FR2682875A1 - Use of poly(2-8)- alpha -sialic acid for the regulation of the action of homeo box peptides - Google Patents

Abstract

The invention relates to poly(2-8)- alpha -sialic acid (colominic acid) as a regulator of the action of homeo box peptides or of their derivatives. The invention consists, in particular, of pharmaceutical compositions containing colominic acid as active principle.

Description

USE OF POLYSIALIC ACID a2-8 FOR THE
REGULATION OF THE ACTION OF HOMEOBOIT PEPTIDES.

 The present invention relates to the regulation of the internalization and intracellular transport of factors having the property of binding to DNA, in particular of homeobox peptides, or of molecules comprising said peptides.

 Several proteins capable of binding to DNA and of regulating the expression of different genes are known: mention may be made, for example, of the so-called finger-zinc proteins (dactyl proteins) including in particular nuclear steroid receptors, proteins with leucine zipper, and the proteins with a helix-turn-helix structure which include homeoproteins.

 Homeoproteins interact with DNA via a specific region, called homeodomain or homeobox domain, having a helix-turn-helix structure, very conserved not only from one homeoprotein to another, but also through the animal kingdom, from one species to another.

The homeobox / DNA peptide link would be the result
- a high affinity bond (KD 10-9 - 10-1 X), involving a consensus sequence
ATTA present in the promoter of different genes
- and a low affinity bond (KD 10-6 - 10-7M), involving the broad groove of the DNA double helix. A publication by KISSINGER et al.

[Cell, 63, 579-590, (1990)] describes a study carried out by crystallography on a "homeodomain engrailed / DNA" complex. This study shows that the part
C-terminal of the homeodomain, comprising in particular the structure called helix 3 (amino acids 42-58 of the engrailed homeodomain), is fixed at the level of the broad groove of the DNA. The bond is essentially ensured by hydrophilic interactions; this binding would be independent of the presence of the consensus sequence.

 The inventors have recently demonstrated that peptides having the sequence of the homeobox domain (such peptides are called in the following: "homeobox peptides") penetrate into neurons in culture and cause an intense differentiation of these cells, and in particular the growth. of their neurites. The discovery of these properties has made it possible to propose the use of these peptides as neurotropic growth factors, and also as transmembrane and / or intracellular transporters of different macromolecules such as polypeptides or oligonucleotides (International Application PCT / FR 91/00444) .

 However, the inventors observed that this penetration phenomenon depended on the cell type, the entry of a homeobox peptide into cells such as fibroblasts is, for example, much less than in nerve cells.

 By continuing their work on the entry mechanisms of the homeobox peptide, the inventors have now highlighted various factors conditioning this entry mechanism.

 They hypothesized the existence of a cellular structure involved in the transmembrane and intra-cellular transport of the homeobox peptide, and have now shown the existence of such a structure, implying a polysaccharide: polysialic acid. a2-8, also known as colominic acid, which is present on the surface of different cells, for example young or regenerating nerve cells, myoblasts, certain populations of T lymphocytes, and which is carried by a membrane protein, the "molecule neuronal adhesion "or NCAM. The inventors have found that the homeobox peptide penetrates into all of these cells, in an amount 10 times greater than into cells lacking this receptor.

They also verified by experiments on the intracellular penetration of the homeobox peptide into neurons, that the entry of said peptide, and consequently its biological effects, were reduced in the presence of colominic acid. They made the same observations
- when the neurons are pretreated with an enzyme, Endo-N, which specifically cleaves the a2-8 bonds of polysialic acid
- when the neurons are preincubated in the presence of an anti-colominic acid antibody.

 These results show the essential role played by colominic acid in the penetration system of the homeobox peptide, and the existence of a specific interaction between the homeobox peptide sequence and colominic acid.

They also established that the effect of colominic acid on the intracellular penetration of the homeobox peptide is comparable to the inhibition by DNA fragments previously observed (Application
PCT / FR 91/00444).

This observation led them to compare the respective three-dimensional structures of colominic acid and double-stranded DNA (under the conformation
B), established by NMR and crystallography.

 FIG. 1 illustrates the results of this comparison: the 4 oxygen atoms of the carboxyl groups of the molecules located respectively in positions 1, 2, 7 and 8 of an octamer of polysialic acid, (FIG. 1 A, on the right) can be superimposed with the 4 oxygen groups of the phosphate groups which delimit the broad groove of the DNA double helix (Figure 1A, left).

On one of the strands, two of said phosphate groups are adjacent to pairs of nucleotides 1 and 2; on the opposite strand, two other groups are adjacent to the nucleotide pairs 7 and 8. This superposition is shown, seen from 2 different angles, in FIGS. 1B and 1C.

 However, it has also been shown that this 1 - 2/7 - 8 configuration of the phosphate groups plays an important role in the low affinity binding (independent of the recognition of a consensus sequence) between the homeobox peptide sequences and 1 ' DNA, by intervening in the contacts between the side chains of the amino acids of helix 3 of the peptide and double-stranded DNA (KISSINGER et al., Publication cited above).

 The term helix 3 denotes a portion of homeobox peptide comprising amino acids 42 to 58 of the peptide sequence. The amino acids are designated according to the numbering used by QUIAN et al.

[Cell, 59, 573-580 (1989]. Some authors also name Helix 3 + 4 this same region of the homeobox peptide.

 The observations made by the inventors make it possible to establish the existence of a structural relationship between the colominic acid and the double helix DNA, and it appears that the site concerned by this relationship plays an important role in the homeobox peptide interaction. / DNA.

 By carrying out mutagenesis experiments on a homeobox peptide, the inventors have further shown that certain structures of helix 3 play an essential role in the intracellular penetration of said peptide.

 The present invention results from this set of results obtained by the inventors.

 The demonstration of the role of colominic acid as a receptor for homeobox peptides, the demonstration of the homeobox peptide / colominic acid interaction, as well as the identification of structures of colominic acid and the homeobox peptide capable of Intervening in this interaction leads to numerous applications, both invivo and in vitro.

 The present invention relates to colominic acid for use as a medicament.

 According to a preferred embodiment of the present invention, said colominic acid comprises at least 8 residues of sialic acid.

 According to a preferred embodiment of the present invention, colominic acid is used as a regulator of the activity of homeobox peptides and their derivatives.

For the purposes of the present invention, the term “homeobox peptide derivatives” means macromolecules comprising structures similar to those identified by the inventors as involved in the binding of these peptides with colominic acid, and in particular
- molecules comprising a peptide part whose sequence is that of a homeobox peptide: these are for example natural homeoproteins, or fusion products between a homeobox or nucleotide peptide, such as those described in the PCT application / FR 91/00444
- molecules comprising or consisting of a peptide part whose sequence is that of helix 3 of a homeobox peptide
- molecules comprising or consisting of a mutant homeobox peptide provided, of course, that said mutation does not abolish the binding capacity with the calominic acid of the peptide: this definition includes mutant peptides which have partially or totally lost their ability to recognition of DNA at the level of a consensus sequence, but having retained their capacity for intracellular penetration.

 Such applications relate in particular to the possibility of extending the field of use of homeobox peptides or their derivatives to all cell types, including those which do not naturally have a receptor for these peptides, as well as that of allowing positive or negative regulation of the activity of said peptides.

 For example, colominic acid molecules, conjugated to a molecule that can be inserted into the cell membrane (lipid, peptide or one of their glycosylated derivatives), can be grafted on the surface of a cell, to create artificial receptors homeobox peptides or their derivatives, with the aim of making it permeable or of increasing its permeability to said peptides or derivatives.

 On the contrary, if one wishes to prevent the effects resulting from the binding between a homeobox peptide and the cellular DNA, colominic acid can be used either at the extracellular level, as a competitive inhibitor of the receptor / homeobox binding or at the intracellular level , as a competitive inhibitor of DNA / homeobox binding.

It is also possible to prevent the entry into the cell of the homeobox peptide using an anti-colominic acid antibody, or by blocking or destroying by endamin-N neuraminisase, the molecules of colominic acid playing the role of receptor.
The invention also encompasses antibodies to colominic acid, for use as inhibitors of intracellular penetration of factors capable of binding to DNA.

 The present invention further relates to the use of colominic acid on cells in culture, in all the applications described above.

In this context, it includes cells whose membrane is modified by insertion of artificial receptors for homeobox peptides or their derivatives, as defined above.

 The invention also includes pharmaceutical compositions comprising, as active ingredient, colominic acid, or an anti-colominic acid antibody.

 The present invention will be better understood using the additional description which follows, which refers to examples demonstrating the role played by colominic acid in the intracellular transport of homeobox peptides and their derivatives.

 It goes without saying, however, that these examples are given solely by way of illustration of the subject of the invention, of which they do not in any way constitute a limitation.

I - DEMONSTRATION OF THE ROLE OF COLOMINIOUE ACID IN THE
TRANSMEMBRANE TRANSPORTATION SYSTEM OF PEPTIDE HOMEO
BOX
PAntp peptides are synthesized, purified, and when necessary, labeled with fluorescein, according to the protocols described in the publication by JOLIOT et al., [Proc. Natl. Acad. Sci. USA 88, 18641866 (1991)] and in PCT Application FR / 91/00444.

 Nerve cells are cultured under the conditions described in the PCT application and the publication by JOLIOT et al. mentioned above.

EXAMPLE 1 Penetration of a chimeric protein comprising a homeobox peptide into cells of different types
A chimeric protein was constructed by genetic recombination. This protein has the following sequence: pAntp-Ser-Arg-rab3Cter. Rab3Cter is made up of the 34 C-terminal amino acids of the protein encoded by the rab3 gene, which is a small protein that binds
GTP (G-protein) and specific to neuroendocrine systems). pAntp-rab3Cter (name of the fusion protein) was contacted with neurons (chicken, rat) or myoblasts (chicken) in culture for 12 hours, then the presence of rab3Cter was checked by immunocytochemistry using 'an antibody to rab3. In all cases, the pAntp-rab3Cter complex is found in the nucleus.

This experience shows that
- the nuclear penetration and addressing system is linked to the presence of PSA (colominic acid) on the surface of the cell.

 - pAntp plays the role of a signal peptide both for entry into the cell, and for nuclear addressing.

EXAMPLE 2 Effect of a treatment with colominic acid on the penetration and the morphological effects of a homeobox peptide
5000 cells are cultured in 10 Rl of culture medium, in TERASAKI type wells covered with a polyornithine substrate (1.5 Rg / ml). After 24 hours, 10 fl of culture medium containing either pAntp alone (400 Fg / ml) or pAntp preincubated with 100 Rg / ml or 1 mg / ml of PSA (colominic acid,
SIGMA), or PSA alone are added. The morphological aspect of the cultures is analyzed 24 hours later. The results of this experiment are as follows
The cells to which the PSA alone was added have a morphology and growth similar to those of the control cells cultured without additive.

 The cells cultured in the presence of pAntp show an increase in neuritic growth, characteristic of the action of this peptide.

 The cells cultured in the presence of PSA (at the two concentrations tested) and pAntp have a morphology and a growth similar to that of the control cells.

 It therefore appears that the presence of PSA alone does not disturb the growth of neurites, and that PSA antagonizes the morphogenetic effects of pAntp.

EXAMPLE 3 Effect of Endo-N Treatment on the Penetration of a Homeobox Peptide
Spinal cord cells of rat embryos (2 × 10 4 cells / 20, μl of culture medium) are spread on glass coverslips coated with polyornithine (SIGMA, PM 40,000, 15 μg / ml). 24 hours later, the cells are incubated for 1 hour in the presence of a solution (107 PFU / ml) of Endo-N neuraminidase from the phage
PK 1A [FINNE and MAKELA, J. Biol. Chem. 260, 12 1265-1270 (1985)], then for 1 hour in the presence of 1Clg of peptide pAntp labeled with fluorescein. A control, in which the Endo-N solution is replaced by culture medium, is also produced.

 At the end of the incubation, the cells are washed, then fixed in an ethanol / acetic acid solution (95/5) (5 minutes, -20 s air-dried, and mounted in moviol.

Examination under the microscope shows that
- In the control cells, a significant fluorescence is observed inside the cell with a particular intensity in the nucleoplasm, which corresponds to the characteristic distribution of the pAntp peptide.

 - in cells pretreated with Endo-N, the intracellular fluorescence is very weak.

EXAMPLE 4 Effect of Treatment with a Colominic Acid Antibody on the Penetration of a Homeobox Peptide
An anti-menigococcal B antiserum (which essentially consists of antibodies directed against colominic acid) is used for this experiment.

 24-hour cultures of rat spinal cord neurons (2,106 cells) are preincubated for 1 hour in the presence of anti-meningococcal antiserum (1/10 dilution). These cells as well as control cells are incubated for 3 hours in the presence of 20 Rg of pAntp peptide.

 The cells are then washed 3 times in PBS, then trypsinized (15 minutes at 37 ° C.) to remove the pAntp peptides which have not penetrated. After 3 washes in PBS, the cells are lysed in the presence of NP 40 (15 minutes, 4 ° C.) and protease inhibitors. The lysate is centrifuged (2000 g, 15 minutes, 4 C) and the pellet containing the nuclei is collected and dissolved in a solution of Nacl 500 mM 1 mM DTT.

The nuclear extract thus obtained is analyzed on a gel
SDS polyacrylamide (15 96).

The results are illustrated in Figure 2
- Profile 1 is that of the nuclear extract obtained from cells preincubated with the anti-serum anti-colominic acid, prior to incubation with pAntp.

 - Profile 2 is that of the extract obtained from cells incubated in the presence of pAntp, itself preincubated with 150 Rg of Hoxp DNA (specific binding sequence of the pAntp peptide).

 - Profile 3 is that of the nuclear extract obtained from cells directly incubated with pAntp.

 The arrow A indicates the histones, the arrow B the peptide pAntp.

 These results show that the penetration of the pAntp peptide is blocked by an anti-colominic acid immune serum as well as by the binding of the peptide to its specific binding sequence.

11. - DEMONSTRATION OF THE ROLE OF THE PROPELLER 3 FROM
HOMEOBOITE PEPTIDE IN TRANSMEMBRANEAN TRANSPORT AND
THE LINK TO THE DNA OF SAID PEPTIDE.

EXAMPLE 5 - Obtaining mutants
4 different mutants were obtained from the pAntp peptide.

 The amino acid sequence of these mutants (code 1 letter) is shown in Figure 3.

- Mutant 48S: loss of Try 48, Pro 49, Gln 50 residues and addition of a Ser 48
- Mutant 50A: replacement of His 36 with
Tyr 36 and replacement of Gln 50 by Ala 50
- Mutant 39S: replacement of Cys 39 by
Ser 39
- Mutant 40P2: introduction of 2 Pro in positions 40 and 41, replacing two Leu residues and
Thr.

 The gel delay experiments demonstrate that the mutants 48S, 50A and 40P2 no longer bind to the Hox-1.3 promoter, while the mutant 39S is still capable of recognizing this sequence. Cell penetration experiments show that the mutants 40P2 and 48S have lost their penetration capacity by more than 80%, while the loss is only 60% for the mutant 50A. The 39S mutant penetrates nerve cells as well as the wild.

Claims (8)

 1) Use of colominic acid for obtaining an agent regulating the action of homeobox peptides and their derivatives in vitro or in vi vo.  2) Use according to Claim 1, characterized in that colominic acid is used for obtaining a competitive inhibitor of intracellular penetration and / or of DNA binding of homeobox peptides or their derivatives.  3) Use according to Claim 1, characterized in that colominic acid is used for obtaining artificial receptors for homeobox peptides or their derivatives.  4) Use according to Claim 3, characterized in that the colominic acid is conjugated to a polypeptide or to a lipid or to one of their glycosylated derivatives.  5) Use according to any one of Claims 1 to 4, characterized in that the colominic acid used comprises at least 8 residues of sialic acid.  6) Artificial receptor for homeobox peptides or their derivatives, which receptor comprises at least one molecule of colominic acid conjugated to a polypeptide or to a lipid.  7) Modified cells, by insertion into their cytoplasmic membrane, of artificial receptors according to Claim 6.  8) Pharmaceutical compositions characterized in that they comprise, as active ingredient, colominic acid.