WO2008053478A2

WO2008053478A2 - Compositions and methods for inhibiting hiv-1 replication and integrase activity

Info

Publication number: WO2008053478A2
Application number: PCT/IL2007/001321
Authority: WO
Inventors: Assaf Friedler; Abraham Loyter; Moshe Kotler; Amnon Hizi; Zvi Hayouka; Joseph Rosenbluh; Aviad Levin; Shoshana Loya
Original assignee: Ramot at Tel Aviv University Ltd; Yissum Research Development Co of Hebrew University of Jerusalem
Current assignee: Ramot at Tel Aviv University Ltd; Yissum Research Development Co of Hebrew University of Jerusalem
Priority date: 2006-10-30
Filing date: 2007-10-30
Publication date: 2008-05-08
Anticipated expiration: 2009-04-30
Also published as: WO2008053478A3

Abstract

The present invention provides isolated peptides comprising a fragment of a LEDGF/p75 protein and use of same for treating HIV-1 infection, inhibiting HIV-1 replication and inhibiting DNA binding and 3'-end processing activity of HIV-1 integrase protein.

Description

COMPOSITIONS AND METHODS FOR INHIBITING HIV l REPLICATION

AND INTEGRASE ACTIVITY

FIELD OF THE INVENTION

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.

BACKGROUND OF THE INVENTION

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. Currently, 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. To overcome the drug-resistance problem, 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. 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). However, 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. However, 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 summary, IN catalyzes the 3' processing as a dimer in the cytoplasm, and mediates the strand transfer as a tetramer in the nucleus.

IN activity requires binding of the cellular protein LEDGF/p75, a member of the hepatoma derived growth factor (HDGF) family. 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 another study, over-expression of a putative LEDGF/p75 IBD at very high concentrations inhibited HIV-I replication at the (nuclear strand) transfer step (18), which was believed to suggest the competitive inhibition of the LEDGF-IN interaction as a new target for anti-HIV drug design. Neither this reference nor any of the other references cited herein disclose or suggest any of the following: (a) the dimer-tetramer equilibrium of IN as a target for anti-HIV drugs; (b) inhibition of binding of IN to the LTR DNA termini; (c) use of smaller fragments of the putative LEDGF/p75 IBD to inhibit 3 '-end processing in the cytoplasm of a target cell. 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).

There is an unmet medical need for new therapeutic modes of treating HIV infection and HIV related disease. Effective IN inhibitors are urgently needed.

The inclusion or description of literary references in this section or any other part of this application does not constitute an admission that the references are regarded as prior art to this invention.

SUMMARY OF THE INVENTION

In one embodiment, 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).

In another embodiment, 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.

In another embodiment, 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. Preferably, 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.

In another embodiment, 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).

In another embodiment, 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.

In another embodiment, 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. Preferably, 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.

In another embodiment, 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).

In another embodiment, 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. In another embodiment, 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. Preferably, 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.

In another embodiment, the present invention provides an isolated 12-mer peptide, wherein the sequence of the 12-mer peptide is KKIRRFVSQVIM (SEQ ID NO: 41).

In another embodiment, the present invention provides an isolated peptide comprising an isolated fragment of a LEDGF/p75 protein of the present invention. Preferably, 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 10-100 amino acids in length. In another embodiment, 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.

In another embodiment, the present invention provides an isolated peptide comprising an isolated mutated fragment of a LEDGF/p75 protein of the present invention.

Preferably, 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

10-100 amino acids in length. In another embodiment, 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.

In another embodiment, the present invention provides an isolated peptide comprising a 12-mer peptide of the present invention. Preferably, the isolated peptide is 13-100 amino acids in length. In another embodiment, 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.

In another embodiment, the present invention provides a pharmaceutical composition, comprising: a plurality of peptides of the present invention and a pharmaceutically acceptable carrier, diluent, or additive. In another embodiment, one of the peptides is a major IN-binding loop-derived peptide and another is a minor IN-binding loop-derived peptide. Each possibility represents a separate embodiment of the present invention.

In another embodiment, 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.

In another embodiment, 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.

In another embodiment, 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.

As provided herein, 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.

It should be understood that the peptides of the invention can be derivatized, as will be explained below, while still maintaining the oligomerization equilibrium-shifting and HIV-I inhibition properties.

In one embodiment, the present invention provides a peptide having the sequence: NSLKIDNLDV (SEQ ID NO: 1).

In another embodiment, 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).

In another embodiment, the present invention provides a peptide having the sequence: KKIRRFVSQVIM (SEQ ID NO: 41).

In one embodiment, the present invention provides a peptide having the sequence: WNSLKIDNLDV (SEQ ID NO: 12).

In another embodiment, the present invention provides a peptide having the sequence: WKKIRRFKVSQVIM (SEQ ID NO: 14).

In another embodiment, the present invention provides a peptide having the sequence: WIHAEIKNSLKIDNLDVNRCIEALD (SEQ ID NO: 46).

In another embodiment, the present invention provides a peptide having the sequence: WKKIRRFVSQVIM (SEQ ID NO: 13).

In another embodiment, 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.

Due to the possibility of synergistic HIV-I replication inhibiting activities of the peptides, it is preferable, in accordance with the invention, that 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.

Preferably, the pharmaceutical compositions are indicated for inhibition of HIV-I replication.

In another embodiment, the pharmaceutical 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.

The term "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.

BRIEF DESCRIPTION OF THE DRAWINGS

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). (C) LEDGF/p75-derived peptides used in Examples 1-6. Figure 2: Ligands binding of IN: fluorescence anisotropy studies. IN was titrated into the following fluorescein-labeled LEDGF/p75 peptides (100 nM): (A). LEDGF/p75 353-378 (circle), (B) LEDGF/p75 361-370 (circle) and LEDGF/p75 401-412 (square). Binding curves were fit to the Hill equation (equation 1), as described in the experimental section. See Table 1 for binding affinities. (C) Effect of LEDGF/p75 peptides on DNA binding of IN. IN was titrated into fluorescein-labeled HIV-I LTR DNA (10 nM) (square). The titration was repeated in the presence of the following LEDGF peptides: LEDGF 361- 370 1 μM (circle), LEDGF 353-378 1 μM (triangle) and LEDGF 401-412 1 μM (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 μM) 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 μM with a Hill coefficient of 2, which is 30 times weaker than the binding in absence of LEGDF/p75.

Figure 3: Effect of ligand binding on the oligomeric state of HIV-I IN. (A)

Analytical ultracentrifuge (AUC) sedimentation equilibrium studies of IN, showing that the oligomerization equilibrium of IN is between high order oligomer, tetramer and dimer, as summarized in figure 3A. (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 μM (left peak), IN 14 μM + LEDGF 361-370 14 μM, IN 14 μM + LEDGF 401-412 14 μM, IN 14 μM +

DNA LTR 14 μM (right peak), IN 14 μM + DNA LTR 14 μM + LEDGF 361-370 14 μM. (C) Oligomerization of IN 52-288 is not affected by ligand binding. The samples were: IN 52-288 15 μM (blue), IN 52-288 + LEDGF/p75 derived peptide 361-370 14 μM (green), IN 52-288 15 μM+LTR DNA 14 μM (grey), IN 52-288 15 μM + LEDGF/p75 derived peptide 361-370 15 μM + LTR DNA 15 μM (magenta).

Figure 4: LEDGF derived peptides are "shiftides" that shift the oligomerization state of IN towards a tetramer. (A) 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. (B) The oligomeric state of IN upon ligand binding. IN binds the LTR DNA as a dimer, and binds the LEDGF peptides as a tetramer. Upon binding the peptides, equilibrium is shifted towards the tetramer and binding to the LTR DNA is inhibited. (C) Model of the "shiftide" mechanism for IN inhibition. LEDGF peptides penetrate into the cells and bind to IN in the cytoplasm. They shift its oligomeric state to a tetramer, reducing the binding affinity to the LTR DNA, and preventing the 3 '-end processing. Strand transfer catalytic activity is also inhibited, because there is no DNA template, due to inhibition of 3 '-end processing.

Figure 5: The LEDGF peptides inhibit the IN catalytic activities in vitro. IN (0.8 μM) 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 μM of fluorescently labeled LEDGF 361-370 (A) or LEDGF 401-412 (B) were incubated for 2 h in 37⁰C with HeLa cells. The cells were then washed three times with phosphate-buffered saline (PBS) and visualized by a fluorescent microscope. (C) 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 β-galactosidase activity. (D) 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.

DETAILED DESCRIPTION OF THE INVENTION

In another embodiment, 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. Preferably, the single amino acid modifications are independently selected from the group consisting of a substitution, an insertion, and a deletion. In another embodiment, the mutated fragment comprises a single amino acid modification relative to SEQ ID NO: 1. In another embodiment, the mutated fragment comprises 2 modifications relative to SEQ ID NO: 1. Each possibility represents a separate embodiment of the present invention. In another embodiment, 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).

In another embodiment, 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. Preferably, the single amino acid modifications are independently selected from the group consisting of a substitution, an insertion, and a deletion. In another embodiment, the mutated fragment comprises a single amino acid modification relative to SEQ ID NO: 2. In another embodiment, the mutated fragment comprises 2 modifications relative to SEQ ID NO: 2. Each possibility represents a separate embodiment of the present invention.

In another embodiment, 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.

In another embodiment, 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. Preferably, the single amino acid modifications are independently selected from the group consisting of a substitution, an insertion, and a deletion. In another embodiment, the mutated fragment comprises a single amino acid modification relative to SEQ ID NO: 3. In another embodiment, the mutated fragment comprises 2 modifications relative to SEQ ID NO: 3. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides an isolated peptide comprising an isolated fragment of a LEDGF/p75 protein of the present invention. Preferably, the isolated peptide is 12-100 amino acids in length. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides an isolated peptide comprising an isolated mutated fragment of a LEDGF/p75 protein of the present invention. Preferably, 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.

In another embodiment, the present invention provides a peptide having the sequence: NSLKIDNLDV (SEQ ID NO: 1).

In another embodiment, 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).

In another embodiment, 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). Preferably, the length of the peptide is 6-100 amino acids. In another embodiment, the isolated peptide consists of the above-described fragment of a LEDGF/p75 protein. In another embodiment, 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.

In another embodiment, 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. Preferably, the length of the isolated peptide is 6-100 amino acids. In another embodiment, the isolated peptide consists of the above- described fragment of a LEDGF/p75 protein. In another embodiment, 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.

In another embodiment, 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.

In another embodiment, 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. In another embodiment, the peptide is an isolated peptide. In another embodiment, the peptide consists of the above- described mutated fragment of a LEDGF/p75 protein. In another embodiment, the fragment of a LEDGF/p75 protein contains residues other than those set forth in SEQ ID NO: 1. In another embodiment, if residues other than those set forth in SEQ ID NO: 1 are present, then 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. In another embodiment, 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.

In another embodiment, 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). Preferably, the length of the peptide is 6-100 amino acids. In another embodiment, the isolated peptide consists of the above-described fragment of a LEDGF/p75 protein. In another embodiment, 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. In another embodiment, 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. Preferably, the length of the peptide is 6-100 amino acids. In another embodiment, the isolated peptide consists of the above-described fragment of a LEDGF/p75 protein. In another embodiment, 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.

In another embodiment, 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. In another embodiment, 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.

In another embodiment, 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 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. In another embodiment, the peptide is an isolated peptide. In another embodiment, the peptide consists of the above-described mutated fragment of a LEDGF/p75 protein. In another embodiment, the fragment of a LEDGF/p75 protein contains residues other than those set forth in SEQ ID NO: 2. In another embodiment, if residues other than those set forth in SEQ ID NO: 2 are present, then 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. In another embodiment, 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 (e.g. the fragment of a LEDGF/p75 protein that comprises SEQ ID NO: 1 or that comprises SEQ ID NO: 2) is, in another embodiment, 7-25 amino acids (AA). In another embodiment, the length of the LEDGF/p75 fragment is 8-25 AA. In another embodiment, the length is 9-25 AA. In another embodiment, the length is 10-25 AA. In another embodiment, the length is 11-25 AA. In another embodiment, the length is 12- 25 AA. In another embodiment, the length is 13-25 AA. In another embodiment, the length is 14-25 AA. In another embodiment, the length is 15-25 AA. In another embodiment, the length is 6-24 AA. In another embodiment, 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. In another embodiment, 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. In another embodiment, 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. In another embodiment, 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.

Defining herein 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. In another embodiment, 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. In another embodiment, the term refers only to the number of unmodified AA in the peptide or peptidomimetic. In another embodiment, 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 (e.g. an isolated peptide comprising a native or mutated version of SEQ ID NO: 1, 2, or 3) is, in another embodiment, 7-100 AA. In another embodiment, the length is 8-100

AA. In another embodiment, 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. In another embodiment, 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. In another embodiment, 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. In another embodiment, 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

13-50 AA. In another embodiment, 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.

In another embodiment, 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 in length, and the sequence of the fragment of a LEDGF/p75 protein comprises the sequence IHAEIKNSLKIDNLDVNRCIEALD (SEQ ID NO: 3).

Preferably, the length of the peptide is 6-100 amino acids. In another embodiment, the isolated peptide consists of the above-described fragment of a LEDGF/p75 protein. In another embodiment, the LEDGF/p75 fragment consists of residues 353-378 from SEQ

ID NO: 40 or a corresponding fragment from a homologous protein. Each possibility represents a separate embodiment of the present invention.

In another embodiment, 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. In another embodiment, the isolated peptide consists of the above-described fragment of a LEDGF/p75 protein. Preferably, the length of the peptide is 6-100 AA. In another embodiment, the isolated peptide consists of the above-described fragment of a LEDGF/p75 protein. In another embodiment, 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. Each possibility represents a separate embodiment of the present invention.

In another embodiment, 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

13-23 AA. In another embodiment, 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. In another embodiment, 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.

In some embodiments, 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

In another embodiment, 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. Preferably, the single AA modification is selected from the group consisting of a substitution, an insertion, and a deletion. In another embodiment, the peptide is an isolated peptide. In another embodiment, the peptide consists of the above-described mutated fragment of a LEDGF/p75 protein. In another embodiment, the fragment of a LEDGF/p75 protein contains residues other than those set forth in SEQ ID NO: 3. In another embodiment, if residues other than those set forth in SEQ ID NO: 3 are present, then 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. In another embodiment, 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.

In another embodiment, 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). Preferably, the length of the isolated peptide is 12-100 amino acids. In another embodiment, the sequence of the isolated peptide is KKIRRFVSQVIM (SEQ ID NO: 41). Each possibility represents a separate embodiment of the present invention.

The term "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. In another embodiment, 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. In another embodiment, the term refers only to the number of unmodified AA in the peptide or peptidomimetic. In another embodiment, 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 (e.g. an isolated peptide comprising a native or mutated version of SEQ ID NO: 41) 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. In another embodiment, 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. In another embodiment, 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.

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 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. Each possibility represents a separate embodiment of the present invention.

A "mutation" of methods and compositions of the present invention is, in another embodiment, a substitution. In another embodiment, the mutation is an insertion. In another embodiment, the mutation is a deletion. In another embodiment, the mutation is an internal deletion. In another embodiment, the mutation is a truncation. Preferably, the term "mutation" refers to an alteration or modification in the sequence of either a peptide or a nucleotide molecule encoding same. In another embodiment, 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.

Each possibility represents a separate embodiment of the present invention. The term "peptide," as used herein, 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.

Preferably, 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. In another embodiment, 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.

Methods for measuring the oligomeric state of HIV-I IN are well known in the art, and include the methods disclosed herein (see, inter alia, Examples 2-3 and the sections entitled "fluorescence anisotropy," "analytical gel filtration," and "analytical ultracentrifugation" hereinbelow). Each method represents a separate embodiment of the present invention.

More preferably, 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. In another embodiment, the inhibition is measured in a physiological solution. Each possibility represents a separate embodiment of the present invention.

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.

More preferably, 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. In another embodiment, the inhibition is measured in a physiological solution. Each possibility represents a separate embodiment of the present invention.

Methods for measuring binding of an HIV-I integrase protein to an HIV-I LTR DNA terminus are well known in the art, and include the methods disclosed herein (see, inter alia, Example 2 and the section entitled "fluorescence anisotropy" hereinbelow). Each method represents a separate embodiment of the present invention.

"Inhibiting binding" refers, preferably, to ability to inhibit IN binding to LTR DNA with an IC₅₀ of 1 nM (nanomolar)-5 mcM (micromolar). In another embodiment, the term refers to ability to inhibit binding with an IC₅₀ of 1 nM-4 mcM. In another embodiment, the term refers to an IC₅₀ range of 1 nM-10 mcM. In another embodiment, the term refers to an IC₅₀ 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 IC₅₀ range of 1 nM-2.5 mcM. In another embodiment, the term refers to an IC₅₀ range of 1 nM-2 mcM. In another embodiment, the term refers to an IC₅₀ range of 1 nM-1.5 mcM. In another embodiment, the term refers to an IC₅₀ 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 IC₅₀ range of 1-500 nM. In another embodiment, the term refers to an IC₅₀ range of 1-300 nM. In another embodiment, the term refers to an IC₅₀ range of 1- 200 nM. In another embodiment, the term refers to an IC_5O range of 1-100 nM. In another embodiment, the term refers to an IC_5O range of 1-70 nM. In another embodiment, the term refers to an IC₅₀ range of 1-50 nM. In another embodiment, the term refers to an IC₅₀ range of 1 nM-30 nM. In another embodiment, the term refers to an IC₅₀ 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. In another embodiment, the term refers to an IC₅₀ range of 2 nM-2.5 mcM. In another embodiment, the term refers to an IC₅₀ range of 2 nM-2 mcM. In another embodiment, the term refers to an IC₅₀ range of 2 nM-1.5 mcM. In another embodiment, the term refers to an IC₅₀ 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 IC₅₀ range of 2- 500 nM. In another embodiment, the term refers to an IC₅₀ range of 2-300 nM. In another embodiment, the term refers to an IC_5O range of 2-200 nM. In another embodiment, the term refers to an IC₅₀ range of 2-100 nM. In another embodiment, the term refers to an IC₅₀ range of 2-70 nM. In another embodiment, the term refers to an IC₅₀ range of 2-50 nM. In another embodiment, the term refers to an IC₅₀ range of 2-30 nM. In another embodiment, the term refers to an IC₅₀ range of 3nM-10mcM. In another embodiment, the term refers to an IC₅₀ 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 IC₅₀ range of 3 nM-3mcM. In another embodiment, the term refers to an IC₅₀ range of 3nM-2.5mcM. In another embodiment, the term refers to an IC₅₀ range of 3 nM-2mcM. In another embodiment, the term refers to an IC₅₀ range of 3nM-lmcM. In another embodiment, the term refers to an IC₅₀ 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 IC₅₀ range of 3-300 nM. In another embodiment, the term refers to an IC₅₀ range of 3-200 nM. In another embodiment, the term refers to an IC₅₀ range of 3- 100 nM. In another embodiment, the term refers to an IC₅₀ range of 3-70 nM. In another embodiment, the term refers to an IC₅₀ 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 IC₅₀ 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 IC₅₀ range of 5nM-3mcM. In another embodiment, the term refers to an IC₅₀ range of 5nM-2mcM. In another embodiment, the term refers to an IC₅₀ range of 5nM-lmcM. In another embodiment, the term refers to an IC₅₀ range of 5-700 nM. In another embodiment, the term refers to an IC₅₀ range of 5-500 nM. In another embodiment, the term refers to an IC₅₀ range of 5-300 nM. In another embodiment, the term refers to an IC₅₀ range of 5-200 nM. In another embodiment, the term refers to an IC₅₀ range of 5-100 nM. In another embodiment, the term refers to an IC₅₀ range of 5-50 nM. In another embodiment, the term refers to an IC₅₀ range of 5-30 nM. In another embodiment, the term refers to an IC₅₀ range of IOnM-lOmcM. In another embodiment, the term refers to an IC_5O range of 10nM-7mcM. In another embodiment, the term refers to an IC₅₀ range of 10nM-5mcM. In another embodiment, the term refers to an IC₅₀ range of 10nM-3mcM. In another embodiment, the term refers to an IC₅₀ 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 IC₅₀ range of 10-700 nM. In another embodiment, the term refers to an IC₅₀ range of 10-500 nM. In another embodiment, the term refers to an IC₅₀ range of 10-300 nM.

In another embodiment, the term refers to an ICs₀ range of 10-200 nM. In another embodiment, the term refers to an IC₅₀ 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 IC₅₀ range of 10-50 nM. In another embodiment, the term refers to an IC₅₀ 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 IC₅₀ range of 20nM-7mcM. In another embodiment, the term refers to an IC₅₀ range of 20nM-5mcM. In another embodiment, the term refers to an IC₅₀ range of 20nM-3mcM. In another embodiment, the term refers to an IC₅₀ range of 20nM-2mcM. In another embodiment, the term refers to an IC₅₀ range of 2OnM-I mcM. In another embodiment, the term refers to an IC₅₀ range of 20- 700 nM. In another embodiment, the term refers to an IC₅₀ range of 20-500 nM. In another embodiment, the term refers to an ICs₀ range of 20-300 nM. In another embodiment, the term refers to an IC₅₀ range of 20-200 nM. In another embodiment, the term refers to an IC_5O range of 20-100 nM. In another embodiment, the term refers to an IC₅₀ range of 20-70 nM. In another embodiment, the term refers to an IC₅₀ 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 IC₅₀ range of 30nM-7mcM. In another embodiment, the term refers to an IC₅₀ range of 30nM-5mcM. In another embodiment, the term refers to an IC₅₀ range of 30nM-3mcM. In another embodiment, the term refers to an IC₅₀ range of 30nM-2mcM. In another embodiment, the term refers to an ICs₀ range of 3OnM- lmcM. In another embodiment, the term refers to an IC₅₀ range of 30- 700 nM. In another embodiment, the term refers to an IC₅₀ range of 30-500 nM. In another embodiment, the term refers to an ICs₀ 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 IC₅₀ range of 30-100 nM. In another embodiment, the term refers to an IC₅₀ range of 30-70 nM. In another embodiment, the term refers to an IC₅₀ range of 30- 50 nM. In another embodiment, the term refers to an IC₅₀ range of 50nM-10mcM. In another embodiment, the term refers to an IC₅₀ range of 50nM-7mcM. In another embodiment, the term refers to an IC₅₀ range of 50nM-5mcM. In another embodiment, the term refers to an IC₅₀ range of 50nM-3mcM. In another embodiment, the term refers to an IC₅₀ range of 50nM-2mcM. In another embodiment, the term refers to an IC₅₀ 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 IC₅₀ range of 50-500 nM. In another embodiment, the term refers to an IC₅₀ range of 50-300 nM. In another embodiment, the term refers to an IC₅₀ range of 50-200 nM. In another embodiment, the term refers to an IC₅₀ range of 50-100 nM. In another embodiment, the term refers to an IC₅₀ range of 50-70 nM. In another embodiment, the term refers to an IC₅₀ range of 70nM-10mcM. In another embodiment, the term refers to an IC₅0 range of 7OnM- 7mcM. In another embodiment, the term refers to an IC₅O range of 70nM-5mcM. In another embodiment, the term refers to an IC₅₀ range of 70nM-3mcM. In another embodiment, the term refers to an IC₅₀ range of 70nM-2mcM. In another embodiment, the term refers to an IC₅₀ range of 7OnM-I mcM. In another embodiment, the term refers to an IC₅₀ range of 70-700 nM. In another embodiment, the term refers to an IC₅₀ range of 70-500 nM. In another embodiment, the term refers to an IC₅₀ range of 70-300 nM. In another embodiment, the term refers to an IC₅₀ 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 IC₅₀ range of lOOnM-lOmcM. In another embodiment, the term refers to an IC₅₀ range of 100nM-7mcM. In another embodiment, the term refers to an IC₅₀ range of 100nM-5mcM. In another embodiment, the term refers to an IC₅₀ range of 100nM-3mcM. In another embodiment, the term refers to an IC₅₀ range of 10OnM- 2mcM. In another embodiment, the term refers to an IC₅₀ range of lOOnM-lmcM. In another embodiment, the term refers to an IC₅₀ 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 IC₅₀ range of 100-300 nM. In another embodiment, the term refers to an IC₅₀ range of 100-200 nM. In another embodiment, the term refers to an IC_5O range of 100-150 nM. In another embodiment, the term refers to an IC₅₀ range of 15OnM- lOmcM. In another embodiment, the term refers to an IC₅₀ range of 150nM-7mcM. In another embodiment, the term refers to an IC₅₀ range of 150nM-5mcM. In another embodiment, the term refers to an IC₅₀ range of 150nM-3mcM. In another embodiment, the term refers to an IC₅₀ range of 150nM-2mcM. In another embodiment, the term refers to an IC₅₀ range of 150nM-lmcM. In another embodiment, the term refers to an IC₅o range of 150-700 nM. In another embodiment, the term refers to an IC₅₀ range of 150-500 nM. In another embodiment, the term refers to an IC₅₀ range of 150-300 nM. In another embodiment, the term refers to an IC₅₀ 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 IC₅₀ range of 200nM-7mcM. In another embodiment, the term refers to an IC₅₀ range of 200nM-5mcM. In another embodiment, the term refers to an IC₅₀ range of 200nM-3mcM. In another embodiment, the term refers to an

IC₅₀ range of 200nM-2mcM. In another embodiment, the term refers to an IC₅₀ range of 20OnM- lmcM. In another embodiment, the term refers to an IC₅₀ range of 200-700 nM. In another embodiment, the term refers to an IC₅₀ range of 200-500 nM. In another embodiment, the term refers to an IC₅₀ range of 200-300 nM. In another embodiment, the term refers to an IC₅₀ 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. In another embodiment, the term refers to an IC_5O range of 300nM-3mcM. In another embodiment, the term refers to an IC₅₀ range of 30OnM- 2mcM. In another embodiment, the term refers to an IC₅₀ range of 30OnM- lmcM. In another embodiment, the term refers to an IC₅₀ 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 IC₅₀ range of 50OnM-I OmcM. In another embodiment, the term refers to an IC₅₀ range of 500nM-7mcM. In another embodiment, the term refers to an IC₅₀ range of 500nM-5mcM. In another embodiment, the term refers to an IC₅₀ 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 IC₅₀ range of 50OnM- lmcM. In another embodiment, the term refers to an IC₅₀ range of 500-700 nM. In another embodiment, the term refers to an IC₅₀ range of 70OnM-I OmcM. In another embodiment, the term refers to an IC₅₀ 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 IC₅₀ range of 700nM-3mcM. In another embodiment, the term refers to an IC₅₀ range of 700nM-2mcM. In another embodiment, the term refers to an IC₅₀ 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 IC₅o range of l-2mcM. In another embodiment, the term refers to an IC_5O range of 1.5- lOmcM. In another embodiment, the term refers to an IC₅O range of 1.5-7mcM. In another embodiment, the term refers to an IC₅₀ range of 1.5-5mcM. In another embodiment, the term refers to an IC₅₀ range of 1.5-3mcM. In another embodiment, the term refers to an IC₅₀ range of 2-1 OmcM. In another embodiment, the term refers to an IC₅o 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. In another embodiment, the term refers to an IC₅₀ range of 3-7mcM. In another embodiment, the term refers to an IC₅₀ range of 3-5mcM. In another embodiment, the term refers to an IC₅₀ range of 5- lOmcM. In another embodiment, the term refers to an IC₅₀ range of 5-7mcM. Each possibility represents a separate embodiment of the present invention.

In another embodiment, 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 μM) 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. In another embodiment, the term refers to ability to inhibit DNA binding by at least 2-fold under the above conditions. Each possibility represents a separate embodiment of the present invention.

"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.

Most preferably, 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," as used herein, may refer to any cell wherein HIV-I is capable of replicating. Preferably, 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.

In another embodiment, a composition of the present invention further comprises a non- naturally occurring amino acid, in addition to the fragment of a LEDGF/p75 protein. In another embodiment, a composition of the present invention further comprises an organic peptidomimetic moiety, in addition to the fragment of a LEDGF/p75 protein. In another embodiment, a side chain of an amino acid of the fragment of a LEDGF/p75 protein has been chemically modified. In another embodiment, a peptidic bond has been replaced by a non- naturally occurring peptidic bond. In another embodiment, one of the amino acids is replaced by the corresponding D- amino acid. In another embodiment, the present invention encompasses an N-methyl variant of the sequence. In another embodiment, the present invention provides a sequence disclosed herein in reverse order, preferably having all D-amino acids {retro inverso). In another embodiment, a derivative of the present invention possesses one of the above modifications at a plurality of locations (e.g. a plurality of residues). In another embodiment, a derivative of the present invention possesses two of the above modifications. In another embodiment, a derivative of the present invention possesses more than 2 of the above modifications. In another embodiment, one of the above modifications is introduced at a location outside the fragment of a LEDGF/p75 protein; i.e. in the surrounding sequence. Each possibility represents a separate embodiment of the present invention.

In another embodiment, 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.

In another embodiment, 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. In another embodiment, 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. In another embodiment, LEDGF/p75 fragment (a) comprises a fragment of SEQ ID NO: 1. In another embodiment, LEDGF/p75 fragment (b) comprises a fragment of a sequence selected from SEQ ID NO: 2 and SEQ ID NO: 41. In another embodiment, both (a) and (b) contain fragments of the respective sequences set forth above. In another embodiment, LEDGF/p75 fragment (a) comprises a mutated version of SEQ ID NO: 1. In another embodiment, LEDGF/p75 fragment (b) comprises a mutated version of a sequence selected from SEQ ID NO: 2 and SEQ ID NO: 41. In another embodiment, both (a) and (b) contain mutated versions of the respective sequences set forth above. In another embodiment, LEDGF/p75 fragment (a) contains a trp (tryptophan) residue adjacent to the amino-terminal (N-terminal) end of the LEDGF/p75 fragment. In another embodiment, LEDGF/p75 fragment (b) contains a trp residue adjacent to the N-terminal end of the LEDGF/p75 fragment. In another embodiment, each of LEDGF/p75 fragments (a) and (b) contains a trp residue adjacent to the N-terminal end of the LEDGF/p75 fragment. Each possibility represents a separate embodiment of the present invention.

In another embodiment, 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. In another embodiment, 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. In another embodiment, LEDGF/p75 fragment (a) comprises a fragment of SEQ ID NO: 3. In another embodiment, LEDGF/p75 fragment (b) comprises a fragment of a sequence selected from SEQ ID NO: 2 and SEQ ID NO: 41. In another embodiment, both (a) and (b) contain fragments of the respective sequences set forth above. In another embodiment, LEDGF/p75 fragment (a) comprises a mutated version of SEQ ID NO: 3. In another embodiment, LEDGF/p75 fragment (b) comprises a mutated version of a sequence selected from SEQ ID NO: 2 and SEQ ID NO: 41. In another embodiment, both (a) and (b) contain mutated versions of the respective sequences set forth above. In another embodiment, LEDGF/p75 fragment (a) contains a trp (tryptophan) residue adjacent to the amino-terminal (N-terminal) end of the LEDGF/p75 fragment. In another embodiment, LEDGF/p75 fragment (b) contains a trp residue adjacent to the N-terminal end of the LEDGF/p75 fragment. In another embodiment, each of LEDGF/p75 fragments (a) and (b) contains a trp residue adjacent to the N-terminal end of the LEDGF/p75 fragment. Each possibility represents a separate embodiment of the present invention. In another embodiment of a pharmaceutical composition of the present invention, the major IN-binding loop-derived peptide (e.g. LEDGF/p75 353-378 or 361-370, a fragment thereof, or a mutated version thereof) and the minor IN-binding loop-derived peptide (e.g. LEDGF/p75 401-412, SEQ ID NO: 41, a fragment thereof, or a mutated version thereof) exhibit synergy in their inhibition of IN DNA binding. In another embodiment, the major IN-binding loop-derived peptide and the minor IN-binding loop- derived peptide exhibit synergy in their inhibition of IN 3 '-end processing activity. In another embodiment, 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.

In another embodiment, the present invention provides a pharmaceutical composition of the present invention for inhibiting replication of an HIV-I in a target cell.

In another embodiment, the present invention provides a pharmaceutical composition of the present invention for treating HIV-I infection in a subject in need thereof.

In another embodiment, 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.

In another embodiment, the present invention provides a pharmaceutical composition of the present invention for inhibiting 3 '-end processing of an HIV-I integrase protein. In another embodiment, the pharmaceutical composition is utilized in an in vitro assay. In another embodiment, the pharmaceutical composition is utilized in a target cell. Each possibility represents a separate embodiment of the present invention.

In another embodiment, 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.

In another embodiment, 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.

In another embodiment, 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. In another embodiment, the pharmaceutical composition is utilized in an in vitro assay. In another embodiment, the pharmaceutical composition is utilized in a target cell. Each possibility represents a separate embodiment of the present invention.

In another embodiment, 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.

In another embodiment, 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.

In another embodiment, 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.

In another embodiment, 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. In another embodiment, the method is performed in an in vitro assay. In another embodiment, the method is performed in a target cell. Each possibility represents a separate embodiment of the present invention.

In another embodiment, a peptide of the present invention is capable of entering a mammalian cell under physiological conditions. In another embodiment, the peptide penetrates the cell membrane of the mammalian cell. In another embodiment, the peptide is actively transported through the cell membrane. In another embodiment, the peptide diffuses through the cell membrane. Each possibility represents a separate embodiment of the present invention.

"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:

MTRDFKPGDLIFAKMKGYPHWPARVDEVPDGAVKPPTNKLPIFFFGTHETAFL GPKDIFPYSENKEKYGKPNKRKGFNEGLWEIDNNPKVKFSSQQAATKQSNASS DVEVEEKETSVSKEDTDHEEKASNEDVTKAVDITTPKAARRGRKRKAEKQVET EEAGVVTTATASVNLKVSPKRGRPAATEVKIPKPRGRPKMVKQPCPSESDIITEE DKSKKXGQEEKQPKKQPKKDEEGQKEEDKPRKEPDKKEGKKEVESKRKNLAK TGVTSTSDSEEEGDDQEGEKKRKGGRNFQTAHRRNMLKGQHEKEAADRKRKQ EEQMETEQQNKDEGKKPEVKKVEKKRETSMDSRLQRIHAEIKNSLKIDNLDVN RCIEALDELASLQVTMQQAQKHTEMITTLKKIRRFKVSQVIMEKSTMLYNKFK NMFLVGEGDSVITQVLNKSLAEQRQHEEANKTKDQGKKGPNKKLEKEQTGSK TLNGGSDAQDGNQPQHNGESNEDSKDNHEASTKKKPSSEERETEISLKDSTLDN (SEQ ID NO: 40).

As provided herein, 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.

In another embodiment, 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.

In another embodiment, a peptide of the present invention further comprises an organic, non-peptidic moiety. In another embodiment, the peptide of the present invention comprises a hydrophobic moiety attached to the end of the LEDGF/p75-derived peptide. In another embodiment, the hydrophobic moiety is a linear hydrocarbon, hi another embodiment, the hydrophobic moiety is a branched hydrocarbon. In another embodiment, the hydrophobic moiety is a linear hydrocarbon. In another embodiment, the hydrophobic moiety is a cyclic hydrocarbon, hi another embodiment, the hydrophobic moiety is a polycyclic hydrocarbon. In another embodiment, the hydrophobic moiety is a heterocyclic hydrocarbon. In another embodiment, the hydrophobic moiety is a hydrocarbon derivative. In another embodiment, the hydrophobic moiety is a protecting group. In another embodiment, the protecting group serves to decrease degradation (e.g. of a linear compound).

In another embodiment, 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.

In another embodiment, 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. In another embodiment, 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. Each possibility represents a separate embodiment of the present invention.

In another embodiment, the additional sequence(s) or moiety(ies) improves a pharmacological property of the peptide, hi another embodiment, the additional sequence(s) or moiety(ies) improves a physiological property of the peptide, hi another embodiment, the property is penetration into cells (e.g. moieties which enhance penetration through membranes or barriers, generally termed "leader sequences"), hi another embodiment, the modified peptides exhibit slower degradation in vivo, hi another embodiment, the modified peptides exhibit slower clearance in vivo, hi another embodiment, the modified peptides exhibit decreased repulsion by various cellular pumps. In another embodiment, the modified peptides exhibit decreased immunogenicity. hi another embodiment, 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.

Preferably, the LEDGF/p75-derived amino acid sequence is in association with (in the meaning described above) a moiety for transport across cellular membranes.

The term "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. Examples of such 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. Other examples of other 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. In the above examples, 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.

These moieties can be cleaved in vivo, either by hydrolysis or enzymatically, inside the cell. (Ditter et al, J. Pharm. Sci. 57:783 (1968); Ditter et al., J. Pharm. Sci. 57:828 (1968); Ditter et al., J. Pharm. Sci. 58:557 (1969); King et al, Biochemistry 26:2294 (1987); Lindberg et al, Drug Metabolism and Disposition 1_7:311 (1989); and Tunek et al, Biochem. Pharm. 37:3867 (1988), Anderson et al, Arch. Biochem. Biophys. 239:538 (1985) and Singhal et al, FASEB J. 1:220 (1987)). 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. In one embodiment, 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.

Examples of 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. Specific examples of 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-. Examples of alkoxy carbonyl and aryloxy carbonyl groups include CH3-O-CO-,

(ethyl)-O-CO-, n-propyl-O-CO-, iso-propyl-O-CO-, n-butyl-O-CO-, sec-butyl-O-CO-, t-butyl-O-CO-, phenyl-O- CO-, substituted phenyl-O-CO- and benzyl-0-CO-, (substituted benzyl)- O-CO-. Adamantan, naphtalen, myristoleyl, tuluen, biphenyl, cinnamoyl, nitrobenzoy, toluoyl, furoyl, benzoyl, cyclohexane, norbornane, Z-caproic. In order to facilitate the N-acylation, one to four glycine residues can be present in the N-terminus of the molecule.

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

-NR2R3) or ester (i.e. the hydroxyl group at the C-terminus is replaced with -OR2). R2 and R3 are independently an aliphatic, substituted aliphatic, benzyl, substituted benzyl, aryl or a substituted aryl group. In addition, 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. Examples of suitable heterocyclic rings include piperidinyl, pyrrolidinyl, morpholino, thiomorpholino or piperazinyl. Examples of C-terminal protecting groups include -NH2, -NHCH3, -N(CH₃^, -NH(ethyl), -N(ethyl)2, -N(methyl)

(ethyl), -NH(benzyl), -N(C₁-C₄ alkyl)(benzyl), -NH(phenyl), -N(Ci-C₄ alkyl) (phenyl), -OCH₃, -O-(ethyl), -O-(n-propyl), -O-(n-butyl), -O-(iso-propyl), -O-(sec- butyl),

-O-(t-butyl), -O-benzyl and -O-phenyl.

Derivatives

There are several types of derivation that can be applied to LEDGF/p75-derived amino acid sequences of the present invention. The derivative may include several types of derivation (replacements and deletions, chemical modification, change in peptidic backbone etc.)

Replacements

Typically 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. Preferably no more than 35%, 30%, 25%, 20%, 15%, 10%, or 5%. 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.

The term "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.

The term "non-naturally occurring amino acid" (amino acid analog) 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. This term 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.

The term "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. Where the 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).

As the naturally occurring amino acids are grouped according to their properties, 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. For producing conservative substitutions by non-naturally occurring amino acids, it is also possible to use amino acid analogs (synthetic amino acids) that are known in the art.

When affecting conservative substitutions, the substituting amino acid should have the same or a similar functional group in the side chain as the original amino acid.

The following are some non-limiting examples of groups of naturally occurring amino acids or of amino acid analogs are listed bellow. Replacement of one member in the group by another member of the group will be considered herein as conservative substitutions:

Group I includes leucine, isoleucine, valine, methionine, phenylalanine, cysteine, and modified amino acids having the following side chains: ethyl, n-butyl, -CH₂CH₂OH, - CH₂CH₂CH₂OH, -CH₂CHOHCH₃ and -CH₂SCH₃. Preferably 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. Preferably, 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 -(CH₂)^COOH, an ester thereof

(substituted or unsubstituted aliphatic, aromatic or benzylic ester), an amide thereof and a substituted or unsubstituted N-alkylated amide thereof. Preferably, 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, β-cycloarginine, μ-hydroxyarginine, N-amidinocitruline and 2-amino-4- guanidinobutanoic acid, homologs of lysine, homologs of arginine and ornithine. Preferably, 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. Preferably, Group VI includes serine, cysteine, and threonine.

The term "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.

In another embodiment, 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. Thus, 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. Examples of 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.

In another embodiment, a functional group may be added to the side chain, deleted from the side chain or exchanged with another functional group. Examples of 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. Examples of such modifications include tryptophan for glycine, lysine for aspartic acid and -(CH2)4COOH for the side chain of serine. These examples are not meant to be limiting. 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). The 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

(1988)); isosteres of amide bonds (Jones etal, Tetrahedron Lett. 29: 3853-3856 (1988));

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. 26:647-650 (1985); Di Maio et al, J. Chem. Soc. Perkin Trans., 1687 (1985); Kahn et al, Tetrahedron Lett. 30:2317 (1989); Olson et al, J. Am. Chem. Soc. 112:323-333 (1990); Garvey et al, J. Org. Chem. 56:436 (1990). Further suitable peptidomimetics include hydroxy- 1,2,3,4-tetrahydroisoquinoline- 3-carboxylate (Miyake et al, J. Takeda Res. Labs 43:53-76 (1989)); 1,2,3,4-tetrahydro- isoquinoline-3-carboxylate (Kazmierski et al, J. Am. Chem. Soc. 133:2275-2283 (1991)); histidine isoquinolone carboxylic acid (HIC) (Zechel et al, Int. J. Pep. Protein Res. 43 (1991)); (2S, 3S)-methyl-phenylalanine, (2S, 3R)-methyl-phenylalanine, (2R, 3S)-methyl- phenylalanine and (2R, 3R)-methyl-phenylalanine (Kazmierski and Hruby, Tetrahedron Lett. (1991)). Chemical modifications:

Typically no more than 40%, preferably 35%, 30%, 25%, 15%, 10%, 5% of the amino acids 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. For example, the side chain may be phosphorylated, glycosylated, fatty acylated, acylated, iondiated or carboxyacylated.

Deletions:

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.

Combination of modifications:

Generally at least 50%, at least 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10, or 5% of the amino acids in the parent sequence of (a)-(b) are maintained so that the combination of the deletions, chemical modifications, and replacements are no more than 50% of the total peptides as long as they retain the property of equilibrium shifting, integrase inhibition or HIV-I replication inhibition.

It should be appreciated that some of the 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)).

Typically "essential amino acids" (as will be detailed below) are maintained or replaced by conservative substitutions while non-essential amino acids may be maintained, deleted or replaced by conservative or non-conservative replacements. Generally, 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. Guidelines for the determination of the deletions, replacements and substitutions are given Other types of derivatives: D-amino acids

The term "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.

Peptidic backbone modifications (peptidomimetics)

The term "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 -CH₂- NH-), alkylation (methylation) on the nitrogen atom, or the bonds have been replaced by, urea bonds, or sulfonamide bond, etheric bond (-CH₂-O-), thioetheric bond (-CH₂-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

Reverse order:

The term "in reverse order" refers to the fact that the 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. peptides that have both a reverse order as explained above, and in addition each and every single one of the amino acids, has been replaced by the non-naturally occurring D- amino acid counterpart, so that the net end result, as regards the positioning of the side chains, (the combination of reverse order and the change from L to D) is zero change. Such retro-inverso peptides, while having similar binding properties to the native peptide, were found to be resistant to degradation

Preparation of peptides of the present invention

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. 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. Methods of solid phase peptide synthesis are described in Aarifield, R.B., Science, 232:341 (1986); Carpino, L.A. and Han, G.Y., J. Org. Chem., 37:3404 (1972); and Gauspohl, H. et al, Synthesis, 5:315 (1992)). The teachings of these references are incorporated herein by reference.

EXAMPLE 1: Design of LEDGF-derived peptides that modulate the IN oligomerization equilibrium

"Shiftides" as used herein, refers to peptides that shift the oligomerization equilibrium of a protein capable of oligomerization, in order to inhibit its activity. In one embodiment, 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. Based on the crystal structure of the IN - LEDGF/p75 complex (PDB ID: 2B4J) (22) (Figure IA-B), three fluorescein-labeled peptides were derived from the IN- binding loops of LEDGF/p75 (Figure 1C). LEDGF/p75 353-378 and LEDGF/p75 361- 370 were derived from the major IN-binding loop and LEDGF/p75 401-412 was derived from the minor IN-binding loop (Figure 1).

EXAMPLE 2: IN binds to the LEDGF-derived peptides as a tetramer and to the

DNA as a dimer

Fluorescence anisotropy was used to test the binding of HIV-I IN to the fluorescein- labeled LEDGF/p75 -derived peptides (Figure 1). IN bound the peptides from the major IN-binding loop (LEDGF 353-378 and LEDGF 361-370) with a K_d of 4 μM. IN binding to the peptide from the minor IN-binding loop was slightly weaker (.Kj = 12 μM). The binding of IN to all three peptides was strongly cooperative, with a Hill coefficient around 4 (Figure 2 and Table 1). Thus, IN binds the LEDGF peptides as a tetramer and with significant affinity. IN binding to a fluorescein labeled 36-BP double-stranded viral LTR DNA was also studied using fluorescence anisotropy. In agreement with the literature (20), IN bound the LTR DNA with K_ά of 37 nM, and a Hill coefficient of 2 (Figure 2, Table 1). This indicates that IN binds the LTR DNA as a dimer.

Table 1. Binding affinity of IN to the LTR DNA and the LEDGF/p75 derived peptides. Experiments were carried out using fluorescence anisotropy. K_d values were obtained by fitting the binding curves depicted in figure 2 to the Hill equation.

EXAMPLE 3: Ligand binding shifts the oligomerization equilibrium of IN

Based on the results of the binding studies, the effect of ligand binding on the oligomerization equilibrium of IN was characterized. Sedimentation equilibrium AUC experiments showed that free IN is in equilibrium between a high order oligomer, a tetramer, and a dimer (Figure 3A,D). Analytical gel filtration was used to study the effect of ligand binding on this equilibrium (Figure 3B-D). As depicted in Figure 3B, Free IN eluted from the column as a high order oligomer. IN was tetrameric in presence of the LEDGF/p75 361-370 peptide, but was dimeric in the presence of the LTR DNA. When incubated with both the LTR DNA and the LEDGF 361-370 peptide at 1:1 ratio, IN was tetrameric as with the peptide only. LEDGF 401-412 also shifted the oligomerization equilibrium of IN to a tetramer.

On the other hand, binding of the peptides or the DNA did not affect the oligomeric state of the truncated IN mutant IN 52-288 (Figure 3C), indicating involvement of the N-terminus of IN. These results are in excellent agreement with the fluorescence anisotropy studies described in the previous Example, since in both cases IN binds the DNA as a dimer and the LEDGF peptides as a tetramer.

EXAMPLE 4: LEDGF-derived peptides inhibit DNA binding of IN

Fluorescence anisotropy was used to test the effect of the peptides on the binding of IN to fluorescein-labeled LTR DNA in the presence of non-labeled LEDGF peptides. The LEDGF peptides derived from the major binding loop inhibited DNA binding of IN, reducing the affinity by 3 -fold to 100 nM, while the peptide derived from the minor binding loop inhibited binding 6-fold, to 200 nM (Figure 2C). Full-length LEDGF inhibited DNA binding of IN 30-fold, to 1 μM (Figure 2D), indicating a synergistic effect between the two IN-binding loops in the context of the full-length protein. Furthermore, since the full-length LEDGF and the peptides were not present simultaneously in any of the experimental systems used, inhibition was not due to the competitive inhibition for the LEDGF-IN interaction.

Thus, inhibition of DNA binding by the peptides identified herein is due to shift of oligomeric state of IN. These results show that these LEDGF peptides act as shiftides, binding IN and shifting its equilibrium to a tetramer that is unable to bind the LTR DNA (schematically depicted in Figure 4).

EXAMPLE 5: The LEDGF peptides inhibit the catalytic activities of IN in vitro

The next experiments 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. Since 3' processing does not take place, there is no DNA template for strand transfer, and this second step does not proceed despite the fact that IN is tetrameric. LEDGF 401-412 was even more potent than LEDGF 361-370 and showed significant inhibition at the lowest concentration tested of 21 μM (Figure 5).

EXAMPLE 6: The LEDGF peptides inhibit HIV-I replication in cell culture by inhibition of in catalytic activity

The next experiments tested whether inhibition of IN activity by the LEDGF peptides leads to inhibition of virus replication in cells. First, the extent of penetration of the peptides into cells was determined. Fluorescein-labeled LEDGF 361-370 and 401-412, but not the longer LEDGF 353-378 peptide, penetrated into HeLa CD4 cultured cells (Figure 6A-B). These peptides were not toxic to cells at the concentrations used, as measured by the MTT test. The effect of the LEDGF 361-370 and LEDGF 401-412 peptides on HIV-I propagation was studied using HeLa MAGI cells, which express the β-galactosidase gene under TAR regulation. Thus, expression of the HIV-I Tat protein in these infected cells turns them blue. Both LEDGF 361-370 and LEDGF 401-412 peptides at concentration of 2.5 μM significantly inhibited expression of HIV-I Tat protein at a concentration-dependent manner (Figure 6C), indicating that these peptides decrease the integration events in the MAGI cells. The peptides inhibited not only transcription of viral genes, but also HIV-I replication: A sharp decrease in the amount of the viral P24 was obtained in infected lymphoid cells incubated with peptides LEDGF 361-370 and LEDGF 401-412, indicating inhibition of production of new virions (Figure 6D). To verify that the inhibition of HIV-I replication was due to reduction of integration events, the provirus number in the cells was estimated using real-time PCR. LEDGF 361-370 and LEDGF 401-412 peptides at concentrations lower than 2.5 μM reduced integration by more than 95% compared to untreated cells (Figure 6E-F). These results confirm that the reduction of viral gene expression observed in MAGI and lymphoid cells is due to inhibition of IN activity.

Thus, excellent agreement was obtained between biophysical, biochemical and cellular studies, all of which showed that the LEDGF peptides tested shift the equilibrium of IN to the tetramer, thereby inhibiting DNA binding by IN and HIV-I replication. EXAMPLE 7: IN binding and inhibition by LEDGF 361-370 and LEDGF 401-412 is not abolished by single amino acid mutations

To define the importance of individual residues of LEDGF 361-370 and LEDGF 401- 412 in IN binding and inhibition, Ala-substitution mutants of shiftides LEDGF 361-370 and LEDGF 401-412 were tested for IN binding by fluorescence anisotropy and IN catalytic activity in a quantitative in vitro assay. 23 peptides were synthesized, wherein each single amino acid of these 2 peptides was substituted by alanine, except for tryptophan (W) in position 1, which was important in determining the exact concentration of the peptides. Truncated peptides LEDGF 365-369 and also LEDGF 405-409 were also synthesized. For binding studies, 60 μM IN was titrated into 100 nM of the fluorescein-labeled Ala scan peptides. For IN catalytic activity measurements, 390 nM IN was incubated in the presence of LEDGF 361-370 Ala scan peptides or LEDGF 401-412 Ala scan peptides at 1 :1 peptide/IN ratios, and the overall integration process was monitored using the quantitative assay system.

AU mutants bound IN to a similar degree as the wild type. Somewhat reduced binding (2-3 fold weaker) was observed for the LEDGF 361-370 mutants S362A, I365A, V370A, LEDGF 365-369, and LEDGF 405-409. The single amino acid substitutions of LEDGF 401-412 did not exhibit greatly altered binding (Figure 7, Table 2). Inhibition of IN activity by the ala-scan mutants was similar to wild-type, while HRP2 483-493 and the truncated LEDGF 365-369 and LEDGF 405-409 peptides exhibited weaker IN inhibition (Table 2). Thus, residues S362, 1365, and V370A are relatively important for binding, and modification of these residues should be performed sparingly. Other single residues can likely replaced by other groups and could provide a location for introducing chemical modifications into the molecule.

The effect of 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).

Binding affinity of HRP2 484-493 to IN was determined to be about lOμM, 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-

493 by the proline residue at position 10 (which is not found in the analogous LEDGF/p75 peptide).

Table 2. Binding affinity to IN and inhibition of IN catalytic activity by modified shiftides.

Table 3: Sequences of the new linear modified peptides.

EXAMPLE 9: Identifying essential and non-essential amino acids in the subsequence chosen

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:

(1) Shortening of sequence:

To determine the minimum sequence of the lead peptide required for IN binding; integrase inhibition and/or HTV-I replication inhibition, 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.

(2) Systematic Amino Acid Replacement: To improve the binding affinity of the lead peptides to IN, a new series of peptides are prepared wherein the residues that do not contribute to IN binding are replaced by other natural and non-natural amino acids. Design of the mutations is based on a bioinformatics search. LEDGF/75 homologous sequences are identified, and amino acids in the lead peptide are mutated according to naturally occurring residues in these positions. In parallel, a combinatorial approach is used to discover which type of amino acid will best fit the mutation positions. Fluorescein-labeled peptides are synthesized, wherein amino acids not important for IN binding are replaced by mixtures of 4-5 natural and non-natural amino acids that have the same character (e.g. polar, hydrophobic etc.). 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.

(3) Increasing peptide stability: To stabilize the lead peptides against proteolysis, non- natural amino acids such as 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. In other experiments, 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.

(4) Conversion into peptidomimetics and Aza-scan: 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 α-carbon of one or more of the amino acid residues is replaced with a nitrogen atom. This reduces the flexibility of the parent linear peptide due to replacement of the rotatable Cα-CO bond by a more rigid urea N-CO structure, and leads to improved pharmacological properties such as increased duration of action, potency, and/or selectivity, as was shown for aza- analogues of angiotensin II and somatostatin and of serine and cysteine protease inhibitors. 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 α-carbon to the α-nitrogen.

(6) Conversion into small molecules: Next, further modifications are introduced into the most potent peptidomimetic shiftide and in its backbone and side chains to further convert it into a small molecule. The modifications are in further modification of the peptide bonds (e.g. reduction of the carbonyl to methylene), and replacement of the side chains by organic groups with similar properties, to fine-tune the interaction with IN.

Omission scan

In other experiments, identification of essential vs. non-essential amino acids in the peptide is achieved by preparing several peptides in which each amino acid is sequentially omitted ("omission-scan"). Amino acids whose loss results in reduction in physiological activity can be defined as "essential," while those whose loss does not cause significant change of activity can be defined as "non-essential" (Morrison et al, Chemical Biology 5:302-307, 2001).

MATERIALS AND EXPERIMENTAL METHODS

Peptide synthesis, labeling and purification

Peptides were synthesized on an Applied Biosystems (ABI) 433A peptide synthesizer using standard Fmoc chemistry. The peptides were labeled using 5¹ (and 6¹) 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.

Protein expression and purification The 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).

Fluorescence anisotropy

Measurements were performed at 1O⁰C by using a PerkinElmer LS-50b luminescence spectrofluorimeter equipped with a Hamilton microlab M dispenser. Fluorescein-labeled peptides or LTR DNA were dissolved in 20 mM Tris buffer pH 7.4, at the desired ionic strength of 190 mM to a final concentration of 0.05-0.1 μM. 1 ml of the labeled ligand solution were placed in a cuvette, and the non-labeled protein (200 μl, -100 μM) was titrated into the labeled ligand in 20 additions of 10 μl at 1-min intervals. Total fluorescence and anisotropy were measured after each addition. The excitation wavelength was 480 nm and the emission wavelength was 530 nm. Bandwidths were changed depending on the amount of the labeled molecule used. Data were fit to the Hill equation (equation 1):

E^qu^ation ^{1 :}

wherein: 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 and K_a is the association constant (1/ *d ).

In competition experiments between peptides and LTR DNA, a mixture of LEDGF/p75 peptide or the full-length LEDGF\p75 (50OnM) and IN (4 μM) was incubated for 1/2 h and then titrated into fluorescein-labeled LTR DNA (1OnM). LTR DNA sequences used were:

F1-U5B36 5'-6-FAM-AGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGT-S' (SEQ ID NO: 4).

U5B365 '-AGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGT-S' (SEQ ID NO: 5).

2U5B36 5'-ACTGCTAGAGATTTTCCACACTGACTAAAAGGGTCT-3 (SEQ ID NO: 6).

Analytical gel filtration

Analytical gel filtration of purified IN 1-288 and IN 52-288 at concentrations of lOμM was performed on AKTA Explorer (Pharmacia) using Superose 12 analytical column 30x1 cm (GE Healthcare - Pharmacia) equilibrated with buffer 20 mM Tris pH 7.4, 1 M NaCl, 10% Glycerol. Proteins were eluted with a flow rate of 1 ml/min at 4⁰C and the elution profile was recorded by continuously monitoring the UV absorbance at 220 nm. The column was calibrated with molecular weight standards (GE Healthcare — Pharmacia). Analytical ultracentrifugation

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.

All experiments were done at 10 °C. Sample volume was 50 μL. Samples were considered to be at equilibrium as was judged by the comparing several scans at each speed. Buffer conditions were 20 mM Tris, pH 7.4, 10% glycerol. Ionic strength of the buffer was adjusted to 190 mM with a stock solution of 3 M NaCl in the same buffer.

Data were processed and analyzed using UltraSpin software (http://www.mrc- cpe.cam.ac.uk).

Cell penetration experiments

The fluorescein-labeled peptides LEDGF 353-378, 361-370 and 401-412 in a final concentration of 10 μM in PBS were incubated with HeLa cells for 2 h at 37⁰C. After three washes in PBS, unfixed cells were visualized by fluorescence microscopy.

In-vitro 3 '-end processing and strand transfer assays

The 3' end processing and the strand transfer activities of the IN were performed as follows:

The following gel-purified oligonucleotides were used in the enzymatic assays of HIV-I IN: A (21-mer), 5'-GTGTGGAAAATCTCTAGCAGT-3'- SEQ ID NO: 42; B (21-mer), 5^I-ACTGCTAGAGATTTTCCACAC-3¹- SEQ ID NO: 43; C (19-mer), 5'- GTGTGGAAAATCTCTAGCA-3'- SEQ ID NO: 44; D (38-mer), 5'- TGCTAGTTCTAGCAGGCCCTTGGGCCGGCGCTTGCGCC-S'- SEQ ID NO: 45. Oligonucleotides A-C correspond to the U5 end of the HIV-I long terminal repeat. Underlined letters indicate the highly conserved CA/TG dinucleotide pair. 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. In order to test the 3'-end processing and the resulting strand transfer activity of IN, the 5 '-end-labeled oligonucleotide A, annealed to its complementary strand, oligonucleotide B (both 21 nucleotides long), was used. 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 μCi Of ³²P-ATP, in a final volume of 50 μl 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.

Assays of the 3 '-End Processing and Strand Transfer (or DNA Joining). In the strand transfer assays described, the labeled 5'-end substrate employed served as both the target and donor DNA leading to an increase in the molecular size of the substrate, whereas in the 3 '-end processing assays, unique cleavage of the labeled substrates was followed. All reactions were performed in 10-μl reaction mixtures with 0.33 pmol of the labeled duplex DNA substrate and the reaction buffer, containing 90 mM NaCl, 7.5 mM MnCl₂, 10 mM DTT, 0.1 mg/ml BSA, 25 mM MOPS (pH 7.2) and 5% glycerol. 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 μl of formamide loading buffer (90% formamide, 10 mM EDTA, 1 mg/ml bromphenol blue, 1 mg/ml xylene cyanole). Samples were heat- denatured, cooled on ice, and loaded onto 6 M urea, 14% polyacrylamide denaturing gels, followed by electrophoresis (urea-PAGE). The gels were dried and subjected to autoradiography at -80 ⁰C or at room temperature to obtain essentially linear exposures.

Cells

Monolayer Adherent HeLa and Hek 293 T cells were grown in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal calf serum (FCS), 0.3 g/1 L- glutamine, 100 U/ml penicillin and 100 U/ml streptomycin (Biological Industries).

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% FCS₅ 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% CO₂ 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 Δenv/VSV-G viruses were harvested from 293T cells 48 and 72 h post transfection with pSVC21 Δenv. Viruses were stored at -75⁰C until titration.

Infection of cultured cells Cultured lymphocytes (1x10⁵) were centrifuged for 5 min at 2000 rpm, the supernatant was aspirated and the cells re-suspended in 0.2-0.5 ml of medium containing virus at multiplicity of infection (m.o.i.) of 0.3 to 2. Following absorption for 1 h at 37° C, the cells were washed to discard unbound virus and incubated for an additional 1-10 days.

HIV-I titration Titration of HIV-I was carried out by the multinuclear activation of a galactosidase indicator (MAGI) assay. TZM-bl cells were transferred into 96-well plates at 10x10³ cells per well. On the following day, the cells were infected with 50 μl of serially diluted virus in the presence of 20 μg/ml of DEAE-dextran (Pharmacia, Sweden). Two days post-infection, cultured cells were fixed with 1% formaldehyde and 0.2% glutaraldehyde in PBS. Following intensive wash with PBS, cells were stained with a solution of 4 mM potassium ferrocyanide, 4 mM potassium ferricyanide, 2 mM MgCl₂ and 0.4 mg/ml of X-GaI (Ornat). Blue cells were counted under a light microscope at a magnification of X 200.

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.

Nested Quantitative PCR analysis

Analysis of HIV-I integrated DNA was preformed as described in Yamamoto et al (Yamamoto, N., Tanaka, C, Wu, Y., Chang, M. O., Inagaki, Y., Saito, Y., Naito, T.,

Ogasawara, H., Sekigawa, I., and Hayashida, Y. (2006) Virus Genes 32(1), 105-113). During first-round PCR, integrated HIV-I sequences were amplified with the HIV-I LTR-specific primer (LTR-TAG-F 5'-

ATGCCACGTAAGCGAAACTCTGGCTAACTAGGGAACCCACTG-B'; SEQ ID NO: 7) and Alu-targeting primers (first-Alu-F 5'-AGCCTCCCGAGTAGCTGGGA-S', SEQ ID NO: 8; and first-Alu-R 5'-TTACAGGCATGAGCCACCG-S', SEQ ID NO: 9) (29) that annealed to conserved regions of the AIu repeat element.

AIu-LTR sequences were amplified from 1/10 of total cell DNA in a 25 μl reaction mixture containing PCR buffer xl, 3.5 mM MgCl₂, 200 μM dNTPs, 300 nM primers, and 0.025 U/μl Taq polymerase. First-round PCR cycle conditions were as follows: a DNA denaturation and polymerase activation step of 10 min at 95⁰C and then 12 cycles of amplification (95⁰C for 15 sec, 60⁰C for 30 sec, 72⁰C for 5 min).

During second-round PCR, 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 (95⁰C for 10 min), followed by 50 cycles of amplification (95⁰C for 15 sec, 60⁰C for 60 sec). SVC21 plasmid containing full-length HIV-1_HXB2 viral DNA was used to generate a standard linear curve at a range of 5ng-0.25fg (R=O.99). DNA samples were assayed with quadruplets of each sample.

MTT assay

Medium was removed and cells were incubated in Earl's solution containing 0.3 mg/ml MTT for one hour. Following incubation, the solution was removed and cells were dissolved in 100 μl DMSO for 10 min at room temperature. DMSO-solubilized cells were transferred onto a 96-well ELISA plate, and OD values were read at a wavelength of 570 nm.

The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without undue experimentation and without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. The means, materials, and steps for carrying out various disclosed functions may take a variety of alternative forms without departing from the invention.

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Claims

CLAIMS:

1. An isolated fragment of a LEDGF/p75 protein, wherein said fragment of a LEDGF/p75 protein is 6-25 amino acids in length, and the sequence of said fragment of a LEDGF/p75 protein comprises either:

a. the sequence NSLKIDNLDV (SEQ ID NO: 1);

b. a portion of the sequence NSLKIDNLDV (SEQ ID NO: 1), wherein said portion is 6-9 amino acids in length; or

c. a mutant of the sequence NSLKIDNLDV (SEQ ID NO: 1), wherein said mutant comprises 1-2 amino acid modifications relative to SEQ

ID NO: 1, wherein each of said amino acid modifications is independently selected from the group consisting of a substitution, an insertion, and a deletion.

2. The isolated mutated fragment of a LEDGF/p75 protein of claim 1, wherein the sequence of said fragment of a LEDGF/p75 protein is set forth in SEQ ID NO: 1 or is a fragment thereof.

3. The isolated mutated fragment of a LEDGF/p75 protein of claim 1, wherein said fragment of a LEDGF/p75 protein is a mutated fragment of a LEDGF/p75 protein that comprises 1-2 amino acid modifications relative to SEQ ID NO: 1, wherein each of said amino acid modifications is independently selected from the group consisting of a substitution, an insertion, and a deletion.

4. An isolated fragment of a LEDGF/p75 protein, wherein said fragment of a LEDGF/p75 protein is 6-25 amino acids in length, and the sequence of said fragment of a LEDGF/p75 protein comprises either:

a. the sequence KKIRRFKVSQVIM (SEQ ID NO: 2); or

b. a portion of the sequence KKIRRFKVSQVIM (SEQ ID NO: 2), wherein said portion is 6-12 amino acids in length.

5. An isolated mutated fragment of a LEDGF/p75 protein, wherein (a) said fragment of a LEDGF/p75 protein is 6-25 amino acids in length; (b) the sequence of said fragment of a LEDGF/p75 protein comprises the sequence KKIRRFKVSQVIM (SEQ ID NO: 2); and (c) said mutated fragment of a LEDGF/p75 protein comprises 1-2 amino acid modifications relative to SEQ ID NO: 2, wherein each of said amino acid modifications is independently selected from the group consisting of a substitution, an insertion, and a deletion.

6. An isolated fragment of a LEDGF/p75 protein, wherein said fragment of a LEDGF/p75 protein is 11-25 amino acids in length, and the sequence of said fragment of a LEDGF/p75 protein comprises either:

a. the sequence IHAEIKNSLKIDNLDVNRCIEALD (SEQ ID NO: 3); or

b. a portion of the sequence IHAEIKNSLKIDNLDVNRCIEALD

(SEQ ID NO: 3), wherein said portion is 11-23 amino acids in length.

7. An isolated mutated fragment of a LEDGF/p75 protein, wherein (a) said fragment of a LEDGF/p75 protein is 11-25 amino acids in length; (b) the sequence of said fragment of a LEDGF/p75 protein comprises the sequence IHAEIKNSLKIDNLDVNRCIEALD (SEQ ID NO: 3); and (c) said mutated fragment of a LEDGF/p75 protein comprises 1-2 amino acid modifications relative to SEQ ID NO: 3, wherein each of said amino acid modifications is independently selected from the group consisting of a substitution, an insertion, and a deletion.

8. An isolated 12-mer peptide, wherein the sequence of said 12-mer peptide is KKIRRFVSQVIM (SEQ ID NO: 41 ).

9. An isolated peptide comprising the isolated fragment of a LEDGF/p75 protein of any of claims 1-7 or the 12-mer peptide of claim 8, wherein said isolated peptide is 12-100 amino acids in length.

10. The isolated peptide or fragment of a LEDGF/p75 protein of any of claims 1-9, wherein, in a physiological solution, said isolated peptide binds a tetramer of an HIV-I integrase protein with a greater affinity than said isolated peptide binds a dimer of said HIV-I integrase protein, thereby increasing the ratio of said tetramer to said dimer in said physiological solution.

11. The isolated peptide or fragment of a LEDGF/p75 protein of any of claims 1-10, wherein said isolated peptide is capable of inhibiting 3'-end processing activity of an HIV-I integrase protein.

12. The isolated peptide or fragment of a LEDGF/p75 protein of any of claims 1-11, wherein said isolated peptide is capable of inhibiting HIV-I replication in a target cell.

13. The isolated peptide or fragment of a LEDGF/p75 protein of any of claims 1-12, wherein said isolated peptide is capable of inhibiting binding of an HIV-I integrase protein to an HIV-I long terminal repeat DNA terminus.

14. The isolated peptide or fragment of a LEDGF/p75 protein of any of claims 1-7 or 9-12, further comprising a tryptophan residue adjacent to the amino-terminal end of said fragment of a LEDGF/p75 protein or said mutated fragment of a LEDGF/p75 protein.

15. The isolated peptide or fragment of a LEDGF/p75 protein of claim 14, wherein said tryptophan residue is the amino-terminal residue of said isolated peptide.

16. A pharmaceutical composition, comprising the isolated peptide or fragment of a LEDGF/p75 protein of any of claims 1-15 and a pharmaceutically acceptable carrier, diluent, or additive.

17. A pharmaceutical composition, comprising: (a) the isolated fragment of a LEDGF/p75 protein of any of claims 1-3 or a peptide comprising said isolated fragment of a LEDGF/p75 protein; (b) the isolated fragment of a

LEDGF/p75 protein of any of claims 4, 5, or 7 or a peptide comprising said isolated fragment of a LEDGF/p75 protein; and (c) a pharmaceutically acceptable carrier, diluent, or additive.

18. A pharmaceutical composition, comprising: (a) the isolated fragment of a LEDGF/p75 protein of any of claims 6-7 or a peptide comprising said isolated fragment of a LEDGF/p75 protein; (b) the isolated fragment of a LEDGF/p75 protein of any of claims 4, 5, or 7 or a peptide comprising said isolated fragment of a LEDGF/p75 protein; and (c) a pharmaceutically acceptable carrier, diluent, or additive.

19. A method of inhibiting replication of an HIV-I in a target cell, comprising administering to said target cell the isolated peptide or fragment of a LEDGF/p75 protein of any of claims 1-15, thereby inhibiting replication of an HIV- 1 in a target cell.

20. A method for treating HIV-I infection in a subject in need thereof, comprising administering to said subject the isolated peptide or fragment of a LEDGF/p75 protein of any of claims 1-15, thereby treating HIV-I infection in a subject in need thereof.

21. A method for inhibiting 3 '-end processing of an HIV-I integrase protein, comprising contacting said HIV-I integrase protein with the isolated peptide or fragment of a LEDGF/p75 protein of any of claims 1-15, thereby inhibiting 3 '-end processing of an HIV-I integrase protein.

22. A method for inhibiting binding of an HIV-I integrase protein to an HIV- 1 long terminal repeat DNA terminus, comprising contacting said HIV-I integrase protein with the isolated peptide or fragment of a LEDGF/p75 protein of any of claims 1-15, thereby inhibiting binding of an HIV-I integrase protein to an HIV-I long terminal repeat DNA terminus.

23. The pharmaceutical composition of any of claims 16-18 for inhibiting replication of an HIV-I in a target cell.

24. The pharmaceutical composition of any of claims 16-18 for treating HIV- 1 infection in a subject in need thereof.

25. The pharmaceutical composition of any of claims 16-18 for inhibiting 3'- end processing of an HIV-I integrase protein.

26. The pharmaceutical composition of any of claims 16-18 for inhibiting binding of an HIV-I integrase protein to an HIV-I long terminal repeat DNA terminus.

27. Use of the isolated peptide or fragment of a LEDGF/p75 protein of any of claims 1-15 in the preparation in a pharmaceutical composition for 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.