DE4239816A1 - Inhibiting glutamate-induced synaptic stimulus transfer - with specific spider toxins, for treating CNS disorders and characterisation of receptor channel sub-types - Google Patents

Abstract

Uses of the toxins (A), Joro Spider Toxin (JSTC), NSTX Spider Toxin and argiotoxin (a) for reversible inhibition or slowing down of glutamate-induced synaptic stimulus transfer in the CNS of mammals; (b) for pharmacological differentiation of various subsets of glutamate receptor channels from AMPA (alpha-amino-hydroxy- 5-methyl-4-isoxazolyl-propionate)/kainate type receptor channels and (c) for pharmacological inhibition of calcium-permeable AMPA/kainate receptor channels are new. USE/ADVANTAGE - (A) are very specific antagonists, affecting the GluR1, GluR3, GluR4 and GluR1,3 receptor subunits, i.e. they antagonist glutamate-mediate current in various types of neurons. (A) can be used to treat CNS diseases (no more details) and also for studying and characterising further receptors. (A) are highly specific and do not compete with kainate for binding sites on the receptor.

Description

The invention relates to the use of a toxin from the Group Joro Spider Toxin (JSTX), Spider Toxin NSTX and Argio toxin for reversible inhibition or slowing down of the gluta mat-induced synaptic transmission of arousal as well appropriate drug.

The synaptic transmission of excitation in the central nervous system of mammals (CNS) is predominantly caused by glutamate AMPA / KA-type receptor channels (α-amino-hydroxy-5-methyl-4- Isoxazole propionate / kainate type) mediated (Collingridge, G.L. and Bliss T.V.P. (1987), TINS 10, (1987), 288; Mayer, M.L., and Westbrook, G.L., Prog. Neurobiol. 28: 197-276 (1987); Monaghan et al., Annu. Rev. Pharmacol. Toxicol. 29 (1989), 356-402). It has recently been found that various ne neurological diseases, such as B. Parkinson's disease and Alzheimer's disease (T. Klockgether, L. Turski, T. Honore, Z.M. Zhang, D.M. Gash, R. Kurlan and J.T. Green amyre, ann. Neurol. 30 (5), (1991), 717; P.A. Loschmann, K.W. Lange, M. Kunow, K.J. Rettig, P. Jahnig, T. Honore, L. Turs ki, H. Wachtel, P. Jenner and C.D. Marsden, J. Neural. Transm. Park. Dis. Dement. Sect. 3 (3), (1991), 203; D. Dewar, D.T. Chalmers, A. Shand, D.I. Graham and J. Mc Culloh, Ann. Neurol. 28 (6), (1990), 805; P.J. Harrison, A.J. Barton, A. Majlerahim and R.C. Pearson, Neuroreport 1 (2), (1990), 149) or some types of epilepsy (J.W. McDonald, E.A. Garo falo, T. Hood, J.C. Sackellares, S. Gilman, P.E. McKeever, J.C. Troncoso and M.V. Johnston, Ann. Neurol. 29 (5), (1991), 529; THERE. Hosford, B.J. Crain, Z. Cao, D.W. Bonhaus, A.H. Friedman, M.M. Okazaki, J.V.N. Adler and J.O. McNamara, J. Neurosci 11 (2), (1991), 428; R. Dingledine, C.J. McBain, J.O. McNamara, Trends Pharmacol. Sci. 11 (8), (1990), 334; S.C. Pedder, R.1. Wilcox, J.M. Tuchek, D.D. Johnson and R.D. Crawford, Eur. J. Pharmacol. 175 (1), (1990)) also with AMPA / KA-type glutamate receptors. So it was  already by Difazio et al., Neurology 42 (2) (1992), 402, a Investigation published that a treatment option Parkinson's disease with AMPA / KA receptor antagonists most occupied.

The object of the present invention was as a medicament suitable further antagonists of the glutamate receptor channels AMPA / KA type. Such antagonists should if possible also specifically for certain receptor channels his.

In the context of the present invention it was found that Joro Spider Toxin (JSTX), the toxin of the Nephila spider clavata, as well as the closely related Athropod toxins NSTX and Argiotoxin as an antagonist of synaptic excitation transmission and of glutamate-mediated currents in various types of neurons (Kawai, N., Miwa, A., Saito, M., Pan- Hou, H.S., Yoshioka, M., J. Physiol. 79: 228-231 (1984); Kawai, N., Niwa, A., Abe, T., Brain Res. 247, 169-171 (1982); Kawai, N., J. Toxicol. Toxin Rev. 10 (2), 131-167 (1991); Akaike, N., Kawai, N., Kiskin, N.I., Kljuchko, E.M., Krishtal, O.A., Tsyndrenko, A.Y., Neurossci. Letters, vol. 79: 326-330 (1987); Saito, M., Sahara, Y., Miwa, A., Shimazaki, K., Najima, T., Kawai N., Brain Res. 481, 16-24 (1989); Ajima, A., Hensch, T., Kado, R.T., Ito, M., Neurosci. Res. 12: 281-286 (1991); Usherwoood, R.N.R. Excitatory amino Acids, New York (1991)) act and thereby mediate kainate Reactions of different subunits one in Xenopus Block oocyte-expressed glutamate receptor channel. The reversible blocking by JSTX, NSTX and argiotoxin specifically for subunits or subunit combinations with a rectifying current-voltage relationship, namely GluR1, GluR3, GluR4 and GluR1,3. These subunits or Subunit combinations are highly calcium permeable.

The blocking already takes place with submicromolar concentrations trations of the toxins used according to the invention in the  carried out experiments. It is also noted been that a change in a single amino acid in the membrane-penetrating region M2 of the receptor channels (Hollmann, M., O'Shea-Greenfield, A., Rogers, S.W., Heine Mann, S., Nature 342: 643 (1989); Boulter, J., Hollmann, M., O'Shea-Greenfield, A., Hartley, M., Deneris, E., Maron, C., Heinemann, S., Science 249, 1033 (1990); Keinänen, K., Wisden, W., Sommer, B., Werner, P., Herb, A., Verdoorn, T.A., Sakmann, B., Seeburg, P.H., Science 249, 556 (1990); Nakanishi, N., Schneider, N.A., Axel, R., Neuron 5, 569-581 (1990); Bettler, B., Egebjerg, J., Sharma, G., Hermans-Borg meyer, I., Moll, C., Stevens, C.F., Heinemann, S., Neuron 8, 257-265 (1992); Egebjerg, J. Bettler, B. Hermans-Borgmeyer, I., Heinemann, S., Nature 351, 745-748 (1991)) to a ver Change in the shape of the voltage curve and thus also to leads to a change in the blockability by the toxins. Calcium permeability is also affected. The bloc The effect of JSTX, NSTX and argiotoxin is reversible and not competitive with Kainat in terms of Binding site of the antagonist.

An object of the present invention is therefore the Ver use of a toxin from the group Joro Spider Toxin (JSTX), Spider toxin NSTX and argiotoxin for inhibition or slowdown of glutamate-induced synaptic excitation transmission in the central nervous system of mammals. Because of your Specificity for special subunits or subunits combinations of glutamate receptor channels are the inventions According to the toxins used clinically valuable drug fe, which allow the excitation effect in specific to reduce neuronal populations of the central nervous system adorn.

Another object of the present invention is Use of a toxin from the Joro Spider Toxin group (JSTX), Spider Toxin NSTX and Argiotoxin for pharmacological differentiation of different subspecies of glutamate  AMPA / KA type receptor channels. Because of the specificity for certain subunits, as stated above, can JSTX, NSTX and Argiotoxin also in research and cha characterization of other receptors.

The functional properties of the AMPA / KA receptor complexes depend crucially on their composition from one another divisions (M. Hollmann et al. Nature 342, (1989), 643; Holl mann, M., Hartley, M., and Heinemann, S., Science 252, (1991), 851-853; K. Keinänen et al., Science 249, (1990), 556; Boulter, J. et al. (1990) Science 249: 1033; Nakanishi, N., Shneider, N.A. and Axel, R. Neuron 5, (1990), 569-581). For example, it was found that AMPA / KA receptors, which contain the subunit GluR2, a linear current show tension and a low calcium permeabi lity, whereas recombinant receptors which were produced from the sub-units GluR1 and GluR3 or the subunit combination GluR1,3 one inside have rectifying current-voltage relationship and a show high permeability for calcium ions (Egebierg, J. Bettler, B., Hermans-Borgmeyer, I. and Heinemann, S. Nature 351: 745-748 (1991); Hume, R.I., Dingledine, R. and Heine mann, S., Science 253, (1991), 1028-1031; Burnashev, N. Monyer, H., Seeburg, P. and Sakmann, B., Neuron Vol. 8 (1992), 189 198). Despite this strong functional sub All subunit combinations of the AMPA / KA Receptors very sensitive to quinoxalinediones, e.g. B. 6-cyano 7-nitroquinoxaline-2,3-dione, CNQX, the best known specific antagonists of glutamatergenic synaptic Transmission of excitation in the brain (T. Honore, S.N. Davies, J. Dreyer, E.J. Fletcher, P. Jacobson, D. Lofge and F.E. Niel sen, Science 241, (1988), 701). In contrast, the Using JSTX as an antagonist to differentiate the different receptor subunits.

In the context of the present invention, it was found that JSTX, NSTX and argiotoxin are potent subunit specific  antagonists for different AMPA / KA receptor subunits units or subunit combinations, namely from high Calcium permeable receptor channels are. You bind with high Affinity to a site in the receptor complex that differs that is from the binding site of classic AMPA / KA recipes tor agonists such as kainate or glutamate.

Another object of the invention is therefore the use Formation of a toxin from the group JSTX, NSTX and argiotoxin to inhibit high calcium permeable AMPA / KA receptor channels.

On the above effects and the fact that AMPA / KA receptors in various neurological diseases play an important role rer object of the present invention, namely a doctor Needy for the treatment of diseases of the central ner vensystems, which as an effective agent has at least one toxin from the group JSTX, NSTX and argiotoxin together with übli Chen contains pharmaceutical carriers and auxiliary substances.

The following examples explain in connection with the Figu ren the invention further.

Fig. 1 shows the results of the experiment for blocking the AMPA / KA-channel subunit combination GluR1 through JSTX.

Figure 2 in conjunction with Table 1 shows both the experimental protocol and the experimental results of the effect of JSTX on different receptor channel subunits.

FIG. 3a shows a comparison of the amino acid sequences of the glutamate receptor subunits GluR1-GluR6 in the presumable region M2 that penetrates the membrane.

Fig. 3b and c show that the wild type receptor combination GluR1,3 is blocked by JSTX, a mutated Rezeptorkom GluR1,3 (Q590R) was not bination.

Examples

The preparation and cultivation of Xenopus oocytes was carried out as von Methfessel et al., Pflügers Arch. 407, (1986), 577 be wrote. Frog oocytes were briefly summarized removed and on the same day with two ng cRNA coding for the different receptor subunits, subunit combination cations or mutants injected. A day later Oocytes in collagenase (Clostridiopeptidase A) from Clostri dium histolyticum type I (Sigma, FRG) incubated for 1 to 4 hours and remaining layers of follicular cells skinned by hand. After two to three days, the skinned oocytes were made for Experiments used.

Current measurements were carried out using the 2-electrode voltage terminal system Turbo Tech 01C ("npi electronic", Tamm, FRG). During the measurements, the recording chamber was continuously rinsed with saline solutions and kept at room temperature (21-24 ° C). Normal saline contained: NaCl 115mM, KCl 2.5mM, CaCl ₂ 1.8mM, 10mM HEPES (pH 7.2, NaOH). Flushing solutions also contained 150 µM nifluic acid (Sigma, FRG) to block the intermediate component of intrinsic Ca ²⁺ activated chloride streams in Xenopus oocytes. The recording electrodes consisted of fine glass pipettes (1-2 MOhm) filled with 2 M KCl. The signals were filtered at 0.1-5 KHz with an 8-pole Bessel filter (Freqency Devices, MA, USA) and recorded on video tapes by a PCM / VCR recording medium (Instrutech, NY, USA). The software used for the analysis was either obtained from Instrutech or Luigs and Neumann (Rattingen, FRG).

example 1 Blocking Cainate-Mediated Currents by AMPA / KA- Receptor channels of type GluR1 through JSTX

Figure 1a shows the chemical structure of the Joro spider toxin used in its synthetic form (RBI, Natick, MA, USA) for the experiments in this patent application. Figure 1b shows kainate-induced currents measured in the two-electrode voltage clamp configuration during administration of 300 µM kainate (KA; left) and 300 µM kainate, 0.5 µM JSTX (KA, JSTX; right). The drug was administered by rapid perfusion in the bath, which led to a complete change of bath solution within 3 seconds. The membrane potential was kept at -100 mV during the experiment. Fig. 1c shows dose-response curves for the receptor GluR1. For this experiment JSTX was used in increasing concentrations starting with 10 nM up to a maximum of 1 µM. The blocking effect was determined 3 minutes after JSTX application. The resulting dose-response curve was adjusted using the least squares method, which resulted in a half-maximum inhibition IC ₅₀ of 40 nM and a Hill coefficient H = 0.98 for this experiment.

Fig. 1d shows the dose-response curves for kainate (10 uM 1 mM, GluR1) in the absence (control) and presence of 0.2 uM JSTX. The maximum kainate-induced current was reduced in the presence of the toxin, but the 1C ₅₀ values were essentially unaffected (control: IC ₅₀ = 29 µM; 0.2 µM JSTX: IC ₅₀ = 20 µM).

Figure 1e shows the blocking effect of JSTX on an oocyte that expresses the AMPA / KA receptor GluR1. The control trace (left) shows the characteristic, non-desensitizing current response obtained by applying kainate to an oocyte to which a voltage of -100 mV was applied. Addition of JSTX to the perfusion medium containing kainate caused a block (right lane of Fig. 1b) that developed over a period of several minutes. A similar slow block development was observed in oocytes preincubated in toxin-containing media a few minutes before the addition of kainate, which is consistent with the earlier assumption that the block is application-dependent (Priestley, T., Woodruff, GN , Kemp, JA, Br. J. Pharmacol. 97, 1315-1323 (1989)). In oocytes maintained at negative voltages, JSTX blocking was only partially reversible (less than 60% recovery after 5 minutes of continuous perfusion with control solution). A more effective recovery (80 to 95% of the control value) was obtained when the oocyte was connected to a voltage source with 0 mV for 60 seconds in the presence of kainate, which suggests a voltage dependency of the blocking effect of JSTX (Priestley, T., Woodruff, GN, Kemp, JA, Br. J. Pharmacol. 97, 1315-1323 (1989)). Further properties of the JSTX blocking are shown in FIGS . 1c and 1d. Figure 1c shows the high affinity of toxin blocking. The concentration that inhibited 50% of the kainate-mediated current response (IC ₅₀ ) by GluR1 was approximately 40 nM JSTX.

Figure 1d shows a dose response relationship for kainate-mediated currents in the absence (control) and presence of 0.2 µM JSTX. The IC ₅₀ values (the concentration of kainate, which gives a half-maximal response) remained practically unaffected by the presence of JSTX (control - 29 µM; 0.2 µM JSTX - 20 µM; GluR1). This suggests that the JSTX binding site does not match the agonist binding site.

Analog effects, as described in this example, were obtained using NSTX and argiotoxin.  

Example 2 Comparison of the blocking effect of JSTX on different ones Receptor subunit combinations

The different receptor combinations were in Xenopus oocytes expressed as described above. In the case of the recipe tor combinations GluR1,2 and GluR3,2, the mRNA in one Ratio of 1:10 injected with a 10 times higher con concentration of GluR2 to the formation of either GluR1 or To prevent GluR3 homooligomeric receptors. The relationship of injected mRNA of the mutant subunit combinations GluR1.3 (Q590R) was 1: 2, for GluR3.3 (Q590R) the ratio was nis 1: 4 to get a linear current-voltage relationship In the case of the receptor types GluR6 (R591) and GluR6 (R691Q) the oocytes were pre-incubated in Concanavalin A (10 mM) to to get an increase in the kainate-induced current response (Egebjerg, J., Bettler, B., Hermans-Borgmeyer, I., Heinemann, S., Nature 351: 745-748 (1991)).

Figure 2 and Table 1 show the subunit specificity of JSTX blocking. Receptors that are composed of the subunits GluR1, GluR3, GluR4 and the subunit combination GluR1,3 were blocked by the toxin with an affinity in the nanomolar concentration range. In contrast, the combinations GluR1.2 (mRNA ratio 1:10), GluR3.2 (1:10) and GluR6 were not blocked by concentrations up to 1 mM JSTX (Table 1). Figure 2 compares the effect of 0.5mM JSTX on kainate-induced currents for the GluR3 and GluR1,2 receptors. The dose response curve of the JSTX blocking for GluR3 is shown in the lower area of Fig. 2a. The IC ₅₀ values for JSTX were measured at 50 nM (n = 4). There was a clear correlation between JSTX-sensitive and non-sensitive AMPA / KA receptor combinations and the shape of their current-voltage curves (Table 1). All subunit combinations with an inward rectifying current-voltage relationship were blocked by nano-molar concentrations of JSTX, while receptor combinations with a linear current-voltage relationship were not affected by such low concentrations of the toxin. Similar subunit-specific effects could be obtained with argiotoxin, NSTX and the synthetic JSTX analogue naphthyl spermine (Naspm).

FIG. 2 also shows: in the upper region of FIG. 2a: control traces (300 μM kainate) and traces which were obtained using 300 μM kainate, 0.5 μM JSTX for receptor GluR3. The currents were blocked 90% using 0.5 µM toxin. The lower area shows, as already mentioned above, the mean dose-response curves for receptor GluR3. Each data point represents an average of 4 experiments; the current values were normalized to their control response (0 μM JSTX). FIG. 2b shows in the upper part kainate-induced currents before and after application of 0.5 μM JSTX to an oocyte which expresses the GluR1.2 receptor subunit combination. In order to suppress the formation of GluR1 homooligomer, the subunit combination GluR1.2 was expressed according to a mRNA ratio GluR1: GluR2 of 1:10. The lower area of FIG. 2b shows the mean current responses which were obtained for the subunit combination GluR1.2 (n = 6) after JSTX application. The small decrease in current responses can easily be explained by a general "run down" of the current response and was also observed in the absence of the toxin.

Table 1

Example 3 An amino acid is decisive for the subunit specific JSTX blocking

Fig. 3 shows the comparison of the amino acid sequences of glutamate receptor subunits GluR1-GluR6 in the assumed, membrane-penetrating region M2 (Hollmann, M., O'Shea-Greenfield, A., Rogers, SW, Heinemann, S., Nature 342, 643 (1989); Boulter, J., Hollmann, M., O'Shea-Green field, A., Hartley, M., Deneris, E., Maron, C., Heinemann, S., Science 249, 1033 (1990); Keinänen, K., Wisden, W., Sommer, B., Werner, P., Herb, A., Verdoorn, TA, Sakmann, B., Seeburg, PH, Science 249, 556 (1990) ; Nakanishi, N., Schneider, NA, Axel, R., Neuron 5, 569-581 (1990); Bettler, B., Egebjerg, J., Sharma, G., Hermans-Borgmeyer, I., Moll, C ., Stevens, CF, Heinemann, S., Neuron 8, 257-265 (1992); Egebjerg, J. Bettler, B. Hermans-Borgmeyer, I., Heinemann, S., Nature 351, 745-748 (1991) ). The amino acid sequence in this region is highly conserved in the GluR1-GluR4 receptor subunits, which belong to a glutamate receptor subfamily. The sequence of GluR6 is less conserved because GluR6 belongs to another receptor subfamily (Egebjerg, J., Bettler, B., Hermans-Borgmeyer, I., Heinemann, S., Nature 351, 745-748 (1991); Bettler, B., Egebjerg, J., Sharma, G., Hermans-Borgmeyer, I., Moll, C., Stevens, CF, Heinemann, S., Neuron 8, 257-265 (1992); Egebierg, J., Bettler , B., Hermans-Borgmeyer, I., Heinemann, S., Nature 351, 745-748 (1991)). By using directed mutagenesis, it could be shown that the exchange of a single amino acid in the membrane-penetrating region M2 is responsible for the shape of the voltage curve, and in some, but not all, glutamate receptors, this regulates the calcium permeability of the glutamate receptor channels (Hume, RI, Ding ledine, R., Heinemann, SF, Science 253, 1028-1031 (1991); Verdoorn, TA, Burnashev, N., Monyer, H., Seeburg, PH, Sakmann, B., Science 252 , 1715-1718 (1991); Egebjerg, J. and Heinemann, S., PNAS, in press; Dingledine, R., Hume, RI, Heinemann, S., J. Neurosci. 12, 4080-4087 (1992)) . GluR2, the subunit which, when coexpressed with GluR1 or GluR3, generates a receptor with a linear current-voltage relationship, contains the positively charged amino acid arginine (Arg, R) at position 586. If this amino acid is replaced by the neutral amino acid glutamine (Gln, Q) this results in a receptor combination GluR1,2 (R586Q) with a rectifying instead of a linear current-voltage curve. Accordingly, the change in the amino acid arginine to glutamine at the analog position in the receptor subunit GluR3 leads to a receptor mutant with a linear instead of a rectifying current-voltage relationship. It was examined whether this amino acid position is also responsible for the JSTX blocking.

FIG. 3b and 3c show that the wild type receptor combination GluR1,3 was blocked by the toxin, but the mutant receptor combination GluR1,3 (Q590R) was not affected. This observation is in line with the hypothesis that JSTX interacts with a location on the AMPA / KA receptor channels that is directly connected to the channel and that determines the ion permeation properties.

The following is shown in detail in FIG. 3: FIG. 3: Schematic drawing of the primary structures of GluRs. The amino acid sequence of the putative transmembrane regions M2 are shown for GluR1-GluR6. Identical amino acids are framed, the amino acid positions that are critical for JSTX blocking are marked with an arrow. The amino acid positions indicate the number of the first and last amino acids shown.

FIG. 3b shows in the upper area of power traces which have been decorated by indu 300 uM KA (left) and after application of 300 uM KA, 0.5 uM JSTX (right) for receptor combination GluR1,3. For this oocyte, the mRNA was injected with a ratio of GluR1: GluR3 of 1: 1. The lower part shows the current-voltage relationship of the subunit combination GluR1.3 for membrane potentials between -100 mV and +60 mV.

Fig. 3c shows the upper region currents obtained from oocytes expressing the mutant subunit combination GluR1,3 (Q590R) (expressed at a mRNA ratio 1: 2) after application of 300 uM KA) left and KA, 0.5 uM Express JSTX (right). The lower part shows the voltage curve for the mutant subunit combination GluR1,3 (Q590R) in the absence of the toxin. All data points in the voltage curves were corrected for the background currents of the oocytes, which were less than 100 nA. The amino acid abbreviations used are: A: alanine, C: cysteine, D: aspartate, E: glutamate, F: phenylalanine, G: glycine, I: isoleucine, L: leucine, M: methionine, N: asparagine, P : Proline, Q: glutamine, R: arginine, S: serine, T: threonine, V: valine, W: tryptophan.

Parallel experiments also carried out for this example with NSTX and argiotoxin show similar results.

Claims (4)

1. Use of a toxin from the Joro Spider Toxin group (JSTX), Spider Toxin NSTX and Argiotoxin for reversible Inhibition or slowdown of the glutamate-induced sy naptic transmission of excitation in the central nervous system stem of mammals. 2. Use of a toxin from the group Joro Spider Toxin (JSTX), Spider Toxin NSTX and Argiotoxin for pharmacolo differentiation of different subspecies from AMPA / Kainate type glutamate receptor channels. 3. Use of a toxin from the Joro Spider Toxin group (JSTX), Spider Toxin NSTX and Argiotoxin for pharmacolo inhibition highly calcium-permeable AMPA / KA recipe gate channels. 4. Medicines for the treatment of diseases of the central len nervous system, characterized, that there is at least one toxin from the Group Joro Spider Toxin (JSTX), Spider Toxin NSTX and Argiotoxin along with usual pharmaceutical carriers contains additives and additives.