Antibacterial and antifungal peptide

Details Antibacterial and antifungal peptide Antibacterial and antifungal peptide

1998-11-16

2000-10-03

Borin, Michael

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Peptide containing doai

514 9, 514 12, 530317, 530324, A61K 3812, A61K 3816, C07K 114, C07K 412, C07K 512

Patent

active

061273368

DESCRIPTION:

BRIEF SUMMARY
The present invention relates to a novel protein-rich peptide with antibacterial and antifungal properties, and to compositions, which can be used in agriculture and in human or animal therapy, containing this peptide as active material. The invention also relates to processes for treating plants using these compositions, as well as to processes for preparing this peptide.
It has been known for a long time that insects have effective resistance to bacteria. This defence is largely based on the rapid synthesis of several families of peptides. This defence is due to the rapid synthesis of several families of peptides with a broad spectrum of activity. This synthesis is induced by a septic wound or by the injection of a low dose of bacteria. Among the antibacterial peptides induced, those best characterized are the insect cecropines and defensines. Several other antibacterial peptides have been partially characterized.
Apart from the insect class, little is known about other arthropods. Scorpions have existed far longer than insects in terms of philogeny.
A peptide has now been isolated, from an induction in the scorpion Androctonus australis, this peptide showing remarkable characteristics as well as antibacterial and antifungal properties.
More particularly, a first aspect of the invention relates to the peptide of formula I SEQ ID NO: 1: ##STR1##
Hereinbelow, the molecule of formula I will be referred to as androctonine. This relatively small molecule contains 4 cysteine residues engaged in two intramolecular bridges.
Another aspect of the invention relates to a first process for obtaining and isolating the above peptide, in which the following are successively carried out: obtained above in contact with an acid medium, with stirring, followed by centrifugation; hydrophilic molecules and elution of the hydrophobic molecules with appropriate components on a separating column;
The hemolymph is taken by incision of the cuticle. It is collected in a tube containing a protease inhibitor. After centrifugation to remove the blood cells, the plasma is stored at -30.degree. C.
In a preferred manner, in the second step (extraction), the Androctonus australis hemolymph is placed in contact with an acidic medium consisting of an acidic solution of an acid (of pH 2). The solution can be a solution of an inorganic or organic acid such as, for example, trifluoroacetic acid. The extract obtained is then centrifuged under cold conditions at a speed of 30,000.times.g at 4.degree. C. for 25 min.
Preferably, in the third step (fractionation), the extract is placed on a reverse-phase cartridge in order to carry out a solid-phase extraction. The water-soluble molecules are washed out with a dilute acid solution and the hydrophobic molecules are eluted with an appropriate eluent. Good results are obtained with trifluoroacetic acid for the washing and an eluent containing increasing amounts of acetonitrile in dilute acid solution.
Preferably, the fourth step (purification) is carried out with a suitable eluent which can be identical to or different from the one in the preceding stage.
Preferably, in the final step (characterization), the nature of the peptide is analyzed by the method of sequencing by Edman degradation (Acta Chemica Scandinavia 10 (1956) pp. 761-768). According to this method, the following structures are obtained SEQ ID NO:2: ##STR2##
No signal was detectable in positions 4, 10, 16 and 20 (Edman degradation). The presence of cysteines in these positions was shown by mass spectrometry, the structure thus obtained being as follows SEQ ID NO: 1: ##STR3##
The measured masses for androctonine above are, respectively:
However, the masses calculated on the basis of sequencing data are, respectively:
The difference in mass corresponds to the formation of two intramolecular disulfide bridges.
In order to establish the connectivity of the disulfide bridges, the molecule was cleaved with an enzyme, Lys-C endoproteinase, which breaks the peptide chain after lysine. The peptides obtained were isolated and mass spectrometry s

REFERENCES:
Cociancich et al. (1993) Biochemical and Biophysical Research Communications 194:17.
Ehret-Sabatier et al. (1996) Journal of Biological Chemistry 271:29537.

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