FR2827046A1 - METHOD FOR SCREENING MOLECULES FOR BINDING TO GP120 PROTEIN FROM IMMUNODEFICIENCY VIRUS - Google Patents

Abstract

The invention relates to a method for screening molecules which can bind to the gp 120 protein of the immunodeficiency virus. The inventive method uses the fluorescene anisotropy technique and the peptide of the following formula (I): TPA- Xaaa- Xaab- Ala or Gln or His - Arg ou Phe - Cys - Xaac- Xaad- Arg - Cys - Lys - Xaae- Xaaf- Xaag- Xaah- Leu ou Lys - Xaai - Lys - Cys - Ala or Gln - Gly or (D)Asp or Ser - Ser or His or Asn - Xaaj- Cys - Thr or Ala - Cys - Xaak-NH2, (I) wherein TPA represents thiopropionic acid, Xaaa, Xaab, Xaac, Xaad, Xaae, Xaaf, Xaag, Xaah, and Xaai are naturals or non natural amino acids which are identical or different, Xaaj represents b-naphtylalanine or phenylalanine or bi-phenylalanine, and Xaak represents Gly or Val or Ile.

Description

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METHOD OF SCREENING MOLECULES FOR BINDING TO
GP120 PROTEIN FROM IMMUNODEFICIENCY VIRUS
DESCRIPTION Technical field
The present invention relates to a method for screening molecules capable of binding to the gp120 protein of the immunodeficiency virus or its analogs. It uses a fluorescence anisotropy technique and a particular family of peptides with high affinity and specificity for the gp120 viral protein.

 There are many immunodeficiency viruses that have a viral envelope protein gp120 or a protein analogous thereto. These include human immunodeficiency virus (HIV), simian immunodeficiency virus (VIS), ovine immunodeficiency virus (VISNA), bovine immunodeficiency virus (VIB) ), and feline immunodeficiency virus (FIV).

 Some of the peptides of the present invention are capable of binding to the gp120 viral protein or its analogs, inhibiting the attachment of the gp120 viral protein to the CD4 receptor, with ICso values between 0, 1 and 400 nM in function. of their sequence.

Some of these molecules are capable of inhibiting T cell infection by the AIDS virus with, for example, ED 50 (effective dose 50%) of 100 to 900 nM. In addition, the binding of these molecules to the recombinant gp120 viral protein induces a variation

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 conformational in it that unmasks new epitopes, with the same efficiency as the soluble CD4 molecule.

 The screening method of the present invention is a powerful research tool for selecting new molecules useful for the manufacture of new anti-AIDS drugs.

 It finds an obvious application in the development of new therapies and new diagnostic means to fight against AIDS.

Prior art
Much research is being conducted to find effective treatments and prevention of infection with the immunodeficiency virus, including human immunodeficiency virus (HIV). This is due in particular to the variability of the virus, the difficulty of understanding the mechanism of its entry into the target cells and the immune response necessary for the protection of the infection by the virus.

 As demonstrated by the analysis of the three-dimensional structure of the CD4-gp120 complex [1] (see appended references), the structural elements of CD4 critical for its binding to the viral glycoprotein gp120 include the amino acids Gly38, Gln40, Gly41, Ser42, Phe43, Thr45, which are included in region 36-47, and arginine-59 of CD4. The 36-47 region of CD4 has been assimilated to the CDR2 region of immunoglobulins and has an ordered orderly structure.

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deuxième brin (C"), correspondant à la séquence 43-47, de former avec le premier une structure ss antiparallèle. Cette structure est stabilisée par des liaisons hydrogène entre l'azote amidique des résidus Gly38, Gln40, Phe43, Thr45, Gly47 du premier brin ss et l'oxygène carbonylique des résidus, respectivement, Thr45, Phe43, Gln40, Gly38, Ile36 du deuxième brin P. hair. This structure is formed of a strand ss (C '), corresponding to the sequence 36-40, followed by a bend corresponding to the sequence 40-43 which allows a

second strand (C "), corresponding to the sequence 43-47, to form with the former an antiparallel structure which is stabilized by hydrogen bonds between the amide nitrogen of residues Gly38, Gln40, Phe43, Thr45, Gly47 of first strand ss and carbonyl oxygen residues, respectively, Thr45, Phe43, Gln40, Gly38, Ile36 of the second strand P.

This hairpin structure plays a central role in the interaction of CD4 with the viral protein gp120 and has a dual function: first, it allows the amino acids Gly38, Gln40, Gly41, Ser42, Phe43 and Thr45 to form a molecular surface complementary to the interaction surface of the viral protein gp120 and, in particular, allows the side chain of Phe43 to insert at the entrance of a hydrophobic cavity into the molecular surface of the viral protein gp120; secondly, it allows a ss-sheet-like interaction between polypeptide backbones of the CD4 strand and the ss15 strand (residues 365-368) of the viral protein gp120.

Arginine 59 (Arg59) also plays a critical role in the CD4-gp120 interaction, since the guanidinium moiety of its side chain forms a double hydrogen bridge with the side chain of the Asp368 residue of the viral protein gp120, allowing the stabilization of the structure-like interaction between CD4 strands and ss15 of the gp120 viral protein.

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 Site-directed mutagenesis experiments demonstrated that all these CD4 residues play a critical role in the interaction with the viral protein gp120 [2], [3].

 The reproduction of this ordered hairpin structure and the conformation of the side chains of its residues is critical in a molecule that must bind to the viral protein gp120 at the site of interaction of CD4. The fact that simple peptide constructs, such as a linear peptide corresponding to the 37-53 sequence and a cyclic peptide corresponding to the 37-46 sequence of CD4, do not have a measurable affinity for the viral protein gp120, is a demonstration of this [ 4].

interagit avec une large dépression de la surface 0 moléculaire (800 A2) de la protéine virale gpl20, en 0 utilisant une large surface moléculaire (742 A2), qui est centrée autour de la région qui a été assimilée à CD4 cell infection by the AIDS virus is mediated by a high affinity interaction between the viral envelope and CD4. Mutagenesis and competition experiments with antibodies made it possible to locate in the D1 domain of CD4, the contact surface with the gp120 protein of the viral envelope. The three-dimensional structure of a functional recombinant form of the gp120 glycoprotein, but deleted from the Vl-V2-V3 loops and the N- and C-terminal regions, in complex with the D1D2 domains of the CD4 and with the Fab fragment of an antibody monoclonal, has recently been resolved by crystallography [1]. In this structure, the CD4

interacts with a large depression of the molecular surface (800 A2) of the viral protein gp120, using a large molecular surface (742 A2), which is centered around the region that has been assimilated to

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 the CDR2 region of immunoglobulins. At the heart of the molecular depression of the viral protein gp120 is a narrow, deep cavity, the "Phe43 cavity", in which the Phe43 side chain at the top of the CDR2 region of CD4 occupies the entrance. The resolution of this structure confirms the previous mutagenesis data, which indicates the Phe43 residue and the entire CDR2 loop as functionally very important, and proposes the latter as a possible target for the inhibition of attachment of HIV-1 virions to cells. The gp120-CD4 interaction allows the attachment of the virus to the target cell membrane and represents the first protein-protein interaction in the mechanism of infection. Nevertheless, other co-receptors are needed for effective entry of the AIDS virus into the cells. This is CXCR-4 [5], [6], which is involved in the entry of lymphotropic viral strains (X4) and CCR-5 [7], [8], which is involved in the entry M-tropic strains (R5) and most primary isolates. Several evidences indicate that the binding of the viral protein gp120 on CD4 would produce conformational changes in the envelope glycoprotein, which would expose new sites, detectable by specific antibodies, called CD4i, and increase the affinity of the envelope. for the chemokine receptors [9], [10], the coreceptors of the input. After fixation of CD4 and co-receptor by the viral protein gp120, other conformational variations would lead to a structural reorganization of gp41, which would lead to

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 the exposure of its fusogenic peptide, then to the fusion of the viral and cellular membranes and finally to the entry of the viral load into the cell.

 The crystallographic structure of the gpl20 viral protein complexed with CD4 has elucidated one of the mechanisms used by the AIDS virus to escape the immune response. It was already known that the gp120 envelope protein had hypervariable and highly glycosylated regions. The crystallographic structure has in fact demonstrated that hypervariable regions and highly glycosylated regions form the exposed molecular surface of viral protein gp120, which also corresponds to the accessible surface of virions. Only a few regions of the molecular surface of the gp120 viral protein are conserved and may be the target of the antibodies. These regions are not normally accessible and are probably protected by the hypervariable loops (VI-V2-V3) which in the crystallized protein have been deleted, and are therefore invisible. One of these regions is a surface near the site of interaction for chemokine receptors, and accessible to CD4i antibodies, after attachment of the CD4 envelope. The CD4 binding site is another exposed and conserved surface: however, this surface is present in a cavity of the gp120 viral protein and is therefore not accessible to the antibodies, but can accommodate CD4 which has a single domain. immunoglobulin unlike antibodies that use two domains for molecular recognition.

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 Inhibition of the interaction of the viral protein gp120 with the main input receptor, CD4, by a soluble recombinant CD4 and / or immunoglobulin chimeras containing CD4 domains, was one of the first suggested and experimental therapeutic approaches for the inhibition of HIV-1 infection. These molecules are effective in vitro in inhibiting viral infection only in the case of laboratory-adapted strains, but are ineffective in many primary isolates. The rapid in vivo degradation of these CD4-derived constructs and their lower affinity for the viral envelope of primary isolates have been proposed as the main causes of their ineffectiveness. However, studies have shown that the affinity for CD4 of recombinant monomeric gp120 from some primary isolates is not significantly different from that of gp120 from laboratory strains. The oligomeric nature of the viral protein gp120 on the surface of the virus, the density and structure of CD4 on the surface of permissive cells and may be the presence of other molecular interactions not yet clearly elucidated or other accessory molecules input could play an important role in the mechanism of entry and represent other causes of the ineffectiveness of soluble CD4 proteins as antagonists of entry. The development of antiviral agents that target the viral protein gp120 presents a major challenge, because of the high genetic variability of the envelope protein,

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 factor that can explain the inefficiency of many inhibitors of the viral protein gp120. Nevertheless, the fact that residues that contribute to forming the core of the gp120 viral protein binding site for CD4 are formed by highly conserved residues suggests that gp120 viral protein inhibitors may be effective against a broad spectrum of genes. HIV-1 isolates.

 Peptide constructs derived from CD4 have been proposed as inhibitors of viral infection: it is a structural mimetic peptide of the CDR2 CD4 loop which incorporates a phenyl group mimicking Phe43 [14] and a cyclic peptide corresponding to the CDR3 loop of CD4 and incorporating additional aromatic residues [15]. However, these constructs have low antiviral activity limited to a laboratory isolate and, although originally designed as structural mimetics of CD4, they are not active in the entry stage [16].

 Currently, a number of gp120-CD4 interaction inhibitors have been proposed and have clinical application. It is for example a large recombinant protein, formed by the genetic fusion of CD4 DID2 domains with the constant domains of an IgG2 immunoglobulin [17].

This PR0542 construct is in the 1/11 clinical phase. This large recombinant protein seems to be well tolerated by the human body, but its effectiveness depends on a high circulating dose difficult to achieve. Smaller molecules have

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oligonucléotide zintevir (AR177) [19], d'un polymère à base de naphtalène sulfonate, PR02000 [20] et d'un dérivé d'amidon, le dextrin 2-sulfate (D2S) [21], qui inhibent des isolats chimiques VIH-1 dans des cultures cellulaires, mais exigent des concentrations élevées. have been proposed as inhibitors of the CD4-gp120 interaction and are in the clinical testing phase. It is: a bis-azo dye FP21399 [18], identified by a combinatorial approach, phosphorothioate

oligonucleotide zintevir (AR177) [19], a naphthalene sulfonate-based polymer, PR02000 [20] and a starch derivative, dextrin 2-sulfate (D2S) [21], which inhibit HIV chemical isolates -1 in cell cultures, but require high concentrations.

Among the inhibitors of entry, there is also SPC3 [22], a synthetic multi-branched peptide construct, which, originally proposed as an inhibitor of the CD4-gp120 interaction, is then found active in a next step the attachment of virions to cells. Other small molecules have been proposed as inhibitors of entry: bicyclam AMD3100 [23], NSC651016 [24], peptide T22 [25] and its more recent version T134 or T140 [26] and TAK779 [27]. These molecules are viral entry inhibitors, active on the coreceptors of the CXCR4 and CCR5 entry, but have no activity in the CD4-gp120 interaction. Peptides derived from the sequence of the C-terminal region of the glycoprotein gp41, peptide T20 (or DP178) [28] and its latest improved version DT1249 [29], are other infection inhibitors that target transient phase of entry, subsequent to the attachment of the viral envelope to CD4 and preceding the fusion of cell virus membranes: these peptides are inactive in the interaction gp120-CD4. Most

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 of these products are currently in phase 1/11 clinical trial, but their therapeutic efficacy remains to be verified.

 The currently used therapeutic anti-AIDS therapeutic regimen, triple therapy, involves a combination of three inhibitors that target two viral enzymes, reverse transcriptase and protease. Although triple therapy has significantly reduced the viral load of many patients, it is not able even in the most favorable cases to eradicate the disease, because dormant reservoirs of infection still persist [30]. The partial success of triple therapy on the one hand and the impossibility of stopping the AIDS infection with the current therapeutic protocols on the other hand, have increased the demand for new drugs that can be active at the level of other viral targets and increase the repertoire and efficacy of current clinical drugs.

 The banks of molecules currently available and potentially active in the inhibition of entry and infection by the AIDS virus are enormous. However, a quick and effective search tool for molecules that are actually active to inhibit the gp120-CD4 interaction necessary for an effective infection with the AIDS virus is lacking.

Current screening methods are slow, inaccurate and require large amounts of reagents. In addition, the costs generated by this research are high.

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Presentation of the invention
The present invention is specifically intended to provide a method of screening for molecules capable of binding with a specific affinity to the gp120 protein of the AIDS virus, including human immunodeficiency virus (HIV).

 The screening method of the present invention includes at least one screening cycle comprising the following steps: a) covalent labeling with a fluorescent probe of a peptide having said affinity determined for the gp120 protein, said peptide comprising the sequence (A) defined below, b) bringing the labeled peptide into contact with the gp120 protein, at a concentration of gp120 which allows between 20 and 50% fixation of the labeled peptide, so as to form a reversible complex labeled peptide-gp120], c) standard measurement of the fluorescence polarization of the labeled peptide-gp120 complex], d) competitive screening test of the labeled peptide-gp120 complex] formed with at least one candidate molecule capable of binding to the gp120 protein at a determined concentration of said molecule (s) and under physicochemical conditions making it possible for competition between said molecule and the labeled peptide to form possible a complex with the gp120 protein, e) measuring the fluorescence polarization emitted by the screening assay, and

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 f) comparing the fluorescence polarization measurement of step e) with the standard measurement of step c) the one or more candidate molecule (s) being considered as a molecule capable of binding to the protein Gp120 when the fluorescence polarization measurement of step e) is less than the standard measurement of step c).

 The molecules sought by the screening method of the present invention are therefore those which compete with the peptide of the present invention.

 Indeed, the method of the present invention is characterized in that it uses a family of particular peptides that mimic CD4 and overcome the aforementioned drawbacks of the prior art. Indeed, it uses a stable peptide, having a very high affinity for the envelope of the AIDS virus, in particular for the gp120 protein.

The peptide of the present invention is characterized in that it comprises the following sequence (A): TPA-P-Cys-P-Cys-P-Cys-Ala or Gln
Gly or (D) Asp or Ser-Ser or His or Asn-Xaaj
Cys-Thr or Ala-Cys-Xaak-NH2 wherein TPA is thiopropionic acid, XaaJ is ss-naphthylalanine, phenylalanine or bi-phenylalanine, Xaak is Gly, Val or Ileu, Pl is 3-6 amino acids , p2 represents 2 to 4 amino acids and p3 represents 6 to 10 amino acids, the amino acids in P1, p2 and p3 being natural or unnatural, identical

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 or different and P1, p2 and p3 having or not a common sequence, said peptide having a hairpin conformation ss whose elbow j3 is formed by the amino acid residues Ala or Gln Gly or DAsp or Ser-Ser or His or Asn -Xaai of its sequence (A).

According to one embodiment of the present invention, the peptide of the present invention is characterized in that it comprises the following sequence (I): TPA-Xaaa-xaab-Ala or Gln or His-Arg or Phe-
1 5
Cys-Xaac Xaad-Cys-Arg-Lys-Xaae-Xaaf-
10
Xaag-Xaah-Leu or Lys Xaai-Lys-Cys-Ala
Or Gln-Gly or (D) Asp or Ser-Ser or His or Asn-
20
XaaJ-Cys-Thr or Ala-Cys-Xaa-NHs,
In which TPA represents thiopropionic acid, Xaaa, Xaab, Xaac, Xaad, Xaae, Xaaf, Xaag, Xaah, and Xaai, are amino acids, natural or unnatural, the same or different, xaaj represents Nal, Phe or Bip , and Xaak represents Gly, Val or Ile.

 The residue (D) Asp represents the optical isomer D of aspartic acid, Nal represents ss-naphthylalanine, and Bip represents bi-phenylalanine.

 The inventors have demonstrated that this peptide defines by the sequence (A), in particular the sequence (I), comprising a thiopropionic acid at the position

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 1 of the sequence, a residue Phe, Nal, or Bip at position 23 of the sequence (Xaaj), and a Gly, Val or Ile residue at position 27 of the sequence (Xaak) has a high affinity and a high binding specificity for the gp120 protein of the viral envelope of the immunodeficiency virus.

 This peptide has only 27 or 28 residues, and has a hairpin structure consisting of two antiparallel strands connected by a bend of four residues. The two antiparallel strands, being at a distance capable of giving intramolecular hydrogen bonding interactions, stabilize the structure.

In particular, the distance between the amido nitrogen and carbonyl oxygen atoms of the peptide bond is 2.5 to 3.6 A. This well-defined and stable structure reproduces critical structural elements of CD4 for its binding to the glycoprotein gp120.

celle des résidus correspondants du CD4, à savoir Gln 40, Ser 42, Phe 43, et Thr 45. En particulier, la chaîne latérale de XaaJ 23 pointe du motif en épingle à cheveux dans une conformation semblable à celle de la The three-dimensional structure of this peptide has been experimentally resolved by nuclear magnetic resonance (NMR). This analysis demonstrates that its three-dimensional organization reproduces the peptide backbone of the hairpin structure of CD4, and the 37-46 region of this peptide can be superimposed on CD4 region 36-37 with a rms deviation of only 1.05. AT. In addition, the side chains of residues Ala or Gln 20, Ser 22, XaaJ 23 (Nal, Phe or Bip), and Thr or Ala have a comparable orientation to

that of the corresponding residues of CD4, namely Gln 40, Ser 42, Phe 43, and Thr 45. In particular, the XaaJ 23 side chain points of the hairpin pattern in a conformation similar to that of the

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 Phe 43 of CD4 to insert at the entrance of a hydrophobic cavity of the viral protein gp120, thus strengthening the link with gp120. Arg and Lys at positions 9 and 18 are topologically equivalent to the Arg59 and Lys35 residues of the CD4 protein.

 TPA and the cysts of the peptide form disulfide bridges which have type 1-3, 2-4 and 3-6 pairing and stabilize the hairpin structure.

 The hairpin structural unit has a surface accessible to the solvent and / or a larger molecule such as a protein, which can promote its interaction with the viral envelope protein.

 The amino acid side chains of the solvent accessible surface of this motif are spatially close and form a continuous molecular surface that mimics the structure of several important residues for the CD4-gp120 linkage.

 The structural platform that contains the structural hairpin pattern also contains other structural regions capable of accommodating additional chemical functionalities in a well-defined spatial position relative to the hairpin structure pattern, thereby enhancing its biological function. binding and / or have labeling groups. For example, a biotin moiety or a fluorescein moiety has been incorporated at the side chain of lysine 11 without causing loss of biological activity of the derivative. The incorporation of these groups allowed

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 the use of these derivatives in interaction tests with the envelope protein, as described below.

k proline lié au résidu Xaak. Un résidu proline ajouté en position 28 stabilise l'extrémité C-terminale et rend moins flexible le brin ss C-terminal du motif structural en épingle à cheveux. According to the invention, the sequence (A), for example the sequence, (I) may further comprise a residue

k proline bound to the Xaak residue. A proline residue added at position 28 stabilizes the C-terminus and makes the C-terminal strand of the hairpin structural pattern less flexible.

 According to the invention, in the sequence (I), Xaai can be Gly.

n 15, ID n 16, ID n 17, ID n 18, ID n 19 et ID n 20 de la liste de séquences annexée. For example, the peptide of the present invention may comprise a sequence selected from ID # 4, ID # 5, ID # 6, ID # 7, ID # 8, ID # 9, ID # 10, ID Noel, ID # 12, ID # 13. ID 14, ID

15, ID 16, ID 17, ID 18, ID 19 and ID 20 of the attached sequence listing.

 The peptide of the present invention can be obtained by solid phase chemical synthesis or genetic recombination. The chemical synthesis can be carried out for example with an automatic peptide synthesizer of the Applied Biosystems type, mod. 433A (trademark). It can be carried out, for example, in Fmoc chemistry which uses the fluoromethyloxycarbonyl group for the temporary protection of the α-amino function of the amino acids.

 The peptide of the invention may also be manufactured by a method comprising the following steps: a) integration of a nucleic acid sequence into an expression vector, said nucleic acid sequence encoding the peptide of the

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 present invention, b) introduction of the expression vector comprising said nucleic acid sequence into a host cell, c) culture of the host cell comprising said nucleic acid under culture conditions for the synthesis of said peptide, d) recovery of the peptide synthesized, and e) grafting TPA in the N-terminal position.

 The technical elements for carrying out this peptide synthesis process are known to those skilled in the art. They are described for example in the book Sombrook, Fritsch and Maniatis, MOLECULAR CLONING, A LABORATORY MANUAL, 2nd edition.

 The grafting of a TPA at the C-terminal position of the peptide may be carried out by means of a conventional organic chemistry method applicable to a peptide.

 The inventors have synthesized a family of peptides that reproduce part of the structure of the CD4 region resembling the CDR2 region of immunoglobulins, which, as demonstrated by mutagenesis and crystallographic analysis, are among the critical regions of CD4 binding to the viral protein gp120. The peptides of the present invention are capable of binding to the region of the glycoprotein gp120 which interacts with CD4, the main receptor for the entry of the AIDS virus, especially HIV-1, into CD4 target cells.

 These peptides constitute a family of inhibitors whose affinity for the viral glycoprotein gp120

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 monomeric recombinant, varies between ICso values ranging from 0.1 nM to 400 nM. They are specific and potent inhibitors of CD4-gp120 interaction based on the structure of CD4. Binding with the recombinant gp120 viral protein competes with soluble CD4 and is specific for the well-structured form.

 In addition, they may be labeled, for example with a fluorescent probe, without the binding affinity to the protein of the viral envelope gp120 being altered.

 These peptides can therefore be used as tracers in the screening method using a fluorescence anisotropy technique according to the present invention.

 This method uses the fluorescence polarization of said peptide covalently labeled with a fluorescent probe.

 The peptide used is that which has an affinity in the range of that of the desired molecules. For example, if one seeks to screen molecules having an affinity of greater than or equal to 10 6 M -1, a labeled peptide having a affinity of 106 M 'will be used. For example also, if one seeks to screen molecules having a higher affinity, for example 109 M 'one will use a peptide having a similar affinity.

 Thus, the screening method of the present invention can be tuned according to the desired affinity by choosing the peptide used.

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 In addition, the affinity for the Gp120 viral protein of the molecules detected by the screening method of the present invention can be determined from that of the peptide according to the invention chosen for the screening.

 The methods and apparatuses usable for carrying out the process of the present invention are those which are accessible to those skilled in the art in the literature relating to the study of molecular interactions, for example in the documents [37], [38], [39], and [40] and in US Pat. No. 5,756,292. Analyzes and simulations can be performed using the BIOEQS program described in documents [41], [42] and [43] or other interaction analysis programs.

 The fluorescent probe should preferably have high quantum efficiency and visible fluorescence for high detection sensitivity.

We have tested rhodamine green, fluorescein and Alexa488 (trademark). Other fluorescent probes with the properties specified above can of course be used.

The fluorescence polarization is calculated from two measurements, the emission intensity of the polarized fluorescence in parallel and perpendicular, with excitation in parallel. Here, the parallel polarization is defined along the z direction of the laboratory axis. The polarization is calculated as follows:

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la relation entre polarisation et anisotropie étant la suivante :

The result can also be expressed in terms of fluorescence anisotropy, calculated as:

the relationship between polarization and anisotropy being as follows:

dans laquelle Ao est l'anisotropie limite du fluorophore, une constante physique déterminée par l'angle entre les dipôles d'absorption et d'émission, T est la durée de vie de fluorescence, et Te est le temps de corrélation rotationnelle. Ce dernier dépend du volume hydraté (Vh) de la molécule ou du complexe moléculaire suivant la relation (5) :

dans laquelle il est la viscosité de la solution, R est la constante de gaz et T, la température en Kelvin. The value of the anisotropy or fluorescence polarization is related to the rotational diffusion rate of the molecule in solution by the following relation (4):

where Ao is the limiting anisotropy of the fluorophore, a physical constant determined by the angle between the absorption and emission dipoles, T is the fluorescence lifetime, and Te is the rotational correlation time. The latter depends on the hydrated volume (Vh) of the molecule or the molecular complex according to relation (5):

in which it is the viscosity of the solution, R is the constant of gas and T, the temperature in Kelvin.

 Thus, when the labeled peptide is alone in solution, its rotational diffusion rate is rapid and therefore the fluorescence anisotropy of the covalently bound fluorophore remains relatively low. On the other hand, when the labeled peptide interacts with gp120, the fluorescence anisotropy of the probe increases considerably since the viral protein gp120 has a molecular weight of 120,000 daltons, and therefore the size of the peptide complex of the present invention gp120 is much higher than that of of the peptide

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 only. So the rotational diffusion rate decreases, and the anisotropy increases.

 In the screening method of the present invention, any molecule capable of inhibiting the interaction between a peptide of the present invention and a viral protein gp120 may represent an inhibitor of the CD4-gp120 interaction, and therefore an inhibitor of the viral infection. It is therefore possible, thanks to the peptides of the present invention to screen in a bank of molecules any molecule capable of interacting with the gp120 protein or a gp120-like protein, in particular molecules with antiviral activity or molecules that are useful for manufacturing of medication.

 The screening method of the present invention operates by competition. The peptide of the present invention, selected and labeled, is brought into the presence of gp120 at a concentration which allows between 20 and 50% fixation. The amount of gp120 should not be saturating, otherwise a large amount of molecules should be used to screen for competition. In addition, the marked peptide-gep201 complex must be reversible to allow competition.

 The concentration conditions of the candidate molecules in the screening assays of the method of the present invention are determined in the same manner as in conventional competition tests for receptor molecules used in molecular biology. The concentration of the candidate molecule (s) is (are) preferably 10 to 100 times greater

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 at the concentration of the labeled peptide of the present invention.

 The process of the present invention is a fast process, especially since it utilizes fluorescence anisotropy techniques, and accurate because it utilizes the peptides of the present invention. In addition, it requires very small amounts of reagents as shown in the examples below which reduces the costs incurred in the search for molecules likely to be used for the manufacture of drugs for the treatment of AIDS compared to the methods of the prior art.

 In addition, the screening method of the present invention is quite suitable for screening libraries of molecules currently available and potentially active in inhibiting the entry and infection of the AIDS virus. It is therefore a tool for a quick and efficient search for molecules that are actually active to inhibit the gp120-CD4 interaction necessary for an effective infection with the AIDS virus.

 For example, according to a first embodiment of the present invention, only one candidate molecule is tested in a screening cycle. In this case, if the fluorescence polarization measurement of step e) is less than the standard measurement of step c), then the candidate molecule tested will be considered as a molecule capable of binding to the viral protein Gp120.

If the fluorescence polarization measurement of step e) is unchanged with respect to the standard measurement of step c), then the candidate molecule tested will not be

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 considered to be able to bind to the Gp120 viral protein. Thus, according to this embodiment, the cycles will have to succeed one another as many times as there are different candidate molecules to be tested, on the basis of one molecule per cycle.

 For example, according to a second embodiment of the present invention, a sample comprising a plurality of different candidate molecules can be tested in a single round of screening. In this case, if the fluorescence polarization measurement of step e) is less than the standard measurement of step c), then the sample will be considered as comprising at least one molecule capable of binding to the viral protein. gpl20. If the fluorescence polarization measurement of step e) is unchanged relative to the standard measurement of step c), then the sample will not be considered as comprising at least one molecule capable of binding to the viral protein Gp120. .

 Thus, according to this second embodiment, a single cycle makes it possible to test a sample comprising several different candidate molecules. If for one of the samples the fluorescence polarization measurement of step e) is lower than the standard measurement of step c), then at least one of the candidate molecules of said sample will be considered as capable of binding to the viral protein. gpl20. The screening can then be refined on the molecules of said sample according to the first embodiment of the present invention to identify the sample molecule (s) capable of binding to the Gp120 viral protein. This second mode of

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e réalisation de la présente invention permet un criblage très rapide de molécules candidates.

The embodiment of the present invention allows for very rapid screening of candidate molecules.

 These first and second embodiments of the present invention may be performed on known candidate molecules currently available and potentially active in inhibiting entry and infection by the AIDS virus.

 According to a third embodiment, the method of the present invention can be applied to unpurified biological fluids such as urine, cell extracts, bacterial, etc.

When the method of the invention makes it possible to consider that such an extract comprises at least one molecule capable of binding to the Gp120 viral protein, this extract can be analyzed by traditional methods of analysis in chemistry to identify the molecules that it contains. A more accurate screening, according to the first embodiment of the present invention, can then be carried out to demonstrate, among all the molecules identified in the extract, the molecule (s) capable of binding to the viral protein Gp120.

 The method of the present invention is therefore a powerful tool for screening, fast, reproducible, sensitive and tunable to a wide range of affinity It is practiced in solution and does not require separating the complexes of free molecules.

 The screening method of the present invention has many advantages that one skilled in the art can easily identify.

 These advantages include the ability of the

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method of being able to select molecules having a predetermined affinity. Indeed, if the conditions of the competition reaction make it possible to keep Kd constant between the labeled peptide of the present invention and the viral protein gp120, the affinity of the molecule selected after the competition step will be at least equal to that marked peptide
Another advantage is that the labeling of the peptide of the present invention can be carried out with different compounds such as Rhodamine green, fluorescein, etc. In addition, the use of compounds such as fluorophores, which absorption and emission properties in the visible may be particularly advantageous for the detection of molecules in unpurified extracts such as urine, cell extracts, bacterial, etc.

 In addition, this method makes it possible to use little viral gp120 protein, as it can take place in the presence of a nonsaturating gp120 concentration, or even preferably at a concentration which allows 20 to 50% fixation.

 In contrast to the Elisa tests commonly performed in laboratories to demonstrate a competition between given molecules, the method of the present invention using the peptides and the anisotropy measurements defined above makes it possible to carry out measurements entirely in solution. In addition, the process of the present invention can be adapted to very small volumes, of a few microliters, and therefore to microwell measurements. It also allows for very fast measurements,

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 one or two minutes for competitions.

 Another advantage of the process of the present invention lies in the reproducibility of the results. Indeed, the anisotropy value for a labeled peptide of the present invention, for example under the conditions defined in the examples below, is always the same.

 Another advantage of the process of the present invention is that the temperature and solution conditions can easily be modified and controlled.

 Yet another advantage is that because the signal-to-noise ratio is very high, the method of the present invention can detect very low levels of fixation.

 Other advantages will become apparent to those skilled in the art upon reading the following examples with reference to the sequence listing and the accompanying figures.

Short description of the sequence listing
The attached sequence listing provides the following peptide sequences: ID sequence n01: sCD4 sequence.

 - ID sequence n02: Scyllatoxin sequence (ScTx).

 - ID sequence n03: CD4M9 sequence: peptide derived from scyllatoxin, not having TPA at position 1 of the sequence.

 sequences ID Nos. 04-16 and 19 and 20: sequences named CD4M9T, CD4M26, CD4M27, CD4M27A, CD4M27B,

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CD4M27C, CD4M30, CD4M31, CD4M32, CD4M33, CD4M35,
K15, K16 and CD4M9BIP and CD4M9V respectively of the present invention.

 sequence ID # 17: a sequence named CD4M3 resulting from scyllatoxin not exhibiting TPA at position 1 of the sequence.

 - ID 18 sequence: sequence named CD4MO from the charybdotoxin.

Brief description of the figures
Figure 1: Inhibition curves of the binding of CD4 to the viral protein gp120, by peptides of the present invention, obtained by ELISA in competition. The peptides tested are: CD4M9, CD4M9T, CD4M27, CD4M32, CD4M33 and CD4M35. Experimental data are reported as percent binding (% F) as a function of peptide concentration.

(séquence ID n03), CD4M33 (séquence ID n 13) et CD4M35 (séquence ID n 14) sont aussi représentées pour comparaison. Les données expérimentales sont reportées en pourcentage de liaison (% F) en fonction de la concentration des peptides. Figure 2: Inhibition curve of the binding of CD4 to the viral protein gp120, by the labeled peptide CD4M33-fluorescein, CD4M33-F, obtained by ELISA in competition. The curves of the CD4M9 peptides

(ID sequence n03), CD4M33 (sequence ID No. 13) and CD4M35 (sequence ID No. 14) are also shown for comparison. Experimental data are reported as percent binding (% F) as a function of peptide concentration.

protéine virale recombinante gpl20-HXB2 avec le peptide CD4M33 (séquence ID n 13) fixé à la surface d'une puce d'un instrument de résonance plasmonique de surface, Les courbes d'association (0-300 s) et de dissociation Figure 3: interaction curves of the

recombinant viral protein gp120-HXB2 with peptide CD4M33 (sequence ID No. 13) attached to the surface of a chip of a surface plasmon resonance instrument, association (0-300 s) and dissociation curves

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 (300-800 s) were recorded after injection of the gp120 viral protein at the concentrations 13.2 (1), 19.8 (2), 29.6 (3), 44.4 (4), 66.6 ( 5) and 100 (6) nM. The data are reported in resonance units (RU) as a function of time (t) in seconds.

 Figure 4 (a) and (b): interaction curve of HIV-1, inactivated by AT-2 [34], with antibody 48d [11] (a) and CG10 [35], (b), fixed on the surface of a chip of a surface plasmon resonance instrument in the presence of the CD4M33 peptide (10 μg / ml), an inactive peptide (Ala23) CD4M9 (Phe23 replaced by Ala in the peptide sequence of CD4M9) ( 10 μg / ml) and in the absence of peptide. The association (0-180 s) and dissociation (180-600 s) curves were recorded after injection of HIV-1, which was preincubated with the peptides at 200C for 1h. The data is reported in resonance unit (RU) as a function of time (t) in seconds.

(séquence ID n010) (figure 4A), CD4M33 (séquence ID n 13) (figure 4B), CD4M35 (séquence ID n 14) (figure 4C) sur la réplication de la souche VIH-1LAI dans les CMSP activées par la PHA-P. Les données sont représentées en pourcentage d'inhibition (% I) de l'activité Transcriptase Inverse (TI) dans les surnageants de culture en fonction de la concentration en nM des peptides. Figures 5 (A) - (C): effects of CD4M30 peptides

(sequence ID No. 10) (FIG. 4A), CD4M33 (sequence ID No. 13) (FIG. 4B), CD4M35 (sequence ID No. 14) (FIG. 4C), on the replication of the HIV-1LAI strain in PBAMs activated by PHA. P. Data are plotted as percent inhibition (% I) of reverse transcriptase (TI) activity in culture supernatants as a function of the nM concentration of the peptides.

 FIGS. 6 (A) and (B): effect of the presence of soluble CD4 and the peptides of the present invention on the interaction of the recombinant viral protein gp120HxB2 with the fixed antibodies CG10 (A) and 48d (B)

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 on the surface of a chip of a surface plasmon resonance instrument. The association (0-180 s) and dissociation (180-400,180-450 s) curves are reported for the gp120 viral protein alone and the gp120 viral protein in the presence of CD4M27 (1.5 equivalents, sequence ID n06). CD4M9 (1.5 equivalents), inactive mutant (Ala23) CD4M9 (1.5 equivalents) and soluble CD4 (1.5 equivalents). The data is reported in resonance unit (RU) as a function of time (t) in seconds.

 Figure 7: Examples of peptide sequences of the present invention shown with the one-letter codes of the amino acid residues. TPA and B represent thio-propionic acid and biphenylalanine respectively and "d" the optical form D of aspartic acid.

 FIGS. 8 a), b) and c): curves illustrating fluorescence anisotropy (An) measurements, expressed in millianisotropy (mA), of the K16 peptide of the present invention labeled with rhodamine green, in solution at a concentration of 2 nM, and put in the presence of viral protein gp120 HXB2 strain at different concentrations [GP120] nanomolar (nM).

 FIGS. 9 and 10: curves illustrating fluorescence anisotropy measurements (An), expressed in millianisotropy (mA), respectively of the CD4M33 and K15 peptides of the present invention labeled with rhodamine green, in solution at a concentration of 2 nM, and placed in the presence of gp120 viral protein of strain HXB2 at different [GP120] nanomolar concentrations (nM).

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 FIG. 11: curve illustrating fluorescence anisotropy (An) measurements, expressed in millianisotropy (mA), of the CD4M9 peptide of the present invention labeled with fluorescein, in solution at a concentration of 6 nM, and put in the presence of protein viral gpl20 strain HXB2 at different concentrations [GP120] nanomolar (nM).

 FIGS. 12a) and 12b): curves illustrating fluorescence anisotropy (An) measurements, expressed in terms of millianisotropy (mA), of the CD4M33 peptide of the present invention labeled with rhodamine green, in solution at a concentration of 6 nM, and brought together with viral protein gp120 or strains HXB2 and SF2 respectively at different concentrations [HXB2] and [SF2] nanomolar (nM).

 FIG. 13: theoretical curves illustrating theoretical values of fluorescence anisotropy (An), expressed in millianisotropy (mA), of a competition between a labeled CD4M33 peptide and an unlabeled CD4M33 peptide, as a function of the nanomolar concentration (nM) of unlabeled CD4M33 peptide, for different nanomolar concentrations (nM) of a viral protein gp120 (HXB2) These curves were calculated from Kd = 14nM of CD4M33 determined by direct titration.

 FIGS. 14 a) to i): curves illustrating fluorescence anisotropy measurements (An), expressed in millianisotropy (mA), of a competition between a fluorescein-labeled CD4M33 peptide and different unlabeled peptides (respectively CD4M33, CD4M35 , CD4M32, CD4, CD4M27, CD4M9, CD4M9TPA, CD4M24 and

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 CD4M9BIP), as a function of the nanomolar concentration (nM) of the unlabeled peptide.

 Examples EXAMPLE 1 SYNTHESIS OF THE PEPTIDES OF THE PRESENT INVENTION

 The peptides of the present invention were made in this example by solid phase chemical synthesis with an Applied Biosystems automatic peptide synthesizer, mod. 433A, and in Fmoc chemistry, which uses the Fluorenylmethyloxycarbonyl (Fmoc) group for the temporary protection of the α-amino function of amino acids.

Protective groups used to prevent side reactions of amino acid side chains in this Fmoc strategy were tert-butyl ether (tBu) for Ser, Thr and Tyr residues; tert-butyl ester (OtBu) for Asp, Glu; trityl (Trt) for Gln, Asn, Cys, His; tert-butyloxycarbonyl (Boc) for Lys and 2,2,5,7,8-pentametylchromane-6-sulfonyl (Pmc) for Arg.

 The coupling reaction proceeds with an excess of 10 equivalents of amino acids (1 mmol) relative to the resin (0.1 mmol). The protected amino acid is dissolved in 1 ml of N-methylpyrollidone (NMP) and 1 ml of a 1M solution of 1-N-hydroxy-7-azabenzotriazole (HOAt) in the NMP solvent. 1 ml of a solution of N, N'-dicyclohexylcarbodiimide (DCC) 1M is then added. After 40 to 50 minutes of activation, the active ester formed is transferred to the reactor which contains

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 resin. Before this transfer and then coupling step, the resin is deprotected from its Fmoc group by a 20% solution of piperidine in NMP. The excess of piperidine is removed by washing with NMP after about 5 to 10 minutes.

 During the deprotection, the detection of dibenzofulvene-piperidine adducts at 305 nm makes it possible to follow the smooth progress of the synthesis. Indeed, the quantification of the adduct makes it possible to estimate the efficiency of the deprotection of the Fmoc group and as a result of the coupling of the last incorporated amino acid. CHEMICAL MODIFICATIONS TO THE PEPTIDES OF THE PRESENT INVENTION.

 A fluorescent probe and a biotin group were coupled on two of the studied peptides of the present invention, CD4M9T (sequence ID No. 4) and CD4M33 (sequence ID No. 13). The incorporation on these two compounds was done on the lysine residue 11, at a face opposite to the binding site of the viral protein gp120. This choice of a lysine has been conditioned by its possibilities of protecting its side chain with a protective group, called orthogonal, such as the group of (1- (4,4-dimethyl-2,6-dioxo-cyclohexylidene) ethyl) (or group Dde). Such a group is in fact stable to the 20% piperidine treatment used for the deprotection of the Fmoc group, but is cleaved specifically by treatment with a 2% hydrazine solution. Once the Dde group was removed, fluorescein and biotin could be coupled.

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INTRODUCTION OF FLUORESCEIN.

peptides K15 (séquence ID n015) ou K16 (séquence ID n 16), respectivement, de la présente invention. Le groupement Dde a donc été utilisé pour la protection de la chaîne latérale de la lysine pendant la synthèse du peptide, et ensuite libéré par deux traitements de 5 min avec 2 % d'hydrazine dans la diméthylformamide (DMF). La molécule de fluorescéine a été couplée directement sur le peptide synthétisé. Le couplage de la sonde a été réalisé à l'aide de l'ester fluorescéine- (5- (6)-carboxylate de Nhydroxysuccinimidyle. Pour cela, à un équivalent de résine sont ajoutés 4 équivalents d'ester activé de fluorescéine en présence de 14 équivalents de diisopropyléthylamine (DIEA). La réaction se déroule dans le solvant NMP pendant une nuit. La déprotection finale est enfin réalisée. The fluorescent group was introduced on the lysine 11 side chain of the peptide of the present invention CD4M33 and lysine 15 or 16 of the present invention.

peptides K15 (sequence ID No. 15) or K16 (sequence ID No. 16), respectively, of the present invention. The Dde group was therefore used for protection of the lysine side chain during peptide synthesis, and then released by two 5 min treatments with 2% hydrazine in dimethylformamide (DMF). The fluorescein molecule was coupled directly to the synthesized peptide. The coupling of the probe was carried out using N-hydroxysuccinimidyl fluorescein- (5- (6) -carboxylate ester.For this, one equivalent of resin are added 4 equivalents of fluorescein activated ester in the presence of 14 equivalents of diisopropylethylamine (DIEA) The reaction is carried out in the NMP solvent overnight, and the final deprotection is finally performed.

INTRODUCTION OF BIOTIN.

 An 8-amino-3, 6-dioctanoic spacer arm was first added to the previously deprotected lysine 11 of the Dde group. The reaction is similar to that described in the previous paragraph. The biotin is then incorporated in the form of an N-hydrosuccinimidyl ester biotinamidocaproate: one equivalent of resin is thus added 4 equivalents of activated ester of biotin in the presence of 14 equivalents of diisopropylethylamine (DIEA). The reaction continues for one night at

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 ambient temperature. The resin is then washed before being treated with the final deprotection solution.

INTRODUCTION OF A THIOL GROUP.

 The CD4M9 peptide is synthesized according to the procedure described above with Lysine at position 11 having as a protective group of the side chain a Dde group. After synthesis of the peptide, the peptide-resin is treated 5 times with a solution of 2% hydrazine in DMF. Coupling of a linker was performed for one hour at room temperature in DMF with 10 equivalents of Fmoc-8-amino-3,6-dioxaoctanoic acid using the HBTU reagent in the presence of diisopropylethylamine. The Fmoc group is then deprotected with 20% piperidine in DMF. The peptide-resin is then treated with 10 equivalents of Traut reagent (2-iminothiolane hydrochloride (Sigma) in the presence of DIEA The peptide is finally released and deprotected as described below.

FINAL DEPROTECTION OF THE RESIN.

 Cleavage of the resin and protecting groups present on the side chains were carried out simultaneously by treatment of the peptide bound to the resin with trifluoroacetic acid (TFA). Before performing the cleavage, the resin was washed several times with dichloromethane (DCM) and finally dried. The reagent used during the cleavage is an acid mixture containing 81.5% of TFA and the phenol (5%), thioanisole (5%), water (5%), ethanedithiol (2.5%) and tri-isopropylsilane ( 1%). The resin was treated with this mixture for three hours with stirring and

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 room temperature, at a rate of 100 ml of solution per gram of resin. The free peptide in solution was recovered by filtration. The peptide was then precipitated and washed cold in diisopropyl ether and then dissolved in 20% acetic acid and lyophilized.

de 2, 0 mg. mil-1. Cette solution a ensuite été ajoutée au goutte-à-goutte, diluée à 0, 2 mg/ml', au tampon de réduction, composé de Tris/HCl 100mM, pH 7,8, et de cystéamine 5 mM. La cystamine (oxydant) 0,5 mM en final, a été ajoutée après 45 minutes de réaction à température ambiante. Le milieu est amené à pH 3,0 après 30 minutes. REPLICATION OF THE PEPTIDES OF THE PRESENT INVENTION BY FORMATION OF DISULFIDE BRIDGES
The peptide recovered after lyophilization, the synthesis crude, is in reduced form, that is, the intrachain disulfide bridges are not formed. The formation of these covalent bonds was performed using the redox cystamine / cysteamine pair. The synthesis crude was taken up in the added water of 0.1% TFA (v / v) and 6M guanidinium chloride to facilitate its dissolution.

2.0 mg. mil-1. This solution was then added dropwise, diluted to 0.2 mg / ml, to the reduction buffer, consisting of 100 mM Tris / HCl, pH 7.8, and 5 mM cysteamine. The final cystamine (oxidant) 0.5 mM was added after 45 minutes of reaction at room temperature. The medium is brought to pH 3.0 after 30 minutes.

 Cysteamine makes it possible to reduce the thiol groups present on the peptide. In the open air, it oxidizes and allows the oxidation of cysteines and thus the folding of the peptide by formation of intrachain disulfide bridges. The cystamine added at the end of the manipulation makes it possible to perfect the folding. The good progress of the oxidation is verified by analytical chromatography by comparing the times of

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 retention of raw and oxidized products, more important for the former.

 The oxidation of the CD4M9 peptide, having a thiol group in position 11, differs somewhat from that described in the paragraph above. The reaction refolding medium, composed of a 20 mM phosphate buffer, 200 mM NaCl, adjusted to pH 7.8, was degassed for a long time with argon and then supplemented with 5 mM cysteamine, 5 mM cytamine. The peptide comprising seven free SH functions is then added and the oxidation reaction is stopped by adding acid after only 10 minutes, sufficient time for folding and formation of the three natural disulfide bridges and sufficiently short to limit product formation. secondary corresponding to higher oxidation states.

PURIFICATION OF THE PEPTIDES OF THE PRESENT INVENTION
The peptides of the present invention were purified by reversed-phase high performance liquid chromatography on a Vydac C18 (Trade Mark) preparative column (1.0 x 25.0 cm). A 0-60% linear gradient of acetonitrile in a 0.1% aqueous trifluoroacetic acid solution was used for 90 minutes. Major peak fractions were analyzed by analytical HPLC; fractions with only one peak were pooled and lyophilized. The product thus obtained was analyzed by mass spectrometry.

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EXAMPLE 2: COMPETITION TESTS BY INDIRECT ELISA

 Inhibition of rgpl20-CD4 interaction was measured by indirect ELISA (Enzyme Linked ImmunoSorbent Assay). 50 ng per well of anti-gp120 antibody, D7324, were immobilized on a 96-well plate (Maxisorb, Nunc) overnight at 4 C. After passivation with bovine serum albumin and three washes with wash buffer (Tris 10). mM, pH 7.8, 0.05% Tween 20), 15 ng of rgpl20HXB2 per well were added. Different concentrations of competitors were then added, after three washes, as well as 0.4 ng of soluble CD4 per well. 100% controls containing only soluble CD4 are carried out. After one night at 40C and three washings, 5 ng of anti-CD4 L120.3 mouse antibody was added per well followed by peroxidase-coupled goat anti-mouse IgG antibody (GAMPO). The revelation was carried out by adding 3,3 ', 5,5'-tetramethylbenzidine, peroxidase fluorescent substrate. The reaction was stopped after 30 minutes by addition of 2M sulfuric acid.

Inhibition of rgpl20-CD4 interaction was calculated by reading absorbance A at 450 nm. Controls without competitor were able to determine the absorbance Aloi% (absorbance in the absence of competitor). The percent inhibition for each peptide concentration of the present invention was calculated by the formula: percent inhibition = 100 x (AlOO% -A) / AlOO%.

The tests were conducted in duplicate and the results expressed as the average of experimental duplicates.

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CI50 présentée par le CD4 soluble (CI50 = 4, 0 x 10-9 M) [4]. The biological activities of the CD4MO peptide (sequence ID No. 18) derived from the charybdotoxin and the CD4M3 peptide (sequence ID No. 17), derived from scyllatoxin (sequence ID No. 2), were tested in an indirect ELISA test. These peptides have the ability to inhibit soluble rgp120LAI-CD4 interaction, with a 50% inhibition concentration (or IC50) of 4.0 x 10-5 M and 2.0 x 10-5 M, respectively ( Table 1) [4]. These values are 10,000 times higher than the

IC 50 presented by soluble CD4 (IC 50 = 4.0 × 10 -9 M) [4].

 The appended FIG. 1 groups together the results obtained in this example with the peptides of the present invention.

 These are representations of the percent fixing (% F) of the viral protein gp120HXB2-CD4 as a function of the molar concentration (C (M)) to the competitor, that is to say to the peptide of the present invention. This figure shows the inhibition of the CD4-gp12 OHXB2 interaction.

 CD4M9 (sequence ID No. 3) was tested in ELISA by competition. It has the ability to inhibit the soluble rgpl20-CD4 interaction with a 50% inhibition concentration (or ICso) of 4.17-7 M (Table I), ie an increase in the inhibition capacity of a factor 50 relative to the peptide of the present invention CD4M3 (sequence ID No. 17). The peptide of the present invention CD4M9, in competition ELISA, is also capable of inhibiting the interaction of CD4 with other recombinant gp120 proteins, derived from T-and M-tropic viruses:

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VIH-lIIB, VIH-lMN, VIH-lBa-L, VIH-1JR-FL, VIH-lw61D, avec une concentration d'inhibition à 50% (CIso) entre 0, 1 et 1, 0 x 10-6 M [4].

HIV-1IIB, HIV-1MN, HIV-1Ba-L, HIV-1JR-FL, HIV-1w61D, with a 50% inhibition concentration (Clso) between 0.1 and 1.0 x 10-6 M [ 4].

This peptide inhibits the soluble gp120HXB2-CD4 interaction at an ICso of 9 x 10 -μM (Figure 1, Table I).

 A CD4M9V mutant, having the substitution of the C-terminal Gly-Pro sequence of CD4M9 by Val, has an ICso of 3.0. 10-7 M in the inhibition of the gp120LAcCD4 interaction (Table 1), which represents a slight increase in inhibitory capacity. The CD4M9Bip mutant has the Phe23Bip substitution with respect to CD4M9; this peptide has an ICso of 1.0- 10-7 M in the inhibition of the gp120LAI-CD4 interaction (Table 1), which represents a further increase of the inhibitory capacity. The CD4M9T mutant (sequence ID No. 4), comprising a thio-propionic acid in substitution of the C-terminal amino acid Cys, has an IC 50 of 3.0. 10-8 M in the inhibition of the gp120LAI-CD4 interaction (Figure 1, the ICso in the gp120HxB2 interaction is 1.1 · 10-s M, Table 1), which represents an increase in the capacity of inhibition by a factor close to 10 with respect to CD4M9.

position N-et C-terminale, respectivement, et ont tous été testés avec la gpl20, issue des virus T tropiques VIH-1HXB2 et VIH-lLAI. The following mutants show a whole thio-propionic residue (TPA) and valine (Val or Ile) in

N-and C-terminal positions, respectively, and have all been tested with gp120, derived from the tropic T viruses HIV-1HXB2 and HIV-1LAI.

The peptide of the present invention CD4M27 (sequence I. D. n06) comprises the Arg5Phe mutations

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 (to remove a non-essential arginine and protect the 6-24 disulfide bridge), Gly27Val and deletion of the C-terminal proline residue (to better stabilize the structure). It shows an increase in the ability to inhibit the CD4-gp12 OHXB2 binding, ICso of 7. 10-9 M, (Fig 1, Table I). The CD4M27 peptides A, B and C are obtained respectively by a Gly21Ser (a), Ser22His (b) and Ser22Asn (c) mutation from the CD4M27 peptide. They exhibit an inhibition capacity comparable to that of the CD4M27 peptide.

la présente invention présente un CIso de 3, 0 nM, dans les tests d'inhibition de l'interaction CD4-gpl20LAI. Le mutant CD4M33 (séquence ID n 13) regroupe les mutations présentes dans les deux peptides précédents, notamment Cys1TPA, Arg5Phe, Phe23Bip, Gly27Val, la délétion d'un résidu en position C-terminale et en plus la mutation Ala4His (pour augmenter la solubilité de la molécule). Ces mutations ont un effet additif et The peptide of the present invention CD4M32 (sequence ID No. 12), compared to CD4M9T, comprises the deletion of the C-terminal proline residue, the Gly27Val mutation and the mutation of Phe23 to biphenylalanine (Bip): the Phe23Bip mutation adds a hydrophobic extension at the side chain of Phe23, to increase the interaction with the hydrophobic cavity of the viral protein gp120. CD4M32 shows an increase in the inhibition of the CD4-gp120HxB2 binding (IC 50 of 10-10 M, Fig. 1). The mutant CD4M30 (sequence ID No. 10), relative to CD4M32, comprises the Arg5Phe mutation (already introduced in CD4M27) and Ala4Gln to increase the solubility of the molecule. This peptide of

the present invention has an ICso of 3.0 nM in the inhibition assays of the CD4-gp120LAI interaction. The mutant CD4M33 (sequence ID No. 13) groups the mutations present in the two previous peptides, in particular Cys1TPA, Arg5Phe, Phe23Bip, Gly27Val, the deletion of a residue in the C-terminal position and in addition the Ala4His mutation (to increase the solubility of the molecule). These mutations have an additive effect and

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 allow the peptide of the present invention CD4M33 to inhibit the CD4-gp120HxB2 and CD4-gp12 OLAI interaction with an IC50 of 1.2 x 10-10 M and 2.5 x 10-10 M (Fig. 1 and Table 1). 1) either an increase in the CD4-gp120 binding capacity by a factor of 1000 relative to CD4M9. An additional peptide, CD4M35 (sequence ID No. 14), was synthesized with, relative to the CD4M33 peptide of the present invention, Val27Ile and Gly21 (D) Asp mutations. These mutations allow the peptide of the present invention to inhibit the CD4-gp120HXB2 interaction with an ICso of 7.0 10-11 M (Fig 1, Table 1).

 Two other peptides K15 (sequence ID No. 15) and K16 (sequence ID No. 16) were synthesized: they contain the substitutions Gln7Val, Leu8Gln, LysllHis, with respect to the CD4M33 peptide of the present invention, and a Lys residue at position 15 or 16 , respectively. A fluorescein fluorescent group was then covalently attached to the side chain of this lysine residue, according to the protocol described in Example 1. The fluorescent peptides K15 and K16 have an ICgo of 5.0. . 10-8 M and 6.0. 10-9 M in the inhibition of the gp120LAI-CD4 interaction, whose affinity for the viral gp120 protein is not modified by labeling.

 In conclusion, the present invention provides a family of peptides that represent potent inhibitors of the CD4-gp120 interaction.

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soluble pour la protéine de l'enveloppe virale gpl20LAI et gpl20HxB2. L'écart type pour chaque valeur est inférieur à 30%.

Table 1
50% inhibition concentration (IC 50) of CD4 mimetic peptides in the interaction of recombinant CD4

soluble for the viral envelope protein gpl20LAI and gp120HxB2. The standard deviation for each value is less than 30%.

<Tb>
<Tb>

Name <SEP> Sequence <SEP> IDn <SEP> IC50 <SEP> (gp120LAI) <SEP> IC50 <SEP> (gp120HXB2)
<tb> CD4MO <SEP> 18 <SEP> 40 <SEP> IIM
<tb> CD4M3 <SEP> 17 <SEP> 20 <SEP> pM
<tb> CD4M9 <SEP> 3 <SEP> 400 <SEP> nM <SEP> 90 <SEP> nM
<tb> CD4M9V-300nM
<tb> CD4M9T <SEP> 4 <SEP> 30nM <SEP> 11 <SEP> nM
<tb> CD4M9Bip <SEP> - <SEP> 100 <SEP> nM
<tb> CD4M27 <SEP> 6 <SEP> 10 <SEP> nM <SEP> 7 <SEP> nM
<tb> CD4M30 <SEP> 10 <SEP> 3, <SEP> 0 <SEP> nM
<tb> CD4M32 <SEP> 12 <SEP> 2, <SEP> OnM <SEP> SOOpM
<tb> CD4M33 <SEP> 13 <SEP> 250 <SEP> pM <SEP> 120 <SEP> pM
<tb> CD4M35 <SEP> 14-70pM
<tb> K15FR <SEP> 15 <SEP> 50nM
<tb> K16FR <SEP> 16 <SEP> 6 <SEP> nM
<Tb>
Example 3 Surface Plasmon Resonance Experiments
Surface plasmon resonance experiments were performed with a Biacore 2000 system (Biacore, Uppsala, Sweden). The peptides to be tested were coupled to biotin (as previously described) and then immobilized on a chip on which streptavidin was previously fixed.

comme suit : la surface de la puce a été d'abord activée par l'injection de 50 ilL d'agent de couplage prévu par le constructeur et pouvant former des liaisons amidiques : N-éthyl-N'- (diméthylaminopropyl) Streptavidin was immobilized on the chip

as follows: the surface of the chip was first activated by the injection of 50 μL coupling agent provided by the manufacturer and can form amidic bonds: N-ethyl-N'- (dimethylaminopropyl)

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5 jlL. min', suivis d'une neutralisation des groupements carboxyliques activés par 2 x 20 p, L d'éthanolamine 1, 0 M, pH 8, 5. Les molécules biotinylées ont été ensuite injectées (10 jj. L. min' dans un tampon Hepes 10 mM, NaCl 0,3 M, pH 7,4) sur trois des quatre pistes de la puce. Sur la première piste, le CD4M9 (10-7 M) a été immobilisé jusqu'à une valeur RU (unité de résonance) 100 ; la deuxième piste a été laissée vierge (témoin) ; la troisième piste a été recouverte avec du CD4 soluble (10-7 M) jusqu'à une valeur de 450 RU ; sur la quatrième piste, le CD4M33 (10-7 M) a été immobilisé à une valeur de 100 RU. Pour chaque analyse, plusieurs concentrations de différentes gpl20 (souches HXB2, BAL, JRFL) ont été injectées, à une vitesse de 50 L.min-1 à 25 C, pour un temps d'association de 4 min. La puce est ensuite rincée avec un tampon de course, 10 mM Hepes, 150 mM NaCl, 3,4 mM EDTA et 0,05 % P20, pH 7,4, pour analyser la phase de dissociation. Après chaque expérience, la puce est régénérée avec 25 go d'acide formique 1,0 M. Les constantes de dissociation sont calculées à partir des constantes cinétiques déterminées par le logiciel Bia-evaluation 3,0. carbodiimide (EDC) / N-hydroxysuccinimide (NHS), 50/50; then, streptavidin gel, 0.2 mg. mL -1 in 10 mM sodium acetate, pH 4.5, were injected

5 jLL. min ', followed by neutralization of the activated carboxylic groups with 2 x 20 μL of 1.0 M ethanolamine, pH 8, 5. The biotinylated molecules were then injected (10 μL / min) into a buffer. 10 mM Hepes, 0.3 M NaCl, pH 7.4) on three of the four tracks of the chip. On the first track, the CD4M9 (10-7 M) was immobilized to a value RU (resonance unit) 100; the second track was left blank (witness); the third lane was covered with soluble CD4 (10-7 M) up to 450 RU; on the fourth track, CD4M33 (10-7 M) was immobilized at 100 RU. For each analysis, several concentrations of different gp120 (strains HXB2, BAL, JRFL) were injected at a speed of 50 L.min-1 at 25 C, for an association time of 4 min. The chip is then rinsed with running buffer, 10 mM Hepes, 150 mM NaCl, 3.4 mM EDTA and 0.05% P20, pH 7.4, to analyze the dissociation phase. After each experiment, the chip is regenerated with 25 g of 1.0 M formic acid. The dissociation constants are calculated from the kinetic constants determined by the Bia-evaluation software 3.0.

Example 4: Antiviral activity of CD4 mimetics.

 The manipulations of the infectious material were carried out in a L3 high security laboratory. In order to be the closest to pathophysiological conditions 1 the study was conducted with the help of

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 primary cultures of human peripheral blood mononuclear cells (PBMCs). In all experiments, the effects of the new molecules were compared to those of AZT.

ISOLATION, CULTURE AND ACTIVATION OF CELLS Culture media
Medium A is composed of RPMI 1640 cell culture medium (Life Technologies) supplemented with 10% fetal calf serum (FCS) decomplemented by heat at +56 ° C for 30 min, 2 mM L-Glutamine. (Roche Product), and a 100 μg / ml solution of three antibiotics (penicillin, streptomycin, neomycin, PSN, Life Technologies). Medium B consists of medium A supplemented with 20 IU / ml of recombinant human IL-2 (Roche Product).

Isolation and activation of PBMCs
The PBMCs are separated from the other elements of the blood by ficoll gradient centrifugation (MSL 2000, Eurobio): 30 ml of blood, a healthy donor, diluted 1/3 are deposited on a 20 ml cushion of ficoll. After 20 min of centrifugation at 850 g, the ring of PBMC is removed and then washed twice with RPMI 1640, after 10 min of centrifugation at 750 g and 5 min at 400 g. The PBMC are then activated for 48 hours with 1 μg / ml of phytohemagglutinin-P (PHA-P, Difco Laboratories). The PBMCs are cultured at +37 ° C., in a saturated humidity atmosphere, at 5% CO 2. At the end of the 48 hours of mitogenic activation, they are cultured in medium B. Throughout the culture, the culture supernatants are removed, and the culture media are renewed every three months.

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 or four days. At each renewal of the culture media, the cell viability is evaluated by microscopic observation.

EVALUATION OF THE ANTIRETROVIRAL ACTIVITY OF MOLECULES
The compounds of the present invention CD4M30, CD4M33 and CD4M35 were solubilized in sterile ppi water (Aguettant), aliquoted and then stored at -20 C. The SAH-CD4 compound (soluble CD4 coupled to human serum albumin supplied by Aventis (Vitry-sur-Seine) was stored at -80 ° C. until use and the solutions and dilutions were then carried out extemporaneously in medium A. The PBMCs were pre-treated with the compounds for 1 hour and then infected with the HIV-1LAI lymphocyte tropism reference isolate or HIV-1LEN clinical isolate.

ombilical (CMSO) activées préalablement par 1 ug/ml de PHA-P et cultivées dans du milieu B. Afin d'éliminer les facteurs solubles tels que les cytokines, les surnageants de culture ont été ultracentrifugés à 360 000 g pendant 5 min, et les culots ont été resuspendus dans du RPMI 1640. Le stock viral ainsi constitué a été ensuite titré à l'aide de CMSP activées par la PHA-P. La TCIDso (50% Tissue Culture Infectious Dose) a été calculée en utilisant la formule de Kärber. The biological characteristics of this isolate are: rapid / high, scyncitia inducing (SI), X4: it is therefore preferentially able to infect lymphocytes. The viral stock was constituted by amplifying this strain in vitro using blood mononuclear cells.

umbilical (CMSO) previously activated with 1 μg / ml of PHA-P and cultured in medium B. In order to eliminate soluble factors such as cytokines, the culture supernatants were ultracentrifuged at 360,000 g for 5 min, and the pellets were resuspended in RPMI 1640. The viral stock thus constituted was then titrated using PHA-P activated PBMCs. TCIDso (50% Tissue Culture Infectious Dose) was calculated using Kärber's formula.

The PBMCs were infected with different viral doses 10-100 TCIDso of the HIV-1LAI strain and by 50

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 TCIDgo of the strain HIV-1LEN (multiplicity of infection m.o.i. = 0.001).

ASSAY OF VIRAL REPLICATION IN CULTIVATION SURNAGEANTS.

 Viral replication was measured at day 7 of the culture by assaying the reverse transcriptase activity in the culture supernatants using the RetroSys (registered trademark) assay kit according to Innovagen's recommendations.

utilisant le logiciel"Dose-effects analysis with microcomputers"mis au point par J. Chou & T. C. Chou. EXEMPLE 5 : PROTOCOLE DE PRODUCTION DE LA PROTEINE VIRALE GP120 RECOMBINANTE. Analysis of the results and determination of the effector doses 50%
The effector doses 50% (DEso) are calculated in

using the software "Dose-effects analysis with microcomputers" developed by J. Chou & TC Chou. EXAMPLE 5 PROTOCOL FOR THE PRODUCTION OF THE RECOMBINANT GP120 VIRAL PROTEIN

gpl20). Le fragment BamHI/PstI ainsi obtenu a été cloné dans le vecteur Bluescript (pBS, Stratagene), conduisant au plasmide pBSm1. La séquence codant l'extrémité N-terminale de la protéine virale gpl20 (Tl à Gll), ainsi que celle codant le peptide signal de l'ecdystéroide glycosyltransférase du baculovirus The fragment coding for the viral protein gp120 (amino acid V12 to R481) was amplified by PCR in the plasmid HIVIIIB / HXB2R, using two primers making it possible to generate a fragment containing the BamHI sites (upstream of the KpnI site of the viral protein gp120) and PstI (downstream of the stop codon added to the end of the viral protein sequence

gpl20). The BamHI / PstI fragment thus obtained was cloned into the Bluescript vector (pBS, Stratagene), leading to plasmid pBSm1. The sequence coding for the N-terminal end of the viral protein gp120 (T1 to G11), as well as that coding for the signal peptide of the baculovirus ecdysteroid glycosyltransferase

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 of Autographa californica, were inserted between the BamHI and KpnI sites of pBSml, leading to pBSm2.

 The BamHI-PstI fragment of pBSm2 was then inserted between the BglII-PstI sites of the p10 baculovirus pll9P transfer vector. Sf9 insect cells were cotransfected with the purified viral DNA of the AcSLP10-modified baculovirus and the recombinant p1P9 gp120 vector DNA. The recombinant viruses are purified by a standard method.

 The Sf9 scla cells (line adapted to growth without serum, deposited in the collection of the Institut Pasteur) are maintained spinner in medium without serum. These cells (5.times.10.sup.5 cells / ml) are then infected with the recombinant viruses at a multiplicity of infection of 1 PFU (unit forming the range) per cell and incubated at 28 C. After six days of infection, the cells are centrifuged. (500 g), and the wild-type gp120 viral protein is concentrated and directly purified from the culture supernatant by affinity chromatography on a Sepharose-bromo-acetylated column coupled to the anti-gp120 antibody D7324.

EXAMPLE 6 FIXATION OF THE VIRAL ENVELOPE

 In order to better characterize its biological activity, the peptide of the present invention CD4M33 (sequence ID No. 13) was labeled with biotin and with the fluorescein fluorescent probe as described in Example 1, at the level of Lys-11 positioned on the opposite surface of the active surface of the peptide of the

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 present invention CD4M33. Its activity remains unchanged in ELISA tests (Figure 2).

du pourcentage de fixation (% F) de peptides de la présente invention à la protéine virale gp120HxB2 en fonction de la concentration molaire de ces peptides (C (M)). Figure 2 summarizes the results obtained in this example. These are curves showing the evolution

the percentage of binding (% F) of the peptides of the present invention to the viral protein gp120HxB2 as a function of the molar concentration of these peptides (C (M)).

 These results show that the CD4M33 peptide can incorporate a labeling group without its biological activity being impaired.

 In order to obtain a first evaluation of the affinity of the peptide of the present invention CD4M33 for the viral protein gp120, an analysis of biospecific interactions (described in Example 3), based on the surface plasmon resonance detection, was carried out. been used. In this technique, the biotinylated CD4M33 peptide of the present invention (prepared as in Example 1) was specifically attached to the carboxylated dextran matrix of a chip that had been pre-grafted with streptavidin. Solutions of different recombinant proteins gp120 (gp120HxB2, gp120BAL and gp120JRFL) were then injected onto this matrix and a signal indicating a specific association and high affinity was then detected. Figure 3 shows the evolution of the plasmon resonance signal as a function of time, after interaction of the gp120HxB2 protein (at different concentrations) with the CD4M33 peptide of the present invention.

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virale gp120BAL et de 8. 5. 10-9 M pour la protéine virale gp12 OJRFL'Ces valeurs sont comparables à la valeur de KD=1, 9xlO-8 M, reportée dans la littérature [34] pour l'interaction CD4-gpl20. La même technique basée sur la détection par résonance plasmonique de surface a été aussi utilisée pour vérifier si le peptide de la présente invention CD4M33 pouvait fixer l'enveloppe virale dans sa forme native. Pour ce faire, une suspension de particules virales inactivées par l'adrithiol-2 [35] a été injectée sur la même puce, greffé avec les anticorps 48d et CG10. Un signal d'interaction spécifique et de haute affinité a été alors clairement détecté, dans le cas où la suspension était incubée avec le peptide de la présente invention CD4M33, mais pas dans le cas où la suspension virale était incubée avec le peptide CD4M9, beaucoup moins actif, ou en absence du peptide (figure 4). After nonlinear regression of the association and dissociation curves obtained, a dissociation constant KD of 2.4 is obtained. 10-9 M for the viral protein gp120HxB2, 7.4. 10-9 M for protein

viral gp120BAL and 8. 5. 10-9 M for the viral protein gp12 OJRFL'Ces values are comparable to the value of KD = 1, 9x10-8 M, reported in the literature [34] for the CD4-gp120 interaction . The same technique based on surface plasmon resonance detection was also used to verify whether the peptide of the present invention CD4M33 could bind the viral envelope in its native form. To do this, a suspension of virus particles inactivated by adrithiol-2 [35] was injected on the same chip, grafted with antibodies 48d and CG10. A specific interaction and high affinity signal was then clearly detected, in the case where the suspension was incubated with the peptide of the present invention CD4M33, but not in the case where the viral suspension was incubated with the CD4M9 peptide, many less active, or in the absence of the peptide (Figure 4).

 Figure 4 shows that the presence of the CD4M33 peptide is essential for the interaction of the viral envelope with the virus envelope-specific antibodies 48d and CG10, and that HIV-1 does not bind to antibodies in its absence. This is an indirect demonstration of the binding of the CD4M33 peptide to the viral envelope of HIV-1.

 These experiments demonstrate that the peptide of the present invention labeled or not labeled CD4M33 has an ability to bind the HIV viral envelope both in its

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 recombinant form isolated and purified only in its native form present on the surface of the virus.

EXAMPLE 7: INHIBITION OF INFECTION BY AIDS VIRUS
In order to evaluate the antiviral capacity of the peptides of the present invention, the inventors conducted experiments, by the HIV-1LAI virus and by the HIV-1Lm chemical isolate, of primary cultures of peripheral blood mononucleosis cells ( CMSP) human. In all experiments, the effects of the new molecules were compared to those of AZT.

The HIV-1LAI virus was added at different viral doses (10-100 TCID50, Table 2) in the presence of varying concentrations of CD4M30, CD4M33, CD4M35 and SAH-CD4. HIV-1LEN virus was added to TCID 50 (Table 4) in the presence of CD4M33. Toxicity tests of CD4 mimetics were also performed on the same cells.

A. TOXICITY OF CD4 MIMETICS. At the doses tested, none of the compounds, AZT, SAH-CD4 or antiCD4 mimetics, decreased the viability of PHA-P-activated PBMCs.

B. ANTI-HIV-LIABILITY ACTIVITY OF CD4 MIMETICS. b. 1. Replication of the HIV-1LAI strain in PBMCs. The HIV-1LAI strain replicates at high level in PHA-P-activated PBMCs. The peak of viral replication is at day 7 of the culture, and the effects of the peptides

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V1H-1uI dans les CMSP. L'AZT inhibe fortement la réplication de la souche VIH-lAI dans les CMSP activées par la PHA-P (Tableau 2). La DEgo est égale à 3-14 nM. b. 3. Effets du composé SAH-CD4 sur la réplication de la soucheVIH-lLAidanslesCMSP. L'activité antirétrovirale du composé SAH-CD4 vis-à-vis des CMSP activées par la PHA-P et infectées par la souche V1H-1LAI se traduit par une inhibition de 8515% de la réplication virale à la concentration de 2, 5 uM. b. 4. Effets des mimétiques CD4 sur la réplication de la souche VIH-ILAI dans les CMSP. Les peptides de la présente invention CD4M30, CD4M33 et CD4M35 ont démontré une activité anti-rétrovirale (Tableau 3) dans les cultures de CMSP activées par la PHA-P et infectées par la souche V1H-1LAI'Le composé CD4M33 est le plus antiviral des trois aux différentes doses virales testées. Son activité est plus faible de celle de l'AZT : DEso = 100-500 nM vs. AZT : DE50 = 3 nM (tableau 3 ci-dessous), mais elle est supérieure d'au moins 1 logarithme de base 10 à celle du dérivé du récepteur CD4, SAH-CD4. CD4M30, CD4M33 and CD4M35 were quantified at this post-infection time. b. 2. Effects of AZT on the replication of the strain

V1H-1uI in the PBMCs. AZT strongly inhibits the replication of the HIV-1AI strain in PHA-P-activated PBMCs (Table 2). The DEgo is equal to 3-14 nM. b. 3. Effects of the SAH-CD4 Compound on the Replication of the HIV-ILI Strain in the CMSP. The antiretroviral activity of the SAH-CD4 compound with PHA-P activated PBMCs infected with the strain V1H-1LAI results in an 8515% inhibition of virus replication at a concentration of 2.5 μM. . b. 4. Effects of CD4 mimetics on the replication of the HIV-ILAI strain in PBMCs. The peptides of the present invention CD4M30, CD4M33 and CD4M35 demonstrated anti-retroviral activity (Table 3) in PHA-P activated PBMC cultures and infected with the V1H-1LAI strain. The CD4M33 compound is the most antiviral of the three at the different viral doses tested. Its activity is lower than that of AZT: DEso = 100-500 nM vs. AZT: DE50 = 3 nM (Table 3 below), but is at least 1 log base 10 greater than that of the CD4, SAH-CD4 receptor derivative.

 FIGS. 5A-C combine the results obtained: percentage inhibition (% I) as a function of the peptide concentration in nM.

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réplication de la souche VIH-1LEN dans les CMSP activées . N dans les CMSP activées par la PHA-P et infectées par l'isolat VIH-1LEN, avec une DEso égale à 2,2 nM (tableau 4). Le degré d'inhibition est identique à celui observé vis-à-vis de la souche de référence à tropisme lymphocytaire VIH- PILAI- c2. Effets du mimétique CD4M33 sur la réplication de l'isolat clinique VIH-1LEN dans les CMSP. L'activité rétrovirale du mimétique CD4M33 vis-à-vis des CMSP activées par la PHA-P et infectées par l'isolat VIH-1LEN se traduit par une diminution dose dépendante de la réplication virale et une DEso égale à 367 nM (tableau 4). Le mimétique CD4M33 conserve donc une activité anti-VIH-1 significative, même si elle s'avère légèrement plus faible par rapport à celle observée avec la souche VIH-1LAI. C. ANTIRETROVIRAL ACTIVITY OF THE CD4M33 COMPOUND WITH RESPECT TO CLINICAL ISOLATE HIV-1NI cl. Effects of AZT on replication of HIV-1LEN isolate in PBMCs. AZT strongly inhibits

replication of the HIV-1LEN strain in activated PBMCs. N in the PHA-P-activated PBMCs infected with the HIV-1LEN isolate, with a DEso of 2.2 nM (Table 4). The degree of inhibition is identical to that observed vis-à-vis the HIV-PILAI-c2 lymphocytic tropism reference strain. Effects of the CD4M33 mimetic on the replication of the HIV-1LEN clinical isolate in PBMCs. The retroviral activity of the CD4M33 mimetic vis-à-vis the PHA-P-activated PBMCs infected with the HIV-1LEN isolate results in a dose dependent decrease in viral replication and an ED 50 equal to 367 nM (Table 4). ). The CD4M33 mimetic therefore retains a significant anti-HIV-1 activity, even if it is slightly lower compared to that observed with the HIV-1LAI strain.

 These experiments demonstrate that these peptides of the present invention are capable of inhibiting cell infection, even at high viral doses, with ED 50 values of 900-35 nM (Table 3 below).

The peptide of the present invention CD4M33 is the most antiviral and its DEso is about 100 nM for standard viral doses of infection (Fig. 4a-c, Table 3 below).

 In addition, the CD4M33 peptide has antiretroviral activity greater than 1 log base 10 as compared to another

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 CD4 receptor, SAH-CD4, and above all showed significant anti-HIV activity against a clinical isolate (Table 4).

 In conclusion, the peptide of the present invention CD4M33, which is capable of binding to the recombinant gp120 viral protein with a Kd of 2.4-8. 0 nM (surface plasmon resonance experiments) and to inhibit the interaction between the CD4-gp120 recombinant proteins in ELISA, with an ICso of 120-250 μM, also has the capacity to inhibit this interaction in primary cultures of human cells, to inhibit the initial stage of entry and thus to block the infection itself of a clinical HIV-1 isolate.

Table 2 Effect of AZT on replication of HIV-LLAI strain in PHA-P-activated PBMCs. The results are expressed as percentages of inhibition + standard deviation:

<Tb>
<tb> AZT <SEP> (nM) <SEP>% <SEP> Inhibition
<tb> 139 + 10%
<tb> 10 <SEP> 59 + 28%
<tb> 100 <SEP> 87 + 18%
<tb> 100 <SEP> 100 <SEP> ~ <SEP> 0%
<tb> 10 <SEP> 000 <SEP> 100 <SEP>: <SEP>! <SEP>: <SEP> 0%
<Tb>

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Table 3 Effective doses 50% (DEso) of the peptides of the present invention and AZT in human phytohemagglutinin-P (PHA-P) -infected and infected cell cultures of peripheral blood mononuclease cells (PBMCs) HIV-LLAI at different infectious doses (TCIDso): 50% Tissue Culture Infectious
Dose):

<Tb>
<tb> DEgo <SEP> (nM)
<tb> Sequences <SEP> 100 <SEP> TCIDso <SEP> 50 <SEP> TCIDso <SEP> 10 <SEP> TCIDso
<tb> CD4M30 <SEP> 894 <SEP> 235 <SEP> 253
<tb> CD4M33 <SEP> 534 <SEP> 133 <SEP> 118
<tb> CD4M35 <SEP> 154 <SEP> 428 <SEP> 266
<tb> AZT <SEP> 14 <SEP> 3 <SEP> 5
<Tb>

Table 4
Effect of AZT and CD4M33 mimetic on the replication of the HIV-1Lm clinical isolate in human phytohemagglutinin-P (PHA-P) activated peripheral blood mononuclease cell cultures (PBMCs). The cells were infected with 50 TCID50 of virus:

<Tb>
<tb> HIV-1LEN <SEP> AZT <SEP> CD4M33
<tb> DEso <SEP> (nM) <SEP> 2, <SEP> 2 <SEP> 367
<tb> DE70 <SEP> (nM) <SEP> 4.8 <SEQ> 542
<tb> DEgo <SEP> (nM) <SEP> 16.5 <SEP> 1006
<Tb>

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 EXAMPLE 8: EXPOSURE OF ANTIGENIC SITES OF THE ENVELOPE

 In order to evaluate the ability of the peptide of the present invention CD4M33 to induce conformational changes in the gp120 protein, characterized by the exposure of epitopes sensitive to neutralizing antibodies, the interaction of such human antibodies 48d and CG10 with the HIV envelope in the presence of CD4M33 and, by comparison, recombinant CD4, was undertaken using the surface plasmon resonance technique. The 48d and CG10 antibodies were covalently fixed on the surface of the chips (bio-chip), then gp120 solutions, in the absence of CD4 and in the presence of an excess of CD4 and CD4M33 were injected. When CD4M33 is added to the viral protein gp12 OHXB2, with a molar excess of 1.5 and 20 fold, the viral protein shows a strong interaction with the antibodies (Figures 6a-b); on the other hand, in its absence (and in the absence of soluble CD4) the envelope protein interacts with the antibodies very weakly (Fig. 6a-b). In addition, the effect of the addition of CD4M33 on increasing the interaction affinity of the viral gp120 protein for antibodies is comparable to that obtained by adding equivalent amounts of soluble CD4.

 The inventors also conducted similar plasmon resonance experiments directly using HIV-1 strain HXB2. FIG. 4 shows the results obtained in these experiments and demonstrates the evolution of the plasmon resonance signal as a function of time after interaction of a suspension of

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 viral particles HIV-1HXB2 with a chip having the immobilized antibodies 48d and CG10: HIV-1 interacts with the antibodies only after incubation with the CD4M33 peptide of the present invention and, in contrast, in its absence or in the presence of the peptide [Ala23 Inactive CD4M9, the interaction is practically nil.

 The surface plasmon resonance technique has thus made it possible to demonstrate that the binding of CD4M33 to the gp120 envelope protein is capable of inducing a modification of the viral protein, which consequently preferentially exposes certain molecular surfaces to Neutralizing antibodies (17b, GC10), isolated from individuals affected by HIV-1 [11], [12], [13].

 The exposure of these cryptic epitopes, induced by the CD4M33 peptide of the present invention, is effective both in the envelope protein in recombinant form and in the envelope of virions. Moreover, in view of recent experiments which show that the CD4-complexed gp120 protein can be a molecular form capable of inducing the formation of neutralizing antibodies [31, 32, 33], the CD4M33-gp120 complex therefore represents a very interesting candidate. as an immunogen for the induction of neutralizing antibodies, especially in a vaccine application.

 CD4M33 has the property of unmasking epitopes of the viral protein gp120, as does soluble CD4, with the advantage that it will not be able to induce an immune response against CD4 and that,

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 moreover, because of its small size, it will allow an even more favorable access to unmasked epitopes of the viral protein gp120.

EXAMPLE 9 SCREENING A) MATERIAL AND METHODS

décrit dans les documents référencés [41], [42] et [43], ainsi que dans le brevet US 5, 756, 292. EQUIPMENT
The equipment used for the measurements is the one

described in referenced documents [41], [42] and [43], as well as in US Pat. No. 5,756,292.

PEPTIDES
Peptides of the present invention, labeled or non-labeled, in the form of 50 μg lyophilized (powder) were solubilized by adding in an Eppendorf (trademark) containing the powder, 50 μl of potassium phosphate buffer, 10 mM , 100 mM KCl, pH 7.0 to obtain a solution concentrated at between 300 and
750 μM.

 Labeling is done with fluorescein (F) or with rhodamine green (R).

 Two other solutions, 1: 10 and 1: 100 dilutions of the above solutions, were prepared with the same buffer.

 These labeled peptides are used first in Gp120 binding experiments to determine their affinity and then are used as tracers in competitive screening experiments of the interaction of these peptides with the gp120 viral protein.

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 The sCD4 (soluble CD4) used is a recombinant protein produced in the Baculovirus expression system by Intracell (trademark) marketed in France by Neosystem Laboratoire (Strasbourg, France).

GP120 VIRAL PROTEINS
The gp120 viral proteins were prepared as solutions at a concentration of 1 mg / ml.

DIRECT TITRAGES
The titrations were carried out in the "opposite" direction.

First, 4 ml of a peptide solution of the present invention, labeled and at a concentration of 6 nM in the phosphate buffer cited above was prepared.

Then, 200 μl of a solution containing 10 μl of gp120 at a concentration of 0.41 μM, 4 μl of fluorescein-labeled peptide of the present invention at a concentration of 6 nM, and 186 μl of buffer was prepared. .

 All anisotropic measurements were made using a Beacon 2000 polarimeter (trademark) (Panvera Corp., Madison WI) with the filter set corresponding to fluorescein.

 The anisotropy of 200 μl of the labeled peptide solution alone was first tested. Depending on the probe used on the peptide and the settings of the apparatus, in particular the gain, the anisotropy of the peptide of the present invention labeled alone varies between 80 and 110 millianisotropy units.

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 Then, the anisotropy of the tube containing the labeled peptide and gp120 at 410 nM was measured.

Generally, the value of the labeled peptide in the presence of this high concentration of gp120 was of the order of 200 millianisotropy.

 The titration was performed by taking 40 μl of the solution and then adding 40 μl of the labeled peptide solution alone at 6 nM. In this way, gp120 was diluted by 0.1 log units per dilution while keeping the concentration of labeled peptide constant. This reverse titration protocol uses as little gp120 as possible.

 The anisotropy measurements were recorded until the signal stabilization. In the case of this interaction, the stabilization is relatively long, since it lasts more than 20 minutes for the first point, but then the dilution points equilibrate faster, in 2 to 4 minutes.

 All assays were performed at 21 C, in a thermostated sample holder of the Beacon 2000 (trademark).

 The labeled peptides showed a tendency to stick to the walls of the containers. Several dilution solutions were therefore made to avoid a gradual decrease in the concentration of labeled peptide.

THE COMPETITIONS
For competitive tests, simulations of the competition of the CD4M33 peptide labeled with

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 fluorescein (CD4M33-F) with the non-labeled CD4M33 peptide were performed. The results of these simulations are presented in the appended FIG.

 These simulations showed that the ideal conditions for competition would be 20mM Gp120 and 6nM labeled peptide.

 Competition was then performed between 10 and 100 nM of unlabeled CD4M33 peptide.

 The competitions were performed experimentally by adding to 200 μl of a 6 nM solution of CD4M33-F peptide and 20 nM gp120 aliquots of 1 μl of unlabeled competitor CD4M33 peptide in the above phosphate buffer to cover concentrations between 10 and 100 nM.

 The values of anisotropies were then measured after each addition.

 The resulting curves of these competitions were analyzed to determine the Kd of the competitor-gp120 interaction by keeping the Kd fixed between the CD4M33-F peptide and the gp120.

 The analyzes and simulations were performed using the BIOESQ program described in documents [41] [42] and [43].

B) RESULTS.

1) MEASUREMENTS OF AFFINITIES WITH DIFFERENT PEPTIDES OF THE PRESENT INVENTION FOR A GP120 PROTEIN
The principle of screening by the method of the present invention is demonstrated in the attached Figures 8a), 8b) and 8c).

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 To obtain the results shown in these figures, a rhodamine green-labeled peptide of the present invention, designated K16-R, in solution at a concentration of 6 nM was titrated with a solution of gp120 strain HXB2.

 In Figure 8a), the gp120 preparation was found to be somewhat less active than that used in Figure 8b). Indeed, in the first case (I K16 / GP120), the affinity (Kd) extracted from the analysis of the data according to a simple fixation model, ie a molecule of gp120 fixing a molecule of peptide of the present invention labeled, was 176 nM, while in the second case (II K16 / GP120), shown in Figure 8b), the affinity was 46 nM.

 A third preparation was thus carried out (III K16 / GP120). The measurements made on this preparation are shown in Figure 8c). In this case the measured affinity (Kd) is 97nM.

 Three other labeled peptides, CD4M33, CD4K15 and CD4M9, were tested for their interaction with gp120 HXB2. The results of these tests are shown in Figures 9, 10 and 11.

Their affinity for the gp120 viral protein was 14 nM, 195 nM, and 323 nM, respectively.

<Desc / Clms Page number 62>

2) AFFINITY MEASUREMENTS CARRIED OUT WITH A PEPTIDE OF THE PRESENT INVENTION FOR DIFFERENT VIRAL PROTEINS GP120
The affinities of the CD4M33 peptide for gp120 proteins from different strains of the virus: HXB2 and SF2, have also been determined.

 They are shown in Figures 12 a) and 12b).

 The 8nM affinity for gp120 HXB2 is very close to the value determined from the results shown in Figure 9, ie 14 nM.

 On the other hand, the gp120 SF2 preparation had a higher affinity for the CD4M33 peptide, of the order of 1 nM.

3) DISPLACEMENT OF A BRAND PEPTIDE OF THE PRESENT INVENTION FIXED WITH THE GPL20 VIRAL PROTEIN BY A NON-BRAND PEPTIDE
Displacement of a labeled peptide having a determined affinity, represented by the CD4M33 peptide, by an unlabeled CD4M33 peptide was performed to simulate screening according to the method of the present invention. The results of this example are shown in Figure 13.

 These results show that a concentration of 20 nM of gp120 allows a fixation approaching 50%, and therefore a relatively high starting anisotropy of about 180 millianisotropy.

 It should be noted that the large change in anisotropy observed during the binding of the peptides of the present invention labeled with gp120, AA-180

<Desc / Clms Page number 63>

 millianisotropy, gives a great dynamic range to this measurement, and allows a very high signal-to-noise ratio. Thus, with a standard deviation of 2 millianisotropy units in the measurements, it is possible to detect changes of 2% in the fixed quantity.

 As a result, competition with a molecule of the same affinity as CD4M33 results in 100% displacement with a competitor concentration approaching 100 nM.

 The inventors have therefore used the competition of labeled CD4M33 with other non-labeled peptides of the present invention to determine their affinity for gp120 HXB2.

The results of these competitions are grouped in Figures 14a) to i)
A CD4M33 labeled competition with unlabeled CD4M33 was first performed to determine if the labeling disrupts the affinity for gp120.

 In direct fixation, shown in Figures 10 and 11, the affinity of the CD4M33 labeled for gp120 HXB2 was 11 + 3 nM.

The analysis of the competition data was carried out by setting the value of the affinity of the labeled CD4M33 to its value of 11 nM and allowing the value of the competitor's affinity to change, in this case the unlabeled CD4M33 peptide. . The value of the corresponding affinity to the line crossing the points in Figure 14a) was 14 nM. It was therefore concluded that the labeling of CD4m33 does not affect its affinity for gp120.

<Desc / Clms Page number 64>

 Then seven other unlabeled peptides of the present invention as well as the soluble domain of human CD4 (sCD4) were tested. The results of these tests are shown in Figures 14b) to 14i).

Their affinity differs by a factor of 40. The results obtained are grouped together in Table 5 below.

 It should be noted that the affinity of the CD4M9 peptide for the viral protein gp120 obtained by competition, Kd = 359 nM, is the same, taking into account the measurement errors estimated at -10%, that that found by direct titration of CD4M9 labeled (Kd = 323 nM).

 Since the screened molecules are not observed directly, but in competition with the labeled peptide of the present invention, this screening test can reveal any molecule, whatever its chemical structure, which would bind to the gp120. competitively with this peptide.

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Table I:

<Tb>
<tb> Peptide <SEP> from <SEP> la
<tb> Affinity <SEP> Kd
<tb> present <SEP> Method <SEP> Gp <SEP> 120
<tb> (nM)
<tb> invention
<tb> K16 <SEP> Direct <SEP> HXB2 <SEP> 176
<tb> K16 <SEP> Direct <SEP> HXB2 <SEP> 46
<tb> K16 <SEP> direct <SEP> HXB2 <SEP> 97
<tb> K15 <SEP> Direct <SEP> HXB2 <SEP> 195
<tb> CD4M9 <SEP> Direct <SEP> HXB2 <SEP> 323
<tb> CD4M9 <SEP> Competition <SEP> HXB2 <SEP> 359
<tb> CD4M33 <SEP> Direct <SEP> HXB2 <SEP> 14
<tb> CD4M33 <SEP> Direct <SEP> HXB2 <SEP> 8
<tb> CD4M33 <SEP> Direct <SEP> SF2 <SEP> 1
<tb> CD4M33 <SEP> Competition <SEP> HXB2 <SEP> 14
<tb> CD4M35 <SEP> Competition <SEP> HXB2 <SEP> 9
<tb> CD4M32 <SEP> Competition <SEP> HXB2 <SEP> 23
<tb> sCD4 <SEP> Competition <SEP> HXB2 <SEP> 9
<tb> CD4M27 <SEP> Competition <SEP> HXB2 <SEP> 148
<tb> CD4M9TPA <SEP> Competition <SEP> HXB2 <SEP> 26
<tb> CD4M9BIP <SEP> Competition <SEP> HXB2 <SEP> 97
<tb> (seq <SEP> ID <SEP># 19)
<tb> CD4M9V <SEP> Competition <SEP> HXB2 <SEP> 320
<tb> (seq <SEP> ID <SEP> n 20)
<Tb>

The peptides of the present invention thus represent in vitro inhibitors of the infection with the AIDS virus, in particular HIV-1.

 The screening method of the present invention which uses the fluorescence anisotropy of a peptide

<Desc / Clms Page number 66>

 The present invention is a novel tool for screening candidate molecules for the manufacture of medicaments for treating AIDS.

<Desc / Clms Page number 67>

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Claims (4)

Cys-Thr or Ala-Cys-Xaak-NH2 wherein TPA is thiopropionic acid, XaaJ is ss-naphthylalanine, phenylalanine or bi-phenylalanine, Xaak is Gly, Val or place, pl is 3-6 amino acids , p2 represents 2 to 4 amino acids and p3 represents 6 to 10 amino acids, the amino acids in Gly or (D) Asp or Ser-Ser or His or Asn-Xaaj TPA-P1-Cys-P2-Cys-P3-Cys 1. A method for screening molecules capable of binding with a specific affinity to the gp120 protein of the immunodeficiency virus, said method including at least one screening cycle comprising the following steps: a) covalent labeling with a fluorescent probe of a peptide having said affinity determined for the gp120 protein, said peptide comprising the following sequence (A):  1 2 3 pl, p2 and p3 being natural or unnatural, identical to 1 2 3 1 or different and pl, p2 and p3 having or not having a common sequence, said peptide having a hairpin conformation ss whose elbow ss is formed by the amino acid residues Ala or Gln-Gly or DAsp or Ser-Ser or His or Asn-Xaai of its sequence (A), b) placed in the presence of the peptide labeled with the gp120 protein, at a concentration of gp120 that allows between 20 and 50% fixation of the labeled peptide, so as to form a reversible complex labeled peptide-gp120], <Desc / Clms Page number 73>  c) standard measurement of the fluorescence polarization of the labeled peptide-gp120 complex], d) competitive screening test of the labeled peptide-gp120 complex] formed with at least one candidate molecule, capable of binding to the gp120 protein, at a determined concentration of said molecule (s) and under physicochemical conditions making it possible for the molecule to compete with the labeled peptide to form a complex with the gp120 protein, e) measure the fluorescence polarization emitted by the screening test, and f) comparing the fluorescence polarization measurement of step e) with the standard measurement of step c), the candidate molecule (s) being considered as a molecule capable of to bind to the Gp120 protein when the fluorescence polarization measurement of step e) is less than the standard measurement of step c).  k proline bound to the Xaak residue.

 The method of claim 1, wherein the sequence (A) may further comprise a residue 3. The method according to claim 1, wherein the sequence is selected from the sequences ID n04, ID n5, ID n6, ID n07, ID n8, ID n09, ID n0 10, ID n ll, ID n 12, ID No. 13, ID No. 14, ID No. 15, ID No. 16, ID No. 17, ID No. 18, ID No. 19, and ID No. 20 of the attached sequence listing. <Desc / Clms Page number 74>  The method of claim 1, wherein the fluorescent probe is a fluorophore having visible absorption properties.