WO2000012755A1

WO2000012755A1 - System for screening compounds with antitumoral, antiviral, antiparasitic and antifungal activity

Info

Publication number: WO2000012755A1
Application number: PCT/ES1999/000275
Authority: WO
Inventors: Juan Carlos Lacal Sanjuan
Original assignee: Consejo Superior De Investigaciones Cientificas
Priority date: 1998-08-28
Filing date: 1999-08-23
Publication date: 2000-03-09
Also published as: AU5518899A

Abstract

Phosphorylcholine (PCho) participates in the transmission of intracellular signals involved in tumoral proliferation. The inhibition of the production of phosphorylcholine (PCho) can be achieved through inhibiting the choline kinase enzyme (Chok). The present invention discloses various alternative and reliable methods for the determination of the activity of the choline kinase ex vivo (in an assay tube, free of cells) or the determination of intracellular levels of Pcho, analysis in vitro (in intact living cells) in the presence or absence of potential compounds having antitumoral activity. The inhibition of the enzyme Chok and the reduction of the levels Pcho would identify said compounds as an antitumoral substance.

Description

TITLE

SCREENING SYSTEM OF COMPOUNDS WITH

ANTITUMORAL, ANTIVIRAL, ANTIPARASITARY AND ACTIVITY

ANTIFUNGICA.

SECTOR OF THE TECHNIQUE

The present invention is related to the design of a new strategy for the identification of both natural and synthetic compounds that exhibit selective activity such as anticancer, antiviral, antiparasitic or antifungal. In summary, the use of phosphorylcholine biosynthesis inhibition is described here, by selective blocking of the choline kinase enzyme, in tumor cells or in cells affected by parasitic infection, as a new strategy for the discovery of compounds that may be used for the treatment of tumors and parasitic diseases or caused by viruses and fungi in animals and humans. The field of invention described herein constitutes a new strategy for the identification of compounds of interest in the treatment of tumors and parasitic, viral and fungal diseases in animals and humans.

LATECH NATIONAL STATE The processes of proliferation, differentiation and programmed cell death

(apoptosis) in higher eukaryotic organisms, three of the phenomena directly related to cell transformation, is performed through communication between cells through complex signal transmission mechanisms (Rozengurt, E. 1986. Science 234: 161-166) . These processes are controlled by diffusible factors that are transported through the bloodstream and are recognized by specific receptors located in the effector cells. In recent years there has been a dramatic advance in the knowledge of the enzyme systems involved in the cascade of signals responsible for the regulation of these cellular functions.

One of the fundamental characteristics of many receptors for growth factors is the receptor-associated tyrosine kinase activity after ligand binding. (Hunter, T. and Cooper, JA 1985. Ann. Rev. Biochem. 54: 897-930; Sibley, DR et al. 1987. Cell 48: 913-922). Through different strategies, numerous intracellular substrates of these receptors have been identified such as PI-PLC, Src, Syp, Shc, GAP-ras and PI3 Kinase (Lacal, JC and Carnero A. 1994. Oncology Reports 1: 677-693). After the connection of these signaling pathways, specific activation of transcription factors, either cytoplasmic or nuclear, that control the expression of the regulatory genes of the cellular response occurs. The oncogenes identified to date can be directly related to the function of any of the structural components of this signal cascade. The cells transformed by these oncogenes show alterations in the signaling pathways used by normal cells, permanently maintaining cascades that under normal conditions are only induced transiently after oncogenic stimulation.

One of the metabolic pathways controlled by growth factors and altered after the oncogenic transformation of more relevance from the point of view of cell proliferation regulation is the metabolism of phospholipids, in particular phosphatidyl inositols (PIs) and phosphatidylcholine ( PC) (Berridge, MJ, and Irvine, RF 1989. Nature 341: 197-205; Pelech, SL and Vanee, DE 1989. Trends in Biochem. Sci. 14: 28-30.). The hydrolysis of phosphatidyl inositol bisphosphate (PIP2) through the action of PLCs causes the formation of inositol-1, 4,5-triphosphate (IP3) and 1,2-diacylglycerol (DAG). The hydrolysis of PC by a PLD originates choline and phosphatidic acid (PA), which are subsequently converted to phosphorylcholine (PCho) and DAG respectively.

Among the various structural components that make up the intracellular signal cascade, the p21-ras protein occupies a prominent place as it functions as a molecular switch that regulates proliferation (Mulcahy, LS et al. 1985. Nature 313: 241-243) and cell transformation (Smith, MR et al. 1986. Nature 320: 540-543).

There is abundant information that establishes a clear connection between growth factors, phospholipid metabolism and oncogenes. Thus, in cells transformed by the ras, src, sis and fms oncogenes, the basal levels of different phospholipid metabolites are, constitutively, above those presented by normal cells (Wolfman, A. and Macara, IG Nature 325: 359-361; Wolfman, A. et al. 1987. J. Biol. Chem. 262: 16546-16552; Lacal, JC et al. 1987. Nature: 330: 269-272; Fleischman, L. et al. 1994. Science 231: 407-410; of Peso L. et al., 1997. Biochem. J. 322: 519-528). On the other hand, PI3K has been identified as an effector of the Ras protein (Rodríguez-Viciana, P. et al. Nature 370: 527-532). These results suggest the existence of different levels of phospholipid metabolism regulation in cells transformed by oncogenes; hence the importance of knowledge of the factors involved in the regulation of these metabolic pathways in cell transformation.

In previous studies of our group it has been observed that in cells transformed by various oncogenes the levels of certain lipid metabolites such as DAG and phosphorylcholine (PCho) are increased with respect to normal cell levels (Lacal, JC et al. 1987, Nature 330: 269-272; Carnero A. et al., 1994. Oncogene 9, 1387-1395; del Peso L. et al., 1997. Biochem. J. 322: 519-528). The production of DAG and PCho comes from the activation of a PC-PLD that generates phosphatidic acid (PA) and choline, which are subsequently converted into DAG and PCho by the action of PA-hydrolase and choline kinase, respectively (Carnero, A ., Square, A., of the Peso, L. and Lacal, JC 1994. Oncogene 9: 1387-1395).

These conclusions are based on the observation that an increase in intracellular phosphorylcholine (PCho) levels occurs in serum stimulated cells or growth factors individually. At short times, counted from the start of the stimulus, (less than 5 minutes) there is an increase of transient PCho, and at long times (more than 3 hours of stimulation) there is an increase greater than about 2-3 times the levels basals (Jiménez, B., del Peso, L., Montaner, S., Esteve, P. and Lacal, JC 1995: J. Cell. Biochem. 57: 141-149). In cells transformed by different oncogenes, it is also appreciated that PCho levels are constitutively increased. The production of PCho after mitogenic stimulation or transformation by oncogenes is due to choline kinase activity, since its total disappearance is achieved through the action of inhibitors of this enzyme (Ram, A., Square, A., Weight, L. and Lacal, JC 1994. Oncogene 9: 1387-1395; Jiménez, B., del Peso, L., Montaner, S., Esteve, P. and Lacal, JC 1995: J. Cell. Biochem. 57: 141-149 ; Cuadrado, A., Carnero, A., Dolfi, F., Jiménez, B. and Lacal JC 1993. Oncogene 8: 2959-2968; Hernández-Alcoceba R., Saniger L., Campos J., Núñez MC, Khaless F., Gallo MA, Espinosa A., and Lacal JC 1997. Oncogene 15, 2289-2301). Choline kinase inhibitors not only completely block phosphorylcholine production induced by growth factors and serum or cells transformed by oncogenes, but drastically inhibit cell proliferation, both in normal and transformed cells. This inhibition is reversed when phosphorylcholine is added to the cells or by the addition of serum, reinforcing the concept of specific inhibition through the enzyme choline kinase (Ram, A., Square, A., Weight, L. and Lacal, JC 1994. Oncogene 9: 1387-1395; Jiménez, B., del Peso, L., Montaner, S., Esteve, P. and Lacal, JC 1995: J. Cell, Biochem. 57: 141-149; Square, A ., Carnero, A., Dolfi, F., Jiménez, B. and Lacal JC 1993. Oncogene 8: 2959-2968).

Given the importance of choline kinase in the mitogenic signal transduction system, the possible role of PCho, product of choline kinase, as a second messenger has also been studied. Our results clearly demonstrate that PCho is necessary for the mitogenic activity of growth factors in NIH 3T3 cells (Jiménez, B., del Peso, L., Montaner, S., Esteve, P. and Lacal, JC 1995: J. Cell. Biochem. 57: 141-149). In the presence of choline kinase inhibitors, cell growth is drastically inhibited in cells stimulated by growth factors such as PDGF or FGF, or in cells transformed by oncogenes such as ras or sis. The addition of insulin, however, reverses this effect (Jiménez, B., del Peso, L., Montaner, S., Esteve, P. and Lacal, JC 1995: J. Cell. Biochem. 57: 141-149; Cuadrado, A., Carnero, A., Dolfi, F., Jiménez, B. and Lacal JC 1993. Oncogene 8: 2959-2968). Also, in the presence of serum, the inhibitory effect of these compounds is not observed. Therefore, the inhibition of the mitogenic capacity of growth factors and oncogenes by choline kinase inhibitors is not due to a nonspecific cellular toxicity process, but to the selective and specific blockage of intracellular signaling pathways. This effect of inhibiting cell proliferation is reversed by the addition of PCho, thereby highlighting the specificity of the system.

In summary, all these previous results demonstrate a high specificity of inhibition of cell proliferation by choline kinase inhibitors. Choline kinase is, therefore, an important step in the regulation of cell proliferation and, as a consequence, all compounds that manifest choline inhibition activity Kinase are excellent candidates for the development of agents with antitumor activity. The design of derivatives that directly affect choline kinase activity may allow the development of effective antitumor therapies.

It is noteworthy that the discovery of compounds with potential antitumor, antiviral, antiparasitic or antifungal activity is based on the analysis of their inhibitory capacity of cell proliferation in cultured cell systems, or complex analysis systems of viral replication inhibition , of parasites or fungi in appropriate crops (XX). It has been proven that these human pathogens can also require the generation of phosphorylcholine for survival (Harnett W., Parkhouse RME, 1995, Perspectives in nematode physiology and biochemistry, pp. 207-242, M / S Narendra Publication House, New Delhi ) and this suggests the possibility of using choline kinase inhibitors to combat the diseases they produce. Therefore, a rapid and economical analysis system that serves to identify compounds capable of inhibiting choline kinase is an effective mechanism for identifying molecules with potential for use as antitumor, antiviral, antiparasitic and antifungal.

DESCRIPTION OF THE INVENTION

The tests to be used for screening compounds with possible antitumor activity are based on their ability to inhibit the production of phosphorylcholine (PCho). The phosphatidylcholine (PC) synthesis scheme can be summarized in the following biological process:

1 2 3 choline -> phosphorylcholine -> CDP-choline -> phosphatidylcholine (Pcho) (PC) where 1 corresponds to choline kinase (EC 2.7.1.32), 2 to citidylyl transferase (EC 2.7.7.15), and 3 to choline phosphotransferase (EC 2.7.8.2).

The original idea, on which the patent application is based, implies that PCho is used by cells for a signal transmission path, alternative and different from that of PC synthesis, essential for the regulation of cell growth. This hypothesis has been published (Jiménez, B., del Peso, L., Montaner, S., Esteve, P. and Lacal, JC 1995: J. Cell. Biochem. 57: 141-149) by the applicant researcher. Finally, we have also shown that cells transformed by various oncogenes have elevated levels of PCho due to the activation of the choline kinase-dependent pathway. These works have been published recently (Square, A. et al., 1993. Oncogene 8: 2959-2968; Carnero A. et al, 1994. Oncogene 9, 1387-1395; del Peso L. et al., 1997. Biochem J. 322: 519-528). Based on these findings, it can be concluded that cell proliferation of tumor lines can be drastically and specifically inhibited by directly inhibiting choline kinase, directly responsible for the production of PCho. Inhibition of PCho production can be achieved by inhibition of the choline kinase enzyme (ChoK). The compounds that are intended to be identified as potentially interesting for study as antitumor agents are choline kinase inhibitors of both natural and synthetic origin. To measure their activity, both ex vivo systems (in test tube, cell-free) and in vitro (in intact living cells) can be used. Various alternative and reliable methods are proposed for the determination of choline kinase activity ex vivo or the determination of intracellular levels of PCho (in vitro analysis) and which are part of the present invention. On the other hand, choline kinase occurs in yeasts, fungi, algae, plants, parasites and in different animal species (mammals, amphibians, fish, insects, ...) so that each of these choline kinase can be used, specific to each organism, in each of the tests described in the present invention and which are part of it. The use of these choline specific kinases of different organisms would allow to identify more specific compounds of each one of these organisms, which will result in a greater efficacy thereof. Within the scope of the aforementioned invention, two tests are described for the analysis of a large number of chemical compounds of both natural origin and synthetic structures for which both the use of the strategy and its design are claimed, and the identification of those compounds with potential subsequent use in the field of chemical therapeutics. In the following examples, although without limiting the possibilities, the general characteristics of both trials are described although they may there are variations of some of the parts thereof that would allow qualitative improvements of the results obtained by them, and which are part of the present invention. These variations may include: 1) the use of choline kinase contained in a cell extract that produces this protein, 2) the use of ion exchange HPLC as an alternative method of detecting Pcho levels, 3) the use of radiolabelled choline or by fluorimetric methods in the analysis of the determination of choline kinase activity, 4) the use of partially or totally purified choline kinase, 5) the tests are carried out in the liquid phase or in the solid-liquid phase.

EXAMPLES

Example 1.- Ex vivo assay of choline kinase inhibitors.

In this assay choline kinase from yeast, commercially available or from mammalian cells, including humans, can be used. The experimental procedure is as described below:

- The 10X reaction buffer (1M Tris pH 8.0, 100 raM MgC12 and 2 mM is prepared

(14C) -methyl-Hill (20 μCi / ml), 100 mM ATP),

A solution of yeast choline kinase (50 mU / ml) is prepared from a commercial preparation of lU / ml, - The different concentrations of the compounds to be analyzed are prepared, with a concentration of 5X in water,

20 ml (milliliters) of buffer + 10 μl of the compound (or water in the controls) + 10 μl water + 10 μl choline kinase are mixed,

Incubate for 45 min. at 37 ° C, - The reaction is stopped by adding 10 μl 500 mM EDTA and the samples are kept on ice,

Equivalent aliquots (30 μl of each sample) are resolved by thin layer chromatography using silica gel plates as support and as mobile phase the following buffer: 0.9% sodium chloride: methanol: ammonium hydroxide (50: 70: 1; v: v: v). Synthetic choline and phosphorylcholine standards are met in parallel radioactive as indicators,

The radioactive spots corresponding to choline and PCho are identified and quantified by the liquid scintillation technique (the corresponding spots are scraped after exposure of a radioactive sensitive film) or by a conventional automatic electronic system,

- The effect of each compound analyzed on the production of PCho is determined by normalizing the radioactivity values of this metabolite against the total radioactivity levels (choline + PCho) in each sample.

Example 2.- In vitro assay of choline kinase inhibitors.

In this assay both mouse cells and available human cells can be used as immortalized cultures:

Mouse or human cells are seeded in 6-well plastic plates in the appropriate medium (DMEM or equivalent supplemented with 10% fetal or newborn calf serum). 200,000 cells are added per well. They are grown until they reach confluence in an incubator with 5% CO2, 95% air and 90% humidity, at 37 ° C,

- Normally at 48 hours the confluence is reached. At this point the plates are washed with TD buffer (50 mM sodium phosphate, 150 mM sodium chloride, 5 mM potassium chloride, pH 7.4) and 2 ml / well of DMEM medium supplemented with 0.5% of calf serum is added. An increasing amount of the compounds to be analyzed, dissolved in the same medium, is added to each well so that an increasing curve is achieved that covers a range of at least 1000 times the lowest concentration used, which will be around 0.1 micromolar (μM). It is incubated for one hour under standard conditions,

0.5 μCi / well of (14C) methylcholine is added as a radioactive precursor. They are incubated under standard conditions of CO2 and humidity for 12-14 hours. The cells are washed with TD buffer and processed for determination of cellular content in PCho. 1 ml / well of a 16% aqueous solution of trichloroacetic acid (TCA) is added). Leave 1 hour in cold room (4 ° C). East processing fixes the cells in the wells and extracts the soluble material in the TCA, containing the metabolites choline and Pcho,

Supernatants are collected in 15 ml tubes resistant to organic solvents. The wells are washed with 1 ml of 16% TCA or mixed with the previous samples,

The cells fixed by the TCA in the wells are dissolved in 0.5 ml of 0.5 N sodium hydroxide and the radioactivity incorporated in the material insoluble in TCA (mainly phosphatidylcholine) is determined by standard methods by counting liquid scintillation in a counter β emissions, - TCA extracts are washed with four volumes of water-saturated ether. When mixing the samples with the ether, two phases are formed. They are vigorously agitated and the upper phase, which is undone, is aspirated. The process is repeated three more times. The samples are neutralized with 50 μl of Tris Base buffer (pH 10) and shaken vigorously to standardize the solution. - The samples are transferred to 1.5 ml eppendorf or similar tubes and dried by lyophilization. in a vacuum centrifugation system, the samples are resuspended in 0.1 ml of distilled water and equivalent aliquots are resolved by thin layer chromatography using silica gel plates as support and as 0.9% sodium chloride: methanol mobile phase : ammonium hydroxide (50: 70: 1). At the same time, synthetic radioactive choline and phosphocholine standards are resolved in parallel as indicators,

The radioactive spots corresponding to choline and PCho are identified and quantified by liquid scintillation (the corresponding spots are scraped after exposure of a radioactive sensitive film) or conventional automatic electronic systems (directly),

- The effect of each compound analyzed on PCho production is determined by normalizing the radioactivity values incorporated in the metabolite (choline or PCho) against the radioactivity levels incorporated in the insoluble material.

Claims

1.- Method of identification of new antitumor, antiviral, antiparasitic and antifungal compounds, characterized by the use of inhibitory activity on choline kinase protein as an identifying criterion.

2. Method according to claim 1 characterized in that choline kinase is of diverse origin, selected from the following group: yeast, fungi, plants, mammalian cells, algae, insects, amphibians and fish.

3. Method according to claim 2 characterized by being an ex vivo analysis system of the inhibitory capacity of said compounds on choline kinase protein and being constituted by the following steps:

- The 10X reaction buffer (1M Tris pH 8.0, 100 mM MgC12 and 2 mM (14C) -methyl-Choline (20 μCi / ml), 100 mM ATP) is prepared,

A solution of yeast choline kinase (50 mU / ml) is prepared from a commercial preparation of lU / ml,

The different concentrations of the compounds to be analyzed are prepared, with a concentration of 5X in water,

Mix 20 ml (milliliters) of buffer + 10 μl of the compound (or water in the controls) + 10 μl water + 10 μl choline kinase, - Incubate 45 min. at 37 ° C,

The reaction is stopped by adding 10 µl 500 mM EDTA and the samples are kept on ice,

Equivalent aliquots (30 μl of each sample) are resolved by thin layer chromatography using silica gel plates as support and as mobile phase the following buffer: 0.9% sodium chloride: methanol: ammonium hydroxide (50: 70: 1; v: v: v). Synthetic standards of radioactive choline and phosphorylcholine are resolved in parallel as indicators,

The effect of each compound analyzed on PCho production is determined by normalizing the radioactivity values of this metabolite against the total radioactivity levels (choline + PCho) in each sample. 4. Method according to claim 2 characterized by being an in vitro analysis system of the inhibitory capacity of said compounds on the choline kinase enzyme and for being constituted by the following steps:

- Normally at 48 hours the confluence is reached. At this point the plates are washed with TD buffer (50 mM sodium phosphate, 150 mM sodium chloride, 5 mM potassium chloride, pH 7,

4) and 2 ml / well of DMEM medium supplemented with 0.5% calf serum are added. An increasing amount of the compounds to be analyzed, dissolved in the same medium, is added to each well so that an increasing curve is achieved that covers a range of at least 1000 times the lowest concentration used, which will be around 0.1 micromolar (μM). It is incubated for one hour under standard conditions,

0.5 μCi / well of (14C) methylcholine is added as a radioactive precursor. They are incubated under standard conditions of CO2 and humidity for 12-14 hours. The cells are washed with TD buffer and processed for determination of cellular content in PCho. 1 ml / well of a 16% aqueous solution of trichloroacetic acid (TCA) is added. Leave 1 hour in cold room (4 ° C). This processing fixes the cells in the wells and extracts the soluble material in the TCA, containing the metabolites choline and Pcho,

Supernatants are collected in 15 ml tubes resistant to organic solvents. The wells are washed with 1 ml of 16% TCA and mixed with the previous samples, The cells fixed by the TCA in the wells are dissolved in 0.5 ml of 0.5 N sodium hydroxide and the radioactivity incorporated in the material insoluble in TCA (mainly phosphatidylcholine) is determined by standard methods by counting liquid scintillation in a counter β emissions, - TCA extracts are washed with four volumes of water-saturated ether. When mixing the samples with the ether, two phases are formed. They are vigorously agitated and the upper phase, which is undone, is aspirated. The process is repeated three more times. The samples are neutralized with 50 μl of Tris Base buffer (pH 10) and shaken vigorously to standardize the solution. - The samples are transferred to 1.5 ml eppendorf or similar tubes and dried by lyophilization. in a vacuum centrifugation system, the samples are resuspended in 0.1 ml of distilled water and equivalent aliquots are resolved by thin layer chromatography using silica gel plates as support and as 0.9% sodium chloride mobile phase: methanol : ammonium hydroxide (50: 70: 1). At the same time, synthetic radioactive choline and phosphocholine standards are resolved in parallel as indicators,

5. Method according to any one of claims 1 to 4 characterized in that the choline kinase is contained in a cell extract that produces the choline kinase protein.

6. Method according to claim 5 characterized in that the production of choline kinase protein is natural or induced by genetic engineering.

7. Method according to any one of claims 1 to 4 characterized in that the method of detection of Pcho levels is performed by ion exchange HPLC.

8. Method according to any one of claims 1 to 4, characterized in that the determination of choline kinase activity is analyzed by radiolabelled choline or by fluorimetric methods.

9. Method according to any one of claims 1 to 4 characterized in that the choline kinase used has been at least partially or totally purified.

10. Method according to any one of claims 1 to 4 characterized in that it is carried out in the liquid phase to form a reaction product that separates from the reaction mixture after completing it.

11. Method according to any one of claims 1 to 4 characterized in that it is carried out with the immobilized choline kinase before the reaction so that the reaction is carried out in a solid-liquid phase.