Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
2000-03-13
2001-12-18
Borin, Michael (Department: 1631)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C514S003100, C514S012200, C530S317000, C530S324000
Reexamination Certificate
active
06331522
ABSTRACT:
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 is 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
Arg Ser Val Cys Arg Gln Ile Lys Ile Cys Arg
Arg
Arg
Tyr Pro Arg Asn Thr Cys Lys Tyr Tyr Cys Gly Gly
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:
a) hemolymph is taken from the scorpion
Androctonus australis;
b) extraction is carried out by placing the
Androctonus australis
hemolymph obtained above in contact with an acid medium, with stirring, followed by centrifugation;
c) the supernatant is fractionated with separation by washing of the hydrophilic molecules and elution of the hydrophobic molecules with appropriate components on a separating column;
d) the extracts are purified;
e) the peptide is characterized.
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° 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×g at 4° 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
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
The measured masses for androctonine above are, respectively:
3076.65±0.24 Da
However, the masses calculated on the basis of sequencing data are, respectively:
3080.65±0.24 Da
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 showed that they were two peptides linked by a disulfide bridge. The sequences deduced were Arg Ser Val Cys Arg Gln Ile Lys plus Cys Thr Asn Arg Asn Pro Tyr and Ile Cys Arg Arg Arg Gly Gly Cys Tyr Tyr.
The connectivity of the disulfide bridges is thus established and cysteine 1 is linked to cysteine 4, cysteine 2 to cysteine 3. This result can be schematized thus: SEQ ID NO: 1
These good correlations confirm the proposed sequences.
The peptides according to the invention can also be obtained without difficulty according to a second process, by FMOC chemical synthesis (Atherton and Sheppard R. C. (1989), Solid Phase Peptide Synthesis (IRL, Oxford, UK)) followed by renaturing in 100 mM ammonium acetate solution at pH 8.5 for 24 hours with stirring at room temperature. The androctonine obtained has the same chromatographic properties as the native molecule and the connectivity of the disulfide bridges is identical to that of the natural molecule. The mass measured after renaturing (3076.61±0.67) is very similar to that of the native molecule. The synthetic molecule has the same antibacterial activity as the native molecule on the bacterium
Micrococcus liteus.
All of the antibacterial and hemolytic tests are carried out with the synthetic molecule.
1. Androctonine has no lytic effect on porcine or bovine red blood cells.
2. These molecules have antibacterial properties on Gram-negative and Gram-negative bacteria (cf. Table 1), phytopathogenic bacteria and phytopathogenic fungi.
REFERENCES:
Cociancich et al. (1993)Biochemical and Biophysical Research Communications197:17.
Ehret-Sabatier et al. (1996)Journal of Biological Chemistry271:29537.
Bulet Philippe
Hetru Charles
Hoffmann Jules
Sabatier Laurence
Aventis CropScience SA
Baker & Botts L.L.P.
Borin Michael
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