Neuropeptides originating in scorpion

Details Neuropeptides originating in scorpion Neuropeptides originating in scorpion

1998-08-26

2004-02-10

Low, Christopher S. F. (Department: 1653)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Peptide containing doai

C514S010100, C514S012200, C530S317000, C530S324000, C424S236100, C435S071300

Reexamination Certificate

active

06689749

ABSTRACT:

TECHNICAL FIELD
The invention relates to novel neuropeptides, more specifically scorpion toxin-related peptides which are obtained from the venom gland of
Heterometrus spinnifer
, a species of scorpion.
BACKGROUND ART
“Scorpion” is a general term for arthropods belonging to the class Arachnida, the order
Scorpionida
and about 600 species of scorpions now inhabit the world. Scorpions have generally believed to be virulently poisonous; however, actually those having fetal toxins are limited to only several species, including
Buthus ustralis
(living in the deserts of Africa),
Centruroides exilicauda
(native to Mexico) and
B. occitauda
(native to South Europe), etc. Up to now, various types of scorpion toxins have been isolated (Dreyer. F., Rev. Physiol. Biochem. Pharmacol., vol.15, pp.94-128, 1990), which are considered to be neurotoxins.
From the studies on pharmacological properties of these toxins, the actions of the toxins against K
+
channels have been drawing attention. For example, margatoxin (MgTX) isolated from the venom gland of
Centruroides margaritatus
(Garcia-Calvo et al., J. Biol. Chem., vol. 268, pp. 18866-18874, 1993) and agitoxin 2 (AgTX2) isolated from the venom gland of
Leiurus quinquestriatus
herbraes (Garcia et al., Biochemistry, vol. 33, pp. 6834-6839, 1994) have been known to act as blockers on voltage-gated K
+
channels. These toxins are peptides composed of 38 amino acid residues and containing three disulfide bridges in their molecules. Recently, also reported is a peptide,
Pandinus imperator
toxin 1 (Pi1), which is a peptide containing four disulfide bridges in the molecule and composed of 35 amino acid residues (Olamendi-Portugal et al., Biochem. J. vol. 315, pp. 977-981, 1996).
On the other hand, it has been considered that production of interleukin-2 (IL-2) by T cells requires Ca
2+
influx into the T cells. Recently, it has been discovered that Kv1.3, which is one type of the K
+
channels, is involved in Ca
2+
influx into T cells accompanied by activation of the T cells (Leonard et al., Proc. Natl. Acad. Sci. USA, vol. 89, pp. 10094-10098, 1992). Further, Lin et al. reported that inhibition of Kv1.3 channels suppresses Ca
2+
influx into T cells, proliferation of T cells and production of IL-2 by T cells (J. Exp. Med., vol. 177, pp.637-645, 1993).
Under these circumstances, scorpion toxins are expected to be applicable to medicines in view of their various pharmacological activities. However, for application to medicines, it is necessary to separate the useful pharmacological activities of the toxins from their undesirable toxicity and, consequently, it becomes necessary to isolate much scorpion toxins and clarify the structure-activity relationship thereof. In these situations, the object of the present invention is to provide novel scorpion toxin-related peptides for contribution to application of scorpion toxins to medicines.
DISCLOSURE OF THE INVENTION
The inventors have made intensive and extensive studies on isolation of novel scorpion toxin-related peptides from the venom gland of a scorpion
Heterometrus spinnifer,
on the basis of the ability to block the rat brain voltage-gated K
+
channels Kv1.3 (also called “RCK3”) expressed in
Xenopus oocytes.
As a result, the inventors have succeeded in isolation and purification of a novel peptide, named HsTX1, represented by SEQ ID NO:1 in which four disulfide bridges are present in the molecule. They have also succeeded in determination of the primary and higher-order structures of HsTX1. It has been found that HsTX1 exhibits much stronger ability to block the K
+
channels compared with the previously reported scorpion toxins and inhibits the IL-2 production by human peripheral blood T cells. Thus, the invention has been accomplished.
According to the present invention, there is provided a peptide represented by an amino acid sequence:
Ala Ser Cys Arg Thr Pro Lys Asp

Cys Ala Asp Pro Cys Arg Lys Glu

Thr Gly Cys Pro Tyr Gly Lys Cys

Met Asn Arg Lys Cys Lys Cys Asn

Arg Cys; (SEQ ID NO:1)
in which 0 to 4 disulfide bridges are present in the molecule and the C terminal may be amidated, as a blocker of voltage-gated K
+
channels or an inhibitor of IL-2 production.
In a specific embodiment, the present invention provides a peptide represented by the above amino acid sequence in which the disulfide bridges are selected from the group consisting of CyS
3
-Cys
24
, Cys
9
-Cys
29
, Cys
13
, Cys
31
and Cys
19
-Cys
34
.
In a most preferred embodiment, the present invention provides a peptide represented by the above amino acid sequence in which the C terminal is amidated and four disulfide bridges, Cys
3
-Cys
24
, CyS
9
-Cys
29
, Cys
13
-Cys
31
and Cys
19
-Cys
34
, are present in the molecule.


REFERENCES:
Ngo et al., ‘Computational Complexity, Potein Structure Prediction, and the Levinthal Paradox,’ The Protein Folding Problem and Tertiary Structuer Prediction. Ed. K. Merz and L. Le Grand. BirkHauser, Boston MA. pp. 491-495, 1994.*
Rogowski, Robert S. et al., “Three new toxins from the scorpion Pandinus imperator selectively block certain voltage-gated K+channels.”, Mol. Pharmacol., 50(5) (1996) , p. 1167-1177.
Gomez-Lagunas, “Two novel toxins from the venom of the scorpion Pandinus imperator show that the N-terminal amino acid sequence is important for their affinities towards Shaker B K+channels.”, J. Membr. Biol., 152 (1) (1996) , p. 49-56.
Sabatier, J.-M, “Synthesis and characterization of Leiurotoxin I analogs lacking one disulfide bridge: evidence that disulfide pairing 3-21 is not required for full toxin activity.”, Biochemistry, 35 (33) (1996) , p. 10641-10647.
Kharrat, R., “Maurotoxin, a four disulfide bridge toxin from Scorpion maurus venom: purification, structure and action on potassium channels.”, FEBS Lett., 406(3) (1997), p. 284-290.
M. Delepierre, “A Novel Potassium Channel Blocking Toxin from the Scorpion Pandinus imperator: A 1H NMR Analysis Using a Nano-NMR probe.”, Biochemistry, 36 (9) (1997), p. 2649-2658.
Kharrat, Ryadh, “Chemical synthesis and characterization of maurotoxin, a short scorpion toxin with four disulfide bridges that acts on K+channels.”, Eur. J. Biochem., 242 (3) (1996), p. 491-498.
Neito, Alejandro, “Noxiustoxin 2, a novel K+channel blocking peptide from the venom of the scorpion Centruroides noxius Hoffmann.”, Toxicon, 34 (8) (1996), p. 913-922.
Blanc, E. “Solution structure of P01, a natural scorpion peptide structurally analogous to scorpion toxins specific for apamin-sensitive potassium channel.”, Proteins, 24 (3) (1996), p. 359-369.

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