WO2014186423A1 - Traitement combiné pour traiter le vih et le sida - Google Patents

Traitement combiné pour traiter le vih et le sida Download PDF

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WO2014186423A1
WO2014186423A1 PCT/US2014/037935 US2014037935W WO2014186423A1 WO 2014186423 A1 WO2014186423 A1 WO 2014186423A1 US 2014037935 W US2014037935 W US 2014037935W WO 2014186423 A1 WO2014186423 A1 WO 2014186423A1
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Prior art keywords
vif
hiv
activator
inhibitor
group
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Harold C. Smith
Ryan P. BENNETT
Kimberly Prohaska
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OYAGEN Inc
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OYAGEN Inc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4745—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/425—Thiazoles
    • A61K31/427—Thiazoles not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/517—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with carbocyclic ring systems, e.g. quinazoline, perimidine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV

Definitions

  • 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.
  • HIV-1 Human immunodeficiency virus type 1
  • AIDS acquired immunodeficiency syndrome
  • 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.
  • 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).
  • structural proteins Gag, Pol and Env
  • essential transacting proteins Tat, Rev
  • Vpr, Vif, Vpu, Nef "auxiliary" proteins that are not required for efficient virus replication in permissive cells
  • STRIBILDTM 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).
  • HAART highly active anti-retroviral therapy
  • I integrase
  • RT reverse transcriptase
  • HIV strains with resistance to some or all of these components had already emerged prior to the availability of STRIBILDTM, thus rendering it ineffective against such strains.
  • HIV developed resistance to STRIBILDTM 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).
  • M46I/L/V in the HIV protease confers resistance to 7 out of 8 inhibitors.
  • the present invention is directed toward overcoming these and other deficiencies in the art.
  • 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.
  • 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.
  • A3G APOBEC3G
  • the Vif inhibitor inhibits Vif-dependent degradation of A3G by disrupting or inhibiting dimerization of Vif in the host cell
  • 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.
  • 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.
  • 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
  • the Vif inhibitor and the A3G activator are examples of the Vif inhibitor and the A3G activator.
  • Vif inhibitors and A3G activators for use in this method are as described herein.
  • 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.
  • 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.
  • the patent or application file may contain at least one drawing executed in color.
  • FIG. 1 is an illustration of the life cycle of an HIV virus in the presence of Vif dimers
  • 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.
  • FIG. 3 is an illustration showing how enabling of the A3G host-defense in a cell can inhibit HIV infection in the cell.
  • 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.
  • CPT camptothecin
  • 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.
  • CPT camptothecin
  • 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.
  • 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)).
  • deoxycamptothecin lactam deoxycamptothecin lactam (denoted as formula 2 in the figure) (corresponds to Formula (I-c)
  • FIG. 8 is a bar graph showing infectivity results for OYA002-16 (Vif inhibitor) combined with SMAA-2.0 (A3G activator).
  • FIG. 9 is a bar graph showing infectivity results for OYA002-16 (Vif inhibitor) combined with SMAA-2.6 (A3G activator).
  • 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
  • the present invention is based, in part, on the discovery that combining a Viral
  • Vif inhibitor 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.
  • 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).
  • 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.
  • 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).
  • 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.
  • Vif inhibitors and A3G activators for use in the methods of the present invention.
  • 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.
  • alkyl is intended to include linear, branched, and cyclic hydrocarbon structures and combinations thereof. A combination would be, for example,
  • 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.
  • 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.
  • 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,
  • aryl and heteroaryl refer to residues in which one or more rings are aromatic, but not all need be.
  • halogen means fluorine, chlorine, bromine or iodine. In one embodiment, halogen may be fluorine or chlorine.
  • heterocyclic group includes within its scope aromatic, non-aromatic, unsaturated, partially saturated and fully saturated heterocyclic ring systems.
  • such groups may be monocyclic or bicyclic and may contain, for example, 3 to 12 ring members, more usually 5 to 10 ring members.
  • 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.
  • Radicals and substituents are generally defined when introduced and retain that definition throughout the specification and in all independent claims.
  • salt forms of the compounds of the present disclosure 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.
  • Suitable pharmaceutically acceptable non-toxic acids or bases including inorganic acids and bases and organic acids and bases.
  • 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, nap
  • 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, ⁇ , ⁇ '-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.
  • 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.
  • 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.
  • N-oxides are the N-oxides of a tertiary amine or a nitrogen atom of a nitrogen-containing heterocycle.
  • 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).
  • an oxidizing agent such as hydrogen peroxide or a per-acid (e.g. a peroxycarboxylic acid).
  • a per-acid e.g. a peroxycarboxylic acid
  • MCPBA m-chloroperoxybenzoic acid
  • Vif inhibitors for use in the methods of the present disclosure are as disclosed herein.
  • 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.
  • Vif dimerization also referred to herein as "Vif multimerization”
  • 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).
  • the Vif inhibitor of the present invention is effective to inhibit Vif from binding to A3G.
  • the Vif inhibitor of the present invention is effective to inhibit Vif-dependent degradation of A3G.
  • 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.
  • 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.
  • camptotechin 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.
  • the present invention provides small molecule compounds that are effective as inhibitors of Vif self-association.
  • a suitable compound that is effective as a Vif inhibitor includes a compound of formula (I):
  • Q is selected from NH, O, and S;
  • R 20a and R 20b are individually selected from hydrogen, hydroxy, and Ci_ 6 alkyl;
  • p is 0, 1, 2, 3, or 4;
  • R 22 is selected from hydrogen and hydroxyl
  • R 23 and R 24 are individually selected from hydrogen and Ci_ 6 alkyl
  • R 25 and R 26 are individually selected from hydrogen and -N0 2 .
  • Q is O.
  • Q is selected from NH and S.
  • Q is NH
  • Q is S.
  • R 20a is Ci_6alkyl.
  • R 20a and R 20b are individually selected from hydrogen, hydroxy, methyl, and ethyl.
  • R 20a is selected from hydrogen and hydroxy.
  • R 20a is hydrogen
  • R 20a is hydroxy
  • R 20b is ethyl
  • R 20a is hydrogen or hydroxy and R 20b is ethyl.
  • R 20a is hydrogen and R 20b is ethyl.
  • R 20a is hydroxy and R 20b is ethyl.
  • R 21 is hydrogen
  • R 21 is -(CH 2 ) P NR 23 R 24 (e.g., -CH 2 N(CH 3 ) 2 ).
  • 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.
  • R 22 is hydrogen
  • R 22 is hydroxyl
  • R 23 and R 24 are individually selected from hydrogen and methyl.
  • R and R are both hydrogen.
  • R 25 and R 26 are both -N0 2 .
  • a suitable Vif inhibitor of formula (I) is a compound selected from the group consisting of
  • 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).
  • 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.
  • APOBEC3G (A3G) activators for use in the methods of the present disclosure are as disclosed herein.
  • 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].
  • HIV viral infectivity factor
  • A3G as the cellular defense factor [3].
  • A3G is incorporated into HIV virions [4, 5] and binds to the viral core [6].
  • 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.
  • 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.
  • A3G-RNP ribonucleoprotein complexes
  • RNA alone binding to A3G is sufficient to inhibit its deaminase activity in vitro [38].
  • 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.
  • HIV In addition to causing A3G's inactivation, HIV expresses the Vif protein, which targets
  • Vif 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].
  • the present invention provides small molecule compounds that are effective as A3G activators.
  • the A3G activators of the present disclosure are effective to disrupt or inhibit A3G:R A interaction in the cell.
  • a suitable compound that is effective as an A3G activator includes a compound of formula (II):
  • 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;
  • R 11 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_ 6 alkyl, cyano, Ci_6alkoxy, COOR 15 , hydroxyCi_6alkyl, and 4- to 6-membered heterocyclic group; n is 0, 1, 2, or 3;
  • R 12 is selected from halo, C h alky!, cyano, Ci_6alkoxy, COOR 15 , and a 4- to 6-membered heterocyclic group;
  • R 15 is selected from hydrogen and Ci_ 6 alkyl.
  • A, B, and C are each CH, forming a phenyl ring.
  • the A, B, C ring is pyridine (i.e., one of A, B and C is
  • 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 R 12 groups:
  • 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:
  • W is absent, such that the center ring of formula (II) is a 5-membered ring.
  • the center (W, X, Y) ring is selected from oxazolyl, thienyl, and thiazolyl.
  • W is absent, X is CH, and Y is S.
  • W is absent, X is N, and Y is S.
  • W is absent, X is N, and Y is O.
  • W is CH or N, such that the center ring of formula (II) is a 6-membered ring.
  • W is CH
  • X is N
  • Y is CH
  • W is N
  • X is N
  • Y is CH
  • Z is CH 2 .
  • R 11 is selected from unsubstituted phenyl and unsubstituted 5- or 6-membered heteroaryl.
  • R 11 is selected from phenyl and 5- or 6-membered heteroaryl, each being substituted with 1, 2, or 3 substituents individually selected from halo, Ci_ 6 alkyl, cyano, Ci_6alkoxy, COOR 15 , hydroxyCi_6alkyl, and 4- to 6-membered heterocyclic group.
  • R 11 is optionally substituted phenyl, where substituents are as described above.
  • R 11 is optionally substituted 5- or 6-membered heteroaryl (e.g., pyrazolyl, thienyl, thiazolyl, oxazolyl), where substituents are as described above.
  • heteroaryl e.g., pyrazolyl, thienyl, thiazolyl, oxazolyl
  • R 11 is substituted pyrazolyl.
  • R 11 is l ,3-dimethyl-lH-pyrazol-5-yl.
  • n is n is 0, 1 , 2, or 3. Where n is greater than 1 , each R 12 group is individually selected (i.e., present R 12 groups may be the same or different).
  • R 15 is selected from hydrogen and Ci_ 6 alkyl. In one subgroup, R 15 is selected from hydrogen and methyl. In a more particular subgroup, R 15 is hydrogen.
  • 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.
  • 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:
  • a suitable A3G activator can be a compound of formula (III)
  • 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.
  • Both R A inhibition of A3G and Vif-mediated A3G degradation represent potential targets for HIV treatment.
  • A3G will remain minimally active due to its interaction with RNA.
  • 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.
  • Vif promotes the sequestration of A3G in large complexes that may involve RNA interactions [55].
  • inhibiting Vif would lead to increased A3G abundance, it may still be predominantly inactive.
  • 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.
  • 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.
  • 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.
  • A3G APOBEC3G
  • the Vif inhibitor inhibits Vif- dependent degradation of A3G by disrupting or inhibiting dimerization of Vif in the host cell
  • 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.
  • 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.
  • 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
  • the Vif inhibitor and the A3G activator are examples of the Vif inhibitor and the A3G activator.
  • Vif inhibitors and A3G activators for use in this method are as described herein.
  • the Vif inhibitor and the A3G activator are administered simultaneously.
  • the A3G activator is administered prior to administration of the Vif inhibitor.
  • the Vif inhibitor is administered prior to administration of the A3G activator.
  • the Vif inhibitor and the A3G activator are administered in multiple alternating doses.
  • 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.
  • HIV reverse transcriptase inhibitors non-nucleoside HIV reverse transcriptase inhibitors
  • HIV protease inhibitors HIV fusion inhibitors
  • HIV attachment inhibitors HIV attachment inhibitors
  • CCR5 inhibitors CXCR4 inhibitors
  • HIV budding or maturation inhibitors HIV integrase inhibitors
  • the HIV infection is due to an HIV virus selected from the group consisting of HIV- 1 and HIV-2.
  • 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.
  • 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.
  • the combination treatment comprises
  • 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.
  • 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.
  • 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.
  • the HIV infection is due to an HIV virus selected from the group consisting of HIV- 1 and HIV-2.
  • 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.
  • 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.
  • 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.
  • contemplated formulations include projected nanoparticles, liposomal preparations, resealed erythrocytes containing the active ingredient, and immuno logically-based formulations.
  • 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.
  • 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.
  • peripheral administration of a pharmaceutical composition includes any route of administration characterized by physical breaching of a tissue of a subject and
  • 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.
  • peritoneal administration is contemplated to include, but is not limited to, subcutaneous, intraperitoneal, intramuscular, intrasternal injection, and kidney dialytic infusion techniques.
  • 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.
  • compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology.
  • 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.
  • compositions suitable for administration to humans 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
  • compositions of the invention include, but are not limited to, humans and other primates, mammals including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, and dogs.
  • Controlled- or sustained-release formulations of a pharmaceutical composition of the invention may be made using conventional technology.
  • 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.
  • 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.
  • 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
  • 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.
  • 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
  • compositions for topical administration may further comprise one or more of the additional ingredients described herein.
  • dosages of the compound of the invention which may be administered to an animal 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.
  • 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.
  • 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.
  • 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.
  • the solvate When water is the solvent, the solvate is referred to as a hydrate.
  • 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.
  • 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.
  • physiologically functional derivative refers to any pharmaceutically acceptable derivative of a compound of the present invention that, upon
  • the term "effective amount” means that amount of a drug or
  • 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.
  • 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.
  • 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.
  • pharmaceutically 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.
  • preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biological Standards.
  • 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.
  • lentivirus 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);
  • HIV simian immunodeficiency virus
  • BIV bovine immunodeficiency virus
  • SHIV chimeric simian/human immunodeficiency virus
  • FV feline immunodeficiency virus
  • HAV human immunodeficiency virus
  • HIV 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.
  • mammal 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
  • 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.
  • “Pharmaceutically acceptable” means physiologically tolerable, for either human or veterinary applications.
  • “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.
  • pharmaceutically acceptable carriers and other components of pharmaceutical compositions see, e.g., Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing Company, 1990.
  • compositions include formulations for human and veterinary use.
  • 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.
  • 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.
  • "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.
  • a "virion” is a complete viral particle; nucleic acid and capsid, further including and a lipid envelope in the case of some viruses.
  • 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.
  • PBMCs Peripheral Blood Mononuclear Cells
  • 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.
  • 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.
  • PBS Dulbecco's phosphate buffered saline
  • 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.
  • Ficoll-Hypaque density gradient Lymphocyte Separation Medium, Cell Grow #85-072-CL, density 1.078+/-0.002 gm/ml
  • PBMCs 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 ⁇ g/mL streptomycin, and 4 ⁇ g/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
  • PBMCs peripheral blood mononuclear cells
  • PBMCs peripheral blood mononuclear cells
  • monocytes-derived-macrophages were depleted from the culture as the result of adherence to the tissue culture flask.
  • 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 ⁇ /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).
  • 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
  • RT reverse transcriptase
  • 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 ⁇ 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 ⁇ 1.0 M EGTA, 125 ⁇ dH20, 125 ⁇ 20% Triton X100, 50 ⁇ 1.0 M Tris (pH 7.4), 50 ⁇ 1.0 M DTT, and 40 ⁇ 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 ⁇ of virus-containing supernatant was added and mixed. The plate was incubated at 37°C for 60 minutes.
  • 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.
  • MTS soluble tetrazolium-based dye
  • CellTiter 96 Reagent CellTiter 96 Reagent, Promega
  • 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.
  • 20-25 ⁇ ⁇ 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.
  • TI 90 90% Therapeutic Index (TC90/IC90)
  • IC50 50% Inhibitory Concentration
  • 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
  • AI Therapeutic index values
  • 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.
  • both wild type and AVif viruses should have high infectivity.
  • VSV-G coat protein vector and V5-A3G in the +A3G conditions with Fugene HD 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 ⁇ ) 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).
  • 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).
  • 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.
  • producer cells were treated with increasing concentrations of OYA002-16 (4, 8, 16, and 33 ⁇ ).
  • A3G-TZM-M cells target cells for infectivity
  • producer cells were treated with 16 ⁇ 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.
  • 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.
  • producer cells were either treated with DMSO solvent (1 st bar, DMSO control set to 100% infectivity) or OYA002-16 (8 ⁇ ; 3 rd bar).
  • A3G-TZM-M cells target cells for infectivity
  • OYA002-16 and SMAA-2.6 treatment causes a greater decrease in HIV infectivity compared to either compound alone at the same concentration.
  • 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.
  • producer cells were treated with either DMSO solvent (1 st bar, DMSO control set to 100% infectivity) or OYA004-06 (8 ⁇ ; 3 rd bar).
  • A3G-TZM-M cells target cells for infectivity
  • OYA002-16 and SMAA-2.6 treatment causes a greater decrease in HIV infectivity compared to either compound alone at the same concentration.
  • Suspene, R., et al, APOBEC3G is a single-stranded DNA cytidine deaminase and functions independently of HIV reverse transcriptase. Nucleic Acids Res, 2004. 32(8): p. 2421-9.
  • HIV-1 FASEBJ, 2009. 23: p. 279-87.
  • the CD4 (T4) antigen is an essential component of the receptor for the AIDS retrovirus. Nature, 1984. 312: p. 763-7.
  • Vif-Cul5 complex that promotes APOBEC3G degradation. Genes Dev, 2004. 18(23): p. 2861-6.

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Abstract

La présente invention concerne l'utilisation d'un inhibiteur de Vif en association avec un activateur d'APOBEC3G (A3G) pour inhiber l'infectivité du VIH dans une cellule hôte et pour traiter ou prévenir l'infection par le VIH ou le SIDA chez un patient. Dans un mode de réalisation, la présente invention concerne un procédé d'inhibition de l'infectivité du VIH dans une cellule hôte, ledit procédé consistant à mettre une cellule hôte comprenant un facteur de défense hôte A3G en contact avec des quantités efficaces d'un point de vue antiviral d'une combinaison composée d'un inhibiteur de Vif et d'un activateur d'A3G, ce qui permet d'inhiber l'infectivité du VIH dans la cellule hôte par l'inhibition de la dégradation d'A3G dépendante de Vif en même tant que l'activation de l'activité A3G déaminase dans la cellule hôte, l'inhibiteur de Vif inhibant la dégradation d'A3G dépendant de Vif par rupture ou inhibition de l'interaction A3G/molécules d'acide nucléique dans la cellule hôte.
PCT/US2014/037935 2013-05-13 2014-05-13 Traitement combiné pour traiter le vih et le sida Ceased WO2014186423A1 (fr)

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WO2018128993A1 (fr) * 2017-01-04 2018-07-12 Oyagen, Inc. Composés, compositions et méthodes pour le traitement du virus de l'immunodéficience humaine
US10588902B2 (en) 2013-06-24 2020-03-17 Oyagen, Inc. Camptothecin derivatives as anti-HIV agents and methods of identifying agents that disrupt Vif self-association

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US20050053977A1 (en) * 2003-06-10 2005-03-10 Greene Warner C. Methods for treating lentivirus infections
WO2011143553A1 (fr) * 2010-05-14 2011-11-17 University Of Rochester Compositions et procédés pour cibler des complexes a3g:arn

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WO1996011005A2 (fr) * 1994-10-06 1996-04-18 Atlas Leon T Utilisation de camptothecine ou de derives de camptothecine dans la fabrication d'un medicament destine au traitement de maladies virales
US20050053977A1 (en) * 2003-06-10 2005-03-10 Greene Warner C. Methods for treating lentivirus infections
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10588902B2 (en) 2013-06-24 2020-03-17 Oyagen, Inc. Camptothecin derivatives as anti-HIV agents and methods of identifying agents that disrupt Vif self-association
WO2018128993A1 (fr) * 2017-01-04 2018-07-12 Oyagen, Inc. Composés, compositions et méthodes pour le traitement du virus de l'immunodéficience humaine
US11116762B2 (en) 2017-01-04 2021-09-14 Oyagen, Inc. Compounds, compositions, and methods for treating human immunodeficiency virus

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