WO1999005522A1

WO1999005522A1 - Use of appropriate cells for screening inhibitors of the tubulin-carboxypeptidase and applications as anti-cancer agents

Info

Publication number: WO1999005522A1
Application number: PCT/FR1998/001645
Authority: WO
Inventors: Didier Job; Laurence Lafanechere; Robert L. Margolis; Jürgen WEHLAND
Original assignee: Commissariat A L'energie Atomique
Priority date: 1997-07-28
Filing date: 1998-07-24
Publication date: 1999-02-04
Also published as: CA2299169A1; FR2766572B1; FR2766572A1

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

USE OF CELLS APPROPRIATE FOR THE SCREENING OF TUBULIN CARBOXYPEPTIDASE INHIBITORS AND APPLICATIONS THEREOF AS ANTI-CANCER.

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 as anti-cancer 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 into tubular structures of about 25 nm in diameter, up to several tens of micrometers in length.

Microtubules play multiple roles in the cell. They play a central role in the genesis and maintenance of the shape of cells. During finterphase, 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 (and β), 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. as its acetylation (Piperno G. et al., J. Cell Biol., 1985, 101, 2085-2094). its phosphorylation (Gard D. et al., 1985. J. Cell Biol, 1985, 100, 764-774), its glutamylation (Eddé B. et al, Science. 1990, 247, 83-85), its polyglycylation (Redeker V. and al., Science, 1994, 266, 1688-1691) or its tyrosination (Barra HS. et al., 1988, 2, 133-143).

This last 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 (detyrosinated tubulin or tubulin-glu); the passage from one to the other of these forms brings into play two specific enzymes, which act at the level of the carboxy-terminal amino acid Tyr of the α subunit 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 approximately 35% of cerebral tubulin, which has been called tubulin Δ2. It includes two fewer amino acids (Glu ⁴⁵⁰ -Tyr ⁴⁵¹ ), 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 Δ2, normally absent from non-neuronal cells. . The generation of Δ2 tubulin, during tumor growth, associated with the suppression of TTL activity is pathological; Δ2 tubulin therefore constitutes a particularly interesting tumor marker.

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 set themselves the goal to use cells for screening substances capable of inhibiting the activity ^* tubulin carboxypeptidase and therefore offset V TTL elimination of activity during growth tumor 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 tubulin-carboxypeptidase (TCP) activity. Said cells producing tubulin-glu in excess, are particularly obtained, or by inhibiting the renewal dynamics of the assembly of tubulin, or by inhibiting ^the activity TTL.

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 the 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.

Moreover, the cells in which ^the TTL activity is inhibited, including an anti-TTL antibodies, but in which the TCP activity is intact, produce tubulin-glue, while when in the presence of inhibitor of TCP, a restoration of normal levels of tubulin-tyr should be observed. The present invention also relates to ^the use of cells which do not produce tubulin-tyr (cells TTL ^"), for a screening or selection ^of inhibitors of tubulin carboxypeptidase.

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

Such cells are in particular cells derived from NIH / 3T3 cells deposited under the ATCC number CRL 1658 (mouse embryonic fibroblasts) and are obtained after multiple passages in culture; these cells produce tubulin-glu and tubulin-Δ2.

The present invention also relates to the use of a crude or semi-purified extract of TCP for the screening or selection of products that inhibit TCP activity. Said crude extract or semi-purified TCP, catalyzing cleavage of the tyrosine of the main polypeptide chain is capable ^of being obtained by cold homogenizing in a selected organ, followed by fractionated precipitation of the proteins and stages of successive separations in particular by adsorption, dialysis and passage through different chromatography columns. Preferably, said selected organ is subjected to the separation steps 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 the tyrosine has been radiolabelled beforehand (for example, ¹⁴ C-tyrosine or ³ H-tyrosine, in accordance with TM Martensen, cited above).

The disappearance of tyrosine tubulin and the appearance of detyrosine tubulin can also be quantified in immunoassays using the antibodies specific for each of these forms of tubulin. 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 cell or extract of TCP, as defined above, and

- the measurement of tubulin-tyr and / or tubulin-glu by any appropriate means.

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 carried out by an immunoassay in the presence of an antibody specific for tubulin-glu. The applicable methods are as follows:

* Fluorimetric methods:

. direct method: the specific anti-tubulin-tyr and anti-tubulin-glu antibodies are labeled with a fluorescent compound and brought into contact with the cell extract;

. indirect method: unlabeled anti-tubulin-tyr and anti-tubulin-glu antibodies 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.

* Enzyme-linked immunoassays

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 β-galactosidase, is visualized by color reaction with the substrate. Several technical methods 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 attached to 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 an inhibitor of tubulin-carboxypeptidase, as defined above, to obtain ^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 method which is the subject of the present invention, with reference to the accompanying drawings, in which: FIG. 1 illustrates the tyrosination-detyrosination cycle of tubulin;

- 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.

- Preparation of cells HeLa cells are cultured routinely in RPMI medium

1640 supplemented with 10% fetal calf serum. - Cell processing

The treatment is carried out at 37 ° C. Taxol (5 μM, 10 μM or 50 μM) or vinblastine (10 μM) 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. Fixed cells are incubated with anti-tubulin-glu antibodies (1/500) and anti-tubulin-tyr antibodies (YL1 / 2, 1/1000), followed by a second incubation with goat anti-rat antibodies conjugated to rhodamine (Jackson) (to detect anti-tubulin-tyr antibodies) 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 peptide used as immunogen sequence is GEEEGEE (7 amino acid residues from ^the C-terminus 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 α 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. An antibody which inhibits TTL is microinjected as described in J. Wehland et al., J. Cell Science, 1987, 88, 185-203. In the absence of an inhibitor, one will essentially measure tubulin-glu, while in the presence of an inhibitor, one will be able to measure the tubulin-tyr that check 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 NIH / 3T3 cells are cultured on DME medium supplemented with 10% calf serum and 1% fetal calf serum.

The 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 Δ2 antibody, in combination with an anti-tubulin-tyr antibody.

The TTL ^" subclones are those which produce tubulin-glu and tubulin Δ2 in abundance. In these cells, the Glu microtubules represent almost all of the cytoplasmic microtubules, instead of the small sub-population usually observed in different cell types: Tubulin-tyr is scarce and diffuse in cytoplasmic microtubules.

In the presence of an inhibitor of TCP, we can measure appeared tubulin-Tyr and check the disappearance of tubulin-glue double-marking as specified in ^Example 1.

As is apparent from the foregoing, the invention in no way limited to those of its methods of implementation, execution and application which have ^just been described more explicitly; on the contrary, it embraces all the variants which may come to the mind of the technician in the matter, without departing from the framework, or the scope, of the present invention.

Claims

1) Use of cells from ^any other unknown tissue, wherein the tyrosination-detyrosination cycle tubulin is modified cells which overproduce tubulin-glu, for selecting or screening for inhibitors of the activity products tubulin-carboxypeptidase (TCP).

2 °) Use according to claim 1, characterized in that said cells are obtained either by inhibition of the renewal dynamic of the tubulin assembly, or by inhibition of TTL activity.

3 °) Use according to claim 1 or claim 2, characterized in that the dynamics of renewal 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 contacting said cells 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 NIH / 3T3 cells deposited under the N ° ATCC 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.

8 °) 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 cell or extract of TCP, according to any one of claims 1 to 7 and

- the measurement of tubulin-tyr and / or tubulin-glue.

9 °) A method of 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, according to claim 7, in which the tyrosine is radiolabelled beforehand, and

- measurement of free radiolabelled tyrosine.

10 °) Use of a tubulin-carboxypeptidase inhibitor, selected according to the method of claim 8 or claim 9, for obtaining an anti-cancer drug.