DE10337904A1 - Using apoptosis-inducing or stimulating agent for treating disease, particularly infections or tumors, acts by interaction with mitochondrial potassium channels - Google Patents

Abstract

Use of an apoptosis-inducing and/or -stimulating substance (AIS) that interacts with mitochondrial potassium channels (MK) to prepare a composition for preventing and/or treating diseases associated with reduced apoptosis. Use of at least one apoptosis-inducing and/or -stimulating substance (AIS) to prepare a composition for prevention and/or treatment of human or animal diseases which involve (or are associated with) excessive reduction in apoptosis and/or, particularly, excessive cell growth and/or division, or in which targeted apoptosis of particular cells is sought. The new feature is that AIS acts by interaction with mitochondrial potassium channels (MK) of the cells concerned. An independent claim is also included for a method for discovery or identification of AIS. ACTIVITY : Parasiticide; Antibacterial; Virucide; Anti-HIV; Hepatotropic; Cytostatic; Apoptotic. No details of tests for these activities are given. MECHANISM OF ACTION : Blocking and/or inhibiting mitochondrial potassium channels; interaction with natural antiapoptotic substances.

Description

The The present invention relates to the use of apoptosis-inducing or apoptosis-stimulating substances (AIS) for the production of medicines or medicines for the prophylactic or curative treatment of diseases of the human or animal body, where a particular excessive reduction of apoptosis or a particular excessive increase in cell growth and / or Cell division occurs or related or in which a therapeutically targeted apoptosis of the respective Cells is intended, and a corresponding treatment method.

Of Furthermore, the present invention relates to a method for inducing or stimulation of apoptosis in cells in which a particular excessive reduction of apoptosis and / or a particular excessive increase in cell growth and / or Cell division occurs or related or in which a therapeutically targeted apoptosis of the cells in question It is intended by influencing mitochondrial potassium channels affected cells by apoptosis-inducing or apoptosis-stimulating Substances.

Finally, concerns the present invention provides a method for finding or identifying apoptosis-inducing or apoptosis-stimulating substances which are selected or that they are by interaction or interaction with mitochondrial potassium channels of the relevant cells induce apoptosis of these cells or stimulate.

apoptosis, sometimes also referred to as programmed or physiological cell death, is a natural process occurring in the organism and poses a genetically fixed form of cell death caused by certain circumstances or factors can be activated. Enable apoptotic processes in a complex, multicellular Organism the controlled maintenance and regulation of the Cell equilibrium, so that these operations in relation to the creation and preservation of life of fundamental Meaning are. In this context, as an important process beispielswei se Embryonic development, in connection with the other Development of irrelevant cells or tissue structures targeted Apoptosis can be eliminated, as for example for the so-called Interdigital folds is the case. Furthermore, the apoptotic Regulation of the immune system, in particular a control certain immune cells takes place, for example, Abwehneaktionen then stop if the corresponding pathogens succeed have been eliminated. The specific death, for example such cells having a viral infection is another effective immunoregulatory action associated with Apoptosis is. But also damaged, Degenerate cells die by targeted apoptotic processes, thus they can not develop a manifest cancer. For example cytotoxic T cells activate an apoptosis program in Cells with altered HLA structure (Humane Leukocyte antigen structure).

Indeed the natural works Control of apoptosis not always, so that serious illnesses result can, which are associated, for example, with increased cellular apoptosis, such as neurodegenerative Diseases or in which there is a decreased rate of apoptosis, as it is z. For example Cancers may be the case.

In It is related to the study of pathological apoptosis processes Therefore, a primary goal, the process of apoptosis in the desired Way to therapeutically influence. A prerequisite for this is that on the factors involved in apoptosis and their functioning are. About that addition, the therapeutic intervention should be due to apoptosis their general meaning advantageously selective and specific be to avoid side effects. In this context is therefore the enlightenment the specific signaling pathways of apoptosis on cell physiology Level, especially at the molecular level.

Here apparently play pro-apoptotic Bc1-2-like proteins, such as Bax, Bad or Bak, in many forms of apoptosis, for example, after stimulation various proapoptotic receptors, treatment with ionizing Radiation or UV light, a crucial role.

Although in almost all forms of apoptosis Bc1-2-like molecules play a crucial role, the cellular mechanisms of action of these molecules are still largely unknown. One can divide Bc1-2-like proteins into two groups, namely pro-apoptotic and anti-apoptotic members of this protein family. Pro-apoptotic Bc1-2-like proteins are, for example, Bax, Bak, Bok, Bad, Bim, Bmf, Bid, Noxa or Puma. Anti-apoptotic family members are, for example, Bc1-2 itself, Bc1-x _L , Bc1-w, Mc1-1, Boo / Diva and A1 / Bf1-1. Some viral proteins, such as. B. E1B19K or BHRF1, are homologous to Bc1-2 and also inhibit apoptosis. All Bc1-2-like proteins contain conserved α-helical domains, called BH domains. A total of four homologous BH domains are known, of which only BH3 is present in all Bc1-2-like proteins, both anti-apoptotic and pro-apoptotic proteins, can be found. The exact molecular function of these BH domains is not yet known, but they seem to serve the interaction between different Bc1-2-like proteins.

Bc1-2-like proteins appear to be regulated by several mechanisms. Thus, the anti-apoptotic molecules Bc1-2 and Bc1-x _{L associate} with pro-apoptotic molecules such as Bax or the BH-3 proteins. As a result, the pro-apoptotic molecules appear to be neutralized in their action and to be blocked by apoptosis. The association of Bc1-2-like proteins with 14-3-3 proteins, the interaction of Bim with dynein, Bmf with the actin cytoskeleton or the binding of phosphorylated bath to certain areas of the cell membrane, the so-called lipid, also act via a similar mechanism rafts. Finally, some pro-apoptotic Bc1-2-like proteins are also inactivated via PKB / Akt-dependent phosphorylation. Dephosphorylation activates the pro-apoptotic activity of these Bc1-2-like proteins.

The most concepts about the mechanisms of action Bc1-2-like Proteins currently assume that these proteins in a corresponding apoptotic stimulus from the cytosol or others cellular Bind compartments to mitochondria and induce apoptosis there. At the moment it is not clear like pro-apoptotic Bc1-2-like proteins act at the molecular level. It has been described that Bax on the adenine nucleotide translocator of mitochondrial permeability Transition pore (PTP), a large and unselective mitochondrial Internal channels, binds and thus regulates the function of PTP. To This idea results from a commitment of Bax to the PTP activation of the channel, as a result, the mitochondrial membrane depolarized and induced apoptosis. The mechanisms of regulation the PTP by Bax and the physiological importance of the interaction for apoptosis are still unknown.

One another concept about the function Bc1-2-like Proteins were derived from the structure of these proteins. structural analysis showed a homology Bc1-2-like Proteins with the diphtheria toxin, which led to the hypothesis that pro-apoptotic Bc1-2-like Proteins in the mitochondrial membrane form its own channel and thereby depolarize the mitochondria. This concept is based on the assumption of the function of Bc1-2-like Proteins as ion channels, but under physiological conditions - at least so far - not approved could be.

The WO 00/06187 A2 relates to the modulation of apoptosis, in particular a method for inducing apoptosis for the treatment of decreased cell apoptosis in related disease states. The WO 00/06187 A2 is based on the previously described, but for physiological cell processes so far unproven concept that proapoptotic proteins and above In addition, anti-apoptotic proteins of the Bc1-2 family have large channels in the Form mitochondrial membrane, in the case of pro-apoptotic Proteins formed, chloride-selective channels cytochrome c from the mitochondria unlike those of anti-apoptotic proteins formed channels potassium selective as well as for Cytochrome c are not permeable. That described in WO 00/06187 A2 The aim of the method is therefore to provide substances form in mitochondrial membranes channels or pores with high cytochrome c permeability and thus cause apoptosis or such substances available represent the properties of channels that are made of anti-apoptotic Proteins are formed, should change so that it finally to Apoptosis of the cell comes. A disadvantage of the in WO 00/06187 A2 described method is in particular to see that it - as already portrayed - on one for physiological cell processes so far not proven concept is based.

WO 02/40025 A1 describes the induction of apoptosis, in particular in inflammation-specific cells, such as eosinophils, for the treatment and prevention of inflammatory diseases, using substances that specifically interact with mitochondrial K _ATP channels (ie mitochondrial, ATP-dependent potassium channels) and lead to the opening of these channels, which should take place as a result of apoptosis in the affected cells. The method described in WO 02/40025 A1 thus aims exclusively at ATP-dependent mitochondrial potassium channels as a pharmacological target; In addition, WO 02/40025 A1 is limited to the treatment of inflammatory diseases such as rhinitis, myocarditis or arthritis.

Ultimately are the molecular mechanisms that govern Bc1-2-like Proteins regulate apoptosis, still unknown. But this protein family of paramount importance in the regulation of apoptosis is possible an identification of these mechanisms completely new approaches to Therapy of diseases of the human or animal body, at an especially excessive reduction of apoptosis and / or a particular excessive increase in cell growth and / or Cell division occurs or related or in which a therapeutically targeted apoptosis of the respective Cells is intended.

The previously described methods of Stan In the prior art, the art does not provide an efficient therapy option with regard to diseases of the human or animal body in which an especially excessive reduction of apoptosis and / or an especially excessive increase of cell growth and / or cell division occurs or which are associated therewith or in which one therapeutically targeted apoptosis of the cells in question is intended.

A The object of the present invention is now to provide an efficient curative or prophylactic treatment option for diseases of the human or animal body to provide a therapeutically targeted apoptosis the cells in question is intended or in which an excessive reduction of apoptosis (i.e., reduced or decreased cellular apoptosis) or an excessive increase of Cell growth or cell division occurs or related herewith or in which a therapeutically targeted apoptosis of the respective Cells is intended.

A Another object of the present invention is a method for finding or identifying corresponding active substances which for use in the context of the treatment of the aforementioned diseases of the human or animal body.

According to one The first object of the present invention relates to the present invention Invention thus the use of at least one apoptosis-inducing or apoptosis-stimulating substance (AIS) for the production of a Drug or drug for prophylactic or curative Treatment of diseases of the human or animal body, at an especially excessive reduction of apoptosis or a particular excessive increase in cell growth or Cell division occurs or related or in which a therapeutically targeted apoptosis of the respective Cells is intended, wherein the apoptosis-inducing and / or apoptosis-stimulating Substance (AIS) selected in such a way or that they are by interaction or interaction with mitochondrial potassium channels (MK) induces apoptosis of these cells or stimulated.

in the The scope of the present invention is under apoptosis induction or Apoptosestimulierung in particular a targeted change or influencing, in particular an increase or increase in the natural To understand apoptosis of the cells in question. Under the term According to the invention, apoptosis induction can be a direct one in particular or active initiation of the apoptosis process in the relevant Cells are understood while the term apoptosis stimulation according to the invention as a particular indirect Initiation of the apoptosis process can be understood, for example as a result, in particular, an irritation or stimulation of the relevant Cells.

Under The term of the relevant cells are within the scope of the present To understand the invention of such cells, by excessively reduced apoptosis or excessive increase of Cell growth or cell division affected or affected or in which a therapeutically targeted apoptosis is intended and involved in the clinical picture or course or determine the clinical picture. For this case, the treatment takes place curative or therapeutic. Continue to count among the cells in question Also, such cells, for example, under a progressive or chronic disease progression in the future from an excessively diminished Apoptosis or excessive elevation of Cell growth or cell division are affected or affected could in this case the treatment is on a prophylactic level, d. H. before the onset of decreased apoptosis or before onset the excessive increase of Cell growth or cell division. For example, to the cells concerned also eukaryotic pathogens, provided in particular mitochondrial potassium channels (MK), in particular the Kv1.3 potassium channel of the inner mitochondrial membrane, own, but also the body's own Cells in which a therapeutically targeted apoptosis intends is, for example, cells that are caused by pathogens, such as bacteria or viruses, are counted become.

Surprisingly, the inventors of the present invention have found that in most cells of the human or animal body, particularly in cells exhibiting or being associated with an excessive reduction in apoptosis or an increase in cell proliferation or cell division associated therewith where a therapeutically targeted apoptosis is intended, mitochondrial, in particular in the inner mitochondrial membrane located, generally voltage-dependent potassium channels, in particular Kv1.3 potassium channels or their homologs exist (ie, natural and permanently present), which - if they interact with pro-apoptotic substances, in particular naturally occurring pro-apoptotic substances, such as pro-apoptotic Bc1-2-like proteins, such as. B. Bax, - lead to the triggering of apoptosis of the cells in question, and that the targeted influence, in particular the inhibition or blockade, these potassium channels with apoptosis-inducing or apoptosis-stimulating Substan zen (AIS) is an efficient measure to induce or stimulate apoptosis of the cells in question, and consequently with excessively reduced apoptosis or excessive increase of cell growth or cell division-related diseases or related diseases which a therapeutically targeted apoptosis of the cells in question is intended to treat. In this way, a very efficient therapy option for such diseases is provided according to the invention, which selectively or selectively intervenes in the apoptotic cell processes. In the context of the present invention, the term interaction of apoptosis-inducing and / or apoptosis-stimulating substances (AIS) on the one hand and mitochondrial potassium channels (MK) on the other hand is understood as influencing the activity of the mitochondrial potassium channels (MK) in the respective cells such that apoptosis is induced or stimulated.

Because the inventors of the present invention have succeeded in demonstrating that the in the mitochondria expressed potassium channels (especially those in the innermitochondrial membrane, in particular voltage-dependent potassium channels, in particular Kv1.3 potassium channels and their homologues) with pro-apoptotic Bc1-2-like proteins, such as. Bax, and Bc1-2-induced (eg, Bax-induced) Mediate apoptosis, so that an "imitation" or activation this interaction or interaction to an induction or Stimulation and thus an increase in apoptosis as such leads and thus an efficient treatment option with regard to the occurrence of excessively reduced apoptosis or excessive increase of Cell growth or cell division related diseases or Diseases in which a therapeutically targeted apoptosis of the intended cells. Pro-apoptotic proteins the Bc1-2 family, such. B. Bax, so interact or interact for example, with Kv1.3 potassium channels of the inner mitochondrial membrane (IMM) and can about this Mechanism induce apoptosis. Here, for example, the Kv1.3 channels the inner mitochondrial membrane (IMM), in particular by the proapoptotic Proteins of the Bc1-2 family, such as. B. Bax, so blocked or inhibits that in induced or stimulated apoptosis of the cells in question becomes. By the term blockade or blocking or inhibiting For the purposes of the present invention, in particular a change of channel-specific, z. B. biophysical parameters, such as a decrease For example, the channel conductance, the channel open time (lifetime) and / or the likelihood of the channel being understood. The interaction or interaction of the pro-apoptotic proteins of the Bc1-2 family, such as B. Bax, especially with the Kv1.3 channels of the inner mitochondrial membrane (IMM) can, for example, a direct, in particular a physical and / or chemical interaction, or an indirect one Be interaction.

Though is the existence of, for example, Kv1.3 channels in the outer cell or cytoplasmic membrane per se prolonged However, it was unknown until the time of the invention that also in Mitochondrial membranes, especially in the inner mitochondrial membranes, such potassium channels are present and that these mitochondrial potassium channels are of paramount importance in terms of apoptotic cellular processes and hence an efficient target for regulation, in particular a trigger represent the cell apoptosis.

In their experiments, the inventors were able to detect the existence of voltage-dependent potassium channels, in particular Kv1.3 potassium channels or homologous channels, in the inner mitochondrial membrane (IMM). In addition, the inventors have been able to show, by means of experiments on complete cell systems and on isolated mitochondria, that the functional expression of these channels in mitochondria is of great importance with regard to apoptotic processes. Thus, it has been demonstrated that the presence of voltage-dependent mitochondrial potassium channels, in particular Kv1.3 potassium channels or homologous channels localized in particular in the inner mitochondrial membrane (IMM), with respect to apoptosis induction, in particular by pro-apoptotic Bc1-2 similar proteins, such as Bax, is necessary. Thus, the inventors were able to show that cells or mitochondria which do not contain these channels because of their genetic disposition, a resistance to pro-apoptotic Bc1-2-like proteins, in particular Bax, and in addition to other substances, which is known that they induce apoptosis, such as C ₆ ceramide or staurosporine, and apoptosis can not be induced in such cells or mitochondria. The inhibition or blockade of these channels on isolated mitochondria led to the induction of apoptosis-specific processes, such as cytochrome c release, changes in mitochondrial membrane potential, in particular depolarization, and release of reactive oxygen species (ROS). In summary, the experimental findings obtained by the inventors of the present invention demonstrate the central importance of the generally voltage-dependent potassium channels, in particular Kv1.3 potassium channels, localized in the mitochondria, in particular in the inner mitochondrial membrane, with respect to apoptotic processes as well a possibility the Apoptoseregulation or Apoptosestimulation about these potassium channels.

As previously described, according to the invention come such apoptosis-inducing and / or apoptosis-stimulating substances (AIS) are used, the selected in this way or that they are through Interaction or interaction with mitochondrial potassium channels (MK) induce apoptosis of these cells or stimulate.

This can be done in several ways, as follows:
A first possibility is that the apoptosis-inducing or apoptosis-stimulating substance (AIS) used according to the invention is such that it interacts or interacts with the mitochondrial potassium channel (MK) in such a way that apoptosis of these cells is induced in the respective cells or is stimulated. In this case, the interaction of the apoptosis-inducing and / or apoptosis-stimulating substance (AIS) with the mitochondrial potassium channel (MK) can be effected in particular by physical and / or chemical binding of the apoptosis-inducing and / or apoptosis-stimulating substance (AIS) to the mitochondrial potassium channel (MK). Preferably, the binding of the apoptosis-inducing and / or apoptosis-stimulating substance (AIS) to the mitochondrial potassium channel (MK) is at least substantially selective, ie advantageously the apoptosis-inducing or apoptosis-stimulating substance (AIS) has an increased relative to the mitochondrial potassium channel (MK) Binding affinity compared to binding affinity to other substances. The interaction or binding of the apoptosis-inducing and / or apoptosis-stimulating substance (AIS) to the mitochondrial potassium channel (MK) can for example take place in the pore area of the mitochondrial potassium channel (MK) in the sense of a channel blocker or at a different binding site, but always with the The proviso that an interaction or binding of the apoptosis-inducing and / or apoptosis-stimulating substances (AIS) with respect to the mitochondrial potassium channel (MK) in the cells in question leads to an induction or stimulation of apoptosis in these cells.

According to one very particularly preferred embodiment this first possibility is the apoptosis-inducing and / or apoptosis-stimulating substance (AIS) selected or procure that they the mitochondrial potassium channel (MK), especially the Kv1.3 potassium channel the inner mitochondrial membrane and / or its homologs blocked or inhibited.

Under The term blocking or inhibiting can according to the invention in particular one through the interaction or interaction of the apoptosis-inducing and / or apoptosis-stimulating substance (AIS) with the mitochondrial Potassium channel (MK), especially the Kv1.3 potassium channel of the inner Mitochondrial membrane, caused change channel-specific, z. B. biophysical parameters, such as the channel conductance, the Open channel life (lifetime) and / or the probability of occurrence of the channel, to be understood. The interaction or interaction the apoptosis-inducing and / or apoptosis-stimulating substance (AIS) with the mitochondrial potassium channel (MK), in particular the Kv1.3 potassium channel of the inner mitochondrial membrane, it can, for example a direct, in particular a physical and / or chemical Interaction, for example in the area of the channel pore, which in particular can lead to a "blocking" or "clogging" of the channel pore, be. About that In addition, according to the invention is also a indirect interaction possible, for example, through changes cell physiological parameters. According to the invention, the term blockade or in particular block complete inactivation of the mitochondrial potassium channel (MK), in particular the Kv1.3 potassium channel of the inner mitochondrial membrane, preferably one with a complete decrease in channel activity, in general with respect to the above-mentioned biophysical channel parameters, to understand, associated with an increase or increase in the natural Apoptosis. The term inhibition may according to the invention in particular understood as an at least partial inactivation of the channel become such that a Decrease or decrease in channel activity, generally with respect to the aforementioned biophysical channel parameters, occurs with an increase or increase in natural Apoptosis.

A second, alternative possibility of interaction between apoptosis-inducing or apoptosis-stimulating substances (AIS) on the one hand and mitochondrial potassium channels (MK) of the respective cells on the other hand is that the apoptosis-inducing and / or apoptosis-stimulating substance (AIS) a change or influence on the physiological cell behavior or the physiological cell parameters, as will be further specified below. According to this embodiment, the apoptosis-inducing and / or apoptosis-stimulating substance (AIS) may be selected so as to influence the physiological parameters of the cells in question such as, in particular, their metabolism, osmolarity, pH, membrane potential and the like, such that the biophysical properties, in particular channel conductance, channel open-time and / or open-channel probability, of the mitochondrial potassium channels (MK) of the cells in question in the Be changed way that induced in the cells in question, apoptosis of these cells and / or stimulated. According to a very particularly preferred embodiment of this second possibility, the physiological cell parameters are influenced by the apoptosis-inducing and / or apoptosis-stimulating substance (AIS) in such a way that the mitochondrial potassium channel (MK), in particular the Kv1.3 potassium channel of the inner mitochondrial membrane, blocks or inhibits becomes.

In addition, the apoptosis-inducing and / or apoptosis-stimulating substance (AIS) may be such that it - in addition to the channel blocking or channel inhibiting effect or in addition to influencing the physical cell parameters - also with anti-apoptotic substances (AAS), as will be defined below the manner interacts or interacts that the anti-apoptotic effect of the anti-apoptotic Substan zen (AAS) is inhibited or at least inhibited. In this case, the anti-apoptotic substance (AAS) can in particular be inactivated, for example, by physical or chemical binding of the apoptosis-inducing and / or apoptosis-stimulating substance (AIS) to the anti-apoptotic substance (AAS). In the context of this mechanism of action, it is thus possible, for example, to reduce the decreased apoptosis, in particular by the anti-apoptotic substance (AAS), to a higher level in the cells in question. The anti-apoptotic substance (AAS) may, for example, be a natural, in particular endogenous substance occurring in the body and / or body cells, in particular an anti-apoptotic peptide, in particular an oligopeptide, a polypeptide or an anti-apoptotic peptide, eg. B. the Bc1-2 family, such as Bc1-2, Mc1-1, A1 / Bf1-1, Bc1-X _L , Boo / diva or Bc1-w, and their homologs or derivatives. With regard to the term homologues and derivatives, reference may be made to the following remarks concerning the apoptosis-inducing and / or apoptosis-stimulating substance (AIS), which apply correspondingly in this context. Characteristic z. For example, for an anti-apoptotic substance (AAS) derived from the Bcl-2 family, the existence of BH domains as structural features is particularly noteworthy, with the anti-apoptotic Bc1-2 proteins in particular having four short BH domains known as BH1 , BH2, BH3 and BH4.

at all previously described embodiments should be the apoptosis-inducing and / or apoptotic-stimulating substance (AIS) are advantageously selected such that they in relation to the cell, especially in relation to the physiological Parameters of the cell in question, is compatible and especially the apoptosis-inducing and / or apoptosis-stimulating substance (AIS) itself does not inhibit or at least inhibit apoptosis. The term compatible In this case, it should be understood that the apoptosis-inducing and / or apoptosis-stimulating substance (AIS) especially the physiological parameters of the cell in question to that effect, that apoptotic Subsequent reactions, such as a cytochrome c release or a Activation of PTP is inhibited (i.e., completely inhibited) or inhibited become.

The used according to the invention apoptosis-inducing or apoptosis-stimulating substance (AIS) an inorganic or organic substance. For example may be the apoptosis-inducing or apoptosis-stimulating substance (AIS) a low molecular weight substance (so-called "Small Drug Molecule") or a high molecular weight, in particular oligomeric or polymeric substance.

For example can the inventively used apoptosis inducing or apoptosis stimulating substance (AIS) Hydrocarbon, a nucleic acid, a lipid or a peptide or a derivative of the aforementioned compounds such as a lipoprotein. In the context of the present The invention is a peptide to be understood as a compound which consists of α-amino acids, which by acid amide bonds (Peptide bonds) chain-shaped connected are; in dependency of Number of amino acids may be between oligopeptides (up to 10 amino acids), polypeptides (10 bis 100 amino acids) and proteins (via 100 amino acids) be differentiated, d. H. The term peptide is according to the invention very far understood and embraced thus oligopeptides, polypeptides and proteins. Under a peptide derivative is any functional variant of the peptide to understand. For example, includes functional variant of a natural occurring peptide z. B. the substitution of one or more amino acids in its sequence, so that the function of the resulting peptide as apoptosis-inducing and / or apoptosis-stimulating Substance (AIS) not affected becomes. A functional variant comprises within the scope of the present invention Invention, for example, a change in the sequence, wherein one or more amino acids removed and / or by another amino acid or modified amino acid or not natural amino acid can be replaced or such an amino acid added can be. Maintaining the apoptosis inducing or apoptosis stimulating Effect can for example, glycosylations or phosphorylations in Regarding the used peptides.

In the context of the present invention, the apoptosis-inducing and / or apoptosis-stimulating substance (AIS) can in particular be a propapoptotically active peptide, in particular an oligopep tid, a polypeptide or a protein, such. B. a member of the pro-apoptotic Bc1-2 family be. These include peptides such as Bax, Bak, Bok, Bim, Bmf, Bad, Bik, Bid, Bc1-x _s , Noxa or Puma and their homologues and derivatives. Such Bc1-2 family peptides may also contain BH domains as structural features, notably the BH4 domain and the BH3 domain. These domains may serve, for example, as potential sites of interaction with anti-apoptotic substance (AAS) (eg, preferably, selective binding of the AIS to the corresponding domain of the AAS for their inactivation). Moreover, in the context of the present invention, the apoptosis-inducing and / or apoptosis-stimulating substance (AIS) can be selected from the group of specific inhibitors or channel blockers of the Kv1.3 potassium channel of the inner mitochondrial membrane (IMM), such as margatoxin and / or shigatoxin, and their homologs and derivatives.

in the The scope of the present invention is the apoptosis-inducing and / or apoptosis stimulating substance (AIS) very particularly preferably in such a way selected and / or procure that they with the Kv1.3 potassium channels and / or their homologues of the inner mitochondrial membranes (IMM) the cells in question interacts or interacts and thereby the Kv1.3 potassium channels and / or their homologs in particular blocked or inhibited, so that apoptosis of the cells in question is induced or stimulated.

As previously described, there is a significant objective of the present Invention in particular, by an interaction between mitochondrial potassium channels (MK) of the cells in question on the one hand and apoptosis-inducing or On the other hand, in these cells apoptosis-stimulating substances (AIS) Induce or stimulate apoptosis.

at the relevant mitochondrial potassium channel (MK) is preferably a particular voltage-dependent potassium channel, which located in the inner mitochondrial membrane (IMM). This Mitochondrial potassium channel (MK) is in particular a Kv1.3 potassium channel or a homologous potassium channel, such as a Kv1.1 potassium channel, Kv1.2 potassium channel, Kv1.4 potassium channel or Kv1.5 potassium channel. Under a homologous Potassium channel is inventively re insbesonde to understand such a channel, which in terms of its specific, in particular biophysical properties and its sequence with the mitochondrial potassium channel (MK) of the inner mitochondrial membrane, especially the Kv1.3 potassium channel. Under a Potassium channel is according to the invention in particular to understand such a channel, the potassium ions at least in the is substantially selective, d. H. whose permeability or Selectivity ratio in referred to potassium ions as larger than other ions 1 is. potassium channels can in particular by channel blockers such as tetraethylammonium chloride (TEACI) be blocked. In the context of the present invention relates the term mitochondrial potassium channel in particular to such Potassium channels, by specific channel blockers, such as Margatoxin (MgTx) or Shigatoxin, can be inhibited especially Kv1.3 potassium channels.

Without in the context of the present invention to a particular theory wanting to determine the cause of triggering apoptosis z. B. by possibly a pro-apoptotic Bc1-2-like peptide explained be that, for example the pro-apoptotic Bc1-2-like Peptide z. B. to the innermitochondrial Kv1.3 potassium channel of the relevant Cell binds and thereby inhibits this Kv1.3 potassium channel, so that as Sequence cell parameters, such as the mitochondrial membrane potential, changed so be that it to trigger comes from apoptosis. According to another theory, but so far under physiological or in vivo conditions not understood could be, for. B. a proapoptotic Bc1-2-like effective Peptide form a new channel or with the Kv1.3 potassium channel or its homologues together a new channel or a new one Training pore.

As previously stated In the context of the present invention, the apoptosis-inducing and / or apoptosis-stimulating substance (AIS) for prophylactic or curative Treatment of diseases of the human or animal body used, where a particular excessive reduction of apoptosis or a particular excessive increase in cell growth or Cell division occurs or related or in which a therapeutically targeted apoptosis of the respective Cells is intended. These can be both acute and chronic Diseases act. For example, the disease to be treated a tumor or cancer, in particular a benign or malignant tumor disease, such as a cancer, e.g. B. colon cancer (Colon carcinoma), breast cancer (breast cancer), ovarian carcinoma, uterine carcinoma, Lung cancer, stomach cancer, liver cancer, pancreatic cancer, kidney cancer, Bladder cancer, prostate cancer, testicular cancer, bone cancer, skin cancer, Kaposi's sarcoma, brain tumor, myosarcoma, neuroblastoma, lymphoma and leukemia, or a parasitic Disease, such as leishmanosis, malaria or giardiasis, or one bacterial disease or a viral disease such as hepatitis or AIDS.

in the Within the scope of the present invention, the treatment of the aforementioned Diseases due to systemic or topical application or administration of the apoptosis-inducing or apoptosis-stimulating substance (AIS) respectively. Here, the apoptosis-inducing or apoptosis-stimulating Substance (AIS) is advantageously used in an amount that is sufficient to provide in the cells the desired interaction between the mitochondrial potassium channels (MK) on the one hand and apoptosis-inducing or apoptosis-stimulating Substance (AIS), on the other hand, sufficient to enter the particular cells to induce apoptosis or to stimulate. The actual site of action of the apoptosis-inducing or apoptosis-stimulating substance (AIS) the relevant cells, in particular the cells in which apoptosis is induced or stimulated should be, preferably the mitochondria or the mitochondrial membranes, in which the previously described innermitochondrial potassium channels (MK) are localized. Thus, the amount of apoptosis-inducing or apoptosis-stimulating substance (AIS) in the context of the present invention to choose such that the Concentration of this substance at the previously defined site of action is sufficient to the desired Effect to achieve.

there may be the apoptosis-inducing or apoptosis-stimulating substance (AIS) as such or else in the form of an appropriate precursor, in particular of a prodrug (prodrug). As part of the present invention is under a prodrug an active ingredient precursor too understand that (for example, against the background of an improved Absorption, increased water solubility, a better taste and / or a lower toxicity etc.) the actual apoptosis-inducing or apoptosis-stimulating substance (AIS) only in the body or in the cells in question, z. B. by physiological activity, in particular in situ, generated from the prodrug. Here are playing in particular chemical and / or physical transformations play a major role. Under a prodrug is not according to the invention only a direct chemical precursor substance but also, for example, one for the apoptosis-inducing or apoptosis-stimulating substance (AIS) coding gene or the corresponding nucleotide sequence, the or the (for example in Framework of a gene therapy approach). B. via suitable vector systems in the body and in particular can be introduced into the cells in question, hereinafter the apoptosis-inducing or apoptosis-stimulating Substance (AIS), in particular as part of protein biosynthesis, is generated.

The Application of the apoptosis-inducing and / or apoptosis-stimulating Substance (AIS) may be systemic, depending on the type of disease being treated and / or topically, wherein the apoptosis-inducing and / or apoptosis stimulating substance (AIS) according to more usual Application forms can be administered. So can the administration for example, orally, lingually, sublingually, buccally, rectally or parenterally, thus bypassing the gastrointestinal tract, such as intravenous, intraarterial, intracardiac, intracutaneously, subcutaneously, transdermally, intraperitoneally or intramuscularly. For certain Diseases, such as intracranial tumors, can also cause an intracerebral and / or intrathecal application may be preferred.

For the application according to the invention become the apoptosis-inducing or apoptosis-stimulating substances (AIS) in a known manner in the usual Translated formulations, such as for example, tablets, dragees, pills, granules, aerosols, syrups, Emulsions, suspensions, solutions, Ointments, creams and gels of all kinds, in particular using Substantially inert and non-toxic, pharmaceutically acceptable excipients or solvent. In this case, the apoptosis-inducing agents used according to the invention should or apoptosis-stimulating substance (AIS) advantageously in a therapeutically effective concentration, d. H. in quantities sufficient to produce the desired effect at the site of action Effect of the apoptosis-inducing or apoptosis-stimulating substance (AIS) to achieve.

The dosage of the apoptosis-inducing or apoptosis-stimulating substance (AIS) depends on numerous factors, such as the type of disease to be treated, the body weight or the route of administration, the individual behavior towards the drug, the formulation, the bioavailability of the apoptosis-inducing or apoptosis-stimulating Substance (AIS), the absorption, distribution, excretion, biotransformation (detoxification, activation) of the apoptosis-inducing or apoptosis-stimulating substance (AIS) and the time or interval at which the administration takes place. For example, an exact dosage may be determined in conjunction with standardized dose-response studies. In addition, the apoptosis-inducing or apoptosis-stimulating substance (AIS) or the formulation containing the apoptosis-inducing or apoptosis-stimulating substance (AIS) may contain carrier substances ("canier") for improving the bioavailability, in particular for altering the permeability of the blood / brain. Barrier may be added or the active ingredients may be integrated or incorporated into corresponding carrier systems. In particular, the apoptosis-inducing or apoptosis-stimulating substance (AIS) should be selected or obtained and / or the formulation should be for apoptosis Ducent or apoptosis-stimulating substance (AIS) be designed so that the apoptosis-inducing or apoptosis-stimulating substance (AIS) is advantageously applied at least substantially selectively with respect to the cells in question or is used. This can be controlled, for example, by the apoptosis-inducing or apoptosis-stimulating substance (AIS), in particular by selecting the apoptosis-inducing or apoptosis-stimulating substance (AIS) and / or by special matching or formulation of the formulation, preferably at least substantially selectively, at least predominantly or more preferably, only in the cells concerned, the desired effect unfolds.

The used in the context of the present invention formulations can in addition to at least one apoptosis-inducing and / or apoptosis-stimulating Substance (AIS) contains other effective drugs.

Farther may also be a treatment of the cells in question outside of the body, especially ex vivo, come into consideration. Where appropriate, where appropriate the cells in question are removed from the body or explanted, and subsequently becomes the apoptosis-inducing or apoptosis-stimulating substance (AIS) on the affected cells. Then there is a (Re) implantation of the remaining cells in the diseased and / or to be treated body. This form of treatment is suitable, for example, if a sometimes toxic apoptosis-inducing or apoptosis-stimulating Substance (AIS) should be used or to achieve the desired therapeutic or prophylactic effect under a systemic or local treatment such a high dose can be used that would have to to be undesirable Cause side effects could. About that In addition, such ex vivo treatment may then be considered if the bioavailability of the active ingredient in topical or systemic treatment at the site of action is not enough to the desired Effect to achieve. The ex-vivo treatment can be used in particular for Treatment of brain cells come into consideration because of the so-called Blood / brain barrier often a transfer of the drug from the bloodstream in the brain prevented. Farther may also be considered an ex-vivo treatment of bone marrow cells come.

• (a) Bereitstellung und/oder Auswahl einer apoptoseinduzierenden und/oder apoptosestimulierenden Substanz (AIS), welche derart ausgewählt und/oder beschaffen ist, daß sie durch Wechselwirkung und/oder Inter aktion mit den mitochondrialen Kaliumkanälen (MK) der betreffenden Zellen eine Apoptose dieser Zellen induziert und/oder stimuliert;
• (b) Verabreichung der apoptoseinduzierenden und/oder apoptosestimulierenden Substanz (AIS) in therapeutisch wirksamen Mengen und/oder Zufuhr der apoptoseinduzierenden und/oder apoptosestimulierenden Substanz (AIS) zu den betreffenden Zellen in zur Erzielung des beabsichtigten Effekts ausreichenden Mengen.

According to a second aspect of the present invention, the present invention relates to a method for inducing and / or stimulating apoptosis in cells in which a particular excessive reduction of apoptosis and / or a particular excessive increase of cell growth and / or cell division occurs or hereby in Or in which a therapeutically targeted apoptosis of the cells in question is intended, by influencing mitochondrial potassium channels (MK) of the cells in question by apoptosis-inducing and / or apoptosis-stimulating substances (AIS), the method comprising the following method steps:

• (a) Provision and / or selection of an apoptosis-inducing and / or apoptosis-stimulating substance (AIS), which is selected and / or designed such that it by interaction and / or interaction with the mitochondrial potassium channels (MK) of the cells concerned an apoptosis of these Induce and / or stimulate cells;
• (b) administering the apoptosis-inducing and / or apoptosis-stimulating substance (AIS) in therapeutically effective amounts and / or delivering the apoptosis-inducing and / or apoptosis-stimulating substance (AIS) to the subject cells in amounts sufficient to achieve the intended effect.

For further Details and embodiments with respect to the inventive method for Induction and / or stimulation of apoptosis in cells according to the second The subject matter of the present invention can be applied to the above statements the use according to the invention according to the first inventive object be referred, which apply here accordingly.

Furthermore, the present invention according to a third aspect of the present invention relates to a method for the prophylactic and / or curative treatment of diseases of the human or animal body, in which a particular excessive reduction of apoptosis or a particular excessive increase in cell growth or cell division occurs or which are associated therewith or in which a therapeutically targeted apoptosis of the cells in question is intended by administering at least one apoptosis-inducing and / or apoptosis-stimulating substance (AIS) in therapeutically effective amounts, wherein the apoptosis-inducing and / or apoptosis-stimulating substance (AIS) is selected such or is designed to induce and / or stimulate apoptosis of these cells by interaction or interaction with mitochondrial potassium channels (MK) (eg, Kv1.3 potassium channels and / or homologs) of the subject cells. For further details and embodiments relating to the inventive method for the prophylactic and / or curative treatment of diseases of the human or animal body, in which a particular excessive reduction of apoptosis or a particular excessive increase of cell growth or cell division occurs or hereby in Related or where a therapeutically targeted Apo ptose the relevant cells is intended, reference may be made to the above statements on the use according to the invention according to the first article of the invention, which apply here accordingly.

• (a) Bereitstellung eines geeigneten Testsystems, mit dem die Wechselwirkung zwischen apoptoseinduzierenden und/oder apoptosestimulierenden Substanzen (AIS) einerseits und mitochondrialen Kaliumkanälen (MK) andererseits erfaßt werden kann;
• (b) Einwirkenlassen der apoptoseinduzierenden und/oder apoptosestimulierenden Substanz (AIS) oder eines entsprechenden Vorläufers auf das in Verfahrensschritt (a) bereitgestellte Testsystem;
• (c) Auswertung mit geeigneten, an das jeweilige Testsystem angepaßten Methoden, insbesondere in Bezug auf das Vorliegen einer Wechselwirkung und/oder Interaktion von apoptoseinduzierender und/oder apoptosestimulierender Substanz (AIS) oder entsprechendem Vorläufer einerseits und Testsystem andererseits.

Finally, according to a fourth aspect of the present invention, the present invention relates to a method for finding or identifying an apoptosis-inducing and / or apoptosis-stimulating substance (AIS), which is such that it can be obtained by interaction and / or interaction with mitochondrial potassium channels ( MK) of the cells in question induces and / or stimulates apoptosis of these cells, the method comprising the following steps:

• (a) provision of a suitable test system with which the interaction between apoptosis-inducing and / or apoptosis-stimulating substances (AIS) on the one hand and mitochondrial potassium channels (MK) on the other hand can be detected;
• (b) exposing the apoptosis-inducing and / or apoptosis-stimulating substance (AIS) or a corresponding precursor to the test system provided in method step (a);
• (c) Evaluation with suitable methods adapted to the respective test system, in particular with regard to the presence of an interaction and / or interaction of apoptosis-inducing and / or apoptosis-stimulating substance (AIS) or corresponding precursor on the one hand and test system on the other hand.

According to one first embodiment the test system provided in method step (a) can be at least have a potassium channel, which may be in an artificial or natural Membrane is located. The membrane may in particular be a lipid bilayer In particular, the potassium channel to be examined may be a voltage-dependent Potassium channel, in particular a Kv1.3 potassium channel or a thereto homologous potassium channel, such as a Kv1.1 potassium channel, Kv1.2 potassium channel, Kv1.4 potassium channel or Kv1.5 potassium channel. In this regard, can on above be referred to, which apply here accordingly. In the context of the present invention can in this embodiment the mitochondrial potassium channels to be examined on the one hand directly from such Cells are isolated in which mitochondrial potassium channels (MK) of course or endogenous, or otherwise isolated from such cells be in the context of an expression system mitochondrial potassium channels, in particular after injection of precursors (such as DNA, cDNA, mRNA etc.), in particular functionally expressed in their membrane, in particular Xenopus oocytes, CTLL-2 cell systems or C8166 B lymphocytes ( "C8166 cell system (s)"). In the context of special Embodiments of the test system are in particular natural or artificial Membranes using mitochondrial potassium channels (MK) as a functional ingredient. These include, for example, mitochondrial Membranes and in particular the inner mitochondrial membranes (IMM), but also cellular Membranes containing mitochondrial potassium channels under a natural Expression system, especially after injection with appropriate precursors, such as the cell membranes of Xenopus oocytes, Membranes of a CTLL-2 cell system or a C8166 cell system.

Farther can artificial membranes are used in the context of the present invention, in which isolated mitochondrial potassium channels (MK) are reconstituted. artificial Membranes can be prepared using appropriate standard methods, they have a defined lipid composition, being naturally occurring Lipids, such as palmitoyloleoylphosphatidylcholine and / or dioleoylphosphatidylethanolamine, or natural Lipid mixtures, such as asolectin or artificial lipids, ie those Lipids that are not in natural membranes occur, can be used.

Natural membranes, the mitochondrial potassium channels (MK) can both as intact membranes of a cellular system, in particular Mitochondria and / or mitoplasts, or used as membrane fragments become.

In Process step (c) may be the evaluation or analysis of a possible Interaction of apoptosis inducing and / or apoptosis stimulating substance (AIS) or a corresponding precursor on the one hand and the Testy system, in particular the mitochondrial potassium channels (MK) on the other hand by means different measuring methods respectively.

These include in particular immunological detection reactions such. B. immunoblotting, wherein proteins, mitochondrial potassium channels (MK) and / or the apoptosis-inducing or apoptosis-stimulating substance (AIS) are separated by electrophoresis, in particular by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis), and on an immobilizing matrix, such as a nitrocellulose filter , z. B. by electrotransmission. Subsequently, an immunological detection reaction can take place, for. B. by enzyme and / or dye-labeled specific and / or unspecific (primary and / or secondary) antibodies, with an interaction of mitochondrial potassium channels (MK) on the one hand and apoptosis-inducing or apoptosis-stimulating substance (AIS) on the other hand are detected can. For example, the interaction or interaction between mitochondrial potassium channels (MK) on the one hand and apoptosis-inducing or apoptosis-stimulating substance (AIS) on the other hand can also be analyzed or detected by coimmunoprecipitation studies. For example, in such studies, in particular when using a Kv1.3 potassium channel, z. For example, commercially available anti-Kv1.3 antibodies (especially antibodies derived from immunizations with GST (glutathione-S-transferase) -coupled Kv1.3 potassium channels) can be used. After immunoprecipitation of the Kv1.3 channels, the precipitates can then be analyzed, for example by Western blot analysis and z. B. with respect to the apoptosis-inducing or apoptosis-stimulating substance (AIS) specific antibodies are analyzed or detected. In addition, it is also possible to use fusion proteins of the apoptosis-inducing or apoptosis-stimulating substance (AIS), which can be obtained in particular by a fusion (coupling) with the GST protein.

Farther For example, such an interaction can also take place via physical-technical detection methods carried out, such as by a crystallographic X-ray structure analysis.

In in terms of natural or artificial Membrane systems of existing mitochondrial potassium channels (MK) can an interaction between these on the one hand and apoptosis-inducing or apoptosis stimulating substance (AIS) on the other hand in addition to physical and / or chemical detection methods also by means of electrophysiological measurement methods to determine biophysical parameters. For this count in particular the patch-clamp method and electrophysiological examinations on planar lipid membranes, in particular in the context of individual or multi-channel experiments an analysis of electrophysiological channel parameters, like ion selectivity and / or channel kinetics, such as. B. channel conductance, open channel, Open probability of the channel, especially under influence the apoptosis-inducing and / or apoptosis-stimulating substance (AIS). For this purpose, for example, a transmembrane Potential difference to be constructed, in particular by applying an external Voltage and / or by using different (eg asymmetric) Internal concentrations with respect to the corresponding membrane sides. As part of the patch-clamp method, the measurements in different Modes are performed especially in the cell-attached, Inside-out mode or in whole-cell or inside-out mode. Especially can patch-clamp analyzes on isolated mitochondria, in particular on Kv1.3-positive mitochondria, for example, in particular transfected CTLL-2 and / or C8166 cell systems, for example in the mitoplast-attached mode, carried out become. It can the experimental approaches apoptosis-inducing or apoptosis-stimulating substances (AIS) be added, and it may be their possible influence in particular the functional properties of mitochondrial potassium channels (MK), such as channel conductance, channel open time and probability of open Channels, to be analyzed. In particular, for example, a Blocking or inhibiting the mitochondrial potassium channel the apoptosis-inducing or apoptosis-stimulating substance (AIS), the z. B. to changes the channel kinetics (for example lower channel open time, lower Channel conductance, etc.) and to a change in the membrane potential to lead can be clearly detected by the methods described above become.

In addition, a change in the channel kinetics, which may result in a change in membrane potential, can also be reconstructed via voltage-dependent dyes which may be associated with the membrane and which, at a certain membrane potential, reduce their affinity for the membrane and are detached from the membrane. such as 3,3'-dihexyloxacarbocyanine iodide ("DioC ₆ (3)").

According to one special design, the test system additionally at least one anti-apoptotic Substance (AAS) contained in the context of a further process step the test system z. B. between process steps (a) and (b) or between the process steps (b) and (c) are supplied can and which is left on the test system. It can an interaction of the antiapoptotic substance (AAS) with the apoptosis-inducing or apoptosis-stimulating substance (AIS) and / or the mitochondrial potassium channels (MK) to further change lead from system parameters, which can be analyzed by the above methods, so that on This way it can be ensured that the apoptosis-inducing or apoptosis-stimulating substance (AIS) even in the presence of a anti-apoptotic substance (AAS) is (still) effective.

In a second, alternative embodiment of the identification or discovery method according to the invention, the test system provided in method step (a) comprises an expression system, wherein the expression system on the one hand a nucleotide sequence (I), which in particular codes for a mitochondrial potassium channel (MK), and on the other hand, a nucleotide sequence (II), which in particular encodes an apoptosis-inducing and / or apoptosis-stimulating substance (AIS). In process step (c), the Interak tion of the gene products (proteins) of the nucleotide sequences (I) and (II), in particular the mitochondrial potassium channel (MK) and the apoptosis-inducing and / or apoptosis-stimulating substance (AIS), using one of the above-mentioned methods, in particular via immunological or physico-chemical Methods are detected.

In a further embodiment of this second embodiment of the invention or identification method, the nucleotide sequences (I) and (II) for the purposes of in step (c) to be performed Evaluation as well each have at least one area, each for at least encodes a protein domain, which are suitable for use in a suitable detection method, especially in the context of a two-hybrid system. It is at least a protein domain a so-called DNA-binding domain and at least one further protein domain an activation domain, wherein the DNA-binding domain on the one hand and the activation domain on the other hand to different Proteins are bound. For example, the DNA-binding domain be bound to the protein encoding the nucleotide sequence (I) is, so z. B. to the mitochondrial potassium channel (MK), or it may be due to the apoptosis-inducing and / or apoptotic-stimulating Substance (AIS) (nucleotide sequence II) be bound.

in the In particular, yeast cells are part of the framework of the two-hybrid system cotransfected corresponding vectors, for example, a first vector the nucleotide sequence (I) and the nucleotide sequence the DNA-binding domain and a second vector corresponding to the nucleotide sequence (II) and the for the activation domain having coding nucleotide sequence. The DNA-binding domain as well the activation domain represent functional components of a yeast transcriptional activator represents, for its function is both domains are essential. The transcriptional activation works here but also if both domains localized on different proteins as long as they are in close contact to be brought. This means that in an interaction of the Proteins encoded by the nucleotide sequence (I) or (II), the DNA-binding domain as well as the activation domain be contacted and thus a transcriptional activation a specific reporter gene can be done. As a transcriptional activator In particular, the GAL4 yeast transcriptional activator can be used. The activated reporter gene is then z. For example, the Lac-Z reporter gene. Activation of this gene leads for the synthesis of β-galactosidase, which the substance X-Gal (5-bromo-4-chloro-3-indolyl-β-D-galactoside) in galactose and the blue dye 5-bromo-4-chloro-3-indole enzymatically implements. It follows that a Interaction of the corresponding proteins and consequently an interaction between mitochondrial potassium channels (MK) on the one hand and apoptosis-inducing ones on the other and / or apoptosis-stimulating substance (AIS) on the other hand then based on a blue coloration the corresponding colonies can be seen. For example to study the interaction or interaction between mitochondrial potassium channels (MK) on the one hand and apoptosis-inducing and / or apoptosis-stimulating Substance (AIS) on the other hand also a mammalian two-hybrid system be used; Such a system offers the advantage that under circumstances as wrong positively detected Protein interactions to a lesser extent than in the case of two-hybrid systems occur on yeast cell basis.

Especially can for the Analysis of the in vivo importance of interaction or interaction between mitochondrial potassium channels (MK) on the one hand and apoptosis-inducing ones on the other and / or apoptosis-stimulating substance (AIS), on the other hand, physiological Models, such. B. knock-out mice, in particular with respect to the apoptosis-inducing or apoptosis-stimulating Substance (AIS) and / or the anti-apoptotic substance (AAS) deficient mice be used. Here, for example, an induction of Apoptosis, in particular by means of the decrease of CD-4 positive cells, the occurrence of apoptosis in the bone marrow, the decrease in peripheral Leukocyte counts, the release of LDH (lactate dehydrogenase) etc. detected or measured.

The Method of finding and / or identifying apoptosis-inducing or apoptosis-stimulating substances (AIS) can be used as part of a preferably automated screening method, in particular one High-throughput screening (HTS), performed being used as part of this automated screening system a high throughput of samples is possible. Furthermore, the apoptosis-inducing or apoptosis-stimulating substances (AIS) or their corresponding precursors provided from a corresponding substance library (substance bank) which is in particular a protein or gene library ("Protein or gene bank") can act.

Further Embodiments, modifications, variations and advantages of the present Invention are for the person skilled in the art readily discernible when reading the description and realizable without him while leaving the scope of the present invention.

The present invention is illustrated by the following embodiments, which, however, by no means limit the present invention.

methods:

Cell Culture:

The cells were grown in RPMI-1640 supplemented with 10% fetal calf serum, 10 mM HEPES (pH 7.4), 2 mM L-glutamine, 1 mM sodium pyruvate, 100 μM nonessential amino acids, 100 units (U) / ml penicillin, 100 μg / ml streptomycin (Life Technologies) and 50 μM β-mercaptoethanol. Murine interleukin-2 (IL-2) (4 U / ml, Roche) was added to the CTLL-2 cells (ATCC, VA, USA) every 2 days. CTLL-2 cells were transfected by electroporation with 40 μg each of pJK / Kv1.3 and pJK plasmids, respectively, and cultured in 800 μg / ml G418 to obtain stably transfected cells. All experiments were performed on a mixed population of transfected cells, avoiding selection of particular clones. In addition, all cultures were re-used after a 4-week growth period from frozen stocks to avoid selection of specific clones. IL-2 was removed prior to the experiments to avoid interaction of IL-2 with pro-apoptotic stimulants. For this purpose, the cells were washed in 132 mM NaCl, 20 mM HEPES (pH 7.4), 5 mM KCl, 1 mM CaCl ₂ , 0.7 mM MgCl ₂ , 0.8 mM MgSO ₄ (H / S) with 500 mM glycine (pH 5.0) was added. After washing, the cells were placed in an IL-2-free cell culture medium for recovery for 3 hours. Under the appropriate conditions and time courses, IL-2 removal did not result in significant apoptosis (5 ± 3% death rate 12 hours after IL-2 removal).

Bax-transfection:

Twenty ⁶ × CTLL-2 / pJK or CTLL-2 / Kv1.3 cells were cotransfected with 10 μg of an expression vector for a GFP-labeled actin and 50 μg pcDNA-Bax. The cells were electroporated at 400 V with 5 pulses, each for 3 ms, and recultivated for 16 hours. Dead cells were removed by a Ficoll gradient and the cells were cultured for an additional 30 hours. Cell culture was performed as described above in the presence of IL-2. To determine Bax-induced apoptosis, cells were washed in H / S as above, supplemented with 500 mM glycine to remove IL-2 from the cells. Subsequently, the cells were washed twice in H / S and cultured in normal cell culture medium (without IL-2) for 12 hours. Transfected cells were identified via expression of GFP-labeled actin using the FITC channel of a fluorescence microscope. These cells were tested for binding of Cy3-annexin to detect apoptosis. Control experiments with propidium iodide excluded necrotic cells.

Patch-clamp experiments:

Whole-cell currents (whole cell currents) were recorded with an EPC-7 amplifier (List) (filtering: 1 kHz, read rate (sampling rate): 5 kHz). Leakage currents were not deducted. The bath solution contained 150 mM NaCl, 5 mM KCl, 2.5 mM CaCl ₂ , 1 mM MgCl ₂ , 10 mM HEPES (pH 7.3). The intracellular solution contained 134 mM KCl, 1 mM CaCl ₂ , 2 mM MgCl ₂ , 10 mM EGTA, 10 mM HEPES (pH 7.3). Mitoplasts were prepared by osmotic shocks of isolated mitochondria in 30 mM Tris / HCl (pH 7.4). The mitoplasts were then equilibrated in 134 mM KCl, 2 mM MgCl ₂ , 1 mM CaCl ₂ , 10 mM EGTA, 10 mM HEPES (pH 7.3). Cells with a diameter of 2-3 microns were selected by video microscopy. Gigaseals (Gigaohm seals) were made on the membrane. The data was recorded at a reading rate of 10 kHz and filtered at 200 Hz. The flows were carried out under asymmetric [K ⁺ ] conditions in a bath solution containing 134 mM KCl, 1 mM CaCl ₂ , 2 mM MgCl ₂ , 10 mM EGTA, 10 mM HEPES / K ⁺ (pH 7.3) and a 50 pipette solution mM KCl, 84 mM NaCl, 1 mM CaCl ₂ , 2 mM MgCl ₂ , 10 mM EGTA, 10 mM HEPES / K ⁺ (pH 7.3). Some experiments were performed under symmetrical [K ⁺ ] conditions with 114 mM K-gluconate, 20 mM KCl, 1 mM CaCl ₂ , 2 mM MgCl ₂ , 10 mM EGTA, 10 mM HEPES / K ⁺ (pH 7.3). In the appropriately labeled patch-clamp experiments, MgTx was used at a concentration of 2 nM and charybdotoxin at a concentration of 50 nM (both toxins are from Alamone Labs, Israel).

Kv1.3 Western:

The Cells were incubated in 25 mM HEPES (pH 7.4), 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton X-100, 125 mM NaCl and 10 mM sodium fluoride each, EDTA and sodium pyrophosphate and 10 μg / ml each of aprotinin and leupeptin (A / L) lysed and centrifuged. The proteins were separated by SDS-PAGE (10%) and blotted (transferred) to nitrocellulose membranes. These membranes were at 1 μg / ml incubated with anti-Kv1.3 antiserum followed by treatment with alkaline phosphatase (AP) coupled anti-rabbit antibodies. The Detection was performed using a Tropix system (TROPIX Inc., Bedford, MA).

Apoptosis experiments:

To determine DNA fragmentation, the cells were treated with propidium iodide and carried out an analysis of the sub-G1 / S fraction by means of flow cytometry. The cells were fixed and permeabilized in 35% ethanol, the RNA was digested and the cells were treated with 5 μg / ml propidium iodide.

To determine caspase-3 activity, the cells were lysed in 50 mM HE-PES (pH 7.4), 0.1% CHAPS, 1 mM DTT, 0.1 mM EDTA, the samples were centrifuged and the supernatants were washed with 1 mM EDTA, 10 mM glycerol and incubated with 20 nM Ac-DEVD-pNA. Caspase-3 activity was subsequently determined by spectrophotometry. The membrane potential (ΔΨ _m) was determined in whole cells by 30 minute incubation in 200 pM _DiOC6 (3) (Molecular Probes, Eugene, Oregon) in H / S. The cells were washed, resuspended in H / S and analyzed by flow cytometry.

To determine the release of mitochondrial cytochrome c, cells were washed after stimulation in cold H / S, for 30 minutes at 4 ° C in 220 mM mannitol, 68 mM sucrose, 50 mM PIPES-KOH (pH 7.3), 50 mM KCl, 2 mM MgCl ₂ , 1 mM DTT, 5 mM succinate-KOH (pH 7.3), 10 μm cytochalasin B, 10 μg / ml A / L incubated and then homogenized. The samples were centrifuged and the supernatant proteins (20 μg) were separated by SDS-PAGE (15%) and transferred to a nitrocellulose membrane. The Western blots were analyzed using a monoclonal mouse-derived anti-cytochrome c antibody (clone 7H8.2C 12, BD Pharmingen, San Diego, CA) and the system Tropix ECL.

electron microscope investigations:

to execution Electron microscopic (EM) studies first became one polyclonal anti-Kv1.3 antibody derived from a rabbit, specifically with the extracellular localized amino acids 204-220 (LPEFRDEKDYPASTSQD) responding. The results were compared with a second polyclonal, rabbit-derived anti-Kv1.3 antibody. Peripheral blood lymphocytes were purified by Ficoll-Hypaque gradient which became cells fixed in 4% paraformaldehyde and 1% glutaraldehyde, in the ethanol gradient dehydrated and in LR white resin (London Resin Company Limited, Berkshire). For an "on-grid" immunochemical Investigation 85 nm sections were made, blocked and washed with PBS containing 0.1% gelatin from cold-water fish skin (Sigma), 0.5 M NaCl and 0.05% Tween 20. The sections were with rabbit-derived anti-Kv1.3 antibody and with goat derived gold-coated (6 nm) anti-rabbit IgG (Aurion, Wageningen, The Netherlands) marked. The samples were treated with uranium acetate and placed on a JEOL 1200EX II electron microscope (JEOL USA, Peabody, MA).

flow cytometry and Western blots on isolated mitochondria

For flow cytometry, the cells were transiently transfected with pEYFP-Mito or pEYFP-ER (Clontech) and sorted after 48 hours. With respect to Western blot experiments, wild-type Jurkat cells, CTLL-2 / pJK or CTLL-2 / Kv1.3 cells were used. For the purification of the mitochondria, the cells were incubated for 30 minutes at 4 ° C. in 0.3 M sucrose, 10 mM TES (pH 7.4), 0.5 mM EGTA and then homogenized. Nuclei and undigested cells were pelleted by centrifugation at 600 x g and 4 ° C for 5 min. The supernatants containing the mitochondria were further purified by a 10 minute Percoll gradient centrifugation (60%, 30%, 18% Percoll in the above buffer, 8500xg) at 4 ° C. Mitochondria located between the 30% and 60% layers were pooled, washed twice, and suspended in 50mM PIPES-KOH (pH 7.4), 50mM KCl, 2mM MgCl ₂ , 5mM EGTA, 1mM DTT, 10 μg / ml A / L, 2 mM ATP, 10 mM phosphocreatine, 50 μg / ml creatine kinase resuspended. A slightly hyperosmotic medium (340 mosmol as measured by osmometer analysis) was used to protect the mitochondria from swelling. For the Western blots or FACS analysis of Kv1.3, equivalents corresponding to 30 μg total protein were used. Western blots were performed using an anti-Kv1.3 antibody, an AP-linked anti-rabbit secondary antibody and the tropix chemiluminescent system. Flow cytometric analyzes were performed on Cy3-coupled anti-Kv1.3 antibodies (Alamone Labs) on intact mitochondria and on permeabilized mitochondria treated with 0.75% Triton X-100.

Cytochrome c release, Membrane potential and release of reactive oxygen species in isolated mitochondria after treatment with recombinant Bax or MgTx:

To determine cytochrome c release from isolated mitochondria, purified mitochondria were prepared in 5 mM succinate, 50 mM PIPES-KOH (pH 7.4), 50 mM KCl, 2 mM MgCl ₂ , 5 mM EGTA, 1 mM DTT, 10 μM A / L, 2mM ATP, 10mM phosphocreatine, 50μg / ml creatine kinase (buffer A), incubated for 20 minutes with 100ng / mg GST-Bax, GST (as control) or 20nM MgTX (Alamone Labs) and as described above with the aid of Western blots.

The membrane potential of the isolated mito Chondria were determined by incubation of purified mitochondria in Buffer A with 1 nM DioC ₆ (3) and addition of 20 nM MgTx. MgTx was added during the experiment, and analysis of membrane potential by flow cytometry was taken immediately after addition of the substance. Depolarization of the mitochondrial membrane was indicated by a left shift of the fluorescence signal caused by a decreased binding of Dio-C ₆ (3). Complete depolarization of the mitochondria was achieved with 1 μM carbonylcyanidechlorophenylhydrazone (CCCP) for 10 minutes at room temperature and served as a control for the integrity of the mitochondria.

Relatively high MgTx concentration was used in these experiments and in isolated mitochondrial studies described below to detect rapid changes in mitochondrial membrane potential and to avoid alteration of mitochondrial activity caused by prolonged in vitro incubation becomes. These concentrations are still significantly lower than the known from the literature MgTx concentrations, which are in the μM range and which are required for influencing Ca ²⁺ -sensitive K ⁺ channels or other Kv channels.

to Determination of the release of reactive oxygen species (ROS) isolated mitochondria were purified mitochondria for 30 minutes at 37 ° C with 0.8 μM Hydroethidine in buffer A, containing 2 mg / ml cytochrome c added has been. The purified mitochondria were subsequently washed with 20 nM MgTx treated and the ROS release by changes of absorption at 550 nm determined by spectrophotometry. The Identification of ROS was over a suppression of the Signal due to sample incubation with dismutase superoxide (20 U / ml), dithiothreitol (DTT, 3 mM) and butylhydroxytoluene (BHT, 50 μM). The Incubation was carried out to degenerate or neutralize ROS. The reagents were Added 10 minutes before the MgTx treatment.

Expression of the functional Kv1.3 in mitochondria from lymphocytes:

To determine the molecular role of Kv1.3 with respect to apoptosis, the subcellular abundance of the channel was first determined. Transmission electron microscopy (TEM) studies on freshly isolated peripheral lymphocytes from human blood confirmed the presence of Kv1.3 in the plasma membrane and in intracellular vesicles, such as the endoplasmic reticulum, but surprisingly also demonstrated the presence of the channel in mitochondria (Fig. 1a ). A closer analysis of the TEM studies showed that Kv1.3 occurs in the inner mitochondrial membrane (IMM) ( 1a ), with the presence of Kv1.3 in fragments of the endoplasmic reticulum possibly associated with the mitochondria excluded.

1a illustrates that Kv1.3 is expressed in mitochondria. An examination by means of transmission electron microscopy on freshly isolated, purified human lymphocytes, which showed the presence of Kv1.3 in mitochondria, can be seen. The arrows are directed to gold particles and point to Kv1.3 in mitochondria (arrows with solid line) and in the cell membrane (arrow with dashed line). Kv1.3 is not recognizable in other cell compartments. The typical double membrane and the presence of cristae are characteristic of the mitochondria. The experiments were performed with two different anti-Kv1.3 antibodies: The upper partial image of 1a Figure 9 shows a Kv1.3 channel labeled with a rabbit-derived polyclonal antibody directed against amino acids 204-220. The lower part illustration of 1a Figure 4 shows the label with polyclonal antibodies derived from a rabbit that are specific to the C-terminus of Kv1.3. The gain was 60,000 times. Control markers using irrelevant antibodies were negative. To demonstrate the functional expression of Kv1.3 in mitochondria, patch clamp experiments were performed on Jurkat cell mitoplasts known to be endogenous in expressing Kv1.3.

These mitoplasts had a cation channel that had biophysical properties similar to those of Kv1.3 from the cell membrane. These characteristics include characteristics known from the literature, such as channel conductance ( 1b - 1d ), Current / voltage relationship (characteristic) ( 1c ), Internal selectivity ( 1d . 1c ) and sensitivity to the Kv1.3-specific inhibitor Margatoxin (MgTx) ( 1d ).

The Kv1.3 inactivation kinetics in mitoplasts could not be studied since applying large voltage jumps across the membrane of the mitoplast to a loss of electrical seal (Seal) led. The data confirm the expression of Kv1.3 in the mitochondria more commonly Cells.

1b - 1d show the functional expression of Kv1.3 in mitochondria. They show single-channel patch-clamp recordings on mitoplasts taken from Jurkat cells were isolated. On the basis of parameters, such as channel conductance ( 1b - 1d ), Current / voltage relationship ( 1c ), Internal selectivity ( 1b . 1c ) and sensitivity to MgTx ( 1d ), the functional expression of Kv1.3 in mitoplasts was proven. 1b shows representative traces of current under asymmetric [K ⁺ ] conditions (134/50 mM K ⁺ ) at +30, 0, -30, and -60 mV. Two channels indicated with status 1 and 2 in relation to the open channels) often work together. An outward net current at 100 mV illustrates potassium selectivity. 1c shows the current-voltage relationship of the channel for symmetric [K ⁺ ] conditions (134 mM, circles) or asymmetric [K ⁺ ] conditions (134/50 mM [K ⁺ ], triangles). 1d shows the treatment of mitochondria from Jurkat cells with the Kv1.3 inhibitor MgTx (2 nM), resulting in the blockage of the canal. Corresponding traces recorded at +10 mV under asymmetric [K ⁺ ] conditions are also shown.

Gentransfektionsexperimente to lead on the presence of Kv1.3 in mitochondria:

In order to avoid measuring another but similar mitochondrial potassium channel, a genetic model was used. Kv1.3-deficient CTLL-2 cells were probed with a Kv1.3 expression vector (pJK-Kv1.3) (cells designated CTLL-2 / Kv1.3) or a control vector (pJK) (designated CTLL-2 / pJK) transfected. Western blots of cell lysates ( 2a ) and patch-clamp investigations in the whole-cell configuration ( 2 B - 2g ) confirm the exclusive expression of Kv1.3 in transfected cells and show that the level of functional expression is comparable to that of Jurkat cells ( 2 B . 2c ). The biophysical properties of Kv1.3 in the plasma membrane of CTLL-2 / Kv1.3 cells were identical to those of endogenous Kv1.3 in Jurkat T lymphocytes ( 2d - 2g ). These data demonstrate the utility of this genetic model for the analysis of mitochondrial expression of Kv1.3.

The results illustrate the expression of Kv1.3 in CTLL-2 cells, wherein 2a Western blots of cell lysates showing the specific expression of Kv1.3 in CTLL-2 / Kv1.3 cells. The Kv1.3 protein (65 kDa) was absent in whole cell lysates of CTLL-2 / pJK cells. The proteins were separated by SDS-PAGE (10%) and blotted onto nitrocellulose and developed using polyclonal rabbit-derived anti-Kv1.3 antibodies using the chemiluminescent system of Tropix. Shown are representative blots from three experiments. 2 B - 2g represent the biophysical properties of Kv1.3 in CTLL-2 / Kv1.3 cells, which resemble the biophysical properties of Kv1.3 in Jurkat cells. 2 B Figure 3 shows the transfection of Kv1.3 into CTLL-2 cells resulting in a mean whole cell current amplitude nearly identical to that detected on the endogenously expressed Jurkat cell line. The mean amplitudes of the whole cell current were determined at 50 mV for Jurkat, CTLL-2 / Kv1.3 and CTLL-2 / pJK cells.

2c shows the distribution of the mean main amplitude currents, which were measured at 50 mV, resulting in a comparable for Jurkat cells and CTLL-2 / Kv1.3 cells expression behavior with respect to Kv1.3.

Representative Whole Cell Currents of CTLL-2 / pJK Cells ( 2d ) or CTLL-2 / Kv1.3 cells ( 2e ) were obtained by changing the membrane potential in the context of 6 discrete 20 mV steps (-70 to 50 mV). The voltage pulses were applied every 45 seconds. The data show the absence of Kv1.3 in CTLL-2 cells ( 2d ) and a typical Kv1.3 current in CTLL-2 / Kv1.3 cells ( 2e ).

2f shows the application-dependent or voltage-dependent inactivation of Kv1.3 in CTLL-2 / Kv1.3 cells, which was determined by instantaneous change of the membrane potential from -70 to 50 mV at intervals of 1 s each. Inactivation kinetics are identical to those measured for Jurkat T cells. In 2g it is shown the addition of 50 nM charybdotoxin (ChTx), a Kv1.3 inhibitor, which inhibits the K ⁺ current in CTLL-2 / Kv1.3 cells. Changing the bath solution containing 5 mM KCl to a medium containing 150 mM KCl results in a shift of the reversal potential from -70 / -80 mV to (0 ± 8) mV, which is the potassium selectivity of Kv1.3 in CTLL-2 /Kv1.3 cells (not shown).

In addition, patch-clamp studies were performed on isolated mitochondria derived from CTLL-2 / Kv1.3 and control CTLL-2 / pJK cells. The patch-clamp studies on mitoplasts from CTLL-2 / Kv1.3 cells show a potassium channel activity that resembles the properties of endogenous Kv1.3 channels in Jurkat mitoplasts ( 3a - 3e ). This current was absent in CTLL-2 / pJK cells (control) ( 3a ). The channel in Kv1.3-transfected CTLL-2 cells had a conductance of about 16 to 25 pS, a weak inward rectification (as expected for Kv1.3), a reversal potential of (-20 ± 6) mV close to at the theoretical equilibrium potential for potassium (E _K ) of -25 mV (indicating the potassium selectivity of the channel), a sensitivity to TEAC1 and a sensitivity to the very specific Kv1.3 inhibitor Margatoxin ( 3a - 3e ). Concrete detection of Kv1.3 currents in mitoplasts of Kv1.3-transfected CTLL-2 cells ( 3a ) shows the expression of Kv1.3 in mitochondria and excludes the detection of another channel.

To further investigate the presence of Kv1.3 in mitochondria, Western blot studies were performed on purified mitochondria of CTLL-2 / Kv1.3, CTLL-2 / pJK and Jurkat cells, which also showed specific expression of Kv1.3 in mitochondria Mitochondria show ( 3f ).

Subsequently, CTLL-2 / Kv1.3 and CTLL-2 / pJK cells transiently transfected with the pEYFP vector encoding a yellow fluorescent protein (EYFP) linked either to the mitochondrial targeting sequence of cytochrome c oxidase (Mito-EYFP) or calreticulin (ER-EYFP), a target sequence of the endoplasmic reticulum, as known from the literature. Transfected cells were sorted by flow cytometry (FACS), the mitochondria were purified, labeled with Cy3-linked anti-Kv1.3 antibodies and analyzed by FACS. The mitochondrial population was mito-EYFP positive and identified by their typical pattern of light scattering. A co-labeling of Mito-EYFP and Cy3-coupled anti-Kv1.3 was detected exclusively in mitochondria that were derived from CTLL-2 / Kv1.3 cells ( 3g ) and from Jurkat cells (not shown) but not detected in those mitochondria derived from CTLL-2 / pJK cells ( 3g ). The transfection experiments with respect to the ER-EYFP construct illustrate the absence of contaminating fragments of the endoplasmic reticulum in the mitochondrial preparations ( 3g ). Mito-EYFP-positive particles did not react with an anti-CD3 antibody, so that it is impossible that Kv1.3-containing cell membrane fragments contaminated the mitochondrial preparation ( 3g ). These data are further evidence for the expression of Kv1.3 in mitochondria.

The Investigations show that the Transfection of Kv1.3 into CTLL-2 cells leads to a functional expression of the potassium channel in mitochondria.

3a - 3e show patch-clamp experiments on isolated mitoplasts from transfected CTLL-2 cells, which demonstrate functional expression of Kv1.3 channels, which resemble those of Jurassic cell mitochondria.

3a shows representative current traces of CTLL-2 / Kv1.3 under symmetric conditions taken at 30 mV. Studies on CTLL-2 / pJK cells under the same conditions show the absence of the Kv1.3 channel. The amplitude histogram shows current amplitudes when the channel is open and closed.

3b refers to the current-voltage relationship of the channel in CTLL-2 / Kv1.3 cells under asymmetric (134/59 mM [K ⁺ ]; triangles) or symmetric (134 mM [K ⁺ ] circles) conditions.

3c . 3d show the treatment of mitochondria isolated from CTLL-2 / Kv1.3 with TEACI (100 mM) ( 3c ) or with MgTx (2 nM) ( 3d ). The channel was blocked in both cases. The currents were at 10 mV ( 3c ) or 100 mV ( 3d ) under asymmetric [K +] conditions. MgTx had no effect on mitoplasts of CTLL-2 / pJK cells (not shown), thus excluding a nonspecific effect of the toxin.

3e shows the time course of the mean current of mitoplasts from CTLL-2 / Kv1.3 and Jurkat cells under the influence of TEACI (closed squares, n = 3), MgTx (closed circles, n = 5) and without inhibitor (open triangles, n = 4). In mitochondria of Kv1.3-deficient cells were comparable activities as those in 3b - 3e described, not found.

3f shows a Western blot study on purified mitochondria from CTLL-2 / Kv1.3 and Jurkat cells, which demonstrates the expression of Kv1.3 in CTLL-2 / Kv1.3 cells, whereas mitochondria from control-transfected CTLL-2 / pJK cells do not have the 65 kDa channel protein. Cell lysates were separated by SDS-PAGE (10%), blotted, and analyzed using a polyclonal rabbit-derived antibody, a second AP-linked anti-rabbit antibody, and the tropix chemiluminescent system.

3g refers to transient transfection of CTLL-2 / Kv1.3 or CTLL-2 / pJK cells with pEYFP-Mito or control pEYFP-ER, demonstrating expression of Kv1.3 in mitochondria. Labeling of purified mitochondria with Cy3-coupled anti-Kv1.3 antibodies revealed expression of Kv1.3 in mitochondria derived from CTLL-2 / Kv1.3 cells and absence of the channel in mitochondria from CTLL-2 / pJK cells. Contamination of the mitochondria with Kv1.3-containing cell membrane fragments or with fragments of the endoplasmic reticulum was fused by labeling with Cy3-coupled anti-CD3 antibodies or by transfection of an EYFP protein fused to a target sequence of the endoplasmic reticulum (pEYFP-ER) , locked out.

Kv1.3 comes together with known mitochondrial ion channels:

Around the presence of Kv1.3 in the inner mitochondrial membrane (IMM) clearly confirm were patches (membrane sections) that had Kv1.3 activity, to the presence of mitochondrial known from the literature ion channels examined. The 107 pS channel and the PTP (Permeability Transition Pore) were used in these studies as indicators in relation to the IMM used. Both channels, which have been shown to occur only in the IMM, but not in the outer mitochondrial Membrane exist were identified by their biophysical and pharmacological Properties identified.

The experiments show a simultaneous activity of Kv1.3 and the 107 pS channel in patches released from CTLL-2 / Kv1.3 cells ( 4a ) and Jurkat mitoplasts (not shown), whereas patches of CTLL-2 / pJK contain only the 107 pS channel and have no Kv1.3 activity (not shown).

To investigate the common occurrence of PTP and Kv1.3, the Ca ²⁺ concentration was increased on the matrix sides of the patches, which were positive with respect to Kv1.3. The [Ca ²⁺ ] increase led to activation of PTP as known from the literature. The data show that 50% of the patches containing Kv1.3 simultaneously have PTP ( 4b ). The common occurrence of both channels was detected only in mitoplasts of CTLL-2 / Kv1.3 and Jurkat cells ( 4b ), whereas mitoplasts from CTLL-2 / pJK cells showed PTP activity, but had no activity with respect to Kv1.3. Several patches of mitoplasts from CTLL-2 / Kv1.3 and Jurkat cells contained all three channels, ie Kv1.3 the 107 pS channel and - after a [Ca ²⁺ ] increase - the PTP. The coexistence of Kv1.3 and the 107 pS channel and / or PTP in mitoplasts of CTLL-2 / Kv1.3 or Jurkat cells illustrates the functional expression of Kv1.3 in the IMM.

The results show the coexistence of Kv1.3 with the 107 pS channel and PTP in the inner mitochondrial membrane. 4a Figure 7 shows Kv1.3 (step 1) and the 107 pS channel (step 1 ") typical of IMM, with current drawn from the same membrane patch. The corresponding trace was recorded at + 60mV under symmetric [K ⁺ ] (134mM K ⁺ ). The amplitude of the small channel was 0.96 pA at a level of 1 pA assumed for Kv1.3 at this potential difference (voltage). A common occurrence of Kv1.3 and the 107 pS channel was found in three out of seven Kv1.3-containing Jurkat cell patches and on two out of seven CTLL-2 / Kv1.3 cell patches.

In 4b was measured in the context of a patch Kv1.3 and then [Ca ²⁺ ] in the bath solution to a concentration of 0.1 mM (150 mM KCl, 0.1 mM CaCl ₂ , 20 mM HEPES / K +, pH 7.3). increased to capture the mitochondrial mega channel in the same patches containing Kv1.3. In such an experiment, the trace shows the activity of the mitochondrial megakanal at a transmembrane potential of -20 mV. Similar results were obtained in two out of seven patches of CTLL-2 / Kv1.3 and five out of seven patches of Jurkatmitoplasts. Subsequently, in two of the seven patches (1 patch to CTLL-2 / Kv1.3 and 1 patch to Jurkat), the common occurrence of all three channels was determined, ie Kv1.3, the 107 pS channel and, after increasing [Ca ²⁺ ], the PTP. The 107 pS channel and the PTP were identified by their typical biophysical and pharmacological properties.

Mitochondrial Kv1.3 is for Bax-induced apoptosis requires:

To analyze the functional importance of mitochondrial Kv1.3 in inducing apoptosis, CTLL-2 / Kv1.3 and control CTLL-2 / pJK cells were transfected with an expression vector for Bax and the apoptosis of cells was determined was induced by the expression of Bax. Transfected cells were identified by co-transfection with a GFP-actin fusion construct. The results show that Kv1.3 was needed for Bax-induced apoptosis. While expression of Bax induced apoptosis in (70 ± 10)% of Kv1.3-positive cells (after 12 hours), Kv1.3-deficient CTLL-2 cells were resistant to Bax ( 5a ). Induction of apoptosis in CTLL-2 / Kv1.3 by cellular expression of Bax was not affected by concomitant treatment with 2 nM MgTx; this corresponds to a dose sufficient to completely inactivate Kv1.3 in the plasma membrane. However, this concentration is not present at the mitochondria, since the peptide is not membrane-permeable. An inhibitor of the K _ATP channel also did not affect Bax-induced apoptosis (not shown).

To unequivocally demonstrate the hypothesis regarding the important function of Kv1.3 in the context of apoptosis, isolated and purified mitochondria were incubated with recombinant GST-Bax or GST as a control. While recombinant Bax induced a rapid release of cytochrome c from Kv1.3-positive mitochondria - indicative of apoptotic changes in these mitochondria - it did not show any effect on Kv1.3-deficient mitochondria ( 5b ). These data show that the prev mitochondrial Kv1.3 is necessary for Bax-induced apoptosis. In contrast, incubation of intact cells with 100 ng / ml GST-Bax did not result in significant apoptosis ( 5b ), so that an important function of the membrane-localized Kv1.3 with respect to apoptosis can be ruled out.

The results illustrate that Kv1.3 is necessary for Bax-induced apoptosis, with 5a refers to an experiment in which cells were transfected with pcDNA-Bax or the control vector by electroporation. Dead cells were removed by Ficoll, and with respect to Bax-induced apoptosis, the decrease in cells was determined 46 hours after transfection. The data show that Kv1.3 is necessary for Bax-induced apoptosis. Kv1.3-deficient cells were resistant to Bax-induced apoptosis. Incubation of intact cells with 2 nM MgTx used to inactivate Kv1.3 in the plasma membrane or with an inhibitor of the K _ATP channel did not affect Bax-induced apoptosis (not shown).

5b demonstrated that incubation of purified Kv1.3-positive mitochondria with 100 ng / ml recombinant GST-Bax for 20 minutes resulted in the release of cytochrome c, indicative of apoptotic changes in these mitochondria. Control GST showed no effect. Kv1.3 deficiency prevented the release of cytochrome c from isolated mitochondria when incubated with recombinant Bax. Isolated mitochondria were treated as described above, the samples were centrifuged for 10 minutes to pellet the mitochondria. Supernatants were separated by SDS-PAGE (2.5%) and treated with anti-cytochrome c antibodies and developed with the Tropix chemiluminescent system.

The inactivation of the mitochondrial Kv1.3 leads to apoptotic changes:

To further study the function of Kv1.3 with respect to apoptosis, Kv1.3-positive CTLL-2 / Kv1.3 cells and Kv1.3-deficient CTLL-2 cells with 100 ng / ml TNFα (tumor Necrosis factor α), 1 μM staurosporine or 20 μM C ₆ -ceramide. While Kv1.3-transfected cells with DNA fragmentation ( 6a . 6b ), Caspase-3 (DEVDase) activation ( 6c ), mitochondrial cytochrome c release ( 6d ), Loss of mitochondrial membrane potential ( 6e ) and morphological changes typical of apoptosis, CTLL-2 / pJK cells showed resistance and did not show any of these changes ( 6a - 6e ).

The investigations show that the expression of Kv1.3 in relation to a TNFα-, Staurosporin- and C ₆ -ceramide-mediated apoptosis is required. Treatment of CTLL-2 / Kv1.3 cells with 20 μM C ₆ -ceramide, 1 mM staurosporine or 100 ng / ml TNFα resulted in DNA fragmentation ( 6a - 6b ), Caspase-3 activation ( 6c ), Cytochrome c release ( 6d ) and change in ΔΨ _m ( 6e ), whereas cells without Kv1.3 do not respond to apoptotic stimulants. Shown are the mean ± SD (standard deviation) or representative results of at least three independent experiments.

To determine the importance of the mitochondrial Kv1.3 channel with respect to apoptosis, purified, isolated mitochondria from CTLL-2 / Kv1.3 or CTLL-2 / pJK cells were used. To mimic the inactivation of Kv 1.3 channels ₆ -ceramide known to occur upon stimulation with CD95 or treatment with cellular C, isolated mitochondria were incubated with 20 nM MgTX, a well-known from the literature, specific inhibitor of the Kv1.3. If mitochondrial Kv1.3 channels are necessary for apoptosis, only mitochondria expressing Kv1.3 should be sensitive to MgTx and, consequently, have such changes as are typical of apoptosis.

The investigations show that the expression of Kv1.3 in relation to a TNFα-, Staurosporin- and C ₆ -ceramide-mediated apoptosis is required. Treatment of CTLL-2 / Kv1.3 cells with 20 μM C ₆ -ceramide, 1 mM staurosporine or 100 ng / ml TNFα resulted in DNA fragmentation ( 6a - 6b ), Caspase-3 activation ( 6c ), Cytochrome c release ( 6d ) and change in ΔΨ _m (CCCP as control) ( 6e ), whereas cells without Kv1.3 do not respond to apoptotic stimulants. Shown are the mean ± SD (standard deviation) or representative results of at least three independent experiments.

MgTx induced a rapid and marked release of cytochrome c from Kv1.3-positive mitochondria, whereas Kv1.3-deficient mitochondria did not react ( 7a ). Treatment of mitochondria with an inhibitor of K _ATP channels did not show any effect on isolated mitochondria and did not induce apoptosis. Corresponding results were found for the mitochondrial membrane potential in MgTx treatment: Kv1.3-positive mitochondria under the influence of MgTx show a transient hyperpolarization that was not present in Kv1.3-deficient mitochondria ( 7b . 7c ). This is consistent with biophysical considerations that result in hyperpolarization with respect to mitochondrial membrane potential upon inhibition of Kv1.3. The hyperpolarization was followed by a distinct depolarization of ΔΨ _m , the by cyclosporin A ( 7c ) was inhibited. Since cyclosporin A is known to inhibit PTP, the observed depolarization is mainly due to activation of PTP. Activation of PTP and ΔΨ _m changes did not occur in Kv1.3-deficient mitochondria treated with MgTx ( 7c ). With respect to untreated mitochondria, ΔΨ _{m was} high and constant during the experiment, indicating that the isolation steps did not damage the mitochondria.

In addition, only Kv1.3-positive mitochondria showed rapid and sustained release of ROS ( 7d ), which occurs in many forms of apoptosis - a response to MgTx. Kv1.3 deficiency in CTLL-2 / pJK cells did not result in ROS release from mitochondria when treated with MgTx ( 7d ). Addition of the radical scavenger butylhydroxytoluene (BHT) or the reducing substance dithiothreitol (DTT) in combination with MgTx led to an inhibition of ROS release ( 7d ) and an inhibition of cyclosporin A-sensitive depolarization (not shown).

These Data show that the Inhibition of mitochondrial Kv1.3 to changes in mitochondria leads, which play a central role in many forms of apoptosis.

The Results clarify that the Inhibition of mitochondrial Kv1.3 to changes in mitochondria which can mediate apoptosis or that for apoptotic Processes are characteristic.

Inhibition of Kv1.3 in isolated mitochondria by MgTx leads to cytochrome c release ( 7a ), a ΔΨ _m hyperpolarization, followed by a depolarization ( 7b . 7c ) and a ROS release ( 7d while Kv1.3-deficient mitochondria from CTLL-2 / pJK showed no response to MgTx ( 7a - 7d ). 7a refers to the incubation of isolated and purified mitochondria in 20 nM MgTx for 30 minutes and shows the release of cytochrome c from mitochondria of CTLL-2 / Kv1.3 or CTLL-2 / pJK determined by Western blotting. The Western blots show the cytochrome c content in mitochondria before and after treatment with MgTx as well as the corresponding content of cytochrome c in the supernatant of the same mitochondrial preparations. 7b shows a representative FACS analysis of ΔΨ _m on isolated mitochondria labeled with 1 nM DioCb (3). Isolated mitochondria were incubated for 0.5 and 20 minutes in 20 nM MgTx and aliquots were analyzed for changes in ΔΨ _m . The FACS data were reproduced four times.

7c shows a quantitative analysis (n = 3) of the mitochondrial membrane potential of isolated mitochondria before and after application of MgTx. The data show an initial hyperpolarization of ΔΨ _m, followed by a marked depolarization. The addition of cyclosporin A, a PTP inhibitor, prevents mitochondrial depolarization, but not the initial hyperpolarization. It follows that PTP secondarily mediates mitochondrial depolarization in relation to the primary hyperpolarization. In mitochondria from CTLL-2 / pJK cells no changes of ΔΨ _m were found after treatment with MgTx. Mitochondrial membrane potential was determined using 1 nM DioCb (3) and confirmed in experiments using TMRE. Complete depolarization using CCCP served as a control of mitochondrial integrity. Shown are mean ± SD of the fluorescence intensities measured by flow cytometry and reported as freely selected fluorescence units. The fluorescence intensities were evaluated using Becton-Dickinson software to determine the mean of 10,000 signals measured per analysis. The tests were repeated 4 times each.

7d shows MgTx-induced ROS release from isolated Kv1.3-positive mitochondria, which was not seen in Kv1.3-deficient mitochondria. ROS was neutralized by preincubation of the isolated mitochondria with 3 mM DTT and 50 μM BHT. Shown are mean ± SD from four independent experiments.

Proof of the interaction or interaction of apoptosis-inducing and / or apoptosis-stimulating Substance (AISI with that in the inner mitochondrial membrane (IMM) localized Kv1.3 potassium channel:

Around the function of located in the inner mitochondrial membrane (IMM) To further analyze Kv1.3 potassium channel was investigated whether Kv1.3 directly with an apoptosis-inducing and / or apoptosis-stimulating Substance (AIS) interacts or interacts. As a test substance was Bax uses a member of the pro-apoptotic Bc1-2 family.

As part of the experimental procedure, Kv1.3-positive cells were stimulated with CD95 and subsequently lysed and Bax immunoprecipitated and the interaction or interaction was tested by means of a Western blot. The results show an interaction or interaction between Kv1.3 and Bax after proapoptotic stimulation ( 8a ).

8a shows the interaction between Kv1.3 and Bax, where the cells were stimulated by CD95. Bax immunoprecipitates were separated by SDS-PAGE (7.5%) and the proteins transferred to a nitrocellulose membrane. After incubation with polyclonal alkaline phosphatase (AP) -coupled secondary antibodies, the evaluation or development was carried out with the chemiluminescence Tropix.

These coimmunoprecipitation experiments complement the results in 5b showing that only Kv1.3-positive but not Kv1.3-deficient mitochondria release cytochrome c after incubation with a GST-Bax fusion protein.

The coimmunoprecipitation experiments were confirmed with an independent method, namely the analysis of molecular interactions in the yeast two-hybrid system ( 8b ). 8b shows an experiment using the yeast two-hybrid system. The interaction or interaction between Kv1.3 and Bax results in the proximity of the fused activation domain and the DNA-binding domain of the yeast transcriptional activator, thereby activating and inducing the expression of β-galactosidase. By staining with β-gal is a blue color (in the black / white representation of 8b shown as gray color) of the colonies in which an interaction or interaction between Kv1.3 and Bax has taken place. In cells transfected with Bax and a control vector no blue colonies could be found.

Claims (26)

Use of at least one apoptosis-inducing and / or apoptosis-stimulating substance (AIS) for the manufacture of a medicament or medicament for the prophylactic and / or curative treatment of diseases of the human or animal body, in which a particularly excessive reduction of apoptosis and / or an especially excessive increase of cell growth and / or cell division occurs or which are associated therewith or in which a therapeutically targeted apoptosis of the cells in question is intended, characterized in that the apoptosis-inducing and / or apoptosis-stimulating substance (AIS) is selected and / or designed such that they interact and / or interaction with mitochondrial potassium channels (MK) of the cells in question induces and / or stimulates apoptosis of these cells. Use according to claim 1, characterized that the apoptosis-inducing and / or apoptosis-stimulating substance (AIS) such is that they interacts with the mitochondrial potassium channel (MK) in such a way and / or interacts, in particular by physical and / or chemical binding of apoptosis-inducing and / or apoptosis-stimulating Substance (AIS) to the mitochondrial potassium channel (MK), that in the relevant cells induced apoptosis of these cells and / or is stimulated. Use according to claim 1 or 2, characterized that the apoptosis-inducing and / or apoptosis-stimulating substance (AIS) selected in this way and / or that they are blocked and / or inhibited the mitochondrial potassium channel (MK). Use according to one of the preceding claims, characterized characterized in that apoptosis-inducing and / or apoptosis-stimulating substance (AIS) the physiological parameters of the cells in question, in particular whose metabolism, osmolarity, pH, membrane potential and the like, so influenced that the biophysical Properties, in particular channel conductance, channel open time and / or Open-Channel Probability of Mitochondrial Potassium Channels (MK) the cells in question are changed in such a way that in the relevant cells induced apoptosis of these cells and / or is stimulated. Use according to one of the preceding claims, characterized characterized in that apoptosis-inducing and / or apoptosis-stimulating substance (AIS) selected in this way and / or that they are additionally with anti-apoptotic substances (AAS), especially in the cells concerned Naturally occurring and / or endogenous anti-apoptotic substances (AAS), in which way interacts and / or interacts that the antiapoptotic effect of antiapoptotic substances (AAS) is inhibited or at least inhibited and / or the antiapoptotic Substances (AAS) are inactivated, in particular by physical and / or chemical binding of apoptosis-inducing and / or apoptosis-stimulating Substance (AIS) to the anti-apoptotic substances (AAS). Use according to one of the preceding claims, characterized characterized in that apoptosis-inducing and / or apoptosis-stimulating substance (AIS) selected in this way and / or that the apoptosis-inducing and / or apoptosis-stimulating substance (AIS) as such has no anti-apoptotic properties and / or in with respect to the cells in question does not inhibit apoptosis or inhibits. Use according to one of the preceding claims, characterized in that the apoptosis-inducing and / or apoptosis-stimulating substance (AIS) is selected from the group of hydrocarbons substances, nucleic acids, lipids or peptides, such as oligopeptides, polypetides and proteins, as well as derivatives of the aforementioned compounds, such as lipoproteins, is selected. Use according to one of the preceding claims, characterized characterized in that apoptosis-inducing and / or apoptosis-stimulating substance (AIS) from the group of specific inhibitors and / or channel blockers of the Kv1.3 potassium channel of the inner mitochondrial membrane (IMM), as Margatoxin and / or Shigatoxin and their homologues and derivatives, selected is. Use according to one of the preceding claims, characterized in that the apoptosis-inducing and / or apoptosis-stimulating substance (AIS) is a pro-apoptotic peptide, in particular oligopeptide, polypeptide or protein, in particular a pro-apoptotic peptide of the Bc1-2 family, such as Bax, Bak, Bok, Bim, Bmf Bad, Bik, Bid, Bc1-x _s, Noxa, Puma, as well as their homologues and derivatives. Use according to one of the preceding claims, characterized characterized in that mitochondrial potassium channel (MK) in the inner mitochondrial membrane (IMM) is localized and / or that the mitochondrial potassium channel (MK) a voltage-dependent Potassium channel is and / or that of mitochondrial potassium channel (MK) a Kv1.3 potassium channel or homologous thereto Potassium channel, in particular a Kv1.1 potassium channel, Kv1.2 potassium channel, Kv1.4 potassium channel or Kv1.5 potassium channel. Use according to one of the preceding claims, characterized characterized in that Treatment of the cells concerned outside the body, in particular ex-vivo especially in that the apoptosis inducing and / or apoptosis stimulating Substance (AIS) is allowed to act on the cells concerned, followed by a (re) implantation of the treated in this way Cells in the diseased and / or to be treated body. Use according to one of the preceding claims, characterized characterized in that apoptosis-inducing and / or apoptosis-stimulating substance (AIS) is administered in an amount sufficient to cause an interaction and / or interaction of this substance with the mi tochondrial potassium channels (MK) to cause such that in induces apoptosis of these cells and / or is stimulated. Use according to one of the preceding claims, characterized characterized in that apoptosis-inducing and / or apoptosis-stimulating substance (AIS) in the form of an appropriate precursor, especially as a prodrug, is applied, which below, in particular in the relevant Cells, in situ the apoptosis-inducing and / or apoptosis-stimulating Substance (AIS) can generate, in particular by chemical and / or physical transformation, gene expression, protein biosynthesis and like. Use according to one of the preceding claims, characterized characterized in that apoptosis-inducing and / or apoptosis-stimulating substance (AIS) systemically and / or topically applied or added. Use according to one of the preceding claims, characterized characterized in that disease to be treated a parasitic disease, a bacterial Disease, a viral disease such as, hepatitis or AIDS, or a tumor or cancer, especially a benign or malignant Tumor disease, especially a cancer, such as colon cancer (Colon carcinoma), breast cancer (breast cancer), ovarian carcinoma, uterine carcinoma, Lung cancer, stomach cancer, liver cancer, pancreatic cancer, kidney cancer, Bladder cancer, prostate cancer, testicular cancer, bone cancer, skin cancer, Kaposi's sarcoma, brain tumor, myosarcoma, neuroblastoma, lymphoma and leukemia. Use according to one of the preceding claims, characterized characterized in that apoptosis-inducing and / or apoptosis-stimulating substance (AIS) selected in this way and / or that they are the Kv1.3 potassium channels the inner mitochondrial membrane (IMM) of the cells in question and / or inhibited. A method for inducing and / or stimulating apoptosis in cells in which a particular excessive reduction of apoptosis and / or a particular excessive increase of cell growth and / or cell division occurs or which are associated therewith or in which a therapeutically targeted apoptosis of the cells in question by influencing mitochondrial potassium channels (MK) of the relevant cells by means of apoptosis-inducing and / or apoptosis-stimulating substances (AIS), characterized by the following method steps: (a) Provision and / or selection of an apoptosis-inducing and / or apoptosis-stimulating substance (AIS) which is selected and / or designed to induce and / or stimulate apoptosis of these cells by interaction and / or interaction with the mitochondrial potassium channels (MK) of the cells in question; (b) administering the apoptosis-inducing and / or apoptosis-stimulating substance (AIS) in therapeutically effective amounts and / or administering the apoptosis-inducing and / or apoptotic stimuli lierenden substance (AIS) to the cells in sufficient quantities to achieve the intended effect. A method according to claim 17, characterized by the features of the characterizing part of one or more of claims 1 to 16th Method for prophylactic and / or curative Treatment of diseases of the human or animal body, at an especially excessive reduction of apoptosis and / or a particular excessive increase in cell growth and / or Cell division occurs or related or in which a therapeutically targeted apoptosis of the respective Cells is intended by administration of at least one apoptosis-inducing and / or apoptosis-stimulating substance (AIS) in therapeutically effective Quantities, characterized in that the apoptosis-inducing and / or apoptosis-stimulating substance (AIS) are selected and / or procured in such a way is, that you by interaction and / or interaction with mitochondrial potassium channels (MK) induces apoptosis of these cells and / or stimulated. A method according to claim 19, characterized by the features of the characterizing part of one or more of claims 1 to 16th Method for finding and / or identifying an apoptosis-inducing and / or apoptosis-stimulating substance (AIS), which is such that by interaction and / or interaction with mitochondrial potassium channels (MK) relevant cells induced apoptosis of these cells and / or stimulated, characterized by the following process steps: (A) Provide a suitable test system with which the interaction between apoptosis-inducing and / or apoptosis-stimulating substances (AIS) on the one hand and mitochondrial potassium channels (MK) on the other can; (b) exposing the apoptosis-inducing and / or apoptosis stimulating substance (AIS) or a corresponding precursor to the test system provided in step (a); (C) Evaluation with suitable methods adapted to the respective test system, in particular with regard to the presence of an interaction and / or Interaction of apoptosis inducing and / or apoptosis stimulating Substance (AIS) or corresponding precursor on the one hand and test system on the other hand. Method according to claim 21, characterized that this in test step (a) provided test system a potassium channel, if necessary in an artificial or natural Membrane, in particular a lipid bilayer, comprises, in particular wherein the potassium channel is a voltage-dependent potassium channel, in particular a Kv1.3 potassium channel or a homologous potassium channel, such as a Kv1.1 potassium channel Kv1.2 potassium channel Kv1.4 potassium channel or Kv1.5 potassium channel, can be, and that in Process step (c) the detection of the interaction of apoptosis-inducing and / or apoptosis stimulating substance (AIS) or equivalent precursor on the one hand and the test system, in particular the potassium channel of the test system, on the other hand by means of electrophysiological measurement methods and / or by means of physical and / or chemical detection methods, in particular voltage-dependent dye reactions, and / or by means of immunological detection reactions, in particular Immunoblotting, done. Method according to claim 22, characterized in that that between the method steps (a) and (b) and / or between the method steps (b) and (c) an additional Intermediate process step, wherein at least one anti-apoptotic substance (AAS) on the test system becomes. Method according to claim 21, characterized that this in test step (a) provided test system an expression system includes, wherein the expression system on the one hand a nucleotide sequence (I), which for one mitochondrial potassium channel (MK), and on the other hand a Nucleotide sequence (II), which for an apoptosis-inducing and / or apoptosis-stimulating substance (AIS) coded, comprises, and that in Process step (c) the interaction of the expressed gene products (Proteins) of the nucleotide sequences (I) and (II) is detected. Method according to Claim 24, characterized that the Nucleotide sequences (I) and (II) for the purposes of in step (c) to be carried out Evaluation as well each have at least one area, each for at least a protein domain which are suitable for use in a suitable detection method, especially in the context of a TWO hybrid system, in particular wherein in the context of a two-hybrid system at least one protein domain a DNA-binding domain and at least one further protein domain is an activation domain, being DNA-binding domain on the one hand and activation domain on the other hand bound to different proteins. Method according to one of claims 21 to 25, characterized in that the method is carried out as part of a preferably automated screening method, in particular high-throughput screening (HTS), and / or that the apoptosis-inducing and / or apoptotic lierenden substances (AIS) or their corresponding precursors from a corresponding substance library (substance bank), in particular protein or gene library (protein / gene bank) are provided.