WO2004040299A2 - Method for identifying compounds which inhibit mechanotransduction in neurons - Google Patents

Abstract

Described is a method for identifying compounds which are capable of inhibiting mechanotransduction in neurons, compounds identified by such a method, pharmaceutical compounds containing the identified compounds and methods for treating pain. Also described is the use of stomatin domain-containing proteins for the identification of compounds which are capable of inhibiting mechanotransduction.

Description

Method for identifying compounds which inhibit mechanotransduction in neurons

The present invention relates to a method for identifying compounds which are capable of inhibiting mechanotransduction in neurons. The present invention also relates to compounds identified by the described method, to a method for the production of a pharmaceutical composition and to the produced pharmaceutical composition. Moreover, the present invention relates to a method for treating pain and to the use of a stomatin domain-containing polypeptide for the identification of a compound which is capable of inhibiting the mechanotransduction in a neuron.

Sensory neurons of the dorsal root ganglia (DRG) innervating the skin, muscle and viscera detect mechanical stimuli in the peripheral tissue. This biological process called sensory mechanotransduction is the basis of the senses of proprioception touch, and of mechanical pain. The ion channels present in sensory neurons that transduce mechanical stimuli are poorly characterized (Gillespie and Walker, 2001 Molecular basis of mechanosensory transduction. Nature 413(6852): 194-202). Recent work has indicated that ion channels belonging to the Degenerin/Enac family may contribute subunits to the transducing ion channels in some types of sensory neurons. For example, the ion channel gene BNC1 (Brain sodium channel 1) contributes to the transduction of touch stimuli in the mouse (Price, et al., Nature 407 (2000), 1007-1011). In addition the DRASIC channel (or ASIC3) is also involved in mechanotransduction in touch and pain detecting sensory neuron in the mouse (Price et al., Neuron 32 (2001), 1071-1083). In both these cases knockout mouse models were generated and the integrity of mechanotransduction in different types of sensory neurons innervating the skin tested using electrophysiological techniques.

The use of gene knockout strategies has so far been successful in identifying potential gene products that contribute to sensory mechanotransduction. However, the same observations indicate that mechanotransduction may be mediated simultaneously by more than one molecular entity. The reason being that deletion of one ion channel subunit has never led to the complete loss of mechanosensory function in single neurons (Price et al. 2000; 2001 , loc. cit.). In addition a mutagenesis screen carried out in the nematode worm has identified many different genes that when deleted lead to loss of touch sensitivity (see Figure 1). It has been hypothesized from this work that gating (opening) of mechanotransduction channels may be mediated via protein/protein interactions (Gillespie and Walker, 2001 , loc.cit).

Up until now just two genes have been directly implicated in the mechanotransduction in vertebrates: BNC1 and DRASIC (see above). When expressed in a cell line (HEK 293 cells), these two proteins can form homomeric channels that can be gated (opened) using a low pH stimulus (protons). The relevance of proton-gating of these channels to mechanotransduction is not clear. For example the endings of sensory neurons in the skin that transduce mechanical stimuli as a rule do not respond to protons. The involvement of the channel or another associated protein in mechanotransduction can at the moment only be unequivocally determined using an ex vivo preparation consisting of the skin and a sensory nerve (Koltzenburg et al., J. Neurophysiology 78 (1997), 1841-1850).

As mentioned above, mechanotransduction is the basis for, inter alia, the sense of mechanical pain. Although the sensation of pain is useful because it alerts the organism to real or impending injury and triggers appropriate protective responses, it can unfortunately also often outlive its usefulness as a warning system and instead become chronic and debilitating. In such cases a treatment is required which allows to ameliorate the sensation of pain in the individual. At the present time only two classes of drugs have been successfully used to treat pain with hyperalgesia and allodynia and these are drugs belonging to the non-steroidal anti- inflammatory (NSAID) class (eg. indomethacin and aspirin) and opiate analgesics. The latter drugs are often of limited usefulness for very common debilitating pain because of their side effects which can include addiction and tolerance because these act within the central nervous system. The NSAID on the other hand are thought to have their major site of action in the periphery and are consequently safe and effective for many conditions. There is an urgent need to find new drug targets in the periphery for analgesic therapy as NSAID have proven to be only of limited effectiveness in relieving completely the pain associated with chronic conditions like rheumatoid arthritis. The use of new molecular targets distinct from that of NSAID may be promising for producing equivalent or better analgesia than with a NSAID. By utilizing a new functionally separate molecular target for pain therapy, it may also be possible to potentiate the analgesic effects of for example NSAID in certain conditions. Thus, there exists a need for means and methods which allow the identification of compounds which can act as analgesics for treating a wide variety of persistent pain conditions.

Thus, the technical problem underlying the present invention is the provision of means and methods for identifying compounds useful in treating pain conditions.

This technical problem is solved by the provision of the embodiments as characterized in the claims.

Accordingly, the present invention relates to a method for identifying a compound capable of inhibiting the mechanotransduction of a neuron comprising the steps of

(a) providing a cell overexpressing a polypeptide selected from the group consisting of stomatin domain-containing proteins;

(b) contacting the cell with a candidate compound;

(c) measuring the current or voltage at the plasma membrane of the cell; and

(d) identifying the candidate compound as a compound capable of inhibiting mechanotransduction if the current or voltage measured in step (c) is altered in comparison to the corresponding current or voltage of a control cell corresponding to the cell provided in step (a) and which has not been contacted with said candidate compound.

The term "mechanotransduction" refers to the signal transduction in which a mechanical stimulus is converted into an action potential. Preferably, the term relates to mechanotransduction that is connected with the sensation of pain, i.e. mechanical nociception (see also Julius and Basbaum, Nature 413 (2001), 203- 210). The term "neuron" refers to any type of neuron that participates in mechanotransduction. These are preferably sensory neurons capable of sensing mechanical or noxious stimuli, preferably neurons which are capable of sensing noxious mechanical stimuli. Preferred are sensory neurons of the dorsal root ganglia (DRG) innervating the skin, muscle and viscera.

The method of the present invention is based on the finding that stomatin domain- containing proteins functionally interact with mechanotransducing ion channels, thereby modulating the activity of said ion channels. This function of the stomatin domain-containing proteins is not described in the prior art. In connection with the present invention, it has been shown that four members of the family of stomatin domain-containing proteins show this function (see Examples). Examples of stomatin domain-containing proteins are stomatin, Nstoml, Nstom2 and MSLP1. The nucleotide sequence encoding mouse stomatin is shown under SEQ ID NO: 1 and the deduced amino acid sequence is shown under SEQ ID NO: 2. The nucleotide sequences encoding Nstoml , Nstom2 and MSLP1 are shown in SEQ ID NOs: 3, 5 and 7, respectively. The corresponding amino acid sequences of Nstoml (rat), Nstom2 (mouse) and MSLP1 (mouse) are shown in SEQ ID NOs: 4, 6 and 8, respectively. The present invention provides, based on the above-mentioned finding, a cell based system that mirrors the effects that stomatin domain-containing proteins have on the gating of mechanotransduction channels and which allows an easy measurement of these effects. This makes it possible to screen for compounds that influence the activity of these proteins, in particular their interaction with and effect on mechanotransduction channels.

The term "stomatin domain-containing proteins" denotes a family of proteins which is characterized by the presence of a stomatin domain.

The term "stomatin domain" refers to an amino acid sequence which comprises the following sequence:

R-x(2)-[LIV]-[SAN]-x(6)-[LIV]-D-x(2)-T-x(2)-W-G-[LIVT]-[KRH]-[LIV]-x-[KRA]-[LIV]-E- [LIV]-[KRQ] (SEQ ID NO: 25; this sequence is also referred to as the "band 7 protein signature" (Prosite database)) or which comprises a protein family signature which has been identified and which is called the SPFH domain (Tavernarakis et al., Trends Biochem Sci 24 (1999), 425-427). "SPFH domain" stands for "stomatin prohibitin flotilin hfl protease homology domain". Examples of stomatin domains according to the meaning of the present invention are shown in Example 5.

Proteins belonging to the familiy of proteins of band 7/SPFH containing proteins includes members of the Prosite:PDOC00977 family, and also proteins with high blast scores to known band 7 proteins,e.g. HflC from E. coli (HFLC_ECOLI), HflK from E. coli (HFLK_ECOLI) and prohibitin family members, such as (PHBJHUMAN). The band 7 domain is catalogued in the Prosite (PDOC00977) and Pfam databases pfamO1145.5, Band_7; see http://www.ncbi.nlm.nih.gov/Structure/cdd/cddsrv.cgi?uid=pfam01145&version=v1.5 8.

Preferably, a stomatin domain-containing protein comprises both of the two above- mentioned sequences (i.e. the band 7 and the SPFH sequence). The band 7 and the SPFH sequence are nearly identical and overlap. However, the SPFH sequence is slightly shorter than the band 7 sequence.

It is particularly preferred that the stomatin domain-containing protein substantially consists of the stomatin domain, especially the stomatin domain of stomatin or Nstom 1. As is shown in Example 5 and Figure 10, the stomatin domain alone can evoke a powerful activating effect on the BNC1 channel.

Preferably, the "stomatin domain-containing protein" is a polypeptide being encoded by a polynucleotide the complementary strand of which hybridises with a nucleotide sequence encoding stomatin or Nstoml or Nstom2 or MSLP1 as shown under SEQ ID NOs: 1 , 3, 5 or 7 and having the activity of interacting with mechanotransducing ion channels, thereby modulating the activity of said ion channels. In this context, the term "hybridization" means hybridization under conventional hybridization conditions, preferably under stringent conditions, as for instance described in Sambrook and Russell (2001), Molecular Cloning: A Laboratory Manual, CSH Press, Cold Spring Harbor, NY, USA. In an especially preferred embodiment, the term "hybridization" means that hybridization occurs under the following conditions: Hybridization buffer: 2 x SSC; 10 x Denhardt solution (Fikoll 400 + PEG

+ BSA; ratio 1 :1 :1); 0.1% SDS; 5 mM EDTA; 50 mM Na2HPO4_;

250 μg/ml of herring sperm DNA; 50 μg/ml of tRNA; or

0.25 M of sodium phosphate buffer, pH 7.2;

1 mM EDTA

7% SDS Hybridization temperature T = 60°C Washing buffer: 2 x SSC; 0.1 % SDS

Washing temperature T = 60°C.

Polynucleotides encoding a stomatin domain-containing protein which hybridize with a nucleotide sequence of SEQ ID NOs: 1 , 3, 5 or 7 can, in principle, be derived from any organism expressing such a protein or can encode modified versions thereof.

Such hybridizing polynucleotides can for instance be isolated from genomic libraries or cDNA libraries of bacteria, fungi, plants or animals. Preferably, such polynucleotides are of mammalian origin, particularly preferred from mouse or human.

Such hybridizing polynucleotides may be identified and isolated by using the polynucleotides described herein or parts or reverse complements thereof, for instance by hybridization according to standard methods (see for instance

Sambrook and Russell (2001), Molecular Cloning: A Laboratory Manual, CSH

Press, Cold Spring Harbor, NY, USA).

The polynucleotides hybridizing with a nucleotide sequence shown under SEQ ID

NOs: 1 , 3, 5 or 7 also comprise fragments, derivatives and allelic variants of one of the polynucleotides encoding a stomatin domain-containing protein as long as the polynucleotide encodes a polypeptide having the above-mentioned activity of a stomatin domain-containing protein. Herein, fragments are understood to mean parts of the polynucleotides which are long enough to encode a polypeptide having said activity. In this context, the term derivative means that the sequences of these polynucleotides differ from the sequence of one of the polynucleotides encoding a stomatin domain-containing protein disclosed herein in one or more positions and show a high degree of homology to these sequences, preferably within sequence ranges that are essential for protein function.

The property of a polynucleotide to hybridize a nucleotide sequence may likewise mean that the polynucleotide encodes a polypeptide, which has a homology, that is to say a sequence identity, of at least 27%, preferably of at least 30%, preferably of at least 40%, more preferably of at least 50%, even more preferably of at least 60% and particularly preferred of at least 70%, especially preferred of at least 80% and even more preferred of at least 90% to an amino acid sequence shown under SEQ ID NOs: 2, 4, 6 or 8. Moreover, the property of a polynucleotide to hybridize a nucleotide sequence may mean that the polynucleotides has a homology, that is to say a sequence identity, of at least 40%, preferably of at least 50%, more preferably of at least 60%, even more preferably of more than 65%, in particular of at least 70%, especially preferred of at least 80%, in particular of at least 90% and even more preferred of at least 95% when compared to the coding sequence shown under SEQ ID NOs: 1 , 3, 5 or 7.

Preferably, the degree of homology is determined by comparing the respective sequence with the coding sequence of SEQ ID NOs: 1 , 3, 5 or 7. When the sequences which are compared do not have the same length, the degree of homology preferably refers to the percentage of nucleotide residues in the shorter sequence which are identical to nucleotide residues in the longer sequence. The degree of homology can be determined conventionally using known computer programs such as the DNAstar program with the ClustalW analysis. This program can be obtained from DNASTAR, Inc., 1228 South Park Street, Madison, Wl 53715 or from DNASTAR, Ltd., Abacus House, West Ealing, London W13 0AS UK (support@dnastar.com) and is accessible at the server of the EMBL outstation. When using the Clustal analysis method to determine whether a particular sequence is, for instance, 80% identical to a reference sequence the settings are preferably as follows: Matrix: blosum 30; Open gap penalty: 10.0; Extend gap penalty: 0.05; Delay divergent: 40; Gap separation distance: 8 for comparisons of amino acid sequences. For nucleotide sequence comparisons, the Extend gap penalty is preferably set to 5.0.

Preferably, the degree of homology of the hybridizing polynucleotide is calculated over the complete length of its coding sequence. It is furthermore preferred that such a hybridizing polynucleotide, and in particular the coding sequence comprised therein, has a length of at least 200 nucleotides, preferably at least 400 nucleotides, more preferably of at least 600 nucleotides, even more preferably of at least 800 nucleotides and most preferably of at least 1000 nucleotides. Preferably, sequences hybridizing to a polynucleotide according to the invention comprise a region of homology of at least 90%, preferably of at least 93%, more preferably of at least 95%, still more preferably of at least 98% and particularly preferred of at least 99% identity to an above-described polynucleotide, wherein this region of homology has a length of at least 400 nucleotides, more preferably of at least 600 nucleotides, even more preferably of at least 800 nucleotides and most preferably of at least 1000 nucleotides.

Homology, moreover, means that there is a functional and/or structural equivalence between the corresponding polynucleotides or the polypeptides encoded thereby. Polynucleotides which are homologous to the above-described molecules and represent derivatives of these molecules are normally variations of these molecules which represent modifications having the same biological function. They may be either naturally occurring variations, preferably orthologs of a polynucleotide comprising the nucleotide sequence of SEQ ID NOs: 1 , 3, 5 or 7, for instance sequences from other alleles, varieties, species, etc., or mutations, and said mutations may have formed naturally or may have been produced by deliberate mutagenesis. The variants, for instance allelic variants, may be naturally occurring variants or variants produced by chemical synthesis or variants produced by recombinant DNA techniques or combinations thereof. Deviations from the above- described polynucleotides may have been produced, e.g., by deletion, substitution, insertion and/or recombination.

The polypeptides encoded by the different variants of the concrete stomatin domain-containing protein-encoding polynucleotides disclosed herein possess certain characteristics they have in common with the polypeptide comprising the amino acid sequence of SEQ ID NOs: 2, 4, 6 or δ.These include for instance biological activity, molecular weight, immunological reactivity, conformation, etc., and physical properties, such as for instance the migration behaviour in gel electrophoreses, chromatographic behaviour, sedimentation coefficients, solubility, spectroscopic properties, stability, pH optimum, temperature optimum etc. The biological activity of a polypeptide of the invention, in particular the activity of interacting with mechanotransducing ion channels, thereby modulating the activity of said ion channels can be tested by methods as described in the Examples. For instance, the capacity of a polypeptide to modulate the activity of a mechanotransducing ion channel may be determined in an experimental set-up similar to those described in the appended Examples. This may involve the measurement of pH-gated ion channel current at cells that are transfected with an expression construct expressing the polypeptide to be tested in the cell and comparing the results with results obtained from pH-gated ion channel current measurements at cells that do not express said polypeptide. If there is a significant difference in the measured current values, preferable by at least 20%, more preferably by at least 50% and even more preferably by at least 200%, then the polypeptide shows the activity of modulating mechanotransducing ion channels.

Preferably, the method of the invention is carried by using any one of the stomatin domain-containing proteins selected of the group consisting of stomatin, neural stomatin-like protein 1 and 2 (Nstoml and Nstom2) and mouse stomatin-like protein 1 (MSLP1) and homologues or derivatives thereof.

Stomatin has first been described as a major membrane protein in the plasma membrane of erythrocytes. The cDNA sequences of stomatin are described e.g. from human (Stewart, Blood 79 (1992), 1593-1601) and mouse (Gallagher, J. Mol. Biochem. 44 (1995), 26358-26363). Some of its biochemical properties have been studied (Stewart GW, Stomatin Int J Biochem Cell Biol 1997 Feb;29(2):271-4). It is an integral membrane protein which has a high sequence homology to the mechanotransduction gene MEC-2, which was discovered in a mutagenesis screen in the nematode worm C. elegans (Tavernarakis and Discroll, Trends Biochem. Sci. 24 (1999), 425-427). It has a hairpin-like shape and the N- as well as the C- terminus are located at the cytosolic side. Stomatin is capable of multimerising with itself. In the experiments underlying the present invention, the nucleotide sequence encoding mouse stomatin as shown under SEQ ID NO: 1 corresponding to the amino acid sequence shown under SEQ ID NO: 2 has been used. The term "stomatin" refers to a protein which shows the above-mentioned characteristics of a stomatin protein. Preferably, the term "stomatin" refers to a protein which shows the amino acid sequence shown in SEQ ID NO: 2 or which is encoded by the nucleotide sequence shown in SEQ ID NO: 1. However, the term also refers to proteins which are encoded by a nucleotide sequence which hybridizes to the nucleotide sequence depicted in SEQ ID NO: 1 and which show the characteristics of a stomatin protein. In this context the meaning of the term "hybridization" is as described hereinabove. It is, for example, also possible to use a sequence encoding human stomatin. A nucleotide sequence encoding human stomatin is depicted in SEQ ID NO: 15. The corresponding amino acid sequence is shown in SEQ ID NO: 16.

Neural stomatin-like protein 1 (also designated "N-stomatin1" or "Nstoml") has for instance been described in WO 00/26362. Rat Nstoml has a sequence identity to rat stomatin on the polypeptide level of 67%. Nstoml has a domain structure that is very similar to that of stomatin. A short cytoplasmic N-terminus is followed by a putative transmembrane loop followed by a longer cytoplasmic polypeptide containing the stomatin/band 7 domain which follows shortly after the transmembrane region. Nstoml is similar to Mec-2, the C.elegans protein involved in mechanotransduction, in that a short hydrophobic stretch is observed in the sequence immediately after the transmemebrane domain (see Figure 9). In the experiments underlying the present invention, the nucleotide sequence encoding rat Nstoml as shown under SEQ ID NO: 3 corresponding to the amino acid sequence shown under SEQ ID NO: 4 has been used. The term "Nstoml" refers to a protein which shows the above-mentioned characteristics of a Nstoml protein. Preferably, the term "Nstoml" refers to a protein which shows the amino acid sequence shown in SEQ ID NO: 4 or which is encoded by the nucleotide sequence shown in SEQ ID NO: 3. However, the term also refers to proteins which are encoded by a nucleotide sequence which hybridizes to the nucleotide sequence depicted in SEQ ID NO: 3 and which show the characteristics of a Nstoml protein. In this context, the meaning of the term "hybridization" is as described hereinabove. The nucleotide sequence of the human Nstoml shown under SEQ ID NO: 19 or a nucleotide sequence encoding a the amino acid sequence of human Nstoml shown under SEQ ID NO: 20 may likewise be of use.

Neural stomatin-like protein 2 (also designated "N-stomatin2" or "Nstom2" or, if it is from human or mouse, "Human/Mouse stomatin-like protein 2") has been identified during preparatory work for the present invention. Nstom2 does not appear to possess a transmembrane loop like stomatin but contains a stomatin-like domain near the middle of the protein (see Figure 9). In mouse there are two splicing- variants of Nstom2, a longer version ("Nstom2-L") which is mainly located in the membrane and a shorter version ("Nstom2-S") mainly located in the cytosol. In the experiments underlying the present invention, the nucleotide sequence encoding mouse Nstom2-L as shown under SEQ ID NO: 5 corresponding to the amino acid sequence shown under SEQ ID NO: 6 has been used. The sequence encoding the shorter version "Nstom2-S" is shown in SEQ ID NO: 9. The corresponding amino acid sequence is shown in SEQ ID NO: 10. The splicing of Nstom2 is schematically shown in Figure 7. The term "Nstom2" refers to a protein which shows the above- mentioned characteristics of a Nstom2 protein. Preferably, the term "Nstom2" refers to a protein which shows the amino acid sequence shown in SEQ ID NO: 5 or which is encoded by the nucleotide sequence shown in SEQ ID NO: 5. However, the term also refers to proteins which are encoded by a nucleotide sequence which hybridizes to the nucleotide sequence depicted in SEQ ID NO: 5 and which show the characteristics of a Nstom2 protein. In this context the meaning of the term "hybridization" is as described hereinabove. Preferably, the Nstom2 protein is encoded by the longer splice variant and is located in the membrane. The nucleotide sequence of the human homolog "human stomatin-like protein 2" (human SLP 2) shown under SEQ ID NO: 13 or a nucleotide sequence encoding a the amino acid sequence of human SLP2 shown under SEQ ID NO: 14 may likewise be of use.

Mouse stomatin-like protein 1 ("MSLP1") or a homologue thereof from another species such as human stomatin-like protein 1 is a further preferred stomatin domain-containing protein for use in the method of the invention. MSLP1 does possess a transmembrane domain which is followed by the stomatin domain. The N- and C-terminal regions of the protein are both probably cytoplasmic. The protein also contains a non-specific lipid transfer protein (nsLTP)-domain at the C-terminal end (Seidel G, Prohaska R Gene 1998 Dec 28; 225 (1-2):23-9). In the experiments underlying the present invention, the nucleotide sequence encoding mouse MSLP1 as shown under SEQ ID NO: 7 corresponding to the amino acid sequence shown under SEQ ID NO: 8 has been used. The MSLP1 nucleotide sequence shown under SEQ ID NO: 7 or a polynucleotide encoding the amino acid sequence shown under SEQ ID NO: 8 may likewise be of use. The term "MSLP1" refers to a protein which shows the above-mentioned characteristics of a MSLP1 protein. Preferably, the term " MSLP1" refers to a protein which shows the amino acid sequence shown in SEQ ID NO: 8 or which is encoded by the nucleotide sequence shown in SEQ ID NO: 7. However, the term also refers to proteins which are encoded by a nucleotide sequence which hybridizes to the nucleotide sequence depicted in SEQ ID NO: 7 and which show the characteristics of a MSLP1 protein. In this context the meaning of the term "hybridization" is as described hereinabove. It is, for example, also possible to use a sequence encoding human stomatin-like protein 1 (human SLP1). A nucleotide sequence encoding human SLP1 is depicted in SEQ ID NO: 11. The corresponding amino acid sequence is shown in SEQ ID NO: 12.

The present invention is based on the finding that stomatin domain-containing proteins functionally interact with mechanotransducing ion channels and that it is possible to identify compounds which inhibit mechanotransduction in a system, which comprises cells expressing the corresponding ion channel or ion channels and overexpressing a stomatin domain-containing protein, by optionally stimulating the cell with acidic pH, and determining the effect of the compound to be tested on the current or voltage at the plasma membrane of the cell. In addition cells overexpressing the stomatin domain containing protein may exhibit a marked depolarization compared to control cells (see Figure 8 (membrane potential); control cells: -55 mV; Nstoml expressing cells exhibit a more depolarized potential of -28mV).

It was in particular found that stomatin is involved in mechanotransduction insofar as it interacts with mechanotransducing ion channels and increases the current or voltage over the plasma membrane, more precisely the proton-activated current or voltage. Compounds which interfere with this interaction and, thus, reduce the influence of stomatin on the proton-induced current or voltage at the plasma membrane can be identified by incubating a cell overexpressing stomatin with the compound, optionally providing an acidic pH stimulus and determining whether the current or voltage at the plasma membrane is decreased in comparison to a control in which the same type of cell is incubated, and optionally stimulated, in the absence of the compound. Compounds identified in this way are thought to have analgesic effects and are, thus, useful in the treatment of pain conditions.

Thus, in one preferred embodiment of the method according to the invention, the polypeptide overexpressed in the cell provided in step (a) is stomatin or a derivative thereof and the compound is identified in step (d) if the current or voltage change measured in step (c) is decreased in comparison to the corresponding current or voltage of the control cell defined in step (d).

The term "decreased" means that the current or voltage in the presence of the compound is statistically significantly lower than in the control. Preferably, the term "decreased" means a decrease of the current or voltage of at least 25%, more preferably of at least 50%, even more preferably of at least 70% and particularly preferred of at least 100% when compared to the control.

Moreover, it was found that apart from stomatin also other stomatin domain- containing proteins, like Nstoml , Nstom2 and MSLP1 , functionally interact with mechanotransducing ion channels. However, in contrast to stomatin, Nstoml and 2 and MSLP1 surprisingly do not act like stomatin but lead, when interacting with mechanotransducing ion channels, to a decrease in the proton-activated current or voltage at the plasma membrane. Compounds which interfere with this interaction can be identified by incubating a cell overexpressing Nstoml , Nstom2 or MSLP1 with a compound, optionally providing an acidic pH stimulus, and determining whether the current or voltage at the plasma membrane is increased in comparison to a control in which the same type of cell is incubated and, optionally stimulated, in the absence of the compound. Compounds identified in this way are thought to inhibit mechanotransduction and to have analgesic effects which makes them useful in the treatment of pain conditions. Thus, in another preferred embodiment of the method according to the invention the polypeptide overexpressed in the cell provided in step (a) is Nstoml or a derivative thereof or Nstom2 or a derivative thereof or MSLP1 or a derivative thereof and the compound is identified in step (d) if the current or voltage measured in step (c) is increased in comparison to the corresponding current or voltage of the control cell defined in step (d).

The term "increased" means that the current or voltage in the presence of the compound is statistically significantly higher than in the control. Preferably, the term "increased" means an increase of the current or voltage of at least 20%, more preferably of at least 50%, even more preferably of at least 100% and particularly preferred of at least 200% when compared to the control.

The Nstoml gene has been inactivated by the present inventors using a knockout mouse. In this mouse, mechanotransduction is severely reduced in many sensory neurons innervating the skin. Thus, the molecular interaction between Nstoml and mechanotransducing ion channels, such as DRASIC, is required for the expression of normal sensory mechanotransduction. Normally sensory neurons do not respond to pH but they do respond very well to mechanical stimuli. The molecular interaction between Nstoml and Deg/Enac channels leads to a reduction in pH sensitivity but can also depolarize the cells perhaps due to a pH-independent activation of the channel.

Sensory neurons that respond to pH in the skin are all nociceptive neurons and the sensation that accompanies pH injection into the skin is painful (Steen et al, J Neurosci 1995 15:3982-9; Reeh PW, Steen KH. Tissue acidosis in nociception and pain. Prog Brain Res 1996;113:143-51). Thus the functional blockade of stomatin domain-containing proteins that increase the pH activation of Deg/Enac channels may be useful in inhibiting inflammatory pain associated with tissue acidosis. Thus, in summary, the pharmacological blockade of both the positive and the negative modulation of Deg/Enac channels by stomatin domain-containing proteins may be very well applicable for the treatment of pain. In a preferred embodiment of the method according to the invention, the measuring of the current or voltage at the plasma membrane takes place upon providing a stimulation by acidic pH.

It was found that already the overexpression of a stomatin domain-containing protein in cells allows it to investigate the effect of a compound on the interaction of this protein with a mechanotransducing ion channel and, thus, its effect on mechanotransduction in the above-described method. However, it can be advantageous to measure the effect on the current or voltage at the plasma membrane after providing an acidic pH stimulus which activates mechanotransducing ion channels present in the cell.

The stimulation by acidic pH can be achieved in any way known to the person skilled in the art which leads to a decrease of the pH value of the medium surrounding the cells to an acidic value. Measures for achieving a decrease in pH value are, e.g., by perfusing single cells with pH6, pH5 and pH4, respectively, buffer by moving an array of outlets over the cell (RSC-200, Biologic, France). Stimulus duration can be set between 1 second up to any given time, with a 1 minute recovery time between each stimulus. In a high throughput procedure, buffered acid solution would be rapidly added to the medium of the cells in a microtitre plate by means of a robotic pipetting system.

The term "acidic pH value" in the context of the present invention means a pH value which is below the neutral pH, i.e. below 7. Preferably, the acidic pH stimulus means that a pH below about 6.5, more preferably in the range of about 4 to about 6 is created in the medium surrounding the cell.

The compounds identified by the method according to the invention as described hereinabove are compounds which, due to their capacity to interfere with the interaction of stomatin domain-containing proteins and mechanotransducing channels, are considered to be able to inhibit mechanotransduction. This property can be verified by submitting the identified compounds to a further test. Accordingly, in a preferred embodiment, the method according to the present invention furthermore comprises the step of:

(e) verifying the capacity of the identified compound of inhibiting mechanotransduction (i) by applying the compound to the receptive field of a sensory neuron of the DRG in a suitable skin-nerve preparation; (ii) measuring the mechanotransduction of said neuron upon a suitable stimulus; and (iii) confirming the capacity of inhibiting mechanotransduction if the measured mechanotransduction is reduced when compared to the mechanotransduction of a corresponding neuron to which said compound was not applied.

Preferably, step (e) of the method of the invention also includes

(iv) testing whether the compound can also inhibit (or potentiate) the acid activation of a subset of nociceptive (pain sensing) sensory neurons. Step (iv) can also be carried out independent of steps (i) to (iii).

This assay using an ex-vivo preparation comprising skin and a sensory nerve allows an unequivocal determination whether the compound inhibits mechanotransduction. The ways to carry out this assay are well known to the person skilled in the art. Such an assay is, e.g., described in Koltzenburg et al. (1997, loc. cit.); Price et al. (2000, loc. cit.) or Price et al. (2001 , loc. cit). In particular, step (iv) mentioned above can be carried out as described in Price et al. (2000, 2001 , loc. cit).

The stimulus applied in step (ii) may depend on the neuron used in the assay. Neurons are recorded and classified according to the scheme outlined in Koltzenburg et al. (1997, loc. cit.). After neurons are classified, a standard indentation stimulus, that is within the normal sensitivity of the sensory neuron, is applied repetitively with a computer controlled piezoelectric device (Nanomotor^tm Kleindieck, Reutlingen Germany). After a control period, the compound is applied to the isolated receptive field of the neuron in the skin. A significant reduction in the mechanosensitivity (identifiable as action potentials per stimulus compared to untreated neurons of the same type) within a few minutes of application would count as a hit. The cell defined in step (a) of the method according to the invention may be any cell which overexpresses a stomatin domain-containing protein. "Overexpressing" in this context means that the expression of such a protein is higher as compared to a corresponding wild-type cell, i.e. it is higher than naturally observed in the corresponding cell type. Preferably the expression is increased by at least 10%, more preferably by at least 20% and even more preferably by at least 50%. The term "expression" refers in this context to the transcription rate, i.e. the amount of mRNA encoding the stomatin domain-containing protein in the cell, the translation rate, i.e. the amount of said stomatin domain-containing protein in the cell, and/or the activity of said protein in the cell. Corresponding methods for measuring the expression at each of these levels are known to the person skilled in the art. The overexpression may, e.g., be due to a mutation in the corresponding cell. Preferably, the overexpression is due to genetic engineering of the cell, in particular by the introduction of a foreign DNA sequence. The DNA sequence may, e.g., be a sequence which is inserted upstream of an endogenously occurring gene encoding a stomatin domain-containing protein, for example, in or close to the expression- regulatory regions of the gene and which leads to an increase of transcription of the gene (in situ activation). Preferably, the cell overexpresses the respective protein due to the introduction of a DNA sequence which encodes the stomatin domain- containing protein. Such a cell can be prepared according to methods well known to the person skilled in the art.

In particular, nucleic acid molecules encoding a stomatin domain-containing protein may be inserted into a suitable vector. Nonlimiting examples for commercially available vectors include plasmid vectors compatible with mammalian cells, such as pUC, pBluescript (Stratagene), pET (Novagen), pREP (Invitrogen), pCRTopo (Invitrogen), pcDNA3 (Invitrogen), pCEP4 (Invitrogen), pMC1 neo (Stratagene), pXT1 (Stratagene), pSG5 (Stratagene), EBO-pSV2neo, pBPV-1 , pdBPVMMTneo, pRSVgpt, pRSVneo, pSV2-dhfr, pUCTag , plZD35, pLXIN and pSIR (Clontech) and plRES-EGFP (Clontech). Baculovirus vectors such as pBlueBac, BacPacz Baculovirus Expression System (CLONTECH), and MaxBacTM Baculovirus Expression System, insect cells and protocols (Invitrogen) are available commercially and may also be used to produce high yields of biologically active protein (see also, Miller (1993), Curr. Op. Genet. Dev., 3, 9; O'Reilly, Baculovirus Expression Vectors: A Laboratory Manual, p. 127). In addition, prokaryotic vectors such as pcDNA2; and yeast vectors such as pYes2 are nonlimiting examples of other vectors suitable for use with the present invention. For vector modification techniques, see Sambrook and Russel (2001), loc. cit. Vectors can contain one or more replication and inheritance systems for cloning or expression, one or more markers for selection in the host, e. g. antibiotic resistance, and one or more expression cassettes.

The coding sequences inserted in the vector can be synthesized by standard methods, isolated from natural sources, or prepared as hybrids. Ligation of the coding sequences to transcriptional regulatory elements (e. g., promoters, enhancers, and/or insulators) and/or to other amino acid encoding sequences can be carried out using established methods.

Furthermore, the vectors may comprise expression control elements, allowing proper expression of the coding regions in suitable hosts. Such control elements are known to the artisan and may include a promoter, translation initiation codon, translation and insertion site or internal ribosomal entry sites (IRES) (Owens (2001), Proc Natl Acad Sci USA 98,1471-1476) for introducing an insert into the vector. Preferably, the nucleic acid molecule encoding a stomatin domain-containing protein is operatively linked to said expression control sequences allowing expression in eukaryotic or prokaryotic cells. Particularly preferred are in this context control sequences which allow for correct expression in neuronal cells and/or cells derived from nervous tissue.

Control elements ensuring expression in eukaryotic and prokaryotic cells are well known to those skilled in the art. As mentioned above, they usually comprise regulatory sequences ensuring initiation of transcription and optionally poly-A signals ensuring termination of transcription and stabilization of the transcript. Additional regulatory elements may include transcriptional as well as translational enhancers, and/or naturally-associated or heterologous promoter regions. Possible regulatory elements permitting expression in for example mammalian host cells comprise the CMV- HSV thymidine kinase promoter, SV40, RSV-promoter (Rous sarcome virus), human elongation factor lα-promoter, CMV enhancer, CaM-kinase promoter or SV40-enhancer. For the expression for example in nervous tissue and/or cells derived therefrom, several regulatory sequences are well known in the art, like the minimal promoter sequence of human neurofilament L (Charron, J. Biol. Chem 270 (1995), 25739- 25745). For the expression in prokaryotic cells, a multitude of promoters including, for example, the tac-lac-promoter, the lacUVδ or the trp promoter, has been described. Beside elements which are responsible for the initiation of transcription such regulatory elements may also comprise transcription termination signals, such as SV40-poly-A site or the tk-poly-A site, downstream of the polynucleotide. In this context, suitable expression vectors are known in the art such as Okayama-Berg cDNA expression vector pcDV1 (Pharmacia), pRc/CMV, pcDNAI , pcDNA3 (In- Vitrogene, as used, inter alia in the appended examples), pSPORTI (GIBCO BRL) or pGEMHE (Promega), or prokaryotic expression vectors, such as lambda gt11. An expression vector as described above is preferably at least capable of directing the replication, and preferably the expression, of the nucleic acids and protein. Suitable origins of replication include, for example, the Col E1 , the SV40 viral and the M13 origins of replication. Suitable promoters include, for example, the cytomegalovirus (CMV) promoter, the iacZ promoter, the gai10 promoter and the Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) polyhedral promoter. Suitable termination sequences include, for example, the bovine growth hormone, SV40, iacZ and AcMNPV polyhedral polyadenylation signals. Examples of selectable markers include neomycin, ampicillin, and hygromycin resistance and the like. Specifically-designed vectors allow the shuttling of DNA between different host cells, such as bacteria-yeast, or bacteria-animal cells, or bacteria-fungal cells, or bacteria-invertebrate cells.

The cell which overexpresses the stomatin domain-containing protein can in principle be any suitable cell which allows carrying out the described method. It may be derived from any organism, such as plant organisms, fungi or bacteria. Preferably, it is a cell which is derived from an animal organism, more preferably from a vertebrate, even more preferably from a mammal, such as a rodent. It is particularly preferred that the cell is a human cell. Examples are HEK293 cells, PC12 cells and Neuroblastoma cells. The cell used in the method according to the invention which overexpresses a stomatin domain-containing protein is preferably a cell which shows a proton-induced inward current. HEK cells are particularly suitable for carrying out the method according to the invention because they have a high current measured under voltage clamp (i.e. 0.9 nA).

Cells of higher organisms in general have a proton-induced inward current. Most of this current is carried by sodium ions. This inward current can easily be verified and measured using well known electrophysiological techniques. For example using patch clamp techniques where whole cell currents can be measured under voltage clamp, in addition membrane voltage changes can be measured in the current clamp mode of the whole cell patch clamp amplifier (Hille 2001 , "Ion channels in excitable membranes" Third edition Sinauer Associates, Massachussets). Alternatively, the ion flux (which always accompanies ion channel opening) can be measured using high throughput fluorescent measurement techniques (Denyer et al., 1998). A cell showing a proton-induced inward current shows a depolarization of the cell upon a pH stimulus when measured in a patch clamp experiment. When measured with patch clamp, even a very small depolarization can be measured, i.e. even a few mV.

In a preferred embodiment of the method according to the invention, the cell used in the method furthermore also overexpresses a mechanotransducing ion channel. As explained above, the cells used in the method show a proton-induced inward current. This current is due to the presence of mechanotransducing ion channels in the membranes of the cells. However, in the context of the described method it can be advantageous to increase the number of such channels in the cells in order to facilitate and/or improve the measurement of the current. The overexpression of a mechanotransducing ion channel in the cells can, e.g., be the result of a mutation or it can be achieved by the introduction of a foreign DNA molecule. In this respect the same applies as already described above in connection with the overexpression of a stomatin domain-containing protein in the cell.

In a preferred embodiment, the overexpressed mechanotransducing ion channel is an ion channel of the Degenerin/Enac family. The members of this family are characterized by possessing two transmembrane domains with the N- and C- termini oriented inside the cell. A large portion of the protein (>50%) is oriented extracellularly and is situated between the two transmembrane domains. These proteins can form selective sodium channels as homomers or heteromeric channels (Kellenberger and Schild, Physiol Rev 82(3), (2002), 735-67). Degenerin/Enac channels are also characterized by having a high ionic selectivity for sodium. On the basis of sequence identity and similarity, these channels are found in various species including Drosophila and C.elegans (see Hille 2001 , loc. cit.).

In a more preferred embodiment, the mechanotransducing ion channel is selected from the group consisting of brain sodium channel 1 (BNC1) and dorsal root acidic sensing channel (DRASIC). The DRASIC protein overexpressed in the cell may in principle be a DRASIC protein from any suitable source. Preferably, it is a DRASIC protein of a vertebrate, more preferably of a mammal, even more preferably of a rodent, such as rat or mouse. In a preferred embodiment, the DRASIC protein is of human origin. In a particularly preferred embodiment the DRASIC protein is a protein comprising the amino acid sequence as shown in SEQ ID NO:22 (human) or 24 (rat) or is encoded by the nucleotide sequence as shown in SEQ ID NO: 21 (human) or 23 (rat). The DRASIC protein may also be encoded by a polynucleotide which hybridizes to a nucleic acid molecule having the sequence of SEQ ID NO: 21 or 23 and which encodes a protein having the characteristics of a DRASIC protein. In this context the meaning of the term "hybridization" is as described hereinabove. The BNC1 protein overexpressed in the cell may in principle be a BNC1 protein from any suitable source. Preferably, it is a BNC1 protein of a vertebrate, more preferably of a mammal, even more preferably of a rodent, such as rat or mouse. In a preferred embodiment, the BNC1 protein is of human origin. The sequence used in the Examples encodes a BNC1 protein from rat. Thus, in a particularly preferred embodiment, the BNC1 protein is a protein comprising the amino acid sequence as shown in SEQ ID NO:18 or is encoded by the nucleotide sequence as shown in SEQ ID NO: 17. The BNC1 protein may also be encoded by a polynucleotide which hybridizes to a nucleic acid molecule having the sequence of SEQ ID NO: 17 and which encodes a protein having the characteristics of a BNC1 protein. In this context the meaning of the term "hybridization" is as described hereinabove. The "providing" of a cell in step (a) can be effected in any way which is suitable to allow the contacting with the compound, the stimulation and the measuring of the current or voltage. Moreover, the step of providing may depend on the mode chosen for measuring the current or voltage in step (c). If, e.g., the measurement is carried out by patch-clamp, the cell may be provided as a single cell. However, if high-throughput screening is chosen, a multitude of cells may be provided, e.g., in the wells of a microtiter plate.

Generally, the cell is provided in vitro, preferably in the form of adherent cell cultures.

The "contacting" of step (b) of the method according to the invention may be carried out in accordance with well-known techniques that involve testing of a compound for a reaction it mediates in a cell. Preferably, said contacting may be brought about by providing the compound to be tested in the medium surrounding the cell.

The measuring of the current or voltage at the plasma membrane according to step (d) of the method according to the invention can be carried out by methods well known to the person skilled in the art. This current or voltage can, e.g., be verified and measured by using well known electrophysiological methods. For example using patch-clamp techniques where whole cell currents can be measured under voltage clamp, in addition membrane voltage changes can be measured in the current clamp mode of the whole cell patch clamp amplifier (Hille 2001 , Textbook entitled Ion channels in excitable membranes). Alternatively, the ion flux (which always accompanies ion channel opening) can be measured using high throughput fluorescent measurement techniques (Denyer et al., (1998) Drug Discovery Today 3, 323-332). With patch-clamp involving single cell methods, even a very small depolarization can be measured, such as a few mV.

The "compound" (in the following also referred to as "candidate agent") to be tested in the method according to the invention can in principle be any molecule, e.g. protein, peptide, nucleic acid, organic or inorganic molecule, etc. Preferably, the compound has a low toxicity for cells of higher organisms, preferably for human cells. It is possible to run a plurality of assays in parallel with different concentrations of the compound to obtain a differential response to the various concentrations. Typically, one of these concentrations serves as a negative control, i.e. at zero concentration or below the level of detection.

Candidate agents encompass numerous chemical classes, though typically they are organic molecules, preferably small organic compounds having a molecular weight of more than 50 and less than about 2,500 daltons. Candidate agents comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups. The candidate agents often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Candidate agents are also found among biomolecules including peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof. Candidate agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides and oligopeptides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc. to produce structural analogs.

The method according to the present invention may be carried out in any way or fashion which meets the requirements of the different steps. For example, the method can be carried out by looking at single cells in a patch-clamp experiment and by measuring the current or voltage at the plasma membrane after providing a pH stimulus in the presence and absence of a compound to be tested. In a preferred embodiment the method according to the present invention is performed as a high-throughput assay. This means that the method is performed in a system designed to allow a large number of compounds to be tested in a short time. This can be done, e.g., in connection with the high-throughput fluorescent measurement techniques described by Denyer et al. (1998), Drug Discovery Today 3, 323-332.

In principle two types of high throughput assays would be suitable for measuring the effects of compounds on the ion channels in the membranes of the cells overexpressing a stomatin domain-containing protein. The first measures sodium ion flux through the membrane and the second measures changes in the membrane potential. Since the ion channels that are gated by protons are highly selective for sodium ions, a sodium flux assay may be used. Two fluorescent probes may be used for detection of sodium - SBFI (as the acetoxymethylester) or a dye called Creedmoor-lib. For most high throughput screening (HTS) applications utilising ion indicator dyes, cells are grown in a microtitre plate (e.g., 96 or 384-well format) for 1 - 2 days and, following removal of the cell culture medium, the dye as the acetoxymethylester (AM) is added. In the AM form, the dyes are cell-permeant (and often poorly fluorescent) and readily enter the cell where they are then hydrolysed and are rendered relatively impermeant and hence cannot readily leave the cytoplasm. Once hydrolysed, the dyes then fluoresce upon binding of the appropriate ion with illumination with light at the correct wavelength. The cells are then washed to remove any remaining extracellular dye. By exciting the cells in a microplate reader and imaging device such as the FLIPR from Molecular Devices with a wavelength of light specific for the dye and monitoring emitted light at a wavelength of light specific for the dye used, changes in intracellular ion concentration can be followed by monitoring the change in fluorescence emission. For opener studies, the ligand (in this case low pH) solution would be applied to the cells within the reader in the presence and absence of the candidate compound. Methods for measuring the membrane potential are also widely used and mostly microtitre plate-based assays are employed using standard microtitre based assays with plate readers such as the FLIPR or Flexstation from Molecular Devices. For normal fluorescence intensity assays, two dye systems are available. Firstly, there is standard DiBAC whereby the dye partitions across the cell membrane depending on the membrane potential and shows increased fluorescence when bound to intracellular proteins. When the cell is depolarised, the intracellular DiBAC concentration increases (the dye carries a negative charge) as the dye repartitions. Unfortunately, this change is slow (several minutes), relatively insensitive and is temperature-sensitive so that small transient changes in the membrane potential can be missed. The other method uses the Membrane Potential dye kit from Molecular Devices. This uses a dye that redistributes very quickly upon changes in the membrane potential and is very simple as the dye can be added directly to the cell culture medium due to the use of a blocking agent to supress extracellular fluorescence. This method allows one to follow transient depolarisations and also small changes (5-10 mV) in the membrane potential. Technical details on this kit can be found at the Molecular Devices web site http://www.moleculardevices.com/. Both of these methods for measuring membrane potential are applicable to measure pH-induced changes in the membrane potential or repolarisation events induced by candidate compounds in cell lines overexpressing stomatin domain- containing proteins which have a depolarized membrane potential (positive with respect to normal resting potential).

As described above, the method according to the present invention allows it to identify compounds which are capable of inhibiting mechanotransduction and which are, therefore, useful as analgetics, i.e. pharmaceuticals to ameliorate and/or treat pain conditions.

Accordingly, the present invention also relates to a method for the production of a pharmaceutical composition comprising the steps of a method according to the invention as described hereinabove and furthermore the step of formulating the compound identified in such a method or a derivative thereof which inhibits mechanotransduction in a pharmaceutically acceptable form.

Moreover, the present invention also relates to a compound identified in a method according to the invention which inhibits mechanotransduction as well as to a pharmaceutical composition comprising such a compound and optionally a pharmaceutically acceptable carrier or a pharmaceutical composition which is obtainable by the above described method. Such pharmaceutical compositions comprise a therapeutically effective amount of the identified compound or a derivative thereof and, optionally, a pharmaceutically acceptable carrier. The pharmaceutical composition may be administered with a physiologically acceptable carrier to a patient, as described herein. In a specific embodiment, the term "pharmaceutically acceptable" means approved by a regulatory agency or other generally recognized pharmacopoeia for use in animals, and more particularly in humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin. Such compositions will contain a therapeutically effective amount of the aforementioned compounds, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration.

In another preferred embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilised powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration. The pharmaceutical composition of the invention can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.

In vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems. Preferably, the pharmaceutical composition is administered directly or in combination with an adjuvant.

The present invention also relates to the use of a compound identified in a method according to the invention for the preparation of a pharmaceutical composition for the treatment of pain.

In another aspect the present invention relates to a method of treating pain comprising administering a therapeutically effective amount of the pharmaceutical composition described hereinabove to a subject in need of such a treatment. In the context of the present invention the term "subject" means an individual in need of a treatment of a neurological disease or of pain. Preferably, the subject is a vertebrate, even more preferred a mammal, particularly preferred a human. The term "administered" means administration of a therapeutically effective dose of the aforementioned pharmaceutical composition comprising the compound to an individual. By "therapeutically effective amount" is meant a dose that produces the effects for which it is administered. The exact dose will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques. As is known in the art and described above, adjustments for systemic versus localized delivery, age, body weight, general health, sex, diet, time of administration, drug interaction and the severity of the condition may be necessary, and will be ascertainable with routine experimentation by those skilled in the art. The methods are applicable to both human therapy and veterinary applications. The compounds described herein having the desired therapeutic activity may be administered in a physiologically acceptable carrier to a patient, as described herein. Depending upon the manner of introduction, the compounds may be formulated in a variety of ways as discussed below. The concentration of therapeutically active compound in the formulation may vary from about 0.1-100 wt %. The agents maybe administered alone or in combination with other treatments. The administration of the pharmaceutical composition can be done in a variety of ways as discussed above, including, but not limited to, orally, subcutaneously, intravenously, intra-arterial, intranodal, intramedullary, intrathecal, intraventricular, intranasally, intrabronchial, transdermally, intranodally, intrarectally, intraperitoneally, intramuscularly, intrapulmonary, vaginally, rectally, or intraocularly. In some instances, for example, in the treatment of wounds and inflammation, the candidate agents may be directly applied as a solution dry spray. The attending physician and clinical factors will determine the dosage regimen. As is well known in the medical arts, dosages for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. A typical dose can be, for example, in the range of 0.001 to 1000 μg; however, doses below or above this exemplary range are envisioned, especially considering the aforementioned factors. The dosages are preferably given once a week, however, during progression of the treatment the dosages can be given in much longer time intervals and in need can be given in much shorter time intervals, e.g., daily. In a preferred case the immune response is monitored using herein described methods and further methods known to those skilled in the art and dosages are optimized, e.g., in time, amount and/or composition. Progress can be monitored by periodic assessment. The pharmaceutical composition of the invention may be administered locally or systemically. Administration will preferably be parenterally, e.g., intravenously. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.

It is also envisaged that the pharmaceutical compositions are employed in co- therapy approaches, i.e. in co-administration with other medicaments or drugs, for example other drugs for preventing, treating or amelioration pain.

The term "pain" includes any condition which is referred to in the broadest sense as pain, in particular physical pain. There are many qualities of pain, e.g., burning pain, stabbing pain, dull aching pain. The quality of the pain is not strictly dependent on the underlying disease or condition, however, almost all types of pain have a mechanical component. Thus, light mechanical stimulation of the affected body part can lead to pain sensations out of proportion to the intensity of the precipitating stimulus. This type of pain is often described as hyperalgesia.

In a preferred embodiment, the pain is a condition selected from the group consisting of chronic pain, allodynia and hyperalgesia.

Chronic pain persists longer than the actual stimulus that has caused the pain. Allodynia is pain which is due to a stimulus which does not normally provoke pain. The term "allodynia" was originally introduced to separate from hyperalgesia and hyperesthesia the conditions seen in patients with lesions of the nervous system where touch, light pressure, or moderate cold or warmth evoke pain when applied to apparently normal skin. In Greek, "allo" means "other" and is a common prefix for medical conditions that diverge from the expected. "Odynia" is derived from the Greek word "odune" or "odyne," which is used in "pleurodynia" and "coccydynia" and is similar in meaning to the root from which words are derived with "-algia" or "- algesia" in them. The words "to normal skin" were used in the original definition but later were omitted in order to remove any suggestion that allodynia applied only to referred pain. "Referred pain" is pain felt in one region of the body, spacially separate from the region originally stimulating such as angina pectoris. Originally, also the pain-provoking stimulus was described as "non-noxious." However, a stimulus may be noxious at some times and not at others, for example, with intact skin and sunburned skin, and also, the boundaries of noxious stimulation may be hard to delimit. Further, allodynia is used with respect to conditions which may give rise to sensitization of the skin, e.g., sunburn, inflammation, trauma. It is important to recognize that allodynia involves a change in the quality of a sensation, whether tactile, thermal, or of any other sort. The original modality is normally non-painful, but the response is painful. There is thus a loss of specificity of a sensory modality. By contrast, hyperalgesia (q.v.) represents an augmented response in a specific mode, viz., pain. With other cutaneous modalities, hyperesthesia is the term which corresponds to hyperalgesia, and as with hyperalgesia, the quality is not altered. In allodynia, the stimulus mode and the response mode differ, unlike the situation with hyperalgesia. This distinction should not be confused by the fact that allodynia and hyperalgesia can be plotted with overlap along the same continuum of physical intensity in certain circumstances, for example, with pressure or temperature. Hyperalgesia is defined as an increased response to a stimulus which is normally painful. Hyperalgesia reflects increased pain on suprathreshold stimulation. For pain evoked by stimuli that usually are not painful, the term allodynia is preferred, while hyperalgesia is more appropriately used for cases with an increased response at a normal threshold, or at an increased threshold, e.g., in patients with neuropathy. It should also be recognized that with allodynia the stimulus and the response are in different modes, whereas with hyperalgesia they are in the same mode. Current evidence suggests that hyperalgesia is a consequence of perturbation of the nociceptive system with peripheral or central sensitization, or both, but it is important to distinguish between the clinical phenomena, which this definition emphasizes, and the interpretation, which may well change as knowledge advances.

Both hyperalgesia and allodynia are symptoms of a very wide variety of diseases and common injuries. Examples of syndromes where pain with hyperalgesia or allodynia require treatment are rheumatoid arthritis, cancer pain, sports injuries, chronic or acute back pain, Herpes Zoster, and post-operative pain.

The present invention also relates to a system useful for identifying a compound capable of inhibiting the mechanotransduction of a neuron comprising

(a) a cell overexpressing a polypeptide selected from the group consisting of stomatin domain-containing proteins; and

(b) a device useful for measuring the current or voltage at the plasma membrane of the cell.

With respect to the cell mentioned in (a) the same applies as described above in connection with the method according to the invention.

The device mentioned under item (b) may be any device suitable to determine and/or measure the current or voltage at the plasma membrane of a cell. One example is a patch-clamp amplifier.

Preferably the system according to the present invention is a system which can be applied in a high-throughput assay, for example in a fluorescent-based method (see above).

In another aspect the present invention relates to a kit comprising cells as defined in step (a) of the method according to the invention, a device useful for measuring the current or voltage at the plasma membrane of a cell, preferably upon stimulation by acidic pH and/or the system according to the invention. With respect to the preferred embodiments, the same applies as what has already been described hereinabove in connection with the method and the system according to the invention, respectively.

Finally, the present invention relates to the use of a polynucleotide encoding a stomatin domain-containing protein, of a polypeptide encoded by said polynucleotide, of cells over-expressing said polypeptide or as defined in step (a) of the method according to the invention or of the system according to the invention for the identification of a compound capable of inhibiting the mechanotransduction of a neuron.

With respect to the preferred embodiments the same applies as what has already been described hereinabove in connection with the method and the system according to the invention, respectively.

These and other embodiments are disclosed and encompassed by the description and examples of the present invention. Further literature concerning any one of the methods, uses and compounds to be employed in accordance with the present invention may be retrieved from public libraries, using for example electronic devices. For example the public database "Medline" may be utilized which is available on the Internet, for example under http://www.ncbi.nlm.nih.gov/PubMed/medline.html. Further databases and addresses, such as http://www.ncbi.nlm.nih.gov/, http://www.infobiogen.fr/, http://www.fmi.ch/biology/research_tooIs.html, http://www.tigr.org/, are known to the person skilled in the art and can also be obtained using, e.g., http://www.google.de. An overview of patent information in biotechnology and a survey of relevant sources of patent information useful for retrospective searching and for current awareness is given in Berks, TIBTECH 12 (1994), 352-364.

Furthermore, the term "and/or" when occurring herein includes the meaning of "and", "or" and "all or any other combination of the elements connected by said term".

The present invention is further described by reference to the following non-limiting figures and examples. The Figures show:

Figure 1 shows the proton-induced current in HEK 293 cells stably expressing BNC1 -tagged with FLAG.

Figure 2 shows the proton-induced current in HEK 293 cells stably expressing BNC1 and stomatin. Stomatin has a small effect on pH-gated inward current. Top is peak transient current and bottom is peak sustained current.

Figure 3 shows the proton-induced current in HEK 293 Nstom-1-BNC1 double transfectants (derived from a BNC1 -expressing HEK cell clone). Peak current in the presence and absence of Nstomatin 1. Top is peak transient current and bottom is peak sustained current.

Figure 4 shows the proton-induced current in HEK 293 N-stomatin-2l-BNC1 double transfectants (derived from a BNC1 -expressing HEK cell clone). The presence of Nstomatin-2 I (full length) inhibits proton- gated inward current. Top is peak transient current bottom is peak sustained current.

Figure 5 shows the proton-induced current in HEK 293 Nstomatin-2s-BNC1 double transfectant (derived from a BNC1 -expressing HEK cell clone). Nstomatin2s has no effect on the peak inward current. Top is peak current measurement and bottom sustained.

Figure 6 shows the proton-induced current in HEK 293 overexpressing Nstomatin 1.

Figure 7 shows schematically the differential splicing of Nstom2. A second sequence of 1004bp was amplified from mouse brain that was identical to Nstom2-l except for a 100bp fragment missing between positions 205 and 305. Analysis of a mouse genomic BAC that contains the Nstom2 gene revealed that the third exon was spliced out in this variant reducing the size by 100bp. Since 2 ESTs (AA475011 and AA213118) are also homologous to this region of the smaller cDNA, it is suggested that Nstom2 (also: "NS2") can form a smaller splice variant that is called Nstom2-s (small). This splice variant has a putative alternative methionine start codon that follows a stop codon inserted at position 212 as a result of the splicing out of exon 3. This putative coding sequence then starts in frame at position 255 and finishes at position 989, thus being translated into a polypeptide of 244 amino acids.

Figure 8 shows the membrane potential in untransformed HEK 293 cells, in HEK 293 cells transformed with BNC1 and in HEK 293 cells overexpressing the different stomatin domain-containing proteins alone or in combination with BNC1.

Figure 9 shows Stomatin family members aligned around their stomatin domain. The stomatin domain is indicated in light grey (with consensus sequence written below) and black boxes show hydrophobic regions.

Figure 10 shows the proton-induced current in HEK293 stomatin domain of stomatin-BNC1 double transfectants (derived from a BNC1- expressing HEK cell clone). The presence of the stomatin domain clearly increases the proton-gate inward current. The diagram shows peak (transient) current.

The following Examples serve to illustrate the invention.

MATERIALS AND METHODS

The following materials and methods were used in the Examples. 1. Molecular biological techniques

Unless stated otherwise in the Examples, all recombinant DNA techniques are performed according to protocols as described in Sambrook and Russell (2001), Molecular Cloning: A Laboratory Manual, CSH Press, Cold Spring Harbor, NY, USA or in Volumes 1 and 2 of Ausubel et al. (1994), Current Protocols in Molecular Biology, Current Protocols.

2. RT-PCR and DNA constructs

Reverse transcription (Superscript II RT, Life Technologies, Gaithersburg MD) was performed using poly(A)RNA prepared from the dorsal root ganglia of mice or rats. The reaction was primed with an oligo dT primer. PCR products were cloned into pCRII (Invitrogen, Netherlands) or pGEM-T Easy for restriction analysis and sequencing.

The cDNA clones containing the full-length sequences of BNC1a (rat), stomatin (mouse), N-stomatin1 (rat), Nstomatin-2 (mouse), and MSLP1 (mouse) were PCR amplified using specific primers directed against the known cDNA sequences. The sequences of the coding regions of these sequences are set forth in the sequence listing. Clones were then modified with either a FLAG or myc tag inserted in frame onto the amino or carboxyl terminus. Modified cDNA clones were then subcloned into pcDNA (Invitrogen) expression vector.

3. Stable cell lines expressing stomatin domain-containing proteins

Standard methods were used to culture HEK293 cells. To passage the cells, they were washed with PBS, which was pipetted up and down to dissociate the cells from the culture dish. They were then centrifuged at 1500rpm for 2 min, resuspended in fresh medium and counted in a Thoma chamber, so that an appropriate number of cells were plated in a new dish. Transfection

HEK293 cells were transfected using the lipofectamine system as described in the manufacturer's instructions. Cells were plated at low density in 10cm culture dishes. The DNA was prepared prior to the transfection. 0.5μg pDNA/μl serum-free medium were combined with a 10% Lipofectamin/serum-free medium solution and incubated for 30min at RT. Cells were rinsed once in the culture dish with serum-free medium. Transfection medium was diluted 1 :10 in serum-free medium prior to applying it to the cells and incubated for 5 hours under normal cell culturing conditions. Transfection medium was carefully removed, covered with normal medium and allowed to grow undisturbed for 3 days.

Cells were passaged, diluting their number 1 :10 before replating. They were plated in a new dish in selection medium, i.e. 500μg/ml geneticin (G418) in normal cell medium. Selection medium was renewed every other day. After 1 week only transfected cells survived, having formed colonies in the culture dish. Using cloning rings, small glass rings, colonies were isolated from the surrounding medium and dissociated by applying 0.05% trypsin and plated separate for further analysis. Clones that showed the highest immunofluorescence using an antibody directed against the FLAG epitope (M2) were tested in Western blots for expression of the appropriate sized full- length protein. The following cell lines that overexpress a single epitope tagged protein were prepared:

BNC1a-N-Flag HEK 293 cells overexpressing BNC1a with an N-terminal FLAG peptide addition. The protein is expressed and pH-gated inward current can be measured.

Nstomatin1-C-myc HEK 293 cells overexpressing BNC1a with an N- terminal FLAG peptide addition. The protein is expressed as determined with Western blots and immunocytochemistry. Stomatin-N-Flag HEK 293 cells overexpressing BNC1a with an N-terminal FLAG peptide addition. The protein is expressed as determined with Western blots and immunocytochemistry.

Stomatin-C-myc HEK 293 cells overexpressing BNC1a with an N-terminal FLAG peptide addition. The protein is expressed as determined with Western blots and immunocytochemistry.

Nstomatin-2l-C-myc HEK 293 cells overexpressing Nstomatin-2l-C-myc with a C-terminal MYC peptide addition. The protein is expressed as determined with Western blots and immunocytochemistry

Nstomatin-2s-C-myc HEK 293 cells overexpressing Nstomatin-2s-C-myc with a C-terminal MYC peptide addition. The protein is expressed as determined with Western blots and immunocytochemistry

MSLP1-C-myc HEK 293 cells overexpressing MSLPI-C-myc with a C- terminal MYC BNC1a with a N-terminal FLAG peptide addition. The protein is expressed as determined with Western blots and immunocytochemistry

5. Double Transfectants

One clone was selected with a high expression of the BNC1 protein. It was also shown that the BNC1a formed functional ion channels in the membrane by recording pH-gated inward currents from these cells (see electrophysiology below). This clone was then used to generate several new cell lines where BNC1a was expressed together with stomatin domain- containing proteins:

BNC1a-N-Flag/Nstomatin-1-C-myc HEK 293 cells overexpressing BNC1a with an N-terminal FLAG peptide addition. Both proteins are expressed. Cells require special culture conditions. Thus, they must be plated on poly-I-lysine coated tissue culture dishes to adhere properly. For harvesting, a cell scraper must be used.

BNC1a-N-Flag/Stomatin-C-myc HEK 293 cells overexpressing BNC1a with an N-terminal FLAG peptide addition. Both proteins are expressed.

BNC1a-N-Flag/Nstomatin-2l-C-myc HEK 293 cells overexpressing BNC1a with an N-terminal FLAG peptide addition. Both proteins are expressed.

BNC1a-N-Flag/Nstomatin-2s-C-myc HEK 293 cells overexpressing BNC1a with an N-terminal FLAG peptide addition. Both proteins are expressed.

BNC1a-N-Flag/mSLP1-C-myc HEK 293 cells overexpressing BNC1a with an N-terminal FLAG peptide addition. Both proteins are expressed.

6. Electrophysiology

Whole-cell recordings were made using glass electrodes (3-5 M Ω resistance) pulled from borosilicate glass (Hilgenberg, Malsfeld, Germany) on a laser micropipette puller (P-2000, Sutter Instruments, Novato, CA). The recording chamber (volume of 500 μl) was superfused continuously (2-3 ml/min) with extracellular solution containing (in mM): NaCl 154, KCI 5.6, CaCI₂ 2, MgCI₂ 1. HEPES 10, glucose 8, pH 7.4; osmolarity= 325 mOsm. Electrodes were filled with solution containing (in mM): KCI 122, Na⁺ 10, MgCI₂ 1. EGTA, 1 , HEPES 10, pH 7.3; osmolarity = 290 mOsm. Cells were visualized at 63* magnification with a Leica DMIRB inverted microscope.

Membrane voltage was clamped using an EPC-9 amplifier run by Pulse software for Windows 2000 (HEKA Electronic, Lambrecht, Germany). Data were filtered with a four-pole Bessel filter (5.0 kHz), and stored for offline analysis. Whole-cell configuration was maintained at 60 mV. Seals ranged from 1.5 to 6.0 G Pipette and cell capacitance artifacts were estimated and corrected according to the procedures described by Sigworth (1995) Electronic design of the patch clamp. In: Single channel recording (Sakmann B, Neher E, eds), pp 3-35. New York: Plenum Press. Short trains of square-wave voltage pulses were applied, and the resulting capacitance transients were averaged, leak-subtracted, and then used to calculate the required corrections to the components of the compensation network. Cells were perfused with pH6, pH5 and pH4 buffer by moving an array of outlets over the cell (RSC-200, Biologic, France). Stimulus duration was 10 seconds with 1 minute recovery time between each stimulus. Peak transient and sustained currents were measured as difference from baseline, current kinetics were measured as current size divided by time to peak (velocity).

Example 1: Stable expression of BNC1a in HEK 293 cells

Cells have a large proton-induced current (>2 nA). This inward current can easily be verified and measured using electrophysiological techniques or alternatively high-throughput fluorescent measurement techniques (Denyer et al. (1998) Drug Discovery Today 3, 323-332). Most of the current is carried by sodium ions (Figure 1). When recordings are made from cells in current clamp mode, then a pH stimulus leads to a large depolarization of the cells (>40 mV). This is important as high- throughput screening methods work most reliably by measuring changes in the membrane potential.

Example 2: Determination of interaction between BNC1 and stomatin domain- containing proteins

We have demonstrated biochemical interactions between stomatin domain- containing proteins and the homomeric BNC1 channels. In these experiments, HEK cells expressing BNC1 with a FLAG epitope tag were transfected with plasmids containing cDNA constructs encoding stomatin or Nstomatin-1 . Antibodies directed against the FLAG epitope attached to the BNC1 channel were used to immunoprecipitate the recombinant BNC1 protein. Western blotting was used to detect stomatin or Nstomatin-1 in the same immunoprecipitates. In this experiment, it was found that stomatin and Nstomatin-1 could be co-immunopreciptated with the BNC1 protein. This experiment suggests that stomatin and Nstomatin-1 can interact with the BNC1 protein in a complex. It is not yet clear from this experiment whether this interaction is direct. Similar co-immunoprecipitation experiments were carried out with Nstomatin-1 and stomatin, which showed that these proteins can also interact with each other. Such biochemical experiments however do not demonstrate whether a stomatin domain-containing protein can change the gating properties of the BNC1 channel protein.

Example 3: Investigation of the proton-gated current in double transfectants

In a new series of experiments, a series of HEK 293 cell lines were generated in which BNC1 homomeric channels are overexpressed together with one of four stomatin domain-containing proteins. Electrophysiological investigation of proton- gated currents in these double transfectants has demonstrated that the kinetics of BNC1 channels' gating is strongly influenced by these proteins. In stomatin-BNC1 double transfectants (derived from a BNC1 -expressing HEK cell clone), the proton-activated current is increased. For example, a 30% increase can be observed with pH5 (see Figure 2).

Normally the proton-gated inward current is very large in BNC1 -expressing HEK cells. When the Nstomatin-1 protein was overexpressed in the same line (BNC1 expressing clones, stable transfectants), then pH became almost completely ineffective at opening the BNC1 ion channel. Immunocytochemical studies revealed that the BNC1 channel protein was still present in large amounts on the membrane. Thus, it appears that the proton-gating of the BNC1 channel was completely inhibited by Nstomatin-1 (see Figure 3).

Nstomatin-2l is the full length form of Nstomatin-2 (human gene hslp2). In this cDNA clone, all exons are spliced into the cDNA. When expressed in HEK cells or in sensory neurons, this form is localized predominantly in the membrane (Western blotting and immunocytochemistry). Double transfectants of BNC1 and Nstomatin-2l display a phenotype almost identical to that of Nstomatin-1 -BNC1 double transfectants. Thus, the peak and sustained proton-gated inward current in these cells is profoundly reduced (see Figure 4).

The Nstomatin-2s form was discovered to be expressed in mouse dorsal root ganglion neurons. In the Nstomatin-2s transcript, one exon has been spliced out of the mRNA (see also Figure 7). As a result an alternative start methionine is utilized which leads to the production of a shorter protein missing the first 100 amino acids of the longer form. The amino acid sequence of the shorter form is however identical to that of the C-terminal portion of the Nstomatin-2l (full length) form. In contrast to Nstomatin-2l, the Nstomatin-2s protein appears to be predominantly localized in the cytoplasmic compartment. Double transfectants of N-stomatin-2s and BNC1 show a normal proton-gated current that is comparable to that in the original BNC1 expressing cell line (see Figure 5).

Mouse stomatin-like protein-1 (MSLP-1) was cloned from DRG total mRNA. The human stomatin-like protein-1 gene sequence has previously been described in the literature (Seidel and Prohaska 1998, loc. cit.). HEK 293 cell lines were generated expressing this gene alone and together with BNC1. MSLP-1 was found to lead to a similar effect on proton induced BNC1 channel gating as was observed for Nstomatin-1 (see above).

Example 4: Endogenous proton-gated inward currents are expressed by HEK cells and are strongly modulated by proteins containing a stomatin-like domain

Normal HEK 293 cells when stimulated with a low pH solution display a large inward current (up to 800pA). When such cells are used to generate a stable cell line where Nstomatin-1 is overexpressed, this endogenous proton-gated current is modulated in a fashion similar to that of the recombinant BNC1 channel (see Figure 6). It is concluded that HEK 293 cells express an endogenous BNC1 channel that is modulated by stomatin-like proteins. Thus, single transformants where stomatin-like proteins are overexpressed could also be used for screening. Example 5: Investigation of the proton-gated current using the stomatin domain of stomatin and Nstoml

In order to determine the effect of the isolated stomatin domain on the proton-gated current of the BNC1 channel, experiments were conducted in which the pH- dependent inward current of HEK 293 cells which were double-transfected with BNC1 and a stomatin domain was measured. In these experiments, the stomatin domain from the Nstoml and stomatin sequence were cloned and tagged with a myc epitope at the C-terminal end using standard molecular cloning techniques. The sequences used were as follows: stomatin domain of stomatin: amino acids 53 to 228 of SEQ ID NO: 2 (total length of 177 amino acids and correspondingly 531 base pairs); NStoml stomatin domain: amino acids 46 to 222 of SEQ ID NO: 4 (total length of 178 amino acids and correspondingly 534 base pairs). In the expression constructs used, we introduced an artificial methionine at the beginning of the stomatin domain to have an artificial start codon. Therefore, the expressed sequences in both cases are one amino acid longer than what is indicated in the sequence. The stomatin domain was introduced into cells stably expressing the ion channel BNC1 and, by applying patch clamp techniques, a large potentiation of pH- gated currents both with the stomatin domain of stomatin and Nstoml was observed. In addition, we observed a significant depolarization of the cells expressing the stomatin domain together with BNC1 indicating that, in the absence , of a pH stimulus, there was some activation of the ion channel. The strong inhibition of the pH-gated current in cells expressing full length Nstoml with BNC1 may arise through a direct effect on the pH-gating properties of the channel that is independent of the activation of the channel in the absence of low pH. Figure 10 shows an example of this potentiation observed with the stomatin domain of stomatin. This potentiation effect of the stomatin domain alone identifies this region as the active part of the protein.

Claims

Claims

1. A method for identifying a compound capable of inhibiting the mechanotransduction of a neuron comprising the steps of

(a) providing a cell overexpressing a polypeptide selected from the group consisting of stomatin domain-containing proteins;

(b) contacting the cell with a candidate compound;

(c) measuring the current or voltage at the plasma membrane of the cell; and

(d) identifying the candidate compound as a compound capable of inhibiting mechanotransduction if the current or voltage measured in step (c) is altered in comparison to the corresponding current or voltage of a control cell corresponding to the cell provided in step (a) and which has not been contacted with said candidate compound.

2. The method of claim 1 , wherein the polypeptide is stomatin or a derivative thereof; and wherein the compound is identified in step (d) if the current or voltage measured in step (c) is decreased in comparison to the corresponding current or voltage of said control cell.

3. The method of claim 1 , wherein the polypeptide is selected from the group consisting of neural stomatin-like protein 1 and 2 (Nstoml and Nstom2) and mouse stomatin-like protein 1 (MSLP1) and homologues or a derivative thereof; and wherein the compound is identified in step (d) if the current or voltage measured in step (c) is increased in comparison to the corresponding current or voltage of said control cell.

4. The method of any one of claims 1 to 3 wherein the measuring of the current or voltage at the plasma membrane in step (c) takes place upon the stimulation by acidic pH.

5. The method of claim 4, wherein stimulation by acidic pH is carried out by a pH-value between pH 4 and 6.

6. The method of any one of claims 1 to 5 furthermore comprising the step of (e) verifying the capacity of the identified compound of inhibiting mechanotransduction

(i) by applying the compound to the receptive field of a sensory neuron of the DRG in a suitable skin-nerve preparation; (ii) measuring the mechanotransduction of said neuron upon a suitable stimulus; and (iii) confirming the capacity of inhibiting mechanotransduction if the measured mechanotransduction is reduced when compared to the mechanotransduction of a corresponding neuron to which said compound was not applied.

7. The method of any one of claims 1 to 6, wherein the cell used in the method is a cell showing a proton-induced inward current.

8. The method of any one of claims 1 to 7, wherein the cell provided in step (a) furthermore overexpresses a mechanotransducing ion channel.

9. The method of claim 8, wherein said mechanotransducing ion channel is a member of the Degenerin/Enac family.

10. The method of claim 9, wherein said mechanotransducing ion channel is selected from the group consisting of brain sodium channel 1 (BNC1) and dorsal root acidic sensing channel (DRASIC).

11. The method of any one of claims 1 to 10, wherein the polypeptide and/or the ion channel is of human origin.

12. The method of any one of claims 1 to 11 which is performed as a high- throughput assay.

13. A method for the production of a pharmaceutical composition comprising the steps of the method of any one of claims 1 to 12 and formulating the compound identified or derivative thereof in pharmaceutically acceptable form.

14. A compound identified by the method of any one of claims 1 to 12.

15. A pharmaceutical composition comprising the compound of claim 14 and optionally a pharmaceutically acceptable carrier or obtainable by the method of claim 13.

16. Use of the compound of claim 14 for the preparation of a pharmaceutical composition for the treatment of pain.

17. A method for the treatment of pain comprising administering an effective amount of the compound of claim 14 or of the pharmaceutical composition of claim 15 to a subject in need for such a treatment.

18. The use of claim 16 or the method of claim 17, wherein said pain is a condition selected from the group consisting of chronic pain, allodynia, acute mechanical pain, and post-operative pain.

19. A system useful for identifying a compound capable of inhibiting the mechanotransduction of a neuron comprising

(a) a cell overexpressing a polypeptide selected from the group consisting of stomatin domain-containing proteins; and

(b) a device useful for measuring the current or voltage at the plasma membrane of the cell.

20. The system of claim 19, wherein the polypeptide is selected from the group consisting of stomatin, neural stomatin-like protein 1 and 2 (Nstoml and Nstom2) and mouse stomatin-like protein 1 (MSLP1) and homologues or derivatives thereof.

21. The system of claim 19 or 20 which is applicable in a high-throughput assay.

22. The system of any one of claims 19 to 21 , wherein the cell of (a) furthermore overexpresses a mechanotransducing ion channel.

23. The system of claim 22, wherein said mechanotransducing ion channel is a member of the Degenerin/Enac family.

24. The system of claim 23, wherein said mechanotransducing ion channel is selected from the group consisting of brain sodium channel 1 (BNC1) and dorsal root acidic sensing channel (DRASIC).

25. The system of any one of claims 19 to 24, wherein the cell of (a) is a cell showing a proton-induced inward current.

26. The system of any one of claims 19 to 25, wherein the polypeptide and/or the ion channel is of human origin.

27. A kit comprising cells as defined in any one of claims 1 to 3, 7 to 10 and 11 , a device useful for measuring the current at the plasma membrane of a cell, and/or the system of any one of claims 19 to 26.

28. Use of a polynucleotide encoding a stomatin domain-containing protein, of a polypeptide encoded by said polynucleotide, of cells overexpressing said polypeptide or as defined in any one of claims 1 to 3, 7 to 10 and 11 or of the system of any one of claims 19 to 26 for the identification of a compound capable of inhibiting the mechanotransduction of a neuron.