FR2766572A1 - USE OF APPROPRIATE CELLS FOR SCREENING TUBULIN-CARBOXYPEPTIDASE INHIBITORS AND APPLICATIONS THEREOF AS ANTI-CANCER - Google Patents

Abstract

The invention concerns the use of cells wherein the tubulin tyrosination-detyrosination cycle is modified, for screening and selecting inhibitors of the tubulin-carboxypeptidase, their use as anti-cancer agents and the screening method using said cells. Said cells, which over-produce tubulin-glu, are adapted for selecting or screening products inhibiting the tubulin-carboxypeptidase activity (TCP).

Description

 The present invention relates to the use of cells, in which the tyrosination-detyrosination cycle of tubulin is modified, for the screening and selection of inhibitors of tubulin-carboxypeptidase, to the application of the latter as that anticancer as well as a screening process using said cells.

 Microtubules, along with actin filaments and intermediate filaments, form the cytoskeleton of eukaryotic cells. Microtubules are protein organelles composed of tubulin molecules, assembled in stru, tubular cells about 25 nm in diameter, with a length of up to several tens of micrometers.

 Microtubules play multiple roles in the cell. They play a central role in the genesis and the maintenance of the shape of the cells. During the interphase, they organize the intracellular space and are responsible for the intracellular transport of organelles. They are essential for cell division, in fact, during mitosis, they reorganize to form the mitotic spindle, responsible for the distribution of chromosomes between the two daughter cells. In the nervous system, microtubules are also directly involved in neurite growth and axonal transport.

 Finally, they play a central role in the motility of differentiated cells, such as, for example, sperm.

In cells, tubulin, formed of two subunits (a and '3), exists in several isoforms. This diversity is generated at two levels
- the selective transcription of one or more genes coding for each subunit,
- a variety of post-translational modifications of tubulin, such as its acetylation (Piperno G. et al., J. Cell Biol., 1985, 101, 2085-2094), its phosphorylation (Gard D. et al., 1985, J. Celi Biol., 1985, 100, 764-774), its glutamylation (Eddé B. et al., Science, 1990, 247, 83-85), its polyglycylation (Redeker V. et al., Science, 1994, 266, 1688-1691) or its tyrosination (Barra HS. Et al., 1988, 2, 133143).

 This latter modification is known as the tyrosination-detyrosination cycle of tubulin and is illustrated in Figure 1.

 In accordance with this cycle, tubulin exists in a tyrosine form (tyrosine tubulin or tubulin-tyr) and in a detyrosine form (detyrosine tubulin or tubulin-glu), the transition from one to the other of these forms involves two specific enzymes, which act at the level of the carboxy-terminal amino acid Tyr of the subunit a of tubulin: the tyrosine residue is cleaved from the main polypeptide chain by a tubulin-carboxypeptidase (TCP) or added by a specialized enzyme, tubulin-tyrosine ligase (TTL) (see Figure 1).

 This cycle, which is absolutely specific to tubulin, includes the synthesis of a peptide bond without the participation of ribosomes.

 Another tubulin isoform, resistant to tyrosination, has also been demonstrated (Paturle-Lafanechère L. et al., J. Cell. Science, 1994, 107, 1529-1543 and Biochemistry, 1991, 30, 10523- 10528). It is a particular form of tubulin, representing about 35% of the cerebral tubulin, which has been called tubulin A2. It includes two fewer amino acids (Glu450-Tyr451), compared to tubulin-tyr (see Figure 2).

 The inventors have now surprisingly found that there is a selective suppression of tubulin-tyrosine ligase (TTL) in tumors, which is directly correlated with the appearance of tubulin A2, normally absent from non-neuronal cells. . The generation of tubulin A2, during tumor growth, associated with the suppression of TTL activity is pathological, tubulin A2 therefore constitutes a tumor marker, which is particularly interesting.

 TTL acts preferentially on free tubulin and does not seem to have any action on tubulin in microtubules, while TCP preferably acts on polymerized tubulin (assembled microtubules). Consequently, the newly assembled microtubules, in interphase, are largely composed of tubulin-tyr while the stable microtubules are largely composed of tubulin-glu.

 The inventors have also found that the absence of TTL is compatible with a residual cycle, in which tyrosine tubulin (tubulin-tyr) comes from the neosynthesis of tubulin, which results in the inhibition of the activity of the tubulin carboxypeptidase helps restore normal levels of tyrosine tubulin.

 This is why the inventors have set themselves the goal of using cells making it possible to screen for substances capable of inhibiting the activity of tubulinecarboxypeptidase and consequently of compensating for the elimination of TTL activity during tumor growth. and therefore inhibit this growth.

 The subject of the present invention is the use of cells originating from any tissue, in which the tyrosination-detyrosination cycle of tubulin is modified, which cells overproduce tubulin-glu, for the selection or screening of products. inhibitors of tubulinecarboxypeptidase (TCP) activity.

 Said cells producing excess tubulin-glu are obtained in particular either by inhibiting the renewal dynamics of the tubulin assembly, or by inhibiting TTL activity.

 The renewal dynamics of the tubulin assembly is inhibited, either by a microtubule stabilizing agent, such as taxol, or by a periwinkle alkaloid, such as vinblastine.

 Indeed, the addition of taxol promotes the polymerization of tubulin, in vitro; added to cells, it causes the assembly of a large proportion of free tubulin into stable microtubules. Vinblastine or vincristine induce the formation of tubulin paracristalline aggregates.

 Such cells, overproducing tubulin-glu, when in the presence of a TCP inhibitor, must see tubulin-glu disappear and tubulin-tyr appear.

In addition, cells in which TTL activity is inhibited, in particular by an anti-TTL antibody, but in which TCP activity is intact, produce tubulin-glu, whereas when they are in the presence of a inhibitor
TCP, a restoration of normal tubulin-tyr levels should be observed.

 The present invention also relates to the use of cells which do not produce tubulin-tyr (TTL cells), for screening or selection of tubulin-carboxypeptidase inhibitors.

Such cells, in which the TCP activity is intact, produce tubulin-glu, whereas when they are in the presence of an inhibitor of
TCP, we must observe the appearance of tubulin-tyr by neosynthesis, and a decrease
or a disappearance of tubulin-glu.

Such cells are in particular cells derived from cells
NIH / 3T3 filed under No. ATCC CRL 1658 (mouse embryonic fibroblasts)
and are obtained after multiple passages in culture, these cells produce tubulin-glu and tubulin-A2.

The present invention also relates to the use of an extract
crude or semi-purified TCP for the screening or selection of products inhibiting TCP activity.

Said crude or semi-purified extract of TCP, catalyzing the cleavage of tyrosine from the main polypeptide chain, is capable of being obtained by homo
cold generation of a selected organ, followed by fractional precipitation of
proteins and successive separation stages, in particular by adsorption, dialysis and
passage on different chromatography columns.

Preferably, said selected organ is subjected to the steps
of separation described by TM Martensen (Methods in Cell Biology, 1982, 24, 265
269).

The enzymatic activity of TCP is measured by the disappearance of the
C-terminal tyrosine present in microtubules, in which tyrosine has been
previously radiolabelled (e.g. 14C-tyrosine or 3H-tyrosine, according to
to TM Martensen, supra).

The disappearance of tyrosine tubulin and the appearance of tubulin
detyrosine can also be quantified in immunoassays using antibodies
specific to each of these forms of tubulin.

The present invention further relates to a screening method
and selection of tubulin-carboxypeptidase inhibitor products, characterized in
that he understands:
- bringing the potential inhibitor into contact with a cell or a
raw or semi-purified extract of TCP, as defined above and
- the measurement of tubulin-tyr and / or tubulin-glue by any means
approved.

For example, the detection of tubulin-tyr can be carried out, either by measuring the incorporation of the radiolabelled tyrosine, or by an immunoassay, in the presence of an antibody specific for tubulin-tyr, the detection of tubulin- glu can be performed by an immunoassay in the presence of an antibody specific to tubulin-glu. The applicable methods are as follows
* Fluorimetric methods:
direct method: the specific anti-tubulin-tyr and antitubulin-glu antibodies are labeled with a fluorescent compound and brought into contact with the cell extract
indirect method: the anti-tubulin-tyr and anti-tubulineglu antibodies, unlabeled, are applied to the cell extract, the labeled antibodies directed against the Ig of the species from which the specific antibody originates, reveal its fixation.

 The measurement is preferably carried out in immunofluorescence by double labeling of the tyrosine and detyrosine isotypes.

* Immunoenzymatic assays, vmatic
The technique is based on the link between tubulin-glu or tubulin-tyr and a standard antibody specific for these tubulins. The antigen or antibody is covalently linked to an enzyme. The enzyme, usually horseradish peroxidase, alkaline phosphatase or ss-galactosidase, is visualized by color reaction with the substrate. Several technical modalities can be used
a competitive technique can be used to measure tubulin-tyr and tubulin-glu. These are the tubulin-tyr and tubulin-glu or peptides reproducing the specific sequences of each of these forms of tubulin, which are always fixed on the microplates. A mixture of cell extract, and small amounts of anti-tubulin-tyr and anti-tubulin-glu antibodies are added. The quantity of antibody fixed on the microplate is all the lower when the tubulin-tyr and tubulin-glu molecules present in the sample to be analyzed have neutralized more antibody molecules.

The subject of the present invention is, moreover, a method for screening and selecting tubulin-carboxypeptidase inhibitor products, characterized in that it comprises:
bringing the potential inhibitor into contact with a crude or semi-purified extract of TCP, as defined above, the tyrosine of which has been radiolabelled beforehand, and
- measurement of free radiolabelled tyrosine.

 The present invention further relates to the use of a tubulin-carboxypeptidase inhibitor, as defined above, for obtaining an anti-cancer drug.

In addition to the foregoing provisions, the invention also comprises other provisions, which will emerge from the description which follows, which refers to examples of implementation of the process which is the subject of the present invention, with reference to the accompanying drawings, in which:
- Figure 1 illustrates the tyrosination-detyrosination cycle of the tubu line;
- Figure 2 illustrates the differences in C-terminal sequences of the different tubulin isoforms.

 It should be understood, 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.

EXAMPLE 1 Use of normal cells overproducing tubulin-glu (action of taxol or vinblastine), to screen for inhibitors of TCP.

- Cell preparation
HeLa cells are cultured routinely in RPMI 1640 medium supplemented with 10% fetal calf serum.

- Cell processing
The treatment is carried out at 370C. Taxol (5 ptM, 10 pLM or 50 pM) or vinblastine (10 CLAM) are added to the culture medium.

- Test:
A TCP inhibitor is added to the culture medium (or it is microinjected into the cells), the cells are fixed by incubation in pure methanol, 6 min, at -20 C. The fixed cells are incubated with anti-tubulin antibodies- glu (1/500) and anti-tubulin-tyr antibodies (YL1 / 2, 1/1000), followed by a second incubation with anti-rat goat antibodies conjugated to rhodamine (Jackson) (to detect anti-tubulin-tyr antibody) and a goat anti-rabbit antibody (Cappel) (1/250) to detect anti-tubulin-glu antibodies.

 The anti-tubulin-glu antibodies are obtained according to the procedure described in Gundersen et al. (Cell, 1984, 38, 779-789). The sequence of the peptide used as immunogen is GEEEGEE (7 amino acid residues of the Cterminal end of tubulin-glu). This peptide is conjugated to KLH and injected into rabbits.

The specificity of the antibodies obtained is verified by ELISA in accordance with the method described by Gundersen et al. cited above. The IgGs obtained are purified according to the method of McKinney et al. (J. Immunol. Meth., 1987, 96, 271-278) and their specificity with respect to tubulin-glu is verified by analysis in western blot.

 The anti-tubulin-tyr monoclonal antibody YL1 / 2 specifically recognizes the carboxy-terminal residue of the a subunit of tyrosine tubulin (Wehland J. et al., J. Cell Biol., 1983, 97, 1467-1475 ), it is obtained according to the procedure described in Kilmartin JV et al., J. Cell Biol., 1982, 93, 576-582).

 In the presence of TCP, detyrosination is observed and only tubulin-glu is detected, whereas when the cells are in the presence of a TCP inhibitor, tubulin-tyr is measured.

EXAMPLE 2 Use of normal cells in which TTL is inhibited.

l
An antibody that inhibits TTL is microinjected as described in J.

Wehland et al., J. Cell Science, 1987, 88, 185-203.

 In the absence of an inhibitor, we will essentially measure tubulineglu, while in the presence of an inhibitor, we will be able to measure the tubulin-tyr that has appeared and verify the disappearance of tubulin-glu by double labeling as specified in example 1.

EXAMPLE 3 Use of TTL cells
The cells derived from the abovementioned MH / 3T3 cells are cultured on DME medium supplemented with 10% calf serum and 1% fetal calf serum.

 TTL subclones are obtained by limiting dilution, after 3 growth cycles.

 They are selected according to the uniformity of their phenotype, by double staining in immunofluorescence, using an anti-tubulin glu antibody, or an anti-tubulin A2 antibody, in combination with an anti-tubulinetyr antibody.

 The TTL subclones are those which produce tubulin-glu and tubulin A2 in abundance.

 In these cells, the Glu microtubules represent almost all of the cytoplasmic microtubules, instead of the small subpopulation usually observed in the different cell types. Tubulin-tyr is scarce and is diffuse in cytoplasmic microtubules.

 In the presence of a TCP inhibitor, it will be possible to measure the tubulin-tyr that has appeared and to verify the disappearance of the tubulin-glu by double labeling as specified in Example 1.

As is apparent from the above, the invention is in no way limited to those of its modes of implementation, embodiment and application which have just been described more explicitly, on the contrary it embraces all of them. variants that can come to mind of the technician in the field, without deviating from the
framework, or scope, of the present invention.

Claims (10)

 1 ") Use of cells from any tissue, in which the tyrosination-detyrosination cycle of tubulin is modified, which cells overproduce tubulin-glu, for the selection or screening of products which inhibit activity tubulin-carboxypeptidase (TCP).  2 ") Use according to claim 1, characterized in that said cells are obtained either by inhibition of the renewal dynamics of the tubulin assembly, or by inhibition of TTL activity.  3) Use according to claim 1 or claim 2, characterized in that the renewal dynamics of the tubulin assembly is inhibited, either by a microtubule stabilizing agent, or by a periwinkle alkaloid.  4) Use according to claim 1 or claim 2, characterized in that the TTL activity is inhibited by bringing said cells into contact with at least one anti-TTL antibody.  5) Use of cells which do not produce TTL, for screening or selection of tubulin-carboxypeptidase inhibitors.  6) Use according to claim 5, characterized in that the TTL cells are cells derived from the NtH / 3T3 cells deposited under the NOATCC CRL 1658, by multiple passages in culture, and in which the Glu microtubules represent almost all of the cytoplasmic microtubules .  7) Use of a crude or semi-purified extract of TCP, for the screening or selection of products that inhibit TCP activity.  - the measurement of tubulin-tyr and / or tubulin-glue.  - bringing the potential inhibitor into contact with a crude or semi-purified cell or extract of TCP, according to any one of claims 1 to 7 and  8) A method of screening and selecting tubulin-carboxypeptidase inhibitor products, characterized in that it comprises:  - measurement of free radiolabelled tyrosine.  bringing the potential inhibitor into contact with a crude or semi-purified extract of TCP, according to claim 7, in which the tyrosine is radiolabelled beforehand, and  9) Method for screening and selecting tubulin-carboxypeptidase inhibitor products, characterized in that it comprises  10) Use of a tubulin-carboxypeptidase inhibitor, selected according to the method of claim 8 or claim 9, for obtaining an anti-cancer medicament.