ES2319838B1 - USE OF INHIBITORS OF THE ACTIVITY OF THE PHILAMINE PROTEIN, FOR THE ELABORATION OF PHARMACEUTICAL COMPOSITIONS, INHIBITING COMPOUNDS OF SUCH ACTIVITY, PHARMACEUTICAL COMPOSITIONS AND THEIR THERAPEUTIC APPLICATIONS AND PROCEDURE IDENTIFICATION OF SUCH INHIBITED COMPOUNDS. - Google Patents

Abstract

Use of inhibitors of the activity of the Filamina A protein for the preparation of compositions pharmaceuticals, compounds that inhibit said activity, pharmaceutical compositions and their therapeutic applications and identification procedure of said compounds inhibitors

The present invention describes for the first time the inducing activity of HIV-1 infection of Filamina A (FLNa) protein as a regulator of interactions of the CD4 protein and of the CXCR4 and CCR5 coreceptors, thus becoming a therapeutic target. Also I know have identified the domains of these regulatory proteins of said interactions identifying inhibitory molecules of same that can be used for the elaboration of therapeutic compositions useful for treatment and prevention of HIV-1 infection, among others diseases. Finally, FLNa can become the basis of a procedure for identifying new compounds HIV-1 infection inhibitors.

Description

Use of inhibitors of the activity of the Filamina A protein for the preparation of compositions pharmaceuticals, compounds that inhibit said activity, pharmaceutical compositions and their therapeutic applications and identification procedure of said compounds inhibitors

Technical field

The present invention is framed in the development of new pharmaceutical compositions for prophylaxis and the treatment of diseases caused by the virus HIV-1 or those with impaired stimulation of the immune system. Therefore, you must subscribe to the sector Pharmacological and biomedical of viral diseases.

State of the art

HIV-1 spreads through the interaction of the virus with a target cell, or by means of Direct transmission between an infected cell with a healthy cell. In both situations, infection of the target cells by HIV-1 requires that the envelope protein (Env) gp120 induces the clustering of numerous molecules of CD4 and of co-receptor on the cell surface. This allows gp41 transmembrane glycoprotein activation of the viral envelope, and the formation of a complex that fuses the membranes containing Env with the membrane of the target cell (1). These interactions depend largely on concentration and distribution of cell receptors, and the content of membrane cholesterol (2-9). Kinetic studies have shown that the grouping of viral receptors in the target cell surface does not occur by passive diffusion, but that need to be actively transported to the contact area Env / receptor by the actin cytoskeleton (10, 11). In fact, the target cell treatment with remodeling inhibitors of F-actin or with protein inhibitors myosin, reduces Env-mediated fusion and / or clustering of receptors (7, 10, 12).

Although the basic elements are known involved in the initial stages of infection, events molecular factors underlying the formation of the fusion complex viral are not clarified (13). For example, they are poorly defined the mechanisms by which HIV stimulates the remodeling of cytoskeleton of the host cell during infection; the Evidence obtained is weak and occasionally contradictory. There is a strong debate about the relative importance of necessary interactions for the grouping of the receptors viral. It has been suggested that the accumulation of F-actin in the zone of contact between the cell target and the virus or infected cell requires the signals mediated by Env-CD4 and Env-co-receptor (10); However, CD4 crosslinking may be sufficient to promote the grouping of chemokine receptors, F-actin, cholesterol and other components of lipid rafts in T cells (11). More important, they are unknown the specific signaling pathways that lead to remodeling of F-actin after HIV binding to receptors, although it is known that neither the signaling of CD4 via p56Lck nor the coupling of co-receptors to G proteins heterotrimerics are necessary for infection (14-16). The identity of activated Rho GTPases by viral receptors it is also a matter of conflict; a study has associated Rac (but not RhoA) with fusion mediated by Env (17), while another has involved RhoA (but not Rac) in said process (18). Finally, although CD4 and co-receptors co-locate in structures rich in ezrin and talin (19), the direct interaction of viral receptors with these proteins of actin binding, nor has the consequences of such hypothetical interactions in the reorganization of F-actin or infection by HIV-1

One possibility is that CD4 and / or co-receptors are bound to a protein adapter that connects these receptors to the actin cytoskeleton and that, at the same time, organize the signaling required for the reorganization of F-actin; said "partner" Molecular has not been described to date. Binding proteins to actin of the family of phylamines can carry out said function. A member of this family, filamina A (FLNa), is present in the periphery of the cytoplasm. FLNa intersects filaments of actin in orthogonal networks and anchor these networks to the membrane cellular and signaling intermediaries (20). FLNa consists in two subunits of 280,000 relative molecular mass, each of which contains 24 tandem repetitions of approximately 96 amino acids with immunoglobulin-like leaf structure. The actin binding domain of FLNa is in the N-terminal while the dimerization domain it is located at repetition 24 in the C-terminal (twenty-one). Both domains are essential for the branching of actin filaments. FLNa interacts with a number of receivers of membrane and intracellular signal transduction proteins, suggesting its function as signaling adapters connecting and coordinating cellular processes with regulation Actin cytoskeleton dynamics (22). The Rho family GTPases (23) and some of their regulatory co-factors are assembled with FLNa which, in turn, regulates GTPase activity, allowing FLNa participate in space-time remodeling of F-actin.

A therapeutic approach would be the use of compounds that interact with HIV-1 receptors, capable of regulating the early events in the reorganization of F-actin induced by the binding of the virus to the cell
Guest.

The initial stages of infection by the human immunodeficiency virus (HIV-1) require the clustering of numerous molecules of the CD4 receptor and of the CXCR4 and CCR5 co-receptors, in a process mediated by the viral glycoprotein gp120 and the cytoskeleton of actin The mechanisms by which HIV-1 induces the reorganization of F-actin (actin filamentous) in the target cell are unknown.

Although it has been described co-location of CD4 and HIV-1 co-receptors in structures rich in ezrin and talin (19), direct interaction between these actin binding proteins and receptors has not been  established.

Patent Description brief description

An object of the present invention constitutes it the use of a protein activity inhibitor compound FLNa (hereinafter, FLNa), hereinafter use of an inhibitor compound of the present invention, for the preparation of a medicament or pharmaceutical composition for the treatment of infection by HIV-1 and diseases that occur with a impaired immune system stimulation.

A particular object of the invention is constitutes the use of a compound that inhibits the activity of the FLNa based on the use of a nucleotide sequence as inhibitor compound, hereinafter FLNa nucleotide sequence of the present invention, which allows the expression of a protein or peptide in human cells, and that is constituted by one or several nucleotide sequences belonging to the following group:

to)

a nucleotide sequence consisting of the nucleotide sequence encoding domain 10 of FLNa human (FLNa D10) of SEQ ID NO1,

b)

a nucleotide sequence consisting of the domain coding nucleotide sequence 13-16 of the human FLNa (FLNa D13-16) of SEQ ID NO9,

C)

a nucleotide sequence analogous to the sequence of a) and b),

d)

a nucleotide sequence, construction genetics, which comprises any sequence belonging to a), b) and c).

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Another particular object of the invention is constitutes the use of a compound that inhibits the activity of the FLNa based on the use of a derived FLNa nucleotide sequence of CD4 as an inhibitor compound that is constituted by one or several nucleotide sequences belonging to the following group:

to)

a nucleotide sequence consisting of the nucleotide sequence encoding the CD4 domain constituted by residues 415-421 (SEQ ID NO7),

b)

a nucleotide sequence analogous to the sequence of a), and

C)

a nucleotide sequence, construction genetics, which comprises any sequence belonging to a) and b).

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Another particular object of the invention is constitutes the use of a compound that inhibits the activity of the FLNa based on the use of a derived FLNa nucleotide sequence of CXCR4 as an inhibitor compound that is constituted by one or several nucleotide sequences belonging to the following group:

to)

a nucleotide sequence consisting of the CXCR4 domain coding nucleotide sequence constituted by SEQ ID NO11,

b)

a nucleotide sequence analogous to the sequence of a), and

C)

a nucleotide sequence, construction genetics, which comprises any sequence belonging to a) and b).

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Another particular object of the invention is constitutes the use of a compound that inhibits the activity of the FLNa based on the use of a derived FLNa nucleotide sequence of CCR5 as an inhibitor compound that is constituted by one or several nucleotide sequences belonging to the following group:

to)

a nucleotide sequence consisting of the CCR5 domain coding nucleotide sequence consisting of waste 325-352 (SEQ ID NO13),

b)

a nucleotide sequence analogous to the sequence of a), and

C)

a nucleotide sequence, construction genetics, which comprises any sequence belonging to a) and b).

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Therefore, another particular object of the invention is constituted by the use of a compound that inhibits FLNa activity based on the use of a sequence of FLNa nucleotides as an inhibitor compound that prevents or decreases the expression of the gene coding for the human FLNa protein consisting of one or several selected nucleotide sequences between:

to)

a specific antisense nucleotide sequence of the sequence of the FLNa protein gene or mRNA,

b)

a specific ribozyme of the protein mRNA FLNa,

C)

a specific mRNA aptamer of the protein FLNa, and

d)

an mRNA-specific interference RNA (iRNA) of the FLNa protein.

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In addition, another particular object of the invention it is constituted by the use of an inhibitor compound of the invention in which the inhibitor compound is one or several proteins or peptides, hereinafter use of the FLNa protein of the present invention, and that It consists of one or several amino acid sequences belonging to the following group:

to)

an amino acid sequence consisting of the amino acid sequence of domain 10 of human FLNa (FLNa D10) (SEQ ID NO2),

b)

an amino acid sequence consisting of the amino acid sequence of domain 13-16 of the Human FLNa (FLNa D13-16) (SEQ ID NO10),

C)

an amino acid sequence analogous to the sequence of a) and b), and

d)

an amino acid sequence comprising a any sequence belonging to a), b) and c).

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Another particular object of the invention is constitutes the use of an inhibitor compound of the invention in the that the inhibitor compound is constituted by one or several amino acid sequences belonging to the following group:

to)

an amino acid sequence consisting of the CD4 domain consisting of residues 415-421 (SEQ ID NO8),

b)

an amino acid sequence analogous to the sequence of a), and

C)

an amino acid sequence comprising a any sequence belonging to a) and b).

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Another particular object of the invention is constitutes the use of an inhibitor compound of the invention in the that the inhibitor compound is constituted by one or several amino acid sequences belonging to the following group:

to)

an amino acid sequence consisting of the CXCR4 domain consisting of SEQ ID NO12,

b)

an amino acid sequence analogous to the sequence of a), and

C)

an amino acid sequence comprising a any sequence belonging to a) and b).

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Another particular object of the invention is constitutes the use of an inhibitor compound of the invention in the that the inhibitor compound is constituted by one or several amino acid sequences belonging to the following group:

to)

an amino acid sequence consisting of the CCRS domain constituted by waste 325-352 (SEQ ID NO14),

b)

an amino acid sequence analogous to the sequence of a), and

C)

an amino acid sequence comprising a any sequence belonging to a) and b).

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On the other hand, another additional object of the The present invention is constituted by the use of cells, whether eukaryotes -preferably human- or prokaryotes, hereafter FLNa cells of the invention, genetically modified and which comprise the nucleotide sequence, the construct or the vector of expression FLNa of the invention - where it can be expressed as suitable form the FLNa peptide or protein of the invention- for the preparation of an inhibitor compound of the invention and its subsequent purification and apication.

On the other hand, several of the compounds FLNa activity inhibitors, such as domains FLNa, CD4, CXCR4 and CCR5 proteins and sequences of nucleotides encoding them, do not exist as such in nature and have been identified and isolated for the first time in the present invention. Therefore, another object of the present invention constitutes it a nucleotide sequence FLNa / domain belonging, by way of illustration and without limiting the scope of the invention, to the following group:

a) the nucleotide sequence FLNa constituted for the minimum domain of FLNa, (FLNa D10) of SEQ ID NO1,

b) the FLNa nucleotide sequence constituted for domain 10-12 of FLNa, (FLNa D10-12) of SEQ ID NO3,

c) the FLNa nucleotide sequence constituted for domain 8-10 of FLNa, (FLNa D8-10) of SEQ ID NO5,

d) the FLNa nucleotide sequence constituted by domain 13-16 of FLNa, (FLNa D13-16) of SEQ ID NO9

e) the nucleotide sequence encoding the CD4 domain consisting of residues 415-421 (SEQ ID NO7),

f) the nucleotide sequence encoding the CXCR4 domain consisting of the sequence SEQ ID NO11,

g) the nucleotide sequence encoding the CCR5 domain consisting of waste 325-352 (SEQ ID NO13)

h) a nucleotide sequence analogous to sequences of a), b), c), d), e), f), and g), and

i) a nucleotide sequence comprising any sequence of a), b), c), d), e), f), g) and h).

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In addition, another object of the present invention is constitutes a sequence of FLNa amino acids / belonging domain, by way of illustration and without limiting the scope of the invention, to the following group:

a) the amino acid sequence consisting of the minimum FLNa domain, (FLNa D10) of SEQ ID NO2,

b) the amino acid sequence consisting of the 10-12 domain of FLNa, (FLNa D10-12) of SEQ ID NO4,

c) the amino acid sequence consisting of FLNa domain 8-10, (FLNa D8-10) of SEQ ID NO6,

d) the amino acid sequence consisting of domain 13-16 of FLNa, (FLNa D13-16) of SEQ ID NO10,

e) the amino acid sequence of the domain of CD4 consisting of waste 415-421 (SEQ ID NO8),

f) the amino acid sequence of the domain of CXCR4 consisting of the sequence SEQ ID NO12,

g) the amino acid sequence of the domain of CCR5 consisting of waste 325-352 (SEQ ID NO14)

h) an amino acid sequence analogous to sequences of a), b), c), d), e), f), and g), and

i) an amino acid sequence comprising any sequence of a), b), c), d), e), f), g) and h).

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In the same way it is part of this invention a gene expression vector, hereinafter vector of FLNa / domain expression, which comprises a nucleotide sequence FLNa / domain. Likewise, a cell forms part of the invention, prokaryotic or eukaryotic, transformed or transfected with the FLNa nucleotide sequence / domain or with the expression vector FLNa / domain.

Another object of the present invention is constitutes a pharmaceutical composition or medication for the treatment of HIV-1 infection and of diseases that occur with an altered system stimulation immune, hereinafter pharmaceutical composition of the present invention, which comprises a compound or agent that inhibits the FLNa activity of the invention, in therapeutically quantity effective together with, optionally, one or more adjuvants and / or pharmaceutically acceptable vehicles.

Another object of the invention is the use of the pharmaceutical composition of the invention, hereinafter use of the pharmaceutical composition of the invention, in a method of treatment or prophylaxis of a mammal, preferably a being human, affected by HIV-1 infection or by diseases that occur with an altered system stimulation immune consisting of the administration of said composition therapeutic in adequate dose.

Another particular object of the invention is constitutes the use of the pharmaceutical composition of the invention in  the one that is treated or prevented is the infection for HIV-1.

Another particular object of the invention is constitutes the use of the pharmaceutical composition of the invention in  that the disease that is treated or prevented is a disease who is studying with an impaired immune system stimulation.

Finally, another object of the invention is constitutes a compound identification procedure inhibitors of the FLNa activity of the human FLNa protein, in hereinafter identification procedure of the invention, based on the interaction of several proteins with FLNa and that includes the Next steps:

to)

generate a biological preparation that mimics the protein-protein interaction that mimics the FLNa interaction with CD4, CXCR4 and CCR5, respectively, or in combination with several of them,

b)

contacting the candidate compound with the preparation of a),

C)

determination of the inhibition of the interaction or not of the FLNa protein with the other proteins mentioned, Y

d)

the identification of an interaction inhibitor of the FLNa protein with the aforementioned proteins when not Look at that interaction.

Detailed description

The present invention is based on the fact that the inventors have observed that CD4 and the two main HIV-1 co-receptors, CXCR4 and CCR5, interact with the actin-binding dimeric protein, filamin A (hereinafter, FLNa) regulating this way its clustering on the cell surface. The binding of FLNa to these receptors is necessary for the HIV-1 gp120 protein to initiate the RhoA \ LeftrightarrowROCK \ LeftrightarrowLIMK \ Leftrightarrowcofilin signaling cascade, thereby integrating the binding of Env protein to viral cell receptors with the signals that control the remodeling of F-actin. Moreover, it has been shown that blocking the interaction of FLNa with HIV-1 receptors prevents
concentration of the Env / CD4 / co-receptor complex in the viral synapse and inhibits infection by viral strains X4 and R5.

In this way it can be said that the protein FLNa is a structural and functional adapter for CD4 and CXCR4 that organize the necessary F-actin rearrangement so that the grouping of the receptors of the HIV-1, in the synapse zone of HIV-1, and cell infection by HIV-1, especially in the initial stages of  infection. These results identify FLNa as a target Therapy for the treatment and prevention of infection by the HIV-1 virus. These data show surprisingly a function previously unknown to the FLNa protein at the entrance of HIV-1, and identifies a cellular mechanism that is usurped by the virus during initial stages of infection.

In this sense, FLNa repeat 10 (FLNa D10, residues 1158-1252: SEQ ID NO1) was identified as the minimum FLNa domain that retains the ability to interact with CD4IC (Figure 3a), which suggested that FLNa D10 interacts with CD4 in vivo . And more specifically, three residues were identified in domain 10 (E1217, F1243 and S1245) that differed from the sequences of the rest of the FLNa domains in the global alignment. By mutagenesis analysis, residues F1243 and S1245 of the specific FPS arrangement of repetition 10 were identified as critical for the binding of FLNa with CD4. The FPS motif involved in the union of FLNa with CD4 is on the opposite side of this common interaction surface. The bioinformatic analysis identified, with a high probability, that residue F of this motif is involved in binding with the ligand. This suggests that the? A and B sheets, which comprise the FPS sequence, would represent an additional binding surface at the FLNa repeat with CD4 (Figure 3d).

The reason for CD4 involved in binding with FLNa is shared by CXCR4, which suggests that there is a determinant Common molecular for interaction with FLNa. In this sense, it identified the binding site of CD4 to FLNa within the residues 415-421 of CD4, near the motive di-leucine involved in CD4 endocytosis, and more specifically, it was observed that amino acids E 416 -K 417, located near the subject involved in the internalization of CD4 L 413 L 414 (31), are critics in the reason of CD4 that interacts with FLNa.

The interaction of CD4 and CXCR4 with repetition FLNa D10 is surprising, since most of the more than 30 known, if not all, proteins that bind directly with FLNa they do it in a region between repetition 15 through 24.

The specific interaction block CXCR4-FLNa for overexpression of the FLNa fragment that contained the domains 10-12 (FLNa D10-12) inhibited the Chemotaxis in Jurkat cells (Figure 5e). These results indicate that FLNa is necessary to activate signaling pathways specific mediated by CXCR4. In addition, blocking the interaction between FLNa and HIV-1 receptors prevents RhoA activation induced by envelope binding  viral, a key step for remodeling to occur F-actin necessary for viral infection. In this In the same way, Env-induced fusion in cells was analyzed target that overexpressed the fragment FLNa D10-12, which specifically blocks the interaction of CD4 and CXCR4. The FLNa interaction block with HIV-1 receptors strongly inhibited the fusion between HEK-293CD4 cells and cells that expressed Env (Figure 6c). On the contrary, the expression of FLNa D8-9 or exchange mutant fragment FLNa D10 (F1243S, \ S1245F}) does not affect the mediated fusion by Env (Figure 6c). These data strongly implicate FLNa as a key molecule in the initial stages of infection by HIV-1, regulating the function of CD4 receptors and CXCR4.

Also, as predicted by the analysis of sequence, the test of two hybrids showed that domain 10 of FLNa (FLNa D10) interacted with CXCR4 but not with CCR5, a receiver in which said motif had not been detected (Figure 5a). Without However, it was observed that CCR5 binds to a FLNa fragment that contains 13-16 protein repeats (Figure 9).

The second important discovery of the present invention is the identification of a cascade of RhoA-mediated signaling that takes place in the steps initials of HIV-1 infection. The results indicate that gp120 binding to cell receptors  of the virus induces the RhoA / ROCK / LIMK / cofilin signaling cascade in cell lines and in primary lymphocytes. Inhibition of ROCK decreases cell-cell fusion mediated by gp41, which shows the importance of this route in the process of Viral infection; on the contrary, blocking PAK-1, another effector molecule of LIMK, has no no effect on the fusion mediated by the envelope, although PAK-1 has been implicated in other steps of the HIV-1 cycle (39, 40). This results confirm and extend the involvement of RhoA, but not Rac, in the HIV-1 infection (18), and point to FLNa and ROCK inhibitors as potential drugs to inhibit virus infection

In summary, it has been identified that the protein FLNa is a therapeutic target for the identification of compounds, which by inhibiting their interaction of FLNa with CD4 and with HIV-1 co-receptors (CXCR4 and CCR5), become inhibitors of cellular entry of HIV-1 and therefore useful as medicines for the prevention and treatment of infection of HIV-1 These inhibitors would prevent the binding of HIV-1 receptors to the actin cytoskeleton, as well as the initiation of a signaling cascade involved in the remodeling of F-actin after activation of HIV-1 receptors by the viral envelope.

CD4, CXCR4 and CCR5 co-add in the immune synapse formed between a T cell and a cell antigen presenter; said co-aggregation of receptors is important for co-stimulation of the T cell (45). It is therefore possible to speculate that the HIV-1 could develop a dependent mechanism of FLNa involved in the activation of the T cell for the subsequent entry into the target cell. Likewise, knowledge detailed of the residues involved in such interaction - be it FLNa-CD4, FLNa-CXCR4 or FLNa-CCR5) - among the four proteins allows use a family of peptides or proteins that inhibit said interaction - constituted or comprising the minimum region involved in such interactions - as medications for prevention and treatment of HIV-1 infection and, more generally, for the prevention and treatment of diseases that occur with the induction or stimulation of immune response from the interaction between a T lymphocyte and an antigen presenting cell. In this same sense, the nucleotide sequences encoding said peptides or proteins They can be used to produce them as well as medications in gene therapy approaches to infection of HIV-1. In this sense, they are useful as HIV-1 infection inhibitors and they could potentially act as immunosuppressants in situations of hyperactivation of the immune system such as diseases autoimmune, or transplant rejection, in addition to those described, nucleotide sequences that inhibit intracellular expression Endogenous FLNa protein in immune system cells.

Therefore, an object of the present invention it is the use of a compound that inhibits the activity of FLNa protein (hereinafter, FLNa), hereafter used a inhibitor compound of the present invention, for the preparation of a drug or pharmaceutical composition for the treatment of HIV-1 infection and diseases with an altered immune system stimulation.

As used in the present invention the term "FLNa activity inhibitor compound" is refers to a molecule that inhibits the stimulation of the response immune from the interaction between a T lymphocyte and a cell antigen presenter, or that inhibits the intensity or decreases the duration of the infection-inducing activity of HIV-1 FLNa protein as described in the present invention. An inhibitor compound may be consisting of a peptide, a protein or a sequence of nucleotides, molecules that allow the expression of a sequence of nucleotides encoding a protein with FLNa activity, a antibody, as well as a chemical compound. In this definition of inhibitor also includes those compounds that prevent or decrease expression of the gene coding for the FLNa protein human, that is, that prevent or reduce the transcription of gene, mRNA maturation, mRNA translation and post-translational modification of the protein. In this sense any peptide or protein constituted or that understand the minimum domains of FLNa, CD4, CXCR4 or CCR5 that allow their interaction - FLNa-CD4 or FLNa-CXCR4 or FLNa-CCR5 - can become an antagonist of it and constitute a drug for Treatment of HIV-1 infection.

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

Thus, a particular object of the invention is constitutes the use of a compound that inhibits the activity of the FLNa based on the use of a nucleotide sequence as inhibitor compound, hereinafter FLNa nucleotide sequence of the present invention, which allows the expression of a protein or peptide in human cells, and that is constituted by one or several  nucleotide sequences belonging to the following group:

to)

a nucleotide sequence consisting of the nucleotide sequence encoding domain 10 of FLNa human (FLNa D10) of SEQ ID NO1,

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b)

a nucleotide sequence consisting of the domain coding nucleotide sequence 13-16 of the human FLNa (FLNa D13-16) of SEQ ID NO9,

C)

a nucleotide sequence analogous to the sequence of a) and b),

d)

a nucleotide sequence, construction genetics, which comprises any sequence belonging to a), b) and c).

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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, for example, by the introduction of conservative or non-conservative nucleotide substitutions conservatives, including the insertion of one or more nucleotides, the addition of one or more nucleotides at any end of the molecule or the deletion of one or more nucleotides in any end or inside the sequence, and that allows the coding of a peptide or protein capable of inhibiting activity FLNa of the human FLNa protein (NM 001456).

From the information described in the present invention and in the state of the art an expert technician in the technical sector you can isolate or build a sequence of nucleotides analogous to those described in the present invention for later use.

In general, an analogous nucleotide sequence is substantially homologous to the nucleotide sequence discussed above. In the sense used in this description, the term "substantially homologous" means that the nucleotide sequences in question have a degree of identity of at least 30%, preferably of at least 85%, or more preferably of At least 95%. Preferred forms of the nucleotide sequence to be used are nucleotide sequences of human origin that comprise domain 10 of FLNa (FLNa D10), possibly
Do be, among other alternatives, any fragment of the human FLNa itself that does not maintain its FLNa activity.

As used in the present invention the term "nucleotide sequence" refers to a sequence of DNA, cDNA or mRNA.

A particular embodiment of the present invention is constituted by the use of a compound that inhibits invention where the FLNa nucleotide sequence is constituted by the minimum domain of FLNa, (FLNa D10) of SEQ ID NO1.

Another particular embodiment of the present invention is constituted by the use of a compound that inhibits invention where the FLNa nucleotide sequence of d) is constituted by domain 10-12 of FLNa, (FLNa D10-12) of SEQ ID NO3.

Another particular embodiment of the present invention is constituted by the use of a compound that inhibits invention where the FLNa nucleotide sequence of d) is constituted by domain 8-10 of FLNa, (FLNa D8-10) of SEQ ID NO5.

Another particular embodiment of the present invention is constituted by the use of a compound that inhibits invention where the FLNa nucleotide sequence is constituted by domain 13-16 of FLNa, (FLNa D13-16) of SEQ ID NO9.

Another particular object of the invention is constitutes the use of a compound that inhibits the activity of the FLNa based on the use of a derived FLNa nucleotide sequence of CD4 as an inhibitor compound that is constituted by one or several nucleotide sequences belonging to the following group:

to)

a nucleotide sequence consisting of the nucleotide sequence encoding the CD4 domain constituted by residues 415-421 (SEQ ID NO7),

b)

a nucleotide sequence analogous to the sequence of a), and

C)

a nucleotide sequence, construction genetics, which comprises any sequence belonging to a) and b).

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Another particular embodiment of the present invention is constituted by the use of a compound that inhibits invention where the FLNa nucleotide sequence is constituted by the nucleotide sequence encoding the CD4 domain consisting of waste 415-421 (SEQ ID NO7).

Another particular object of the invention is constitutes the use of a compound that inhibits the activity of the FLNa based on the use of a derived FLNa nucleotide sequence of CXCR4 as an inhibitor compound that is constituted by one or several nucleotide sequences belonging to the following group:

to)

a nucleotide sequence consisting of the CXCR4 domain coding nucleotide sequence constituted by SEQ ID NO11,

b)

a nucleotide sequence analogous to the sequence of a), and

C)

a nucleotide sequence, construction genetics, which comprises any sequence belonging to a) and b).

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Another particular embodiment of the present invention is constituted by the use of a compound that inhibits invention where the FLNa nucleotide sequence is constituted by the nucleotide sequence coding for the CXCR4 domain constituted by SEQ ID NO11.

Another particular object of the invention is constitutes the use of a compound that inhibits the activity of the FLNa based on the use of a derived FLNa nucleotide sequence of CCR5 as an inhibitor compound that is constituted by one or several nucleotide sequences belonging to the following group:

to)

a nucleotide sequence consisting of the CCR5 domain coding nucleotide sequence consisting of waste 325-352 (SEQ ID NO13),

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b)

a nucleotide sequence analogous to the sequence of a), and

C)

a nucleotide sequence, construction genetics, which comprises any sequence belonging to a) and b).

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Another particular embodiment of the present invention is constituted by the use of a compound that inhibits invention where the FLNa nucleotide sequence is constituted by the nucleotide sequence coding for the CCR5 domain consisting of waste 325-352 (SEQ ID NO13).

On the other hand, there may be capable molecules to prevent or reduce the expression of the gene coding for the human FLNa protein, that is, that prevent or decrease the gene transcription, mRNA maturation, translation of MRNA and post-translational modification of the protein. By way of illustration, said molecules can be a nucleotide sequence encoding a nucleotide sequence specific antisense of the gene sequence or mRNA of the FLNa protein, or a nucleotide sequence that encodes a specific ribozyme of the FLNa protein mRNA, or a nucleotide sequence encoding a specific aptamer of the FLNa protein mRNA, or a nucleotide sequence that encodes an interference RNA ("small interference RNA" or siRNA) specific to the FLNa protein mRNA. These sequences of nucleotides mentioned can be used in a process of gene therapy in which by any technique or procedure their integration into the cells of a human patient This objective can be achieved through administration to these cells of a gene construct that comprises one or more of said nucleotide sequences mentioned in order to transform said cells allowing their  expression within them so that the FLNa protein expression. Advantageously, said construction gene may be included within a vector, such as, by example, an expression vector or a transfer vector.

Therefore, another particular object of the invention is constituted by the use of a compound that inhibits FLNa activity based on the use of a sequence of FLNa nucleotides as an inhibitor compound that prevents or decreases the expression of the gene encoding the human FLNa protein constituted by one or several nucleotide sequences selected from:

a) an antisense nucleotide sequence sequence specific for the gene or protein mRNA FLNa,

b) a ribozyme specific to the mRNA of the FLNa protein,

c) a specific mRNA aptamer of the FLNa protein, and

d) an interference RNA (iRNA) specific to FLNa protein mRNA.

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Another particular embodiment of the present invention is constituted by the use of a compound that inhibits invention where the FLNa nucleotide sequence is constituted by an FLNA-specific interference RNAi (RNAi) as per For example, by way of illustration and without limiting the scope of the invention, those described in the following list: SEQ ID NO19, SEQ ID NO20; SEQ ID NO21, SEQ ID NO22, SEQ ID NO23, SEQ ID NO24; SEQ ID NO25 and SEQ ID NO26. (see Example 2.1; Table III).

The nucleotide sequences a) -d) mentioned above prevent the expression of the gene in mRNA or mRNA in the FLNa protein, and therefore cancel its biological function, and can be developed by an expert in the genetic engineering sector in role of existing knowledge in the state of the art on transgenesis and cancellation of gene expression (Clarke, AR (2002) Transgenesis Techniques. Principles and Protocols, 2nd Ed. Human Press, Cardiff University; US20020128220 Patent. Gleave, Martin. TRPM-2 antisense therapy; Puerta-Ferández E et al . (2003) Ribozymes: recent advances in the development of RNA tools. FEMS Microbiology Reviews 27: 75-97; Kikuchi, et al., 2003. RNA aptamers targeted to domain II of Hepatitis C IRES virus that bind to its apical loop region J. Biochem. 133, 263-270; Reynolds A. et al ., 2004. Rational siRNA design for RNA interference. Nature Biotechnology 22 (3): 326-330).

The defined FLNa nucleotide sequence previously as a gene construction FLNa corresponds to a gene construct that allows the expression of a FLNa protein. This FLNa gene construct of the invention can also understand, if necessary and to allow for better isolation, detection or secretion outside the peptide cell 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 a construction FLNa genetics comprising, in addition to the nucleotide sequence FLNa of the invention, any other nucleotide sequence coding for a peptide or peptide sequence that allows the isolation, detection or secretion outside the cell peptide cell expressed, for example, by way of illustration and without limiting the scope of the invention, a sequence of polyhistidine (6xHis), a peptide sequence recognizable by a monoclonal antibody (for example, for 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 FLNa nucleotide sequence and FLNa genetic construct described previously can be isolated and obtained by an expert by employing techniques widely known in the state of the art (Sambrook et al . `` Molecular cloning, a Laboratory Manual 2 nd) Aed., Cold Sping Harbor Laboratory Press, NY, 1989 vol 1-3). Said nucleotide sequences can be integrated into a gene expression vector that allows the regulation of the expression thereof under suitable conditions inside the cells.

Therefore, another particular object of the present  invention is constituted by the use of a compound that induces invention where the FLNa nucleotide sequence is a vector of expression, hereinafter FLNa expression vector of the invention, comprising a FLNa nucleotide sequence or a construct FLNa genetics, described in the present invention, and which allows the  expression of a protein or peptide capable of inhibiting infection of HIV-1 in human cells. An example of a particular embodiment, by way of illustration and without limiting the scope of the invention, is constituted by expression vectors elaborated in the present invention: for example retroviruses recombinant pRV-FLNa D8-9 IRESGFP or pRV-FLNa D10-12 -IRESGFP (see Examples, Material and Methods).

In general, an expression vector comprises, in addition to the FLNa nucleotide sequence or the FLNa genetic construct described in the invention, a promoter that directs its transcription (e.g., pT7, plac, ptrc, ptac, pBAD, ret , 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 ( tlt2 , etc.) , polyadenylation signal, origin of replication, ribosome binding sequences (RBS), 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 or a viral vector. 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]. One strategy could be to use lentivirus to infect the target cells, as it is already being tried in other therapies (Ralph GS, Binley K, Wong LF, Azzouz M, Mazarakis ND (2006) Gene therapy for neurodegenerative and ocular diseases using lentiviral vectors Clin Sci (Lond) 110: 37-46).

In addition, another particular object of the invention it is constituted by the use of an inhibitor compound of the invention in which the inhibitor compound is one or several proteins or peptides, hereinafter use of the FLNa protein of the present invention, and that It consists of one or several amino acid sequences belonging to the following group:

to)

an amino acid sequence consisting of the amino acid sequence of domain 10 of human FLNa (FLNa D10) (SEQ ID NO2),

b)

an amino acid sequence consisting of the amino acid sequence of domain 13-16 of the Human FLNa (FLNa D13-16) (SEQ ID NO10),

C)

an amino acid sequence analogous to the sequence of a) and b), and

d)

an amino acid sequence comprising a any sequence belonging to a), b) and c).

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In the sense used in this description, the term "analogous" is intended to include any sequence of amino acids that can be isolated or constructed based on the sequence shown herein, for example, by the introduction of conservative amino acid substitutions or not conservatives, including the insertion of one or more amino acids, the addition of one or more amino acids at any end of the molecule or the deletion of one or more amino acids in any end or inside the sequence, and that inhibits the FLNa activity, HIV infection in human cells, of FLNa human

From the information described in the Present invention and a technician skilled in the art sector can isolate or build an amino acid sequence analogous to described in the present invention.

In general, an analogous amino acid sequence is substantially homologous to the amino acid sequence commented above. In the sense used in this description, the expression "substantially homologous" means that the amino acid sequences in question have a degree of identity of at least 30%, preferably of at least one 85%, or more preferably, at least 95%.

Another particular embodiment of the present invention is constituted by the use of a compound that inhibits invention in which the inhibitor compound is an FLNa protein whose amino acid sequence is constituted by domain 10 of human FLNa (FLNa D10) (SEQ ID NO2).

Another particular embodiment of the present invention is constituted by the use of a compound that inhibits invention in which the inhibitor compound is an FLNa protein whose amino acid sequence is constituted by the domain 10-12 of the human FLNa protein (FLNa D10-12) (SEQ ID NO4).

Another particular embodiment of the present invention is constituted by the use of a compound that inhibits invention in which the inhibitor compound is an FLNa protein whose amino acid sequence is constituted by the domain 8-10 of the human FLNa protein (FLNa D8-10) (SEQ ID NO6).

Another particular embodiment of the present invention is constituted by the use of a compound that inhibits invention in which the inhibitor compound is an FLNa protein whose amino acid sequence is constituted by the domain 13-16 of the human FLNa protein (FLNa D13-16) (SEQ ID NO10).

Another particular object of the invention is constitutes the use of an inhibitor compound of the invention in the that the inhibitor compound is constituted by one or several amino acid sequences belonging to the following group:

to)

an amino acid sequence consisting of the CD4 domain consisting of residues 415-421 (SEQ ID NO8),

b)

an amino acid sequence analogous to the sequence of a), and

C)

an amino acid sequence comprising a any sequence belonging to a) and b).

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Another particular embodiment of the present invention is constituted by the use of a compound that inhibits invention in which the inhibitor compound is an FLNa protein whose amino acid sequence is constituted by the domain of CD4 consisting of waste 415-421 (SEQ ID NO8).

Another particular object of the invention is constitutes the use of an inhibitor compound of the invention in the that the inhibitor compound is constituted by one or several amino acid sequences belonging to the following group:

to)

an amino acid sequence consisting of the CXCR4 domain consisting of SEQ ID NO12,

b)

an amino acid sequence analogous to the sequence of a), and

C)

an amino acid sequence comprising a any sequence belonging to a) and b).

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Another particular embodiment of the present invention is constituted by the use of a compound that inhibits invention in which the inhibitor compound is an FLNa protein whose amino acid sequence is constituted by the domain of CXCR4 constituted by SEQ ID NO12.

Another particular object of the invention is constitutes the use of an inhibitor compound of the invention in the that the inhibitor compound is constituted by one or several amino acid sequences belonging to the following group:

to)

an amino acid sequence consisting of the CCR5 domain consisting of waste 325-352 (SEQ ID NO14),

b)

an amino acid sequence analogous to the sequence of a), and

C)

an amino acid sequence comprising a any sequence belonging to a) and b).

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Another particular embodiment of the present invention is constituted by the use of a compound that inhibits invention in which the inhibitor compound is an FLNa protein whose amino acid sequence is constituted by the domain of CCR5 consisting of waste 325-352 (SEQ ID NO14).

On the other hand, another additional object of the The present invention is constituted by the use of cells, whether eukaryotes -preferably human- or prokaryotes, hereafter FLNa cells of the invention, genetically modified and which comprise the nucleotide sequence, the construct or the vector of expression FLNa of the invention - where it can be expressed as suitable form the FLNa peptide or protein of the invention- for the preparation of an inhibitor compound of the invention and its subsequent purification and apication. These cells can be transformed, infected or transfected by said nucleotide sequences by genetic engineering techniques known to a person skilled in the art. [Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989). Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory] and are part of the present invention Gene expression systems can allow or not the integration of the new genetic material in the genome of the host cell. These cells can be useful for the production of peptides with inhibitory activity of the HIV-1 infection of human cells that can Be the basis of a pharmaceutical composition.

In addition, both the nucleotide sequence, gene construct or FLNa expression vector can be used as a medicament to protect human cells, preferably human cells of the immune system, in a method of treatment and prophylaxis of gene therapy of a patient affected by an infection. of HIV-1, in which the gene expression of these nucleotide sequences is carried out in the cells of the immune system which would allow said cells to prevent HIV-1 infection. Gene therapy procedures can be applied directly to the patient by administering one of these nucleotide sequences as a medicament, or cells from the immune system could be extracted from a human being and once ex vivo they can be transformed with nucleotide sequences and subsequently returned to the being. human or keep them ex vivo for later use.

In this same sense, proteins or peptides and the cells themselves can become biopharmaceuticals.

On the other hand, several of the compounds FLNa activity inhibitors, such as domains FLNa, CD4, CXCR4 and CCR5 proteins and sequences of nucleotides encoding them, do not exist as such in nature and have been identified and isolated for the first time in the present invention. Therefore, another object of the present invention constitutes it a nucleotide sequence FLNa / domain belonging, by way of illustration and without limiting the scope of the invention, to the following group:

a) the nucleotide sequence FLNa constituted for the minimum domain of FLNa, (FLNa D10) of SEQ ID NO1,

b) the FLNa nucleotide sequence constituted for domain 10-12 of FLNa, (FLNa D10-12) of SEQ ID NO3,

c) the FLNa nucleotide sequence constituted for domain 8-10 of FLNa, (FLNa D8-10) of SEQ ID NO5,

d) the FLNa nucleotide sequence constituted by domain 13-16 of FLNa, (FLNa D13-16) of SEQ ID NO9

e) the nucleotide sequence encoding the CD4 domain consisting of residues 415-421 (SEQ ID NO7),

f) the nucleotide sequence encoding the CXCR4 domain consisting of the sequence SEQ ID NO11,

g) the nucleotide sequence encoding the CCR5 domain consisting of waste 325-352 (SEQ ID NO13)

h) a nucleotide sequence analogous to sequences of a), b), c), d), e), f), and g), and

i) a nucleotide sequence comprising any sequence of a), b), c), d), e), f), g) and h).

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In addition, another object of the present invention is constitutes a sequence of FLNa amino acids / belonging domain, by way of illustration and without limiting the scope of the invention, to the following group:

a) the amino acid sequence consisting of the minimum FLNa domain, (FLNa D10) of SEQ ID NO2,

b) the amino acid sequence consisting of the 10-12 domain of FLNa, (FLNa D10-12) of SEQ ID NO4,

c) the amino acid sequence consisting of FLNa domain 8-10, (FLNa D8-10) of SEQ ID NO6,

d) the amino acid sequence consisting of domain 13-16 of FLNa, (FLNa D13-16) of SEQ ID NO10,

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e) the amino acid sequence of the domain of CD4 consisting of waste 415-421 (SEQ ID NO8),

f) the amino acid sequence of the domain of CXCR4 consisting of the sequence SEQ ID NO12,

g) the amino acid sequence of the domain of CCR5 consisting of waste 325-352 (SEQ ID NO14)

h) an amino acid sequence analogous to sequences of a), b), c), d), e), f), and g), and

i) an amino acid sequence comprising any sequence of a), b), c), d), e), f), g) and h).

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As used in the present invention the term "FLNa / domain" refers to any sequence of nucleotides or amino acids derived or isolated from FLNa, CD4, CXCR4 and CCR5, for example, any fragment of said proteins that do not they exist as such in nature, and that is able to inhibit the Human FLNa protein activity.

In the same way it is part of this invention a gene expression vector, hereinafter vector of FLNa / domain expression, which comprises a nucleotide sequence FLNa / domain. Likewise, a cell forms part of the invention, prokaryotic or eukaryotic, transformed or transfected with the FLNa nucleotide sequence / domain or with the expression vector FLNa / domain.

Another object of the present invention is constitutes a pharmaceutical composition or medication for the treatment of HIV-1 infection and of diseases that occur with an altered system stimulation immune, hereinafter pharmaceutical composition of the present invention, which comprises a compound or agent that inhibits the FLNa activity of the invention, in therapeutically quantity effective together with, optionally, one or more adjuvants and / or pharmaceutically acceptable vehicles.

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 agent or compound capable of inhibiting infection of HIV-1 and diseases that occur with a impaired immune system stimulation, calculated to produce the desired effect and, in general, will be determined, among others causes, due to the characteristics of the compounds, including the age, condition of the patient, the severity of the alteration or disorder, and of the route and frequency of administration.

In another particular embodiment, said therapeutic composition is prepared in the form of a solid form or aqueous suspension, in a pharmaceutically acceptable diluent. The therapeutic composition provided by this invention may be administered by any appropriate route of administration, for which said composition will be formulated in the pharmaceutical form appropriate to the route of administration chosen. In one embodiment in particular, the administration of the therapeutic composition provided by this invention is carried out parenterally, by orally, intraperitoneally, subcutaneously, etc. A review of the different pharmaceutical forms of administration of medications and of the excipients necessary to obtain they can be found, for example, in the "Treaty of Farmacia Galenica ", C. Faulí i Trillo, 1993, Luzán 5, S.A. Editions, Madrid.

Another particular object of the invention is constitutes a pharmaceutical composition of the invention in which the compound or inhibitory agent is constituted by one or several nucleotide sequences belonging to the following group:

to)

a nucleotide sequence consisting of the nucleotide sequence encoding domain 10 of FLNa human (FLNa D10) of SEQ ID NO1,

b)

a nucleotide sequence consisting of the domain coding nucleotide sequence 13-16 of the human FLNa (FLNa D13-16) of SEQ ID NO9,

C)

a nucleotide sequence analogous to the sequence of a) and b), and

d)

a nucleotide sequence, construction genetics, which comprises any sequence belonging to a), b) and c).

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Another particular embodiment of the present invention constitutes the pharmaceutical composition of the invention  in which the FLNa nucleotide sequence is constituted by the nucleotide sequence encoding domain 10 of FLNa human (FLNa D10) of SEQ ID NO1.

Another particular embodiment of the present invention constitutes the pharmaceutical composition of the invention  in which the FLNa nucleotide sequence of d) is constituted for domain 10-12 of FLNa, (FLNa D10-12) of SEQ ID NO3.

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Another particular embodiment of the present invention constitutes the pharmaceutical composition of the invention  in which the FLNa nucleotide sequence of d) is constituted for domain 8-10 of FLNa, (FLNa D8-10) of SEQ ID NO5.

Another particular embodiment of the present invention constitutes the pharmaceutical composition of the invention  in which the FLNa nucleotide sequence is constituted by the domain 13-16 of FLNa, (FLNa D13-16) of SEQ ID NO9.

Another particular object of the invention is constitutes a pharmaceutical composition of the invention in which the compound or inhibitory agent is constituted by one or several nucleotide sequences belonging to the following group:

to)

an amino acid sequence consisting of the CD4 domain consisting of residues 415-421 (SEQ ID NO8),

b)

an amino acid sequence analogous to the sequence of a), and

C)

an amino acid sequence comprising a any sequence belonging to a) and b).

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Another particular embodiment of the present invention constitutes the pharmaceutical composition of the invention  in which the FLNa nucleotide sequence is constituted by the nucleotide sequence encoding the CD4 domain constituted by residues 415-421 (SEQ ID NO8).

Another particular object of the invention is constitutes a pharmaceutical composition of the invention in which the compound or inhibitory agent is constituted by one or several nucleotide sequences belonging to the following group:

to)

a nucleotide sequence consisting of the CXCR4 domain coding nucleotide sequence constituted by SEQ ID NO11,

b)

a nucleotide sequence analogous to the sequence of a), and

C)

a nucleotide sequence, construction genetics, which comprises any sequence belonging to a) and b).

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Another particular embodiment of the present invention constitutes the pharmaceutical composition of the invention  in which the FLNa nucleotide sequence is constituted by the CXCR4 domain coding nucleotide sequence constituted by SEQ ID NO11.

Another particular object of the invention is constitutes a pharmaceutical composition of the invention in which the compound or inhibitory agent is constituted by one or several nucleotide sequences belonging to the following group:

to)

a nucleotide sequence consisting of the nucleotide sequence coding for the CCR5 domain constituted for waste 325-352 (SEQ ID NO13),

b)

a nucleotide sequence analogous to the sequence of a), and

C)

a nucleotide sequence, construction genetics, which comprises any sequence belonging to a) and b).

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Another particular embodiment of the present invention constitutes the pharmaceutical composition of the invention  in which the FLNa nucleotide sequence is constituted by the CCR5 domain coding nucleotide sequence consisting of waste 325-352 (SEQ ID NO13).

A particular object of the invention is constitutes a pharmaceutical composition of the invention in which the FLNa nucleotide sequence as an inhibitor compound is a nucleotide sequence that prevents or decreases the expression of gene coding for the human FLNa protein and that is constituted by one or several nucleotide sequences selected from:

a) an antisense nucleotide sequence sequence specific for the gene or protein mRNA FLNa,

b) a ribozyme specific to the mRNA of the FLNa protein,

c) a specific mRNA aptamer of the FLNa protein, and

d) an interference RNA (iRNA) specific to FLNa protein mRNA.

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Another particular embodiment of the present invention constitutes pharmaceutical composition in which the nucleotide sequence of d) is constituted by an RNA of FLNa-specific interference (RNAi) such as by way of illustrative and without limiting the scope of the invention, the described in the following list: SEQ ID NO19, SEQ ID NO20; SEQ ID NO21, SEQ ID NO22, SEQ ID NO23, SEQ ID NO24; SEQ ID NO25 and SEQ ID NO26. (see Example 2.1; Table III).

Another particular embodiment of the present invention constitutes a pharmaceutical composition of the invention in which the nucleotide sequence is a vector of FLNa expression, which comprises one or more of the sequence of FLNa nucleotides described in the present invention.

Another particular object of the invention is constitutes a pharmaceutical composition of the invention in which FLNa protein or peptide as an inhibitor compound is consisting of one or more of the amino acid sequences belonging to the following group:

to)

an amino acid sequence consisting of the amino acid sequence of domain 10 of human FLNa (FLNa D10) (SEQ ID NO2),

b)

an amino acid sequence consisting of the amino acid sequence of domain 13-16 of the Human FLNa (FLNa D13-16) (SEQ ID NO10),

C)

an amino acid sequence analogous to the sequence of a) and b), and

d)

an amino acid sequence comprising a any sequence belonging to a) and b).

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Another particular embodiment of the present invention constitutes the pharmaceutical composition of the invention  in which the amino acid sequence is the sequence of amino acids from domain 10 of human FLNa (FLNa D10) (SEQ ID NO2).

Another particular embodiment of the present invention constitutes the pharmaceutical composition of the invention  in which the amino acid sequence of d) is the sequence of amino acids constituted by the 10-12 domain of the human FLNa protein (FLNa D10-12) (SEQ ID NO4).

Another particular embodiment of the present invention constitutes the pharmaceutical composition of the invention  in which the amino acid sequence of d) is the sequence of amino acids constituted by domain 8-10 of the human FLNa protein (FLNa D8-10) (SEQ ID NO 6).

Another particular embodiment of the present invention constitutes the pharmaceutical composition of the invention  in which the amino acid sequence is the sequence of amino acids constituted by domain 13-16 of the human FLNa protein (FLNa D13-16) (SEQ ID NO10).

Another particular object of the invention is constitutes a pharmaceutical composition of the invention in which FLNa protein or peptide as an inhibitor compound is consisting of one or several amino acid sequences belonging to the following group:

to)

an amino acid sequence consisting of the CD4 domain consisting of residues 415-421 (SEQ ID NO8),

b)

an amino acid sequence analogous to the sequence of a), and

C)

an amino acid sequence comprising a any sequence belonging to a) and b).

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Another particular embodiment of the present invention constitutes the pharmaceutical composition of the invention  in which the amino acid sequence is constituted by the CD4 domain consisting of residues 415-421 (SEQ ID NO8).

Another particular object of the invention is constitutes a pharmaceutical composition of the invention in which FLNa protein or peptide as an inhibitor compound is consisting of one or several amino acid sequences belonging to the following group:

to)

an amino acid sequence consisting of the CXCR4 domain consisting of SEQ ID NO12,

b)

an amino acid sequence analogous to the sequence of a), and

C)

an amino acid sequence comprising a any sequence belonging to a) and b).

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Another particular embodiment of the present invention constitutes the pharmaceutical composition of the invention  in which the amino acid sequence is constituted by the CXCR4 domain constituted by SEQ ID NO12.

Another particular object of the invention is constitutes a pharmaceutical composition of the invention in which FLNa protein or peptide as an inhibitor compound is consisting of one or several amino acid sequences belonging to the following group:

to)

an amino acid sequence consisting of the CCR5 domain consisting of waste 325-352 (SEQ ID NO14),

b)

an amino acid sequence analogous to the sequence of a), and

C)

an amino acid sequence comprising a any sequence belonging to a) and b).

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Another particular embodiment of the present invention constitutes the pharmaceutical composition of the invention  in which the amino acid sequence is constituted by the CCR5 domain consisting of waste 325-352  (SEQ ID NO14).

Another particular embodiment of the present invention constitutes the pharmaceutical composition of the invention  wherein the inhibitor compound is constituted by a combination any of the possible mixtures of the sequences of nucleotides, any combination of possible mixtures of amino acid sequences or any possible mixture of both types of sequences described in the present invention.

Another object of the invention is the use of the pharmaceutical composition of the invention, hereinafter use of the pharmaceutical composition of the invention, in a method of treatment or prophylaxis of a mammal, preferably a being human, affected by HIV-1 infection or by diseases that occur with an altered system stimulation immune consisting of the administration of said composition therapeutic in adequate dose.

The pharmaceutical composition of the present invention can be used in a form treatment method isolated or in conjunction with other pharmaceutical compounds.

Another particular object of the invention is constitutes the use of the pharmaceutical composition of the invention in  the one that is treated or prevented is the infection for HIV-1.

Another particular object of the invention is constitutes the use of the pharmaceutical composition of the invention in  that the disease that is treated or prevented is a disease who is involved with an impaired immune system stimulation belonging, by way of illustration and without limiting the scope of the invention, to the following group:

-

acute or chronic inflammatory disorder or an immune disorder, for example an autoimmune disease selected from the group consisting of: rheumatoid arthritis, lupus systemic erythematosus, scleroderma, Sjögren's syndrome, diabetes autoimmune, thyroiditis, and other immune diseases organ-specific, including the psoriasis;

-

neurological type disorder selected from the following group: multiple sclerosis, myasthenia severe, and other neurological immune diseases,

-

gastrointestinal disorder selected from the following group: Crohn's disease, colitis ulcerative, celiac disease, and hepatitis,

-

respiratory type disorder selected from the following group: emphysema, tract infections respiratory,

-

cardiovascular disorder selected from the following group: atherosclerosis, cardiomyopathy, rheumatic fever, endocarditis, vasculitis, and other diseases cardiological of an immune nature,

-

allergic type disorder or a reaction of hypersensitivity (types I, II, III, and IV), including asthma, rhinitis, and other hypersensitivity reactions mediated by immune system;

-

disorder of the rejection type of a transplant or graft, and

-

disorder or condition pertaining to next group: acute lung damage, distress syndrome acute respiratory, arthritis (e.g., CIA), bronchitis, fibrosis cystic, hepatitis, inflammatory bowel disease, damage from reperfusion, nephritis, pancreatitis, arterial occlusion, accident cerebrovascular, systemic lupus erythematosus, damage induced by ultraviolet light, vasculitis and sarcoidosis.

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Finally, another object of the invention is constitutes a compound identification procedure inhibitors of the FLNa activity of the human FLNa protein, in hereinafter identification procedure of the invention, based on the interaction of several proteins with FLNa and that includes the Next steps:

to)

generate a biological preparation that mimics the protein-protein interaction that mimics the FLNa interaction with CD4, CXCR4 and CCR5, respectively, or in combination with several of them,

b)

contacting the candidate compound with the preparation of a),

C)

determination of the inhibition of the interaction or not of the FLNa protein with the other proteins mentioned, Y

d)

the identification of an interaction inhibitor of the FLNa protein with the aforementioned proteins when not Look at that interaction.

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Another particular object of the invention is It constitutes the procedure for identifying inhibitors of FLNa activity where the interaction of the mentioned proteins, or fragments thereof, is analyzed by a two test hybrids, preferably in yeasts, of immunoprecipitation between proteins or analyzing specific biological activities of CD4, CXCR4 and CCR5.

Another particular embodiment of the present invention is the method of identifying inhibitors of FLNa activity where FLNa-CD4 interaction is analyzed in vivo by cross immunoprecipitation between both proteins; or by activation of CD4 induced by anti-CD4 antibodies and subsequently analyzing the phosphorylation of FLNa in serine residues, or by locating and size of the CD4 cluster in the membrane (see Example 1 and Figure 1).

Another particular embodiment of the present invention is the identification procedure of FLNa activity inhibitors where the interaction is analyzed FLNa-CXCR4 in stimulation tests with its ligand, CXCL12, in cells that stably express a CXCR4 receiver labeled, for example with GFP; or analyzing its action on Ca 2 flows (Figure 5c) or on the formation of F-actin (Figure 5d) or its activity Chemotoxic

Another particular embodiment of the present invention is the method of identifying inhibitors of FLNa activity where the role of FLNa in the formation of the HIV-1 synapse (stable adhesive bond between a cell containing a viral envelope protein) is analyzed. and the host cell membrane) by forming conjugates between effector cells that express the viral envelope protein, for example env IIIB, and target cells expressing labeled CD4 and / or CXCR4, for example with GFP, and where the accumulation of CXCR4 in the contact zone between the target and effector cell is analyzed, as well as the accumulation of FLNa and actin at the contact site.

On the other hand, any average expert in this technical sector and with the information provided in the present invention can develop and fine-tune other examples of the activity inhibitor identification procedure FLNa of the invention.

Description of the figures

Figure 1. Identification of FLNa as a CD4 binding protein . a, Yeasts were co-transfected with GAD-LCK (positive control), GAD-MyoD, GAD-ALL, GAD-HCI, GAD-FLNa and GAD-p53 (negative control) with GBD-CD4IC (396-433). The co-transformants were seeded in medium plates without leucine or tryptophan (-LW) or in medium without adenine, histidine, leucine and tryptophan (-LHAW). Yeast growth was analyzed after 72 h and colonies were tested for β-galactosidase activity; The blue color indicates the specific interaction of the two proteins. b, CD4 and FLNa interact in a precipitation test. GST or GST-CD4IC proteins were incubated with total protein extracts and added. The upper panels show the immunoblot of GST-bound proteins and cell extracts (Input), using an anti-FLNa antibody; the lower panels show the staining of the gel with Coomassie blue (representative of 3 experiments). c, Co-immuno-isolation of CD4 and FLNa. Jurkat cells were incubated with magnetic particles upholstered with anti-CD4; After nitrogen cavitation, affinity purified membranes (left) and cell lysates (right) were tested with anti-p56Lck and anti-FLNa antibodies (representative of two experiments). d, Interaction of endogenous CD4 and FLNa. Equal amounts of lysates of MT-2 cells were immunoprecipitated (IP) with control IgG, anti-CD4 or anti-FLNa antibodies, and analyzed for CD4 and FLNa (representative of 4 experiments). e, Antibody induced CD4 activation causes phosphorylation of FLNa. MT-2 cells were incubated with an anti-CD4 activating antibody for the indicated times, and after lysing the cells, CD4 was immunoprecipitated from identical amounts of cell extracts. These precipitates were resolved in SDS-PAGE and the membranes were sequentially tested with anti-phosphoserine (P-Ser), anti-FLNa and anti-CD4 antibodies (representative of three experiments).

Figure 2. FLNa regulates the clustering of CD4 . a, Co-redistribution of endogenous CD4 and FLNa. Jurkat cells were incubated with spheres upholstered with IgG or anti-CD4; the cells were fixed and stained with an anti-FLNa antibody (red). The combination of transmission images and red fluorescence is shown. Representative cells of more than 30 analyzed in two experiments are shown: bar = 10 \ Boxm. be, The expression of FLNa regulates the clustering of CD4. The A7-CD4 and M2-CD4 cells were sequentially incubated with anti-CD4 and mouse Cy2-anti-IgG antibodies (4 ° C), then transferred at 37 ° C (b) or at 12 ° C (d) for 20 min. The cells were fixed and analyzed by confocal microscopy. The size of the aggregates was quantified (c, e) by Image J from the cells recorded in two independent experiments (n = 85 data from 25 A7-CD4 cells at 37 ° C; n = 115 (20 cells) M2-CD4 a 37 ° C; n = 64 (19 cells) A7-CD4 at 12 ° C; n = 128 (15 cells) M2-CD4 at 12 ° C). Bar = 5 \ Boxm in b, d.

Figure 3. Identification of FLNa residues that interact with CD4 . a, Growth of yeasts co-transfected with the selected FLNa fragments plus CD4IC in -LW or -LHAW medium. The activity \ Box-galactosidase (blue colonies) indicates the interaction between the two proteins. b, In vivo interaction of CD4 and FLNa D10 Jurkat cells were transfected with the FLNa D8-9 or FLNa D10 fragments fused to GFP, and CD4 crosslinking was induced by sequential incubation with GP120 and anti-GP120 (red). CD4 was stained with an antibody that recognizes the intracellular domain of CD4 (blue), and FLNa fragments were detected with GFP fluorescence (green); (n = 14 recorded cells). Bar = 10 \ Boxm. c, F1243, S1245 residues of FLNa are critical for interaction with CD4. The interaction was analyzed by co-transfection of yeasts with specific FLNa mutants in pGAD and pGBD-CD4IC. The growth and blue staining of the colonies confirmed the interactions. d, Prediction of the binding regions. The Flna domain 17 was used as a master structure to predict the binding sites in multiple sequence alignments of the 24 FLNa domains (see Methods). The panel on the left shows the joint surface constructed with the sheets-? C and D, while the predicted joint region constructed with the sheets-? A and B is shown in the panel on the right (180º rotation on the Z axis). Predicted residues on the junction surface are shown as filled spaces, as well as important amino acids in CD4 binding. The amino acid numbering follows the PBD numbering for chain A of domain 17. The colors indicate the degree of consistency of the prediction: red is the most likely residues, followed by violet; Less consistent predictions are shown in yellow. The cyan color indicates the residues involved in binding to CD4; residue F1948 of domain 17 of FLNa was detected with high probability, but is shown in cyan due to its involvement in the interaction with CD4. The asterisks mark the residues exchanged in domain 10 (S 1946 in domain 17 corresponds to F 1243 in domain 10, and F 1948 in domain 17 corresponds to S 1245 in domain 10).

Figure 4. Identification of CD4 residues that interact with FLNa . a, Analysis of the interaction of CD4IC mutants with domain 10 of FLNa by the two yeast hybrid system. Asterisks indicate the termination of the protein; Individual amino acid substitutions are indicated in red. (+) indicates colony growth and (-) non-growth; all colonies that grew in selective medium-LHAW were positive for β-galactosidase activity. b, FLNa and p56Lck simultaneously bind to CD4. Jurkat cells were stimulated for 5 min with an anti-CD4 antibody or were not treated (-). Cell lysates were immunoprecipitated (IP) with anti-CD4 or anti-FLNa antibodies and proteins resolved by SDS-PAGE analyzed with anti-p56Lck and anti-FLNa antibodies by immunoblot. c, Structure of the fragments of CD4 and p56Lck (pbd code 1Q68). The CD4 is shown in gray and p56Lck in blue; The yellow ball is the molecule of Zn 2+ that coordinates both proteins. The CD4 residues involved in the FLNa interaction are represented in solid in green (E 416) and red (K 417).

Figure 5. FLNa interacts with CXCR4 . a, Yeasts were co-transfected with GAD-FLNa D8-9 or GAD-FLNa D10 plus GBD-CXCR4 (314-360). Co-transformants were seeded on selective media -LW or -LHAW, growth was analyzed at 72 h and colonies were tested for Box-galactosidase activity. b, Interaction of endogenous FLNa and CXCR4. Similar amounts of cell lysates were immunoprecipitated with control IgG or anti-CXCR4 or anti-FLNa antibodies, and immunoblotting was performed with specific anti-CXCR4 and anti-FLNa antibodies (n = 4 independent experiments). c, d, The expression of FLNa is necessary for specific signals induced by CXCR4. M2-CXCR4-GFP and A7-CXCR4-GFP cells were tested for Ca 2+ mobilization induced by CXCL12 (c) with the Fluo-3, AM probe, or F-actin formation (d) measured as incorporation of phalloidin. For Ca 2+ mobilization, a representative histogram of three independent experiments is shown; For the formation of F-actin, the data represent the mean ± SEM of triplicates in one of three experiments. e, Blocking the interaction between FLNa and CXCR4 inhibits chemotaxis induced by CXCL12. MT2 cells transfected with the indicated FLNa fragments were tested for chemotaxis against CXCL12 in transwell assays. The migration index was calculated by dividing the number of cells that migrated in response to the agonist by the number of cells that migrated in response to the basal environment for each condition. The data is the mean ± SEM of a representative experiment of three.

Figure 6. FLNa is necessary for cell-cell fusion induced by Env . a, HEK-293-CD4 target cells were transfected with control RNAi or specific for FLNa; at the indicated times, the target cells were mixed with the HEK-293-Env_ {IIIb} effector. The effector-target cell fusion was quantified as the renilla / firefly luciferase activity ratio and the data are expressed as percent inhibition, using the value for cells transfected with an empty vector as a reference. Data are mean ± SEM of triplicates in a representative experiment of two. b, FLNa expression was analyzed by immunoblot in total lysates of the target cells in a. The immunoblot with transferrin anti-receptor was used as a loading control. c, HEK-293-Env IIIb effector cells were mixed with HEK-293-CD4 target cells transfected with different amounts of empty vector, FLNa D10-12, FLNa D8-9 or FLNa ^ {D10 (F1243S, \ S1245F)} The data are mean ± SEM of triplicates in a representative experiment of three performed.

Figure 7. The formation of the HIV synapse requires the binding of FLNa to CD4 and CXCR4 . a, FLNa is necessary for co-receptor clustering induced by Env. A7-CD4-CXCR4-GFP, M2-CD4-CXCR4-GFP or A7-CXCR4-GFP cells were mixed with the HEK-293-Env_ {IIIb} effector cells. The conjugates were stained with anti-FLNa antibodies (red); green staining corresponds to CXCR4-GFP. b, FLNa-mediated CD4 signals are necessary for the accumulation of CD4 and the co-receptor in the target-effector cell contact zone. The A7-CXCR4-GFP cells were transiently transfected with wild CD4 or the CD4 Δ416 mutant, which does not interact with FLNa, and then mixed with the HEK-293-Env IIIb cells. The conjugates were stained with the anti-gp120 scFv-m9 antibody (red). c, CXCR4 signals regulate the redistribution of FLNa and CD4. Conjugates formed between HEK-293-EnvIIIb and HEK-293 cells expressing CD4-YFP (green) untreated or treated with AMD3100; the conjugates were stained with an anti-FLNa antibody (red). (a, b and c, 10 \ Boxm bar). In all cases, the images are representative of at least 20 cells in two independent experiments.

Figure 8. FLNa mediates the inactivation of ADF / cofilin via RhoA / ROCK induced by gp120 . a, b, FLNa mediates RhoA activation and gp120-induced cofilin inactivation. HEK-293-CD4 cells transfected with the empty vector, FLNa D10-12 or FLNa D8-9 were incubated with microspheres upholstered with gp120 IIIB for the indicated times. Identical amounts of cell extracts were analyzed with the indicated antibodies. RhoA-GTP (active) levels were analyzed by precipitation with the rhotekin binding domain coupled to GST, followed by immunoblotting with anti-RhoA antibodies. The quantification of the relative levels of Rho-GTP and phospho-cofilin are shown in b; Each point represents the mean ± SEM of the densitometry values obtained in two independent experiments. c, FLNa regulates the formation of F-actin induced by gp120. The cells in a were analyzed for F-actin formation by measuring the incorporation of phalloidin; the data are mean ± SEM of triplicates in a representative experiment of three. d, RhoA-dependent signaling pathways regulate Env-mediated cell-cell fusion. HEK-293-CD4 cells were transfected with the indicated expression plasmids or treated with the ROCK inhibitor Y-27632 or with the CXCR4 antagonist AMD3100 (control). The percent inhibition was calculated using non-transfected cells or cells treated with DMSO (for Y-27632) as references. The data are mean ± SEM of the inhibition values obtained in two independent experiments. e, The activity of ROCK is necessary for the redistribution of FLNa and the co-receptor to the HIV synapse induced by Env. Conjugates formed between effector cells and A7-CD4-CXCR4-GFP target cells pretreated with vehicle or Y-27632. FLNa was stained with anti-FLNa antibodies (red) and CXCR4 was visualized as a fusion protein with GFP (green). Representative cells are shown (n = 15; 10 \ Boxm bar). f, The phosphorylation of cofilin and FLNa induced by gp120 depends on the activity of ROCK. Jurkat cells were treated with vehicle or Y-27632 before incubation with spheres upholstered with gp120. Equal amounts of cell lysates with anti-phospho-cofilin, cofilin, phosphoserine and FLNa antibodies were analyzed by immunoblot. The migration of the 250 kDa marker is shown on the left in the phosphoserine and FLNa hybridizations. g, Quantification of the relative levels of phospho-cofilin and phosphoserine in f. Data are mean ± SEM of densitometry values (n = 3).

Figure 9. FLNa binds to CCR5 and mediates R5 virus infection . a, Yeasts were co-transfected with the indicated FLNa fragments cloned into pGAD and pGBD-CCR5 (325-352). Co-transformants were seeded on selective media -LW or -LHAW, growth was analyzed at 72 h. Colonies that grew in the -LHAW medium were positive for \ Box-galactosidase activity. b, Interaction of endogenous CCR5 and FLNa. Identical amounts of Jurkat-GFP-CCR5 cell lysates were immunoprecipitated with control (IgG), anti-CCR5 or anti-FLNa antibodies, and the filters were hybridized with anti-CCR5 and anti-FLNa (representative of 4 independent experiments). c, M2-GFP-CCR5 and A7-GFP-CCR5 cells were tested for COLS-induced mobilization with the Fluo-3, AM probe. Arrows indicate stimulation with the agonist. d, The chemotaxis of M2-GFP-CCR5 and A7-GFP-CCR5 cells was tested against baseline (white bars), CCL5 (black bars) or FCS (striped bars) in transwells. The data are mean ± SEM of the migration index for each cell line. e, Jurkat-GFP-CCR5 cells were transfected with control RNAi or FLNa specific; at 48 h, the cells were infected with HIV-1 replication-deficient viruses pseudotyped with the VSV-G envelopes or an R5 envelope of a clinical isolate of an HIV-1 infected patient. Cellular infection is proportional to luciferase activity (in arbitrary units) measured in cell extracts. Data are mean ± SEM of triplicates in a representative experiment of three. f, FLNa expression was analyzed by immunoblot in total cell lysates in e. Hybridization with an anti-actin antibody was used as a loading control.

Figure 10.- Multiple sequence alignments of FLNa domains . The alignments are based on the published information. The amino acids in repetition 10 that differ from the remaining sequences are indicated in red. The structural alignment of FLNa repeat 17 is shown below (B, residue in an isolated beta bridge; E, extended beta sheet; G, helix 310; T, hydrogen-bound spin; S, beta sheet). The leucine residue that interacts with phenylalanine is shown in red on a gray background.

Figure 11.- Multiple sequence alignments of the C-terminal CXCR4 region of different species . Sequence names are SwissProt numbers. The two lower lines indicate the sequence and secondary structure of the C-terminal domain of CD4-HUMAN (pbd code 1Q68). Lowercase CD4 amino acids indicate regions that do not align, while amino acids in the motif shared between CD4 and CXCR4 are indicated in uppercase. The E416 and K417 residues of CD4 involved in the interaction with FLNa are located in a helix turn (the nomenclature corresponds to structure 1Q68; S, beta sheet; H, alpha-helix; T, turn).

Figure 12.- Regulation of the HIV synapse by FLNa . a, FLNa is necessary for actin clustering and the Env-induced co-receptor. FLNa A7-CD4-CXCR4-GFP cells, FLNa M2-CD4-CXCR4-GFP-deficient cells, or A7-CXCR4-GFP cells were transfected with actin-RFP and mixed with effector cells. The red and green stains correspond to actin and CXCR4, respectively. b, The signaling of the co-receptor is necessary for the redistribution of FLN to the contact zone between the target and effector cell. A7-CD4-CXCR4-GFP target cells were pretreated with the CXCR4 antagonist AMD-3100 and conjugates with effector cells expressing Env were stained with the anti-FLNa antibody (red); CXCR4 was visualized in green by GFP fluorescence. Bar = 10 \ Boxm.

Figure 13.- gp120 induces RhoA activation and inactivation of ADF / cofilin in primary target cells . a, Activated peripheral blood human lymphocytes were incubated with gp120 spheres during the indicated times, and identical amounts of cell extracts were analyzed for activated RhoA in precipitation assays with the rhotekin binding domain bound to GST. The filters were hybridized with anti-RhoA, anti-phospho-cofilin and cofilin antibodies. b, c, Quantification of the relative levels of RhoA-GTP and phospho-cofilin of the cells in a; Each point represents the mean ± SEM of the densitometry values obtained in two independent experiments.

Figure 14.- The activity of ROCK is necessary for the redistribution of actin and the co-receptor to the HIV synapse induced by Env . Conjugates formed between the untreated or treated A7-CD4-CXCR4-GFP target cells, and the effector cells. Actin was stained with phalloidin-rhodamine (red) and CXCR4 was visualized as a fusion protein with GFP (green). The cells shown are representative of 22 (bar = 10 \ Boxm).

Examples of realization Example 1 Identification of the minimum domain of the FLNa and CD4 protein or CXCR4, respectively, involved in the interaction between these protein 1.1.- CD4 interacts with FLNa

To identify new interaction proteins with CD4, the binding of proteins encoded by a cDNA expression library generated on the Jurkat T cell line with the intracellular domain of CD4 (CD4IC) by an assay of Two hybrids in yeasts. The sequencing of positive clones identified previously described proteins (p56Lck) and some new interaction proteins with CD4 not previously described, as a fragment of the MyoD family inhibitor (Gen Bank Accession Number: BC007836), a protein that contained the domain I-mfa (HIC; AY196485), the protein kinase 2 that interacts with homeodomain (HIPK2; NM-O22740), and two independent clones of the FLNa actin binding protein (NM-001456). The specificity of the interaction is verified by transformation "one by one"; the co-transfection of CD4IC with MyoD, HIC, HIPK2 or FLNa gave the transformed colonies the ability to grow in medium without adenosine, histidine, tryptophan and leucine, and to dye blue in a rehearsal of α-galactosidase (Figure 1a). Of this new CD4 binding proteins were decided to analyze FLNa.

The FLNa cDNA fragment rescued in the two hybrid assay encoded for amino acids 968-1252, corresponding to the repetitions 8-10 of FLNa. To examine the interaction FLNa-CD4, protein binding was tested for CD4IC-GST fusion (glutathione S-transferase) with FLNa in cell lysates Jurkat cell totals. CD4IC-GST, but not GST, efficiently interacted with FLNa protein in lysates (Figure 1b). It was also found that the CD4 protein interacted with the FLNa fragment (968-1252) fused to GST (not shown).

FLNa-CD4 interaction was analyzed in vivo by affinity isolation of cell membranes containing CD4, using magnetic microspheres upholstered with anti-CD4 antibodies after nitrogen cavitation of Jurkat cells. In unstimulated cells, p56Lck and a small fraction of FLNa co-precipitated with anti-CD4 microspheres; cross-linking of CD4 increased the co-purification of FLNa with CD4 and p56Lck (Figure 1c). Direct interaction of FLNa and CD4 was confirmed by cross immunoprecipitation (Figure 1d). Activation of antibody-induced CD4 caused phosphorylation of FLNa in serine residues (Figure 1e), but not in tyrosines (not shown), although FLNa has been described as a p56Lck substrate in vitro (25). Together, these results indicate that CD4 interacts with FLNa in vivo .

1.2.- FLNa regulates the grouping of CD4

Since the clustering of CD4 depends on the integrity of the actin cytoskeleton (11), the effect was studied of the expression of FLNa in the mobility of CD4. First it was analyzed the distribution of FLNa and CD4 expressed endogenously in Jurkat cells incubated with antibody-coated microspheres anti-CD4. In 98% of the conjugates microsphere-cell, FLNa staining was concentrated in the contact zone between the cell expressing CD4 and the microsphere (Figure 2a). Next the grouping was studied of CD4 in M2 cells (FLNa-deficient cells) and A7 (in clone of M2 stably expressing FLNa (26)) both transfected with CD4 The size of the CD4 aggregates at 37 ° C was strongly reduced in M2-CD4 cells compared to A7-CD4 cells (Figure 2b). The quantification of The data showed that in M2-CD4, CD4 cells It was distributed in small aggregates with an average area of 0.32 ± 0.06 \ Boxm2, while the average size of the aggregates of CD4 in A7-CD4 cells was 1,125 ± 0.22 Box2. The size range of CD4 aggregates it was also significantly different between M2 and A7 cells (chi2 = 27.5, p = 0.001, 4 degrees of freedom; Figure 2c). When the cross-linking was performed at 12 ° C, a temperature in the which prevents cellular metabolism (27), the size of CD4 aggregates were similar between M2 and A7 cells (areas means: 0.146 ± 0.02 \2 for M2-CD4; 0.155 ± 0.022 ± 2 for A7-CD4; Figure 2 of).

1.3.- Characterization of the FLNa region that interact with CD4

The ability of FLNa shortening mutants to interact with the CD4IC was examined using the two hybrid system, and FLNa repeat 10 (FLNa D10, residues 1158-1252) was identified as the minimum FLNa domain that retained the interaction with CD4IC (Figure 3a). All shortening mutants that lacked this repetition lost interaction with CD4IC (not shown). The GFP-FLNa D10 domain co-located with the CD4-gp120 complexes, while the distribution of the GFP-FLNa D8-9 fragment was nucleocytosolic and did not co-localize with CD4-gp120 (Figure 3b); this suggested that FLNa D10 interacts with CD4 in vivo . Sequence analysis of the 24 domains using the structural information of the complex between domain 17 of FLNa and the GPIbα protein (28), identified three residues in domain 10 (E1217, F1243) and S 1245) that differed from the sequences of the rest of the FLNa domains in the global alignment (Figure 10). The replacement of E1217 with proline (FLNa D10 (E1217P)}, the amino acid found in all other domains, did not affect the FLNa-CD4 interaction (Figure 3c). The specific arrangement of residues F1243 and 51245 in the domain 10 (F 1243 P 1244 S 1245) differs from the "classic" serine-proline-phenylalanine (SPF) motif found in the other repeats (Figure 10). An exchange mutant was found, in which residues F 1243 and S 1245 were changed to cause SPF (FLNa D10 (F1243S, \ S1245F)), lost interaction with CD4IC (Figure 3c).

The FLNa domain 17 structure was used to  model domain 10 (using WHATIF servers and SWISSMODEL) and estimate the interaction surfaces (29). Be predicted with high probability two main regions as important in binding to other proteins. One involved the leaves ? C and D, which interact with GPIb? and the cytosolic domain of β7 integrin (28, 30); this is considered the general joint surface (30). The second interaction surface involved the? A and B e sheets included residues F 1243 P 1244 S 1245 that had been predicted in the interaction with CD4 (Figure 3d). The prediction of this second interaction surface therefore supports the union FLNa-CD4.

1.4.- Identification of CD4 residues that interact  with FLNa

To locate the CD4 region responsible for interaction with FLNa, sequence analysis was combined with directed mutagenesis (Figure 4a). The interaction of three was analyzed mutants in which parts of the C-terminal (CD4IC Δ429, CD4IC Δ421 and CD4IC Δ416) with FLNa D10 using the two system hybrids This analysis identified the FLNa binding site within  of waste 415-421 of CD4, near the ground di-leucine involved in CD4 endocytosis (31). This region has also been implicated in the interaction of CD4 with p56Lck, in addition to the CxCP conserved motive that directs the union of high kinase affinity (32, 33). The mutation of the CxCP motif prevented the interaction of CD4IC with p56Lck, but not with FLNa (Figure 4a). FLNa also co-precipitated with p56Lck after cross-linking of CD4 (Figure 4b), suggesting the simultaneous binding of CD4 with p56Lck and FLNa. Based on the available 3D structure of the p56Lck-CD4 complex (PBD code 1Q68), the CD4 residues that interact were eliminated with p56Lck. This allowed the identification of amino acids E416 and K417 in the turn of the \ Box-propeller, as potential interaction sites of CD4 with FLNa by n-capping (Figure 4c). In fact, the replacement of E 416 -K 417 by alanines (CD4IC E416A, K417A) prevented the interaction CD4-FLNa, while replacing the waste 412-415 (CD4IC 412-415A) or K 418 (CD4IC k418A) by alanines did not affect the binding of FLNa (Figure 4a). These results indicate that amino acids E 416 -K 417 are critical in the reason for CD4 that interacts with FLNa.

1.5.- FLNa domain 10 also interacts with CXCR4

A small motif with similar load to that found in CD4 was identified in CXCR4 (Figure 11); This led to study the possible interaction of FLNa with this co-receptor of HIV-1. As predicted by the sequence analysis, the two-hybrid assay showed that FLNa D10 interacted with CXCR4 but not with CCR5, a receptor in which the motif had not been detected (Figure 5a). Cross-co-precipitation of endogenous FLNa and CXCR4 confirmed the FLNa-co-receptor interactions in vivo (Figure 5b).

To analyze the influence of FLNa on the CXCR4 functionality, signaling events were analyzed mediated by CXCR4 after stimulation with its ligand, CXCL12, in M2 and A7 cells that stably expressed a CXCR4 receptor merged in the C-terminal to GFP. CXCL12 induced comparable Ca 2+ flows in M2-CXCR4 cells and A7-CXCR4 (Figure 5c), an event mediated by heterotrimeric G proteins; however, the formation of F-actin induced by CXCL12 was strongly inhibited in M2-CXCR4 cells deficient in FLNa (Figure 5d). The specific interaction block CXCR4-FLNa for overexpression of the FLNa fragment that contained the domains 10-12 (FLNa D10-12) inhibited the Chemotaxis in Jurkat cells (Figure 5e, p <0.001, Student t-test). These results indicate that FLNa is necessary to activate specific signaling pathways mediated by CXCR4.

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Example 2 FLNa is a key molecule of infection by HIV-1 and, therefore, a therapeutic target 2.1.- FLNa is necessary for cellular infection due to strains X4 of HIV-1

Next, the function of FLNa was analyzed in the initial stages of HIV-1 infection. The Env-mediated membrane fusion in cells with levels Physiological CD4 and co-receptors is a process Actin and cholesterol dependent (9); However, the co-receptor overexpression suppress these requirements (7). Thus, the function of FLNa in membrane fusion decreasing FLNa expression with small interfering RNA (RNAi) in cells HEK-293CD4, which express endogenous CXCR4 and ectopically CD4 (5). As effector cells were used HEK-293 transiently transfected with env111B, a wrap of a strain X4. The decrease in the expression of FLNa in the target cell with specific RNAi reduced fusion Env-mediated cell (Figure 6a). Approximately one was observed 50% inhibition of Env-induced fusion at 48h after of transfection of HEK-293CD4 cells with RNAi, compared to cells transfected with control RNAi. This inhibition was similar to the decrease in the expression of the FLNa protein in transfected cells (Figure 6b). The similar strong results in infection experiments with HIV-1 non-replicative pseudotyped with the envelope X4 (not shown).

FLNa interacts with many proteins cytosolic and membrane, and therefore it is possible that the effect observed in the RNAi experiments could be independent of the interaction of FLNa with CD4 and CXCR4. Therefore, the Env-induced fusion in target cells that overexpressed the fragment FLNa D10-12, which specifically blocks the interaction of CD4 and CXCR4. The FLNa interaction block with HIV-1 receptors inhibited consistently fusion between cells HEK-293CD4 and the cells expressing Env (Figure 6c). On the contrary, the expression of the fragment FLNa D8-9 or exchange mutant FLNa D10 (F1243S, S1245F)} did not affect the fusion mediated by Env (Figure 6c). These data strongly implicate FLNa as a key molecule in the initial stages of infection by HIV-1, regulating the function of CD4 receptors and CXCR4.

2.1.- FLNa is necessary to group CD4 and CXCR4 in the HIV-1 synapse

Cellular infection depends on the recruitment of CD4 and Env-induced co-receptors, resulting in a stable adhesive bond between the cell that contains the viral envelope and the host cell membrane, called the HIV synapse (10, 35 ). To determine the role of FLNa in the formation of the HIV synapse, conjugates were formed between the HEK-293 effector cells expressing env IIIB and the M2-CD4-CXCR4-GFP or A7-CD4-CXCR4- target cells. GFP The expression of CXCR4-GFP in 100% of the target cells allowed their discrimination of the effector cells. It was found that the expression of FLNa was necessary for the accumulation of CXCR4-GFP in the contact zone between the target and effector cell; This polarization of CXCR4 was parallel to the accumulation of FLNa (Figure 7a) and actin (Figure 12a) at the contact site. Redistribution of CXCR4-GFP, FLNa (Figure 7a) and actin (Figure 12a) was not observed in conjugates formed with A7-CXCR4-GFP cells that did not express CD4. These results indicated that the redistribution of the co-receptor was dependent on FLNa and required the interaction of Env with CD4 and / or the co-receptor.

To determine which of these interactions initiated the redistribution of the receptors, were formed conjugates in the target cells A7-CXCR4-GFP transfected transiently with wild CD4 or the mutant CD4? 416, the which does not interact with FLNa. To display gp120 joined with CD4 m9 antibody, a single chain Fv (scFv) derived from a Fab anti-gp120 (X5) with a greater affinity for CD4-gp120 complexes than by gp120 alone (36). The CXCR4-GFP and scFv m9 staining was concentrated in the zone of contact between effector cells and target cells that expressed wild CD4 while these markers showed diffuse staining in cells expressing CD4? 416 (Figure 7b). These results suggested that the FLNa-CD4 interaction was necessary for the redistribution of the co-receptor during training of the HIV-1 synapse. The treatment of target cell with the CXCR4 antagonist AMD3100 prevented the accumulation of CXCR4-GFP and FLNa in the area of contact between the effector and target cell (Figure 12b), as well as the redistribution of CD4 to said region (Figure 7c), indicating that the CXCR4 signals are also necessary for the formation of the synapse of HIV-1.

2.3.- FLNa organizes the signals for the remodeling of F-actin initiated by the receptors of the HIV-1

To study the mechanism by which FLNa influence the cellular infection, the activation of the Rho-GTPases induced by viral envelope in Jurkat cells transfected with different FLNa fragments. Be simulated cross-linking of CD4 and CXCR4 with upholstered particles with gp120_ {IIIB}. Previous studies with complete viruses indicated that HIV-1 binding induces activation RhoA specific (18); here, the microspheres upholstered with gp120 they also induced RhoA activation in Jurkat cells transfected with the empty vector or with the fragment FLNa D8-9, while the fragment overexpression FLNa D10-12 inhibited RhoA activation induced by gp120 (Figure 8a, quantification at 8b).

RhoA signals through two effectors, mDia and p160ROCK. One of the target of ROCK is the kinase LIM (LIMK), the which in turn phosphorylates and inactivates the depolymerization factor of actin (ADF) / cofilin, which leads to the stabilization of F-actin (35). It was found that the binding of gp120 induced cofilin phosphorylation in transfected cells with the empty table or with the fragment FLNa D8-9, with a peak at 30 seconds after  addition of gp120. The cofilin phosphorylation was severely decreased in cells that overexpressed the FLNa D10-12 fragment, in which only observed a slight increase at long incubation times (Figure 8a, quantification in 8b). Compatible with the kinetics of RhoA activation and phosphorylation / inactivation of cofilin, the gp120 binding increased F-actin levels after 30 seconds in the cells expressing the fragment FLNa D8-9; however, in the cells that overexpressed the fragment FLNa D10-12 a net loss of F-actin after gp120 binding (Figure 8c). These results suggest a function for cofilin phosphorylation induced by gp120 in the stabilization of F-actin The union of gp120 to CD4 and CXCR4 also induced phosphorylation in serines in cells transfected with the empty vector or with the FLNa D8-9 fragment (Figure 8a). FLNa phosphorylation was not observed in the cells expressing the FLNa D10-12 fragment (Figure 8a), indicating that said fragment effectively blocked the interaction between FLNa with CD4 / CXCR4. RhoA activation and cofilin phosphorylation were also observed in lymphocytes of peripheral blood (PBLs) isolated from healthy individuals incubated with the microspheres upholstered with gp120 (Figure 13), indicating that HIV-1 induces this signaling pathway in cells primary target.

The LIMK / cofilin pathway can be initiated by ROCK or by the Rac effector, PAK-1. To determine the relevance of these two kinases in infection by HIV-1, fusion assays were performed using target cells expressing the dominant negative mutants RacN17, RhoAN19, or PAK-1, R229, H83L, H86L (PAK-DN, with mutations in the kinase domains and binding to GTPase), or treated with the ROCK inhibitor Y-27632. The loss of function of Rac or PAK-1 did not affect the merger mediated by Env, while the overexpression of RhoAN19 or the treatment of target cells with Y-27632 inhibited cell fusion (Figure 8d). The treatment of target cells with Y-27632 prevented the accumulation of CXCR4, FLNa and actin at the HIV-1 synapse (Figure 8e, Figure 14), and inhibited phosphorylation of cofilin and FLNa induced by gp120 (Figure 8f, g), indicating that these Signaling events are dependent on ROCK. The results therefore they imply that the activation of the signaling pathway RhoA \ LeftrightarrowROCK \ LeftrightarrowLIMK \ Leftrightarrowcofilin dependent on FLNa, it is a fundamental event in the cluster of HIV-1 receptors and in the membrane fusion mediated by gp41.

2.3.- FLNa is also a necessary cofactor for the R5 virus input

Finally, it was analyzed if the FLNa requirements for HIV-1 infection were limited to X4 viral strains. The two hybrid system was used to analyze the interaction of the C-terminal domain of CCR5 (amino acids 325-352) with the FLNa fragments that cover domains 8 to 24 (Figure 9a). The analysis showed that CCR5 specifically interacted with the FLNa fragment that contained repeats 13-16. Cross-precipitation of endogenous FLNa with CCR5 confirmed the FLNa-CCR5 interaction in vivo (Figure 9b). As with CXCR4, the expression of FLNa did not regulate the flow of Ca 2+ mediated by CCR5 (Figure 9c). However, CCR5-mediated chemotaxis was significantly decreased in M2-CCR5-GFP (FLNa-deficient) cells compared to A7-CCR5-GFP cells expressing FLNa (p <0.005, Student's t-test; Figure 9d ); This severe reduction in chemotaxis was not observed when fetal calf serum was used as chemoattractant.

To analyze the role of FLNa in infection of R5 virus, infections with the variant of the HIV-1 NL4.3 replication defective, pseudotyped with the envelope R5 (LMP0608), or with the envelope of the virus of the vesicular stomatitis (VSV-G). The cells Jurkat-CCR5, an established cell line that express CCR5 fused to GFP (37), were transfected with RNAi irrelevant or specific FLNa and were used as target. Be observed approximately 50% reduction in the expression of FLNa at 48 hours after transfection with RNAi to FLNa compared to cells transfected with the control RNAi (Figure 9e). Despite the partial reduction in FLNa levels and the non-physiological expression of CCR5 in this cell line, the FLNa RNAi significantly inhibited cell infection with R5 virus compared to cells transfected with RNAi control (Figure 9f). Infection of the target cells with the pseudotyped viruses with VSV-G was comparable in Both conditions. These results demonstrate that the expression of FLNa also regulates the entry of R5 strains from HIV-1 in the target cells.

Materials and methods Cloning and generation of CD4 and FLNa mutants

Plasmid pSRαpuro-CD4 and pCD4-YFP were provided by G. Del Real (National Center for Biotechnology, Madrid) and TJ Hope (Department of Microbiology and Immunology, University of Illinois, Chicago, IL, USA), respectively. The FLNa D8-9 and FLNa D10-12 fragments were amplified by PCR using primers 5'-GCGAATTCCTGGACCTCAGCAAG-3 '(SEQ ID NO15) / 5'-GCGGATCCGCGGGAACCACGTGGGC-3' (SEQ ID NO16) and 5'-GCGAATTCTTTGACGCATCCAAA-3 '(SEQ ID NO17) / 5'-GCGGATCCCTTGAAAGGACTGCCT
GG-3 '(SEQ ID NO18), respectively, and were cloned into pEGFPN1 and pcDNA3.1. These FLNa fragments were also subcloned in the bicistronic plasmid pRV-IRESGFP (Genetrix, Madrid, Spain). Specific amino acids of CD4IC or FLNa D10 were mutated by the Quick Change (Stratagene) mutagenesis kit, with appropriate primer pairs (Table I) and the pfs Turbo DNA polymerase for PCR amplification. The products were digested with DpnI to eliminate parental chains. The resulting plasmids were transformed into supercompetent Epicurian Coli XL1-Blue bacteria (Stratagene) and the sequence of the selected clones was verified in the sequencing service of the National Center for Biotechnology. The CD4 and FLNa mutants were subcloned into pGBKT7 and pGADT7, respectively, for the assay of two yeast hybrids, and pcDNA3.1 for expression in mammalian cells.

TABLE I PCR primers used for the generation of CD4 and FLNa mutants

one

Cell culture and transfections

Peripheral blood lymphocytes will purified on Ficoll-Hypaque gradients (Amersham Biosciences) and were activated for two days with Phytohemagglutinin (PHA, 1 / / ml) and interleukin 2 (IL2, 50 ng / ml). MT-2 cells, Jurkat and cells Jurkat-CCR5-GFP (37) remained in RPMI 1640, 10% FCS, 1 mM sodium pyruvate, 2 mM L-glutamine, 100 U / ml penicillin and 100 µg / ml streptomycin. HEK-293 cells were maintained in DMEM, 10% FCS, 2 mM L-glutamine and antibiotics; HEK-293-CD4 cells are cultured as HEK-293 cells, with the addition of G418 (1 mg / ml). M2 and A7 cells were maintained as described (26). M2 and A7 cells were transfected with SRαpuro-CD4; after selection with puromycin, the resulting clones were infected with retroviruses pRV-CXCR4-GFP recombinants or pRV-GFP-CCR5 (40) and GFP cells positives were selected by cytometry in a Coulter Epics Altra (Beckman Coulter). The cells were maintained in G418 and puromycin, and selected by flow cytometry every two months to maintain the expression of CXCR4 and CCR5. M2 and A7 cells too were infected with recombinant retroviruses pRV-CXCR4-GFP or pRV-GFP-CCR5 to generate the M2 and A7 cells that stably express these receptors of chemokines HEK-293 cells were transfected transiently with the FLNa D8-9 fragments and FLNa D10-12 cloned in pEGFPN1 and pcDNA3.1 using JetPei (Poly Transfection). Jurkat cells became infected with recombinant retroviruses pRV-FLNa D8-9 IRESGFP or pRV-FLNa D10-12 -IRESGFP  and infected cells were selected by cytometry of flow.

Essay of the library by two hybrids

CD4, CXCR4 and CCR5 baits were constructed by amplifying the CD4IC (amino acids 396-433), the C-terminal of CXCR4 (amino acids 314-360) and the C-terminal of CCR5 (amino acids 325-352) by PCR with specific primers (Table II), and the products were subcloned on the sites EcoRI-SalI of the vector with the binding domain to Ga14 (pGBKT7, Clontech). CD4IC was used to investigate proteins binding to CD4 in a Jurkat cDNA library, which was generated with the SMART cDNA Synthesis (Clontech), merged into the domain of Ga14 activation (pGADT7-Rec, Clontech). The positive clones were isolated and sequenced, and the positive results by transformation one-on-one agar plates without  adenine, histidine, tryptophan and leucine, and using a test of β-galactosidase with X-? -Gal (Clontech) as substratum.

TABLE II PCR primers used for amplification of the baits used in the two hybrid assay

2

FLNa D8-9 fragments, D10-12, D13-16, D16-20 and D20-24 were amplified by PCR with specific primers (Table I) and cloned at sites EcoRI-BamHI (D8-9, D10-12, D16-20), EcoRI-XhoI (D13-16) and BamHI-XhoI (D20-24) Vector pGADT7. Yeasts were co-transformed with these fragments and baits CD4IC, CXCR4 or CCR5, and interactions Positive were analyzed as before.

Immunoaisulation of plasma membrane fragments

Magnetic microspheres were upholstered M-450 goat anti-mouse immunoglobulins (Ig) (Dynal) with an anti-CD4 monoclonal antibody (Caltag Laboratories) following the manufacturer's instructions. Jurkat cells (3 x 10 7) were incubated (4 ° C, 2 min) with the anti-CD4 microspheres (microsphere / cell ratio 1: 2) in RPMI with 1% FCS. The conjugates microspheres-cells were incubated at 37 ° C for 0, 3 and 7 minutes, cold washed once with H buffer (10 mM Hepes sodium, pH 7.2, 250 mM sucrose, 2 mM MgCl2, 10 mM NaF, and 1 mM orthovanadate) and were resuspended in 1 ml of buffer H with 0.2 mM pervanadate and with the CLAP protease inhibitor (Sigma-Aldrich). The cells were cavitated with nitrogen (4ºC, 50 bar, 7 min) by means of a cavitation pump nitrogen (Parr Instrument Company). The homogenate was carried up 10 ml with buffer H and the microspheres were collected with a magnet (Dynal) After washing 3 times with 10 ml of buffer H (1 minute each, at 4 ° C), cell lysates were concentrated by ultracentrifugation (100,000 x g, 4 ° C, 20 min) in one TL100 ultracentrifuge table (Beckman Coulter). The microspheres and cell concentrates were analyzed by immunoblot.

Analysis of FLNa interactions in vitro and in vivo

Precipitation tests were performed with GST incubating (4 ° C, 4 h) similar amounts of GST, GST-CD4IC and GST-FLNaD8-10 immobilized in GST spheres (Amersham) with Jurkat cell extracts. After of washing with lysis buffer, bound proteins were eluted with 2x loading buffer and resolved by electrophoresis in gels polyacrylamide (SDS-PAGE). A fraction of the eluted proteins were analyzed by immunoblot with antibodies anti-FLNa (Neo Markers) or anti-CD4 (Santa Cruz Biotech); the rest were stained with Coomassie blue then electrophoresis to confirm the expression of the fusion protein

FLNa interactions in vivo were analyzed by immunoprecipitation. For interaction with CD4, MT-2 cells (1 x 10 7) cultured in the absence of serum were incubated (20 min, 4 ° C) with anti-CD4 antibody HP2.6; an anti-mouse Ig antibody was added to the tubes and the tubes were transferred at 37 ° C for the indicated times. The reaction was terminated by placing the tubes on ice and the cell aggregates washed immediately in cold PBS buffer (Phosphate Buffered Saline). The cells were lysed with RIPA buffer and 300 g of the extracts were immunoprecipitated (3 h, 4 ° C) with anti-CD4, anti-FLNa or anti-mouse IgG (control) antibodies, followed by incubation (45 min, 4 ° C) with G-Sepharose protein. The precipitated proteins were resolved in SDS-PAGE and analyzed in immunoblot with anti-CD4, anti-FLNa or anti-phosphoserine (Biosource) antibodies. For the interactions of CXCR4 and CCR5, 2 x 10 7 Jurkat-GFP-CCR5 cells were lysed and 200 µg of the extracts were immunoprecipitated with anti-CCR5 antibodies (MAB1801, R&D Systems), anti-CXCR4 (H118, Santa Cruz Biotech.), Anti-FLNa or anti-IgG (control), followed by G-Sepharosa protein. The precipitated proteins were resolved in SDS-PAGE and analyzed in immunoblot with anti-CCR5, anti-CXCR4 and anti-FLNa antibodies.

Immunofluorescence and confocal studies

To study the redistribution of the receptors to the HIV synapse, A7 and M2 cells stably expressing CD4 and CXCR4-GFP were mixed (20 min, 37 ° C) with HEK-293 cells expressing the envelope of HIV-1 Env IIIb}. The complexes formed were fixed with 4% paraformaldehyde (PFA, 5 min, 4 ° C), permeabilized with Triton X-100 (0.1%, 10 min, 4 ° C), and stained with anti-FLNa, actin-Rhodamine antibodies (Molecular Probes) and scFv-m9, followed by suitable secondary antibodies labeled with fluorophores (Jackson ImmunoResearch Laboratories). In some cases, the target cells were transiently transfected with the fusion protein between actin and the fluorescent red protein (actin-RFP; a gift from L. Rajendran, Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany).

For the CD4 clustering experiments, A7-CD4 and M2-CD4 cells are incubated (30 min, 4 ° C) with anti-CD4 antibody HP2.6. After removing unbound antibody, cells are incubated (20 min) with an anti-IgG antibody of mouse coupled to Cy2 (Jackson ImmunoResearch Laboratories) at 37 ° C or 12 ° C The cells were washed with cold PBS, fixed with PFA and analyzed ten random fields for each condition in a confocal Leica with a 63x objective (NA 1.2). The images are quantified using Image J analysis software. In some experiments, clustering of CD4 was induced by incubation (ratio 1: 1) Jurkat cells with 4.5 m carboxylated microspheres (Polysciences, Inc.) upholstered with antibodies anti-CD4. The cells were fixed with PFA, they were permeabilized and stained with antibodies anti-FLNa and secondary antibodies labeled with Cy3

Calcium mobilization assays

2 x 10 6 M2 and A7 cells expressing CXCR4-GFP and GFP-CCR5 were incubated with Fluo-3, AM (Molecular Probes) (300 mM in DMSO, 10 / / 106 cells). The cells were resuspended in medium complete with 2 mM CaCl2, and kept at 37 ° C until addition of CXCL12 or CCL5 (20 nM, Peprotech). The release of Ca 2+ was determined (37 ° C, 525 nm) in an EPICS flow cytometer XL (Beckman Coulter).

Chemotaxis Assays

To analyze the function of FLNa in the CXCR4-induced chemotaxis, MT-2 cells will transiently transfected with GFP, GFP-FLNa D8-9 or GFP-FLNa D10-12, and cells Greens were selected by cytometry. After 24 h, it 3 x 10 5 cells grown without serum were seeded in the chamber superior of a "transwell" of 0.8 \ Boxm (Cost); the chambers were filled with serum-free medium alone or with CXCL12 (25 nM). After incubation (4 h, 37 ° C), the number was estimated of cells in the lower chamber by flow cytometry. Be calculated the migration rate as the number of cells that migrate in response to CXCL12 regarding those that migrate in response to medium without chemoattractant. In the case of chemotaxis mediated by  CCR5, M2-CCR5-GFP and A7-CCR5-GFP grown in the absence of serum were seeded in 0.8 µm transwells upholstered with fibronectin; the lower chambers were filled with basal medium serum free, or containing CCL5 (100 nM), or containing 1% FCS. After incubation, the cells in the upper well are eliminated; the filters were stained and the number of cells that They had migrated was calculated as described (46).

Preparation of gp120 microspheres

Microspheres upholstered with streptavidin (6 Box, Polysciences, Inc.) were sequentially incubated at 4 ° C with  a biotinylated Fab'2 fragment of an antibody obtained in rabbit against sheep IgGs (Jackson ImmunoResearch Laboratories), a antibody obtained in sheep against gp120 (D7324, Alto Bio Reagents) and recombinant gp120_IIIB (NIH AIDS Research and Reference Reagent Program). Primary human lymphocytes activated with PHA (2 x 10 5) or Jurkat cells (5 x 10 6) infected with recombinant retroviruses for fragments FLNa D10-12 or FLNa D8-9 is stimulated with the spheres of gp120 (2: 1 ratio for lymphocytes, 1: 1 for Jurkat) during the indicated times. For some experiments, these cells were lysed with RIPA buffer and the Cell extracts were analyzed for active RhoA and Rac by immunoprecipitation with domains GST-Rhotekin and GST-PAK, respectively, or for immunoblot with antibodies anti-RhoA, anti-Rac (Santa Cruz Biotechnology), anti-phosphoserine, anti-phosphophilin (Biosource), cofilin (Cell Signaling) or anti-FLNa.

F-actin formation

To analyze the formation of F-actin induced by gp120, 2 x 10 5 cells Jurkat expressing the fragments FLNa D10-12 or FLNa D8-9 in RPMI 1640/1% FCS / 10 mM Hepes (pH 7.5), were stimulated with the spheres of gp120 at the indicated times. Aliquots of cells before stimulation with gp120 to determine the basal levels of F-actin. The cells were fixed immediately with PFA, they were permeabilized and stained (15 min, 4 ° C) with FITC-phalloidin (Molecular Probes). The Phalloidin incorporation was measured by flow cytometry (LSR, Beckton Dickinson). A similar protocol was used to measure F-actin in response to CXCL12 (50 nM, 37 ° C) in M2- and A7-CXCR4-GFP cells.

Env-induced cell-cell fusion

Cell-cell fusion assays were performed as described (5). Basically, the env IIIB gene of HIV-1 was introduced into HEK-293 effector cells by infection (1 h, 37 ° C) with a recombinant vaccinia virus. HEK-293-CD4 transfected with pSC-luc, which codes for the firefly luciferase gene under the control of vaccinia virus promoter 7.5 (provided by D. Rodríguez, National Center for Biotechnology, Madrid, Spain), was used as target cells. , and the plasmid without promoter for renilla luciferase. Where indicated, these cells were also transfected with an empty vector, FLNa D10-12, FLNa D8-9, FLNa D10 (F1243S, S1245F)}, RhoA-GFP, RhoAN19-GFP, Rac -GFP, RacN17-GFP, PAK-1, or PAK-DN (a gift from MA del Pozo, National Center for Cardiovascular Research, Madrid, Spain). In some experiments, the target cells were pretreated with vehicle, the ROCK inhibitor Y-27632 (10 µM, Calbiochem) or with the CXCR4 antagonist AMD3100 (10 µM, Sigma-Aldrich) before mixing with the effector cells. For RNAi experiments, cells were co-transfected with a specific RNAi duplex pool for FLNa (Table III) or control (100 nM; SMARTpool, Dharmacon), and with a control RNAi duplex labeled with Cy3 to determine efficiency of transfection by flow cytometry. The silencing was confirmed 48 h post-transfection by immunoblot.

TABLE III Specific RNAi duplex pool for FLNa

3

Fusion was performed by mixing 10 5 cells effectors grown in 100 µg / ml of rifampicin with 2 x 10 5 target cells (6 h, 37 ° C), and the activities of the Luciferase renilla and firefly were measured in cell lysates (25 mM Tris-phosphate, pH 7.8, 1% Triton X-100, 1 mM EDTA, 1 mM DTT, 8 mM MgCl2, 15% glycerol).

Cellular infections with replication-deficient viruses

The lentiviral vector pNL4.3.Luc.R-E-, obtained from Dr. Nathaniel Landau (47) through the NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH, was used for the production of HIV-1 pseudotypes through transfection HEK-293T transient with envelopes VSV-G or an R5 envelope cloned from an isolate of a patient infected with HIV-1 (LMP0608,  GenBank DQ448819). Supernatants were obtained 48 hours later of the transfection, filtered (0.45 µm pore) and stored frozen (-80ºC) until use. Cells were infected Jurkat-GFP-CCR5 48 hours after FLNa-specific RNAi transfection or control to an M.O.I. of 0.1. Infectivity was measured 48 hours after infection with a luciferase assay (Promega) on a Berthold luminometer Sirius (Berthold).

Computational analysis

The complete protein sequences CD4-Human (Uniprot) and FLNa were used as questions against the NCBI databases, using PSI-BLAST to get all the sequences available. The multiple alignments shown are performed with T-COFFEE and analyzed manually.

Molecular models

A structural model of the FLNa domain 10 was generated based on the available structure of domain 17 (not shown). The model was based on the published crystal structure of the FLNa-domain 17-GPIbα complex (PDB code 2BP3) and was obtained using WHATIF (29) and SWISS-MODEL (49). The model was evaluated using PSQS ( http://wwwl.jcsg.org/psqs/ ) and WHATIF.

Statistical analysis

In the CD4 clustering experiments, the images were analyzed blindly to the conditions of treatment. For each experimental condition, they were taken randomly confocal images of 20-30 cells, in at least three independent experiments. The data is analyzed using appropriate statistical tests; the distribution of the size of aggregates of CD4 was analyzed using the test chi 2, while chemotaxis data were compared using the Student t-test. P values that They reached statistical significance are indicated in the text.

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Claims (21)

1. Use of a compound that inhibits the activity of the human FLNa protein characterized by: a) the nucleotide sequence consisting of the nucleotide sequence encoding the domain 10-12 of the human FLNa protein (FLNa D10-12), comprised by SEQ ID NO3, b) nucleic acid molecules whose chain complementary hybrid with the sequence of a), c) nucleotide sequence that differs from the a), due to the degeneracy of the genetic code or any of its combinations, for the preparation of a drug or pharmaceutical composition for the treatment and prevention of infection by HIV-1 and diseases that occur with a altered system stimulation immune.

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2. Use of an inhibitor compound that prevents or decreases the expression of the gene encoding the human FLNa protein characterized by containing at least one interference RNA (iRNA) specific to the FLNa protein mRNA, characterized by the sequences specified below. : SEQ ID NO19, SEQ ID NO20; SEQ ID NO21, SEQ ID NO22, SEQ ID NO23, SEQ ID NO24; SEQ ID NO25 and SEQ ID NO26. 3. Use of a human FLNa protein inhibitor compound characterized by: a) the amino acid sequence consisting of the amino acid sequence of domain 10-12 of the human FLNa protein (FLNa D10-12) (SEQ ID NO4), b) amino acid sequences with an identity at least 99%, 98%, 95%, 90%, or 80% with the sequence SEQ ID NO4.

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4. Nucleotide sequence FLNa / domain characterized because its expression allows the inhibition of FLNa activity and because it belongs to the following group: a) the nucleotide sequence FLNa constituted for domain 10-12 of FLNa, (FLNa D10-12) of SEQ ID NO3, b) nucleic acid molecules whose chain complementary hybrid with the sequence of a), c) nucleotide sequence that differs from the a) due to the degeneracy of the genetic code.

         \ vskip1.000000 \ baselineskip
      

5. FLNa amino acid sequence / domain characterized because it inhibits FLNa activity and because it belongs to the following group: a) the amino acid sequence consisting of the 10-12 domain of FLNa, (FLNa D10-12) of SEQ ID NO4, b) amino acid sequences with an identity of at least 99%, 98%, 95%, 90%, or 80% with the following sequences: SEQ ID NO4.

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6. Gene expression vector characterized in that it comprises a FLNa nucleotide sequence / domain according to claim 4. 7. Cell, either prokaryotic or eukaryotic, transformed or transfected characterized in that it comprises the FLNa / domain nucleotide sequence according to claim 4 or the FLNa / domain expression vector according to claim 6. 8. Use of the cell according to claim 7 for the manufacture of an amino acid sequence according to the claim 5. 9. Use of the eukaryotic cell according to claim 7 for the preparation of a medicament or pharmaceutical composition of gene therapy for treatment and prevention of HIV-1 infection and of diseases that occur with an altered system stimulation immune. 10. Pharmaceutical composition or medicament for the treatment of HIV-1 infection and of diseases that occur with an altered stimulation of the immune system characterized in that it comprises one or more inhibitor compounds according to claims 1 to 5, in therapeutically effective amount together with , optionally, one or more pharmaceutically acceptable adjuvants and / or vehicles. 11. Pharmaceutical composition according to claim 10 characterized in that the inhibitor compound is constituted by one or more sequences belonging to the following group:

to)

nucleotide sequence constituted by the domain coding nucleotide sequence 10-12 of the human FLNa protein (FLNa D10-12), comprised by SEQ ID NO3,

b)

nucleic acid molecules whose chain complementary hybrid with the sequence of a),

C)

nucleotide sequence that differs from a), due to the degeneracy of the genetic code

or any of your combinations

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12. Pharmaceutical composition according to claim 10 wherein the inhibitor compound is a nucleotide sequence that prevents or decreases the expression of the gene encoding the human FLNa protein characterized by containing at least one interference RNA (iRNA) specific to the FLNa protein mRNA , characterized by the sequences specified below: SEQ ID NO19, SEQ ID NO20; SEQ ID NO21, SEQ ID NO22, SEQ ID NO23, SEQ ID NO24; SEQ ID NO25 and SEQ ID NO26. 13. Pharmaceutical composition according to claim 10 characterized in that the inhibitor compound is an expression vector according to claim 6. 14. Pharmaceutical composition according to claim 10 wherein the inhibitor compound contains one of the following elements:

to)

amino acid sequence consisting of the amino acid sequence of the 10-12 domain of the human FLNa protein (FLNa D10-12), (SEQ ID NO4),

b)

amino acid sequences with an identity of at minus 99%, 98%, 95%, 90%, or 80% with SEQ ID NO4.

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15. Use of the pharmaceutical composition according to  claims 10 to 14 in a method of treatment or prophylaxis of a mammal, preferably a human being, affected for an HIV-1 infection or for diseases that they have a consistent impaired immune system stimulation in the administration of said therapeutic composition in doses adequate. 16. Use according to claim 15 characterized in that the disease that occurs with an altered stimulation of the immune system belongs to the following group:

-

acute or chronic inflammatory disorder or an immune disorder, for example an autoimmune disease selected from the group consisting of: rheumatoid arthritis, lupus systemic erythematosus, scleroderma, Sjögren's syndrome, diabetes autoimmune, thyroiditis, and other immune diseases organ-specific, including the psoriasis;

-

neurological type disorder selected from the following group: multiple sclerosis, myasthenia severe, and other neurological immune diseases,

-

gastrointestinal disorder selected from the following group: Crohn's disease, colitis ulcerative, celiac disease, and hepatitis,

-

respiratory type disorder selected from the following group: emphysema, tract infections respiratory,

-

cardiovascular disorder selected from the following group: atherosclerosis, cardiomyopathy, rheumatic fever, endocarditis, vasculitis, and other diseases cardiological of an immune nature,

-

allergic type disorder or a reaction of hypersensitivity (types I, II, III, and IV), including asthma, rhinitis, and other hypersensitivity reactions mediated by immune system;

-

disorder of the rejection type of a transplant or graft, and

-

disorder or condition pertaining to next group: acute lung damage, distress syndrome acute respiratory, arthritis (e.g., CIA), bronchitis, fibrosis cystic, hepatitis, inflammatory bowel disease, damage from reperfusion, nephritis, pancreatitis, arterial occlusion, accident cerebrovascular, systemic lupus erythematosus, damage induced by ultraviolet light, vasculitis and sarcoidosis.

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17. Method for identifying compounds that inhibit FLNa activity characterized in that it is based on the interaction of several proteins with the FLNa protein and that it comprises the following steps:

to)

generation of a biological preparation that mimic the protein-protein interaction that mimic the FLNa interaction with CD4, CXCR4 and CCR5, respectively, or in combination with several of them,

b)

contacting the candidate compound with the preparation of a),

C)

determination of the inhibition of the interaction or not of the FLNa protein with the other proteins of a), and

d)

the identification of an interaction inhibitor of the FLNa protein with the aforementioned proteins when not Look at that interaction.

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18. Identification method according to claim 17, characterized in that the interaction of said proteins, or fragments thereof, is analyzed by a two-hybrid assay, preferably in yeast, of immunoprecipitation between proteins or by analyzing specific biological activities of CD4, CXCR4 and CCR5. 19. Identification method according to claim 17, characterized in that the FLNa-CD4 interaction is analyzed in vivo by cross-immunoprecipitation between both proteins; or by activating CD4 induced by anti-CD4 antibodies and subsequently analyzing the phosphorylation of FLNa in serine residues, or by locating and determining the size of the CD4 cluster in the membrane. 20. Identification method according to claim 17, characterized in that the FLNa-CXCR4 interaction is analyzed in stimulation assays with its ligand, CXCL12, in cells stably expressing a labeled CXCR4 receptor, for example with GFP; or analyzing its action on Ca 2 flows or on the formation of F-actin or its chemotactic activity. 21. Identification method according to claim 17, characterized in that the role of the human FLNa protein in the formation of the HIV-1 synapse is analyzed by the formation of conjugates between effector cells that express the viral envelope protein, for example env IIIB, and target cells expressing labeled CD4 and / or CXCR4, for example with GFP, and where the accumulation of CXCR4 in the contact zone between the target and effector cell is analyzed, as well as the accumulation of FLNa and actin on the contact site.