AU754894B2 - Novel peptides having biological activity - Google Patents

Description

WO 99/37664 PCT/KR99/00036 NOVEL PEPTIDES HAVING BIOLOGICAL ACTIVITY TECHNICAL FIELD The peptides of the present invention have stronger antimicrobial activities than conventional peptides and have the activity at high salt concentrations.

BACKGROUND ART The present invention relates to biologically active peptides. Every animal on earth possesses biophylaxis systems to defend or protect itself from the infection by virus or bacteria. One of such systems is a non-specific immunity using antimicrobial peptides.

Antimicrobial peptides are considered as a new type of drug due to the following outstanding properties. Firstly, antimicrobial peptides show stronger antimicrobial activities than conventional antibiotics against a broad spectrum of microorganisms. Secondly, antimicrobial peptides have a high industrial applicability which is beneficial to the human body since the antimicrobial peptides show antimicrobial activity against foreign pathogens without destroying the host cells. Thirdly, there is a smaller chance to develop microbial resistance since the antimicrobial peptides show their activity by a mechanism that is totally different from that of the conventional antibiotics, which have serious problems of developing resistance. Studies on antimicrobial peptides began by isolating cecropin from an insect which has an under-developed immune system. After the first finding, magainin, bombinin WO 99/37664 PCT/KR99/00036 from amphibians, defensins from mammals were isolated. The studies on antimicrobial peptides are actively performed, and to date, about 2,000 antimicrobial peptides have been identified and reported from species ranging from microorganisms to human.

However, there are several barriers to develop the above mentioned antimicrobial peptides as drugs. Firstly, the conventional antimicrobial peptides act at relatively high concentrations. For instance, in case of magainin, an antimicrobial peptide isolated from epidermis of an amphibian, the active concentration is 50-200 g/ml (Zasloff M. (1987) Proc. Natl. Acad. Sci. USA, 84: 5449-5453) even though it is effective against Gram-positive and Gramnegative bacteria and fungi. This concentration range is quite high considering that the conventional antibiotics act against a specific microorganism in the range 0.1-1 gg/ml. Secondly, the antimicrobial activity of the antimicrobial peptides is sensitive to salt concentration. In case of cystic fibrosis that invades the human lung, for instance, the antimicrobial peptide was not effective due to an abnormal increase of the salt concentrations at the site of invasion (Goldman, M. J. et al. (1997) Cell, 88: 553-560).

Antimicrobial peptides isolated from Korean toad were reported by the present inventors in Biochemical and Biophysical Research Communications 218, 408- 413 (1996). These antimicrobial peptides known as buforin I and buforin II showed strong antimicrobial activities against a broad-spectrum of microorganisms including Gram-positive and Gram-negative bacteria and fungi.

Buforin I and buforin II also have antimicrobial activities at a concentration of 1-4 gg/ml, which is stronger than that of conventional antimicrobial peptides.

WO 99/37664 PCT/KR99/00036 These antimicrobial peptides, however, are also sensitive to salt concentrations. Therefore, it has been desired to develop antimicrobial peptides that have an enhanced antimicrobial activities and are not sensitive to salt concentrations to have antimicrobial activities in vivo.

DISCLOSURE OF THE INVENTION It is an object of the present invention to provide novel biologically active peptides.

Another object of the present invention is to provide peptides that have antimicrobial activities against a wide variety of microorganisms with stronger antimicrobial activities.

It is another object of the present invention to provide peptides that are insensitive to salt concentrations in potentiating the antimicrobial activity.

A further object of the present invention is to provide a secondary structure of peptides that are not sensitive to salt concentration in potentiating the antimicrobial activity.

Another object of the present invention is to provide a precursor peptide that could prepare biologically active peptides.

Still another object of the present invention is to provide cDNA that can code for biologically active peptides.

WO 99/37664 PCT/KR99/00036 BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a secondary structure of buforin II determined by NMR spectrometry in the presence of 50 trifluoroethanol as a structure-forming agent.

Figure 2 is a graph showing the minimal inhibitory concentration as a function of a salt concentration.

DETAILED DESCRIPTION OF THE INVENTION The peptide of the present invention comprises a peptide having an amphiphilic a-helix structure.

Also the peptide of the present invention comprises a peptide that has an altered secondary structure of buforin II (Biochemical and Biophysical Research Communications 218, 408-413 (1996)).

The present inventors, have shown that the secondary structure of buforin II comprises a random coil (1-4 residue), extended helix (5-10 residue) and normal a-helix (11-21 residue) structures, starting from the N-terminus.

In the structure of buforin II, the peptide sequence having normal a-helix structure (11-21 residue), PVGRVHRLLRK has a strong antimicrobial activity. The present inventors have identified that a peptide, especially a WO 99/37664 PCT/KR99/00036 peptide with at least the sequence forming the random coil structure (1-4 residue) is removed, has a very strong antimicrobial activity. Therefore, the group of peptides according to the present invention consists of peptides that contain an a-helix structure of buforin II, especially those having the PVGRVHRLLRK sequence. These a-helix forming sequences, for instance the sequence PVGRVHRLLRK, can additionally have amino acids at the C- or Nterminus preferably amino acids forming extended helix or normal helix at the N-terminus or an amidated peptide at the C-terminus.

Another group of peptides according to the present invention comprises a peptide having a repeat unit of [RLLR], (n is an integer between 1 and 6), (RLLR being the specific repeat pattern found in the amino acid sequence of buforin II) and preferably peptides where The peptides can include additional amino acids at the C- or N-terminus, and the amino acid sequence at the N-terminus can include those that do not form a random coil, preferably those forming an extended helix. The group of amino acid sequence, for instance, includes RAGLQFPVG[RLLR]i,

RAGLQFPVG[RLLR]

2

RAGLQFPVG[RLLR]

3 [RLLR}b, [RLLR] [RLLR], and etc.

The peptides according to the present invention can be synthesized by wellknown techniques in the field, for instance, by using an automatic peptide synthesizer or by using a genetic engineering technique. For instance, the peptide can be produced by constructing fusion gene composed of fusion partner and the peptide genes, transforming it into host microorganism, WO 99/37664 PCT[KR99/00036 expressing the fusion protein in the host, cleaving the fusion protein with proteolytic enzyme or chemical agent, and purifying the antimicrobial peptide.

For this purpose, for instance, a DNA sequence can be inserted between fusion partner and peptide genes to introduce a sequence encoding processing site which can be cleaved by proteases such as factor Xa and enterokinase, or by chemical agents such as CNBr and hydroxylamine.

To introduce DNA sequence encoding CNBr cleavage site, for instance, fusion partner and antimicrobial peptide genes can be in-frame fused by ligating the fusion partner gene digested at its 3 -end with a restriction enzyme whose recognition sequence contains Met codon (ATG) in their recognition sequence, such as Aflll, Bsml, BspHI, BspLU11I, Ncol, Ndel, Nsil, Ppul01, Sphl, Styl, or their isoschizomers, and the peptide gene digested at its 5 -end with a restriction enzyme whose cleavage site is compatible with the cleavage site of fusion partner. For another example, to introduce DNA sequence encoding hydroxylamine cleavage site, a DNA sequence encoding Asn-Gly.,can be introduced between fusion partner and peptide genes. For instance, fusion partner and peptide genes can be in-frame fused by ligating fusion partner gene digested at its 3 -end with a restriction enzyme or its isoschizomer whose recognition sequence contains Asn codon in its recognition sequence, and the peptide gene digested at its 5 -end with a restriction enzyme whose cleavage sequence containing Gly codon can be in-frame fused to the 3 -end of fusion partner by compatible cohesive or blunt end.

The gene structure in the present invention can be introduced into host cell by WO 99/37664 PCT/KR99/00036 cloning it into an expression vector such as plasmid, virus, or other conventional vehicle in which the gene can be inserted or incorporated.

The peptides according to the present invention contain C-terminal amidated forms.

The peptides according to the present invention show strong antimicrobial activities against a wide variety of microorganisms including Gram-negative and Gram-positive bacteria, fungi and protozoa.

The peptides according to the present invention can be administered with other biologically active pharmaceutical preparations such as biologically active chemicals, other peptide, and etc.

The amino acids in the present invention are abbreviated according to the IUPAC IUB nomenclature as below.

amino acid Alanine Arginine Asparagine Aspartic acid Cysteine Glutamic acid Glutamine Glycine abbreviation

A

R

N

E

C

D

Q

G

WO 99/37664 PCT/KR99/00036 Histidine

H

Isoleucine I Leucine

L

Lysine

K

Methionine M Phenylalanine F Proline P Serine S Threonine T Tryptophane W Tyrosine Y Valine V The invention will be further illustrated by the following examples. It will be apparent to those having conventional knowledge in the field that these examples are given only to explain the present invention more clearly, but the invention is not limited to the examples given.

EXAMPLE 1. Preparation of peptides According to the sequence given in Table 1, a variety of peptides were synthesized by using an automatic peptide synthesizer and were purified by using a C18 reverse phase high performance liquid chromatography (Waters Associates, USA).

WO 99/37664 PTK9/03 PCT/KR99/00036 Table 1. Amino acid sequense of buforin 11 and its derivatives Peptide Amino acid sequence SEQ ID NO. 1 RAGLQFPVGRVHRLLRK SEQ ID NO. 2 AGLQFPVGRVHRLLRK SEQ ID NO. 3 GLQFPVGRVHRLLRK SEQ ID NO. 4 LQFPVGRVHRLLRK SEQ ID NO. 5 QFPVGRVHRLLRK SEQ ID NO. 6 FPVGRVHRLLRK SEQ ID NO. 7 PVGRVHRLLRK SEQ ID NO. 8 TRSSRAGLQFPVGRVHR SEQ ID NO. 9 RAGLQFPVGRVHRLLR SEQ ID NO. 10 RAGLQFPVGRVHRLL SEQ ID NO. 11 RAGLQFPVGRVHRL SEQ ID NO. 12 RKGLQKLVGRVHRLLRK SEQ ID NO. 13 RLLRRLLRRLLRRLLRRLLR SEQ ID NO. 14 RVHRLLRRVHRLLRRVHRLLR SEQ ID NO. 15 RAGLQFPVGRLLRRLLRRLLR SEQ ID NO. 16 RAGLQFPVGRVHRLLRK-NH 2 SEQ ID NO. 17 RAGLQFPVGRLLR SEQ ID NO: 18 RAGLQFPVGRLLRRLLR SEQ ID NO. 19 RLLRRLLRRLLR SEQ ID NO. 20 RLLRRLLRRLLRRLLR WO 99/37664 PCT/KR99/00036 EXAMPLE 2. Estimation of antimicrobial activity By using the peptides as in Example 1, the minimal inhibitory concentration of the peptides were determined against a variety of microorganisms. Bacteria and fungi were incubated overnight in Mller-Hinton and Saboraud media, respectively, at 37 and 30 oC, respectively, and were inoculated in media for 2 hours to a midlogarithmic phase. After diluting the bacteria and fungi to 10 per 1 ml, they were inoculated into a 96-well plate containing serially diluted peptides and incubated for additional 18 hours. The minimal inhibitory concentration was determined at a concentration that inhibits the growth of the microorganisms by measuring the absorbance. The results are shown in Table 2.

Table 2. Antimicrobial Activity of the peptides Minimal Inhibitory Concentrations (pg/ml) Microorganisms Buforin Seq Seq Seq Seq Seq Seq Seq Seq Seq Seq Seq II No.1 No.2 No.3 No.4 No.5 No.6 No.7 No.8 No.9 No.10 No.11 Gram-positive Bacillus subtilis 2 1 4 4 8 18 32 25 >200 12 50 100 Staphylococcus aureus 4 2 8 8 18 62 32 50 >200 50 200 200 Streptococcus mutans 2 1 4 4 8 36 32 25 >200 25 50 100 Streptococcus pneumoniae 4 2 4 4 18 18 32 50 >200 25 100 100 Gram-negative Escherichia coli 4 2 2 2 8 36 32 25 >200 12 50 200 Serratia sp. 1 2 2 2 4 18 16 25 >200 12 25 100 Psudomonasputida 4 2 2 2 8 36 32 50 >200 25 50 200 Salmonella typhimurium 2 1 4 4 18 18 64 50 >200 25 50 200 Fungi Candida albicans I I 8 8 36 62 32 50 >200 50 >200 >200 Cryptococcus neoformans I I 8 8 62 62 >100 50 >200 50 100 200 Saccharomyces cerevisiae 1 1 8 8 36 62 >100 50 >200 100 >200 >200 Table 2. continued Minimal Inhibitory Concentrations (p/mi) Microorganisms Buforin Seq fSeq [Seq Seq Seq Marg- BuQI5-13) Buf(5-13) (RLLR), (RLLR), 11 No. 12 [No. 13 No. 14 No. 15 No.16 ainin 2 lRLLRJ 11111-11 2 Gram-positive_____ Bacillus sublilis 2 18 2 6 1 1 50 32 4 16 2 Staphylococcus aureus 4 18 1 50 1 1 50 64 8 16 1 Streptococcus mutans 2 36 2 25 0.5 0.5 100 16 16 16 2 Streptococcus pneumoniae 4 18 2 00 I I 50 328 16 2 Gram-negative_____ Escherichia ccli 4 18 2 100 1 2 100 32 8 32 2 Serralia sp. 1 4 I 3 1 I 25 32 8 16 1 Psudomonas putida 4 36 2 50 1 2 50 32 16 16 2 Salmonella tvp/imttriuni 2 18 2 5TI 2 50 16 4 16 2 Fungi Candida albicans I 16 f 8 1>2001 25 32 4 32 0 '.0 '.0 cJJ -4 0% 0% Cryptocaccus neoformans Saccharomyces cerevisiae WO 99/37664 PCT/KR99/00036 The peptide, RAGLQFPVG(RLLR) 3 that has a fused amino acid sequence forming extended <alpha>-helix at the N-terminus of (RLLR) 3 showed an especially potent antimicrobial activity, and the peptide (RLLR) 4 and (RLLR) 5 which has 4 and 5 repetitions of RLLR,respectively, also showed strong antimicrobial activities.

The peptide that had a deletion of the sequence forming a random coil structure from buforin II also showed a potent antimicrobial activity, and the peptide that had an amidation at the N-terminus showed a more potent antimicrobial activity.

EXAMPLE 3. Estimation of antimicrobial activity as a function of salt concentrations The minimal inhibitory concentration of the peptides was measured as a function of salt concentrations to determine whether the antimicrobial activity is dependent on the salt concentrations. The method of estimating the minimal inhibitory concentration was identical as in Example 2 except that the concentration of NaCI was changed. The result is shown in Figure 2. The antimicrobial activity of the peptides according to the present invention did not vary as a function of salt concentration whereas that of buforin and magainin changed sensitively as a function of salt concentrations.

Editorial Note 19846/99 The following Sequence Listing pages 1 to 8 are part of the Description.

The Claims pages follow starting from page number 14.

WO 99/37664 PTK9/03 PCT[KR99/00036 SEQUENCE LISTING <110> SAL4YANG GENEX CORPORATION; KIM, Sun-Chang <120> BIOLOGICALLY ACTIVE PEPTIDES <130> PA/SYG99049 <150> KR 1998-1797 <151> 1998-01-22 <160> <170> KOPATIN <210> 1 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> PEPTIDE <400> 1 Arg Ala Gly Leu Gin Phe Pro Val Gly Arg Val His Arg Leu Leu Arg 1 5 10 1s Lys <210> <211> <212> <213> <220> <223> <400> Ala Gly 1 2 16

PRT

Artificial Sequence PE PT IDE 2 Leu GIn Phe Pro Val Gly Arg Val His Arg Leu Leu Arg Lys 5 10 -1I WO 99/37664 PTK9/03 PCT/KR99/00036 SEQUENCE LISTING <210> <211> <212> <213> <220> <223> <4 00> Gly Leu

I

3

PRT

Artificial Sequence

PEPTIDE

3 Gin Phe Pro Val Gly Arg Val His Arg Leu Leu Arg Lys 5 10 <210> <2.11> <212> <213> <220> <223> <400> Leu Gin 1 <210> <211> <212> <213> <220> <223> <400> Gin Phe 1 4 14

PRT

Artificial Sequence

PEPTIDE

4 Phe Pro Val Gly Arg Val His Arg Leu Leu Arg Lys 5 13

PRT

Artificial Sequence

PEPTIDE

Pro Val Gly Arg Val His Arg Leu Leu Arg Lys -2 WO 99/37664 PTK9/03 PCT[KR99/00036 <210> <211> <213> <220> <223> <400> Phe Pro 1 <210> <211> <2 12 <213> <220> <223> <400> Pro Val 1 <2110 <211> <212> <213> <220> <223> <400> Thr Arg 1 SEQUENCE LISTING 6 12

PRT

Artificial Sequence

PEPTIDE

6 Val Gly Arg Val His Arg Leu Leu Arg Lys 5

PRT

Artificial Sequence

PEPTIDE

Gly Arg Val His Arg Leu Leu Arg Lys 5 17

PRT

Artificial Sequence

PEPTIDE

8 Ser Ser Arg Ala Gly Leu Gln Phe Pro Val Gly Arg Val His 5 10 3- WO 99/37664 PTK9/03 PCTIKR99/00036 SEQUENCE LISTING <210> <211> <212> <213> <220> <223> <400> Arg Ala 1 9 16

PRT

Artificial Sequence

PEPTIDE

9 Gly Leu Gin Phe Pro Val. Gly Arg Val His Arg Leu Leu Arg 5 10 <210>' <211>' <212>' <213>' <220>' <223>' <400> Arg Ala 1 <210>' <211> <212> <213> <220> <223>

PRT

Artificial Sequence

PEPTIDE

Gly Leu Gin Phe Pro Val Gly Arg Val His Arg Leu Leu 5 10 11 14

PRT

Artificial Sequence PE PT IDE <400> 11 Arg Ala Gly Leu Gin Phe Pro Val Gly Arg Val His Arg Leu 1 5 -4 WO 99/37664 WO 9937664PCT/KR99/00036 <210> <211> <212> <213> <220> <223> <400> Arg Lys 1 SEQUENCE LISTING 12 17

PRT

Artificial Sequence

PEPTIDE

12 Gly Leu Gin Lys Leu Val Gly Arg Val His Arg Leu Leu Arg 5 10 <210> <211> <212> <213> <220> <223> <400> Arg Leu 1 13

PRT

Artificial Sequence

PEPTIDE

13 Leu Arg Arg Leu Leu Arg Arg Leu Leu Arg Arg Leu Len Arg 5 10 Arg Leu Leu Arg <210> 14 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> PEPTIDE <400> 14 5 WO 99/37664 PTK9/03 PCT[KR99/00036 SEQUENCE LISTING Arg Val His Arg Leu Leu Arg Arg Val His Arg Leu Leu Arg Arg Val His Arg Leu Leu Arg <210> <211> 21 <212> PRT <213> Artificial Sequence <220> <223> PEPTIDE <400> Arg Ala Gly Leu Gin Phe Pro Val Gly Arg Leu Leu Arg Arg Leu Leu 10 Arg Arg Leu Leu Arg <210> 16 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> PEPTIDE, AMIDATION <400> Arg Ala 1 Gly Leu GIn Phe Pro 5 Val Gly Arg Val His Arg Leu Leu Arg 10 Arg Lys <210> 17 <211> 13 -6 WO 99/37664 PTK9/03 PCT[KR99/00036 SEQUENCE LISTING <212> PP.T <213> Artificial Sequence <220> <223> <400> Arg Ala 1 <210> <211> <212> <213> <220> <223> <400> Arg Ala 1 PE PTIDE 17 Gly Leu Gln Phe Pro Val Gly Arg Leu Leu Arg 5 18 17

PRT

Artificial Sequence PE PT IDE 18 Gly Leu Gin Phe Pro Val Gly Arg Leu Leu Arg Arg Leu Leu 5 10 Arg <210> 19 <211> <212> <213> <220> <223> <400> Arg Leu 1 12

PRT

Artificial Sequence

PEPTIDE

19 Leu Arg Arg Leu Leu Arg Arg Leu Leu Arg 5 <210> -7 WO 99/37664 WO 9937664PCT[KR99/00036 SEQUENCE

LISTING

<211> 16 <212> PRT <213> Artificial Sequence <220> <223> PEPTIDE <400> Arg Leu Leu Arg Arg Leu Leu Arg Arg Leu Leu Arg Arg Leu Leu Arg 1 5 10 8-

Claims (16)

1. A purified peptide consisting of sequence [RLLR]n, wherein n is an integer between 2 and 6.

2. A purified peptide comprising sequence [RLLR]n, wherein n is an integer between 2 and 6, and a Gly residue is fused to the N-terminus of the peptide.

3. A purified peptide comprising sequence [RLLR]n, wherein n is an integer between 2 and 6, and the C-terminus of the peptide is amidated.

4. A purified peptide consisting of sequence RAGLQFPVG[RLLR]n, wherein n is an integer between 1 and 6. oo* o 15

5. A purified peptide comprising sequence RAGLQFPVG[RLLR]n, wherein n is an integer between 1 and 6, and a Gly residue is fused to the N-terminus of the peptide.

6. A purified peptide comprising sequence RAGLQFPVG[RLLR]n, wherein n 20 is an integer between 1 and 6, and the C-terminus of the peptide is amidated.

7. A purified peptide comprising sequence RAGLQFPVGRVHRLLRK or PVGRVHRLLRK, wherein a Gly residue is fused to the N-terminus of the Speptide.

8. A purified peptide comprising sequence RAGLQFPVGRVHRLLRK or PVGRVHRLLRK, wherein the C-terminus of the peptide is amidated.

9. A purified peptide comprising RKGLQKLVGRVHRLLRK, wherein the peptide forms an alpha-helix. A purified peptide comprising RKGLQKLVGRVHRLLRK, wherein the RA", peptide forms an alpha-helix and a Gly residue is fused to the N-terminus of the peptide. peptide.

W:\Fiona\Niger\621327\62L327_SPECIdoc

11. A purified peptide comprising RKGLQKLVGRVHRLLRK, wherein the peptide forms an alpha-helix and the C-terminus of the peptide is amidated.

12. A purified peptide consisting of the sequence RAGLQFPVGRVHRLLRK or PVGRVHRLLRK, wherein the peptide has antimicrobial activity.

13. A method for inhibiting the growth of bacteria or fungi with an effective amount of peptide consisting of sequence [RLLR]n, wherein n is an integer between 1 and 6.

14. A method for inhibiting the growth of bacteria or fungi with an effective amount of peptide consisting of sequence RAGLQFPVG[RLLR]n, wherein n is an integer between 1 and 6.

15. A method according to claim 13 or 14, wherein a Gly residue is fused to the N-terminus of the peptide. *o

16. A method according to claim 13 or 14, wherein the C-terminus of the peptide is amidated. Dated: 9 September 2002 PHILLIPS ORMO E &FITZPATRICK Attorneys for dG1A4tS t.t SSAMYANG GENEX CORPORATION and KOREA ADVANCED INSTITUTE OF SCIENCE AND TECHNOLOGY