pharmaceutical compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. This suspension or solution may be

formulated according to the known art, and may comprise, in addition to the active ingredient, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable formulations may be prepared using a non-toxic peritoneally-acceptable diluent or solvent, such as water or 1,3 -butane diol, for example. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or di-glycerides. Other parentally-administrable formulations which are useful include those which comprise the active ingredient in micro crystalline form, in a liposomal preparation, or as a component of a biodegradable polymer systems. Compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt.

[00126] Formulations suitable for topical administration include, but are not limited to, liquid or semi-liquid preparations such as liniments, lotions, oil-in- water or water-in-oil emulsions such as creams, ointments or pastes, and solutions or suspensions. Topically-administrable formulations may, for example, comprise from about 1% to about 10% (w/w) active ingredient, although the

concentration of the active ingredient may be as high as the solubility limit of the active ingredient in the solvent. Formulations for topical administration may further comprise one or more of the additional ingredients described herein.

[00127] Typically, dosages of the compound of the invention which may be administered to an animal, preferably a human, will vary depending upon any number of factors, including but not limited to, the type of animal and type of disease state being treated, the age of the animal and the route of administration.

[00128] The compound can be administered to an animal as frequently as several times daily, or it may be administered less frequently, such as once a day, once a week, once every two weeks, once a month, or even less frequently, such as once every several months or even once a year or less. The frequency of the dose will be readily apparent to the skilled artisan and will depend upon any number of factors, such as, but not limited to, the type and severity of the disease being treated, the type and age of the animal, and the like. Preferably, the compound is, but need not be, administered as a bolus injection that provides lasting effects for at least one day following injection. The bolus injection can be provided intraperitoneally.

[00129] Publications discussed herein are provided solely for their disclosure prior to the filing date of the described application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed. [00130] In considering the functional derivatives of the small compounds of the present invention, one of ordinary skill in the art can readily determine various structural changes that can enhance the therapeutic characteristics of the compounds while maintaining their functionality as Vif inhibitors (e.g., effective as inhibitors of Vif self-association) or A3G activators (e.g., effective to disrupt or inhibit A3G:RNA interaction in the cell). As noted above, with regard to such

determinations, the definitions provided herein may apply unless otherwise indicated. For purposes of this invention, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th Ed. Additionally, general principles of organic chemistry are described in "Organic Chemistry", Thomas Sorrell, University Science Books, Sausalito: 1999, and "March's Advanced Organic Chemistry", 5th Ed., Ed.: Smith, M. B. and March, J., John Wiley & Sons, New York: 2001, which are herein incorporated by reference in their entirety.

[00131] As used herein, and as would be understood by the person of skill in the art, the recitation of "a compound" - unless expressly further limited - is intended to include salts, solvates and inclusion complexes of that compound. Unless otherwise stated or depicted, structures depicted herein are also meant to include all stereoisomeric (e.g., enantiomeric, diastereomeric, and cis-trans isomeric) forms of the structure; for example, the R and S configurations for each asymmetric center, (Z) and (E) double bond isomers, and (Z) and (E) conformational isomers. Therefore, single stereochemical isomers as well as enantiomeric, diastereomeric, and cis-trans isomeric (or conformational) mixtures of the present compounds are within the scope of the invention. Unless otherwise stated, all tautomeric forms of the compounds of the invention are within the scope of the invention. Additionally, unless otherwise stated, structures depicted herein are also meant to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structures except for the replacement of hydrogen by deuterium or tritium, or the replacement of a carbon by a ¹³C- or ¹⁴C-enriched carbon are within the scope of this invention. Such compounds are useful, for example, as analytical tools or probes in biological assays. The term "solvate" refers to a compound in the solid state, wherein molecules of a suitable solvent are incorporated in the crystal lattice. A suitable solvent for therapeutic administration is physiologically tolerable at the dosage administered. Examples of suitable solvents for therapeutic administration are ethanol and water.

When water is the solvent, the solvate is referred to as a hydrate. In general, solvates are formed by dissolving the compound in the appropriate solvent and isolating the solvate by cooling or using an antisolvent. The solvate is typically dried or azeotroped under ambient conditions. Inclusion complexes are described in Remington: The Science and Practice of Pharmacy 19^th Ed. (1995) volume 1, page 176-177, which is incorporated herein by reference. The most commonly employed inclusion complexes are those with cyclodextrins, and all cyclodextrin complexes, natural and synthetic, are specifically encompassed within the claims.

[00132] While it may be possible for the compounds of the invention to be administered as the raw chemical, it is preferable to present them as a pharmaceutical composition. According to a further aspect, the present invention provides a pharmaceutical composition comprising a compound of the invention or a pharmaceutically acceptable salt or solvate thereof, together with one or more pharmaceutical carriers thereof and optionally one or more other therapeutic ingredients. 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.

[00133] As used herein, the term "physiologically functional derivative" refers to any pharmaceutically acceptable derivative of a compound of the present invention that, upon

administration to a mammal, is capable of providing (directly or indirectly) a compound of the present invention or an active metabolite thereof. Such derivatives, for example, esters and amides, will be clear to those skilled in the art, without undue experimentation. Reference may be made to the teaching of Burger's Medicinal Chemistry And Drug Discovery, 5 ^th Edition, Vol 1 : Principles and Practice, which is incorporated herein by reference to the extent that it teaches physiologically functional derivatives.

[00134] As used herein, the term "effective amount" means that amount of a drug or

pharmaceutical agent that will elicit the biological or medical response of a tissue, system, animal, or human that is being sought, for instance, by a researcher or clinician. The term "therapeutically effective amount" means any amount which, as compared to a corresponding subject who has not received such amount, results in improved treatment, healing, prevention, or amelioration of a disease, disorder, or side effect, or a decrease in the rate of advancement of a disease or disorder. The term also includes within its scope amounts effective to enhance normal physiological function. For use in therapy, therapeutically effective amounts of a compound of the present invention, as well as salts, solvates, and physiological functional derivatives thereof, may be administered as the raw chemical. Additionally, the active ingredient may be presented as a pharmaceutical composition. [00135] Pharmaceutical compositions of the present invention comprise an effective amount of one or more compound of the present invention, or additional agent dissolved or dispersed in a pharmaceutically acceptable carrier. The phrases "pharmaceutical or pharmacologically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, such as, for example, a human, as appropriate. The preparation of a pharmaceutical composition that contains at least one compound of the present invention, or additional active ingredient will be known to those of skill in the art in light of the present disclosure, as exemplified by Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference. Moreover, for animal (e.g., human) administration, it will be understood that preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biological Standards.

[00136] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated herein by reference). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the pharmaceutical compositions is contemplated.

[00137] The term "lentivirus" as used herein may be any of a variety of members of this genus of viruses. The lentivirus may be, e.g., one that infects a mammal, such as a sheep, goat, horse, cow or primate, including human. Typical such viruses include, e.g., Vizna virus (which infects sheep);

simian immunodeficiency virus (SIV), bovine immunodeficiency virus (BIV), chimeric simian/human immunodeficiency virus (SHIV), feline immunodeficiency virus (FIV) and human immunodeficiency virus (HIV). "HIV," as used herein, refers to both HIV-1 and HIV-2. Much of the discussion herein is directed to HIV or HIV-1; however, it is to be understood that other suitable lentiviruses are also included.

[00138] The term "mammal" as used herein refers to any non-human mammal. Such mammals are, for example, rodents, non-human primates, sheep, dogs, cows, and pigs. The preferred non-human mammals are selected from the rodent family including rat and mouse, more preferably mouse. The preferred mammal is a human. [00139] As used herein, the terms "peptide," "polypeptide," and "protein" are used

interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids which can comprise a protein's or peptide's sequence.

Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. "Polypeptides" include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptide, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.

[00140] "Pharmaceutically acceptable" means physiologically tolerable, for either human or veterinary applications. In addition, "pharmaceutically acceptable" is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administered to a subject without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained. Essentially, the

pharmaceutically acceptable material is nontoxic to the recipient. The carrier would naturally be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as would be well known to one of skill in the art. For a discussion of pharmaceutically acceptable carriers and other components of pharmaceutical compositions, see, e.g., Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing Company, 1990.

[00141] As used herein, "pharmaceutical compositions" include formulations for human and veterinary use.

[00142] As used herein, the terms "prevent," "preventing," "prevention," "prophylactic treatment" and the like refer to reducing the probability of developing a disorder or condition in a subject, who does not have, but is at risk of or susceptible to developing a disorder or condition.

[00143] As used herein, the terms "treat," "treating," "treatment," and the like refer to reducing or ameliorating a disorder and/or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated. [00144] "Viral infectivity" as that term is used herein means any of the infection of a cell, the replication of a virus therein, and the production of progeny virions therefrom.

[00145] A "virion" is a complete viral particle; nucleic acid and capsid, further including and a lipid envelope in the case of some viruses.

EXAMPLES

[00146] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the described invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.), but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.

EXAMPLE 1

Live HIV Virus Testing Using Combination of Vif Inhibitor and A3G Activator

[00147] This example relates to various experiments conducted to evaluate the antiviral efficacy and cytotoxicity of four compounds, two Vif inhibitors (OYA002-16 and OYA004-06) and two A3G activators (SMAA-2.0 and SMAA-2.6). These compounds were tested in a standard PBMC cell-based microtiter anti-HIV assay against HIV-1 isolates representing different viral subtypes, and co -receptor tropisms.

Drug Preparation

[00148] The solubilized stocks were stored at -20°C until the day of the assay. Stocks are thawed at room temperature on each day of assay setup (dates are listed on the individual graph pages) and are used to generate working drug dilutions used in the assays. Working dilutions are made fresh for each experiment and are not stored for re -use in subsequent experiments performed on different days. The compounds were tested at a 100 μΜ high-test concentration with 8 additional serial half-log dilutions in the PBMC assays (concentration range = 10 nM to 100 μΜ). Zidovudine (AZT; Nucleoside Reverse Transcriptase Inhibitor; NRTI) was included in the PBMC assays as a positive control antiviral compound using a 1.0 μΜ (1,000 nM) high-test concentration with eight additional serial half- log dilutions (concentration range = 100 pM to 1.0 μΜ).

Efficacy Evaluation in Human Peripheral Blood Mononuclear Cells (PBMCs)

A. Virus Isolates

[00149] Five virus isolates were selected for use in these experiments. The viruses were chosen to include isolates from each of the seven HIV-1 Group M envelope subtypes A, B, D, and G. All of the virus isolates were obtained from the NIH AIDS Research and Reference Reagent Program.

[00150] Low passage stocks of each virus was prepared using fresh human PBMCs and stored in liquid nitrogen. Pre-titered aliquots of each virus were removed from the freezer and thawed rapidly to room temperature in a biological safety cabinet immediately before use.

B. Anti-HIV Efficacy Evaluation in Fresh Human PBMCs

[00151] Fresh human PBMCs were isolated from screened donors, seronegative for HIV and HBV (Biological Specialty Corporation, Colmar, PA). Cells were pelleted/washed 2-3 times by low speed centrifugation and resuspension in Dulbecco's phosphate buffered saline (PBS) to remove contaminating platelets. The leukophoresed blood was then diluted 1 : 1 with PBS and layered over 14 mL of Ficoll-Hypaque density gradient (Lymphocyte Separation Medium, Cell Grow #85-072-CL, density 1.078+/-0.002 gm/ml) in a 50 mL centrifuge tube and then centrifuged for 30 minutes at 600 X g. Banded PBMCs were gently aspirated from the resulting interface and subsequently washed 2X with PBS by low speed centrifugation.6 After the final wash, cells were enumerated by trypan blue exclusion and re-suspended at 1 x 10 cells/mL in RPMI 1640 supplemented with 15 % Fetal Bovine Serum (FBS), 2 mM L-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, and 4 μg/mL Phytohemagglutinin (PHA; Sigma, St. Louis, MO; catalog #L1668). The cells were allowed to incubate for 48-72 hours at 37°C. After incubation, PBMCs were centrifuged and resuspended in RPMI 1640 with 15% FBS, L-glutamine, penicillin, streptomycin, non-essential amino acids

(MEM/NEAA; Hyclone; catalog # SH30238.01), and 20 U/mL recombinant human IL-2 (R&D Systems Inc., Minneapolis, MN; catalog #202IL). PBMCs were maintained in this medium at a concentration of 1-2 x 106 cells/mL, with twice-weekly medium changes until they were used in the assay protocol. Monocytes-derived-macrophages were depleted from the culture as the result of adherence to the tissue culture flask.

[00152] For the standard PBMC assay, PHA stimulated cells from at least two normal donors were pooled (mixed together), diluted in fresh medium to a final concentration of 1 x 106 cells/mL, and plated in the interior wells of a 96 well round bottom microplate at 50 μΕ/well (5 x 104 cells/well) in a standard format developed by the Infectious Disease Research department of Southern Research Institute. Pooling (mixing) of mononuclear cells from more than one donor is used to minimize the variability observed between individual donors, which results from quantitative and qualitative differences in HIV infection and overall response to the PHA and IL-2 of primary lymphocyte populations. Each plate contains virus control wells (cells plus virus) and experimental wells (drug plus cells plus virus). Test drug dilutions were prepared at a 2X concentration in microtiter tubes and 100 of each concentration was placed in appropriate wells using the standard format. 50 of a predetermined dilution of virus stock was placed in each test well (final MOI = 0.1). Separate plates were prepared identically without virus for drug cytotoxicity studies using an MTS assay system

(described below; cytotoxicity plates also include compound control wells containing drug plus media without cells to control for colored compounds that affect the MTS assay). The PBMC cultures were maintained for seven days following infection at 37°C, 5% C02. After this period, cell-free

supernatant samples were collected for analysis of reverse transcriptase activity, and compound cytotoxicity was measured by addition of MTS to the separate cytotoxicity plates for determination of cell viability. Wells were also examined microscopically and any abnormalities were noted.

[00153] All compounds were tested in triplicate from 0.01 to 100 μΜ in ½-log increments. A3G activator compounds were added to cells 3 hours prior to infection, while Vif dimerization antagonists were added at the time of infection.

C. Reverse Transcriptase Activity Assay

[00154] A microtiter plate-based reverse transcriptase (RT) reaction was utilized (Buckheit et al,

AIDS Research and Human Retroviruses 7:295-302, 1991). Tritiated thymidine triphosphate (3H-TTP, 80 Ci/mmol, NEN) was received in 1 : 1 dH20:Ethanol at 1 mCi/ml. Poly rA:oligo dT template:primer (Pharmacia) was prepared as a stock solution by combining 150 μΐ poly rA (20 mg/ml) with 0.5 ml oligo dT (20 units/ml) and 5.35 ml sterile dH20 followed by aliquoting (1.0 ml) and storage at -20°C. The RT reaction buffer was prepared fresh on a daily basis and consisted of 125 μΐ 1.0 M EGTA, 125 μΐ dH20, 125 μΐ 20% Triton X100, 50 μΐ 1.0 M Tris (pH 7.4), 50 μΐ 1.0 M DTT, and 40 μΐ 1.0 M MgC12. The final reaction mixture was prepared by combining 1 part 3H-TTP, 4 parts dH20, 2.5 parts poly rA:oligo dT stock and 2.5 parts reaction buffer. Ten microliters of this reaction mixture was placed in a round bottom microtiter plate and 15 μΐ of virus-containing supernatant was added and mixed. The plate was incubated at 37°C for 60 minutes. Following incubation, the reaction volume was spotted onto DE81 filter- mats (Wallac), washed 5 times for 5 minutes each in a 5% sodium phosphate buffer or 2X SSC (Life Technologies), 2 times for 1 minute each in distilled water, 2 times for 1 minute each in 70% ethanol, and then dried. Incorporated radioactivity (counts per minute, CPM) was quantified using standard liquid scintillation techniques.

D. MTS Staining for PBMC Viability to Measure Cytotoxicity

[00155] At assay termination, the uninfected assay plates were stained with the soluble tetrazolium-based dye MTS (CellTiter 96 Reagent, Promega) to determine cell viability and quantify compound toxicity. MTS is metabolized by the mitochondria enzymes of metabolically active cells to yield a soluble formazan product, allowing the rapid quantitative analysis of cell viability and compound cytotoxicity. This reagent is a stable, single solution that does not require preparation before use. At termination of the assay, 20-25 μΐ_^ of MTS reagent was added per well and the microtiter plates are then incubated for 4-6 firs at 37°C, 5% C02 to assess cell viability. Adhesive plate sealers were used in place of the lids, the sealed plate was inverted several times to mix the soluble formazan product and the plate was read spectrophotometrically at 490/650 nm with a Molecular Devices SPECTRAmax plate reader.

Data Analysis

[00156] Results of this experiment are summarized in Table 1 below

Table 1

IC90 = 90% Inhibitory Concentration TC90 = 90% Toxicity Concentration

TI 90 = 90% Therapeutic Index (TC90/IC90) IC50 = 50% Inhibitory Concentration

TC50 = 50% Toxicity Concentration TI 50 = 50% Therapeutic Index (TC50/IC50)

OYA002-16 aka CPT-lactam OYA004-06 aka 12, 14 dinitro CPT-lactam

[00157] Using an in-house computer program, the PBMC data analysis includes the calculation of IC50 (50% inhibition of virus replication), IC90 (90% inhibition of virus replication), IC95 (95% inhibition of virus replication), TC50 (50% cytotoxicity), TC90 (90% cytotoxicity), TC95 (95% cytotoxicity) and therapeutic index values (TI =TC/IC; also referred to as Antiviral Index or AI). Raw data for both antiviral activity and toxicity with a graphical representation of the data are provided in a printout summarizing the individual compound activity.

EXAMPLE 2

Pseudotyped HIV Virus Testing Using Combinations of Vif Inhibitors and A3G Activators

[00158] Single cycle pseudotyped infectivity experiments were done in 6 well format in order to obtain enough virus to do viral particle purifications for western blot detection of A3G in the viral particle. The antiviral activity of the hits in a single-round infection with pseudotyped HIV were conducted using HEK293T producer cells +/- A3G and viruses that are +/- Vif. The wild type HIV proviral vector codes for all HIV genes except nef (replaced with EGFP) and env. The AVif proviral vector is identical to wild type except that it contains a stop codon early within the Vif gene. AVif +A3G is a strong positive control for this assay because without Vif present, A3G is able to be encapsidated within viral particles and have strong antiviral activity. Alternatively, in the absence of A3G, both wild type and AVif viruses should have high infectivity.

[00159] Single-round infectivity assays utilized transient transfection of the viral vectors with

VSV-G coat protein vector and V5-A3G in the +A3G conditions with Fugene HD (Promega). Proviral DNA: VSV-G: A3G were added to cells with a ratio of 1 :0.5:0.08 which establishes levels of A3G that are comparable to endogenous A3G. These virus producer cells were dosed with OYA002-16 or OYA004-06 (4-33 μΜ) four hours after transfection and viral particles were harvested from the media 24 hours after transfecting by filtering through a 0.45 -micron syringe filter. Viral load was then normalized with a p24 ELISA (Perkin Elmer).

[00160] The infections utilized A3G-TZM-bl reporter cells that contain stably integrated luciferase that is driven by the HIV-LTR promoter; therefore luciferase is expressed upon successful HIV infection. A3G-TZM-bl cells were treated with increasing concentrations of SMAA-2.0 or SMAA-2.6 for 3 hours prior to infection (75-625 nM for SMAA-2.0, 35-250 nM for SMAA-2.6).

Triplicate infections in 96-well plates at 10,000 cells/well with 500 pg p24/well proceeded for 24 hours, at which point cells received a second dose of SMAA-2.0 or SMAA-2.6. Infectivity was determined 48 hours after infection by the addition of SteadyGlo™ Reagent (Promega) to each well for 30 minutes. Luminescence was measured as a quantitative metric for changes in infectivity with each compound as compared to controls, in which relative luminescence units (RLU) with no compounds are set to 100%.

[00161] In summary, various combinations of Vif inhibitors and A3G activators were analyzed for their effects on HIV infectivity inhibition, including those combinations noted in below Table 2:

Table 2

[00162] FIG. 8 is a bar graph showing the infectivity results for OYA002-16 (Vif inhibitor) combined with SMAA-2.0 (A3G activator). Pseudotyped virus was produced in HEK 293T cells (producer cells) with A3G co-expression. During viral production, producer cells were treated with increasing concentrations of OYA002-16 (4, 8, 16, and 33 μΜ). A3G-TZM-M cells (target cells for infectivity) were treated with increasing concentrations of SMAA-2.0 (0.125, 0.250, 0.500, and 1 μΜ) for 3 hours prior to infection. For the co -treatment conditions shown, producer cells were treated with 16 μΜ OYA002-16 and target cells were treated with 250 nM SMAA-2.0, which are the EC50 values previously determined for each compound. Untreated or SMAA-2.6 pre -treated A3G-TZM-bl cells were infected with 500 pg of p24-normalized pseudotyped virus that was either untreated or treated with increasing concentrations of OYA002-16. As a control for maximum A3G antiviral activity, cells were infected with -Vif virus (+A3G). All values are the averages of triplicate wells ±standard deviation. The horizontal line represents the compound concentrations corresponding to a 50% decrease in viral infectivity (EC50) and the arrow points out the increased antiviral effect of combining OYA002-16 and SMAA-2.0 treatment. These results demonstrate that combining OYA002-16 and SMAA-2.0 treatment at their EC50 values causes >50% decrease in HIV infectivity.

[00163] FIG. 9 is a bar graph showing infectivity results for OYA002-16 (Vif inhibitor) combined with SMAA-2.6 (A3G activator). Pseudotyped virus was produced in HEK 293T cells (producer cells) with A3G co-expression. During viral production, producer cells were either treated with DMSO solvent (1^st bar, DMSO control set to 100% infectivity) or OYA002-16 (8 μΜ; 3^rd bar). A3G-TZM-M cells (target cells for infectivity) were treated with SMAA-2.6 (75 nM; 4^th bar) for 3 hours prior to infection. These same concentrations were tested for the co -treatment experiment (5^th bar). Untreated or SMAA-2.6 pre-treated A3G-TZM-bl cells were infected with 500 pg of p24- normalized pseudotyped virus that was either untreated or treated with OYA002-16. As a control for maximum A3G antiviral activity, cells were infected with -Vif virus (+A3G; 2^nd bar). All values are the averages of triplicate wells ±standard deviation. These results demonstrate that combining

OYA002-16 and SMAA-2.6 treatment causes a greater decrease in HIV infectivity compared to either compound alone at the same concentration.

[00164] FIG. 10 is a bar graph showing infectivity results for OYA004-06 (Vif inhibitor) combined with SMAA-2.6 (A3G activator). Pseudotyped virus was produced in HEK 293T cells (producer cells) with A3G co-expression. During viral production, producer cells were treated with either DMSO solvent (1^st bar, DMSO control set to 100% infectivity) or OYA004-06 (8 μΜ; 3^rd bar). A3G-TZM-M cells (target cells for infectivity) were treated with SMAA-2.6 (75 nM; 4^th bar) for 3 hours prior to infection. These same concentrations were tested for the co -treatment experiment (5^th bar). Untreated or SMAA-2.6 pre-treated A3G-TZM-bl cells were infected with 500 pg of p24- normalized pseudotyped virus that was either untreated or treated with OYA004-06. As a control for maximum A3G antiviral activity, cells were infected with -Vif virus (+A3G, 2^nd bar). All values are the averages of triplicate wells ±standard deviation. These results demonstrate that combining

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[00166] While the present invention has been described with reference to the specific

embodiments thereof it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adopt a particular situation, material, composition of matter, process, process step or steps, to the objective spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto.

Claims

WHAT IS CLAIMED IS:

1. A method for inhibiting infectivity of HIV in a host cell, said method comprising:

contacting a host cell comprising APOBEC3G(A3G) host defense factor with a combination of antiviral-effective amounts of a Vif inhibitor and an A3G activator,

thereby inhibiting infectivity of HIV in the host cell by simultaneously inhibiting Vif- dependent degradation of A3G and activating A3G deaminase activity in the host cell,

wherein the Vif inhibitor inhibits Vif-dependent degradation of A3G by disrupting or inhibiting dimerization of Vif in the host cell, and

wherein the A3G activator activates A3G deaminase activity by disrupting or inhibiting A3G:nucleic acid molecule interaction in the host cell.

2. The method according to claim 1, wherein the Vif inhibitor comprises a compound of formula (I):

(I) or a pharmaceutically acceptable salt thereof, wherein:

Q is selected from NH, O, and S;

R^20a and R^20b are individually selected from hydrogen, hydroxy, and Ci_₆alkyl; R²¹ is selected from hydrogen, -NHC(=0)(CH₂)_pNR²³R²⁴, and -(CH₂)_PNR²³R²⁴; p is 0, 1 , 2, 3, or 4;

R²² is selected from hydrogen and hydroxyl;

R²³ and R²⁴ are individually selected from hydrogen and Ci_₆alkyl; and R²⁵ and R²⁶ are individually selected from hydrogen and -N0₂.

3. The method according to claim 2, wherein the Vif inhibitor of formula (I) is a compound selected from the group consisting of

(I-c) (I-d)

(i-g)

4. The method according to claim 1, wherein the A3G activator comprises a compound of formula (II):

or a pharmaceutically acceptable salt thereof, wherein:

A, B, and C are each individually selected from CH and N;

W is absent or is selected from CH and N;

X is selected from CH and N;

Z is selected from C(=0) and CH₂;

R¹¹ is selected from phenyl and 5- or 6-membered heteroaryl, wherein said phenyl or 5- or 6- membered heteroaryl is substituted with 0, 1, 2, or 3 substituents individually selected from halo, Ci_ ₆alkyl, cyano, Ci_6alkoxy, COOR¹⁵, hydroxy Ci_6alkyl, and 4- to 6-membered heterocyclic group; n is 0, 1, 2, or 3;

R¹² is selected from halo, C_halky!, cyano, Ci_6alkoxy, and COOR¹⁵, and a 4- to 6-membered heterocyclic group; and

R¹⁵ is selected from hydrogen and Ci_₆ alkyl.

5. The method according to claim 4, wherein the A3G activator of formula (II) is a compound selected from the group consisting of

and

(Il-a) (Il-b)

6. The method according to claim 1, wherein the Vif inhibitor is a compound selected from the group consisting of

(I-a) (I-b)

(I-c) (I-d)

and wherein the A3G activator is a com ound selected from the group consisting of

(Il-a) (Il-b)

7. The method according to claim 1, wherein the Vif inhibitor is selected from the group consisting of camptotechin, topotecan, irinotecan, and analogs thereof and having a related chemical scaffold (chemotype).

8. The method according to claim 1, wherein the A3G activator is selected from the group consisting of

(III) (IV) and analogs thereof and having a related chemical scaffold (chemotype).

9. A method for treating or preventing HIV infection or AIDS in a patient, said method comprising:

administering to a patient in need of such treatment or prevention a combination of a therapeutically effective amount of a Vif inhibitor and an APOBEC3G (A3G) activator,

wherein the Vif inhibitor is administered at least once at a dosage effective to disrupt or inhibit multimerization of Vif in a cell of the patient,

wherein the A3G activator is administered at least once at a dosage effective to disrupt or inhibit A3G:nucleic acid molecule interaction in a cell of the patient, and

wherein the Vif inhibitor and the A3G activator are administered in combination and up to the maximum tolerated dose of each agent.

10. The method according to claim 9, wherein the Vif inhibitor is effective to inhibit Vif dimerization by direct or indirect inhibition of binding of Vif dimers at the Vif dimerization domain, said Vif dimerization domain comprising the amino acid sequence of pro line -pro line- leucine-proline (PPLP).

11. The method according to claim 9, wherein the Vif inhibitor is effective to inhibit Vif from binding to A3G.

12. The method according to claim 9, wherein the Vif inhibitor is effective to inhibit Vif-dependent degradation of A3G.

13. The method according to claim 12, wherein the Vif inhibitor is effective to inhibit Vif- dependent degradation of A3G by inhibiting interaction of Vif with one or more enzymes selected from the group consisting of Cullin 5, Elongin B, and Elongin C, thereby inhibiting ubiquitination of A3G.

14. The method according to claim 9, wherein the A3G activator is effective to disrupt or inhibit A3G:RNA interaction in the cell.

15. The method according to claim 9, wherein the Vif inhibitor comprises a compound of formula (I):

(I) or a pharmaceutically acceptable salt thereof, wherein:

Q is selected from NH, O, and S;

R ^a and R are individually selected from hydrogen, hydroxy, and Ci_₆alkyl; R²¹ is selected from hydrogen, -NHC(=0)(CH₂)_pNR²³R²⁴, and -(CH₂)_PNR²³R²⁴; p is 0, 1 , 2, 3, or 4;

R²² is selected from hydrogen and hydroxyl;

16. The method according to claim 15, wherein the Vif inhibitor of formula (I) is a compound selected from the group consisting of

(I-c) (I-d)

(i-g)

The method according to claim 9, wherein the A3G activator comprises a compound of

or a pharmaceutically acceptable salt thereof, wherein:

A, B, and C are each individually selected from CH and N;

W is absent or is selected from CH and N;

X is selected from CH and N;

Z is selected from C(=0) and CH₂;

R¹⁵ is selected from hydrogen and Ci_₆ alkyl.

18. The method according to claim 17, wherein the A3G activator of formula (II) is a compound selected from the group consisting of

and

(Il-a) (Il-b)

19. The method according to claim 9, wherein the Vif inhibitor is a compound selected from the group consisting of

(I-a) (I-b)

(I-c) (I-d)

(Il-a) (Il-b)

20. The method according to claim 9, wherein the Vif inhibitor is selected from the group consisting of camptotechin, topotecan, irinotecan, and analogs thereof and having a related chemical scaffold (chemotype).

21. The method according to claim 9, wherein the A3G activator is selected from the group consisting of

22. The method according to claim 9, wherein the Vif inhibitor and the A3G activator are administered simultaneously.

23. The method according to claim 9, wherein the A3G activator is administered prior to administration of the Vif inhibitor.

24. The method according to claim 9, wherein the Vif inhibitor is administered prior to

administration of the A3G activator.

25. The method according to claim 9, wherein the Vif inhibitor and the A3G activator are administered in multiple alternating doses.

26. The method according to claim 9 further comprising:

administering a therapeutically effective amount of at least one other agent for treating HIV selected from the group consisting of HIV reverse transcriptase inhibitors, non-nucleoside HIV reverse transcriptase inhibitors, HIV protease inhibitors, HIV fusion inhibitors, HIV attachment inhibitors, CCR5 inhibitors, CXCR4 inhibitors, HIV budding or maturation inhibitors, and HIV integrase inhibitors.

27. The method according to claim 9, wherein the HIV infection is due to an HIV virus is selected from the group consisting of HIV-1 and HIV-2.

28. A method of designing a personalized treatment regimen for treating or preventing HIV infection or AIDS in a patient, said method comprising:

determining the therapeutically effective dosage range of a Vif inhibitor in a patient; and determining the therapeutically effective dosage range of an APOBEC3G (A3G) activator, wherein determining the therapeutically effective dosage ranges of the Vif inhibitor and the A3G activator is based on a combination treatment of these two agents.

29. The method according to claim 28, wherein the Vif inhibitor is effective to inhibit Vif dimerization by direct or indirect inhibition of binding of Vif dimers at the Vif dimerization domain, said Vif dimerization domain comprising the amino acid sequence of pro line -pro line- leucine-proline (PPLP).

30. The method according to claim 28, wherein the Vif inhibitor is effective to inhibit Vif from binding to A3G.

31. The method according to claim 28, wherein the Vif inhibitor is effective to inhibit Vif- dependent degradation of A3G.

32. The method according to claim 18, wherein the Vif inhibitor is effective to inhibit Vif- dependent degradation of A3G by inhibiting interaction of Vif with one or more enzymes selected from the group consisting of Cullin 5, Elongin B, and Elongin C, thereby inhibiting ubiquitination of A3G.

33. The method according to claim 28, wherein the A3G activator is effective to disrupt or inhibit A3G:RNA interaction in the cell.

34. The method according to claim 28, wherein the Vif inhibitor comprises a compound of formula (I):

(I) or a pharmaceutically acceptable salt thereof, wherein:

Q is selected from NH, O, and S;

R ^a and R are individually selected from hydrogen, hydroxy, and Ci_₆alkyl; R²¹ is selected from hydrogen, -NHC(=0)(CH₂)_pNR²³R²⁴, and -(CH₂)_PNR²³R²⁴; p is 0, 1, 2, 3, or 4; selected from hydrogen and hydroxyl;

R and R are individually selected from hydrogen and Ci_₆alkyl; and

R and R are individually selected from hydrogen and -N0₂.

35. The method according to claim 34, wherein the Vif inhibitor of formula (I) is a compound selected from the group consisting of

-55-

(i-g)

The method according to claim 28, wherein the A3G activator comprises a compound of

(II)

or a pharmaceutically acceptable salt thereof,

wherein:

A, B, and C are each individually selected from CH and N;

W is absent or is selected from CH and N;

X is selected from CH and N;

Y is selected from CH, O, and S;

the dotted line is either a single bond or a double bond; Z is selected from C(=0) and CH₂;

R¹⁵ is selected from hydrogen and Ci_₆ alkyl.

37. The method according to claim 36, wherein the A3G activator of formula (II) is a compound selected from the roup consisting of

and

(Il-a) (Il-b)

38. The method according to claim 28, wherein the Vif inhibitor is a compound selected from the group consisting of

(I-a) (I-b)

(i-g)

and wherein the A3G activator is a compound selected from the group consisting of

and

(Il-a) (Il-b)

39. The method according to claim 28, wherein the Vif inhibitor is selected from the group consisting of camptotechin, topotecan, irinotecan, and analogs thereof and having a related chemical scaffold (chemotype).

40. The method according to claim 28, wherein the A3G activator is selected from the group consisting of

41. The method according to claim 28, wherein the combination treatment comprises

simultaneously administering the Vif inhibitor and the A3G activator.

42. The method according to claim 28, wherein the combination treatment comprises administering the A3G activator prior to the Vif inhibitor.

43. The method according to claim 28, wherein the combination treatment comprises administering the Vif inhibitor prior to the A3G activator.

44. The method according to claim 28, wherein the combination treatment comprises administering the Vif inhibitor and the A3G activator in multiple alternating doses.

45. The method according to claim 28, wherein the combination treatment further comprises: administering a therapeutically effective amount of at least one other agent for treating HIV selected from the group consisting of HIV reverse transcriptase inhibitors, non-nucleoside HIV reverse transcriptase inhibitors, HIV protease inhibitors, HIV fusion inhibitors, HIV attachment inhibitors, CCR5 inhibitors, CXCR4 inhibitors, HIV budding or maturation inhibitors, and HIV integrase inhibitors.

46. The method according to claim 28, wherein the HIV infection is due to an HIV virus is selected from the group consisting of HIV-1 and HIV-2.