# Patent

## Title
Group of synthetic antimicrobial peptides

## Abstract

A group of new synthetic antimicrobial peptides are disclosed, which demonstrate stronger bactericidal activity than native antimicrobial peptides. The present synthetic antimicrobial peptides can be produced by solid-phase chemical synthesis or gene expression and be used to prepare the medicines for treating the diseases induced by bacteria, viruses and fungi, as well as the anticancer drugs.

## Background
This application is a national stage filing under 35 U.S.C. § 371 of PCT International application PCT/CN03/00522, filed Jul. 1, 2003, and claims priority under 35 U.S.C. §119(a) to China Patent Application number 02136766.6 filed Sep. 2, 2002.
FIELD OF THE INVENTION
This invention relates generally to antimicrobial peptides, the methods for preparing these peptides and methods for inhibiting the growth of microbes such as bacteria, yeast and viruses utilizing the peptides of the invention. The peptides are also useful for inhibiting tumor cell.
BACKGROUND OF THE INVENTION
The recent research data have revealed that, besides their immune systems, almost all living organisms have an additional defense system against invasion of pathogenic microorganisms. They produce antimicrobial peptides in their bodies and use them as a defense means against pathogenic microorganisms. Antimicrobial peptides are composed of 20-60 amino acids, and their molecular weights are about 2000-7000 D. Thus far, as many as about 200 antimicrobial peptides have been found from amphibians, insects, mammals, plants, microorganisms and fishes. The importance of peptides as physiologically active materials has been greatly recognized. These antimicrobial peptides are showing antimicrobial activity against a broad spectrum of microorganisms, including Gram-negative bacteria, Gram-positive bacteria, protozoa and fungi. Some of them are effective against both cancer cells and viruses. Most of the antimicrobial peptides kill target cells rapidly and specifically, and have unusually broad activity spectra.
By researching the primary structure and higher-order structure of the antimicrobial peptide, a lot of researcher find that when the antimicrobial peptide is in the hydrophobic environment of mimic membrane, its antimicrobial activity is related with the α-helix proportion. Another research result indicates that antimicrobial peptide kills the microorganisms by destructing the intact membrane of the microorganisms, which makes the membrane of the microorganisms leakage (Nakajima Y. et al., J. Biol. Chem, 262:1665-1669; Zasloff M. Nature, 2002, 415:389-395). So someone tries to search the antimicrobial peptides having stronger antimicrobial activity by increasing α-helix structure or heightening the positive charge amino acids proportion in the antimicrobial peptides (Broth W. B. et al., Antimicrobial Agents Chemotherapy, 2001, 45:1894-1895; Hong S. Y. et al., Peptides, 2001, 22:1669-1674).
DISCLOSURE OF THE INVENTION
The present invention provides a group of synthetic antimicrobial peptides. They were designed on the basis of analysis of the native antimicrobial peptides. The sequences of the peptides of the invention are provided as follows:








Arg Phe Arg Leu Val Arg Arg Ile Val Leu Ala (A1-A2-A3-A4)(A1′-A2′-A3′-A4′)



(SEQ ID NO: 1)


 


Arg  Phe  Arg  Leu  Val  Arg  Arg  Ile  Val  Leu  Ala


(A1-A2-A3-A4)(A1′-A2′-A3′-A4′)(A1′′-A2′′-A3′′-A4′′)


(SEQ ID NO: 2)


 


Arg Phe Lys Leu Val Arg Arg Ile Val Leu Ala (A1-A2-A3-A4)(A1′-A2′-A3′-A4′)


(SEQ ID NO: 3)


 


Arg  Phe  Lys  Leu  Val  Arg  Arg  Ile  Val  Leu  Ala


(A1-A2-A3-A4)(A1′-A2′-A3′-A4′)(A1′′-A2′′-A3′′-A4′′)


(SEQ ID NO: 4)


 


Arg Phe Lys Leu Val Lys Arg Ile Val Leu Ala (A1-A2-A3-A4)(A1′-A2′-A3′-A4′)


(SEQ ID NO: 5)


 


Arg  Phe  Lys  Leu  Val  Lys  Arg  Ile  Val  Leu  Ala


(A1-A2-A3-A4)(A1′-A2′-A3′-A4′)(A1′′-A2′′-A3′′-A4′′)


(SEQ ID NO: 6)


 


Arg Phe Lys Leu Val Lys Lys Ile Val Leu Ala (A1-A2-A3-A4)(A1′-A2′-A3′-A4′)


(SEQ ID NO: 7)


 


Arg  Phe  Lys  Leu  Val  Lys  Lys  Ile  Val  Leu  Ala


(A1-A2-A3-A4)(A1′-A2′-A3′-A4′)(A1′′-A2′′-A3′′-A4′′)


(SEQ ID NO: 8)


 


Lys Phe Lys Leu Val Lys Lys Ile Val Leu Ala (A1-A2-A3-A4)(A1′-A2′-A3′-A4′)


(SEQ ID NO: 9)


 


Lys  Phe  Lys  Leu  Val  Lys  Lys  Ile  Val  Leu  Ala


(A1-A2-A3-A4)(A1′-A2′-A3′-A4′)(A1′′-A2′′-A3′′-A4′′)


(SEQ ID NO: 10)


 


Arg Phe Arg Leu Phe Arg Arg Ile Leu Val Gly (A1-A2-A3-A4)(A1′-A2′-A3′-A4′)


(SEQ ID NO: 11)


 


Arg  Phe  Arg  Leu  Phe  Arg  Arg  Ile  Leu  Val  Gly


(A1-A2-A3-A4)(A1′-A2′-A3′-A4′)(A1′′-A2′′-A3′′-A4′′)


(SEQ ID NO: 12)


 


Arg Phe Lys Leu Phe Arg Arg Ile Leu Val Gly (A1-A2-A3-A4)(A1′-A2′-A3′-A4′)


(SEQ ID NO: 13)


 


Arg  Phe  Lys  Leu  Phe  Arg  Arg  Ile  Leu  Val  Gly


(A1-A2-A3-A4)(A1′-A2′-A3′-A4′)(A1′′-A2′′-A3′′-A4′′)


(SEQ ID NO: 14)


 


Arg Phe Lys Leu Phe Lys Arg Ile Leu Val Gly (A1-A2-A3-A4)(A1′-A2′-A3′-A4′)


(SEQ ID NO: 15)


 


Arg  Phe  Lys  Leu  Phe  Lys  Arg  Ile  Leu  Val  Gly


(A1-A2-A3-A4)(A1′-A2′-A3′-A4′)(A1′′-A2′′-A3′′-A4′′)


(SEQ ID NO: 16)


 


Arg Phe Lys Leu Phe Lys Lys Ile Leu Val Gly (A1-A2-A3-A4)(A1′-A2′-A3′-A4′)


(SEQ ID NO: 17)


 


Arg  Phe  Lys  Leu  Phe  Lys  Lys  Ile  Leu  Val Gly


(A1-A2-A3-A4)(A1′-A2′-A3′-A4′)(A1′′-A2′′-A3′′-A4′′)


(SEQ ID NO: 18)


 


Lys Phe Lys Leu Phe Lys Lys Ile Leu Val Gly (A1-A2-A3-A4)(A1′-A2′-A3′-A4′)


(SEQ ID NO: 19)


 


Lys  Phe  Lys  Leu  Phe  Lys  Lys  Ile  Leu  Val  Gly


(A1-A2-A3-A4)(A1′-A2′-A3′-A4′)(A1′′-A2′′-A3′′-A4′′)


(SEQ ID NO: 20)


 


Arg Phe Arg Gly Val Arg Arg Ile Leu Val Gly (A1-A2-A3-A4)(A1′-A2′-A3′-A4′)


(SEQ ID NO: 21)


 


Arg  Phe  Arg  Gly  Val  Arg  Arg  Ile  Leu  Val  Gly


(A1-A2-A3-A4)(A1′-A2′-A3′-A4′)(A1′′-A2′′-A3′′-A4′′)


(SEQ ID NO: 22)


 


Arg Phe Arg Gly Val Lys Arg Ile Leu Val Gly (A1-A2-A3-A4)(A1′-A2′-A3′-A4′)


(SEQ ID NO: 23)


 


Arg  Phe  Arg  Gly  Val  Lys  Arg  Ile  Leu  Val  Gly


(A1-A2-A3-A4)(A1′-A2′-A3′-A4′)(A1′′-A2′′-A3′′-A4′′)


(SEQ ID NO: 24)


 


Arg Phe Arg Gly Val Lys Lys Ile Leu Val Gly (A1-A2-A3-A4)(A1′-A2′-A3′-A4′)


(SEQ ID NO: 25)


 


Arg  Phe  Arg  Gly  Val  Lys  Lys  Ile  Leu  Val  Gly


(A1-A2-A3-A4)(A1′-A2′-A3′-A4′)(A1′′-A2′′-A3′′-A4′′)


(SEQ ID NO: 26)


 


Lys Phe Arg Gly Val Lys Lys Ile Leu Val Gly (A1-A2-A3-A4)(A1′-A2′-A3′-A4′)


(SEQ ID NO: 27)


 


Lye  Phe  Arg  Gly  Val  Lys  Lys  Ile  Leu  Val  Gly


(A1-A2-A3-A4)(A1′-A2′-A3′-A4′)(A1′′-A2′′-A3′′-A4′′)


(SEQ ID NO: 28)


 


Arg Trp Arg Ile Gly Arg Arg Ile Val Leu Ala (A1-A2-A3-A4)(A1′-A2′-A3′-A4′)


(SEQ ID NO: 29)


 


Arg  Trp  Arg  Ile  Gly  Arg  Arg  Ile  Val  Leu  Ala


(A1-A2-A3-A4)(A1′-A2′-A3′-A4′)(A1′′-A2′′-A3′′-A4′′)


(SEQ ID NO: 30)


 


Arg Trp Arg Ile Gly Lys Lys Ile Val Leu Ala (Al-A2-A3-A4)(A1′-A2′-A3′-A4′)


(SEQ ID NO: 31)


 


Arg  Trp  Arg  Ile  Gly  Lys  Lys  Ile  Val  Leu  Ala


(A1-A2-A3-A4)(A1′-A2′-A3′-A4′)(A1′′-A2′′A3′′-A4′′)


(SEQ ID NO: 32)


 


Lys Trp Arg Ile Gly Lys Lys Ile Val Leu Ala (A1-A2-A3-A4)(A1′-A2′-A3′-A4′)


(SEQ ID NO: 33)


 


Lys  Trp  Arg  Ile  Gly  Lys  Lys  Ile  Val  Leu  Ala


(A1-A2-A3-A4)(A1′-A2′-A3′-A4′)(A1′′-A2′′-A3′′-A4′′)


(SEQ ID NO: 34)


 


Lys Trp Lys Ile Gly Lys Lys Ile Val Leu Ala (A1-A2-A3-A4)(A1′-A2′-A3′-A4′)


(SEQ ID NO: 35)


 


Lys  Trp  Lys  Ile  Gly  Lys  Lys  Ile  Val  Leu  Ala


(A1-A2-A3-A4)(A′-A2′-A3′-A4′)(A1′′-A2′′-A3′′-A4′′)


(SEQ ID NO: 36)


 


Arg Trp Arg Leu Phe Arg Arg Ile Gly Ile Gly (A1-A2-A3-A4)(A1′-A2′-A3′-A4′)


(SEQ ID NO 37)


 


Arg  Trp  Arg  Leu  Phe  Arg  Arg  Ile  Gly  Ile  Gly


(A1-A2-A3-A4)(A1′-A2′-A3′-A4′)(A1′′-A2′′-A3′′-A4′′)


(SEQ ID NO: 38)


 


Arg Trp Arg Leu Phe Lys Arg Ile Gly Ile Gly (A1-A2-A3-A4)(A1′-A2′-A3′-A4′)


(SEQ ID NO: 39)


 


Arg  Trp  Arg  Leu  Phe  Lys  Arg  Ile  Gly  Ile  Gly


(A1-A2-A3-A4)(A1′-A2′-A3′-A4′)(A1′′-A2′′-A3′′-A4′′)


(SEQ ID NO: 40)


 


Arg Trp Arg Leu Phe Lys Lys Ile Gly Ile Gly (A1-A2-A3-A4)(A1′-A2′-A3′-A4′)


(SEQ ID NO: 41)


 


Arg  Trp  Arg  Leu  Phe  Lys  Lys  Ile  Gly  Ile  Gly


(A1-A2-A3-A4)(A1′-A2′-A3′-A4′)(A1′′-A2′′-A3′′-A4′′)


(SEQ ID NO: 42)


 


Arg Trp Lys Leu Phe Lys Lys Ile Gly Ile Gly (A1-A2-A3-A4)(A1′-A2′-A3′-A4′)


(SEQ ID NO: 43)


 


Arg  Trp  Lys  Leu  Phe  Lys  Lys  Ile  Gly  Ile  Gly


(A1-A2-A3-A4)(A1′-A2′-A3′-A4′)(A1′′-A2′′-A3′′-A4′′)


(SEQ ID NO: 44)


 


Lys Trp Lys Leu Phe Lys Lys Ile Gly Ile Gly (A1-A2-A3-A4)(A1′-A2′-A3′-A4′)


(SEQ ID NO: 45)


 


Lys  Trp  Lys  Leu  Phe  Lys  Lys  Ile  Gly  Ile  Gly


(A1-A2-A3-A4)(A1′-A2′-A3′-A4′)(A1′′-A2′′-A3′′-A4′′)


(SEQ ID NO: 46)


 


Arg Phe Arg Val Ile Arg Arg Ile Leu Val Gly (A1-A2-A3-A4)(A1′-A2′-A3′-A4′)


(SEQ ID NO: 47)


 


Arg  Phe  Arg  Val  Ile  Arg  Arg  Ile  Leu  Val  Gly


(A1-A2-A3-A4)(A1′-A2′-A3′-A4′)(A1′′-A2′′-A3′′-A4′′)


(SEQ ID NO: 48)


 


Arg Phe Arg Val Ile Arg Lys Ile Leu Val Gly (A1-A2-A3-A4)(A1′-A2′-A3′-A4′)


(SEQ ID NO: 49)


 


Arg  Phe  Arg  Val  Ile  Arg  Lys  Ile  Leu  Val  Gly


(A1-A2-A3-A4)(A1′-A2′-A3′-A4′)(A1′′-A2′′-A3′′-A4′′)


(SEQ ID NO: 50)


 


Arg Phe Arg Val Ile Lys Lys Ile Leu Val Gly (A1-A2-A3-A4)(A1′-A2′-A3′-A4′)


(SEQ ID NO: 51)


 


Arg  Phe  Arg  Val  Ile  Lys  Lys  Ile  Leu  Val  Gly


(A1-A2-A3-A4)(A1′-A2′-A3′-A4′)(A1′′-A2′′-A3′′-A4′′)


(SEQ ID NO: 52)


 


Lys Phe Lys Val Ile Lys Lys Ile Leu Val Gly (A1-A2-A3-A4)(A1′-A2′-A3′-A4′)


(SEQ ID NO: 53)


 


Lys  Phe  Lys  Val  Ile  Lys  Lys  Ile  Leu  Val  Gly


(A1-A2-A3-A4)(A1′-A2′-A3′-A4′)(A1′′-A2′′-A3′′-A4′′)


(SEQ ID NO: 54)






(A1-A2-A3-A4)(A1′-A2′-A3-A4′) or (A1-A2-A3-A4)(A1′-A2′-A3′-A4′)(A1′′-A2′′-A3′′-A4′′) is the core structure of the antimicrobial peptides. A1, A1′ or A1′′ is one selected from the group consisting of Lys and Arg. A2, A2′ or A2′′ is one selected from the group consisting of Gly, Ala, Val, Leu, Ile and Phe. A3, A3′ or A3′′ is one selected from the group consisting of Gly, Ala, Val, Leu, Ile and Phe. A4, A4′ or A4′′ is one selected from the group consisting of Lys and Arg. The N-terminal end of the core structure (A1-A2-A3-A4) is linked with a sequence having 11 amino acids. The first or third or sixth or seventh amino acid of the sequence is one selected from the group consisting of Lys and Arg. The second amino acid of the sequence is one selected from the group consisting of Trp and Phe. The fourth or fifth or eighth or ninth or tenth or eleventh amino acid of the sequence is one selected from the group consisting of Leu, Ile, Ala, Val and Gly.
The synthetic peptides provided by the invention comprise their functional analogs derived from amino acid substitution, cyclization, replacement of L-amino acid with D-amino acid, deletion or addition.
One method for producing the peptides provided by the invention is solid-phase peptide synthesis. The other method for producing the peptide is expressing a nucleic acid sequence encoding the peptide in a host cell transformed with a recombinant vector. Then the peptide is expressed in the host cell. The vector is one selected from the group consisting of plasmid and virus. The host cell can be a prokaryotic cell, including Escherichia coli and Bacillus subtilis. The host cell also can be a eukaryotic cell, including yeast cell, plant cell, insect cell and mammal cell. The peptide can be detected by mass-spectroscopy.
In order to research the relation between the structure and the function of the antimicrobial peptides provided by the invention, we used peptide synthesizer, which was purchased from ABI, to produce a group of peptides. Presented below are examples of the solid phase synthesis of these peptides. The sequences of GK-1, GK-2, GK-3 are provided as below.








GK-1:



Lys Trp Lys Leu Phe Lys Lys Ile Gly Ile Gly Arg


 


Leu Leu Lys Arg Gly Leu Arg Lys Leu Leu Lys


 


(SEQ ID NO: 55)


 


GK-2:


Lys Trp Lys Leu Phe Lys Lys Ile Gly Ile Gly Arg


 


Leu Leu Arg Arg Leu Leu Arg Arg Leu Leu Arg


 


(SEQ ID NO: 56)


 


GK-3:


Arg Trp Arg Leu Phe Lys Arg Ile Gly Ile Gly Arg


 


Leu Leu Lys Arg Gly Leu Arg


 


(SEQ ID NO: 57)






To assay the MIC of three peptides GK-1, GK-2 and GK-3 of the present invention, 96-well microtiter plate was used (In Yup Park et al; FEBS Letters; 437 (1998) 258-262), cecropin A1 and buforin II as control. The result indicated that the bactericidal activity of the peptides provided by the invention were stronger than the two native antimicrobial peptides.
Synthesis and detection the MIC of the functional analogs of the peptides were provided by the invention, which were deletion derivative and cyclization derivative. The result indicated that the bactericidal activity of the peptides provided by the invention were stronger than the two native antimicrobial peptides.
When the antimicrobial peptides kill the bacteria, they may act on the higher organism, include human cells. The reason is that the antimicrobial peptide kills the microorganisms by destructing the intact membrane of the microorganisms, which makes the leakage of the membrane of the microorganisms. So the hemolytic activity of the antimicrobial peptides is one of the standards of the toxicity of the antimicrobial peptides. If the peptides make the hematoglobin release, then the number of the OD490 can confirm the intensity of the toxicity of the antimicrobial peptides. This experiment in the invention detected the hemolytic activity on the human red blood cells of the antimicrobial peptides. The results indicated that the hemolytic rate was very small, to confirm that the hemolytic activity of antimicrobial peptide was very low.
The results of acute toxicity test in Kunming mouse of the antimicrobial peptides indicated that the peptides had no toxicity. The experiment of the efficacy of antimicrobial peptide against the Staphylococcus aureus infection in mice demonstrated that antimicrobial peptide provided by the invention was effective against S. aureus infection in an acute infection model in mice.
The invention provides a group of new synthetic antimicrobial peptides. They can be produced by the method of solid-phase peptide synthesis. The other method for producing the peptide is expressing a nucleic acid sequence encoding the peptide in a host cell transformed with a recombinant vector. Then the peptide is expressed in the host cell. The antimicrobial peptides have broad spectral bactericidal activity on Gram-positive bacteria, Gram-negative bacteria, fungi. The bactericidal activity of the peptides are stronger than the native antimicrobial peptides. The peptides have no toxicity to animal and plant cells. The peptides protected completely from the Staphylococcus aureus infection in mice when administered at a dosage of 0.25 mg/kg, while Vancomycin, which was a specific medicine for Staphylococcus aureus, was 100% effective only at the dosage of 4.5 mg/kg. The results demonstrated that antimicrobial peptide provided by the invention was effective against S. aureus infection in an acute infection model in mice. The antimicrobial peptides provided by the invention can be used to produce the drug to treat the diseases induced by Gram-positive bacteria, Gram-negative bacteria, and fungi.
The peptides of the present can be used to prepare a drug for treating the pathologic microbes, fungus and/or virus infectious diseases.
The peptides of the present invention can also be used to prepare an antitumor drug.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is mass-spectrogram for the antimicrobial peptide GK-2.



EXAMPLES
Example 1
Preparation and Purification of Antimicrobial Peptide
Prepare GK-1, GK-2 and GK-3. Prepare cecropin A1 and buforin II as control.
Sequence of cecropin A1 (see Morishima, I., etc, Comp. Biochem. Physiol., 1990, B 95 (3), 551-554):








Arg Trp Lys Leu Phe Lys Lys Ile Glu Lys Val Gly



 


Arg Asn Val Arg Asp Gly Leu Ile Lys Ala Gly Pro


 


Ala Ile Ala Val Ile Gly Gln Ala Lys Ser Leu


 


(SEQ ID NO: 58)






Sequence of buforin II (see Park, C. B., Biochem. Biophys. Res. Commun. 1996, 218 (1), 408-413):










Thr Arg Ser Ser Arg Ala Gly Leu Gln Phe Pro




 



Val Gly Arg Val His Arg Leu Leu Arg Lys



 



(SEQ ID NO: 59)






Presented below are examples of the solid phase synthesis of these peptides. The peptide synthesizer was purchased from ABI, USA. After cleaving with high concentration TFA, the peptide was purified by reverse-phase column. The purified peptide was then analyzed by MS. The procedures in detail are provided as follows:
1. Preparation of antimicrobial peptide (GK-2, 0.1 mmol)
All reagents are purchased from ABI, USA.
The sequence of peptide GK-2 is








N-Lys Trp Lys Leu Phe Lys Lys Ile Gly Ile Gly Arg



 


Leu Leu Arg Arg Leu Leu Arg Arg Leu Leu Arg-C.


 


(SEQ ID NO: 56)






The Pioneer Peptide Synthesis System performs solid-phase synthesis, in which peptide chains are assembled on a solid support from the C-terminus, one amino acid at a time, elongating the chain toward the N-terminus. Calculate the amount of support (Fmoc-Arg(Pbf)-PEG-PS, purchased from ABI, loading factor 0.19 mmole/g) needed for the synthesis. Weigh the support and transfer it to the column. Removal of the Fmoc (9-fluorenyl-methyloxycarbonyl) protecting group from the terminal amine or the resin is accomplished by treating the resin with 20% solution of piperidine in N,N-Dimethylformamide (DMF). The required volume of the 20% solution of piperidine in DMF is calculated automatically on the scale of the reaction being run. The resin is then washed with DMF. The 9-fluorenyl-methyloxycarbonyl (Fmoc) protected amino acid was dissolved in O-(7-Azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium exafluorophosphate (HATU)/diisopropylethylamine (DIPEA). The solution was recycled through the column for 30 minutes. The resin is then washed with DMF. Repeat the steps from removal to coupling till the end of synthesis (see Pioneer Peptide Synthesiser User's manual for details).
The resulting peptides were cleaved as follows:
After reaction, the resin was removed, to which was added B type cleavage cocktail (88% TFA, 5% phenol, 5% water, 2% TIPS), continue to reaction for about 2 hours at room temperature. Filtering, and to the filtrate was added 10-fold volume of pre-cold absolute ether. The precipitate was collected by centrifugation at 4000 rpm for 10 minutes, and dried at room temperature.
2. Purification of antimicrobial peptide
Weigh an amount of dried peptide, resolved in 0.1% TFA. The peptide was purified by reverse-phase column (elution: 80% acetonitrile/0.1% TFA). Collect the elution fraction.
3. Identification of antimicrobial peptide
As shown in FIG. 1, the molecular weight of antimicrobial peptide GK-2 is analyzed and calculated by MS:

734.8×4=2939.2, 2939.2−4=2935.2  (1)

979.1×3=2937.3, 2937.3−3=2934.3  (2)

1468×2=2936, 2936−2=2934  (3)

The calculated MW of GK-2 is 2934. The theory value calculated from the peptide sequence is 2932.74. The peptide prepared proved to be the designed GK-2 antimicrobial peptide. The certified antimicrobial peptide is stored for further use.
Antimicrobial peptide GK-1, GK-3 and natural antimicrobial peptides cecropin A1 and buforin II were prepared similarly to the preparation of GK-2 antimicrobial peptide.
Example 2
Expression of Antimicrobial Peptide GK-1 Gene in E. coli 
The bacterial expression vector pGEX-4T1 is used for bacterial expression in this example (Amersham Pharmcia Biotech). Antimicrobial peptide gene GK-1 was designed and synthesized and cloned into pGEX-4T1, then the expression vector containing GK-1 was transformed into E. coli JM109, GST-GK-1 fusion protein was expressed by IPTG inducing, GK-1 was obtained after cleaving by thrombin.
ATP, IPTG, T4 polynucleotide kinase, T4DNA ligase, Klenow Fragment, Restriction endonucleases are products of BIOLAB except for special indication. The agarose gel DNA extraction kit is product of shanghai sangon, primers for PCR amplification were synthesized by shanghai sangon. Thrombin cleavage kit from sigma.
With respect to the methods of DNA separation, purification, PCR reaction enzyme cleavage, plasmid transformation, fragment collection, link reaction etc. are referred to Molecular Cloning: A Laboratory Manual (edited by Joe Sambrook, David Russell, Cold Spring Harbor Lab (CSHL) Press, 2001). E. coli JM109 was cultured in LB liquid or solid medium.
We use E. coli bias coden design GK-1 gene sequence, the sequence as following: For cloning the mature protein, the 5′ primer containing the BamHI (GGATCC) restriction site, The 3′ primer containing the stop coden (TAG), the sequence contain 78 bp.
The sequence of GK-1 gene was synthesized by DNA synthesizer. A DNA segment was amplified by PCR reaction. A pair of primers were P1: 5′-CCTAGGTTTACCT-3(SEQ ID NO: 60) P2: 3′-CCGCCTGCTGAA-5′(SEQ ID NO: 61). PCR reaction was as following: 94° C. 30 seconds; 45° C., 45 seconds; 72° C., 30 seconds; 30 cycles. The PCR product was cleaved by BamHI after it reacted with Klenow fragment. The fragment collected by agarose gel DNA extraction kit (procedure see the kit). The recycled fragment linked with pGEX-4T1 vector which was cleaved by BamHI and SmaI, the recombinant plasmid transformed E. coli JM109, then transformants identified by SmaI. The fusion protein GST-GK-1 was induced to be expressed by IPTG. The fusion protein was purified by GST affinity column, and GK-1 antimicrobial peptide was obtained after it cleaved by thrombin. For the procedure, please see the kit.
GK-1 polypeptide sequence:








Lys Trp Lys Leu Phe Lys Lys Ile Gly Ile Gly Arg



 


Leu Leu Lys Arg Gly Leu Arg Lys Leu Leu Lys


 


(SEQ ID NO: 55)






GK-1 gene sequence:








GGATCCAAATGGAAACTGTTTAAAAAAATTGGCATTGGCCGCCTGCTGA



 


ACGCGGCCTGCGCAAGCTGCTGAAATAG


 


(SEQ ID NO: 62)






Example 3
Expression of Antimicrobial Peptide GK-1 Gene in Yeast
ATP, IPTG, T4 polynucleotide kinase, T4DNA ligase, Klenow Fragment, Restriction Endonucleases are products of BIOLAB except for special indication. The agarose gel DNA extraction kit is product of shanghai sangon, primers for PCR amplification were synthesized by shanghai sangon. Thrombin cleavage kit is available from sigma.
The DNA sequence of GK-1 gene which was cleaved by BamHI linked with the DNA sequence of GST, then the linked gene was cloned into pBluescriptSKII (from Stratagene company, USA). Recombinant plasmid was transformed into E. coli DH5α (from CMCC, Wuhan, P.R.C). The plasmid was identified by DNA sequencing. The plasmid was cleaved by EcoRI and XhoI, then linked to yeast expression vector pPIC9. pPIC9 is used for yeast expression in this example (from invitrogen). The expression vector containing GK-1 was then transformed into KM71 (from Invitrogen company, USA), GST-GK-1 fusion protein was induced to be expressed by methyl alcohol, and GK-1 was obtained after cleavage by thrombin.
With respect to the methods of DNA separation, purification, PCR reaction, enzyme cleavage, plasmid transformation, fragment collection, ligase reaction etc. are referred to Molecular Cloning: A Laboratory Manual (edited by Joe Sambrook, David Russell, Cold Spring Harbor Lab (CSHL) Press, 2001). KM71 was cultured in BMGY liquid or solid medium. When GST-GK-1 fusion protein was expressed, BMMY medium was used. BMMY medium supplied methyl alcohol to 1% every 24 hours.
We use yeast bias coden design GK-1 and GST gene sequence, the sequence as follows: For cloning the mature protein, the 5′ primer containing the BamHI (GGATCC) restriction site, The 3′ primer containing the stop coden (TAG) and EcoRI (GAATCC) restriction site, the sequence contain 84 bp. Additional a XhoI (CTCGAG) restriction site at 5′-terminal of the GST was supplied.
Preparation of the sequence of GK-1 gene: the sequence of GK-1 gene was synthesized by DNA synthesizer. Amplify a DNA segment by PCR reaction. A pair of primer were P3: 5′CCTAGGTTTACCT3′ (SEQ ID NO: 60) and P4: 5′AAGTCGTCCGCC 3′ (SEQ ID NO: 61). PCR reaction is performed as follows: 94° C., 30 seconds; 45° C., 45 seconds; 72° C., 30 seconds; 35 cycles.
Preparation of the sequence of GST gene: designed a pair of primers, the sequences were as follows:








5′-CTCGAGATGTCCCCTATACTAGGTT-3′;
(SEQ ID NO: 63)


 


5′-CAGTGCTACGCCGGCGAG-3′.
(SEQ ID NO: 64)






Amplify the GST gene segment of pGEX-4T1 vector by PCR reaction with P5 and P6. PCR reaction is performed as follows: 94° C., 30 seconds; 45° C., 45 seconds; 72° C., 30 seconds; 30 cycles.
Link the PCR products to plasmid: GK-1 PCR products is cleaved by BamHI/EcoRI after reacting with Klenow fragment. The fragment was collected by agarose gel DNA extraction kit (see the kit manual for details). The recycled fragment was linked with pBluescriptSKII vector which was cleaved by XhoI/EcoRI and GST PCR products which were cleaved by BamHI/XhoI. E. coli DH5α was transformed by the recombinant plasmid. The transformants were identified by antibiotic resistance test, restriction endonucleases etc, and then was identified further by DNA sequencing and cleaved by XhoI/EcoRI. The expression plasmid pPIC9-gst-gk1 was constructed by linking the plasmid to pPIC9 vector which was cleaved by XhoI/EcoRI. The recombinant plasmid transformed E. coli DH5α. Scan transformants by Ampicillin resistance test. Prepare the KM71 competent cell (Clare J J, et al., Gene, 1991, 105:205-212). Then transported (1.5 KV, 22.5 uF) the recombinant plasmid pPIC9-gst-gk1 cleaved with SacI into KM71 cell. Spreaded the electroporated yeast onto YPD plate, scanned for the fusion protein positive clone after being cultured two days at 30° C.
The fusion protein GST-GK-1 was induced to be expressed by methanol in transformant. The fusion protein was purified by GST affinity column and obtained GK-1 antimicrobial peptide after cleavage with thrombin. See the kit manual for details.
GST-GK-1 fusion protein sequence:
Leu Glu Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile Lys Gly Leu Val Gln Pro Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu Glu His Leu Tyr Glu Arg Asp Glu Gly Asp Lys Trp Arg Asn Lys Lys Phe Glu Leu Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Ile Asp Gly Asp Val Lys Leu The Gln Ser Met Ala Ile Ile Arg Tyr Ile Ala Asp Lys His Asn Met Leu Gly Gly Cys Pro Lys Glu Arg Ala Glu Ile Ser Met Leu Glu Gly Ala Val Leu Asp Ile Arg Tyr Gly Val Ser Arg Ile Ala Tyr Ser Lys Asp Phe Glu The Leu Lys Val Asp Phe Leu Ser Lys Leu Pro Glu Met Leu Lys Met Phe Glu Asp Arg Leu Cys His Lys The Tyr Leu Asn Gly Asp His Val The His Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp Val Val Leu Tyr Met Asp Pro Met Cys Leu Asp Ala Phe Pro Lys Leu Val Cys Phe Lys Lys Arg (SEQ ID NO: 65)
Ile Glu Ala Ile Pro Gln Ile Asp Lys Tyr Leu Lys Ser Ser Lys Tyr Ile Ala Trp Pro Leu Gln Gly Trp Gln Ala The Phe Gly Gly Gly Asp His Pro Pro Lys Ser Asp Leu Val Pro Arg Gly Ser Lys Trp Lys Leu Phe Lys Lys Ile Gly Ile Gly Arg Leu Leu Lys Arg Gly Leu Arg Lys Leu Leu Lys (SEQ ID NO: 66)
GST-GK-1 gene sequence:
TTAGAAATGTCTCCTATTTTAGGTTATTGGAAAATTAAAGGTTTAGTTCAACC TACTCGTTTATTATTAGAATATTTAGAAGAAAAATATGAAGAACATTTATATGA ACGTGATGAAGGTGATAAATGGCGTAATAAAAAATTTGAATTAGGTTTAGAAT TTCCTAATTTACCTTATTATATTGATGGTGATGTTAAATTAACTCAATCTATGG CTATTATTCGTTATATTGCTGATAAACATAATATGTTAGGTGGTTGTCCTAAA GAACGTGCTGAAATTTCTATGTTAGAAGGTGCTGTTTTAGATATTCGTTATGG TGTTTCTCGTATTGCTTATTCTAAAGATTTTGAAACTTTAAAAGTTGATTTTTT ATCTAAATTACCTGAAATGTTAAAAATGTTTGAAGATCGTTTATGTCATAAAAC TTATTTAAATGGTGATCATGTTACTCATCCTGATTTTATGTTATATGATGCTTT AGATGTTGTTTTATATATGGATCCTATGTGTTTAGATGCTTTTCCTAAATTAGT TTGTTTTAAAAAACGTATTGAAGCTATTCCTCAAATTGATAAATATTTAAAATC TTCTAAATATATTGCTTGGCCTTTACAAGGTTGGCAAGCTACTTTTGGTGGTG GTGATCATCCTCCTAAATCTGATTTAGTTCCTCGTGGTTCTAAATGGAAATTA TTTAAAAAAATTGGTATTGGTCGTTTATTAAAACGTGGTTTACGTAAATTATTA AAATGAGAATTT (SEQ ID NO: 67)
Example 4
MIC Assay of Several Invention Peptides
All strains used in the following examples were purchased from NICPBP.
To assay the MIC of three peptides GK-1, GK-2 and GK-3 of the present invention, 96-well microtiter plate was used, cecropin A1 and buforin II as control.
The minimum inhibitory concentrations (MIC) of the invention peptides were determined using methods described below:
The strain was recovered, inoculated into sloped medium, and grown overnight at 37° C. Typical clone selected were grown overnight at 37° C. in LB culture, diluted in the same medium to give concentrations of about 104-105 CFU/ml. The broth dilutions were set up in a 96-well microtiter plate by putting 100 μl of LB-S in every well. Added diluted peptide to every well (10 ul per well), cultured overnight at 37° C. The next day, the plates were scored for growth in the wells, and the MIC determined (In Yup Park et al.; FEBS Letters; 437 (1998) 258-262). Results were summarized in table 1.
When the ratio of the growth concentration for the bacteria with antimicrobial peptides to that for the bacteria without antimicrobial peptides is greater than 90%, the concentration of antimicrobial peptides is the minimum inhibitory concentration (The minimum inhibitory concentration (MIC) is defined as the minimal concentration when the growth of bacteria is significantly inhibited).







TABLE 1







Compare of MIC against different bacteria of five antimicrobial


peptides









MIC of some antimicrobial peptides



(ug/ml)












Strain
cecropin A1
buforin
<−1
GK-2
<−3
















G+
Staphylococcus
16
4
0.4
0.2
0.5



aureaus



CMCC26003



Bacillus subtilis
12
6
4
4
5



DB430



Bacillus pumilus
50
6
0.5
1
0.8



CMCC63202



Micrococcus
50
8
1.0
0.8
1.2



lysoleikticus



S1.634



Micrococcus lutea
30
8
2
4
3



CMCC28001


G−
Escherichia coli
20
16
1
0.5
1.6



ATCC8099



Klebsiella
16
20
2
0.8
2



pneumoniae



CMCC46117



B Subacute
12
14
4
1
6



sclerosing



panencephalitis



CMCC50094



Pseudomonas
18
20
10
12
1.8



aeruginosa



CMCC10104


Fungi
Candida albicans
50
30
8
10
11



ATCC10231



Saccharomyces
50
20
14
12
12



cerevisiae



ATCC9736









Lower MIC value means higher antimicrobial activity.
Example 5
MIC of Functional Analogs of Invention Peptides Derived from Cyclization, Deletion
Design and synthesis functional analogs of invention peptides: GK-19 (deletion derivative) and GK-20 (cyclization derivative). Synthesis is performed on Pioneer Peptide Synthesiser. See Pioneer Peptide Synthesiser User's manual for details. After purification by reverse-phase column (see Example 1), the analogs were then subjected to MIC test (see Example 4). Results were summarized in Table 2. Sequences of GK-19 (deletion derivative) and GK-20 (cyclization derivative):
GK-19:
Arg Phe Lys Leu Phe Lys Lys Ile Pro Arg Leu Leu Arg Arg Gly Leu Arg Lys Val Leu Lys (SEQ ID NO: 68)
GK-20:
Lys Trp Lys Leu Phe Lys Lys Ile Gly Ile Gly Arg Leu Leu Lys Arg Gly Leu Arg Lys Leu Leu Lys (SEQ ID NO: 69)







TABLE 2







MIC of functional analogs of invention peptides GK-19 and GK-20









MIC(ug/ml)









Strain
GK-19
GK-20













G+
Staphylococcus aureaus CMCC26003
1.0
0.8



Bacillus subtilis DB430
2
2



Bacillus pumilus CMCC63202
10
6



Micrococcus lysoleikticus S1.634
4
2



Micrococcus lutea CMCC28001
10
4


G−
Escherichia coli ATCC8099
2
8



Klebsiella pneumoniae CMCC46117
2
10



B Subacute sclerosing panencephalitis
1
8



CMCC50094



Pseudomonas aeruginosa
4
10



CMCC10104


Fungi
Candida albicans ATCC10231
16
18



Saccharomyces cerevisiae ATCC9736
12
20









Lower MIC value means higher antimicrobial activity.
Example 6
In-Vitro Hemolytic Activity
This experiment was to detect the hemolytic activity of the antimicrobial peptides. The references were Cecropin A1 and buforin II, which were solid-phase peptide synthesized by our company. Blood sample was normal human blood.
The test step was shown below:
Human red blood cells was washed by PBS (PBS:35 Mm phosphate buffer/0.15 m NaCl, pH7.0). Suck 100 ul 8% red blood cells suspension to 96-well plate, add 100 ul antimicrobial peptide (including cecropin A1, buforin II, GK-1, GK-2, GK-3) to each well, then incubated at 37° C., after 1 hour, 1500 rpm centrifuged for 5 minutes. Suck 100 ul 4% red blood cells suspension to new 96-well plate, detect the hematoglobin releasing under 414 nm by microplate reader. The negative control was PBS, the positive control was TritonX-100. The result was summarized in table 3:







TABLE 3







Results of hemolytic activity of five antimicrobial peptides








Concentration



of antimicrobial


peptide
Hemolytic rate(%)












(ug/ml)
cecropin A1
buforinll
GK-1
GK-2
GK-3















12.5
0
0
0
0
0


25
0
0
0
0
0


50
0
0
0
0
0


100
1.2
0
0.5
0.2
0.6


200
3
0.5
0.8
1.0
1.1


500
10
1.7
1.5
2
1.9









The number of hemolytic rate in table 3 was smaller, the hemolytic activity of antimicrobial peptide was lower.
Example 7
Acute Toxicity Test in Kunming Mouse
The test was to detect the toxicity to animal of antimicrobial peptides including GK-1, GK-2, GK-3, provided by the invention. The references were Cecropin A1 and buforin II, which were solid-phase peptide synthesized by our company.
60 Kunming mouse, half was female, half was male, weight was 33.5±0.25 g. The dosage of antimicrobial peptide was 1 mg/kg, intramuscular injecting one time per day, consecutive 7 days. We observed the reaction of the mouse under the maximum dosage. The result of the test demonstrated that the mice were normal and no abnormal reaction after 7 days intramuscular injection. It can be concluded that the antimicrobial peptides provided by the invention have no toxicity.
Example 8
Comparison of the Efficacy of Antimicrobial Peptide and Vancomycin Against the Staphylococcus aureus Infection in Mice
The infection model was Staphylococcus aureus infection in the Kunming mouse.
The test step was shown below:
S. aureus CMCC26003 was cultured overnight, with moderate agitation, in Veal Infusion broth (Difco) and diluted in broth containing 5% hog gastric mucin (Difco). Male Kunming mice weighing approximately 20 grams were infected intraperitoneally with 106-107 viable cells. There were 3 mice in each treatment group. Antimicrobial peptide GK-1 was administered intravenously (in 0.1 ml 5% dextrose for injection), within 10 minutes of infection. Vancomycin was administered subcutaneously.







TABLE 4







Efficacy of antimicrobial peptide and vancomycin against Staphylococcus


aureus infection in mice










Inhibitory rate (%)










Dose
GK-1
Vancomycin


(mg/kg)
i.v.
s.c.












0
0



0.125
20


0.25
100


0.5
100
0


1.0
100
40


2.0

80


4.0

100


8.0

100









As shown in Table 4, GK-1 protected 100% of the infected mice when administered at a dosage of 0.25 mg/kg intravenously. Vancomycin was 100% effective only at the dosage of 4.5 mg/kg. All of the untreated mice died in less than 24 hours.
This example demonstrated that antimicrobial peptide provided by the invention was effective against S. aureus infection in an acute infection model in mice using a highly virulent challenge dose of bacteria.
Example 9
Determination of the Inhibitory Activity Against Tumor Cells
To determine the inhibitory activity of the peptides against tumor cells, a MTT colorimetric assay was performed. Fifty percent inhibitory concentrations of the antimicrobial peptides against tumor cells and normal fibroblasts were determined. Tumor cell K562 (human chronic myeloid leukemia cell), Bcap-37 (human breast cancer cell), QGY-7703 (human hepatocellular carcinoma cell), LOVO colon cancer cell, and mouse NIH-3T3 fibroblast were selected. These cells were provided by the College of Life Sciences, Fudan University. Cells were grown in RPMI1-1640, containing 10% inactivated calf serum. Cells were transferred into the 96-well plate at 2*104 cells/well, and 150 μl was added in each well. After the 96-well plate were incubated overnight at 37, in 5% carbon dioxide, 20 μl diluted peptide solution was added to each well, then incubated for 3 days, 20 μl MTT solution was added to each well, and incubated at 37° C. for 4 hours, 40 μl, 002M HCl solution containing 20% SDS was added to each well to solve the purple crystal, and incubated overnight at 37° C. The absorbance at 570 nm was determined.







TABLE 5







Result of the inhibitory activity against tumor cells (IC50)











GK-1 (ug/ml)
GK-2 (ug/ml)
GK-3 (ug/ml)














K562
45
56
13


Bcap-37
52
47
25


QGY-7703
38
44
21


LOVO
35
32
31


mouse
>100
>100
>100


NIH-3T3


fibroblast









The results show that the antimicrobial peptides of GK-1, GK-2 and GK-3 all have antitumor activities, and the effect of GK-3 is the best.

## Claims

1. A synthetic antimicrobial peptide comprising an amino acid sequence as set forth in SEQ ID NO:46.

2. The synthetic antimicrobial peptide of claim 1, wherein the amino acid sequence is Lys Trp Lys Leu Phe Lys Lys Ile Gly Ile Gly Arg Leu Leu Lys Arg Gly Leu Arg Lys Leu Leu Lys (SEQ ID NO: 55).

3. The synthetic antimicrobial peptide of claim 2, wherein the Lysine at position 1 is covalently bound to the Lysine at position 23 to form a cyclization derivative.

4. The synthetic antimicrobial peptide of claim 1, wherein the amino acid sequence is Lys Trp Lys Leu Phe Lys Lys Ile Gly Ile Gly Arg Leu Leu Arg Arg Leu Leu Arg Arg Leu Leu Arg (SEQ ID NO: 56.

5. A method for producing a synthetic antimicrobial peptide, comprising:
performing solid-phase chemical synthesis to produce the peptide of claim 1.

6. A method for treating a subject having an infectious disease induced by bacteria and/or fungi, comprising:
administering to the subject the synthetic antimicrobial peptide of claim 1 in an amount effective for treating the infectious disease.