ES2326348A2 - USE OF AGONIST COMPOUND OF THE TACTICAL ACTIVITY TUBULINA DEACETILASA OF THE HDAC6 PROTEIN IN THE PREPARATION OF PHARMACEUTICAL COMPOSITIONS, SUCH PHARMACEUTICAL COMPOSITIONS AND ITS APPLICATIONS IN THE TREATMENT OF VIRAL INFECTIONS. - Google Patents

Abstract

The invention relates to the use of HDAC6 protein tubulin deacetylase activity agonist compounds in the preparation of pharmaceutical compositions, to said pharmaceutical compositions and to applications of same in the treatment of viral infections. The invention is based on the fact that the acetylation/deacetylation equilibrium of the microtubules is key for modulating the entry of fusogenic viruses into eucaryotic cells, preferably the entry of the HIV-1 virus into T cells, and which is regulated by the HDAC6 protein, thereby enabling novel therapeutic approaches to be defined for the prophylactics and treatment of viral infections. The aforementioned therapeutic approaches are based on the use of compounds that activate the activity of the HDAC6 protein and of the pharmaceutical compositions that contain same in methods for the treatment of said viral infections.

Description

Use of agonist compounds of the activity HDAC6 protein tubulin deacetylase in the preparation of pharmaceutical compositions, said pharmaceutical compositions and its applications in the treatment of viral infections.

The present invention is based on the fact that the balance of acetylation / deacetylation of microtubules It is key to modulate the entry of spherogenic viruses in the eukaryotic cells, preferably virus entry HIV-1 in T cells, and that is regulated by the HDAC6 protein, which allows defining new approaches Therapeutics for prophylaxis and treatment of infections viral. These therapeutic approaches are based on the use of compounds activating the activity of said HDAC6 protein and of the pharmaceutical compositions containing them in methods of Treatment of such viral infections.

Technical sector

The present invention is framed in the development of new pharmaceutical compositions for prophylaxis and the treatment of diseases caused by spherogenic viruses, and more preferably for the HIV-1 virus. For the therefore, it must be attached to the pharmacological and biomedical sector of the viral diseases.

State of the art

The process of infection of eukaryotic cells by enveloped or spherogenic viruses requires the fusion of the plasma or endosomal membranes of the host cell with the viral one. This reaction is catalyzed by specific proteins in the virus envelope, called fusion proteins, which allow the energy barriers of the process to be overcome. For example, Retrovirus penetration into permissive cells for infection can occur by membrane fusion dependently or independently of pH. The first case is the influenza virus (T. Stegmann, A. Helenius. Influenza Virus Fusion: from models towards a mechanism, in: J. Bentz (Ed.). Viral Fusion Mechanisms, CRC Press, Boca Raton, FL (1993, pp. 89-111)), of the Semliki Forest virus (R. Bron, et al ., 1993. EMBO J. 12: 693-701), and the second case of the human immunodeficiency virus (HIV) and Apes (SIV) (MO McClure, et al ., 1988. EMBO J. 7: 513-518) or Sendai virus (D. Hoekstra et al ., 1985. Biochemistry 24: 4739-4745). It should be noted that both processes are mechanistically similar, except that membrane fusion occurs in endosomes or on the target cell surface, respectively (I. Martín, et al ., 1999. Advance Drug Delivery Reviews 38: 233-255 ). During the fusion process the viral fusion proteins interact simultaneously with the cell and viral membranes, favoring the formation of the fusion pore (TH Sollner, 2004. Current Opinion in Cell Biology 16: 429-435). Most known viral fusion proteins interact with the viral envelope through its transmembrane domain, which connects the intraviral region (or cytoplasmic, in infected cells) with the ectodomain of the fusion protein. On the other hand, it is accepted that the interaction of the viral fusion protein with the membrane of the target cell involves a highly hydrophobic domain, of about 15 amino acid residues, called fusion peptide (SG Peisajovich, et al ., 2003. BBA 1614 : 122-129). This segment is located in the N-terminal region of fusion proteins, as in most orthomyxoviruses, paramyxoviruses and other related retroviruses (J. White et al ., 1983. Q. Rev. Biopohys. 16: 151 -195; BM Blumberg et al ., 1985. J. Gen. Virol. 66: 317-331; WR Gallaher. 1987. Cell 50: 327-328), or inside the fusion protein ectodomain as in the cases of Rous Sarcoma virus (E. Hunter et al ., 1983. J. Virol. 46: 920-936), of Vesicular Stomatitis virus (MA Whitt, et al ., 1990. J. Virol. 64: 4907-4913) and Ebola virus (WR Gallaher. 1996. Cell 85: 477-478). It is generally accepted that each viral fusion protein contains a single fusion peptide, which is directly responsible for destabilization of the cell membrane and initiator of the formation of the fusion pore, although the participation of other regions of Viral fusion proteins in fusion pore formation (SG Peisajovich, et al ., 2003. BBA 1614: 122-129).

Fusion proteins are classified in two main groups: Type I (eg influenza virus, Ebola, HIV and Measles) and type II (Dengue virus, yellow fever and encephalitis) (Reviews in T.H. Sollner. 2004. Current Opinion in Cell Biology 16: 429-435; A.E. Smith and A. Helenius, 2004. Science 304: 237-242). The Type I proteins consist of glycoproteins that are assembled in homotymers, which are associated with each other through structures elongated spiral-shaped coil, in whose region N-terminal fusion peptide is located. The release of energy that occurs during the insertion of the peptide in the cell membrane, allows the approximation of viral and cellular membranes in a focused way, generating the pore of fusion (Review in, A.E. Smith and A. Helenius, 2004. Science 304: 237-242). Type II proteins are their own of Flavivirus and alpha virus (Review in, A.E. Smith and A. Helenius, 2004. Science 304: 237-242), is characterized by having internal fusion sequences, and synthesize and assemble on the viral surface as heterodimers with other membrane proteins. At low pH, there is a change in the quaternary structure of heterocomplexes, allowing the dissociation of the heterodimer viral fusion protein, and favoring the regrouping of fusion proteins together to form active and functional homotymers (Review in, A.E. Smith and A. Helenius, 2004. Science 304: 237-242).

On the other hand, Human Immunodeficiency Virus (HIV) is the etiologic agent of Acquired Immunodeficiency Syndrome (AIDS) (F. Barre-Sinoussi, et al ., 1983. Science 200: 868-871). Type 1 HIV (HIV-1) primarily infects CD4 + cells such as T lymphocytes, monocytes and dendritic cells, at the level of lymphoid organs and mucous membranes of viral entry regions (AS Fauci, 1988. Science 239: 617-622 ; S. Patterson, et al ., 1994. Res. Virol. 145: 171-176). HIV-1 infection requires a high affinity interaction between the virus envelope glycoproteins, gp120 / gp41, with the CD4 receptor (AG Dalgleish, et al ., 1984. Nature 312: 763-767; PJ Maddon, et al ., 1986. Cell 47: 333-348) and also requires association with chemokine receptors, mainly CCR5 (H. Choe, et al ., 1996. Cell 85: 1135-1148, H. Deng, et al. ., 1996. Nature 381: 661-666; T. Dragic, et al ., 1996. Nature 381: 667-673) and CXCR4 (Y. Feng, et al ., 1996. Science 272: 872-877), referred to as co-recipients Thus, it has been proposed that the sequential binding of the gp120 viral protein with CD4, and with one of the co-receptors, induces specific conformational changes in gp41, thus allowing viral fusion with the cellular plasma membrane and the formation of the fusion pore (H. Choe, et al ., 1996. Cell 85: 1135-1148). The co-receptor used by HIV-1 determines the tropism of the viral strain. Consequently, viruses that use the CCR5 receptor are called R5 tropics and those that use CXCR4, X4 tropics (EA Berger, et al ., 1999. Annu. Rev. Immunol. 17: 657-700).

From a clinical point of view, HIV-1 R5 viruses infect monocytes and macrophages, and are involved in horizontal (MT Roos, et al ., 1992. J. Infect. Dis. 165: 427-432) and vertical ( G. Scarlatti, et al ., 1993. Virology 197: 624-629) of the virus, prevailing in the asymptomatic seropositive phase of the disease. In advanced stages of the disease, HIV-1 X4 viruses, which primarily infect CD4 T lymphocytes (EA Berger, et al ., 1999. Annu. Rev. Immunol. 17: 657-700), become the predominant strain, being detected in 50% of patients infected with HIV-1 (M. Tersmette, et al ., 1988. J. Virol. 62: 2026-2032; RI Connor, et al ., 1997. J. Exp. Med. 185: 621-628). The disease progression phase is further characterized by an increase in viral load (viremia) and a rapid and dramatic decrease in CD4 + T cells (G. Pantaleo and AS Fauci, 1996. Annu. Rev. Microbiol. 50: 825-854), which is related to the general collapse of the immune system, and is considered a key factor in the evolution of AIDS (JD
Lifson, et al ., 1986. Science 232: 1123-1127; H. Blaak, et al ., 2000. Proc. Natl Acad. Sci. USA 97: 1269-1274).

Lymphoid tissue, in which activated and permissive immunocompetent cells for HIV-1 infection are located, represents one of the main reservoirs of VIM-1 and a favorable environment for viral replication. During the presentation processes of the antigen the infected cell-host cell contacts are favored. This results in cell fusion, generating multinucleated giant cells (syncytia), and favoring viral propagation. Fused infected cells die by apoptosis, quickly after their formation. This process is related to the degeneration of the lymphoid environment, the dramatic depletion of CD4 + T cells, and is associated with the appearance of tropic HIV-1 X4 strains (AS Fauci, 1996. Nature 384: 529-539; AB Van't Wout , et al ., 1998. J. Virol. 72: 5099-5107). Fusion and death of infected cells, together with lysis of individual cells, are considered the acute cytopathic effects of HIV (DD Ho, et al ., 1995. Nature 373: 123-126; X. Wei, et al ., 1995. Nature 373: 117-122).

The process of fusion and infection of CD4 + T cells by the HIV-1 virus is established by cooperative interaction of the viral protein gp120, not covalently associated with the transmembrane viral protein gp41, with the CD4 receptor and the CXCR4 or CCRS correceptor of the target cell (RW Doms, 2000. Virology 276: 229-37; SE Kuhmann, et al ., 2000. J. Virol. 74: 7005-15). During this first stage of virus-cell interaction, HIV induces the co-distribution and / or association of the CD4 receptor with the CXCR4 or CCR5 correceptor, in the areas of contact between the virus and the target cell. Consequently, blocking the codistribution of the CD4 and CXCR4 / CCR5 receptors hinders the formation of the fusion pore, inhibiting the stages of fusion and viral entry (DS Dimitrov, et al ., 1999. Virology 259: 1-6). More specifically, different studies suggest that each Env trimer of the HIV-1 virus interacts with multiple molecules of CD4, CXCR4 or CCR5 (H. Choe, et al ., 1996. Cell 85: 1135-1148) and that several trimers are necessary for the formation of the fusion pore (SE Kuhmann, et al ., 2000. J. Virol. 74: 7005-7015). The fusion of the cellular and viral membranes, ultimately governed by the gp41 protein, necessarily involves the reorganization of the cytoskeleton of the target cell, modifications of the cell morphology and the induction of cellular signals.

However, and although there is evidence of the importance of the cytoskeleton for viral infection, there is little data on its reorganization processes that take place during HIV-1 infection. On the other hand, the cellular components of the cytoskeleton are involved in the early events during infection by different viruses, regulating the entry of the virus and the release of its genome as well as promoting the movement of viral capsids and the integration of the viral genome. Thus, actin and tubulin cytoskeletons must be intact for effective entry of herpes simplex virus and simian virus 40, respectively (H. Deng, et al ., 1996. Nature 381: 661-666; T. Dragic, et al ., 1996. Nature 381: 667-673). In the case of the HIV-1 virus, it has been described that infection and fusion with the host cell can be completely inhibited by disruption of the actin network (Y. Feng, et al ., 1996. Science 272: 872- 877; EA Berger, et al ., 1999. Annu. Rev. Immunol. 17: 657-700), probably by interfering with the co-localization of the CD4 and CXCR4 receptors (Y. Feng, et al ., 1996. Science 272: 872-877). Along these lines, it has also been described that actin and cytoskeleton tubulin are involved in the transmission and secretion of the HIV-1 virus (R. Pearce-Pratt, et al ., 1994. J. Virol. 68: 2898-2905 ; C. Jolly, et al ., 2004. J. Exp. Med. 199: 283-293).

Therefore, the molecular events that take place during cell fusion and the fusion pore formation mediated by the HIV-1 virus are key phenomena in the infectious process of said virus and the development of human acquired immunodeficiency disease (AIDS) . However, there are a number of still unknown aspects of the fusion process that takes place in an infectious process between a virus and its host cell, and more preferably of the HIV-1 virus infectious process, and the role of the cytoskeleton in this process. Similarly, there is also not much information on how the regulation of the infectious process of the HIV-1 virus can occur through interference in the dynamic state of the host cell's plasma membrane, although it has been observed that the HIV virus Env- 1 induces the reorganization of the actin cytoskeleton via Rac-1 (SE Pontow, et al ., 2004. J. Virol. 78: 7138-7147).

On the other hand, the binding of the cytoskeleton to the cellular plasma membrane provides a support mechanism for said membrane that regulates its rigidity, strength and elasticity (J. Dai and MP Sheetz, 1999. Biophys. J. 77: 3363-3370). The association between the cell membrane and the cytoskeleton is complex and includes cytoskeleton interactions with membrane proteins and lipids. Acetylated α-tubulin constitutes the hydrophobic form of tubulin that is predominantly associated with the membrane (M. Nunez-Fernandez, et al ., 1997. Mol. Cell Biochem. 170: 91-98) by its direct interaction with the ubiquitous cell surface protein Na +, K + -ATPase (CH Casale, et al ., 2003. FEBS Lett. 534: 115-118).

Experimental results indicate that acetylation of short cortical microtubules, of α-tubulin, is directly involved in the control of plasma membrane dynamics, and cell migration (C. Gubert, et al ., 2002. Nature 417 : 455-458), as well as in the stabilization of the different intracellular structures (BJ Jasmin, et al ., 1990. Nature 344: 673-675). In addition, acetylated α-tubulin protein is involved in the stability and functionality of microtubules (G. Piperno, et al ., 1987. J. Cell Biol. 104: 289-302). Stable microtubules regulate actin-dependent plasma membrane folding and cell mobility by activating Rac-1 (CM Waterman-Storer, et al ., 1999. Curr. Opin. Cell Biol. 11: 61-67) or Rho (G. Piperno, et al ., 1987. J. Cell Biol. 104: 289-302).

Acetylation of the α-tubulin protein is under the control of the enzyme histone deacetylase 6 (HDAC6) (C. Hubbert, et al ., 2002. Nature 417: 455-458; A. Matsuyama, et al ., 2002 EMBO J. 21: 6820-6831), belonging to class II of the HDACs family of proteins, which is mainly located in the cytoplasm of eukaryotic cells, and is inhibited by trichostatin (TSA) (C. Hubbert, et al ., 2002. Nature 417: 455-458; A. Matsuyama, et al ., 2002. EMBO J. 21: 6820-6831) and tubacina (SJ Haggarty, et al ., 2003. Proc. Natl. Acad. Sci USA 100: 4389-4394).

Recently, it has been described that the HDAC6 enzyme, through its tubulin deacetylase activity, controls the organization in the cell membrane of CD3 and LFA-1 surface receptors, the translocation of the microtubule organizing center (MTOC) and the activation of T lymphocytes (JM Serrador, et al ., 2004. Immunity 20: 417-28).

However, the role of the tubulin protein of the cortical cytoskeleton of the host cell on the capacity of viral fusion and fusion pore formation is unknown. Moreover, there is no data available on the possible implication of the acetylated forms of α-tubulin in the HIV-1 virus infection and fusion.

Description of the invention

The present invention is based on the fact that the balance of acetylation / deacetylation of microtubules It is key to modulate the entry of spherogenic viruses in the eukaryotic cells, preferably virus entry HIV-1 in T cells, and that is regulated by the HDAC6 protein, which allows defining new approaches Therapeutics for disease prophylaxis and treatment caused by fusogenic viruses, preferably caused by acquired human immunodeficiency virus (HIV-1). These therapeutic approaches are based in the use of activating compounds of said activity HDAC6 protein and pharmaceutical compositions that they contain.

Thus, an object of the present invention is constitutes the use of a compound or regulatory agent, preferably an activator, inducer or agonist of the activity HDAC6 protein tubulin deacetylase in the preparation of pharmaceuticals or pharmaceutical compositions for prophylaxis and treatment of diseases, disorders or pathologies of mammals, whether human or other animals, caused by the entrance and Eukaryotic cell infection by a spherogenic virus.

Another object of the present invention is constitutes a pharmaceutical composition or a medicine for the treatment of diseases, disorders or pathologies that occur with the entry and infection of a spherogenic virus in a cell eukaryotic, preferably in human cells, and more preferably in T lymphocytes comprising a compound or agent activator of the HDAC6 protein, in therapeutically quantity effective together with, optionally, one or more adjuvants and / or pharmaceutically acceptable vehicles and that is able to inhibit the entry of said virus into the eukaryotic cell. Another object of the present invention constitutes the use of the composition Pharmaceutical of the present invention in a treatment method of a mammal, preferably a human being, affected by a disease, disorder or pathology caused by the infection of a Fusonogenic virus consisting of the administration of said therapeutic composition in appropriate dose that modifies the state of tubulin acetylation and acting by inhibiting the entry of virus inside the cell.

Detailed description

The invention faces the problem of provide new effective tools for identifying  new pharmaceutical compounds and new therapies for prophylaxis and treatment of viral infections, preferably of spherogenic viruses, and more preferably of the virus HIV-1

The solution provided by this invention is based on the fact that the results described show that the acetylation / deacetylation of protein α-tubulin represents a key regulator of susceptibility of eukaryotic host cells to merge - fusion pore formation - and be infected by a fusogenic virus, without affecting expression and distribution in the cell surface of the receptors involved in the phase of specific recognition of the infective process of the cell host, specifically, infection of the T lymphocyte by the virus HIV-1 through CD4, CCR5 and CXCR4 receptors. This fact indicates that said route of regulation of the mechanisms general membrane fusion inherent to the fusion mechanism of a whole series of enveloped viruses, through the enzyme HDAC6, is key to allow infection of eukaryotic cells for these fusogenic viruses.

In the present invention, the stabilization of acetylation of the α-tubulin protein is obtained by silencing enzyme expression HDAC6 using interference RNAs (siRNA) or through its chemical inhibition (TSA). It should be noted that although the compound TSA can inhibit other nuclear HDCAs, other chemical inhibitors broad spectrum such as sodium butyrate (NaBut) or trapoxin B not affect tubulin deacetylase HDAC6 activity (C. Gubert, 2002. Nature 417: 455-458). Thus, the quantification of the acetylated α-tubulin protein in presence of TSA compared to NaBut, allows monitoring the effect of specific inhibition of tubulin activity HDAC6 enzyme deacetylase. It should be noted that the stabilization of acetylated α-tubulin by inhibiting the HDAC6 enzyme it was strongly associated with increased ability of cells to fuse and be infected for the HIV-1 virus.

On the contrary, the target cells that overexpress the active form "wild-type" (wt) of the enzyme HDAC6, coupled or not to the green GFP protein, they had reduced levels of acetylated α-tubulin as well as a minor susceptibility to merge and to be infected with the virus HIV-1, suggesting that acetylation of α-tubulin can regulate deformability of the cell membrane. However, the overexpression of the active form (wt) of HDAC6 not altered the expression of CD4 or CXCR4, nor its distribution in HIV-1 induced pseudopods. Besides, the redistribution of the HDAC6 enzyme in the clustering region CXCR4 / CD4 decreases the amount of α-tubulin  acetylated in the cell / virus contact areas HIV-1, as noted herein work, being able to be associated with a lower deformability of the membrane by the HIV-1 virus. Therefore, it proposes that the connection between the plasma membrane and the protein acetylated α-tubulin is found influenced by the activity of the HDAC6 enzyme, regulating the dynamics and elasticity of the plasma membrane, and therefore strongly influences the fusion capacity of proteins viral with their host cells, and specifically Env proteins gp41 / gp120 of HIV-1 virus with T lymphocyte.

How the HDAC6 enzyme is recruited in the areas contact cell / virus, overexpression of this enzyme can represent a cellular protection mechanism against HIV-1 virus infection, by altering the α-tubulin protein accumulation acetylated in the cell / HIV-1 contact areas. Is therefore plausible that the negative effect of the enzyme HDAC6 on the HIV-1 virus fusion and infection out more relevant the greater the amount of HDAC6 protein is expressed in host cells, which could easily act in the ways α-tubulin acetylates that concentrate on the cell / HIV-1 contact areas. To this respect, rich areas ("hot spots") generated by the action of HIV-1, where CD4 and the co-receptor, did not contain acetylated α-tubulin in the cells that overexpress the active enzyme HDAC6.

Taking into account all these evidences experimental, it can be postulated that enzyme inhibition HDAC6 represents a putative mechanism for positive regulation of the fusion capacity of gp41 / gp120 proteins, which could be dependent on the cell membrane capacity of the host to be deformable, and directly related to the state of the cortical cytoskeleton of tubulin. In this model, the Tubulin is involved in HIV-1 infection in the initial stages of infection, when the protein acetylated α-tubulin confers to the membrane cellular the dynamic state necessary for the fusion between the cell membrane-virus.

In summary, the present invention is based on the fact that the balance of acetylation / deacetylation of microtubules is key to modulate the entry of spherogenic viruses in eukaryotic cells, preferably the entry of the HIV virus in T cells, and that is regulated by the HDAC6 protein, which allows to define new therapeutic approaches for prophylaxis and treatment of diseases caused by viruses fusogenic, preferably caused by the virus Acquired human immunodeficiency (HIV-1). These therapeutic approaches are based on the use of compounds activators of the activity of said HDAC6 protein and of the pharmaceutical compositions that contain them.

HDAC6 activator or agonist compounds could prevent the fusion and entry of spherogenic viruses in eukaryotic cells, preferably fusion and virus entry HIV-1 in T lymphocytes. In this sense, in addition, gene transfer technology could, through vectors suitable, be useful in a treatment procedure of gene therapy to express, preferably in a way tissue-specific enzyme HDAC6 in the T lymphocytes of patients suffering from this type of disease.

Thus, an object of the present invention is constitutes the use of a compound or regulatory agent, preferably an activator, inducer or agonist of the activity tubulin deacetylase of the HDAC6 protein, hereinafter use of a compound of the present invention, in the preparation of pharmaceuticals or pharmaceutical compositions for prophylaxis and treatment of diseases, disorders or pathologies of mammals, whether human or other animals, caused by the entrance and Eukaryotic cell infection by a spherogenic virus.

As used in the present invention the term "compound / activating agent or agonist" refers to a molecule that when bound or interacts with the HDAC6 protein, or with functional fragments thereof, increases the intensity or prolongs the duration of the biological activity of said protein. This definition also includes those compounds or molecules that allow to increase the expression of the gene coding for the HDAC6 protein, either the naturally occurring endogenous form in a cell or an exogenous form that is artificially introduced in said cell. An activating agent may be constituted by a peptide, a protein, a nucleic acid, carbohydrates, a antibody, a chemical compound, or any other type of molecule that increases the effect and / or duration of the activity of the HDAC6 protein.

As used herein, the term "fusogenic virus" refers to a virus that infects eukaryotic cells, preferably from human and non-human mammals, by fusing the plasma or endosomal membrane of said host cell and the viral envelope. belonging, by way of illustration and without limiting the scope of the present invention, to the following group: the influenza virus (Review in, T. Stegmann and A. Helenius. Influenza virus fusion: fusion models toward a mechanism, in: J Bentz (Ed.), Viral Fusion Mechanisms, CRC Press, Boca Raton, FL, 1993, pp: 89-111), Semliki Forest virus (R. bron, et al ., 1993. EMBO J. 12: 693- 701), human immunodeficiency virus (HIV), simian immunodeficiency virus (SIV) (MO McClure, et al ., 1988. EMBO J. 7: 513-518), Sendai virus (D. Hoekstra, et al ., 1985. Biochemistry 24: 4739-4745), Rous Sarcoma virus (Review in, SG Peisajovich and Y. Shai. 2003. BBA 1614: 122-129 ), Vesicular Stomatitis virus (Review in, SG Peisajovich and Y. Shai. 2003. BBA 1614: 122-129), Ebola virus (Review in, SG Peisajovich and Y. Shai. 2003. BBA 1614: 122-129), Measles virus, Dengue virus, yellow fever virus and encephalitis (Review in, TH Sollner. 2004. Current Opinion in Cell Biology 16: 429-435; Review in, AE Smith and A. Helenius 2004. Science 304: 237-242).

A particular object of the present invention is  constitutes the use of a compound of the invention in which the Pharmaceutical composition is used for prophylaxis and treatment of acquired human immunodeficiency disease caused by the HIV-1 virus.

As used in the present invention The term "HIV-1 virus" refers to any of the existing strains of HIV-1 virus and HIV-2.

In this sense, in the state of the art there are described agonist compounds of the activity of the HDAC proteins, which can be used in the field of present invention as agonists of the HADC6 protein, and that indicated below for illustrative purposes without that meaning a limitation on the scope of the present invention: xanthines (US Patent 20030134865).

Thus, another particular object of the present invention constitutes the use of a compound of the present invention in which the agonist compound is constituted by a Xanthine or its derivatives.

Another particular object of the present invention it is constituted by the use of a compound of the present invention in which the compound is constituted by a genetic construction, hereinafter HDAC6 gene construct of the present invention, which allows the expression of a protein or peptide with activity tubulin deacetylase that mimics tubulin activity human HDAC6 protein deacetylase inside cells mammalian, preferably human, and more preferably in T lymphocytes, similar to genetic constructs elaborated in the present invention (see Example 3) and comprising one or more HDAC6 nucleotide sequences belonging to the following group:

to)

a nucleotide sequence consisting of the nucleotide sequence human tubulin deacetylase protein coding HDAC6,

b)

a nucleotide sequence analogous to the sequence of a),

C)

a fragment of any one of the sequences of a) and b), Y

d)

a nucleotide sequence comprising any sequence belonging to a), b) and c).

In the sense used in this description, the term "analogous" is intended to include any sequence of nucleotides that can be isolated or constructed based on the sequence shown herein (SEQ ID NO1), by example, by introducing nucleotide substitutions conservative or non-conservative, including the insertion of one or more nucleotides, the addition of one or more nucleotides in any of the ends of the molecule or the deletion of one or more nucleotides at any end or inside the sequence, and that allows the coding of a peptide or protein capable of mimic the tubulin deacetylase action of human HDAC6.

The nucleotide sequence, full length, coding for the HDAC6 human tubulin deacetylase protein that used in the present invention (SEQ ID NO1) is identified by access number AF132609 in the NCBI.

In general, an analogous nucleotide sequence is substantially homologous to the nucleotide sequence commented above. In the sense used in this description, the expression "substantially homologous" means that the nucleotide sequences in question have a degree of identity of at least 30%, preferably of at least one 85%, or more preferably, at least 95%. We must indicate that the nucleotide sequence of human HDAC6 (SEQ ID NO1) presents a variable homology with respect to other forms existing in other species.

Another particular embodiment of the present invention is the use of the compound of the present invention in which the nucleotide sequence of a) of the HDAC6 gene construct is the HDAC6 nucleotide sequence characterized by SEQ ID NO1 encoding the tubulin deacetylase protein human HDAC6 (SEQ ID NO2). A particular embodiment of the present invention is the use of the compound of the present invention in which the HDAC6 gene construct comprises the HDAC6 nucleotide sequence characterized in that it is a fragment of b) constituted by a nucleotide sequence encoding at least the sequence of amino acids comprised between the two histine residues 216 and 611 of human HDAC6 (SEQ ID NO2), key to its functional activity. It should be noted that the mutation in these two histines produces an inactive HDAC6 protein (see Example 3; Serrador JM et al ., 2004. HDAC6 deacetylase activity links the tubulin cytoskeleton with immune synapse organization. Immunity 20: 417-428) so This domain may be necessary for a fragment of HDAC6 to be functionally active.

The HDAC6 gene construct of the invention you can also understand, if necessary and to allow a better isolation, detection or secretion to the cytoplasm of the peptide expressed, to a nucleotide sequence that codes for a peptide capable of being used for isolation purposes, detection or secretion of said peptide. Therefore, another object Particular of the present invention is the use of a compound of the present in which the agonist compound is a HDAC6 genetic construct described above comprising, in addition to the nucleotide sequence HDAC6, any other nucleotide sequence encoding a peptide or sequence peptide that allows isolation, detection or secretion to the cellular cytoplasm of the peptide expressed, for example, by way of illustrative and without limiting the scope of the invention, a polyhistidine sequence (6xHis), a peptide sequence recognizable by a monoclonal antibody (for example, for its identification, or any other that serves to purify the fusion protein resulting by immunoaffinity chromatography: tag peptides such as c-myc, HA, E-tag) (Using antibodies: a laboratory manual. Ed. Harlow and David Lane (1999). Cold Spring Harbor Laboratory Press. New York Chapter: Tagging proteins. Pp. 347-377).

The HDAC6 nucleotide sequence and the HDAC6 genetic construct described previously can be obtained by one skilled in the art by employing techniques widely known in the state of the art (Sambrook et al . `` Molecular cloning, a Laboratory Manual 2 nd ed., Cold Sping Harbor Laoratory Press, NY, 1989 vol 1-3). Said nucleotide sequences may be integrated into a gene expression vector that allows regulation of the expression thereof under appropriate conditions.

Therefore, another particular object of the present  invention constitutes the use of a compound of the present invention in which the compound is constituted by a vector of gene expression, hereinafter HDAC6 expression vector, which comprises an HDAC6 nucleotide sequence or a construct HDAC6 genetics described in the present invention and allowing the expression of a protein or peptide with tubulin activity deacetylase that mimics the tubulin deacetylase activity of the human HDAC6 protein inside mammalian cells, preferably human cells, and more preferably in T lymphocytes.

In general, an expression vector comprises, in addition to the HDAC6 nucleotide sequence or the HDAC6 genetic construct described in the invention, a promoter that directs its transcription (eg, pT7, plac, ptrc, ptac, ABAD, Box Boxret , etc.), to which it is operatively linked, and other necessary or appropriate sequences that control and regulate said transcription and, where appropriate, the translation of the product of interest, for example, transcription initiation and termination signals ( t1t2 , etc), polyadenylation signal, origin of replication, ribosome binding sequences (RES), coding sequences of transcriptional regulators, (enhancers), transcriptional silencers (silencers), repressors, etc. Examples of appropriate expression vectors can be selected according to the conditions and needs of each specific case among expression plasmids, viral vectors (DNA or RNA), cosmids, artificial chromosomes, etc. which may also contain markers that can be used to select cells transfected or transformed with the gene or genes of interest. The choice of the vector will depend on the host cell and the type of use to be performed. Therefore, according to a particular embodiment of the present invention said vector is a plasmid. The obtaining of said vector can be carried out by conventional methods known to those skilled in the art, as well as for the transformation of microorganisms and eukaryotic cells different widely known methods can be used - chemical transformation, electroporation, microinjection, etc. - described in various manuals [ Sambrook, J., Fritsch, EF, and Maniatis, T. (1989). Molecular cloning: a laboratory manual, 2 nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY].

Gene expression systems may or may not allow the integration of the new genetic material into the genome of the host cell. Thus, both the nucleotide sequence, gene construct or the HDAC6 expression vector can be used as a medicament to protect host cells, preferably T lymphocytes, against infection of a spherogenic virus, preferably by the HIV-1 virus, in a procedure for treatment and prophylaxis of in vitro gene therapy of human T lymphocytes against HIV-1. Biopharmaceutical tools and gene therapy procedures are sufficiently known by an expert in the art sector.

Finally, the origin of these compounds agonist regulators of HDAC protein activity can be varied, so that they can be natural origin (by example, of plant, bacterial, viral, animal or eukaryotic microorganisms) or synthetic.

Another object of the present invention is constitutes a pharmaceutical composition or a medicine for the treatment of diseases, disorders or pathologies that occur with the entry and infection of a spherogenic virus in a cell eukaryotic, preferably in human cells, and more preferably in T lymphocytes, hereinafter composition Pharmaceutical of the present invention, comprising a compound or activating agent of the HDAC6 protein, in therapeutically quantity  effective together with, optionally, one or more adjuvants and / or pharmaceutically acceptable vehicles and that is able to inhibit the entry of said virus into the eukaryotic cell.

Pharmaceutical adjuvants and vehicles acceptable that can be used in said compositions are adjuvants and vehicles known to those skilled in the art and commonly used in the preparation of compositions therapeutic

In the sense used in this description, the expression "therapeutically effective amount" refers to the  amount of the agonist regulatory agent or compound of the HDAC6 protein tubulin deacetylase activity, calculated to produce the desired effect and, in general, will be determined, among other causes, due to the characteristics of the compounds, including age, patient's condition, severity of the alteration or disorder, and of the route and frequency of administration. In another particular embodiment, said composition Therapeutic is prepared in the form of a solid form or suspension aqueous, in a pharmaceutically acceptable diluent. The composition Therapeutic provided by this invention can be administered by any appropriate route of administration, for which said Composition will be formulated in the appropriate pharmaceutical form to the route of chosen administration. In a particular embodiment, the administration of the therapeutic composition provided by this invention is carried out parenterally, orally, orally intraperitoneal, subcutaneous, etc. A review of the different pharmaceutical forms of drug administration and of excipients necessary to obtain them can found, for example, in the "Pharmacy Treaty Galénica ", C. Faulí i Trillo, 1993, Luzán 5, S.A. Ediciones, Madrid.

Another particular object of the present invention it constitutes a pharmaceutical composition of the invention in the that the agonist compound is a xanthine or its derivatives.

Another particular object of the present invention it constitutes a pharmaceutical composition of the invention in the that the activating compound is a genetic construct or a vector of HDAC6 expression that allows the expression of a protein or peptide with tubulin deacetylase activity that mimics the Tubulin deacetylase activity of human HDAC6 protein in the inside mammalian cells, preferably human, and more preferably in T lymphocytes, similar to those genetic constructs elaborated in the present invention (see example 3) and comprising one or more of the sequences of HDAC6 nucleotides belonging to the following group:

to)

a nucleotide sequence consisting of the sequence of nucleotides encoding the human tubulin deacetylase protein HDAC6,

b)

a nucleotide sequence analogous to the sequence of a),

C)

a fragment of any one of the sequences of a) and b), Y

d)

a nucleotide sequence comprising any sequence belonging to a), b) and c).

Another particular embodiment of the present invention constitutes the pharmaceutical composition of the invention in which the nucleotide sequence of a) is the sequence of nucleotides SEQ ID NO1 coding for tubulin protein HDAC6 human deacetylase (SEQ ID NO2).

Another particular embodiment of the present invention constitutes the pharmaceutical composition of the invention in which the nucleotide sequence of b) is a fragment consisting of a nucleotide sequence encoding at least of the amino acid sequence between the two residues of histine 216 and 611 of the HDAC6 (SEQ ID NO2).

Another object of the present invention is constitutes the use of the pharmaceutical composition of the present invention, hereinafter use of the pharmaceutical composition of the present invention, in a method of treatment or prophylaxis of a mammal, preferably a human being, affected by a disease, disorder or pathology caused by the infection of a Fusonogenic virus consisting of the administration of said therapeutic composition in appropriate dose that modifies the state of  tubulin acetylation and acting by inhibiting the entry of virus inside the cell.

In this way, this agonist compound of the HDAC6 can favor the deacetylation of tubulin by increased HDAC6 protein tubulin deacetylase activity inside eukaryotic cells and therefore prevent virus entry and infection.

The use of the pharmaceutical composition of the The present invention can be used in a method of treating in isolation or in conjunction with other compounds Pharmacists

Another particular object of the present invention is the use of the pharmaceutical composition of the invention in a method of treating a disease caused by a spherogenic virus that infects eukaryotic cells, preferably human and non-human mammalian cells, belonging, by way of illustration. and without limiting the scope of the present invention, to the following group: influenza virus (Review in, T. Stegmann and A. Helenius. Influenza virus fusion: fusion models toward a mechanism, in: J. Bentz (Ed. ), Viral Fusion Mechanisms, CRC Press, Boca Raton, FL, 1993, pp: 89-111), the Semliki Forest virus (R. bron, et al ., 1993. EMBO J. 12: 693-701), the virus of human immunodeficiency (HIV), simian immunodeficiency virus (SIV) (MO McClure, et al ., 1988. EMBO J. 7: 513-518), Sendai virus (D. Hoekstra, et al ., 1985 Biochemistry 24: 4739-4745), Rous Sarcoma virus (Review in, SG Peisajovich and Y. Shai. 2003. BBA 1614: 122 -129), Vesicular Stomatitis virus (Review in, SG Peisajovich and Y. Shai. 2003. BBA 1614: 122-129), Ebola virus (Review in, SG Peisajovich and Y. Shai. 2003. BBA 1614: 122-129), Measles virus, Dengue virus, yellow fever virus and encephalitis (Review in, TH Sollner. 2004. Current Opinion in Cell Biology 16: 429-435; Review in, AE Smith and A. Helenius 2004. Science 304: 237-242).

Another particular embodiment of the present invention constitutes the use of the pharmaceutical composition of the  invention in a method of treating disease of Acquired human immunodeficiency caused by the virus HIV-1 spherogenic.

Description of the figures

Figure 1.- HDAC6 inhibition increases the cell-cell fusion mediated by Env proteins of the HIV-1 virus. (a) The marking X-Gal of HeLa P5 cells fused with cells HeLa ADA in absence (control) or presence of TSA (10, 100 and 300 nM), NaBut (2 µM), RANTES (300 and 700 nM) or antibody monoclonal (Acm) anti-CD4 (5 µg / ml). (b) Β-Gal quantification of the experiments of Env-mediated cell fusion of tropic strains R5 and tropic X4 in HeLa P5 cells treated with TSA, and in the presence or absence of anti-CD4 monoclonal antibody. (c) Western blot for kinetic analysis of protein acetylation acetylated α-tubulin in HeLa P5 cells treated with TSA (10, 100 and 300 nM) or NaBut (500 µM). The figures indicate how many times the protein is increased acetylated α-tubulin with respect to cells untreated (d) Analysis by flow cytometry of the expression of CD4, CXCR4 and CCR5 in HeLa P5 cells treated with 300 nM of TSA 2 µM NaBut or untreated cells (e) Western blot for the kinetic analysis of protein deacetylation α-tubulin after treatment with TSA (300 nM, 4 hrs.) in clarified HeLa P5 cells. The figures indicate the amount of acetylated α-tubulin protein after eliminating the TSA, and referring to the zero moment (4 hrs. of treatment with TSA 300 nM). (f) Quantification β-Gal of fusion experiments of HeLa ADA cells with HeLa P5 cells pretreated with TSA or Nabut for 4 hrs at 37 ° C before the addition of HeLa ADA cells. The effect of a monoclonal antibody is also shown. anti-CD4. (g) The X-Gal marking of HeLa ADA cells fused with untreated (control) or with HeLa P5 cells pre-treated with 300 nM TSA, in presence or absence of a monoclonal antibody anti-CD4.

Figure 2.- The inhibition of the HDAC6 enzyme increases cell fusion in protein-mediated T lymphocytes Env of the VHI-1 virus. (a) Cytometry analysis of fused syncytia flow between J77 cells and T cells Hxbc2-Env in the presence or absence of TSA or NaBut A Western blot of kinetic analysis of the acetylation of the α-tubulin protein in J77 cells treated with TSA (2 µM). The values indicate the increased α-tubulin acetylation in comparison with those defined in the control band of α-tubulin. (b) Images of syncytiums formed between J77 and Hxbc2 cells under TSA treatments and NaBut. In addition, the effect of a monoclonal antibody is shown. anti-CD4. (c) Flow cytometry analysis of J77 cells expressing CD4 and CXCR4 treated with TSA (300 nM), NaBut (2 µM) or untreated cells. (D) Internalization of CXCR4 induced by SDF-1α in the absence of TSA or in the presence of 300 nM of TSA. (and) acetylated α-tubulin in treated J77 cells with TSA (300 nM) or NaBut (2 mM) and in untreated cells. The data from fluorescence images and western blots are representative of 3 independent experiments (mean ± SE; n = 4).

Figure 3.- The silencing of the expression of HDAC6 increases fusion with cells expressing Env proteins of the HIV-1 virus. (a) Western quantification blot of the expression of the enzyme HDAC6 and of acetylated α-tubulin in HeLa P5 cells treated with siRNA. Fluorescence images show high levels of acetylated α-tubulin in cells where the expression of HDAC6 was silenced (arrowheads). (b) DIC images of syncytia formed between HeLa ADA cells fused with HeLa P5 cells treated with siRNA (1 and 2) or without treat (4) or with HeLa P5 cells transfected with a siRNA such as negative control and specificity (5). It also shows the effect of an anti-CD4 monoclonal antibody on untreated cells (3) or cells treated with siRNA-HDAC6 (6). (c) Quantification of β-Gal of Env-mediated cell fusion between HeLa P5 cells, treated with siRNA (yes) or not, with cells HeLa expressing Env of strains R5- or X4-tropics, in the presence or absence of a anti-CD4 monoclonal antibody.

Figure 4.- The silencing and inhibition of HDAC6 promotes HIV-1 virus infection. (to) Effect of TSA or NaBut on virus infection HIV-1 HeLa P5 cell with viral strains HIV-1_ {Bal} and HIV-1_ {NL43}. The infection was measured by producing β-Gal, 52 hrs after infection. (b) Viral production (measure of p24) in PBL activated with PHA pretreated with TSA or NaBut. Immunofluorescence images and Western blot analyzes show the levels of acetylated α-tubulin in PBL activated with PHA, after 2 hrs. of treatment with TSA (300 nM) or Nabut (2 µM). The effect of AZT, a neutralizer of the anti-CD4 monoclonal antibody, RANTES and SDF-1α in panels A and B. (c) Effect of silencing of siRNA HDAC6 in virus infection HIV-1 HeLa P5 cell. Infection is measured through β-Gal production. Also I know shows the effect of a neutralizing monoclonal antibody.

Figure 5.- The overexpression of HDAC6 decreases the ability of cells to fuse and be infected by the HIV-1 virus. (a) Expression of the wild (wt) and mutated (dm) forms of the enzyme HDAC6-GFP in HeLa P5 cells. (b) Analysis of western blot of wild enzyme expression (wt) and mutated (dm) HDAC6-GFP and its effects on the levels of acetylated α-tubulin protein. (c) Analysis of flow cytometry of the expression of CD4, CXCR4 and CCR5 in HeLa P5 cells not transfected or transfected with the form wild (wt) or mutated (dm) of the HDAC6-GFP enzyme. (d) Quantification (production of β-Gal) of the Env-mediated cell fusion between ADA and HeLa cells transfected (control), or between HeLa P5 cells transfected with the wild (wt) or mutated (dm) form of the enzyme HDAC6-GFP. DIC images of the syncytia formed after 16 hours of fusion. (e) Infection of HIV-1 virus in non-transfected HeLa P5 cells (control) or in transfected with the wild (wt) or mutated form (dm) of the HDAC6-GFP enzyme with the strain HIV-1_ {BaL}. In addition, the effect of a monoclonal antibody anti-CD4 and AZT. The images Western blot and DIC are representative of three experiments independent (mean ± SEM; n = 4).

Figure 6.- The overexpression of HDAC6 alters HIV-1 virus infection and the redistribution of α-tubulin protein acetylated in the cell / virus contact areas HIV-1 (a) Flow cytometry analysis of MT-2 cells that express the wild (wt) and mutated (md) of the HDAC6-GFP enzyme and protein GFP (b) Flow cytometry analysis of CD4 and CXCR4 in cells Parental MT-2, expressing wt-HDAC6 or dm-HDAC6. (c) Analysis of entry and infection of the HIV-1 virus in MT-2 cells transfected with retrovirus with the wild (wt) and mutated forms (md) of the HDAC6-GFP enzyme by means of DNA quantification HIV-1-gag intracellular, with respect to control MT-2 cells transfected or not with GFP (control). These results are representative of 3 independent experiments. (d) Location of acetylated α-tubulin and enzyme wild (wt) or mutated (dm) HDAC6-GFP in cells MT-2, before and during the infection of HIV-1 (control, left panel and HIV-1_ {NL43} in right panel, respectively).

Figure 7.- The HIV-1 virus recruit HDAC6 enzyme and protein acetylated α-tubulin in the contact areas  cell / virus, where CD4 and CXCR4 are co-distributed. (a) Redistribution of the α-tubulin protein acetylated in cell fusion models with HeLa cells and T lymphocytes (see arrowhead) in contact areas Syncytium cell / cell. (b) Expression and location of the endogenous enzyme HDAC6 and protein acetylated α-tubulin in cells Parental MT-2 infected by the virus HIV-1 and in uninfected cells. (c) Analysis of fluorescence microscopy of CD4, CXCR4 and wild enzyme (wt) or mutated (dm) HDAC6-GFP in cells MT-2 not infected (left panel) or infected (right panel).

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Examples of realization Example 1 The HDAC6 enzyme block promotes syncytium formation gp41 / gp120 dependent

In order to evaluate the effect of the stabilization of the α-tubulin protein on the fusion capacity of the envelope proteins of the HIV-1 gp41 / gp120 virus, chemical HDAC6 inhibitors were used. Thus, the effect of the general inhibitor of HDACs, TSA proteins, which also inhibits the tubulin deacetylase activity of HDAC6, and sodium butyrate (NaBut) in different cell-cell fusion models mediated by the HIV-1 virus was tested. First, a cell fusion model mediated by Env proteins was used in HeLa 243 cells or in ADA cells that were fused with HeLa P5 cells (Schwartz, O. et al ., 1994. Virology 198: 360-365; Pleskoff, O. et al ., 1997. Science 276: 1874-1878). It was observed that TSA strongly promotes the formation of giant polynucleated cells (syncytium) in a dose-dependent manner (Figure 1A, 1B), while NaBut did not cause such a result (Figure 1A). The syncytia formed by the cells treated with TSA were greater than those obtained in cells not treated or treated with NaBut (Figure 1A), and this effect was observed with both Env proteins of the tropic strains X4 and R5, with a maximum dose activity of 300 nM of TSA (Figure 1B). A clear correlation was observed between the effect of TSA on the fusion capacity of gp41 / gp120 (Figure 1B) and the amount of acetylated α-tubulin protein (Figure 1C), both being dose dependent (Figure 1B and 1C, which present 300 nM of TSA) and time. On the contrary, it did not observe any effect on acetylation of the α-tubulin protein with different doses and treatment times with NaBut (Figure 1C). As expected, the effect of TSA on syncytium formation was reversed by anti-CD4 neutralizing monoclonal antibodies (Figure 1A and 1B), or by RANTES (Figure 1A) and SDF-1α (data not shown). Moreover, the expression levels of CD4, CXCR4 and CCR5 in HeLa P5 cells were not significantly altered by the addition of TSA (Figure 1D). These data suggest that TSA favors the passage of fusion pore formation during the cell fusion process, causing changes in membrane deformability without altering the initial interaction of Env proteins with the CD4 receptor.

To assess the effect of TSA on the activation of LTR-lacZ , a series of cell-cell fusion experiments were performed by pretreating the HeLa P5 cells with TSA, and removing it before the addition of the HeLa Env + cells. It was observed that TSA stabilizes an important set of acetylated α-tubulin, which decreases to baseline levels after 2 hours after removing TSA (Figure 1E). The amount of acetylated α-tubulin removed at 30 and 60 minutes was 60% and 30% higher, respectively, than those observed in untreated cells or in NaBut treated cells (Figure 1E). As once contact with the HIV-1 virus occurs, membrane fusion and viral entry take place in approximately 30-60 minutes (Reeves, JD et al ., 2002. Proc. Natl. Acad. Sci. USA 99: 16249-54; Raviv, Y. et al ., 2002. Virology 293: 243-51), these data suggest that microtubule acetylation translates into greater permissibility of fusion cells. Therefore, treatment of HeLa P5 cells with TSA increases syncytium formation with respect to ADA treatment in HeLa cells (Figure 1F) and in HeLa 243 cells (data not shown). The effect of TSA or NaBut on the transcription of LTR-lacZ , after eliminating drugs, was excluded due to the absence of β-Gal production in non-fused HeLa P5 control cells, or in NaBut treated cells (Figure 1F, 1G)

In order to transfer these observations to T lymphocytes, a cell-cell fusion model based on Jurkat cells expressing X4-tropic Hxbc2-Env was used (Cao, J., et al ., 1996. J Virol 70, 1340-54 ), co-cultured with J77 cells (CXCR4 + / CD4 +). Treatment of J77 cells with TSA clearly stabilizes an intense acetylated α-tubulin protein cortex (Figure 2A, 2E) and induces the formation of large syncytia, while NaBut produced no effect (Figure 2A and 2B). As observed in HeLa cells, the expression of CXCR4 and CD4 proteins was not significantly modified by treatment with TSA or with NaBut (Figure 2C), nor was internalization of CXCR4 induced by SDF-1α affected by TSA (Figure 2D).

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Example 2 Silencing the HDAC6 protein increases the input and HIV-1 infection

To further assess the role of acetylated α-tubulin protein in fusion and HIV-1 infection, selective silencing of HDAC6 expression was performed by specific inhibition of its mRNA with interfering RNAs (siRNA) (Hubbert C., et al ., 2002. Nature 417: 455-458; Serrador JM, et al ., 2004. Immunity 20: 417-28). Inhibition of endogenous HADC6 expression in HeLa P5 cells induced a 3-fold increase in the α-tubulin protein, without affecting the total levels of this protein or other cytoskeleton components, such as vimentin (Figure 3A) or the expression of CXCR4, CCR5 and CD4 (data not shown). Specific HDAC6 silencing generated more permissible HeLa P5 cells, which exhibit greater fusion activity with both Env R5-tropic and X4-tropic (Figure 3B, 3C).

In fusion models cell-cell, the contact areas presented by the cells expressing Env were greater than those observed in cell infection models, through the use of purified HIV-1 viral particles. The next question that was asked was whether the effect of inhibition of HDAC6 and, therefore, the subsequent stabilization of the protein cortical acetylated α-tubulin, influences the HIV-1 virus infection using cells HeLa P5 or peripheral cell lymphocytes activated with PHA (PHA-activated PBL). Virus infection HIV-1 in HeLa P5 cells was assessed by β-Gal production and activation of peripheral blood lymphocytes by PHA by determination of the p24 antigen in the cell-free supernatant (Figure 4A and 4B, respectively). It was observed that TSA increased infection by the HIV-1 virus in a dose-dependent manner in HeLa P5 cells and in PBL (Figure 4A and 4B) with any of the strains used: R5-tropic HIV-1_ {Bal} and X4-tropic HIV-1 NL-43 (Figure 4A and 4B). TSA increased HIV-1 infection through the cytosolic HDAC6 enzyme inhibition and stabilization of cortical acetylated α-tubulin protein (Figure 4B), which was maintained after the removal of TSA (Figure 4A and 4B), which excludes the transcriptional effects of TSA in the β-Gal production and in the replication of HIV-1 virus. As in fusion models cell-cell, the increased infection of the HIV-1 virus in HeLa P5 cells (Figure 4A) and in PBL cells activated with PHA correlated with levels of acetylated α-tubulin protein (Figure 1E and 4B). Also, HDAC6 silencing produced HeLa P5 cells more permissive, which were more effectively infected with viral particles of tropic R5- or X4 strains of HIV-1 (Figure 4C).

These results suggest that the levels of acetylated α-tubulin protein exert a clear effect on the susceptibility of target cells to fuse and be infected by the HIV-1 virus.

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Example 3 Overexpression of HDAC6 alters cell fusion and HIV-1 viral infection

The effect of overexpression of the active wild form of HDAC6 (wild-type, wt) (Figure 5A and 5B) on the fusion capacity of gp120 / gp41 proteins and HIV-1 virus infection was also assessed. . As a control, a mutated form of the inactive enzyme HDAC6 deacetylase (dm) was used in parallel (Figure 5A and 5B) in which two histidine residues key to deacetylase activity were mutated to alanine (Serrador JM, et al ., 2004. Immunity 20: 417-28). It was observed that i) the fusion capacity of HeLa P5 cells transformed with the wild form of the HDAC6-GFP protein with HeLa ADA cells (Figure 5D) or with 243 cells (data not shown), and ii) the ability to be infected by viral strains of HIV-1 tropics R5 and X4 (Figure 5E and data not shown, respectively) were drastically reduced with respect to that observed with parental cells or HeLa P5 cells transfected with the inactive form of HDAC6-GFP (dm).

Similar results were observed in HIV-1 virus infection experiments (Figure 6C) with the T lymphocyte cell line MT-2 expressing the wild or mutated form of HDAC6-GFP, translated by viral spinoculation (Figure 6A, about 70% of the cells express the two enzyme forms). Acetylated cortical microtubules are poorly detected in MT-2 cells that express the wild form of the HDAC6-GFP protein (Figure 6D, determined by the absence of fluorescence in cells white (marked with arteriscus) when compared to cells parental or MT-2 cells that express the form mutated HDAC6-GFP protein (Figure 6D, see the fluorescence in cells at the bottom of the figure). Be observed that the wild enzyme HDAC6-GFP alters the HIV-1 virus entry and infection (Figure 6C, about 75% inhibition) by determination quantitative proviral DNA HIV-1-gag content in cells MT-2 infected 12 hours after infection with the HIV-1 virus (Figure 6C). On the other hand, the Overexpression of the HDAC6 enzyme did not modify the levels of CD4, CCR5 or CXCR4 on the cell surface (Figure 5C and 6B).

Therefore, the susceptibility of cells to being infected by the HIV-1 virus seems to be directly related to your protein levels α-tubulin acetylated, and inversely linked to the activity of the HDAC6 enzyme (Figure 5E and 6C.)

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Example 4 The HIV-1 virus recruits the enzyme HDAC6 and the acetylated α-tubulin protein in the areas of virus / cell contact

As previously mentioned, cooperative interaction must take place between the viral envelope and the cellular receptor and the co-receptor molecules for the entry of the virus (Layne SP, et al ., 1990. Nature 346: 277 -9; Kuhmann SE, et al ., 2000. J. Virol 74: 7005-15) Modification of the co-distribution of CD4 and co-receptor molecules in the cell / virus contact areas can alter the process of fusion pore formation (Dimitrov DS, et al ., 1999. Virology 259: 1-6) and, therefore, regulate HIV-1 virus infection.

Therefore, we proceeded to determine if the HDAC6 enzyme and protein concentration deacetylated α-tubulin located in the cell / cell contact area during virus infection HIV-1 induced syncytium formation. In both transformed cells, HeLa and MT-2, the form wild (wt) and mutated (dm) of the HDAC6-GFP enzyme they were located in the cytoplasm (Figure 5A, 6D and 7C), so similar to that observed with the endogenous enzyme (Figure 7B). The HeLa P5 and MT-2 cells expressing the two forms of the HDAC6 enzyme did not undergo significant morphological changes and as indicated above, the levels of CD4, CXCR4 and CCR5 in the cell surface remained unchanged (Figure 5C and 6B). Interestingly, it was observed that the protein acetylated α-tubulin was concentrated mainly in the contact area of the cells using both Env R5-tropic and X4-tropic cells (Figure 7B, indicated by arrows.)

Next, the location of the HDAC6 enzyme during cell infection and it was observed that the HIV-1 virus induced redistribution of the enzyme HDAC6-GFP towards pseudopodia structures, co-locating c on CXCR4 and CD4 (Figure 7C), what which indicates that the HDAC6 enzyme is recruited in the areas of HIV-1 cell / virus contact. This redistribution of the HDAC6 enzyme was independent of its enzymatic activity, since that both the wild and the mutated form presented a similar location (Figure 7C). Moreover, the protein acetylated α-tubulin is also concentrated in HIV-1 virus-induced pseudopodia, although only in cells that express the inactive form (dm) of the HDAC6-GFP enzyme (Figure 6D, see fluorescence in the lower figures of the right panel) or in T cells not treated with low levels of endogenous enzyme HDAC6 (Figure 7B), while no such concentration is observed in cells that overexpress the active form (wt) of the enzyme HDAC6-GFP (Figure 6D, evaluated by the absence of fluorescence, white cells in the right panel, see asterisk).

The ability of the enzyme tubulin deacetylase HDAC6 to regulate the co-distribution of CXCR4 and CD4 induced by the HIV-1 virus was analyzed during initial stages of the infective process. The virus strain HIV-1NL43 Tropic X4 induced clustering of CXCR4 and CD4 after 90 minutes of cell infection MT-2 transformed with either the wild form (wt) or with the mutated form (dm) of the enzyme HDAC6-GFP (Figure 7C). Overexpression of the active form (wt) of the enzyme HDAC6-GFP had no effect on the co-distribution of CD4-CXCR4 (Figure 7C). These data indicate that the HDAC6 enzyme alters the input and HIV-1 virus infection by a mechanism that does not seem to alter virus interactions HIV-1 / receptor-co-receptor  in the cell membrane.

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Material and methods Antibodies and reagents

The anti-CD4 monoclonal antibody (mAb) HP2 / 6 (IgG1) that inhibits the gpl20 / CD4 interaction has already been described previously (Carrera AC, et al ., 1987. Eur. J. Immunol. 17: 179-86); anti-CD4 v4-phycoerythrin (PE) (IgG1) mAb that does not react with the gp120 junction point was provided by Becton Dickinson (San Jose, CA). The polyclonal antibody sc-5255 anti-HDAC6 was provided by Santa Cruz Biotechnology Inc (Santa Cruz, CA), and the anti-α-tubulin mAb B-5-1-2, the acetylated anti-tubulin 6-11B-1 and anti-vimentin were provided by Sigma Chemical Co. (St. Louis, MO). TSA and sodium butyrate (Nabut) were purchased from Calbiochem (San Diego, CA). SDF-1α was kindly provided by Dr. F. Baleux (Pasteur Institute, Paris, France), RANTES was obtained from R&D (Marina del Rey, CA). The fluorescent locators of CMTMR and CMAC cells were obtained from Molecular Probes (Eugene, OR).

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Cells

Jurkat J77 human T cell lines, MT-2 cells and Jurkat T cells expressing the envelope of HIV-1 (Env) -Hxbc2 virus (Jurkat Hxbc2) were grown in RPMI 1640 culture medium supplemented with 10% of FCS The HeLa P5 cell clone, stably transformed with the cDNA of human CD4 and CCR5 proteins and with a β-Gal reporter gene directed by the HIV LTR (Pleskoff O., et al ., 1997. Science 276: 1874 -8) was provided by Dr. M. Alizon (Hópital Cochin, Paris, France). HeLa 243 and ADA cells were also provided by Dr. M. Alizon, and co-express the Tat and Env proteins of the HIV-1 virus (Schwartz O., et al ., 1994. Virology 198: 360-5; Pleskoff O., et al ., 1997. Science 276: 1874-8). Human peripheral blood lymphocytes (PBL) were obtained and isolated from healthy donors using a density gradient by Ficoll-Hypaque centrifugation (Amersham Biosciences, Buckinghamshire, UK). PBL cells were activated for 3 days with 1 µg / mL of PHL (Murex Diagnostics Corp. Norcross, GA) and were then cultured with IL-2 (6 units / mL).

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HDAC6 recombinant DNA constructs and technique lipo-cellular transfection

The wild construction (wt) -HDAC6-GFP and its mutant (dm) -HDAC6-GFP, which has a double mutation (H216A / H611A) that causes inactivation deacetylase have been previously described (Serrador JM, et al ., 2004. Immunity 20: 417-28). These two expression vectors were transfected into HeLa P5 cells using Megafectin-20 (Qbiogene Inc. Carlsbad, CA) according to the manufacturer's instructions.

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HDAC6 retroviral transduction in T cells

MT-2 and Jurkat J77 cells expressing GFP, the wild (wt) or mutated (dm) form of HDAC6-GFP were obtained by infection with a modified Moloney murine virus (MLV), which is defective for replication. Briefly, GFP, wt-HDAC6-GFP and dm-HDAC6-GFP were cloned into the bicistronic vector pLZR-IRES / GHR (kindly provided by Dr. Antonio Bernad, National Center for Biotechnology (CSIC) Madrid, Spain). Retroviral production and cell transduction was carried out as described previously (Abad JL, et al ., 2002. J. Gene. Med. 4: 27-37).

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Flow cytometry analysis and tests of internalilzación

Cells not treated and treated with TSA or NaBut were washed and incubated with anti-CXCR4 or anti-CCR5 antibodies labeled with PE-12G5, or with anti-CD4 labeled with FITC for 30 minutes at room temperature. As controls other mAbs were used against irrelevant domains. For the CXCR4 internalization assays, J77 cells were incubated with different concentrations of SDF-1α for 1 hour at 37 ° C and subsequently the cells were analyzed as described (Valenzuela-Fernandez A., et al ., 2001. J. Biol. Chem. 276: 26550-8).

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Immunofluorescence

HeLa P5, MT-2 and Jurkat J77 cells were fixed and permeabilized for 3 minutes in PBS 2% formalde-
Hybrid - 0.5% Triton X-100, and immunostained for α-tubulin, acetylated α-tubulin or HDAC6 as described previously (Grozinger CM, et al ., 1999. Proc. Natl. Acad. Sci. USA 96 : 4868-73; Serrador JM, et al ., 2004. Immunity 20: 417-28). The cells were visualized using a Leica DMR photomicroscope (Leica, Heidelberg, Germany) and a Leica TCS-SP confocal microscope.

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Western blot analysis

The cells were resuspended in 60 µL of a MES solution (10 mM MES at pH 7.4, 150 mM CiNa, 5 mM EGTA, 5 mM MgCl2) which also contains a set of protease inhibitors (Roche Diagnostics GMBH , Mannheim, Germany), were sonicated twice for 30 seconds at 4 ° C and boiled in 5% β-mercaptoethanol solution for 1 minute at 100 ° C. Cell lysates were analyzed on a 12% SDS-PAGE gel and immunolabelled for the HDAC6, α-tubulin, acetylated α-tubulin and vicentin protein as described (Serrador et al . (2004) HDAC6 deacetylase activity links the tunulin cytoskeleton with immune synapse organization. Immunity 20: 417-28).

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Determination of cell-cell cell fusion Env-mediated HIV-1 virus

The β-galactosidase cell fusion assays were performed as described (Valenzuela-Fernandez A., et al ., 2001. J. Biol. Chem. 276: 26550-8). Briefly, HeLa 243 or HeLa ADA cells were co-incubated with HeLa P5 cells arranged in 96-well plates in a 1: 1 ratio for 16 hours. Then, the fused cells were washed, lysed (50 µL of lysis solution) and the enzymatic activity was evaluated by chemiluminescence (β-gal reporter gene assay, Roche Diagnostics GMBH). When indicated, HeLa P5 cells were pre-treated with TSA or NaBut. X-Gal staining of the syncytiums formed, after 16 hours of cell co-culture was performed by fixing the cells with 0.5% glutaraldehyde and labeling with the substrate of β-galactosidase X-gal (5-bromo-4 -chloro-3-indoyl-? -D-galactopyranoside), as described above (Pleskoff O., et al ., 1997. Science 276: 1874-8).

The double fluorescence cell fusion assay was performed as described (Valenzuela-Fernandez A., et al ., 2001. J. Biol. Chem. 276: 26550-8). Briefly, Jurkat Hxbc2 cells, which express the Hxbc2 Env of the tropic strain X4 of the HIV-1 virus, labeled with CMAC were co-cultured with TJ77 cells pre-loaded with CMTMR. The cellular syncytium, labeled with two chromophores, was detected 16 hours later by flow cytometry or fluorescent microscopy. When indicated, the cells were pre-incubated with TSA, Nabut or anti-CD4 mAb.

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Preparation of HIV-1 virus

Highly infectious preparations of HIV-1NL43 and HIV-1BAL virus strains were generated by several consecutive passes of the original HIV-1 isolates isolated from peripheral blood mononuclear cells (PBMC) as described ( Valenzuela A., et al ., 1997. J. Virol. 71: 8289-98). Briefly, PBMC cells were infected with a synchronous dose of HIV-1 NL43 and HIV-1 BAL virus, culture supernatants (centrifuged at 5,000 xg for 10 minutes) were recovered three days later and kept at 70 ° C. Fresh defrosted aliquots were filtered through a 0.22 µm pore size filter before use.

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HIV-1 virus infection and entry

Infection and entry of the HIV-1 NL43 virus was tested in PBL cells activated by PHA or in MT-2 cells. When indicated, T cells pre-treated with anti-CD4 mAbs (5 µg / mL), SDF-1α (300 nM), AZT (5 µg / mL), TSA or NaBut. The entry of HIV-1 virus into MT-2 cells was monitored for 12 hours after infection with HIV-1 with the measurement of HIV-1 gag proviral DNA by Taqman-PCR using specific primers as described above ( Zhao Y., et al ., 2002. J. Clin. Microbiol. 40: 675-8). HIV-1 infection of PBL cells activated with PHA were evaluated by measuring the concentration of p24 in the culture supernatant by ELISA (INNOTEST HIV-1 antigen aAb Innogenetic NY Ghent, Belgium) as described (Valenzuela A., et al ., 1997. J. Virol. 71: 8289-98).

Infection with virus strains HIV-1_ {NL43} and HIV-1_ {BAL}, of HeLa P5 cells were performed in 96-well plates for 5 hours at 37 ° C. The virus was removed by washing (PBS) and the Infected cells were treated with trypsin (5 minutes at 37 ° C). HeLa P5 cells were cultured during the next 52 hours. Then, the infected cells were lysed, and the HIV-1 infection levels were evaluated by measurement of β-galactosidase. When indicate, the cells were pre-treated with TSA or NaBut for 2 hours at 37 ° C before infection with HIV-1

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HDAC6 Mute

A double stranded siRNA was generated against the HDAC6 sequence between nucleotides 211-231 (Eurogenentec, Seraing, Belgium) as described (Serrador JM, et al ., 2004. Immunity 20: 417-28). HeLa P5 cells were transfected with this siRNA at a final concentration of 200 nM using Oligofectamine (Invitrogen Corp. Carslbad, CA) according to the manufacturer's instructions. Negative control transfection with reagent only, or with transfection mixtures with a double stranded siRNA (Eurogenentec, Seraing, Belgium) that do not recognize any eukaryotic protein, were used as controls. The cells were transfected twice for 3 days and collected 24 hours after the last transfection. Total cell lysates (40 µg) were analyzed by Western blotting with anti-HDAC6, acetylated anti-α-tubulin and anti-α-tubulin antibodies.

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Grouping of CXCR4 and CD4 proteins during infection with HIV-1

Jurkat J77 and MT-2 cells transfected with the wild form (wt) of HDAC6-GFP or mutated (dm) HDAC6-GFP were co-cultivated with the strain HIV-1 NL43 (MOI, 1) for 90 minutes at 37 ° C. Then, the viral particles were cleared and the cells set with 2% paraformaldehyde. The co-distribution of CD4 and CXCR4 in structures of pseudopodia was analyzed by immunofluorescence of the cells labeled with a non-neutralizanet monoclonal antibody PE-v4 and with Alexa 350-streptavidin against a biotinylated primary monoclonal antibody anti-CXCR4.

<110> UNIVERSITY AUTONOMA DE MADRID

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<120> USE OF AGONIST COMPOUNDS OF THE HDAC6 TUBULINA DEACETILASE ACTIVITY IN THE ELABORATION OF PHARMACEUTICAL COMPOSITIONS, SUCH PHARMACEUTICAL COMPOSITIONS AND ITS APPLICATIONS IN THE TREATMENT OF VIRAL INFECTIONS

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<130> P574 / 2005

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<160> 2

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<170> PatentIn version 3.3

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<210> 1

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<211> 3648

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<212> DNA

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<213> Homo sapiens

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<220>

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<221> CDS

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<222> (1) .. (3648)

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<223> HDAC6 coding sequence

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<400> 1

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<210> 2

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<211> 1215

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<212> PRT

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<213> Homo sapiens

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<400> 2

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2. 3

Claims (5)

1. Use of an agonist compound of the activity tubulin deacetylase of the HDAC6 protein, consisting of a xanthine or its derivatives, in the preparation of medicines or Pharmaceutical compositions for the prophylaxis and treatment of diseases of mammals, whether human or other animals, caused by the entry and infection of a spherogenic virus of eukaryotic cells. 2. Use of a compound according to claim 1 characterized in that the fusogenic virus belongs to the following group: influenza virus, Semliki Forest virus, human immunodeficiency virus (HIV), simian immunodeficiency virus (SIV) Sendai virus, Rous Sarcoma virus, Vesicular Stomatitis virus, Ebola virus, Measles virus, Dengue virus, yellow fever virus and encephalitis. 3. Use of a compound according to claim 1 characterized in that the fusogenic virus is the HIV-1 virus. 4. Use of a compound according to the preceding claims , characterized the eukaryotic cell of a mammal is a human T cell. 5. Pharmaceutical composition or a medicine for the treatment of diseases, disorders or pathologies that occur with the entry and infection of a spherogenic virus in a eukaryotic cell characterized in that it comprises an agonist compound of the human HDAC6 protein, consisting of a xanthine or its derivatives , in therapeutically effective amount together with, optionally, one or more pharmaceutically acceptable adjuvants and / or vehicles.