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WO2008053478A2 - Compositions et procédés pour inhiber la réplication et l'activité de l'intégrase du vih-1 - Google Patents

Compositions et procédés pour inhiber la réplication et l'activité de l'intégrase du vih-1 Download PDF

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Publication number
WO2008053478A2
WO2008053478A2 PCT/IL2007/001321 IL2007001321W WO2008053478A2 WO 2008053478 A2 WO2008053478 A2 WO 2008053478A2 IL 2007001321 W IL2007001321 W IL 2007001321W WO 2008053478 A2 WO2008053478 A2 WO 2008053478A2
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WIPO (PCT)
Prior art keywords
ledgf
another embodiment
protein
fragment
hiv
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PCT/IL2007/001321
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English (en)
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WO2008053478A3 (fr
Inventor
Assaf Friedler
Abraham Loyter
Moshe Kotler
Amnon Hizi
Zvi Hayouka
Joseph Rosenbluh
Aviad Levin
Shoshana Loya
Original Assignee
Yissum Research Development Company Of The Hebrew University Of Jerusalem
Ramot At Tel-Aviv University Ltd.
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Application filed by Yissum Research Development Company Of The Hebrew University Of Jerusalem, Ramot At Tel-Aviv University Ltd. filed Critical Yissum Research Development Company Of The Hebrew University Of Jerusalem
Publication of WO2008053478A2 publication Critical patent/WO2008053478A2/fr
Publication of WO2008053478A3 publication Critical patent/WO2008053478A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention provides isolated peptides comprising a fragment of a LEDGF/p75 protein and use of same for treating HIV-I infection, inhibiting HIV-I replication and inhibiting DNA binding and 3 '-end processing activity of HIV-I integrase protein.
  • HIV-I causes acquired immunodeficiency syndrome (AIDS), one of the most widespread and lethal epidemics of the last decades.
  • Current anti-HIV therapy is aimed mainly at inhibiting HIV-I entry into cells or inhibiting the viral enzymes reverse transcriptase (RT) or protease.
  • RT reverse transcriptase
  • inhibitors of all three types are approved by the FDA as anti-HIV drugs (1).
  • the major difficulty with the currently used anti-HIV therapy is the high mutation rate that the virus undergoes which results in the emergence of drug-resistant virus strains.
  • it is essential to identify new drug targets that are essential for virus propagation and to develop new approaches for designing drugs against these targets.
  • the integrase protein (IN) of HIV-I is the only viral enzyme against which there is no approved drug.
  • IN is a 32-kDa protein, which catalyzes the integration of the reverse transcribed viral DNA into the host genome.
  • the integration proceeds in two steps: (1) 3' processing, in which IN removes a pGT di-nucleotide from the 3' end of each viral long terminal repeat (LTR) (2), which occurs after reverse transcription in the cytoplasm. Strand transfer, a concerted nucleophilic attack by the 3'-hydroxyl residues of the viral DNA on the phosphodiester bridges located on either side of the major groove in the target DNA, which takes places in the nucleus.
  • LTR viral long terminal repeat
  • IN exists in solution in equilibrium between monomers, dimers and tetramers (3-6). High-order oligomers of IN were also observed, and detergents such as CHAPS induced dissociation of these oligomers (6). DNA binding also induced dissociation of the multimeric form of HIV-I integrase to monomer-dimer equilibrium (3). The physiological role of the high-order oligomers is yet unknown. Different oligomeric states of IN were observed at various stages of its activity and in different cellular compartments. A dimeric IN is required to bind at each end of the viral DNA during the 3' processing in the cytoplasm (7).
  • a dimer is unlikely to catalyze the strand transfer step since it is too small to mediate concerted integration of both Viral DNA ends (8).
  • a tetrameric IN is required for strand transfer activity (9).
  • IN tetramer bound to the cellular protein LEDGF/p75 was able to catalyze the full-site integration of the two viral LTR ends into a target DNA in vitro.
  • the isolated dimeric form of the enzyme is involved in the processing and integration of only one viral DNA terminus (3). When two DNA-bound dimers approach one another, they form a tetramer and integration proceeds.
  • IN catalyzes the 3' processing as a dimer in the cytoplasm, and mediates the strand transfer as a tetramer in the nucleus.
  • LEDGF/p75 acts in vivo as a tethering factor of IN, which tethers IN to the chromosomes (10-11).
  • LEDGF/p75 is an abundant DNA-binding nuclear protein that binds IN and enhances its in-vitro catalytic activity (12-14).
  • LEDGF/p75 has an evolutionarily conserved IN-binding domain (IBD) of 80 amino acids (residues 347—429). Recently, the crystal structure of the dimeric catalytic core domain of HIV-I IN complexes with the IBD of LEDGF/p75 was solved (15).
  • Recombinant LEDGF/p75 enhanced the strand-transfer activity of the recombinant HIV-I IN in vitro in a mini HIV-based IN assay (16).
  • Cross-linking experiments revealed that IN binds LEDGF as a tetramer in the nucleus (3).
  • the effect of LEDGF/p75 on IN activity depends on the concentration of LEDGF/p75 used. Low concentrations of LEDGF/p75 were found to stimulate the DNA-binding of HIV-I IN to DNA in fluorescent correlation spectroscopy experiments (17).
  • IN is a highly-specific drug target because of its essential role in the HIV-I proliferation (19) and since it has no mammalian homologues (20). Attempts to develop IN inhibitors to date thus far have been aimed primarily at inhibiting the 3' processing and strand transfer catalytic activities of IN. To date, the best IN inhibitors suitable for clinical evaluation are the strand transfer inhibitors DKAs and their derivatives such as the naphthyridine compounds (e.g., MK-0518 STI) and the dihydroquinoline carboxylic acid compounds (GS-9137, STI) (1).
  • the naphthyridine compounds e.g., MK-0518 STI
  • GS-9137, STI dihydroquinoline carboxylic acid compounds
  • the present invention provides isolated peptides comprising a fragment of a LEDGF/p75 protein and use of same for treating HIV-I infection, inhibiting HIV-I replication and inhibiting DNA binding and 3 '-end processing activity of HIV-I integrase protein.
  • the present invention provides an isolated fragment of a LEDGF/p75 protein, wherein the fragment of a LEDGF/p75 protein is 6-25 amino acids in length, and the fragment of a LEDGF/p75 protein comprises the sequence NSLKIDNLDV (SEQ ID NO: 1).
  • the present invention provides an isolated fragment of a LEDGF/p75 protein, wherein the fragment of a LEDGF/p75 protein is 6-25 amino acids in length, and the fragment of a LEDGF/p75 protein comprises a portion of the sequence NSLKIDNLDV (SEQ ID NO: 1), wherein the portion is 6-9 amino acids in length.
  • the present invention provides an isolated mutated fragment of a LEDGF/p75 protein, wherein (a) the fragment of a LEDGF/p75 protein is 6-25 amino acids in length; (b) the sequence of the fragment of a LEDGF/p75 protein comprises the sequence NSLKIDNLDV (SEQ ID NO: 1); and (c) the mutated fragment of a LEDGF/p75 protein comprises 1-2 amino acid modifications relative to SEQ ID NO: 1.
  • the single amino acid modifications are independently selected from the group consisting of a substitution, an insertion, and a deletion. Each possibility represents a separate embodiment of the present invention.
  • the present invention provides an isolated fragment of a LEDGF/p75 protein, wherein the fragment of a LEDGF/p75 protein is 6-25 amino acids in length, and the sequence of the fragment of a LEDGF/p75 protein comprises the sequence KKIRRFKVSQVIM (SEQ ID NO: 2).
  • the present invention provides an isolated fragment of a LEDGF/p75 protein, wherein the fragment of a LEDGF/p75 protein is 6-25 amino acids in length, and the sequence of the fragment of a LEDGF/p75 protein comprises a portion of the sequence KKIRRFKVSQVIM (SEQ ID NO: 2), wherein the portion is 6- 12 amino acids in length.
  • the present invention provides an isolated mutated fragment of a LEDGF/p75 protein, wherein (a) the fragment of a LEDGF/p75 protein is 6-25 amino acids in length; (b) the sequence of the fragment of a LEDGF/p75 protein comprises the sequence KKIRRFKVSQVIM (SEQ ID NO: 2); and (c) the mutated fragment of a LEDGF/p75 protein comprises 1-2 amino acid modifications relative to SEQ ID NO: 2.
  • the single amino acid modifications are independently selected from the group consisting of a substitution, an insertion, and a deletion. Each possibility represents a separate embodiment of the present invention.
  • the present invention provides an isolated fragment of a LEDGF/p75 protein, wherein the fragment of a LEDGF/p75 protein is 11-25 amino acids in length, and the sequence of the fragment of a LEDGF/p75 protein comprises the sequence IHAEIKNSLKIDNLD VNRCIEALD (SEQ ID NO: 3).
  • the present invention provides an isolated fragment of a LEDGF/p75 protein, wherein the fragment of a LEDGF/p75 protein is 11-25 amino acids in length, and the sequence of the fragment of a LEDGF/p75 protein comprises a portion of the sequence IHAEIKNSLKIDNLDVNRCIEALD (SEQ ID NO: 3), wherein the portion is 11-23 amino acids in length.
  • the present invention provides an isolated mutated fragment of a LEDGF/p75 protein, wherein (a) the fragment of a LEDGF/p75 protein is 11-25 amino acids in length; (b) the sequence of the fragment of a LEDGF/p75 protein comprises the sequence IHAEIKNSLKIDNLDVNRCIEALD (SEQ ID NO: 3); and (c) the mutated fragment of a LEDGF/p75 protein comprises 1-2 amino acid modifications relative to SEQ ID NO: 3.
  • the single amino acid modifications are independently selected from the group consisting of a substitution, an insertion, and a deletion. Each possibility represents a separate embodiment of the present invention.
  • the present invention provides an isolated 12-mer peptide, wherein the sequence of the 12-mer peptide is KKIRRFVSQVIM (SEQ ID NO: 41).
  • the present invention provides an isolated peptide comprising an isolated fragment of a LEDGF/p75 protein of the present invention.
  • the isolated peptide is 7-100 amino acids in length.
  • the isolated peptide is 8-100 amino acids in length.
  • the isolated peptide is 9- 100 amino acids in length.
  • the isolated peptide is 10-100 amino acids in length.
  • the isolated peptide is 11-100 amino acids in length.
  • the isolated peptide is 12-100 amino acids in length.
  • the present invention provides an isolated peptide comprising an isolated mutated fragment of a LEDGF/p75 protein of the present invention.
  • the isolated peptide is 7-100 amino acids in length. In another embodiment, the isolated peptide is 8-100 amino acids in length. In another embodiment, the isolated peptide is 9-100 amino acids in length. In another embodiment, the isolated peptide is
  • the isolated peptide is 11-100 amino acids in length. In another embodiment, the isolated peptide is 12-100 amino acids in length. Each possibility represents a separate embodiment of the present invention.
  • the present invention provides an isolated peptide comprising a 12-mer peptide of the present invention.
  • the isolated peptide is 13-100 amino acids in length.
  • the present invention provides a pharmaceutical composition, comprising an isolated peptide or LEDGF/p75 fragment of the present invention and a carrier, diluent, or additive.
  • the present invention provides a pharmaceutical composition, comprising: a plurality of peptides of the present invention and a pharmaceutically acceptable carrier, diluent, or additive.
  • a pharmaceutically acceptable carrier diluent, or additive.
  • one of the peptides is a major IN-binding loop-derived peptide and another is a minor IN-binding loop-derived peptide.
  • the present invention provides a pharmaceutical composition of the present invention for inhibiting replication of an HIV-I in a target cell, treating HIV-I infection in a subject in need thereof, inhibiting binding of an HIV-I integrase protein to an HIV-I long terminal repeat DNA terminus, or inhibiting 3 '-end processing of an HIV-I integrase protein.
  • the present invention provides use of a peptide of the present invention in the preparation of a medicament for inhibiting replication of an HIV-I in a target cell, treating HIV-I infection in a subject in need thereof, inhibiting binding of an HIV-I integrase protein to an HIV-I long terminal repeat DNA terminus, or inhibiting 3 '-end processing of an HIV-I integrase protein.
  • the present invention provides methods of inhibiting replication of an HIV-I in a target cell, treating HIV-I infection in a subject in need thereof, inhibiting binding of an HIV-I integrase protein to an HIV-I long terminal repeat DNA terminus, or inhibiting 3 '-end processing of an HIV-I integrase protein, comprising administering a peptide of the present invention to the target cell.
  • peptides of the present invention possess a superior ability to inhibit HIV-I replication and HIV-I viral integrase 3 '-end processing activity, both in vitro and in cells. Further, as provided herein, other embodiments of peptides of the present invention inhibited integration of viral DNA and HIV-I replication in cell culture by 2 orders of magnitude. Thus, peptides of the present invention possess a number of superior properties relative to previously known methods of combating HIV-I infection. In some embodiments, the HIV-I replication-inhibiting activity of the peptides of the invention works via the oligomerization equilibrium shifting mechanism as described herein.
  • peptides of the invention can be derivatized, as will be explained below, while still maintaining the oligomerization equilibrium-shifting and HIV-I inhibition properties.
  • the present invention provides a peptide having the sequence: NSLKIDNLDV (SEQ ID NO: 1).
  • the present invention provides a peptide having the sequence: KKIRRFKVSQVIM (SEQ ID NO: 2).
  • the present invention provides a peptide having the sequence: IHAEIKNSLKIDNLDVNRCIEALD (SEQ ID NO: 3).
  • the present invention provides a peptide having the sequence: KKIRRFVSQVIM (SEQ ID NO: 41).
  • the present invention provides a peptide having the sequence: WNSLKIDNLDV (SEQ ID NO: 12).
  • the present invention provides a peptide having the sequence: WKKIRRFKVSQVIM (SEQ ID NO: 14).
  • the present invention provides a peptide having the sequence: WIHAEIKNSLKIDNLDVNRCIEALD (SEQ ID NO: 46).
  • the present invention provides a peptide having the sequence: WKKIRRFVSQVIM (SEQ ID NO: 13).
  • the present invention further provides derivatives of the above peptides.
  • the present invention further concerns a compound comprising the peptide as defined above (e.g. a peptide of SEQ ID NO: 1-3) attached in at least one of its terminals either to an organic, non-peptidic moiety, or to an amino acid sequence, with the proviso that the amino acid sequence is other that the contiguous sequence naturally occurring in the LEDGF/p75 protein.
  • the present invention further concerns pharmaceutical compositions comprising a pharmaceutically acceptable carrier and as an active ingredient at least one of the peptides or the compounds as defined above.
  • the pharmaceutical composition comprises a combination of the two peptides and/or a combination of the derivatives of the peptides and/or fragments as defined herein, or a combination of the compounds comprising the peptides with an addition at the N- or C-terminal.
  • the pharmaceutical compositions are indicated for inhibition of HIV-I replication.
  • compositions of the present invention are indicated for treatment of HIV-I infection.
  • the present invention further concerns a method for the treatment of HIV-I infection, comprising administering to a subject in need of such treatment a therapeutically effective amount of at least one peptide, or at least one compound of the invention as defined above.
  • treatment of HIV infection refers to improvement in at least one clinical parameter associated with the HTV infection compared to non-treated control subjects.
  • Clinical parameters include, but are not limited to, viral load (number of particles in the blood) and depletion of CD4-bearing white blood cells.
  • the improvement may be actual reduction in the viral load, but may also be manifest by impeding the rate of increase of the viral load or impeding physical deterioration and/or side effects associated with AIDS, or AIDS-related syndrome.
  • Figure 1 Design of the LEDGF/p75 derived peptides.
  • A Crystal structure of the complex between LEDGF IBD IN dimer.
  • B Zoom into the interface of the LEDGF-IN complex and the design of the LEDGF derived peptides: LEDGF residues 353-378, LEDGF residues 361-370, LEDGF residues 401-412. Coordinates taken from Protein Data Bank (PDB) ID: 2B4J (Cherepanov et al., 2005).
  • PDB Protein Data Bank
  • C LEDGF/p75-derived peptides used in Examples 1-6.
  • Figure 2 Ligands binding of IN: fluorescence anisotropy studies.
  • LEDGF 361- 370 1 ⁇ M (circle), LEDGF 353-378 1 ⁇ M (triangle) and LEDGF 401-412 1 ⁇ M (X). Binding affinities to the DNA in presence of the peptides were between 50 - 200 nM, with coefficients of around 2.2 (See Table 1).
  • D full- length LEDGF/p75 decreases the affinity of IN to the LTR DNA. DNA binding was measured using fluorescence anisotropy. A mixture of LEDGF/p75 (50OnM) and IN (4 ⁇ M) was incubated for 1 h and then titrated into fluorescein-labeled LTR DNA (1OnM). Binding data were fit to the Hill equation and was estimated to be 1 ⁇ M with a Hill coefficient of 2, which is 30 times weaker than the binding in absence of LEGDF/p75.
  • B Binding of LEDGF 361-370, LEDGF 401-412 or the LTR DNA forces the IN out of the high order oligomer to dimeric or tetrameric form. Oligomerization of full-length IN (i.e. residues 1-288) in the presence of various ligands was studied using analytical gel filtration. Samples were IN 14 ⁇ M (left peak), IN 14 ⁇ M + LEDGF 361-370 14 ⁇ M, IN 14 ⁇ M + LEDGF 401-412 14 ⁇ M, IN 14 ⁇ M +
  • LEDGF derived peptides are "shiftides” that shift the oligomerization state of IN towards a tetramer.
  • a proposed mechanism of action for the LEDGF/p75 protein ESf is in equilibrium between its dimeric and tetrameric forms in the cytoplasm. These forms participate in different stages of the integration reaction. The binding of IN to LTR DNA is taking place in the cytoplasm during the 3 '-end processing reaction. The next step is the strand transfer, which taking place in the nucleus.
  • the LEDGF/p75 is predominantly located in the nucleus and we propose that it promotes the strand transfer by binding to IN and shifting its oligomeric state from dimer to tetramer.
  • FIG. 5 The LEDGF peptides inhibit the IN catalytic activities in vitro.
  • IN 0.8 ⁇ M was incubated with the LEDGF derived peptides and the 3 '-end processing and strand transfer enzymatic activities were analyzed as described in Materials and methods.
  • Figure 6 Inhibition of HIV-I replication by LEDGF/p75 derived peptides
  • A-B Cellular uptake of the LEDGF/p75 derived peptides. 10 ⁇ M of fluorescently labeled LEDGF 361-370 (A) or LEDGF 401-412 (B) were incubated for 2 h in 37 0 C with HeLa cells. The cells were then washed three times with phosphate-buffered saline (PBS) and visualized by a fluorescent microscope.
  • PBS phosphate-buffered saline
  • LEDGF-derived peptides inhibit TAR- mediated transcription of HIV-I genes. TZM-Bl cells were incubated with the LEDGF derived peptides at the indicated concentrations and tested for ⁇ -galactosidase activity.
  • LEDGF-derived peptides inhibit HIV-I replication in cell culture. T-lymphoid cells were incubated with the indicated peptides and the total content of new virions was estimated based on the P24 protein content.
  • E-F Inhibition of integration by the LEDGF peptides in cells. Depicted are real-time PCR after incubation with LEDGF 361-370 (E) and LEDGF 401-412 (F). Results are depicted as percent of integrated viral DNA.
  • Figure 7 Fluorescence anisotropy binding studies, (a) LEDGF 361-370 Ala scan peptides (b) LEDGF 401-412 Ala scan peptides. Data were fit to the Hill equation. Binding affinities and Hill coefficients are depicted in Table 2.
  • the present invention provides isolated peptides comprising a fragment of a LEDGF/p75 protein and use of same for treating HIV-I infection, inhibiting HIV-I replication and inhibiting DNA binding and 3 '-end processing activity of HIV-I integrase protein.
  • the present invention provides an isolated fragment of a LEDGF/p75 protein, wherein the fragment of a LEDGF/p75 protein is 6-25 amino acids in length, and the fragment of a LEDGF/p75 protein comprises the sequence NSLKIDNLDV (SEQ ID NO: 1).
  • the present invention provides an isolated fragment of a LEDGF/p75 protein, wherein the fragment of a LEDGF/p75 protein is 6-25 amino acids in length, and the fragment of a LEDGF/p75 protein comprises a portion of the sequence NSLKIDNLDV (SEQ ID NO: 1), wherein the portion is 6-9 amino acids in length.
  • the present invention provides an isolated mutated fragment of a LEDGF/p75 protein, wherein (a) the fragment of a LEDGF/p75 protein is 6-25 amino acids in length; (b) the sequence of the fragment of a LEDGF/p75 protein comprises the sequence NSLKIDNLDV (SEQ ID NO: 1); and (c) the mutated fragment of a LEDGF/p75 protein comprises 1-2 amino acid modifications relative to SEQ ID NO: 1.
  • the single amino acid modifications are independently selected from the group consisting of a substitution, an insertion, and a deletion.
  • the mutated fragment comprises a single amino acid modification relative to SEQ ID NO: 1.
  • the mutated fragment comprises 2 modifications relative to SEQ ID NO: 1.
  • the present invention provides an isolated fragment of a LEDGF/p75 protein, wherein the fragment of a LEDGF/p75 protein is 6-25 amino acids in length, and the sequence of the fragment of a LEDGF/p75 protein comprises the sequence KKIRRFKVSQVIM (SEQ ID NO: 2).
  • the present invention provides an isolated fragment of a LEDGF/p75 protein, wherein the fragment of a LEDGF/p75 protein is 6-25 amino acids in length, and the sequence of the fragment of a LEDGF/p75 protein comprises a portion of the sequence KKIRRFKVSQVIM (SEQ ID NO: 2), wherein the portion is 6- 12 amino acids in length.
  • the present invention provides an isolated mutated fragment of a LEDGF/p75 protein, wherein (a) the fragment of a LEDGF/p75 protein is 6-25 amino acids in length; (b) the sequence of the fragment of a LEDGF/p75 protein comprises the sequence KKIRRFKVSQVIM (SEQ ID NO: 2); and (c) the mutated fragment of a LEDGF/p75 protein comprises 1-2 amino acid modifications relative to SEQ ID NO: 2.
  • the single amino acid modifications are independently selected from the group consisting of a substitution, an insertion, and a deletion.
  • the mutated fragment comprises a single amino acid modification relative to SEQ ID NO: 2.
  • the mutated fragment comprises 2 modifications relative to SEQ ID NO: 2.
  • the present invention provides an isolated fragment of a LEDGF/p75 protein, wherein the fragment of a LEDGF/p75 protein is 11-25 amino acids in length, and the sequence of the fragment of a LEDGF/p75 protein comprises the sequence IHAEIKNSLKIDNLD VNRCIEALD (SEQ ID NO: 3).
  • the present invention provides an isolated fragment of a LEDGF/p75 protein, wherein the fragment of a LEDGF/p75 protein is 11-25 amino acids in length, and the sequence of the fragment of a LEDGF/p75 protein comprises a portion of the sequence IHAEIKNSLKIDNLD VNRCIEALD (SEQ ID NO: 3), wherein the portion is 11-23 amino acids in length.
  • the present invention provides an isolated mutated fragment of a LEDGF/p75 protein, wherein (a) the fragment of a LEDGF/p75 protein is 11-25 amino acids in length; (b) the sequence of the fragment of a LEDGF/p75 protein comprises the sequence IHAEIKNSLKIDNLDVNRCIEALD (SEQ ID NO: 3); and (c) the mutated fragment of a LEDGF/p75 protein comprises 1-2 amino acid modifications relative to SEQ ID NO: 3.
  • the single amino acid modifications are independently selected from the group consisting of a substitution, an insertion, and a deletion.
  • the mutated fragment comprises a single amino acid modification relative to SEQ ID NO: 3.
  • the mutated fragment comprises 2 modifications relative to SEQ ID NO: 3.
  • the present invention provides an isolated 12-mer peptide, wherein the sequence of the 12-mer peptide is KKIRRFVSQVIM (SEQ ID NO: 41).
  • the present invention provides an isolated peptide comprising an isolated fragment of a LEDGF/p75 protein of the present invention.
  • the isolated peptide is 12-100 amino acids in length. Each possibility represents a separate embodiment of the present invention.
  • the present invention provides an isolated peptide comprising an isolated mutated fragment of a LEDGF/p75 protein of the present invention.
  • the isolated peptide is 12-100 amino acids in length. Each possibility represents a separate embodiment of the present invention.
  • Isolated fragment of a LEDGF/p75 protein preferably refers to a LEDGF/p75 fragment that is isolated from contiguous LEDGF/p75 protein sequences. In another embodiment, the term refers to a LEDGF/p75 fragment that is isolated from additional LEDGF/p75 protein sequence other than the recited sequence. The term is not intended to exclude peptides that comprise, in addition to the recited LEDGF/p75 fragment, additional non-LEDGF/p75 amino acid residues, either naturally occurring or non- naturally occurring.
  • the present invention provides a peptide having the sequence: NSLKIDNLDV (SEQ ID NO: 1).
  • the present invention provides a peptide having the sequence: KKIRRFKVSQVIM (SEQ ID NO: 2). In another embodiment, the present invention provides a peptide having the sequence: IHAEIKNSLKIDNLDVNRCIEALD (SEQ ID NO: 3).
  • the present invention provides a peptide having the sequence: KKIRRFVSQVIM (SEQ ID NO: 41).
  • the present invention provides a peptide having the sequence: WNSLKIDNLDV (SEQ ID NO: 12).
  • the present invention provides a peptide having the sequence: WKKIRRFKVSQVIM (SEQ ID NO: 14).
  • the present invention provides a peptide having the sequence: WIHAEIKNSLKIDNLDVNRCIEALD (SEQ ID NO: 46).
  • the present invention provides a peptide having the sequence: WKKIRRFVSQVIM (SEQ ID NO: 13).
  • the present invention provides an isolated peptide comprising a fragment of a LEDGF/p75 protein, wherein the fragment of a LEDGF/p75 protein is 6- 25 amino acids in length, and the sequence of the fragment of a LEDGF/p75 protein comprises the sequence NSLKIDNLDV (SEQ ID NO: 1).
  • the length of the peptide is 6-100 amino acids.
  • the isolated peptide consists of the above-described fragment of a LEDGF/p75 protein.
  • the LEDGF/p75 fragment consists of residues 361-370 from SEQ ID NO: 40 or a corresponding fragment from a homologous protein. Each possibility represents a separate embodiment of the present invention.
  • the present invention provides an isolated peptide comprising a fragment of a LEDGF/p75 protein, wherein the fragment of a LEDGF/p75 protein is 6- 25 amino acids in length, and the fragment of a LEDGF/p75 protein comprises a portion of the sequence NSLKIDNLDV (SEQ ID NO: 1), wherein the portion of SEQ ID NO: 1 is 6-9 amino acids in length.
  • the length of the isolated peptide is 6-100 amino acids.
  • the isolated peptide consists of the above- described fragment of a LEDGF/p75 protein.
  • the LEDGF/p75 fragment consists of a portion of residues 361-370 from SEQ ID NO: 40 or a corresponding fragment from a homologous protein. Each possibility represents a separate embodiment of the present invention.
  • the length of the portion of SEQ ID NO: 1 contained in the fragment of a LEDGF/p75 protein is 7-9 amino acids. In another embodiment, the length is 8-9 AA. In another embodiment, the length is 6-8 AA. Each possibility represents a separate embodiment of the present invention.
  • the present invention provides a peptide comprising a mutated fragment of a LEDGF/p75 protein, wherein (a) the fragment of a LEDGF/p75 protein is 6-25 amino acids in length; (b) the sequence of the fragment of a LEDGF/p75 protein comprises the sequence NSLKIDNLDV (SEQ ID NO: 1); and (c) the mutated fragment of a LEDGF/p75 protein comprises a single amino acid modification relative to SEQ ID NO: 1.
  • the single amino acid modification is preferably selected from the group consisting of a substitution, an insertion, and a deletion.
  • the peptide is an isolated peptide.
  • the peptide consists of the above- described mutated fragment of a LEDGF/p75 protein.
  • the fragment of a LEDGF/p75 protein contains residues other than those set forth in SEQ ID NO: 1.
  • the isolated peptide optionally comprises 1-2 additional single amino acid mutations in the residues other than those set forth in SEQ ID NO: 1.
  • the additional single amino acid mutations are independently selected from the group consisting of a substitution, an insertion, and a deletion. Each possibility represents a separate embodiment of the present invention.
  • the present invention provides an isolated peptide comprising a fragment of a LEDGF/p75 protein, wherein the fragment of a LEDGF/p75 protein is 6- 25 amino acids in length, and the sequence of the fragment of a LEDGF/p75 protein comprises the sequence KKIRRFKVSQVIM (SEQ ID NO: 2).
  • the length of the peptide is 6-100 amino acids.
  • the isolated peptide consists of the above-described fragment of a LEDGF/p75 protein.
  • the LEDGF/p75 fragment consists of residues 401-412 from SEQ ID NO: 40 or a corresponding fragment from a homologous protein. Each possibility represents a separate embodiment of the present invention.
  • the present invention provides an isolated peptide comprising a fragment of a LEDGF/p75 protein, wherein the fragment of a LEDGF/p75 protein is 6- 25 amino acids in length, and the fragment of a LEDGF/p75 protein comprises a portion of the sequence KKIRRFKVSQVIM (SEQ ID NO: 2), wherein the portion of SEQ ID NO: 2 is 6-12 amino acids in length.
  • the length of the peptide is 6-100 amino acids.
  • the isolated peptide consists of the above-described fragment of a LEDGF/p75 protein.
  • the LEDGF/p75 fragment consists of a portion of residues 401-412 from SEQ ID NO: 40 or a corresponding fragment from a homologous protein. Each possibility represents a separate embodiment of the present invention.
  • the length of the portion of SEQ ID NO: 2 contained in the fragment of a LEDGF/p75 protein is 7-12 AA. In another embodiment, the length is 8- 12 AA. In another embodiment, the length is 9-12 AA. In another embodiment, the length is 10-12 AA. In another embodiment, the length is 11-12 AA. In another embodiment, the length is 6-11 AA. In another embodiment, the length is 7-11 AA. In another embodiment, the length is 8-11 AA. In another embodiment, the length is 9-11 AA. In another embodiment, the length is 10-11 AA. In another embodiment, the length is 6-10 AA. In another embodiment, the length is 7-10 AA. In another embodiment, the length is 8-10 AA. In another embodiment, the length is 9-10 AA.
  • the length is 6-9 AA. In another embodiment, the length is 7-9 AA. In another embodiment, the length is 8-9 AA. In another embodiment, the length is 6-8 AA. In another embodiment, the length is 7-8 AA. In another embodiment, the length is 6-7 AA. Each possibility represents a separate embodiment of the present invention.
  • the present invention provides a peptide comprising a mutated fragment of a LEDGF/p75 protein, wherein (a) the fragment of a LEDGF/p75 protein is
  • the sequence of the fragment of a LEDGF/p75 protein comprises the sequence KKIRRFKVSQVIM (SEQ ID NO: 2); and (c) the mutated fragment of a LEDGF/p75 protein comprises a single amino acid modification relative to SEQ ID NO: 2.
  • the single amino acid modification is preferably selected from the group consisting of a substitution, an insertion, and a deletion.
  • the peptide is an isolated peptide.
  • the peptide consists of the above-described mutated fragment of a LEDGF/p75 protein.
  • the fragment of a LEDGF/p75 protein contains residues other than those set forth in SEQ ID NO: 2.
  • the isolated peptide optionally comprises 1-2 additional single amino acid mutations in the residues other than those set forth in SEQ ID NO: 2.
  • the additional single amino acid mutations are independently selected from the group consisting of a substitution, an insertion, and a deletion. Each possibility represents a separate embodiment of the present invention.
  • the length of the fragment of a LEDGF/p75 protein contained in peptides of the present invention is, in another embodiment, 7-25 amino acids (AA).
  • the length of the LEDGF/p75 fragment is 8-25 AA.
  • the length is 9-25 AA.
  • the length is 10-25 AA.
  • the length is 11-25 AA.
  • the length is 12- 25 AA.
  • the length is 13-25 AA.
  • the length is 14-25 AA.
  • the length is 15-25 AA.
  • the length is 6-24 AA.
  • the length is 6-23 AA. In another embodiment, the length is 6-22 AA. In another embodiment, the length is 6-21 AA. In another embodiment, the length is 6-20 AA. In another embodiment, the length is 6-19 AA. In another embodiment, the length is 6-18 AA. In another embodiment, the length is 6-17 AA. In another embodiment, the length is 6-16 AA. In another embodiment, the length is 6-16 AA. In another embodiment, the length is 6-14 AA. In another embodiment, the length is 6-13 AA. In another embodiment, the length is 6-12 AA. In another embodiment, the length is 7-24 AA. In another embodiment, the length is 7-23 AA. In another embodiment, the length is 7-22 AA.
  • the length is 7-21 AA. In another embodiment, the length is 7-20 AA. In another embodiment, the length is 7-19 AA. In another embodiment, the length is 7-18 AA. In another embodiment, the length is 7-17 AA. In another embodiment, the length is 7-16 AA. In another embodiment, the length is 7-16 AA. In another embodiment, the length is 7-14 AA. In another embodiment, the length is 7-13 AA. In another embodiment, the length is 7-12 AA. In another embodiment, the length is 8-24 AA. In another embodiment, the length is 8-23 AA. In another embodiment, the length is 8-22 AA. In another embodiment, the length is 8-21 AA. In another embodiment, the length is 8-20 AA.
  • the length is 8-19 AA. In another embodiment, the length is 8-18 AA. In another embodiment, the length is 8-17 AA. In another embodiment, the length is 8-16 AA. In another embodiment, the length is 8-16 AA. In another embodiment, the length is 8-14 AA. In another embodiment, the length is 8-13 AA. In another embodiment, the length is 8-12 AA. In another embodiment, the length is 9-24 AA. In another embodiment, the length is 9-23 AA. In another embodiment, the length is 9-22 AA. In another embodiment, the length is 9-21 AA. In another embodiment, the length is 9-20 AA. In another embodiment, the length is 9-19 AA. In another embodiment, the length is 9-18 AA.
  • the length is 9-17 AA. In another embodiment, the length is 9-16 AA. In another embodiment, the length is 9-16 AA. In another embodiment, the length is 9-14 AA. In another embodiment, the length is 9-13 AA. In another embodiment, the length is 9-12 AA. In another embodiment, the length is 10-24 AA. In another embodiment, the length is 10-23 AA. In another embodiment, the length is 10-22 AA. In another embodiment, the length is 10-21 AA. In another embodiment, the length is 10-20 AA. In another embodiment, the length is 10-19 AA. In another embodiment, the length is 10-18 AA. In another embodiment, the length is 10-17 AA. In another embodiment, the length is 10-16 AA. In another embodiment, the length is 10-16 AA. In another embodiment, the length is 10-14 AA. In another embodiment, the length is 10-13 AA. In another embodiment, the length is 10-12 AA. Each possibility represents a separate embodiment of the present invention.
  • a "length" in terms of a number of “amino acids” or "AA” preferably refers to peptide or peptidomimetic containing the number of total AA specified, including both naturally occurring AA and AA modified in any manner disclosed herein.
  • the term refers only to the number of L-amino acids (i.e. AA having the naturally occurring stereo configuration) in the peptide or peptidomimetic.
  • the term refers only to the number of unmodified AA in the peptide or peptidomimetic.
  • the term refers only to the number of AA in the peptide or peptidomimetic wherein the side chain is unmodified (i.e. AA preceded by or following a modified peptide bond would be included in the count).
  • an isolated peptide of methods and compositions of the present invention e.g. an isolated peptide comprising a native or mutated version of SEQ ID NO: 1, 2, or 3
  • the length of an isolated peptide of methods and compositions of the present invention is, in another embodiment, 7-100 AA. In another embodiment, the length is 8-100
  • the length is 9-100 AA. In another embodiment, the length is 10-100 AA. In another embodiment, the length is 11-100 AA. In another embodiment, the length is 12-100 AA. In another embodiment, the length is 13-100 AA. In another embodiment, the length is 15-100 AA. In another embodiment, the length is 17-100 AA. In another embodiment, the length is 20-100 AA. In another embodiment, the length is 6-90 AA. In another embodiment, the length is 7-90 AA. In another embodiment, the length is 8-90 AA. In another embodiment, the length is 9-90 AA. In another embodiment, the length is 10-90 AA. In another embodiment, the length is 11- 90 AA.
  • the length is 12-90 AA. In another embodiment, the length is 13-90 AA. In another embodiment, the length is 15-90 AA. In another embodiment, the length is 17-90 AA. In another embodiment, the length is 20-90 AA. In another embodiment, the length is 6-80 AA. In another embodiment, the length is 7- 80 AA. In another embodiment, the length is 8-80 AA. In another embodiment, the length is 9-80 AA. In another embodiment, the length is 10-80 AA. In another embodiment, the length is 11-80 AA. In another embodiment, the length is 12-80 AA. In another embodiment, the length is 13-80 AA. In another embodiment, the length is 15-80 AA. In another embodiment, the length is 17-80 AA.
  • the length is 20-80 AA. In another embodiment, the length is 6-70 AA. In another embodiment, the length is 7-70 AA. In another embodiment, the length is 8-70 AA. In another embodiment, the length is 9-70 AA. In another embodiment, the length is 10-70 AA. In another embodiment, the length is 11-70 AA. In another embodiment, the length is 12-70 AA. In another embodiment, the length is 13-70 AA. In another embodiment, the length is 15-70 AA. In another embodiment, the length is 17-70 AA. In another embodiment, the length is 20-70 AA. In another embodiment, the length is 6-90 AA. In another embodiment, the length is 7-90 AA. In another embodiment, the length is 8-60 AA.
  • the length is 9-60 AA. In another embodiment, the length is 10-60 AA. In another embodiment, the length is 11-60 AA. In another embodiment, the length is 12-60 AA. In another embodiment, the length is 13-60 AA. In another embodiment, the length is 15-60 AA. In another embodiment, the length is 17-60 AA. In another embodiment, the length is 20-60 AA. In another embodiment, the length is 6- 50 AA. In another embodiment, the length is 7-50 AA. In another embodiment, the length is 8-50 AA. In another embodiment, the length is 9-50 AA. In another embodiment, the length is 10-50 AA. In another embodiment, the length is 11-50 AA. In another embodiment, the length is 12-50 AA. In another embodiment, the length is
  • the length is 15-50 AA. In another embodiment, the length is 17-50 AA. In another embodiment, the length is 20-50 AA. In another embodiment, the length is 6-40 AA. In another embodiment, the length is 7-40 AA. In another embodiment, the length is 8-40 AA. In another embodiment, the length is 9-40 AA. In another embodiment, the length is 10-40 AA. In another embodiment, the length is 11-40 AA. In another embodiment, the length is 12-40 AA. In another embodiment, the length is 13-40 AA. In another embodiment, the length is 15-40 AA. In another embodiment, the length is 17-40 AA. Each possibility represents a separate embodiment of the present invention.
  • the present invention provides an isolated peptide comprising a fragment of a LEDGF/p75 protein, wherein the fragment of a LEDGF/p75 protein is 11 -
  • the sequence of the fragment of a LEDGF/p75 protein comprises the sequence IHAEIKNSLKIDNLDVNRCIEALD (SEQ ID NO: 3).
  • the length of the peptide is 6-100 amino acids.
  • the isolated peptide consists of the above-described fragment of a LEDGF/p75 protein.
  • the LEDGF/p75 fragment consists of residues 353-378 from SEQ ID NO: 1
  • ID NO: 40 or a corresponding fragment from a homologous protein. Each possibility represents a separate embodiment of the present invention.
  • the present invention provides an isolated peptide comprising a fragment of a LEDGF/p75 protein, wherein the fragment of a LEDGF/p75 protein is 11- 25 amino acids (AA) in length, and the fragment of a LEDGF/p75 protein comprises a portion of the sequence IHAEIKNSLKIDNLDVNRCIEALD (SEQ ID NO: 3), wherein the portion of SEQ ID NO: 3 is 11-23 AA in length.
  • the isolated peptide consists of the above-described fragment of a LEDGF/p75 protein.
  • the length of the peptide is 6-100 AA.
  • the isolated peptide consists of the above-described fragment of a LEDGF/p75 protein.
  • the LEDGF/p75 fragment consists of a portion of residues 353-378 from SEQ ID NO: 40 or a corresponding fragment from a homologous protein.
  • the length of the portion of SEQ ID NO: 3 contained in the fragment of a LEDGF/p75 protein is 12-23 AA. In another embodiment, the length is
  • the length is 14-23 AA. In another embodiment, the length is 15-23 AA. In another embodiment, the length is 11-22 AA. In another embodiment, the length is 12-22 AA. In another embodiment, the length is 13-22 AA. In another embodiment, the length is 14-22 AA. In another embodiment, the length is 15-22 AA. In another embodiment, the length is 11-21 AA. In another embodiment, the length is 12-21 AA. In another embodiment, the length is 13-21 AA. In another embodiment, the length is 14-21 AA. In another embodiment, the length is 15-21 AA. In another embodiment, the length is 11-20 AA. In another embodiment, the length is 12-20 AA. In another embodiment, the length is 13-20 AA.
  • the length is 14-20 AA. In another embodiment, the length is 15-20 AA. In another embodiment, the length is 11-18 AA. In another embodiment, the length is 12-18 AA. In another embodiment, the length is 13-18 AA. In another embodiment, the length is 14-18 AA. In another embodiment, the length is 15-18 AA. In another embodiment, the length is 11-16 AA. In another embodiment, the length is 12-16 AA. In another embodiment, the length is 13-16 AA. In another embodiment, the length is 14-16 AA. In another embodiment, the length is 15-16 AA. Each possibility represents a separate embodiment of the present invention.
  • those mutations and fragments of SEQ ID NO: 1, 2, 3, and 41 are those that exhibit substantially similar activity to the peptide from which it was derived (e.g. SEQ ID NO: 1, 2, 3, and 41) in one or more of the following: inhibiting binding of an HIV-I integrase protein to an HIV-I long terminal repeat DNA terminus, inhibiting replication of an HIV-I in a target cell, treating HIV-I infection in a subject in need thereof, or inhibiting 3 '-end processing of an HIV-I integrase protein
  • the present invention provides a peptide comprising a mutated fragment of a LEDGF/p75 protein, wherein (a) the fragment of a LEDGF/p75 protein is 11-25 amino acids (AA) in length; (b) the fragment of a LEDGF/p75 protein comprises the sequence IHAEIKNSLKIDNLDVNRCIEALD (SEQ ID NO: 3); and (c) the mutated fragment of a LEDGF/p75 protein comprises a single AA modification relative to SEQ ID NO: 3.
  • the single AA modification is selected from the group consisting of a substitution, an insertion, and a deletion.
  • the peptide is an isolated peptide.
  • the peptide consists of the above-described mutated fragment of a LEDGF/p75 protein.
  • the fragment of a LEDGF/p75 protein contains residues other than those set forth in SEQ ID NO: 3.
  • the isolated peptide optionally comprises 1-2 additional single amino acid mutations in the residues other than those set forth in SEQ ID NO: 3.
  • the additional single amino acid mutations are independently selected from the group consisting of a substitution, an insertion, and a deletion. Each possibility represents a separate embodiment of the present invention.
  • the present invention provides an isolated peptide comprising a 12-mer peptide, wherein the sequence of the 12-mer peptide is KKIRRFVSQVIM (SEQ ID NO: 41).
  • the length of the isolated peptide is 12-100 amino acids.
  • the sequence of the isolated peptide is KKIRRFVSQVIM (SEQ ID NO: 41).
  • 12-mer peptide preferably refers to a peptide or peptidomimetic containing 12 total AA, including both naturally occurring AA and AA modified in any manner disclosed herein.
  • the term refers only to the number of L-amino acids (i.e. AA having the naturally occurring stereo configuration) in the peptide or peptidomimetic.
  • the term refers only to the number of unmodified AA in the peptide or peptidomimetic.
  • the term refers only to the number of AA in the peptide or peptidomimetic wherein the side chain is unmodified (i.e. AA preceded by or following a modified peptide bond would be included in the count).
  • Each possibility represents a separate embodiment of the present invention.
  • the length of an isolated peptide of methods and compositions of the present invention is, in another embodiment, 13-100 AA. In another embodiment, the length is 14-100 AA. In another embodiment, the length is 15-100 AA. In another embodiment, the length is 20-100 AA. In another embodiment, the length is 25-100 AA. In another embodiment, the length is 30-100 AA. In another embodiment, the length is 40-100 AA. In another embodiment, the length is 50-100 AA. In another embodiment, the length is 12-90 AA. In another embodiment, the length is 13-90 AA. In another embodiment, the length is 14-90 AA.
  • the length is 15-90 AA. In another embodiment, the length is 20-90 AA. In another embodiment, the length is 25-90 AA. In another embodiment, the length is 30-90 AA. In another embodiment, the length is 40-90 AA. In another embodiment, the length is 50-80 AA. In another embodiment, the length is 12-80 AA. In another embodiment, the length is 13-80 AA. In another embodiment, the length is 14-80 AA. In another embodiment, the length is 15-80 AA. In another embodiment, the length is 20-80 AA. In another embodiment, the length is 25-80 AA. In another embodiment, the length is 30-80 AA. In another embodiment, the length is 40-80 AA. In another embodiment, the length is 50-80 AA.
  • the length is 12-70 AA. In another embodiment, the length is 13-70 AA. In another embodiment, the length is 14-70 AA. In another embodiment, the length is 15-70 AA. In another embodiment, the length is 20-70 AA. In another embodiment, the length is 25-70 AA. In another embodiment, the length is 30-70 AA. In another embodiment, the length is 40-70 AA. In another embodiment, the length is 50-70 AA. In another embodiment, the length is 12-90 AA. In another embodiment, the length is 13-60 AA. In another embodiment, the length is 14-60 AA. In another embodiment, the length is 15-60 AA. In another embodiment, the length is 20-60 AA. In another embodiment, the length is 25-60 AA. In another embodiment, the length is 30-60 AA. In another embodiment, the length is 40-60 AA. In another embodiment, the length is 50-60 AA. Each possibility represents a separate embodiment of the present invention.
  • an isolated peptide of methods and compositions of the present invention further comprises a tryptophan residue adjacent to the amino-terminal end of the LEDGF/p75 protein fragment. In another embodiment, an isolated peptide of methods and compositions of the present invention further comprises a tryptophan residue adjacent to the amino-terminal end of the mutated LEDGF/p75 protein fragment. Preferably, the tryptophan residue is the amino-terminal residue of the isolated peptide.
  • a “mutation" of methods and compositions of the present invention is, in another embodiment, a substitution.
  • the mutation is an insertion.
  • the mutation is a deletion.
  • the mutation is an internal deletion.
  • the mutation is a truncation.
  • the term "mutation” refers to an alteration or modification in the sequence of either a peptide or a nucleotide molecule encoding same.
  • a peptide of methods and compositions of the present invention comprises multiple AA mutations, in some cases multiple AA mutations relative to the fragment of a LEDGF/p75 protein.
  • peptide refers to either a peptide or a peptidomimetic.
  • peptidomimetic refers to a moiety derived from a peptide and having any of the modifications described herein, either singly or in combination. Each possibility represents a separate embodiment of the present invention.
  • the isolated peptide or fragment of a LEDGF/p75 protein of methods and compositions of the present invention binds, in a physiological solution, a tetramer of an HIV-I integrase protein with a greater affinity than the isolated peptide or fragment binds a dimer of the HIV-I integrase protein, thereby increasing the ratio of the tetramer to the dimer in the physiological solution.
  • the isolated peptide or fragment binds a tetramer of an HIV-I integrase protein under physiological conditions with a greater affinity than the isolated peptide or fragment binds a dimer of the HIV-I integrase protein under the same conditions.
  • Each possibility represents a separate embodiment of the present invention.
  • the isolated peptide or fragment of a LEDGF/p75 protein of methods and compositions of the present invention is capable of inhibiting 3 '-end processing activity of an HIV-I integrase protein.
  • the inhibition is measured in a physiological solution.
  • Methods for measuring the 3 '-end processing activity of HIV IN are well known in the art, and include the methods disclosed herein (see, inter alia, the section entitled “In- vitro 3 '-end processing and strand transfer assays” hereinbelow) and methods known in the art, e.g. those described in Craigie (1991) Nucleic Acid Res. 19:2729-34; and Hwang (2000) Nucleic acid Res. 28:4884-92. Each method represents a separate embodiment of the present invention.
  • the isolated peptide or fragment of a LEDGF/p75 protein of methods and compositions of the present invention is capable of inhibiting binding of an HIV-I integrase protein to an HIV-I long terminal repeat (LTR) DNA terminus.
  • the inhibition is measured in a physiological solution.
  • “Inhibiting binding” refers, preferably, to ability to inhibit IN binding to LTR DNA with an IC 50 of 1 nM (nanomolar)-5 mcM (micromolar). In another embodiment, the term refers to ability to inhibit binding with an IC 50 of 1 nM-4 mcM. In another embodiment, the term refers to an IC 50 range of 1 nM-10 mcM. In another embodiment, the term refers to an IC 50 range of 1 nM-8 mcM. In another embodiment, the term refers to an IC 5O range of 1 nM-3 mcM. In another embodiment, the term refers to an IC 50 range of 1 nM-2.5 mcM.
  • the term refers to an IC 50 range of 1 nM-2 mcM. In another embodiment, the term refers to an IC 50 range of 1 nM-1.5 mcM. In another embodiment, the term refers to an IC 50 range of 1 nM-1 mcM. In another embodiment, the term refers to an IC 5O range of 1-700 nM. In another embodiment, the term refers to an IC 50 range of 1-500 nM. In another embodiment, the term refers to an IC 50 range of 1-300 nM. In another embodiment, the term refers to an IC 50 range of 1- 200 nM. In another embodiment, the term refers to an IC 5O range of 1-100 nM.
  • the term refers to an IC 5O range of 1-70 nM. In another embodiment, the term refers to an IC 50 range of 1-50 nM. In another embodiment, the term refers to an IC 50 range of 1 nM-30 nM. In another embodiment, the term refers to an IC 50 range of 2 nM-10 mcM. In another embodiment, the term refers to an IC 5O range of 2 nM-7 mcM. In another embodiment, the term refers to an IC 5O range of 2 nM-5 mcM. In another embodiment, the term refers to an IC 5O range of 2 nM-3 mcM.
  • the term refers to an IC 50 range of 2 nM-2.5 mcM. In another embodiment, the term refers to an IC 50 range of 2 nM-2 mcM. In another embodiment, the term refers to an IC 50 range of 2 nM-1.5 mcM. In another embodiment, the term refers to an IC 50 range of 2 nM-1 mcM. In another embodiment, the term refers to an IC 5O range of 2-700 nM. In another embodiment, the term refers to an IC 50 range of 2- 500 nM. In another embodiment, the term refers to an IC 50 range of 2-300 nM. In another embodiment, the term refers to an IC 5O range of 2-200 nM.
  • the term refers to an IC 50 range of 2-100 nM. In another embodiment, the term refers to an IC 50 range of 2-70 nM. In another embodiment, the term refers to an IC 50 range of 2-50 nM. In another embodiment, the term refers to an IC 50 range of 2-30 nM. In another embodiment, the term refers to an IC 50 range of 3nM-10mcM. In another embodiment, the term refers to an IC 50 range of 3nM-7mcM. In another embodiment, the term refers to an IC 5O range of 3nM-5mcM. In another embodiment, the term refers to an IC 50 range of 3 nM-3mcM.
  • the term refers to an IC 50 range of 3nM-2.5mcM. In another embodiment, the term refers to an IC 50 range of 3 nM-2mcM. In another embodiment, the term refers to an IC 50 range of 3nM-lmcM. In another embodiment, the term refers to an IC 50 range of 3-700 nM. In another embodiment, the term refers to an IC 5O range of 3-500 nM. In another embodiment, the term refers to an IC 50 range of 3-300 nM. In another embodiment, the term refers to an IC 50 range of 3-200 nM. In another embodiment, the term refers to an IC 50 range of 3- 100 nM.
  • the term refers to an IC 50 range of 3-70 nM. In another embodiment, the term refers to an IC 50 range of 3-50 nM. In another embodiment, the term refers to an IC 5O range of 5nM-10mcM. In another embodiment, the term refers to an IC 50 range of 5nM-7mcM. In another embodiment, the term refers to an IC 5O range of 5nM-5mcM. In another embodiment, the term refers to an IC 50 range of 5nM-3mcM. In another embodiment, the term refers to an IC 50 range of 5nM-2mcM. In another embodiment, the term refers to an IC 50 range of 5nM-lmcM.
  • the term refers to an IC 50 range of 5-700 nM. In another embodiment, the term refers to an IC 50 range of 5-500 nM. In another embodiment, the term refers to an IC 50 range of 5-300 nM. In another embodiment, the term refers to an IC 50 range of 5-200 nM. In another embodiment, the term refers to an IC 50 range of 5-100 nM. In another embodiment, the term refers to an IC 50 range of 5-50 nM. In another embodiment, the term refers to an IC 50 range of 5-30 nM. In another embodiment, the term refers to an IC 50 range of IOnM-lOmcM.
  • the term refers to an IC 5O range of 10nM-7mcM. In another embodiment, the term refers to an IC 50 range of 10nM-5mcM. In another embodiment, the term refers to an IC 50 range of 10nM-3mcM. In another embodiment, the term refers to an IC 50 range of 10nM-2mcM. In another embodiment, the term refers to an IC 5O range of IOnM-lmcM. In another embodiment, the term refers to an IC 50 range of 10-700 nM. In another embodiment, the term refers to an IC 50 range of 10-500 nM. In another embodiment, the term refers to an IC 50 range of 10-300 nM.
  • the term refers to an ICs 0 range of 10-200 nM. In another embodiment, the term refers to an IC 50 range of 10-100 nM. In another embodiment, the term refers to an IC 5O range of 10-70 nM. In another embodiment, the term refers to an IC 50 range of 10-50 nM. In another embodiment, the term refers to an IC 50 range of 10- 30 nM. In another embodiment, the term refers to an IC 5O range of 20nM-10mcM. In another embodiment, the term refers to an IC 50 range of 20nM-7mcM. In another embodiment, the term refers to an IC 50 range of 20nM-5mcM.
  • the term refers to an IC 50 range of 20nM-3mcM. In another embodiment, the term refers to an IC 50 range of 20nM-2mcM. In another embodiment, the term refers to an IC 50 range of 2OnM-I mcM. In another embodiment, the term refers to an IC 50 range of 20- 700 nM. In another embodiment, the term refers to an IC 50 range of 20-500 nM. In another embodiment, the term refers to an ICs 0 range of 20-300 nM. In another embodiment, the term refers to an IC 50 range of 20-200 nM. In another embodiment, the term refers to an IC 5O range of 20-100 nM.
  • the term refers to an IC 50 range of 20-70 nM. In another embodiment, the term refers to an IC 50 range of 20- 50 nM. In another embodiment, the term refers to an IC 5O range of 30nM-10mcM. In another embodiment, the term refers to an IC 50 range of 30nM-7mcM. In another embodiment, the term refers to an IC 50 range of 30nM-5mcM. In another embodiment, the term refers to an IC 50 range of 30nM-3mcM. In another embodiment, the term refers to an IC 50 range of 30nM-2mcM. In another embodiment, the term refers to an ICs 0 range of 3OnM- lmcM.
  • the term refers to an IC 50 range of 30- 700 nM. In another embodiment, the term refers to an IC 50 range of 30-500 nM. In another embodiment, the term refers to an ICs 0 range of 30-300 nM. In another embodiment, the term refers to an IC 5O range of 30-200 nM. In another embodiment, the term refers to an IC 50 range of 30-100 nM. In another embodiment, the term refers to an IC 50 range of 30-70 nM. In another embodiment, the term refers to an IC 50 range of 30- 50 nM. In another embodiment, the term refers to an IC 50 range of 50nM-10mcM.
  • the term refers to an IC 50 range of 50nM-7mcM. In another embodiment, the term refers to an IC 50 range of 50nM-5mcM. In another embodiment, the term refers to an IC 50 range of 50nM-3mcM. In another embodiment, the term refers to an IC 50 range of 50nM-2mcM. In another embodiment, the term refers to an IC 50 range of 50nM-lmcM. In another embodiment, the term refers to an IC 5O range of 50- 700 nM. In another embodiment, the term refers to an IC 50 range of 50-500 nM. In another embodiment, the term refers to an IC 50 range of 50-300 nM.
  • the term refers to an IC 50 range of 50-200 nM. In another embodiment, the term refers to an IC 50 range of 50-100 nM. In another embodiment, the term refers to an IC 50 range of 50-70 nM. In another embodiment, the term refers to an IC 50 range of 70nM-10mcM. In another embodiment, the term refers to an IC 5 0 range of 7OnM- 7mcM. In another embodiment, the term refers to an IC 5 O range of 70nM-5mcM. In another embodiment, the term refers to an IC 50 range of 70nM-3mcM. In another embodiment, the term refers to an IC 50 range of 70nM-2mcM.
  • the term refers to an IC 50 range of 7OnM-I mcM. In another embodiment, the term refers to an IC 50 range of 70-700 nM. In another embodiment, the term refers to an IC 50 range of 70-500 nM. In another embodiment, the term refers to an IC 50 range of 70-300 nM. In another embodiment, the term refers to an IC 50 range of 70-200 nM. In another embodiment, the term refers to an IC 5O range of 70-100 nM. In another embodiment, the term refers to an IC 50 range of lOOnM-lOmcM. In another embodiment, the term refers to an IC 50 range of 100nM-7mcM.
  • the term refers to an IC 50 range of 100nM-5mcM. In another embodiment, the term refers to an IC 50 range of 100nM-3mcM. In another embodiment, the term refers to an IC 50 range of 10OnM- 2mcM. In another embodiment, the term refers to an IC 50 range of lOOnM-lmcM. In another embodiment, the term refers to an IC 50 range of 100-700 nM. In another embodiment, the term refers to an IC 5O range of 100-500 nM. In another embodiment, the term refers to an IC 50 range of 100-300 nM. In another embodiment, the term refers to an IC 50 range of 100-200 nM.
  • the term refers to an IC 5O range of 100-150 nM. In another embodiment, the term refers to an IC 50 range of 15OnM- lOmcM. In another embodiment, the term refers to an IC 50 range of 150nM-7mcM. In another embodiment, the term refers to an IC 50 range of 150nM-5mcM. In another embodiment, the term refers to an IC 50 range of 150nM-3mcM. In another embodiment, the term refers to an IC 50 range of 150nM-2mcM. In another embodiment, the term refers to an IC 50 range of 150nM-lmcM. In another embodiment, the term refers to an IC 5 o range of 150-700 nM.
  • the term refers to an IC 50 range of 150-500 nM. In another embodiment, the term refers to an IC 50 range of 150-300 nM. In another embodiment, the term refers to an IC 50 range of 150-200 nM. In another embodiment, the term refers to an IC 5O range of 200nM-10mcM. In another embodiment, the term refers to an IC 50 range of 200nM-7mcM. In another embodiment, the term refers to an IC 50 range of 200nM-5mcM. In another embodiment, the term refers to an IC 50 range of 200nM-3mcM. In another embodiment, the term refers to an
  • IC 50 range of 200nM-2mcM refers to an IC 50 range of 20OnM- lmcM. In another embodiment, the term refers to an IC 50 range of 200-700 nM. In another embodiment, the term refers to an IC 50 range of 200-500 nM. In another embodiment, the term refers to an IC 50 range of 200-300 nM. In another embodiment, the term refers to an IC 50 range of 30OnM-I OmcM. In another embodiment, the term refers to an IC 5O range of 300nM-7mcM. In another embodiment, the term refers to an IC 5O range of 300nM-5mcM.
  • the term refers to an IC 5O range of 300nM-3mcM. In another embodiment, the term refers to an IC 50 range of 30OnM- 2mcM. In another embodiment, the term refers to an IC 50 range of 30OnM- lmcM. In another embodiment, the term refers to an IC 50 range of 300-700 nM. In another embodiment, the term refers to an IC 5O range of 300-500 nM. In another embodiment, the term refers to an IC 50 range of 50OnM-I OmcM. In another embodiment, the term refers to an IC 50 range of 500nM-7mcM. In another embodiment, the term refers to an IC 50 range of 500nM-5mcM.
  • the term refers to an IC 50 range of 500nM-3mcM. In another embodiment, the term refers to an IC 5O range of 50OnM- 2mcM. In another embodiment, the term refers to an IC 50 range of 50OnM- lmcM. In another embodiment, the term refers to an IC 50 range of 500-700 nM. In another embodiment, the term refers to an IC 50 range of 70OnM-I OmcM. In another embodiment, the term refers to an IC 50 range of 700nM-7mcM. In another embodiment, the term refers to an IC 5O range of 700nM-5mcM. In another embodiment, the term refers to an IC 50 range of 700nM-3mcM.
  • the term refers to an IC 50 range of 700nM-2mcM. In another embodiment, the term refers to an IC 50 range of 70OnM- lmcM. In another embodiment, the term refers to an IC 5O range of 1-lOmcM. In another embodiment, the term refers to an IC 5O range of l-7mcM. In another embodiment, the term refers to an IC 5O range of l-5mcM. In another embodiment, the term refers to an IC 5O range of l-3mcM. In another embodiment, the term refers to an IC 5 o range of l-2mcM. In another embodiment, the term refers to an IC 5O range of 1.5- lOmcM.
  • the term refers to an IC 5 O range of 1.5-7mcM. In another embodiment, the term refers to an IC 50 range of 1.5-5mcM. In another embodiment, the term refers to an IC 50 range of 1.5-3mcM. In another embodiment, the term refers to an IC 50 range of 2-1 OmcM. In another embodiment, the term refers to an IC 5 o range of 2-7mcM. In another embodiment, the term refers to an IC 5O range of 2- 5mcM. In another embodiment, the term refers to an IC 5O range of 2-3mcM. In another embodiment, the term refers to an IC 5O range of 3-1 OmcM.
  • the term refers to an IC 50 range of 3-7mcM. In another embodiment, the term refers to an IC 50 range of 3-5mcM. In another embodiment, the term refers to an IC 50 range of 5- lOmcM. In another embodiment, the term refers to an IC 50 range of 5-7mcM.
  • IC 50 range of 3-7mcM In another embodiment, the term refers to an IC 50 range of 3-5mcM. In another embodiment, the term refers to an IC 50 range of 5- lOmcM. In another embodiment, the term refers to an IC 50 range of 5-7mcM.
  • the term refers to ability to inhibit DNA binding by at least 3- fold, when pre-incubated with IN at a concentration of 500 nM peptide under physiological conditions (e.g. with IN at a concentration of 4 ⁇ M) and DNA at a concentration of 1OnM. In another embodiment, the term refers to ability to inhibit
  • DNA binding by at least 6-fold under the above conditions refers to ability to inhibit DNA binding by at least 2-fold under the above conditions.
  • the term refers to ability to inhibit DNA binding by at least 2-fold under the above conditions.
  • physiological conditions are well known to those skilled in the art, and include, for example, 0.2 M Tris, pH 7.4, with 0.15 M NaCl. Another example of “physiological conditions” is found herein in the section entitled “fluorescence anisotropy.” Those skilled in the art will be readily able to discern physiological conditions appropriate for DNA binding assays, 3 '-end processing activity, etc. Each possibility represents a separate embodiment of the present invention.
  • the isolated peptide or fragment of a LEDGF/p75 protein of methods and compositions of the present invention is capable of inhibiting HIV-I replication in a target cell.
  • Target cell may refer to any cell wherein HIV-I is capable of replicating.
  • the target cell is a human cell or an immortalized cell line derived from a human cell. Each possibility represents a separate embodiment of the present invention.
  • Methods for measuring HIV-I replication in a target cell are well known in the art, and include the use of HeLa MAGI cells (see, inter alia, Example 6 and the sections entitled “Infection of cultured cells,” “HIV-I titration,” and “p24 assay,” and hereinbelow). Each method represents a separate embodiment of the present invention.
  • a composition of the present invention further comprises a non- naturally occurring amino acid, in addition to the fragment of a LEDGF/p75 protein.
  • a composition of the present invention further comprises an organic peptidomimetic moiety, in addition to the fragment of a LEDGF/p75 protein.
  • a side chain of an amino acid of the fragment of a LEDGF/p75 protein has been chemically modified.
  • a peptidic bond has been replaced by a non- naturally occurring peptidic bond.
  • one of the amino acids is replaced by the corresponding D- amino acid.
  • the present invention encompasses an N-methyl variant of the sequence.
  • the present invention provides a sequence disclosed herein in reverse order, preferably having all D-amino acids ⁇ retro inverso).
  • a derivative of the present invention possesses one of the above modifications at a plurality of locations (e.g. a plurality of residues).
  • a derivative of the present invention possesses two of the above modifications.
  • a derivative of the present invention possesses more than 2 of the above modifications.
  • one of the above modifications is introduced at a location outside the fragment of a LEDGF/p75 protein; i.e. in the surrounding sequence.
  • the present invention provides a pharmaceutical composition, comprising an isolated peptide isolated peptide or fragment of a LEDGF/p75 protein of the present invention and a carrier, diluent, or additive.
  • the present invention provides a pharmaceutical composition, comprising: (a) an isolated LEDGF/p75 fragment with a sequence set forth in SEQ ID NO: 1 or a peptide comprising the LEDGF/p75 fragment; (b) an isolated LEDGF/p75 fragment with a sequence selected from the sequences set forth in SEQ ID NO: 2 and SEQ ID NO: 41, or a peptide comprising the LEDGF/p75 fragment; and (c) a pharmaceutically acceptable carrier, diluent, or additive.
  • the pharmaceutical composition comprises (a) a peptide comprising a LEDGF/p75 fragment from the region 361-370 or a corresponding region of a homologous LEDGF/p75 protein; (b) a peptide comprising a LEDGF/p75 fragment from the region 401-412 or a corresponding region of a homologous LEDGF/p75 protein; and (c) a pharmaceutically acceptable carrier, diluent, or additive.
  • LEDGF/p75 fragment (a) comprises a fragment of SEQ ID NO: 1.
  • LEDGF/p75 fragment (b) comprises a fragment of a sequence selected from SEQ ID NO: 2 and SEQ ID NO: 41.
  • both (a) and (b) contain fragments of the respective sequences set forth above.
  • LEDGF/p75 fragment (a) comprises a mutated version of SEQ ID NO: 1.
  • LEDGF/p75 fragment (b) comprises a mutated version of a sequence selected from SEQ ID NO: 2 and SEQ ID NO: 41.
  • both (a) and (b) contain mutated versions of the respective sequences set forth above.
  • LEDGF/p75 fragment (a) contains a trp (tryptophan) residue adjacent to the amino-terminal (N-terminal) end of the LEDGF/p75 fragment.
  • LEDGF/p75 fragment (b) contains a trp residue adjacent to the N-terminal end of the LEDGF/p75 fragment.
  • each of LEDGF/p75 fragments (a) and (b) contains a trp residue adjacent to the N-terminal end of the LEDGF/p75 fragment.
  • the present invention provides a pharmaceutical composition, comprising: (a) an isolated LEDGF/p75 fragment with a sequence set forth in SEQ ID NO: 3 or a peptide comprising the LEDGF/p75 fragment; (b) an isolated LEDGF/p75 fragment with a sequence selected from the sequences set forth in SEQ ID NO: 2 and SEQ ID NO: 41, or a peptide comprising the LEDGF/p75 fragment; and (c) a pharmaceutically acceptable carrier, diluent, or additive.
  • the pharmaceutical composition comprises (a) a peptide comprising a LEDGF/p75 fragment from the region 353-378 or a corresponding region of a homologous LEDGF/p75 protein; (b) a peptide comprising a LEDGF/p75 fragment from the region 401-412 or a corresponding region of a homologous LEDGF/p75 protein; and (c) a pharmaceutically acceptable carrier, diluent, or additive.
  • LEDGF/p75 fragment (a) comprises a fragment of SEQ ID NO: 3.
  • LEDGF/p75 fragment (b) comprises a fragment of a sequence selected from SEQ ID NO: 2 and SEQ ID NO: 41.
  • both (a) and (b) contain fragments of the respective sequences set forth above.
  • LEDGF/p75 fragment (a) comprises a mutated version of SEQ ID NO: 3.
  • LEDGF/p75 fragment (b) comprises a mutated version of a sequence selected from SEQ ID NO: 2 and SEQ ID NO: 41.
  • both (a) and (b) contain mutated versions of the respective sequences set forth above.
  • LEDGF/p75 fragment (a) contains a trp (tryptophan) residue adjacent to the amino-terminal (N-terminal) end of the LEDGF/p75 fragment.
  • LEDGF/p75 fragment (b) contains a trp residue adjacent to the N-terminal end of the LEDGF/p75 fragment.
  • each of LEDGF/p75 fragments (a) and (b) contains a trp residue adjacent to the N-terminal end of the LEDGF/p75 fragment.
  • the major IN-binding loop-derived peptide e.g. LEDGF/p75 353-378 or 361-370, a fragment thereof, or a mutated version thereof
  • minor IN-binding loop-derived peptide e.g.
  • the major IN-binding loop-derived peptide and the minor IN- binding loop-derived peptide exhibit synergy in their inhibition of HIV-I replication.
  • Each possibility represents a separate embodiment of the present invention.
  • the present invention provides a pharmaceutical composition of the present invention for inhibiting replication of an HIV-I in a target cell.
  • the present invention provides a pharmaceutical composition of the present invention for treating HIV-I infection in a subject in need thereof.
  • the present invention provides a pharmaceutical composition of the present invention for inhibiting binding of an HIV-I integrase protein to an HIV-I long terminal repeat DNA terminus.
  • the present invention provides a pharmaceutical composition of the present invention for inhibiting 3 '-end processing of an HIV-I integrase protein.
  • the pharmaceutical composition is utilized in an in vitro assay.
  • the pharmaceutical composition is utilized in a target cell.
  • the present invention provides a use of a peptide of the present invention in the preparation of a medicament for inhibiting replication of an HIV-I in a target cell.
  • the present invention provides a use of a peptide of the present invention in the preparation of a medicament for treating HIV-I infection in a subject in need thereof. In another embodiment, the present invention provides a use of a peptide of the present invention in the preparation of a medicament for inhibiting binding of an HIV-I integrase protein to an HIV-I long terminal repeat DNA terminus.
  • the present invention provides a use of a peptide of the present invention in the preparation of a medicament for inhibiting 3 '-end processing of an HIV-I integrase protein.
  • the pharmaceutical composition is utilized in an in vitro assay.
  • the pharmaceutical composition is utilized in a target cell.
  • the present invention provides a method of inhibiting replication of an HIV-I in a target cell, comprising administering a peptide of the present invention to the target cell, thereby inhibiting replication of an HIV-I in a target cell.
  • the present invention provides a method of treating HIV-I infection in a subject in need thereof, comprising administering a peptide of the present invention to the subject, thereby treating HIV-I infection in a subject in need thereof.
  • the present invention provides a method of inhibiting binding of an HIV-I integrase protein to an HIV-I long terminal repeat DNA terminus, comprising contacting the HIV-I integrase protein with a peptide of the present invention, thereby inhibiting binding of an HIV-I integrase protein to an HIV-I long terminal repeat DNA terminus.
  • the present invention provides a method of inhibiting 3 '-end processing of an HIV-I integrase protein, comprising contacting the HIV-I integrase protein with a peptide of the present invention, thereby inhibiting 3 '-end processing of an HIV-I integrase protein.
  • the method is performed in an in vitro assay.
  • the method is performed in a target cell.
  • a peptide of the present invention is capable of entering a mammalian cell under physiological conditions.
  • the peptide penetrates the cell membrane of the mammalian cell.
  • the peptide is actively transported through the cell membrane.
  • the peptide diffuses through the cell membrane.
  • LEDGF/p75 protein refers, in another embodiment, to a protein having the sequence set forth in SwissProt Accession Number 075475 or a homologue, variant, or isoform of this sequence. In another embodiment, the sequence of the LEDGE/p75 protein is:
  • peptides of the present invention possess a superior ability to inhibit HIV-I replication and HIV-I viral integrase 3 '-end processing activity, both in vitro, in cells, and in vivo. Further, as provided herein, other embodiments of peptides of the present invention inhibited integration of viral DNA and HIV-I replication in cell culture by 2 orders of magnitude. Thus, peptides of the present invention possess a number of superior properties relative to previously known methods of combating HIV- 1 infection.
  • a peptide of the present invention further comprises an additional (i.e. non-LEDGF/p75) peptide sequence, attached to an end of the LEDGF/p75-derived peptide, hi another embodiment, the additional peptide sequence is attached to the N- terminal end of the LEDGF/p75 -derived peptide. In another embodiment, the additional peptide sequence is attached to the C-terminal end of the LEDGF/p75 -derived peptide. In another embodiment, the additional peptide sequences are attached to the N-terminal and C-terminal ends of the LEDGF/p75-derived peptide. Each possibility represents a separate embodiment of the present invention.
  • a peptide of the present invention further comprises an organic, non-peptidic moiety.
  • the peptide of the present invention comprises a hydrophobic moiety attached to the end of the LEDGF/p75-derived peptide.
  • the hydrophobic moiety is a linear hydrocarbon, hi another embodiment, the hydrophobic moiety is a branched hydrocarbon.
  • the hydrophobic moiety is a linear hydrocarbon.
  • the hydrophobic moiety is a cyclic hydrocarbon, hi another embodiment, the hydrophobic moiety is a polycyclic hydrocarbon.
  • the hydrophobic moiety is a heterocyclic hydrocarbon.
  • the hydrophobic moiety is a hydrocarbon derivative.
  • the hydrophobic moiety is a protecting group. In another embodiment, the protecting group serves to decrease degradation (e.g. of a linear compound).
  • the non-peptidic moiety is attached to the N-terminal end of the LEDGF/p75-derived peptide, hi another embodiment, the non-peptidic moiety is attached to the C-terminal end of the LEDGF/p75-derived peptide. In another embodiment, the non- peptidic moieties are attached to the N-terminal and C-terminal ends of the LEDGF/p75- derived peptide. Each possibility represents a separate embodiment of the present invention.
  • an additional peptide sequence is attached to the N-terminal end of the LEDGF/p75-derived peptide and a non-peptidic moiety is attached to the C-terminal end.
  • an additional peptide sequence is attached to the C-terminal end of the LEDGF/p75 -derived peptide and a non-peptidic moiety is attached to the N- terminal end.
  • the additional sequence(s) or moiety(ies) improves a pharmacological property of the peptide
  • the additional sequence(s) or moiety(ies) improves a physiological property of the peptide
  • the property is penetration into cells (e.g. moieties which enhance penetration through membranes or barriers, generally termed "leader sequences")
  • the modified peptides exhibit slower degradation in vivo
  • the modified peptides exhibit slower clearance in vivo
  • the modified peptides exhibit decreased repulsion by various cellular pumps.
  • the modified peptides exhibit decreased immunogenicity.
  • the modified peptides exhibit improved administration to a subject in need, hi another embodiment, the modified peptides exhibit improved penetration through an in vivo barriers (e.g. the gut), hi another embodiment, the modified peptides exhibit increased specificity for HIV-I integrase. hi another embodiment, the modified peptides exhibit increased affinity for HIV-I integrase. In another embodiment, the modified peptides exhibit decreased toxicity. In another embodiment, the modified peptides exhibit improvement in another pharmacological or physiological property. In another embodiment, the modified peptides exhibit improvement in ability to be imaged using an existing technology. Each possibility represents a separate embodiment of the present invention.
  • the association between the amino acid sequence component of the compound and other components of the compound may be by covalent linking, by non-covalent complexion, for example, by complexion to a hydrophobic polymer, which can be degraded or cleaved producing a compound capable of sustained release; by entrapping the amino acid part of the compound in liposomes or micelles to produce the final compound of the invention.
  • the association may be by the entrapment of the amino acid sequence within the other component (liposome, micelle) or the impregnation of the amino acid sequence within a polymer to produce the final compound of the invention.
  • the LEDGF/p75-derived amino acid sequence is in association with (in the meaning described above) a moiety for transport across cellular membranes.
  • moiety for transport across cellular membranes refers to a chemical entity, or a composition of matter (comprising several entities) that causes the transport of members associated (e.g. a LEDGF/p75-derived amino acid sequence) with it through phospholipidic membranes.
  • moieties are hydrophobic moieties such as linear, branched, cyclic, polycyclic or hetrocyclic substituted or non-substituted hydrocarbons.
  • Another example of such a moiety are short peptides that cause transport of molecules attached to them into the cell by, gradient derived, active, or facilitated transport.
  • non-peptidic moieties known to be transported through membranes are glycosylated steroid derivatives, which are well known in the art.
  • the moiety of the compound may be a polymer, liposome or micelle containing, entrapping or incorporating the amino acid sequence therein.
  • the compound of the invention is the polymer, liposome micelle etc. impregnated with the amino acid sequence.
  • Suitable functional groups for increasing transport across cellular membranes are described in Green and Wuts, "Greene 's Protective Groups in Organic Synthesis,” John Wiley and Sons, 2007, the teachings of which are incorporated herein by reference.
  • Preferred protecting groups are those that facilitate transport of the compound attached thereto into a cell, for example, by reducing the hydrophilicity and increasing the lipophilicity of the compounds.
  • Hydroxyl protecting groups include esters, carbonates and carbamate protecting groups.
  • Amine protecting groups include alkoxy and aryloxy carbonyl groups, as described above for N-terminal protecting groups.
  • Carboxylic acid protecting groups include aliphatic, benzylic and aryl esters, as described above for C-terminal protecting groups.
  • the carboxylic acid group in the side chain of one or more glutamic acid or aspartic acid residue in a compound of the present invention is protected, preferably with a methyl, ethyl, benzyl or substituted benzyl ester, more preferably as a benzyl ester.
  • N-terminal protecting groups include acyl groups (-CO-R1) and alkoxy carbonyl or aryloxy carbonyl groups (-CO-O-R1), wherein Rl is an aliphatic, substituted aliphatic, benzyl, substituted benzyl, aromatic or a substituted aromatic group.
  • acyl groups include acetyl, (ethyl)-CO-, n-propyl-CO-, iso-propyl-CO-, n-butyl-CO-, sec-butyl-CO-, t-butyl-CO-, hexyl, lauroyl, palmitoyl, myristoyl, stearyl, oleoyl phenyl-CO-, substituted phenyl-CO-, benzyl-CO- and (substituted benzyl)-CO-.
  • alkoxy carbonyl and aryloxy carbonyl groups include CH3-O-CO-,
  • the carboxyl group at the C-terminus of the compound can be protected, for example, by an amide (i.e., the hydroxyl group at the C-terminus is replaced with -NH 2, -NHR2 and
  • R2 and R3 are independently an aliphatic, substituted aliphatic, benzyl, substituted benzyl, aryl or a substituted aryl group.
  • R2 and R3 taken together with the nitrogen atom, R2 and R3 can form a C4 to C8 heterocyclic ring with from about 0-2 additional heteroatoms such as nitrogen, oxygen or sulfur.
  • suitable heterocyclic rings include piperidinyl, pyrrolidinyl, morpholino, thiomorpholino or piperazinyl.
  • C-terminal protecting groups include -NH2, -NHCH3, -N(CH 3 ⁇ , -NH(ethyl), -N(ethyl)2, -N(methyl)
  • the derivative may include several types of derivation (replacements and deletions, chemical modification, change in peptidic backbone etc.)
  • no more than 40% of the amino acids are replaced by a naturally or non- naturally occurring amino acid or with a peptidomimetic organic moiety.
  • the replacement may be by naturally occurring amino acids (both conservative and non-conservative substitutions), by non- naturally occurring amino acids (both conservative and non-conservative substitutions), or with organic moieties which serve either as true peptidomimetics (i.e. having the same steric and electrochemical properties as the replaced amino acid), or merely serve as spacers in lieu of an amino acid, so as to keep the spatial relations between the amino acid spanning this replaced amino acid. Guidelines for the determination of the replacements and substitutions are provided below. Preferably no more than, 35%, 30%, 25%, 20%, 15%, 10%, or 5% of the amino acids are replaced.
  • naturally occurring amino acid refers to a moiety found within a peptide and is represented by -NH-CHR-CO-, wherein R is the side chain of a naturally occurring amino acid.
  • non-naturally occurring amino acid is either a peptidomimetic, or is a D or L residue having the following formula: -NH-CHR-CO-, wherein R is an aliphatic group, a substituted aliphatic group, a benzyl group, a substituted benzyl group, an aromatic group or a substituted aromatic group and wherein R does not correspond to the side chain of a naturally-occurring amino acid.
  • R also refers to the D-amino acid counterpart of naturally occurring amino acids.
  • Amino acid analogs are well-known in the art; a large number of these analogs are commercially available. Many times the use of non-naturally occurring amino acids in the peptide has the advantage that the peptide is more resistant to degradation by enzymes which fail to recognize them.
  • conservative substitution in the context of the present invention refers to the replacement of an amino acid present in the native sequence in the specific peptide with a naturally or non-naturally occurring amino or a peptidomimetic having similar steric properties.
  • side-chain of the native amino acid to be replaced is either polar or hydrophobic
  • the conservative substitution should be with a naturally occurring amino acid, a non-naturally occurring amino acid or with a peptidomimetic moiety which is also polar or hydrophobic (in addition to having the same steric properties as the side-chain of the replaced amino acid).
  • conservative substitutions by naturally occurring amino acids can be easily determined, bearing in mind the fact that, in accordance with the invention, replacement of charged amino acids by sterically similar non-charged amino acids are considered as conservative substitutions.
  • amino acid analogs synthetic amino acids
  • the substituting amino acid should have the same or a similar functional group in the side chain as the original amino acid.
  • Group I includes leucine, isoleucine, valine, methionine, phenylalanine, cysteine, and modified amino acids having the following side chains: ethyl, n-butyl, -CH 2 CH 2 OH, - CH 2 CH 2 CH 2 OH, -CH 2 CHOHCH 3 and -CH 2 SCH 3 .
  • Group I includes leucine, isoleucine, valine and methionine.
  • Group II includes glycine, alanine, valine, cysteine, and a modified amino acid having an ethyl side chain.
  • Preferably Group II includes glycine and alanine.
  • Group III includes phenylalanine, phenylglycine, tyrosine, tryptophan, cyclohexylmethyl, and modified amino residues having substituted benzyl or phenyl side chains.
  • Preferred substituents include one or more of the following: halogen, methyl, ethyl, nitro, methoxy, ethoxy and -CN.
  • Group III includes phenylalanine, tyrosine and tryptophan.
  • Group IV includes glutamic acid, aspartic acid, a substituted or unsubstituted aliphatic, aromatic or benzylic ester of glutamic or aspartic acid (e.g., methyl, ethyl, n-propyl iso-propyl, cyclohexyl, benzyl or substituted benzyl), glutamine, asparagine, CO-NH-alkylated glutamine or asparagine (e.g., methyl, ethyl, n-propyl and iso-propyl) and modified amino acids having the side chain -(CH 2 ) ⁇ COOH, an ester thereof
  • Group FV includes glutamic acid, aspartic acid, glutamine, asparagine, methyl aspartate, ethyl aspartate, benzyl aspartate and methyl glutamate, ethyl glutamate and benzyl glutamate.
  • Group V includes histidine, lysine, arginine, N-nitroarginine, ⁇ -cycloarginine, ⁇ -hydroxyarginine, N-amidinocitruline and 2-amino-4- guanidinobutanoic acid, homologs of lysine, homologs of arginine and ornithine.
  • Group V includes histidine, lysine, arginine, and ornithine.
  • a homolog of an amino acid includes from 1 to about 3 additional methylene units in the side chain.
  • Group VI includes serine, threonine, cysteine and modified amino acids having C1-C5 straight or branched alkyl side chains substituted with -OH or -SH.
  • Group VI includes serine, cysteine, and threonine.
  • non-conservative substitutions concerns replacement of the amino acid as present in the native peptide by another naturally or non-naturally occurring amino acid, having different electrochemical and/or steric properties, for example as determined by the fact the replacing amino acid is not in the same group as the replaced amino acid of the native peptide sequence.
  • Those non-conservative substitutions which fall under the scope of the present invention are those which still constitute a compound having integrase inhibiting activities.
  • a "non-conservative substitution” is a substitution in which the substituting amino acid (naturally occurring or modified) has significantly different size, configuration and/or electronic properties compared with the amino acid being substituted.
  • the side chain of the substituting amino acid can be significantly larger (or smaller) than the side chain of the native amino acid being substituted and/or can have functional groups with significantly different electronic properties than the amino acid being substituted.
  • non-conservative substitutions of this type include the substitution of phenylalanine or cycohexylmethyl glycine for alanine, isoleucine for glycine, or -NH-CH[(-CH2)5_COOH]-CO- for aspartic acid.
  • a functional group may be added to the side chain, deleted from the side chain or exchanged with another functional group.
  • non-conservative substitutions of this type include adding an amine or hydroxyl, carboxylic acid to the aliphatic side chain of valine, leucine or isoleucine, exchanging the carboxylic acid in the side chain of aspartic acid or glutamic acid with an amine or deleting the amine group in the side chain of lysine or ornithine, hi yet another alternative, the side chain of the substituting amino acid can have significantly different steric and electronic properties from the functional group of the amino acid being substituted.
  • a "peptidomimetic organic moiety" can be substituted for amino acid residues in the compounds of this invention both as conservative and as non-conservative substitutions. These peptidomimetic organic moieties either replace amino acid residues of essential and non-essential amino acids or act as spacer groups within the peptides in lieu of deleted amino acids (of non-essential amino acids).
  • peptidomimetic organic moieties often have steric, electronic or configurational properties similar to the replaced amino acid and such peptidomimetics are used to replace amino acids in the essential positions, and are considered conservative substitutions. However, such similarities are not necessarily required.
  • the only restriction on the use of peptidomimetics is that the peptides retain their integrase inhibiting properties or HTV-replication inhibiting properties/ or equilibrium shifting properties as defined above.
  • Peptidomimetics are often used to inhibit degradation of the peptides by enzymatic or other degradative processes.
  • the peptidomimetics can be produced by organic synthetic techniques. Examples of suitable peptidomimetics include D amino acids of the corresponding L amino acids, tetrazol (Zabrocki et al, J. Am. Chem. Soc. 110:5875-5880
  • LL-3-amino-2-propenidone-6-carboxylic acid (LL- Acp) ⁇ Kemp et al, J. Org. Chem. 50:5834-5838 (1985)). Similar analogs are shown in Kemp et al, Tetrahedron Lett. 29:5081-5082 (1988) as well as Kemp et al, Tetrahedron Lett. 29:5057-5060 (1988), Kemp et al, Tetrahedron Lett. 29:4935-4938 (1988) and Kemp et al, J. Org. Chem. 54:109-115 (1987). Other suitable peptidomimetics are shown in Nagai and Sato, Tetrahedron Lett.
  • the amino acids typically have their side chains modified.
  • the modification means the same type of amino acid residue, but to its side chain a functional group has been added.
  • the side chain may be phosphorylated, glycosylated, fatty acylated, acylated, iondiated or carboxyacylated.
  • the deletion may be of terminal or non-terminal amino acids to result either in deletion of non terminal amino acid or in a fragment having at least 3,4,5,6,7,8,9,10,11,12 amino acids.
  • derivatives are not active. Those derivatives that fall under the scope of the invention are those that can inhibit the HIV-I replication, preferably those that inhibit the viral integrase activity, most preferably those that can cause a shift in the oligeramization equilibrium shift in a similar manner to the parent protein of (a)-(b)).
  • essential amino acids are maintained or replaced by conservative substitutions while non-essential amino acids may be maintained, deleted or replaced by conservative or non-conservative replacements.
  • essential amino acids are determined by various Structure-Activity-Relationship (SAR) techniques (for example amino acids when replaced by Ala cause loss of activity) are replaced by conservative substitution while non-essential amino acids can be deleted or replaced by any type of substitution.
  • SAR Structure-Activity-Relationship
  • corresponding D-amino acid refers to the replacement of the naturally occurring L-configuration of the natural amino acid residue by the D-configuration of the same residue.
  • At least one peptidic backbone has been altered to a non-naturally occurring peptidic backbone means that the bond between the N- of one amino acid residue to the C- of the next has been altered to non-naturally occurring bonds by reduction (to -CH 2 - NH-), alkylation (methylation) on the nitrogen atom, or the bonds have been replaced by, urea bonds, or sulfonamide bond, etheric bond (-CH 2 -O-), thioetheric bond (-CH 2 -S-), or to -CS-NH-.
  • the side chain of the residue may be shifted to the backbone nitrogen to obtain N-alkylated-Gly (a peptoid) as well as aza peptides
  • sequence of (a) to (c) may have the order of the amino acids as it appears in the native protein, or may have the reversed order (as read in the C-to N-direction). It has been found that many times sequences having such a reverse order can have the same properties, in small peptides, as the "correct” order, probably due to the fact that the side chains, and not the peptidic backbones are those responsible for interaction with other cellular components. Particularly preferred, are what is termed "retro inverso" peptides - i.e.
  • Peptide sequences for producing any of the sequence of the compounds of the invention can be synthesized by solid phase peptide synthesis (e.g., t-BOC or F-MOC) method, by solution phase synthesis, or by other suitable techniques including combinations of the foregoing methods.
  • solid phase peptide synthesis e.g., t-BOC or F-MOC
  • F-MOC solid phase peptide synthesis
  • the t-BOC and F-MOC methods which are established and widely used, are described in Merrifield, J Am. Chem. Soc, 88:2149 (1963); Meienhofer, Hormonal Proteins and Peptides, CH. Li, Ed., Academic Press, 1983, pp. 48-267; and Barany and Aarifield, in The Peptides, E. Gross and J. Meienhofer, Eds., Academic Press, New York, 1980, pp. 3-285.
  • EXAMPLE 1 Design of LEDGF-derived peptides that modulate the IN oligomerization equilibrium
  • shiftides refers to peptides that shift the oligomerization equilibrium of a protein capable of oligomerization, in order to inhibit its activity.
  • a shiftide binds preferentially to the inactive oligomeric state and shifts the oligomerization equilibrium of the protein towards it.
  • Shiftide HIV-I IN inhibitors were discovered as follows. IN exists in the cell in equilibrium between tetramer, dimer and monomer. Each of these oligomeric states is in an additional equilibrium between LEDGF/p75- and DNA- bound and unbound states. A screen was performed to identify peptides that perturb the oligomerization equilibrium of IN by shifting it towards the tetramer, which is unable to bind DNA and as a result unable to catalyze the 3' processing reaction. The screen measured ability of LEDGF/p75-derived peptides to bind preferentially to the tetrameric state of IN, relative to its dimer.
  • EXAMPLE 2 IN binds to the LEDGF-derived peptides as a tetramer and to the
  • EXAMPLE 5 The LEDGF peptides inhibit the catalytic activities of IN in vitro
  • LEDGF-peptides 361-370 and LEDGF 401- 412 were tested the ability of LEDGF-peptides 361-370 and LEDGF 401- 412 to inhibit the enzymatic activities of IN, specifically the 3' processing and strand transfer activities of IN. Both LEDGF 401-412 and LEDGF 361-370 strongly inhibited the 3 '-end processing activity of IN in vitro. Further, the peptides inhibited strand transfer activity of IN when a DNA that did not undergo 3' processing was used as template. The inhibitory effect of the peptides is due to inhibition of the cytoplasmic 3' processing step of the integration mechanism, which is caused by the shift in the equilibrium of IN oligomerization.
  • LEDGF 401-412 was even more potent than LEDGF 361-370 and showed significant inhibition at the lowest concentration tested of 21 ⁇ M ( Figure 5).
  • EXAMPLE 6 The LEDGF peptides inhibit HIV-I replication in cell culture by inhibition of in catalytic activity
  • LEDGF 361-370 and LEDGF 401-412 peptides at concentrations lower than 2.5 ⁇ M reduced integration by more than 95% compared to untreated cells (Figure 6E-F).
  • Lysine 413 was also determined by constructing a peptide wherein a lysine residue was inserted at position 407 (as in the native sequence); this peptide was designated "LEDGF 401-413.” Inclusion of this amino acid did not affect IN binding or inhibition.
  • EXAMPLE 8 Testing of a human LEDGF/p75 homologue-derived peptide
  • a peptide homologous to LEDGF 361-370 was designed from the homologous human protein hepatoma-derived growth factor related protein 2 (HRP2).
  • LEDGF and HRP2 have a conserved predicted Integrase Binding Domain (IBD), and they both stimulate IN activity in vitro. The full sequence of those proteins has 29% identity and 49% similarity.
  • the IBD of HRP2 exhibits 50% identity and 77% similarity to the LEDGF IBD.
  • the sequence similarity to LEDGF/p75 IBD was used to design a labeled peptide derived from HRP2 IBD, which "HRP2 484-493" (Table 3).
  • HRP2 484-493 Binding affinity of HRP2 484-493 to IN was determined to be about lO ⁇ M, and the Hill coefficient was 4.5 ( Figure 7A).
  • HRP2 484-493 inhibited IN catalytic activity in vitro by about 20%, which is much less than the parent LEDGF/p75 derived shiftides, which inhibited the IN activity by 80% at the same concentration (Table 2). This difference in level of inhibition is likely due to prevention of conformational flexibility of HRP2 484-
  • non-essential amino acids Deletion and replacement by non-conservative amino acids generally is performed in "non-essential" amino acids, while essential amino acids should be maintained or replaced by conservative substitutions. To identify essential vs. non-essential amino acids, the following techniques are utilized:
  • a series of truncated partly overlapping peptides derived from LEDGF 361-370 and LEDGF 401-412 is prepared. Peptides are shortened by one residue at a time from the N terminus and from the C terminus. The short peptides are labeled with fluorescein for the fluorescence anisotropy and cellular uptake studies.
  • Non-natural amino acids will be primarily utilized, since their incorporation increases peptide stability. Binding of the peptides to IN is tested using fluorescent anisotropy, mixtures that bind IN are separated by HPLC, and binding of individual peptides to ESf is tested. To identify synergy between the different mutations, peptides are synthesized with multiple amino acid replacements, wherein mutations resulting in tightest IN binding are combined.
  • N-methyl amino acids and D-amino acids are introduced into the peptide sequence.
  • a D-amino acid scan of the lead peptides is performed, and a series of peptides is synthesized wherein each amino acid is systematically replaced by its D enantiomer.
  • an N-methyl amino acids scan of the lead peptides is performed (similarly to the D-amino acid scan). N-methylation is known to stabilize peptide to enzymatic degradation and increase their oral bioavailability due to the lack of the amide protons, which reduces their polarity.
  • the lead peptides at this stage bear an optimized side chain composition with only the required pharmacophores present, have a shorter sequence and are stable against proteolysis. Next, backbone modifications are introduced. The lead peptides are subject to AZA scan, to improve the peptide stability and its binding affinity and specificity.
  • Aza peptides are peptide analogs in which the ⁇ -carbon of one or more of the amino acid residues is replaced with a nitrogen atom.
  • each amino acid in the lead peptides is systematically replaced by the corresponding Aza amino acid, followed by assays for binding IN and inhibiting IN activity and HIV-I replication as described above, hi other experiments, the backbone amide bonds are each systematically converted into a peptoid bond, wherein the side chain of the peptide is moved from the ⁇ -carbon to the ⁇ -nitrogen.
  • Peptides were synthesized on an Applied Biosystems (ABI) 433A peptide synthesizer using standard Fmoc chemistry.
  • the peptides were labeled using 5 1 (and 6 1 ) carboxyfluorescein succinimidyl ester (Molecular Probes) at the N-terminus using 4- fold excess of the fluorescein and 4-fold excess of hydroxybenzotriazole (HoBt). All amino acids were purchased from NOVAbiochem.
  • Peptides were purified on a Gilson HPLC using a reverse-phase C 8 semi-preparative column (ACE) with a gradient from 5% to 60% buffer B in buffer A [buffer A, 0.001% (vol/vol) trifluoroacetic acid (TFA) in water and buffer B 0.001% (vol/vol) TFA in acetonitrile]. Peptides were analyzed using MALDI TOF Mass Spectroscopy on a Voyager DE-Pro instrument (Applied Biosystems). Peptide concentration before each experiment was determined using a UV spectrophotometer (Shimadzu) according to the method of Gill and Von Hippel (21). Sequences of the peptides synthesized in this study are shown in Figure 1C and Table 3.
  • His-tagged IN expression vector was provided by Dr. A. Engelman, Harvard Medical School, and its expression and purification were performed as described (22). His-tagged LEDGF/p75 was expressed in BL21(DE3) pLysS cells (Novagen, Inc.) and was purified as described (23).
  • R is the measured fluorescence anisotropy value
  • AR is amplitude of the fluorescence anisotropy change from the initial value (peptide only) to the final value (peptide in complex)
  • [IN] is the added integrase concentration
  • RQ is the starting anisotropy value, corresponding to the free peptide
  • K a is the association constant (1/ *d ).
  • F1-U5B36 5'-6-FAM-AGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGT-S' (SEQ ID NO: 4).
  • the equilibrium sedimentation experiments were performed on a Beckman XL-I ultracentrifuge using Ti-60 rotor and 6-sector cells at speed of 30,000 and 40,000 rpm.
  • the fluorescein-labeled peptides LEDGF 353-378, 361-370 and 401-412 in a final concentration of 10 ⁇ M in PBS were incubated with HeLa cells for 2 h at 37 0 C. After three washes in PBS, unfixed cells were visualized by fluorescence microscopy.
  • oligonucleotides A-C correspond to the U5 end of the HIV-I long terminal repeat.
  • Oligonucleotide C is identical to A, after the removal of the GT dinucleotides from its 3'-end and thus after annealing to oligonucleotide B, creating a dinucleotide overhang at the 5'-end of oligonucleotide B.
  • Oligonucleotide D termed "dumbbell,” folds to form a structure mimicking the integration intermediate.
  • oligonucleotide A annealed to its complementary strand
  • oligonucleotide B both 21 nucleotides long
  • the duplex of oligonucleotides C and B were used for assaying the 3 '-end processing activity.
  • 5 '-End Labeling and Substrate Preparations Fifty pmol of oligonucleotides A, C, or D were 5 '-end-labeled using 1 unit of T4 polynucleotide kinase and 50 ⁇ Ci Of 32 P-ATP, in a final volume of 50 ⁇ l of the appropriate buffer (supplied by the manufacturer) for 30 min at 37 °C. The samples were then heat-inactivated. 5 '-end-labeled oligonucleotides A or C were annealed each to an equimolar amount of oligonucleotide B in 55 mM Tris- HCl (pH 7.5) and 0.27 M NaCl.
  • HIV-I IN 500 ng of HIV-I IN (which equals 8 pmol, assuming IN dimers of the 32-kDa subunits) was assayed.
  • the HIV-I IN was pre-incubated on ice for 5 min the absence or the presence of increasing concentrations of the tested peptides. Reactions were initiated after adding the labeled DNA substrate in the reaction buffer, incubated for 30 min at 37 °C, and then stopped by adding 10 ⁇ l of formamide loading buffer (90% formamide, 10 mM EDTA, 1 mg/ml bromphenol blue, 1 mg/ml xylene cyanole).
  • DMEM Dulbecco's Modified Eagle's Medium
  • FCS fetal calf serum
  • FCS fetal calf serum
  • Biological Industries 100 U/ml penicillin and 100 U/ml streptomycin
  • the T-lymphocyte cell line Sup Tl and H9 provided by the NIH Reagent Program (Division of AIDS, NIAID, NIH, USA), was grown in RPMI 1640 medium supplemented with 10% FCS 5 100 U/ml penicillin, 100 U/ml streptomycin and 2 mM L- glutamine (Biological Industries).
  • TZM-bl indicator cells were obtained through the NIH Reagent Program (Division of AIDS, NIAID, NIH, USA), and were grown in DMEM supplemented with 10% FCS, 100 U/ml penicillin, 100 U/ml streptomycin and 2 raM L-glutamine (Biological Industries). Cells were incubated at 37°C in a 5% CO 2 atmosphere.
  • Viruses HIV-I wild type virus was generated by transfection (33) of 293T cells with pSVC21 plasmid containing the full-length HIV-1 HXB2 viral DNA (34). Wild type and ⁇ env/VSV-G viruses were harvested from 293T cells 48 and 72 h post transfection with pSVC21 ⁇ env. Viruses were stored at -75 0 C until titration.
  • HIV-I titration Titration of HIV-I was carried out by the multinuclear activation of a galactosidase indicator (MAGI) assay.
  • MAGI galactosidase indicator
  • TZM-bl cells were transferred into 96-well plates at 10x10 3 cells per well. On the following day, the cells were infected with 50 ⁇ l of serially diluted virus in the presence of 20 ⁇ g/ml of DEAE-dextran (Pharmacia, Sweden). Two days post-infection, cultured cells were fixed with 1% formaldehyde and 0.2% glutaraldehyde in PBS.
  • p24 assay Assays utilized an HIV-I p24 antigen capture assay kit (SAIC, AIDS Vaccine Program, Frederick, MD), in accordance with the standards and instructions supplied by the manufacturers.
  • SAIC HIV-I p24 antigen capture assay kit
  • AIu-LTR sequences were amplified from 1/10 of total cell DNA in a 25 ⁇ l reaction mixture containing PCR buffer xl, 3.5 mM MgCl 2 , 200 ⁇ M dNTPs, 300 nM primers, and 0.025 U/ ⁇ l Taq polymerase.
  • First-round PCR cycle conditions were as follows: a DNA denaturation and polymerase activation step of 10 min at 95 0 C and then 12 cycles of amplification (95 0 C for 15 sec, 60 0 C for 30 sec, 72 0 C for 5 min).
  • the first-round PCR product could be specifically amplified by using the tag specific primer (tag-F 5'-ATGCCACGTAAGCGAAACTC-S'; SEQ ID NO: 10) and the LTR primer (LTR-R 5'-AGGCAAGCTTTATTGAGGCTTAAG-S'; SEQ ID NO: 11) that was designed by PrimerExpress® (Applied Biosystems) using default settings.
  • the second-round PCR was performed on 1/25 of the first-round PCR product in a mixture containing 300 nM of each primer, 12.5 microliter (mcl) of 2X SYBR green master mix (Applied Biosystems) at a final volume of 25 mcl were run on an ABI PRIZM 7700 (Applied Biosystems).
  • the second-round PCR cycles began with a DNA-denaturation and polymerase-activation step (95 0 C for 10 min), followed by 50 cycles of amplification (95 0 C for 15 sec, 60 0 C for 60 sec).
  • DMSO-solubilized cells were transferred onto a 96-well ELISA plate, and OD values were read at a wavelength of 570 nm.

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Abstract

La présente invention concerne des peptides isolés comprenant un fragment d'une protéine LEDGF/p75 et leur utilisation pour traiter l'infection par le VIH-1, inhiber la réplication du VIH-1 et inhiber la liaison de l'ADN et l'activité de traitement de l'extrémité 3' de la protéine intégrase du VIH-1.
PCT/IL2007/001321 2006-10-30 2007-10-30 Compositions et procédés pour inhiber la réplication et l'activité de l'intégrase du vih-1 WO2008053478A2 (fr)

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013177198A1 (fr) * 2012-05-21 2013-11-28 The Regents Of The University Of Colorado, A Body Corporate Peptides ledgf et formulations en contenant pouvant être utilisés en vue du traitement d'affections dégénératives
US8987250B2 (en) 2012-04-20 2015-03-24 Gilead Sciences, Inc. Therapeutic compounds
US9006229B2 (en) 2011-04-21 2015-04-14 Gilead Sciences, Inc. Benzothiazole compounds and their pharmaceutical use
US9102614B2 (en) 2010-07-02 2015-08-11 Gilead Sciences, Inc. Naphth-2-ylacetic acid derivatives to treat AIDS
US9163067B2 (en) 2008-10-06 2015-10-20 Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd HIV-1 integrase derived stimulatory peptides interfering with integrase—Rev protein binding
US9284323B2 (en) 2012-01-04 2016-03-15 Gilead Sciences, Inc. Naphthalene acetic acid derivatives against HIV infection
US9296758B2 (en) 2010-07-02 2016-03-29 Gilead Sciences, Inc. 2-quinolinyl-acetic acid derivatives as HIV antiviral compounds
US9376392B2 (en) 2012-01-04 2016-06-28 Gilead Sciences, Inc. 2-(tert-butoxy)-2-(7-methylquinolin-6-yl) acetic acid derivatives for treating AIDS
US10563254B2 (en) 2007-01-23 2020-02-18 Cambridge Enterprise Limited Nucleic acid amplification and testing

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
CHEREPANOV PETER ET AL.: "Identification of an evolutionarily conserved domain in human lens epithelium-derived growth factor/transcriptional co-activator p75 (LEDGF/p75) that binds HIV-1 integrase" JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 279, no. 47, 19 November 2004 (2004-11-19), pages 48883-48892, XP002478701 ISSN: 0021-9258 *
DATABASE PDB [Online] 98 amino acids 24 September 2005 (2005-09-24), CHEREPANOV P., AMBROSIO A.L., RAHMAN S., ELLENBERGER T., ENGELMAN A.: "Structural basis for the recognition between HIV-1 integrase and LEDGF/p75" XP002478702 retrieved from EBI accession no. PDB:2B4J Database accession no. 2B4J cited in the application -& CHEREPANOV PETER ET AL.: "Structural basis for the recognition between HIV-1 integrase and transcriptional coactivator p75" PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, vol. 102, no. 48, November 2005 (2005-11), pages 17308-17313, XP002478700 ISSN: 0027-8424 cited in the application *
DE RIJCK JAN ET AL.: "Overexpression of the lens epithelium-derived growth factor/p75 integrase binding domain inhibits human immunodeficiency virus replication"[Online] 20 September 2006 (2006-09-20), XP002478856 Retrieved from the Internet: URL:http://jvi.asm.org/cgi/reprint/JVI.00801-06v1> [retrieved on 2008-04-30] *
HAYOUKA ZVI ET AL.: "Inhibiting HIV-1 integrase by shifting its oligomerization equilibrium" PNAS, vol. 104, no. 20, 15 May 2007 (2007-05-15), pages 8316-8321, XP002478699 *
LATAILLADE MAX ET AL.: "The hunt for HIV-1 integrase inhibitors." AIDS PATIENT CARE AND STDS JUL 2006, vol. 20, no. 7, July 2006 (2006-07), pages 489-501, XP009099694 ISSN: 1087-2914 cited in the application *

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US11447821B2 (en) 2007-01-23 2022-09-20 Cambridge Enterprise Limited Nucleic acid amplification and testing
US10563254B2 (en) 2007-01-23 2020-02-18 Cambridge Enterprise Limited Nucleic acid amplification and testing
US9738878B2 (en) 2008-10-06 2017-08-22 Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd. HIV-1 integrase derived peptides and compositions
US9163067B2 (en) 2008-10-06 2015-10-20 Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd HIV-1 integrase derived stimulatory peptides interfering with integrase—Rev protein binding
US9102614B2 (en) 2010-07-02 2015-08-11 Gilead Sciences, Inc. Naphth-2-ylacetic acid derivatives to treat AIDS
US9296758B2 (en) 2010-07-02 2016-03-29 Gilead Sciences, Inc. 2-quinolinyl-acetic acid derivatives as HIV antiviral compounds
US9006229B2 (en) 2011-04-21 2015-04-14 Gilead Sciences, Inc. Benzothiazole compounds and their pharmaceutical use
US9284323B2 (en) 2012-01-04 2016-03-15 Gilead Sciences, Inc. Naphthalene acetic acid derivatives against HIV infection
US9376392B2 (en) 2012-01-04 2016-06-28 Gilead Sciences, Inc. 2-(tert-butoxy)-2-(7-methylquinolin-6-yl) acetic acid derivatives for treating AIDS
US9096586B2 (en) 2012-04-20 2015-08-04 Gilead Sciences, Inc. Therapeutic compounds
US8987250B2 (en) 2012-04-20 2015-03-24 Gilead Sciences, Inc. Therapeutic compounds
RU2617964C2 (ru) * 2012-05-21 2017-04-28 Дзе Риджентс Оф Дзе Юниверсити Оф Колорадо, Э Боди Корпорэйт Пептиды ledgf и их композиции для лечения дегенеративных заболеваний
US9526760B2 (en) 2012-05-21 2016-12-27 The Regents Of The University Of Colorado, A Body Corporate LEDGF peptides and formulations thereof for treatment of degenerative disorders
US10221226B2 (en) 2012-05-21 2019-03-05 The Regents Of The University Of Colorado, A Body Corporate LEDGF peptides and formulations thereof for treatment of degenerative disorders
WO2013177198A1 (fr) * 2012-05-21 2013-11-28 The Regents Of The University Of Colorado, A Body Corporate Peptides ledgf et formulations en contenant pouvant être utilisés en vue du traitement d'affections dégénératives

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