WO2011070533A1

WO2011070533A1 - Peptides and their derivatives inhibiting extracellular release of hiv-1 tat protein and hiv-1 replication

Info

Publication number: WO2011070533A1
Application number: PCT/IB2010/055715
Authority: WO
Inventors: Silvia Agostini; Mauro Giacca
Original assignee: International Centre For Genetic Engineering And Biotechnology (Icgeb); Scuola Normale Superiore Di Pisa
Priority date: 2009-12-10
Filing date: 2010-12-10
Publication date: 2011-06-16
Also published as: IT1397569B1; ITRM20090650A1

Abstract

A protein comprising a single or a combination of three intracytoplasmatic peptide stretches of the C- terminal domain of the Na+, K+-ATPase alpha sub-unit is herein disclosed, all said peptide stretches binding HIV-1 Tat protein. Said protein is the fusion of said three stretches, corresponding to the 824-843, 939-951 and 1007-1023 regions of said domain and have the sequence NH2 - YEQAESDIMKRQPRNPKTDK- COOH, NH2 - TRRNSVFQQGMKN- COOH, NH2 - LIIRRRPGGWVEKETYY- COOH. The invention also discloses a mammal expression vector expressing said protein and the corresponding DNA. The use of said substance is also disclosed for inhibiting HIV-1 Tat protein transactivation, inhibiting HIV-1 replication and treating HIV-1 infection and related diseases.

Description

Peptides and their derivatives inhibiting extracellular release of HIV-1 Tat protein and HIV-1 replication

The present invention relates to the medical and pharmacological field, in particular to the treatment of infectious diseases, more in particular to the treatment of infections from the HIV-1 virus. Background of the invention

Introduction of modern a nti retroviral therapy, known as highly active antiretroviral therapy (HAART), has dramatically changed the natural history of HIV/AIDS. HAART is based on the simultaneous administration of multiple drugs (usually two drugs against the viral reverse transcriptase and one against the viral protease), in order to minimize the probability of occurrence of simultaneous mutations rendering the virus insensitive to treatment. HAART-treated patients live significantly longer in the absence of major signs of immunodeficiency. This treatment, however is fraught with at least two major problems: on one hand, it appears unable to eradicate the infection while, on the other hand, it has major toxicity problems, which often determine poor compliance of the patients to therapy. In addition, the emergence of viral escape mutants resistant to multiple drugs is often reported in patients treated for prolonged periods of time with HAART. For all these reasons, the development of novel drugs and the identification of novel therapeutic targets for HIV-1 infection and AIDS is mandatory.

In this context, the HIV-1 Tat protein appears as a very appealing target for therapeutic intervention. In cell culture experimental models, the protein appears essential for HIV-1 replication, since deletion or point-mutations impairing its activity have a strong detrimental effect on viral replication. This is essentially due to the role of Tat as a transcriptional activator of the viral genes, acting by binding the TAR RNA sequence transcribed from the integrated viral LTR promoter. Additionally, the protein possesses the unusual property of being released by the expressing cells and internalized by neighboring cells; as outlined above, extracellular Tat has been reported to exert a number of pleiotropic activities both in the extracellular environment and in different cell types. Mounting evidence indicates that these activities might be involved in the pathogenesis of HIV-1 disease, including the progressive development of immunodeficiency and the specific toxic damage to the brain in the course of AIDS-associated neuropathy, a very frequent condition that accompanies HIV-1 infection. Thus, the development of drugs capable to block Tat function, either intracellular^ or extracellularly, or, better, in both compartments, appears highly desirable. So far however, the attempts aimed at designing anti-Tat drugs have led to largely disappointing results. The protein has no known enzymatic activity and possesses a highly flexible structure - no Tat crystals have been obtained yet, indicative of its flexible conformation adapting to different cellular protein partners -, which prevents the easy possibility of designing or selecting highly effective drugs. Of the few small compounds that, in the past years, have been reported to inhibit Tat or block Tat-TAR interaction, none has so far advanced toward clinical experimentation. Use of a decoy RNA sequence, consisting of a multimerized TAR element, has been described as a means to divert Tat from its natural target at the HIV-1 promoter. This decoy however, which is one of the therapeutic genes used in a few gene therapy trials, requires to be expressed intracellular^ upon delivery using viral vectors, with obvious limitations in terms of applicability. Finally, anti-Tat vaccination has been proposed as a means for both preventing HIV-1 replication and targeting extracellular Tat. However, the experimentation in this respect has so far generated very poor results.

Most secreted proteins contain an N-terminal signal peptide promoting their trafficking and subsequent delivery to the cell surface by vesicular transport through the endoplasmic reticulum (ER) and Golgi apparatus. For poorly understood reasons, however, a heterogeneous group of extracellular proteins does not make use of signal peptide-dependent secretory transport, and exit cells by a process termed "unconventional" or "non-classical" protein secretion (Nickel, W 2007. J Cell Sci 120, 2295). This group comprises pro-angiogenic mediators, among which FGF-1 and FGF-2, inflammatory cytokines (IL-lb and MIF); molecules of the extracellular matrix, including galectin-1; viral or parasitic proteins, such as Leishmania HASPB); reviewed in: Backhaus, R, et al. 2004. J Cell Sci 117, 1727; Nickel, W 2005. Traffic 6, 607; Nickel, W 2007. J Cell Sci 120, 2295; Nickel, W, et al. 2008. Annu Rev Cell Dev Biol; Schafer, T, et al. 2004. J Biol Chem 279, 6244; Seelenmeyer, C, et al. 2008. FEBS Lett 582, 1362). Of interest, in several instances the release process appears to be somehow regulated, for example by heat shock in the case of FGF-1 (Prudovsky, I, et al. 2002. J Cell Biol 158, 201; Landriscina, M, et al. 2001. J Biol Chem 276, 25549; Jackson, A, et al. 1992. Proc Natl Acad Sci U S A 89, 10691); LPS treatment for MIF (Flieger, O, et al. 2003. FEBS Lett 551, 78) or monocyte activation for IL-lb (Auron, PE, et al. 1987. J Immunol 138, 1447). Also HOV-1 Tat I unconventionally secreted through the plasma membrane (Rayne F. et al., 2009, Cell Biology International, vol. 34, no. 4, 409-413.

Despite the growing number of different proteins shown to be released from the cell through non- canonical secretion, both the extracellular release mechanisms and the molecular identity of the secretory machine(s) involved have remained elusive. Multiple independent evidence indicates that various kinds of distinct non-classical export routes may exist, ranging from vesicular transport to direct membrane translocation.

The Tat protein of the human immunodeficiency virus type 1 (HIV-1) is a small (101 aa in several clinical strains, or 86 aa in the widely utilized HXB2 laboratory strain), acting as a powerful transcriptional activator of viral gene expression. At the long terminal repeat (LTR) promoter, the protein binds a cis- acting RNA element (trans-activation-responsive region, TAR) present at the 5'-end of each viral transcript (Berkhout, B, et al. 1989. Cell 59, 273). Through this interaction, Tat activates HIV-1 transcription by promoting the assembly of transcriptionally active complexes at the LTR by multiple protein-protein interactions (reviewed in: Marcello, A, et al. 2001. IUBMB Life 51, 175; Brigati, C, et al. 2003. FEMS Microbiol. Lett. 220, 57). Besides its action on the regulation of HIV-1 gene expression, almost 20 years ago it was first demonstrated that Tat also possesses the unusual property of entering the cells when present in the extracellular milieu. Since these early experiments were performed by assessing the capacity of the protein to transactivate an LTR-reporter gene cassette, their results also implied that not only the protein enters the cells, but also that it was transported to the nucleus in a transcriptionally active form (Frankel, AD, et al. 1988. Cell 55, 1189; Green, M, et al. 1988. Cell 55, 1179). This property was later extensively characterized and shown to depend on a 9-amino acid long ((aa 49-57), arginine-rich sequence corresponding to the Tat basic domain, which also mediates nuclear transport and TAR binding; work performed in different laboratories over the last few years has shown that short peptides corresponding to this amino acid stretch can be used as a biotechnological tool for the intracellular delivery of heterologous proteins, drugs, viral vectors, siRNAs and nanoparticles (reviewed in: Fittipaldi, A, et al. 2005. Adv Drug Deliv Rev 57, 597). The present inventors have previously shown that extracellular Tat binds heparin, and that heparin/Tat interaction requires the integrity of the Tat basic domain (Rusnati, M, et al. 1997. J. Biol. Chem. 272, 11313; Rusnati, M, et al. 1999. J. Biol. Chem. 274, 28198; Rusnati, M, et al. 1998. J. Biol. Chem. 273, 16027; Hakansson, S, et al. 2001. Protein Sci 10, 2138; Ziegler, A, et al. 2004. Biophys J 86, 254). In particular, we first demonstrated that membrane-bound heparan-sulphate proteoglycans (HSPG) are the cell surface receptor for Tat internalization, since cells genetically impaired in the synthesis of these molecules fail to internalize the extracellular protein (Tyagi, M, et al. 2001. J. Biol. Chem. 276, 3254). We also found that Tat internalization occurs through an active endocytosis process, mainly involving the caveolar endocytosis route (Ferrari, A, et al. 2003. Mol. Ther. 8, 284; Fittipaldi, A, et al. 2003. J. Biol. Chem. 278, 34141). Of notice, a few of the studies that elucidated the molecular mechanisms of extracellular Tat internalization also noticed that the cells constitutively expressing Tat release the protein in the cell culture supernatant (Chang, HC, et al. 1997. AIDS 11, 1421; Tyagi, M, et al. 2001. J. Biol. Chem. 276, 3254; Tasciotti, E, et al. 2005. Hum Gene Ther 16, 1389), which remains concentrated on the cell surface and protected from proteolytic degradation (Tasciotti, E, et al. 2003. Cancer Gene Ther. 10, 64; Marchio, S, et al. 2005. Blood 105, 2802). From this location, the protein was shown to be able to both modulate HIV-1 virus infection and enter in a transcriptionally active form into neighboring cells.

The mechanisms underlying Tat release from the expressing cells have however remained largely elusive. The protein does not contain a N-terminal signal peptide driving its secretion from the ER-Golgi pathway and, accordingly, protein export appears insensitive to drugs which disrupt the integrity of such organelles (Chang, HC, et al. 1997. Aids 11, 1421). Similar to entry into the cells when present in the extracellular milieu, Tat release also depends upon the integrity of the basic region of the protein (Tasciotti, E, et al. 2005. Hum Gene Ther 16, 1389; Tasciotti, E, et al. 2003. Cancer Gene Ther 10, 64). However, release appears to be independent of HSPG production and trafficking, since it also occurs in cells genetically defective in all proteoglycan biosynthesis (Tyagi, M, et al. 2001. J Biol Chem 276, 3254). Multiple experimental evidence indicates that extracellular Tat export might play an essential role in HIV- 1 disease development, and thus represent a potential target for novel therapeutic intervention. Extracellular Tat promotes the production of cytokines and cytokine receptors; modulates the survival, proliferation and migration of different cell types; exerts angiogenic activity in vitro and in vivo; inhibits antigen-specific lymphocyte proliferation (reviewed in: Fittipaldi, A, et al. 2003. J. Biol. Chem. 278, 34141; Giacca, M 2004. Curr Drug Targets Immune Endocr Metabol Disord 4, 277). It is likely that some of the above-mentioned effects of extracellular Tat could have important implications in HIV-1 disease pathogenesis, with particular reference to the neuropathogenic role of HIV-1 infection in the central nervous system (Kolson, DL, et al. 1993. AIDS Res. Hum. Retroviruses 9, 677; Philippon, V, et al. 1994. Virology 205, 519; Sabatier, JM, et al. 1991. J. Virol. 65, 961; Weeks, BS, et al. 1995. J. Neurosci. Res. 42, 34). Since the envelope of HIV-1 virions is formed upon budding from the cell membrane, another intriguing possibility is that extracellular, membrane-bound Tat might confer still unexplored properties to the HIV virions. During the physiological events encompassing HIV-1 infection, among the remarkable properties of the virus is its ability to cross the mucosal and the blood-brain barriers. The former event takes place during primary infection; infection of the brain is one of the still unexplained hallmarks of the disease, occurring at the clinical or subclinical level in the majority of patients (Resnick, L, et al. 1988. Neurology 38, 9). Indeed, HIV-1 is able to pass through brain microvascular cells using the same pathway as cholera toxin does, which depends on the integrity of lipid rafts, without disruption of the tight junctions and in the absence of productive infection of endothelial cells (Liu, NQ, et al. 2002. J Virol 76, 6689). Of notice, this process resembles transcytosis, the characteristic epithelial transcellular vesicular pathway that occurs through caveolar endocytosis (Bomsel, M 1997. Nat. Med. 3, 42).

For all the above mentioned reasons, the inhibition of Tat function, both inside and outside the cells, has been seen as a potential therapeutical target for the treatment of HIV-1 infections and related diseases. Inhibition of Tat protein is a possible way to stop viral replication, control or suppress the infection or block the molecular mechanisms involved in HIV-1 disease development of specific organs or cell types, such as those the central nervous system.

Many strategies have been proposed for the inhibition of Tat. EP0386563, to Bayer, discloses Tat inhibiting antisense nucleotides.

CA2074188, to United States of America, discloses a DNA construct for use in gene therapy to inhibit HIV-1 replication.

WO90/08780, to United States of America provides a composition of matter TAR RNA and Tat protein

W091/15224, to Smith Kline Beecham, provides agent inhibiting RGD-mediated Tat cell adhesion of peptide mimic nature.

WO 92/02228, to Medical Research Council, discloses an oligonucleotide comprising three unpaired residues including a uridine residue corresponding to U₂3 in the sequence of HIV TAR RNA and as flanking base pairs corresponding to G26-C39 in the sequence of HIV TAR RNA. US 5,597,895, to University of Texas, as well as US 5,889,175, to Transgene S.A., disclose transdominant Tat mutants capable of inhibiting HIV-1 replication.

WO 96/40759, of Ciba-Geigy describes peptoids which inhibit the Tat/TAR interaction.

US 5,821,046, of RiboTargets Holdings, PLC provides RNA oligonucleotides that bind HIV Tat protein. US 5,686,264, to University of Texas, discloses transdominant HIV Tat substitution and truncated gene mutants, capable of inhibiting the expression of the HIV-1 virus in the presence of an equimolar concentration of the wild type Tat protein in vitro.

WO 98/47913, to University of New Jersey teaches inhibition replication by a Tat RNA-binding domain peptide analog R-Arg-Lys-Lys-Arg-Arg_Gln-Arg-Arg-Arg-X-(biotin)-NH₂. Again, University of New Jersey discloses in WO 99/02488 oligocarbamate derivatives as Tat inhibiting agents. See also WO 00/43332 for Tat-derived oligourea compounds.

Another method for inhibiting HIV-1 replication is provided by this University in WO 03/72709 with variants of Tat protein.

WO 99/42441 of CNRS discloses aromatic compounds having at least one proton donor or acceptor function capable of allosteric inhibition of Tat protein.

WO 99/46991 of University of Maryland teaches the treatment of HIV-1 associated dementia by administering Tat inhibitors.

Rana and Huq disclose in US 6,468,969 polypeptides with D-amino acids as Tat inhibitors.

Backbone cyclic peptide analogs functionally mimicking nuclear localization signal are provided in US 6,664,368, to Hebrew University of Jerusalem.

A different strategy is taught by Malfroy-Camine, in US 6,703,019, by means of cationized antibodies against Tat.

Hur and Chong, US 2004/0123877, disclose a fusion protein repressing HIV transcription, comprising a) a transcription inhibitory polypeptide or compound thereof selected from the group consisting of polypeptide strongly repressing the activity of Spl or NF-kB transcription factor, polypeptide repressing transcription activity by condensing chromatin, and a polypeptide having binding activity to promoter; and b) Tat protein. See also Giacca, 2009, Retrovirology, vol. 6, no. 2, page 18

WO 2004/084805, of The J. David Gladstone Institutes, provides a method of inducing an immune response to HIV-1 Tat protein by administering an acetylated Tat protein. Mathews et al. disclose in US 2005/221288 a variant of wt HIV-1 Tat protein bearing a 23Thr/Asn mutation and capable of inhibiting viral transcription.

Aguilar-Cordova et al., US 2005/260717, teach a method for inhibiting HIV propagation by transfecting cells with a double transdominant fusion gene, which is contructed by linking Tat and Rev transdominant mutants. siRNA strategy is disclosed in US 2007/082864 by Haren Bellan et al.

Despite many attempts, the search of an effective agent endowed with the desired inhibiting activity is still ongoing.

Na⁺,K⁺-ATPase is an enzymatic complex built into the plasma membrane that catalyzes ATP hydrolysis coupled with Na⁺ and K⁺ transfer through the membrane against the electrochemical gradient. The complex is composed of two essential subunits: the alpha subunit hydrolyzes ATP coupled with Na⁺ and K⁺ transport, whereas the beta subunit is required for protein folding and modulates substrate affinity; an auxiliary subunit modulates substrate affinity but is not required for enzymatic activity. In all animal cells Na⁺,K⁺-ATPase participates in the creation of resting membrane potential and in maintenance and regulation of cell volume, in addition to more specialized functions in tissues kidney epithelium, muscle and neurons (reviewed in: Kabakov, A 1994. J Theor Biol 169, 51). The enzymatic function of the Na,K- ATPase is specifically inhibited by cardiac glycosides of plant origin, such as digoxin and ouabain, which were first used for the treatment of heart failure over 200 years ago. At low, non-toxic concentration, inhibition of the Na⁺,K⁺-ATPase by these molecules exerts a positive inotropic effect on cardiac contractility. The site of cardiac glycoside binding is located on the extracellular part of the Na⁺,K⁺- ATPase alpha-subunit (Lingrel, JB, et al. 1994. Kidney Int Suppl 44, S32). Of notice, cells sensitivity to these inhibitors differs depending on animal species; minimal sensitivity is characteristic for the rat enzyme (Emanuel, JR, et al. 1988. J Biol Chem 263, 7726; Lane, LK, et al. 1993. J Biol Chem 268, 17930). Zampar Guillermo G et al., 1999 Biochemical Journal, vol. 422, no. 1, 129-137 show interaction of acetylated tubulin with the fifth cytoplasmatic domain of the Na⁺/K⁺-ATPase (NKA), advancing the idea that NKA may act as a microtubule-plasma membrane anchorage site. Canfield V. et al., 1998, Biochemistry, vol.37, no. 20, 7509-2960 study the regions of Na⁺/K⁺-ATPase and H⁺/K⁺-ATPase responsible of specific ion transport. Yoon T. et al., 2006, FEBS LETTERS, vol. 580, no. 14, 3558-3564 disclose interaction between nexin 6 and translationally controlled tumor protein in the regulation of Na⁺/K⁺-ATPase activity.

WO2008/055530 to Unibioscreen disclose methods and reagents for the treatment of proliferative diseases, such as tumors, by targeting alpha-1 and/or alpha-3 subunits of Na⁺/K⁺-ATPase.

WO02/00200677 discloses the peptide sequence LRMYPLKPTWWFCAPYSSLIIRRRPGGWVEKETY-Y as an ovarian polypepride useful in prevention, treatment and diagnosis of cancer, immune disorders, cardiovascular disorders and neurological diseases. While exploring the mechanisms responsible for intercellular Tat trafficking, we discovered that the protein specifically binds the catalytic subunit of the Na⁺,K⁺-ATPase and that this interaction is essential to mediate extracellular release of Tat. We also found that ouabain blocks the interaction between the two proteins, a finding that is consistent with previous observations that this drug impairs secretion of Tat (Chang, HC, et al. 1997. Aids 11, 1421), in addition to that of FGF-2 (Dahl, JP, et al. 2000. Biochemistry 39, 14877; Florkiewicz, RZ, et al. 1998. J Biol Chem 273, 544).

WO 98/37880 and WO 98/37881, to Ciblex Corporation describes small molecules with inhibiting action on leaderless protein export. A number of leaderless proteins are described, among which HIV Tat. The inhibitors interfere between the leaderless protein and the transport molecule, for example Na⁺/ ⁺ATPase. This reference, however, does not specifically address the mechanism of inhibition, does not provide specific insights into the relevance of inhibition for HIV-1 Tat export, does not explore the effectiveness of Tat export inhibition on Tat function and HIV-1 replication, and, finally, does not imply relevance of Tat export on HIV-1 disease development. In addition, this reference postulates that a direct link exists between drug treatment, Na⁺/ ⁺ATPase inhibition and leaderless protein export, which is not the case for HIV-1 Tat, since, for example, Na⁺/ ⁺ATPase mutants that are enzymatically inactive are still efficient in mediating Tat export.

In our work, the present inventors discovered that HIV-1 Tat export specifically depends upon the physical interaction of Tat and the Na⁺/ ⁺ATPase. Binding between the two proteins was found to require integrity of the basic domain of Tat and of the C-terminal region of the Na⁺,K⁺-ATPase alpha subunit. In particular, the latter region contains three short intracytoplasmic peptide stretches that are juxtaposed in the protein tridimensional structure, which are all required to bind Tat.

As a consequence of our discovery, we provide, and it is an object of the present invention, active agents endowed with the activity of inhibiting the HIV-1 Tat protein, therefore making available a method for the inhibition of all the functions exerted by Tat, either in the cells expressing this protein (in particular, inhibition of HIV-1 transcription and HIV-1 replication) or upon its extracellular release, hence a method for the treatment of HIV-1 infection and HIV-l-related diseases.

Summary of the invention

It is an object of the present invention a peptide corresponding to one of the three intracytoplasmic peptide stretches of the C-terminal domain of the Na⁺,K⁺-ATPase alpha sub-unit, or of their combination, said peptide stretches being required for binding HIV-1 Tat protein. In particular, said peptides correspond to region 824-843, region 939-951 and region 1007-1023 of C-terminal domain of the Na⁺,K⁺- ATPase alpha sub-unit, respectively.

It is another object of the present invention is one of the above peptides, or a mixture thereof, for use in the inhibition of HIV-1 virus replication, in the treatment of HIV-1 virus infection and in the treatment of HIV-l-induced diseases. A further object of the present invention is a protein comprising one or a combination of the above described three intracytoplasmic peptide stretches of the C-terminal domain of the Na⁺,K⁺-ATPase alpha subunit, which are all required for binding HIV-1 Tat protein. Said protein is obtained as a recombinant protein or is expressed in the cells upon transfer of the relative DNA coding sequence. The present invention also provides the above protein and/or peptides for use as medicament.

Still another object of the present invention is a pharmaceutical composition comprising the above protein and/or peptide as active ingredients for the inhibition of HIV-1 virus replication and in the treatment of HIV-1 virus infection or HIV-1 induced disease.

These and other objects of the invention will be disclosed in detail in the foregoing specification also by means of figures and examples.

Figure 1 A. Schematic representation of the Na⁺,K⁺-ATPase alpha subunit at the cell membrane. The transmembrane portions of the protein are shown by black rods; the extracellular and intracellular loops by gray lines. Numbering refers to NCBI Reference Sequence with accession number NM_000701.7 (Na+/K+ -ATPase alpha 1 subunit isoform). B. Na⁺,K⁺-ATPase domains. The arrows indicate the regions corresponding to the PI, P2 and P3 peptides. C. Amino acid sequence of the PI, P2 and P3 peptides of human, pig and rat. Numbering corresponds to NCBI Reference Sequences: NM_000701.7, NM_214249.1 and NM_012504.1 respectively. D. Amino acid sequence of the P1-P3-P3 fusion protein. X indicates any amino acid in the linker region between the peptides. E. DNA sequence coding for the P1-P2-P3 fusion protein. Y corresponds to any nucleotide sequence coding for the linker amino acids between the PI, P2 and P3 peptide sequences. F. Schematic representation of a transcriptional cassette for the intracellular expression of the P1-P2-P3 fusion protein. Pr: any promoter. Pa: any polyadenylation signal.

Figure 2A. Tat binds the three cytoplasmic peptides in the C-terminus of the Na⁺,K⁺-ATPase alpha 1 protein. Biotinylated peptides were bound to streptavidin beads, incubated with [S³⁵]-Tat86, extensively washed, and then resolved by SDS-PAGE. The panel shows the gel exposed to a phosphoimager. The graph shows the amount of bound proteins expressed as percentages of radiolabeled input. B. Expression of the C-Terminal cytoplasmic loops of Na⁺,K⁺-ATPase impair with transactivation. Hela cells stably expressing an LTR-driven luciferase gene were transfected with different amounts of both Tat and FLAG-P1-P2-P3, in order to assess the possible effect of an interaction between the two proteins in the transactivating activity of Tat. HeLa cells were transfected using a construct containing the U3 and R sequences of the HIV-1 LTR (or a CMV promoter in the negative controls) upstream of the Firefly luciferase gene as a reporter, and pcDNA3-Tat86 as an effector, in the presence of various amounts of FLAG-P1-P2-P3 construct; A plasmid (pCI-Neo, Promega) expressing the Renilla Luciferase gene under the control of a CMV promoter was co-transfected into cells to standardize each experiment. The measured activities were standardized by the activities of Renilla; the histograms represent the mean values of three independent experiments, together with the standard error. C. Cell treatment with PI, P2, P3 peptides block Tat86-TK release. Cells were transfected with Tat86-TK and, after 36 h, washed with heparin and then treated with 2-15 μς/ιηΙ of an equimolar mixture of the three peptides (Pmix); presence of Tat86-TK in the cell culture supernatant was analyzed by western blotting after 4 h incubation. Lane 2 shows the results of a similar experiment in which the P1-P2-P3 fusion protein was expressed intracellularly. The levels of intracellular protein expression was assessed by western blotting on whole cell lysates (WCL). D. Treatment with PI, P2 and P3 peptides impairs HIV-1 infection in a single-round cell infection assay. HOS CCR5+ cells were infected with a BAL-pseudotyped pNL4-3-luciferase HIV-1 that, prior to infection, was incubated with an equimolar mix of PI, P2 and P3 peptides (Pmix) at 15 Mg/ml concentration, at 37°C for 1 h; cells were incubated with the virus for 4 h, washed, and fresh medium containing 15 mg/ml Pmix was added; after 24 hours, the cells were harvested and the expression level of luciferase was tested. E. Inhibition of HIV-1 replication upon cell treatment with PI, P2 and P3 peptides. CEM T-cells were infected with HIV-1_{B U} for 4 h in the presence of a mixture of the three peptides (Pmix) or of a control FLAG peptide. After the infection, the medium containing the virus was washed and substituted with fresh medium, containing the corresponding peptide preparation. At t=0 and subsequently, every 3 days until the 12th day, the supernatants were tested for RT activity, while cells were diluted 1:2 and fresh peptides were added to the culture media.

Detailed description of the invention

It is a first object of the present invention a protein comprising a single or a combination of three intracytoplasmic peptide stretches of the C-terminal domain of the Na⁺,K⁺-ATPase alpha subunit, said peptide stretches all bind HIV-1 Tat protein. It is a second object of the present invention a peptide, even synthetic, corresponding to one of the three intracytoplasmic peptide stretches of the C-terminal domain of the Na⁺,K⁺-ATPase alpha sub-unit, these peptide stretches all binding HIV-1 Tat protein. According to this object, a preferred peptide is one selected from the group consisting of the peptide corresponding to region 824-843 of C-terminal domain of the human Na⁺,K⁺-ATPase alpha sub-unit; the peptide corresponding to region 939-951 of C-terminal domain of the human Na⁺,K⁺-ATPase alpha sub-unit and the peptide corresponding to region 1007-1023 of C-terminal domain of the human Na⁺,K⁺-ATPase alpha sub-unit.

Quite advantageously, the peptides, or derivatives thereof, of the present invention corresponding to one of the three intracytoplasmic peptide stretches of the C-terminal domain of the Na⁺,K⁺-ATPase alpha sub- unit, all cross the cell membrane and enter the cells upon extracellular delivery. This property, unpredictable a priori, makes the peptides and their derivatives advantageous for the administration, without need of further modifications or special pharmaceutical formulations to intracellular delivery.

The present invention also refers to all the possible medicinal chemistry modifications of the aforementioned peptides, introduced to overcome the shortcomings of native peptides in terms of short duration of action, biodistribution, oral absorption and systemic bioavailability. These modifications include, but are not limited to substitution of individual amino acids with other L-amino acids, D-amino acids, non natural amino acids or amino acid metabolites or intermediates (e.g. agmatine, desaminotyrosine), N-terminal modification, fatty acid acylation, introduction of conformational restrictions, introduction of chemical moieties inducing peptide multimerization, modification by addition of further moieties improving pharmacodynamic properties of the peptides (e.g. modification by PEGylation, glycosylation, or conjugation with hyaluronic acid, conjugation or fusion to cell penetrating peptides) and the like. The present invention also refers to the possibility of exploiting the structural information derived from the aforementioned peptides for the synthesis of chemical molecules resembling their structure (e.g. derivation of peptidomimetics and the like) using computational methods. These modifications are intended to solve the above mentioned shortcomings, but they should not affect or alter the biological activity discovered in the present invention, in particular the inhibition of HIV-1 Tat protein transactivation, the inhibition of HIV-1 replication and their use in the treatment of HIV-1 infection and/or HIV-l-associated diseases.

In particular the present invention provides also transforming the above peptides into pharmaceutically acceptable salts. Said salts are well-known to the skilled person and do not need particolar descriptions. As an exemplary reference see Remington's Pharmaceutical Sciences Handbook. All these modifications are intended to be comprised in the terms "derivative" or "derivatives".

It is a third object of the present invention the above protein and/or the above peptides, either alone or in mixture among themselves for use in the inhibition of HIV-1 Tat protein transactivation. Also, the present invention provides the above protein and/or the peptides for use in the inhibition of HIV-1 replication, consequently for use in the treatment of HIV-1 infection. The same substances are for use in the treatment of HIV-l-associated diseases.

A fourth object of the present invention is a mammalian expression vector, such as for example a plasmid, virus or other vector expressing the above protein or the individual peptides.

In general, as further object of the present invention, the above protein and/or vector and/or peptide(s) are for use as medicament. It is also another object of the present invention a pharmaceutical composition comprising the protein and/or vector and/or peptide(s) disclosed herein.

The intracellular expression of the fusion protein of the present invention blocks Tat transactivation.

Most notably, CD4⁺ T-cell treatment with a mixture of soluble, even synthetic peptides of the invention impair both Tat extracellular release and HIV-1 replication. Different embodiments of the invention

A preferred embodiment of the present invention is the protein having the sequence

NH₂-YEQAESDIMKRQPRNPKTDK (X)_n TRRNSVFQQGMKN (X)_n LIIRRRPGGWVEKETYY-COOH (SEQ ID: 1), where (X)_n is a peptide stretch of any length (n) composed of any amino acid (X), with n varying from 0 to 1,000.

Another preferred embodiment of the present invention is a DNA sequence coding for the above protein is: 5'-TATGAGCAGGCTGAGAGTGACATCATGAAGAGACAGCCCAGAAATCCCAAAACAGACAAA-(Y)_m- ACCAGGAGGAATTCGGTCTTCCAGCAGGGGATGAAGAAC-(Y)_m-

CTCATCATCAGGCGACGCCCTGGCGGCTGGGTGGAGAAGGAAACCTACTAT-3' (SEQ ID: 2), where (Y)_m is a DNA sequence of any length (m) composed of any amino acid-coding nucleotide triplet (Y), with n varying from 0 to 3,000. and the hybridizing nucleotide sequences, also under stringent hybridization, thereof. In this contest the terms "hybridization" and "stringent" refer to the conventional hybridization techniques well known to the person skilled in this field (Buzdin A and Lukyanov S (eds) Nucleic Acids Hybridization Kluwer Academic Publishers Netherlands 2007).

This sequence is another object of the present invention, together with other equivalent DNA sequences that code for the aforementioned amino acid sequences according to the genetic code (due to the degeneration of the genetic code).

Another preferred embodiment of the present invention is a peptide selected from the group consisting of:

NH₂-YEQAESDIMKRQPRNPKTDK-COOH (SEQ ID: 3) NH2-TRRNSVFQQGMKN-COOH (SEQ ID: 4)

NH₂-LIIRRRPGGWVEKETYY-COOH (SEQ ID: 5)

The protein of the present invention is prepared according to conventional techniques, part of the general common knowledge of the ordinary skilled in this art. Reference can be made to general textbooks, see for example Sambrook, Molecular Cloning a Laboratory Manual, Third Edition, Cold Spring Harbor, NY, 2001. The protein is produced and purified from a transformed microorganism, for example BL21 bacteria. The microorganism is transformed with the corresponding vector. For further details see the Methods sections in the Examples. Bacterial cultures are grown in appropriate media and protein production is induced, for example with isopropyl β-D-l-thiogalactopyranoside (IPTG) or another inducer. Bacteria are then lysed, for example in a typical lysis buffer (such as 50 mM Tris-HCI pH 8.0, 100 mM NaCI, 5% glycerol, 2 mM dithiothreitol), with usual means, for example sonication. Protein purification is carried out using conventional procedures (see Sambrook above). One example of purification technique is the well-known method with glutathione cross-linked agarose beads for GST-fusion proteins. Other techniques for the preparation of the fusion protein can be used.

Synthetic peptides of the present invention can be obtained by any conventional method, such as for example solid phase synthesis, for example the one based on Fmoc/t-Bu chemistry.

The peptide-resin complex is cleaved/deprotected using a typical reagent. In an embodiment of the invention, modified Reagent H mixture (trifluoroacetic acid 80%, phenol 3%, thioanisole 3%, 3,6-dioxa- 1,8-octanedithiol 8%, water 2.5%, methylethylsulphide 2%, hydroiodic acid 1.5% w/w) is used. Peptides are isolated, for example by precipitation with suitable means, for example diethylether, washed and freeze-dried. The crude peptides are purified by preparative RP-HPLC or other conventional technique. The purified fractions can be pooled and freeze-dried. The present invention provides proteins and peptides as active agents for use in the treatment of HIV infections. The treatment can be carried out thanks to the inhibition of HIV-1 Tat protein transactivation by the protein and/or peptides of the present invention. As result, the above protein and/or the peptides inhibit HIV-1 replication. The DNA sequence encoding for the protein can also be used in the above treatment. Accordingly, the present invention also provides a method for treating a subject suffering from HIV infection comprising administering to said subject an effective amount of the protein and/or the peptide(s) and/or the DNA sequence.

The methods of the present invention are suitable for treating individuals who have an immunodeficiency virus infection. Such individuals include those who have been diagnosed as having an HIV infection. In some cases, the individual is CD4⁺ deficient, or CD4⁺ low. The terms "CD4⁺- deficient" and "CD4⁺-low" are used interchangeably herein, and, as used herein, refer to a state of an individual in whom the number of CD4⁺ T lymphocytes is reduced compared to an individual with a healthy, intact immune system. CD4⁺ deficiency includes a state in which the number of functional CD4⁺T lymphocytes is less than about 600 CD4⁺T cells/mm³ blood; a state in which the number of functional CD4⁺T cells is reduced compared to a healthy, normal state for a given individual; and a state in which functional CD4⁺ T cells are completely absent.

As used herein, a"CD4⁺-deficient individual" is one who has a reduced number of functional CD4⁺-T cells, regardless of the reason, when compared to an individual having a normal, intact immune system. In general, the number of functional CD4⁺-T cells that is within a normal range is known for various mammalian species.

Also suitable for treatment according to the present invention are individuals who are at risk of contracting an immunodeficiency virus infection. Such individuals include, but are not limited to, individuals with healthy, intact immune systems, but who are at risk for becoming HIV infected ("at-risk" individuals). At-risk individuals include, but are not limited to, individuals who have a greater likelihood than the general population of becoming HIV infected.

Individuals at risk for becoming HIV infected include, but are not limited to, individuals at risk for HIV infection due to sexual activity with HIV-infected individuals; intravenous drug users; individuals who may have been exposed to HIV-infected blood, blood products, or other HIV- contaminated body fluids; babies who are born from HIV-infected mothers.

Also suitable for treatment with a subject method are individuals who were treated for an immunodeficiency virus infection, but who relapsed, e. g. , whose CD4⁺ T cell count was increasing in response to anti-viral therapy for HIV, but whose CD4⁺ T cell counts subsequently began to fall. The methods of the present invention are suitable for treating individuals who failed treatment with previous anti-viral therapy for the treatment of an HIV infection, ("treatment failure patients"). Such treatment failure patients include individuals who have undergone previous HAART or STI treatment regimens.

The methods of the present invention are also suitable for treating individuals who, due to HIV-1 infection, suffer of other HIV-1 concurrent diseases, including however not limited to, HIV-l-induced neuropathy or Kaposis' sarcoma.

Also suitable are individuals who were previously treated with anti-retroviral therapy for the treatment of an HIV infection, and in whom drug-resistant HIV has emerged.

The medicament which is an object of the present invention is known to the expert in the field and does not require any particular description. By way of an example, the reader is referred to the patent literature cited above.

Conveniently, said medicament is in the form of a preparation for parenteral administration, but other forms are equally suitable for carrying out the present invention. The person skilled in the art will decide the effective time of administration, depending on the patient's conditions, degree of severity of the disease, response of the patient and any other clinical parameter within the general knowledge of this matter.

The pharmaceutical compositions will contain at least one of the protein, DNA coding for the protein, peptide, or expression vector disclosed above as an active ingredient, in an amount such as to produce a significant therapeutic effect. The compositions covered by the present invention are entirely conventional and are obtained with methods which are common practice in the pharmaceutical industry, such as, for example, those illustrated in Remington's Pharmaceutical Science Handbook, Mack Pub. N. Y. - last edition, see also the patent literature cited in this description. According to the administration route chosen, the compositions will be in solid or liquid form, suitable for oral, parenteral or intravenous administration. Gene therapy is also another embodiment. The compositions according to the present invention contain, along with the active ingredient, at least one pharmaceutically acceptable vehicle or excipient. These may be particularly useful formulation coadjuvants, e.g. solubilising agents, dispersing agents, suspension agents, and emulsifying agents.

The active agents for use in the present invention can be administered as a medicament, i. e., a pharmaceutical composition. The composition contains at least one active agent of the present invention with a suitable carrier. A variety of administrative techniques may be utilized, among them parenteral techniques such as subcutaneous intravenous and intraperitoneal injections, catheterizations and the like. Average quantities of the active agent may vary and in particular should be based upon the recommendations and prescription of a qualified physician. The pharmaceutical compositions used in the methods of this invention for administration to animals and humans comprise the active compound in combination with a pharmaceutical carrier or excipient.

The medicament can be in the form of tablets (including lozenges and granules), dragees, capsules, pills, ampoules, intranasal sprays, or suppositories comprising the compound of the invention.

"Medicament" as used herein means physically discrete coherent portions suitable for medical administration. "Medicament in dosage unit form" as used herein means physically discrete coherent units suitable for medical administration, each containing a daily dose or a multiple or a sub-multiple of a daily dose of the active compound of the invention in association with a carrier and/or enclosed within an envelope. Whether the medicament contains a daily dose, or, for example, a half, a third or a quarter of a daily dose will depend on whether the medicament is to be administered once, or, for example, twice, three times or four times a day, respectively.

Advantageously, the compositions are formulated as dosage units, each unit being adapted to supply a fixed dose of active ingredients. Tablets, coated tablets, capsules, ampoules, intranasal sprays and suppositories are examples of preferred dosage forms according to the invention. It is only necessary that the active ingredient constitute an effective amount, i. e., such that a suitable effective dosage will be consistent with the dosage form employed in single or multiple unit doses. The exact individual dosages, as well as daily dosages, will, of course, be determined according to standard medical principles under the direction of a physician.

The active compound can also be administered as suspensions, solutions and emulsions of the active compound in aqueous or non-aqueous diluents, syrups, granulates or powders. Diluents that can be used in pharmaceutical compositions (e. g., granulates) containing the active compound adapted to be formed into tablets, dragees, capsules and pills include the following : (a) fillers and extenders, e. g., starch, sugars, mannitol and silicic acid: (b) binding agents, e. g., carboxymethyl cellulose and other cellulose derivatives, alginates, gelatine and polyvinyl pyrrolidone; (c) moisturizing agents, e. g., glycerol; (d) disintegrating agents, e. g., agar-agar, calcium carbonate and sodium bicarbonate : (e) agents for retarding dissolution, e. g., paraffin; (f) resorption accelerators, e. g., quaternary ammonium compounds : (g) surface active agents, e. g., cetyl alcohol, glycerol monostearate; (h) adsorptive carriers, e. g., kaolin and bentonite; (i) lubricants, e. g., talc, calcium and magnesium stearate and solid polyethylene glycols. The skilled person can bring variants to the exemplary embodiments. The tablets, dragees, capsules and pills comprising the active compound can have the customary coatings, envelopes and protective matrices, which may contain opacifiers.

They can be so constituted that they release the active ingredient only or preferably in a particular part of the intestinal tract, possibly over a period of time. The coatings, envelopes and protective matrices may be made, for example, from polymeric substances or waxes. The active ingredient can also be made up in microencapsulated form together with one or several of the above-mentioned diluents.

The diluents to be used in pharmaceutical compositions adapted to be formed into suppositories can, for example, be the usual water-soluble diluents, such as polyethylene glycols and fats (e. g., cocoa oil and high esters, (e. g., C, 4-alcohol withC, 6-fatty acid]) or mixtures of these diluents. The pharmaceutical compositions which are solutions and emulsions can, for example, contain the customary diluents, such as solvents, dissolving agents and emulsifiers. Specific non-limiting examples of such diluents are water, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1, 3-butylene glycol, dimethylformamide, oils (for example, ground nut oil, glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitol or mixtures thereof.

For parenteral and intranasal administration, solutions and suspensions should be sterile, e. g., water or arachis oil contained in ampoules and, if appropriate, blood-isotonic.

The pharmaceutical compositions which are suspensions can contain the usual diluents, such as liquid diluents, e. g., water, ethyl alcohol, propylene glycol, surface active agents (e. g., ethoxylated isostearyl alcohols, polyoxyethylene sorbitols and sorbitan esters), microcrystalline cellulose, aluminum metha hydroxide, bentonite, agar-agar and tragacanth, or mixtures thereof.

The pharmaceutical compositions can also contain colouring agents and preservatives, as well as perfumes and flavouring additions (e. g., peppermint oil and eucalyptus oil, and sweetening agents, (e. g., saccharin and aspartame). The pharmaceutical compositions will generally contain from 0.5 to 90% of the active ingredient by weight of the total composition. In addition to the active compound, the pharmaceutical compositions and medicaments can also contain other pharmaceutically active compounds.

Any diluent in the medicaments of the present invention may be any of those mentioned above in relation to the pharmaceutical compositions. It is envisaged that this active compound will be administered perorally, intranasally, parenterally (for example, intramuscularly, intrathecally, intraperitoneally, subcutaneously, transdermal^ or intravenously), rectally or locally, preferably intranasally or parenterally, especially perlingually, or intravenously. Most preferably, the peptide of formula I or its analogue or salt, is administered by the intranasal or intravenous route. The dosage rate, is preferably in the range of 0.01 to 20 mg/kg of body weight, and most preferably in the range of 0.05 to 5 mg/kg of body weight, and will be a function of the nature and body weight of the subject to be treated, the individual reaction of this subject to the treatment, type of formulation in which the active ingredient is administered, the mode in which the administration is carried out and the point in the progress of the disease or interval at which it is to be administered. Thus, it may in some case suffice to use less than a minimum dosage rate while in other cases an upper limit must be exceeded to achieve the desired results. Where larger amounts are administered, it may be advisable to divide these into several individual administrations over the course of the day. In this regard, the intranasal administration may utilize metered dose devices known in the art.

The following examples further illustrate the invention. EXAMPLE 1

HIV-1 Tat binds the alphal subunit of the cellular Na⁺,K⁺-ATPase

Previous experiments have indicated that extracellular release of some of the proteins that do not follow the canonical, ER-Golgi-mediated pathway of secretion, such as FGF2 (Dahl, JP, et al. 2000. Biochemistry 39, 14877; Florkiewicz, RZ, et al. 1998. J Biol Chem 273, 544) and HIV-1 Tat (Chang, HC, et al. 1997. Aids 11, 1421) is inhibited by the cardiac glycoside ouabain. Since the specific cellular target of ouabain is the alpha subunit of Na+,K+-ATPase, we wanted to investigate whether Tat might directly interact with this protein and whether this interaction might be involved in extracellular Tat release.

To test this hypothesis, we performed co-immunoprecipitation experiments between a fusion protein corresponding to the 86 amino acid form of Tat fused to the thymidine kinase enzyme (Tat86-TK) and the endogenous, ubiquitously expressed, Na⁺,K^÷-ATPase alphal subunit in HEK293T cells expressing Tat86-TK. We found that alphal was present in the anti-Tat86-TK immunoprecipitate and, most notably, that cell treatment with 25 μΜ ouabain for 4 h prior of cell harvesting completely abolished the interaction between the two proteins. Since the rat Na⁺,K⁺-ATPase is know to be insensitive to ouabain treatment, due to the presence of natural amino acid variations in its alphal subunit (Emanuel, JR, et al. 1988. J Biol Chem 263, 7726), we wondered whether this protein still interacted with Tat. Human HEK293T cells were transfected with vectors expressing rat alphal together with vectors expressing either Tat86-TK or a TK reporter protein fused to 11 amino acids in the Tat basic domain (aa 48-58; Tatll-TK). When alphal was immunoprecipitated with a specific antibody, either Tat-fusion protein was also present in the immunoprecipitates. Of notice, however, and in contrast with human alphal, the addition of ouabain did not modify the interaction between rat alphal and the Tat proteins. In addition, Tat also bound the catalytically inactive mutant 716N of the Na⁺,K⁺-ATPase alphal subunit, which bears an Asp to Asn mutation in the ATP binding site imparing the catalytic activity of the enzyme.

Collectively, these results indicate that Tat binds human and rat Na⁺,K⁺-ATPase subunit irrespective of the enzymatic activity of the proteins.

METHODS

Constructs expressing TK, Tatll-TK and Tat86-TK (Tasciotti, E, et al. 2003. Cancer Gene Ther 10, 64), Tat86, Tatll-GFP (Fittipaldi, A, et al. 2003. J Biol Chem 278, 34141), Tat86(R5A) (Demarchi, F, et al. 1996. J. Virol. 70, 4427) were already described. The rat alphal point mutant bearing a missense point mutation (G to A) at nucleotide 2447 (leading to the substitution of the Asp at position 716 with Asn, in the third, large cytoplasmic loop of the protein) was introduced by two-step PCR mediated mutagenesis, using PCR primers carrying the designed substitution; the second step was performed using the same primers described for the deletion mutants. The amplicon was then digested with Hind III and Not I, and cloned into pcDNA3 (Invitrogen Co). The FLAG epitope was inserted as follows: complementary oligonucleotides encoding the FLAG sequence lacking the ATG were designed, annealed, and the resulting dsDNA was cloned into the alphal mutant vectors (as well as the pcDNA3 vector encoding the wild type alphal) between the Not I and Bam HI sites, resulting in vectors expressing wild type, catalytic mutant or deletion mutant alphal proteins tagged with the FLAG epitope at the C-terminus.

The constructs, expressing the larger cytoplasmic regions of rat Na⁺,K⁺-ATPase alphal subunit as fusion proteins with glutathione-S-transferase (GST) in the prokaryotic vector pGEXIT were a gift from P. Devarajan (Devarajan, P, et al. 1994. Proc Natl Acad Sci U S A 91, 2965). The construct encoding a single chain Ig tagged with the SV5 peptide (Sc-VHD16-SV5), in the eukaryotic vector pcDNA3, was a kind gift from O. Burrone (Sepulveda, J, et al. 2003. J Mol Biol 333, 355).

Polyclonal antiboby against HSV-TK was purchased from William C. Summers Laboratory (Yale University, US); monoclonal antibody against tubulin was from Sigma; monoclonal antibody against SV5 peptide was a kind gift from Prof. Oscar Burrone (ICGEB, Trieste, Italy); monoclonal antibody against HIV-1 Tat (5A5.3) was obtained through the AIDS Research and Reference Reagent Program (Division of AIDS, NIAID, NIH from Dr. Jon Karn); monoclonal antibody against Na⁺,K⁺-ATPase subunit was from Upstate (Lake Placid, NY); monoclonal antibody against GM-130 was from Transduction Laboratories (Lexington, KY); monoclonal anti-FLAG antibody and anti-FLAG conjugated agarose were from Sigma; Horseradish peroxidase (HRP)-conjugated secondary antibodies for western blotting were from Santa Cruz biotechnology and Alexa 488-coupled secondary antibodies used for confocal microscopy were purchased from Molecular Probes Inc. (Eugene, OR). The protease inhibitor cocktail was from Roche (Strasbourg, France). All other reagents were from Sigma unless otherwise specified.

For immunoprecipitation, cell pellets were lysed in NHEN buffer containing protease inhibitors (Roche). The protein concentration of the extracts was determined by the Bradford assay (BioRad). Anti-TK and anti-alphal antibodies were incubated overnight at 4°C with cell extracts (2-3 mg). Following incubation, Protein-A Trisacryl beads (Pierce Technologies Inc., Rockfors, IL) was used to bind the immunocomplexes, according to the manufacturer's instructions. After incubation, beads were washed 5 times with NHEN buffer, then processed for SDS-PAGE and western blotting using appropriate antibodies.

Anti-FLAG M2-Agarose beads (Sigma) were incubated overnight at 4°C with cell extracts (2.5-4.5 mg). After incubation, the immunocomplexes were analyzed by western blotting using appropriate antibodies.

EXAMPLE 2 Interaction of Tat with the alphal subunit of the cellular Na⁺,K⁺-ATPase is crucial for Tat secretion and transcellular Tat transactivation

Since rat alphal binds Tat in a ouabain-insensitive manner, we wondered whether its overexpression might rescue the inhibition that ouabain imposes on the secretion of Tat-fusion proteins in human cells. To ascertain this possibility, HEK293T cells were transfected with Tat86-TK or Tatll-TK, together with the rat alphal subunit. Indeed, the overexpression of this protein restored the cellular release of the Tat proteins in cells treated with ouabain. Of notice, the 716N alphal mutant was equally effective as the wild type protein in rescuing ouabain inhibition, again indicating that the effect of alphal on Tat release is independent from its enzymatic activity. Secretion of the ScVH16-SV5 was left unaffected by any of these treatments. Superimposable results were also obtained when studying the release of Tat86-TK under exactly the same experimental conditions.

We also wanted to verify whether the marked inhibition that ouabain exerts on the release of Tat-fusion proteins also impairs the transcellular transactivation properties of wild type Tat. For this purpose, we transiently transfected HEK293T cells with an expression vector for the 86 aa form of Tat (Tat86); 24 h after transfection, cells were either left untreated or treated with 25 mM ouabain for 4 h, and the resulting cell conditioned media were collected and added to human HL3T1 cells, a HeLa cell derivative cell line carrying a silent HIV-1 LTR-CAT construct. Conditioned medium from cells expressing Tat86 activated LTR transcription in the reporter cell line, as assessed by CAT quantification, while ouabain treatment completely abrogated such effect; no decrease of CAT activity in the reporter cell line after ouabain treatment was observed when the Tat86-expressing cells were co-transfected with a vector encoding rat alphal, either wild type of catalytically inactive. Collectively, the results so far described are consistent with the conclusions that the Tat basic domain specifically recognizes the Na⁺,K⁺-ATPase alphal subunit and that this interaction, rather than the catalytic activity of the enzyme, is essential to mediate the release of active Tat or Tat-fusion proteins into the extracellular environment. METHODS

HEK 293T or CHO A-745 cells were seeded in 6-cm plates (lxlO⁶) one day prior transfection; cells were transiently transfected with either Tatll-TK, Tat86-TK or TK constructs and with the Sc-VHD16-SV5 construct as a positive control of classical secretion. The calcium-phosphate-DNA complexes were incubated for 12 hours, then the medium was replaced with fresh DMEM, and the cells were incubated for additional 24 hours. The secretion assays were performed by washing the cells (3 washes of 10 min each) with Optimem containing 20 g/ml heparin to prevent secreted Tat protein binding to the extracellular heparan sulfate proteoglycans upon transfection (Tyagi, M, et al. 2001. J Biol Chem 276, 3254), and incubating the cells with 2 ml of Optimem plus heparin for the indicated times. Following incubation, cell culture supernatants were collected and concentrated using Amicon Ultra 10 concentrator (Millipore) according to the manufacturer's instructions; the concentrated fraction was then processed for western blotting. As a control of protein expression, the cellular fraction was collected, lysed in NHEN buffer (20 mM Hepes pH 7.5, 300 mM NaCI, 0.5% NP-40, 20% glycerol, 1 mM EDTA) containing protease inhibitors (Roche). Total protein concentration was assessed by the Bradford assay (BioRad), and 30 pg of each sample was processed for Western Blotting. CHO Kl and PsgA-745 (Rostand, KS, et al. 1997. Infect Immun 65, 1) were obtained from the American Type Culture Collection (ATCC; Manassas, VA) and mantained in Kaighn's modification of Ham's F12 medium. HEK 293T and U20S (obtained from the ATCC) and HL3T1 cells (a HeLa cells derivative stably transfected with a silent LTR-CAT cassette), a kind gift of B. Felber (Felber, BK, et al. 1988. Science 239, 184), were mantained in DMEM. All culture media were supplemented with 10% fetal calf serum, 2 mM L-glutamine, and 50 g/ml gentamicin.

U20S cells (5xl0⁴) were seeded in four-well glass chamber slides (LabTek II-Nalge Nunc) and transfected with the Polyfect transfection kit (Qiagen) for 24 h; in all transfections, an adequate quantity of empty pcDNA3 vector was added to to achieve a similar DNA amount. HEK 293T cells were transfected using standard calcium phosphate precipitation (Sambrook, J, et al. Molecular Cloning. A Laboratory Manual/II Edition, Cold Spring Harbor Laboratory, 1989), incubated for 36 h, and then processed for secretion or co-immunoprecipitation experiments.

To test the biological activity of secreted Tat protein, HEK 293T cells were transfected with a pcDNA3 construct expressing Tat86 and a pcDNA3 expressing either wild type of one of the deleted alphal mutants; after 24 h incubation, cells were washed and the medium substituted with fresh medium plus heparin 20 g/ml and, for the secretion inhibition experiments, supplemented with 25 μΜ ouabain. After 4 h, the supernatant was collected and added to HL3T1 cells, containing an integrated bacterial chloramphenicol-acetyltransferase gene (CAT) gene under the control of the HIV-1 LTR, in the presence of 100 μΜ chloroquine. After 24 h incubation, Tat-driven CAT expression was assayed by quantifying the levels of CAT protein using a CAT ELISA kit (Roche Diagnostics, Meylan, France).

Other reagents and procedures are the same as described in the Methods to Example 1. EXAMPLE 3

The Tat basic region binds the C-terminal cytoplasmic loops of Na⁺,K⁺-ATPase alphal subunit.

We wanted to define the regions of Tat and Na+,K+-ATPase alphal subunit involved in the interaction between the two proteins. Alphal is a large, integral membrane protein with 10 transmembrane- spanning domains plus cytoplasmic N- and C-terminal regions (Lingrel, JB, et al. 1994. Kidney Int Suppl 44, S32; Morth, JP, et al. 2007. Nature 450, 1043). We focused our attention on the cytoplasmic domains of the protein which were the most likely candidates for Tat binding, as the rest of the protein is either embedded in the phospholipidic bilayer or protrudes into the extracellular space. GST-fused alphal fragments corresponding to the three main cytoplasmic regions (Devarajan, P, et al. 1994. Proc Natl Acad Sci U S A 91, 2965), as well a fusion of the three C terminal short cytoplasmic domains of the rat aphal subunit, named P1-P2-P3, were used in GST-pull down experiments using in vitro translated [35S]- labeled Tat, or a mutated [35S]-Tat mutant - Tat86(R5A) -, in which the arginine residue of the Tatll peptide had been replaced by alanines (Demarchi, F, et al. 1996. J. Virol. 70, 4427). Labeled Tat did not bind to any of the large cytoplasmic loops of Na+,K+-ATPase alphal; in contrast, showed strong binding with the P1-P2-P3 protein, corresponding to the fusion of the three short, C-term loops. The Tat86(R5A) mutant failed to interact with any of the alphal regions.

To further characterize the region of binding between Tat and the C-terminus of the Na+,K+-ATPase alphal subunit, we analyzed the in vitro binding of Tat with three synthetic peptides of the present invention, corresponding to the three short cytoplasmic sequences (residues 824-843, 939-951 and 1007- 1023, named PI, P2 and P3 respectively) in the C-terminal domain of the alphal protein. To assess binding, synthetic biotinylated peptides were coupled to streptavidin beads, and subsequent pull down assays were performed employing radiolabeled, in vitro translated Tat.

Following incubation and extensive washes, the Tat protein showed marked binding to the beads coated with any of the three peptides, in particular with the P2 peptide, however not with uncoupled beads or beads coated with an irrelevant biotinylated peptide (Figure 2A). The Tat86(R5A) mutant did not show detectable binding to any of the three peptides.

Of notice, the amino acid sequence of the three C-terminal intracytoplasmic loops of the Na+,K+-ATPase alphal subunit appears very conserved, with a perfect homology existing between sequences of rat, human and pig alphal, for which the crystal structure was recently resolved (Morth, JP, et al. 2007. Nature 450, 1043). METHODS

The alphal ACTERM lacks the fragment spanning amino acids 784-3040 (comprising the last two C- terminal cytoplasmic fragments as well as a transmembrane region between them); the lower primer was designed containing a nonsense mutation (i.e. a Stop codon) at amino acid position 784 (corresponding to nucleotide 2352) and the restriction site for Not I. The truncated cds of rat alphal was amplified from the pcDNA3-alphal vector with these primers and an upper primer, containing an Hind III site. The Alphal deletion mutant was cloned in pcDNA3 vector between Hindlll and Notl sites.

The DNA segment encoding for the fusion of the three C-terminal cytoplasmic stretches of the alphal subunit, linked by a glycine residue (P1-P2-P3), was obtained by annealing two long oligodeoxynucleotides:

5' TATGAGCAGGCTGAGAGTGACATCATGAAGAGACAGCCCAGAAATCCCAA

AACAGACAAAGGTACCAGGAGGAATTCGGTCTTCCAGCA 3' [SEQ ID: 6] and

5' ATAGTAGGTTTCCTTCTCCACCCAGCCGCCAGGGCGTCGCCTGATGATGA

GACCGTTCTTCATCCCCTGCTGGAAGACCGAATTCCTCC 3' [SEQ ID: 7] encoding amino acid sequences 824-843, 939-951 and 1007-1023 of the alphal subunit, followed by PCR amplification of the fragments using external primers containing appropriate restriction sites for cloning. In particular, the Pl-P2-P3-encoding DNA segment was then cloned into pFLAG-CMV2 vector, between Not I and EcoR I sites, obtaining the PFLAG-P1-P2-P3 construct, and into the pGEX-2T vector, between Hind III and Eco RI sites, obtaining the pGEX-Pl-P2-P3 construct. The recombinant fusion protein corresponding to GST-Tat, GST, alphal cytoplasmic portions 1, 2 and 3 (GST-PI, -P2, -P3 respectively), and the GST-P1-P2-P3 fusion protein were produced and purified from BL21 bacteria transformed with the corresponding vectors. Bacterial cultures were grown in terrific broth plus ampicillin and protein production was induced with IPTG 0.5 mM for 3 h at 30°C until an OD₆oo between 0.6 and 0.8 was obtained. Bacteria were then resuspended in lysis buffer (50 mM Tris-HCI pH 8.0, 100 mM NaCI, 5% glycerol, 2 mM dithiothreitol) and sonicated by 4 pulses of 30 seconds each. Bacterial lysates were mixed with a 50% (vol/vol) slurry of glutathione cross-linked agarose beads and the GST-fusion proteins were allowed to bind the beads at 4°C on a rotating wheel for 1 h. The suspension was loaded on an empty plastic column (Bio-Rad, Richmond, CA), letting the unbound proteins pass through, and the beads were washed with 400 bed volumes of lysis buffer. The fusion proteins bound to GST agarose beads were then resuspended in an adequate amount of lysis buffer and stored at -80°C prior to use. The integrity and purity of the proteins were assessed by sodium-dodecyl- sulphate gel electrophoresis (SDS-PAGE) followed by Coomassie staining. The pcDNA3-Tat86 and the pcDNA3-Tat86(R5A) (Tat86 mutant in the basic region) were used as templates to produce the ³⁵S-labeled Tat86 and Tat86(R5A) proteins for in vitro binding by using the TNT Reticulocyte Lysate System (Promega, Madison, WI).

Binding of GST-fused alpha 1 fragments to ³⁵S-Tat86 and ³⁵S-Tat86(R5A) was performed as follows. Briefly, 1 pg of recombinant proteins, after pretreatment in a solution containing DNase I 0.25 U/μΙ and RNase 0.2 g/μΙ to remove contaminant bacterial nucleic acids, were incubated with 600 c.p.m. of in vitro translated Tat in a solution containing 0.2 mg/ml ethidium bromide. Following extensive washes, the reaction mixture was resolved by SDS-PAGE electrophoresis and analyzed by Phospholmager.

Synthetic peptides corresponding to residues 824-843, 939-951 an 1007-1023 of alphal C-terminus (PI, P2 and P3, respectively) were obtained from the Peptide Synthesis Facility at ICGEB Trieste. Peptides were synthesized on solid phase (preloaded NovaSyn TGT, Novabiochem) by Fmoc/t-Bu chemistry using a home-built automatic synthesizer based on a Gilson Aspec XL SPE system. The peptide-resin were cleaved/deprotected using a modified Reagent H mixture (trifluoroacetic acid 80%, phenol 3%, thioanisole 3%, 3,6-dioxa-l,8-octanedithiol 8%, water 2.5%, methylethylsulphide 2%, hydroiodic acid 1.5% w/w) for 3 h. The peptides were then precipitated by diethylether, washed and freeze-dried. The crude peptides were purified by preparative RP-HPLC on a 25x300 mm column (Load&Lock system, Varian) packed with VariTide RPC resin (Polymer Laboratories - Varian) using a gradient from 0.1% TFA in water to 0.1% TFA in acetonitrile. The purified fractions were checked by ESI-MS, pooled and freeze- dried. For all the three peptides, N-biotinylated, N-fluoresceinated and non-conjugated forms were synthesized.

Synthetic biotinylated peptides, corresponding to PI, P2 and P3, were used for binding assays as follows: 25 μΙ of streptavidin resin (UltraLink Plus streptavidin beads, Pierce), were pretreated with 3 mg of BSA for 10 min at room temperature, and subsequently incubated with 50 pg of peptide at 22°C for 1 h; after 3 washes, the peptide-coated beads were incubated with either 600 c.p.m. of in vitro translated Tat as previously described, or with 250 ng of recombinant GST-Tat, for 2 h at 4°C. Following extensive washes, the reaction mixture was resolved by SDS-PAGE electrophoresis and analyzed either by Phospholmager, or blotted and analyzed by western analysis using an anti-GST antibody.

Other reagents and procedures are the same as described in the Methods to Examples 1 and 2.

EXAMPLE 4 A fusion protein corresponding to the Na⁺,K⁺-ATPase alphal C-terminal loops (P1-P2-P3 protein) binds Tat and impairs transactivation

To test binding of Tat to the three C-terminal, short cytoplasmic loops of the Na⁺,K⁺-ATPase alphal subunit in vivo, a plasmid expression vector was obtained, encoding for the P1-P2-P3 fusion protein, corresponding to the Na⁺,K⁺-ATPase alphal C-terminal loops linked by a glycine residue, under the control of the constitutive CMV promoter. The P1-P2-P3 encoding plasmid was co-expressed in HEK293T cells together with either Tat86-TK or Tatll-TK, and the lysates were used in co-immunoprecipitation experiments. Western blot using an anti-TK antibody showed interaction between the tripartite fusion protein and either the 86- and the 11-aa versions of Tat, but not with TK control. Since these co-immunoprecipitation experiments indicated that the P1-P2-P3 fusion protein, once expressed intracellular^, was able to bind the basic region of Tat, we wondered whether the overexpression of this protein might also affect Tat transactivation. To test this possibility, HeLa cells expressing an LTR-driven luciferase gene were transfected with different amounts of both a Tat- expressing plasmid (1, 5 and 25 ng) and a plasmid expressing the P1-P2-P3 protein (300, 600 ng). These results revealed that, at any of the three Tat plasmid concentrations, the co-expression of the higher dose of the P1-P2-P3 plasmid significantly inhibited transcriptional activation of the LTR-reporter construct. Cell viability was unaffected by P1-P2-P3 transfection at the highest concentration.

These results support the general conclusion that binding between Tat and the tripartite P1-P2-P3 protein occurs inside the cells, and is consistent with the possibility that the overexpression of the fusion protein diverts Tat from its transactivation function at the HIV-1 LTR promoter, thus acting as a competitive inhibitor.

METHODS

Luciferase assays were performed using a construct containing the U3 and R sequences of the HIV-1 LTR (or a CMV promoter in the negative controls) upstream of the firefly luciferase gene as a reporter, and pcDNA3-Tat86 as an effector, in the presence of various amounts of the FLAG-tagged P1-P2-P3 construct (see above). Plasmid pCI-Neo-Renilla (Promega) expressing the renilla luciferase gene under the control of the CMV promoter was co-transfected into cells to standardize transfection efficiency in each experimental point.

HeLa cells were transfected using Lipofectamine 2000 Reagent (Invitrogen, according to manufacturer's protocol), with 150 ng of pLTR-luciferase or CMV-luciferase, 10 ng of pCI-Neo-Renilla, 1, 5 or 25 ng of pcDNA3-Tat86 and 300 or 600 ng of Pl-P2-P3-encoding plasmid. The total amount of transfected DNA was normalized according to pcDNA3 plasmid.

Cells were lysed and harvested 48 h post transfection. Reagents for dual luciferase assays were purchased from PJK GmbH, Germany; luminescence was measured using the Perkin Elmer EnVision 2104 Multilabel Reader, with an integration time of 10 sec.

The measured activities were standardized by the levels of renilla; as a control of the basal activity of LTR-luciferase, with or without FLAG-P1-P2-P3, mock experiments were included where the effector was not transfected.

Other reagents and procedures are the same as described in the Methods to Examples 1-3. EXAMPLE 5

Synthetic peptides corresponding to the Na⁺,K⁺-ATPase alphal C-terminal loops block Tat export

The observation that the fusion protein P1-P2-P3 was able to decrease Tat transactivation, albeit at a high molar ratio to Tat, prompted us to wonder what the effect of short peptides corresponding the alphal C-terminal loops might be on Tat extracellular release. At difference with the endogenously expressed P1-P2-P3 protein, peptides can be administered exogenously at high concentration and their binding to Tat should not be subject to possible structural constrains imposed by their fusion in the Pl- P2-P3 construct. In addition, our in vitro binding data showed that any of the three C-terminal intracytoplasmic loops of the alphal subunit was able to independently bind Tat.

We tested the effects of cell treatment with the individual peptides or with Pmix on the release of Tat. Cells were transfected with Tatll-TK together with the single chain antibody ScVH16-SV5 and treated with 5 or 10 g/ml of the PI, P2 and P3 peptides. Cell treatment with any of the three peptides, at the higher concentration, showed marked inhibition of extracellular Tat-fusion protein release, while leaving the secretion of the control scFv antibody undisturbed; an almost complete block was observed when using Pmix at the lower concentration. Based on these results, we tested the effects of a broader Pmix concentration range (from 2 to 15 pg/ml) on the secretion of Tat86-TK. Cell treatment with Pmix inhibited extracellular Tat release in a dose-dependent manner, while canonical secretion of ScVH16-SV5 was unaffected. Of notice, transfection of the P1-P2-P3 tripartite fusion protein was ineffective in this assay, consistent with the possibility that the affinity of this protein for the Tat basic domain might be lower than that of the individual peptides.

METHODS

All reagents and procedures are the same as described in the Methods to Examples 1-4.

EXAMPLE 6 Synthetic peptides corresponding to the Na⁺,K⁺-ATPase alphal C-terminal loops block HIV-1 replication

Next we wondered whether the exogenous administration of the Pmix peptides might affect HIV-1 infection. A first set of experiments was performed by a single round infection assay in HOS CCR5 cells using an HIV-l-NL4-Luc virus carrying the M-tropic Bal envelope. When cells were pre-incubated with Pmix, however not with a control FLAG peptide (10 g/ml each), before viral infection a significant decrease in luciferase activity, measured 24 h post-infection, was observed.

In a subsequent experiment, we wanted to analyze the effect of the Pmix and FLAG (control) peptides on the infection of CEM CD4⁺ T cells with wild type HIV-1_{B U}- Over the course of 12 days after infection, half of the cell medium was replaced every second day with fresh medium containing either of the two peptides at a concentration of 10 g/ml. Viral infection was monitored by measuring reverse transcriptase activity in the cell culture supernatant. The Pmix peptides were found to markedly suppress viral infection, while the FLAG peptide control (Pcontrol) had no relevant effect. These results collectively indicate that the suppression of Tat extracellular release and of Tat function by peptides derived from the Na⁺,K⁺-ATPase alphal subunit C-terminal domain markedly impairs HIV-1 infection.

METHODS

Stocks of the HIV-lBAL-luciferase vector were prepared by the standard calcium-phosphate metod of transfection in HEK293T cells using pNL4.3-luciferase plasmid (He, J, et al. 1995. J Virol 69, 4587; Connor, RI, et al. 1996. J Virol 70, 5306) and the M-tropic Bal envelope encoding plasmid at a ratio 3: 1. The supernatant containing virions were collected 48 H after transfection, centrifuged 5 min at 1,500 rpm and filtered with 45 mm Millipore filter. Cells were infected for 4 h.

Stocks of the viral clones HIV-1BRU (Petit, C, et al. 1999. J Virol 73, 5079) were prepared by the standard calcium-phosphate method of transfection in HEK293T cells. The supernatant containing virions were collected 48 h after transfection, centrifuged 5 min at 1,500 rpm and filtered with 45 mm Millipore filter. Before infection viral stocks were treated with Dnase I (Invitrogen) 40U/ml for 1 hour at room temperature. Cells were infected for 4 h.

For single-round infections, HOS CCR5⁺ cells were infected with pNL4.3-luciferase pseudotyped with the M-tropic Bal envelope. Prior to infection, the virus was pre-incubated with an equimolar mix of PI, P2 and P3 peptides (Pmix) at 15 g/ml concentration, at 37°C for 1 h; cells were incubated with the virus for 4 h, washed, and fresh medium containing 15 mg/ml Pmix was added; after 24 hours, the cells were harvested and the expression level of luciferase was tested with DualGlo Luciferase assay kit (Promega, Madison, WI) according to manufacturer's instructions. Luciferase activity was calculated after normalization against the activity of the co-transfected Renella luciferase. For infections with wild type virus, CEM cells (0.5xl0⁶/ml) were infected with HIV-1_BRU at a MOI of 0.05, for 4 h; where indicated, infection was performed in the presence of 10 g/ml of Pmix, or of FLAG peptide, as a negative control. After infection, the medium containing the virus was washed and substituted with fresh medium, containing the corresponding peptide preparation. At t=0 and subsequently, every 3 days until the 12^th day, the supernatants were collected and tested, while cells were counted, diluted to 0.5xl0⁶/ml and fresh peptides were added to the culture media. The amount of virus was evaluated by measuring the RT activity in the supernatants following standard protocols, and normalizing the RT values over the number of cells.

Other reagents and procedures are the same as described in the Methods to Examples 1-5.

EXAMPLE 7 Synthetic peptides derived from the Na⁺K⁺-ATPase C-terminal region are internalized by the cells when administered exogenously

To text the efficacy of intracellular internalization of the individual PI, P2 and P3 peptides, these were synthesized conjugated to fluorescein and administered to HEK293T cells at the concentrations of 2, 5 or 10 g/ml. After 4 h, the cells were submitted to prolonged trypsinization and extensively washed to remove extracellular fluorescence, and then analyzed by flow cytometry. All three peptides showed a remarkable capacity of dose-dependent cell internalization. Of notice, the flow cytometry profiles show increase of fluorescence in the vast majority of cells, indicating that internalization of Pmix is a general cell property. METHODS

For the fluorescent peptide uptake experiment, synthetic biotinylated peptides, fluorescein-labeled PI, P2 and P3, or an equimolar mix of the three peptides named Pmix, were diluted in Optimem and incubated with cells for 4 h at 37°C. Cells were then trypsinized, washed once with DMEM and twice with PBS, and analyzed by flow cytometry. To visualize interacellular fluorescence, HEK293T cells were seeded on poly- Lysine-treated chamber slides; after 4 h incubation with fluorescent peptides, cells were briefly trypsinized, washed twice with PBS, an fixed with PFA 2%. Slides were analyzed upon mounting with Vectashield medium. Fluorescent images were acquired using a TCS-SL Leica confocal microscope.

Claims

1. Protein comprising a single or a combination of three intracytoplasmic peptide stretches of the C- terminal domain of the Na⁺,K⁺-ATPase alpha subunit, said peptide stretches all bind HIV-1 Tat protein.

2. Protein according to claim 1, having the sequence NH₂-YEQAESDIMKRQPRNPKTDK (X)_n TRRNSVFQQG M KN (X)_n LIIRRRPGGWVEKETYY-COOH, wherein (X)_n is a peptide stretch of any length (n) composed of any amino acid (X), with n varying from 0 to 1,000.

3. DNA coding for protein of claim 2, in particular a DNA having the sequence 5'- TATGAGCAGGCTGAGAGTGACATCATGAAGAGACAGCCCAGAAATCCCAAAACAGACAAA-(Y)_m- ACCAGGAGGAATTCGGTCTTCCAGCAGGGGATGAAGAAC-(Y)_m-

CTCATCATCAGGCGACGCCCTGGCGGCTGGGTGGAGAAGGAAACCTACTAT-3', wherein (Y)_m is a DNA sequence of any length (m) composed of any amino acid-coding nucleotide triplet (Y), with n varying from 0 to 3,000.

4. Peptide, or a derivative thereof, corresponding to one of the three intracytoplasmic peptide stretches of the C-terminal domain of the Na⁺,K⁺-ATPase alpha sub-unit, said peptide stretches all binding HIV-1 Tat protein.

5. Peptide or derivative thereof according to claim 4 , corresponding to a region of C-terminal domain of the human Na⁺,K⁺-ATPase alpha sub-unit, said region being selected from the group consisting of region 824-843; region 939-951 and region 1007-1023.

6. Peptide or derivative thereof according to claim 5, selected from the group consisting of peptide selected from the group consisting of:

NH₂-YEQAESDIMKRQPRNPKTDK-COOH (SEQ ID: 1)

NH₂-TRRNSVFQQGMKN-COOH (SEQ ID: 2)

NH₂-LIIRRRPGGWVEKETYY-COOH (SEQ ID: 3)

7. Mammalian expression vector expressing protein of claim 1 or 2.

8. Protein of claim 1 or 2 for use in the inhibition of HIV-1 Tat protein transactivation.

9. Protein of claim 1 or 2 and/or the DNA of claim 3 and/or a peptide or derivative thereof of any one claims 4-6 and/or the vector of claim 7 for use in the inhibition of HIV-1 replication.

10. Protein of claim 1 or 2 and/or the DNA of claim 3 and/or a peptide or derivative thereof of any one claims 4-6 and/or the vector of claim 7 for use in the treatment of HIV-1 infection.

11. Protein of claim 1 or 2 and/or the DNA of claim 3 and/or a peptide or derivative thereof of any one claims 4-6 and/or the vector of claim 7 for use in the treatment of HIV-l-associated diseases.

12. Protein of claim 1 or 2 and/or the DNA of claim 3 and/or a peptide or derivative thereof of any one claims 4-6 and/or the vector of claim 7 for use as medicament.

13. Pharmaceutical composition comprising the protein of claim 1 or 2 and/or the DNA of claim 3 and/or a peptide or derivative thereof of any one claims 4-6 and/or the vector of claim 7.