FR2850397A1 - Method for identifying antidepressants, based on ability of test compounds to inhibit potassium channels that contain two pore domains, also use of antibodies against these channels for treating depression - Google Patents

Abstract

Method for identifying compounds (I) suitable for treating depression comprises in vitro contact between a test compound and a potassium channel (A) that has two P (pore) domains, or equivalent channels, and measuring any modulation of basal channel activity caused by the test compound. Independent claims are also included for the following: (1) a kit for the method comprising cells that overexpress (A) at the surface and buffers for measuring basal channel activity; and (2) use of antibodies (Ab) directed against (A) to prepare a composition for treatment and/or prevention of depression.

Description

METHOD FOR IDENTIFYING BIOLOGICALLY ACTIVE COMPOUNDS IN THE

TREATMENT OF DEPRESSION

  The present invention relates to the field of the treatment of depression and, more particularly, the implementation of new methods for identifying biologically active compounds in the treatment of said pathology.

  Depression is one of the most common psychological disorders. The risk of developing depression during life varies between 5 and 25%, depending on the study and depending on the subjects tested (men or women).

  Despite this high frequency, this pathology remains unrecognized and seems to involve biological, psychological and / or social factors. Depression is diagnosed when different symptoms are present in patients.

  Among the criteria used in the diagnosis of depression are, in particular, depressed mood, loss of interest or pleasure, unexplained weight loss (or gain), insomnia (or hypersomnia), low energy or fatigue, low self-esteem, etc.

  The most recent and currently the most prescribed antidepressant molecules are selective inhibitors of reuptake of monoamines and especially of serotonin. Antidepressants are generally effective across the spectrum of depressive episodes. However, the response rate is only 70% in the case of mild depression, and this rate decreases with the severity of the episodes (Mental health: new conceptions, new hopes. World Health Report. WHO 2001 , Geneva). In addition, the antidepressants available on the market have side effects. These common side effects of antidepressants are: orthostatic hypotension, drowsiness, insomnia, dizziness, nausea, weight loss or gain, loss of libido .... There is therefore a real need to identify new antidepressants that may be prescribed for patients who are resistant to available treatments and others, and who have fewer side effects than those described above.

  The potassium channel subunits (about 80 genes in mammals) can be grouped into 3 major structural families depending on whether they have 2, 4 or 6 10 transmembrane segments (STM) according to the topology predicted by sequence analysis genetic. The characteristic common to all the potassium channel subunits is the presence of a very conserved motif, the "P" domain, so called because it enters into the constitution of the pore that the channel forms through the plasma membrane. The subunits of the K + channels belonging to the 2 and 6 STM families have only one P domain and assemble in tetramer to form a functional channel. The 4 STM subunits have 2 P domains and operate in dimers (Lesage et al. 1996 EMBO 20 J. 15, 6400-7). It is to this structural family that the channels concerned by the present invention belong.

  The family of potassium channels with 2 P domains comprises 14 members whose corresponding genes are designated 25 KCNK1 to KCNK17 (Lesage & Lazdunski, 2000, Am J Physiol. 279: 793-801): - channels having little inward rectification properties marked, channels TWIK-1 and TWIK-2 belong to this group; - channels activated by unsaturated fatty acids and by stretching, channels TREK-1, TREK-2 (for "TWIKRelated K + channel" and TRAAK (for "TWIK-Related Arachidonic Acid-stimulated K + channel") belong to this group - pH dependent channels activated by alkalosis, TALK1 and TALK2 channels (for "TWIK-related ALkalinactivated K + channel") belong to this group; pH dependent channels inhibited by acidosis, 5 channels TASK1, TASK2 and TASK3 (for "TWIK-Acid-sensitive background K + channel") belong to this group.

  2 P-domain potassium channels were detected in all of the tissues examined, whether in humans, mice or rats. Each channel has its own expression profile giving each tissue a unique combination of potassium channels with 2 P domains. However, studies of expression of channels with 2 P domains carried out in humans and animals, reveal differences in tissue expression depending on the species. As an example, we can cite the expression of the TREKi channel in mouse and human hearts (Lesage & Lazdunski, 2000, Am J Physiol. 279: 793-801).

  Certain potassium channels with 2 P domains, such as the TREK-1, TREK-2 and TASK-1 channels, are activated by volatile anesthetics and therefore constitute potential targets for the identification and development of new anesthetic molecules (Patel et al., 1999, Nature Neuroscience, vol. 2, pages 422-6). This use is the subject of the international patent application published on August 17, 2000 under the number WO 00/47738.

  The inventors have created a line of genetically modified mice lacking potassium channels of the TREK-1 type. They have shown that when these mice are subjected to behavioral tests intended to measure the effectiveness of antidepressant molecules, they exhibit behavior comparable to that of wild mice treated with existing antidepressants. These observations strongly suggest that the potassium channels with 2 P domains, and more particularly the K + channels with 2 P domains chosen from the TREK-1, TREK-2 and TRAAK channels constitute new potential targets for the development of antidepressant molecules.

  Consequently, the present invention relates to the description of molecular targets such as the potassium channels with two P domains and, more particularly, the TREK-1, TREK-2 and TRAAK channels which can be used to identify new biologically active compounds in the treatment of depression. The invention also relates to the methods to be used using these targets for the discovery of new drugs.

  By "biologically active compound", in the context of the present invention, is meant any natural or synthetic chemical molecule as by way of example and in a non-exhaustive manner proteins, peptides, lipoproteins, polysaccharides, chemical compounds simple different from macromolecules, etc. The validation of the antidepressant biological effect of said compound can be carried out using an animal model (including the genetically modified mice described in the present invention), such as mice or rats that undergo recognized tests such as the forced swimming test and the "hanging by the tail" test. These tests are explained in more detail in the experimental part below.

  The present invention relates to a method for identifying a biologically active compound in the treatment of depression comprising the following steps: a) contacting, advantageously in vitro, said biologically active compound with a potassium channel with 2 P domains or a functionally channel equivalent; b) measuring the possible modulation of the basal activity of the potassium channel with 2 P domains or of the functionally equivalent channel by said biologically active compound.

  In a preferred embodiment, step (a) of the method which is the subject of the present invention comprises the following steps: i) culturing cells which express a potassium channel with 2 P domains or a functionally equivalent channel ; ii) incubating said cells with said biologically active compound.

  Advantageously, the cells cultivated in stage (i) of the method which is the subject of the present invention are cells which overexpress a potassium channel with 2 P domains or a functionally equivalent channel. Any cell which can overexpress on its surface, transiently or constitutively, a potassium channel with 2 P domains in a conformation suitable for the transport of potassium is usable within the framework of the present invention.

  Such cells include eukaryotic cells such as yeast, invertebrate cells and vertebrate cells, especially mammalian cells. Mammalian cells which can be used in the context of the invention are, by way of example but not exhaustive, cells from primary cultures, neuronal cells, fibroblasts, cardiac cells, COS cells, cells CHO (for "Chinese Hamster Ovary"), embryonic cells, HELA cells. Vertebrate cells other than mammals which can be used in the context of the invention are, by way of example but not exhaustive, oocytes of amphibians such as oocytes of Xenopus laevis. Invertebrate cells which can be used in the context of the invention are, by way of example but not exhaustive, insect cells such as the Sf9 cells of Spodoptera frugiperda. Yeast cells which can be used in the context of the invention are, by way of example but not limited to, Saccharomyces cerevisiae cells.

  A nucleic acid molecule encoding a potassium channel with 2 P domains or a functionally equivalent channel or a vector as described below can be used to transfect cells which overexpress, transiently or constitutively, a potassium channel at 2 P domains. The introduction of a nucleic acid into cells so as to obtain the expression of the protein encoded by said nucleic acid can be carried out by any method known to those skilled in the art such as transfection methods in cells of suitable nucleic acid constructs or methods of microinjection of RNA encoding said protein into cells. The transfection methods which can be used in the context of the invention are, by way of example and in a non-exhaustive manner, electroporation, calcium phosphate precipitation, lipofection, viral infection, etc.

  By way of examples, a method which can be used in the context of the present invention is briefly described below.

  Those skilled in the art can make any modification to the protocol described without however departing from the scope of the present invention. The sequence encoding a potassium channel with two P domains is inserted between the 5 ′ and 3 ′ non-coding sequences of a Xenopus laevis protein (such as globin) in an appropriate vector such as the pEXO vector (Lingueglia et al. ., 1994, J. Biol. Chem., Vol. 269, pages 13726-39). The construct is introduced into an appropriate cell type for the replication of said vector and / or the transcription of RNA. Alternatively, the vector can be used as a template for in vitro transcription.

  The complementary RNA is transcribed and injected into an appropriate cell, such as an Xenopus laevis oocyte.

  As indicated above, any potassium channel with 2 P domains can be used in the context of the present invention. Advantageously, the potassium channel with 2 usable P domains is chosen from the group comprising the TREK-1 channel, the TREK-2 channel and the TRAAK channel. Consequently, the nucleic acid molecule encoding a potassium channel with 2 P domains or a functionally equivalent channel is, in particular, chosen from a molecule encoding a TREK-1 channel, a molecule encoding a TREK-2 channel and a molecule encoding a TRAAK channel. This nucleic acid molecule can be, by way of example and in a non-exhaustive manner, a mammalian nucleic acid molecule such as a molecule of human, mouse, rat ...

  Those skilled in the art are capable of identifying in databases such as Genbank, nucleic acid molecules which can be used in the context of the present invention.

  In Table 1 below, the references, in Genbank, of potassium channels with two P domains used in the context of the invention and of human origin are presented. 25

Table 1.

  I References Channel TWIK-1 NM 002245 Channel TWIK-2 NM 004823 Channel TWIK-3 NM_033456 NM_033455 NM_033347 NM_033348 NM 005717 Channel TREK-1 NM 014217 Channel TREK-2 NM_138318 NM_138317 NM 021161 Channel TASK-1 NM 002246 NM channel 003724 TRAAK channel NM_016611 NM_033311 NM 033310 TALK-1 channel NM 032115 A person skilled in the art is able to identify, from the sequences of potassium channels with two P domains known in the prior art, sequences of homologs of these 5 channels not yet described. In this identification, a person skilled in the art can use primers specific for each of the channels to identify by PCR the sequence coding for this channel in another species.

  Due to the redundancy of the genetic code, other sequences than the sequences described above and encoding identical amino acid sequences can be used in the context of the invention. These sequences include, but are not limited to sequences comprising all or a portion of a sequence encoding a P-domain potassium channel which has been modified by substitution of different codons which encode equivalent amino acids, thereby producing a silent mutation . By "equivalent amino acid" is meant, for example, an amino acid which belongs to the same class of amino acids as the wild amino acid. Those skilled in the art know the classes of amino acids which include: polar amino acids (hydropobes), - neutral polar amino acids, - positively charged amino acids (basic), - negatively charged amino acids (acids) .

  By "channel functionally equivalent to a potassium channel with 2 P domains>, in the context of the present invention, is meant a channel which has an amino acid sequence close to that of a potassium channel with 2 P domains chosen. This sequence of amino acids has at least 90% identity, preferably at least 95% identity and, most particularly, at least 99% identity with the amino acid sequence of the potassium channel with 2 P domains chosen. In addition, the structural and functional properties between the two channels are identical, those skilled in the art are capable, on the basis of the articles relating to each of the potassium channels with 2 P domains usable in the context of the present invention, of defining protocols. in order to confirm the identity of the properties between these two types of channels.

  The vector which can be used within the framework of the present invention to transform cells overexpressing a potassium channel with two P domains or a functionally equivalent channel comprises at least one nucleic acid molecule encoding a potassium channel with two P domains or a functionally equivalent channel as described above, advantageously associated with control sequences adapted to the method of expression or production of said channels in a cellular host. The vector used is chosen according to the host (cultured cells) into which it is to be transferred; it can be any vector such as a plasmid. The example protocol given above describes the vector pEXO which can be used for the transfection of Xenopus laevis oocytes. The preparation of these vectors as well as the production or the expression of a potassium channel with two P domains in a cellular host can be carried out by all the techniques of molecular biology and genetic engineering well known to those skilled in the art. job.

  The effective overexpression on the surface of a cell transfected with a nucleic acid molecule encoding a potassium channel with two P domains or a functionally equivalent equivalent can be easily verified using antibodies specific for said potassium channel.

  This verification may employ common in situ hybridization techniques.

  A compound capable of modulating the basal activity of an ion channel and, more particularly, of a potassium channel with 2 P domains is a compound which "opens" or "closes" said channels and therefore modulates the ion currents passing to through these channels. A compound capable of opening an ion channel is also called an activator or agonist. A compound that closes an ion channel is called an inhibitor or antagonist. In order to study the modulation of the basal activity of a potassium channel with two P domains, it is necessary to study the modulation of the ionic currents passing through these channels. In the context of potassium channels with two P domains, the ion currents studied are potassium currents. Consequently, the aim of step (b) of the method which is the subject of the present invention is to measure, by all appropriate means, the modulation of the ion currents.

  The results obtained by the inventors on mice in which the gene coding for the TREK-1 protein has been inactivated presented below suggest that compounds capable of "closing" potassium channels with 2 P domains, and, more particularly, of "closing" the potassium channels with 2 P domains chosen from the TREK-1, TREK-2 and TRAAK channels are likely to be used in the treatment of depression. Thus, advantageously, the modulation of 11 the activity of potassium channels with two P domains or of functionally equivalent channels measured in step (b) of the method object of the present invention is an inhibition of the activity of these canals. This inhibition can be partial or complete depending on the biologically active compound tested.

  Those skilled in the art know different techniques capable of measuring the ion currents crossing ion channels such as, for example, the imposed voltage technique, the double microelectrode technique or the patchclamp technique used in the attached cell, whole cell configuration, excised patch or perforated patch. These techniques are known to those skilled in the art who will be perfectly capable of implementing them in the context of the present invention by using cells capable of overexpressing on their surface a potassium channel with two P domains or a channel functionally equivalent (Maingret et al., 2000, J. Biol.

  Chem., Vol. 275, pages 10128-33; "Principles and techniques in electrophysiology", Extract from the thesis of Fabrice Duprat, 20 www.ipmc. cnrs.fr/-duprat/cyberfac/documents/electrophy.pdf).

  All techniques making it possible to detect the activity of an ion channel in vivo or in vitro, by direct or indirect visualization using fluorescent (imaging) compounds radioactive (flow techniques) or others (biosensor, cell death) are also usable. in the context of the present invention.

  It is obvious that the measurement of the modulation of the basal activity of potassium channels with two P domains usable in the context of the present invention requires measuring, first of all, the activity of said channels in the absence of the biologically active compound. . Once this measurement has been made, a measurement under the same experimental conditions but in the presence of the biologically active compound to be tested must be carried out in order to measure the modulation of the basal activity.

  The present invention also relates to a kit 5 for implementing a method for identifying a biologically active compound in the treatment of depression. This kit advantageously comprises cells capable of overexpressing on their surface a potassium channel with two P domains and more particularly a potassium channel with two P domains chosen from the TREK-1, TREK-2 and TRAAK channels. This kit can also contain any buffer necessary for measuring the basal activity of potassium channels.

  The present invention also relates to methods for treating or preventing depression using compounds identified in the methods described above.

  The present invention therefore relates to the use of potassium channel inhibitors with two P domains for the preparation of a medicament intended for the treatment and / or prevention of depression. Advantageously, the present invention relates to the use of potassium channel inhibitors with two P domains chosen from TREK-1 channels, TREK-2 channels and TRAAK channels. By way of examples and in a non-exhaustive manner, we can envisage using as inhibitors of a potassium channel an antibody directed against said channel and, preferably, a monoclonal antibody.

  Other advantages and characteristics of the invention will become apparent on reading the examples which follow concerning the study of depression in mice whose gene kcnk2 coding for the protein TREK-1 has been inactivated. These examples are given by way of illustration and should not be interpreted as limiting the scope of the invention. They refer to the appended figures in which: - Figure 1 shows the duration of immobility of wild TREK1 + / + control mice and Knock Out mice (KO TREK1 - / -) in the "tail suspension" test; - Figure 2 shows the duration of immobility of wild TREK1 + / + control mice and Knock Out mice (KO TREK1 - / -) in the forced swimming test; FIG. 3 compares the duration of immobility, in the context of the forced swimming test, obtained for wild mice not treated or treated with imipramine at 15 mg / kg or with fluoxetine at 16 mg / kg.

I. KO TREK-1 mouse - / -.

  In order to study the physiological role of potassium channels with two P domains, the inventors carried out inactivation (Knock Out, KO) of the kcnk2 gene coding for the TREK-1 protein in mice. The KO mice (TREK1 - / -) were subjected to various tests making it possible to evaluate their behavior.

  The genetically modified mice lacking a TREK-1 channel (TREK-1 KO) were produced by homologous recombination in ES cells (Embryonic Stem cells) of 129 mice, according to techniques of molecular and cellular biology 25 and embryology, well known to those skilled in the art (Hogan et al. 1994. Manipulating the Mouse Embryo 2d ed., Cold spring Harb. Press).

  The targeting vector is constructed from a Bgl2 / EcoRl restriction fragment of mouse genomic DNA 30 129 containing exons 1 to 3 of the KCNK2 gene. The gene is inactivated by deletion of exon 3, which codes in particular for the first transmembrane segment of the TREK1 subunit. The introduction into the targeting vector of the neomycin resistance gene under the control of an independent promoter (PGK Promoter of Phospho Glycetate Kinase) allows the selection of recombination events in clones of ES cells. The vector thus constructed is introduced into ES cells by electroporation. The ES cells are then cultured in a selective medium (antibiotic: G-418) for 8 days. The homologous recombination events are identified by analysis (PCR for Polymerase Chain Reaction and Southern blot technique) of the DNA of the clones of resistant ES cells. Male chimeric mice are generated by injecting positive ES cells into blastocysts of C57Bl / 6 mice. These mice are then mated with C57Bl / 6 females to ensure the germline transmission of the mutation and to found the TREK-1 KO mouse line.

  All of the behavioral studies, and in particular those presented below, were carried out on male TREK-1 KO mice, aged 8 to 10 weeks, originating from the 8th generation of successive crosses on a C57Bl / 6 genetic background. Each animal was subjected to only one test and for only one test.

  II. Depression assessment procedure.

  In mice, tests to assess the state of depression are based on resignation behavior. They measure the inability of the animal to seek an escape from an adversary situation. This absence of will, this resignation, translates a desperation of the animal assimilable to a depressive state. The two tests currently recognized and used to predict the effectiveness of antidepressant substances in humans or animals are: - the forced swimming test or Porsolt test (Porsolt et al., 1977, Arch. Int. Pharmacodyn. , vol. 229, pages 327-336) - the "tail-hanging" test (Crawley, 1999, Brain Research, vol. 935, pages 18-26; Crawley & Paylor, 1997, Hormone and Behavior, vol. 31 , pages 197-211; Cryan et al., 2002, TIPS, vol. 23, pages 1238-45; Nestler et al., 2002, Neuron, vol. 34, pages 13-25). Knock Out TREK1 - / mice were subjected to these two tests.

1. The forced swimming test.

  The mouse is placed in a plexiglass cylinder 30 cm high and 15 cm in diameter, 2/3 filled with water at 28WC, and the swimming behavior of the animal is observed over a period of 6 minutes. After a brief moment of panic, the animal begins to swim. Throughout the duration of the test, the mouse will thus alternate periods of active swimming with periods during which it allows itself to float. The cumulative measurement of these floatation periods during the last 4 15 minutes of the test reflects the depressed state of the animal, in the sense that they correspond to a renunciation to seek an escape from this situation.

  In wild mice, conventional antidepressants such as imipramine (Tofranil) or fluoxetine 20 (Prozac) reduce the flotation time and therefore increase the swimming time.

  2. The "suspension by the tail" test.

  The mouse is suspended by the end of the tail. In this situation, the animal alternates the periods of activity where it struggles while trying to recover and the periods of inactivity where it lets itself hang without movement.

  According to the same principle as in the previous test, the cumulative inactivity time over a period of 6 minutes is measured.

  Also in this test, the antidepressants used in therapy reduce the time of inactivity in favor of the time of activity.

III. Results.

  The results obtained in these various tests are presented in FIGS. 1 to 3. These results show a significant difference between the KO TREK1 - / - mice and the wild control mice (TREKI + / +). This difference 5 is expressed in favor of a difference in the immobility time in each of the tests (Figures 1 and 2).

  In the forced swimming test, the difference in behavior between KO TREK1 - / - mice and control mice is very comparable to that which can be observed during the same test between wild mice, treated with antidepressants. and untreated wild mice. KO TREK1 - / - mice in fact exhibit identical behavior to that of wild mice treated with antidepressants (Figure 3).

  These results therefore indicate the important physiological role that the TREK-1 channel plays in the neuronal processes which generate depressive behaviors and make this channel appear as a target for the development of future antidepressant molecules.

Claims (14)

  1) Method for identifying a biologically active compound in the treatment of depression comprising the following steps: a) bringing said biologically active compound into vitro with a potassium channel with 2 P domains or a functionally equivalent channel; b) measuring the possible modulation of the basal activity of the potassium channel with 2 P domains or of the functionally equivalent channel by said biologically active compound.   2) Method according to claim 1, characterized in that said step (a) comprises the following steps: i) culturing cells which express a potassium channel with 2 P domains or a functionally equivalent channel; ii) incubating said cells with said biologically active compound.   3) Method according to claim 2, characterized in that the cells cultivated in step (i) are cells which overexpress a potassium channel with 2 P domains or a functionally equivalent channel.   4) Method according to any one of claims 2 or 3, characterized in that said cells comprise eukaryotic cells such as yeasts, invertebrate cells and vertebrate cells, in particular mammals.   5) Method according to any one of claims 2 to 4, characterized in that said cells are transfected with a nucleic acid molecule encoding a potassium channel with 2 P domains or a functionally equivalent channel.   6) Method according to any one of claims 2 to 5, characterized in that said cells are transfected with a vector.   7) Method according to claim 6, characterized in that said vector comprises at least one nucleic acid molecule encoding a potassium channel with two P domains or a functionally equivalent channel, advantageously associated with suitable control sequences.   8) Method according to any one of the preceding claims, characterized in that the potassium channel with 2 usable P domains is chosen from the group comprising the TREK-1 channel, the TREK-2 channel and the TRAAK channel.   9) Method according to any one of the preceding claims, characterized in that said step (b) consists in measuring the modulation of the ion currents.   10) Method according to any one of the preceding claims, characterized in that said step (b) consists in measuring the inhibition of the activity of potassium channels with two P domains or of functionally equivalent channels.   11) Method according to any one of the preceding claims, characterized in that said step (b) consists in measuring the closure of potassium channels with two P domains or functionally equivalent channels.   12) Kit for the implementation of a method according to any one of the preceding claims, characterized in that it comprises cells capable of overexpressing on their surface a potassium channel with two P domains and any buffer 5 necessary for the measurement of the basal activity of potassium channels.   13) Use of an antibody directed against a potassium channel with two P domains for the preparation of a medicament intended for the treatment and / or prevention of depression.   14) Use according to claim 13, characterized in that said antibody is an antibody directed against a potassium channel with two P domains chosen from TREK-1 channels, TREK-2 channels and TRAAK channels.