U.S. patent number 4,810,777 [Application Number 07/021,493] was granted by the patent office on 1989-03-07 for antimicrobial compounds.
This patent grant is currently assigned to The United States of America as represented by the Department of Health. Invention is credited to Michael A. Zasloff.
United States Patent |
4,810,777 |
Zasloff |
March 7, 1989 |
Antimicrobial compounds
Abstract
A new class of broad spectrum antibiotic polypeptides termed
"Magainin" have been described. These peptides have a molecular
weight of about 2500 or less, are highly water soluble,
non-cytolyic to animal cells including red-blood cells and are
amphiphilic.
Inventors: |
Zasloff; Michael A. (Rockville,
MD) |
Assignee: |
The United States of America as
represented by the Department of Health (Washington,
DC)
|
Family
ID: |
21804544 |
Appl.
No.: |
07/021,493 |
Filed: |
March 4, 1987 |
Current U.S.
Class: |
530/326; 930/10;
930/190; 930/DIG.811; 930/DIG.821 |
Current CPC
Class: |
C07K
14/46 (20130101); A61K 38/00 (20130101); Y10S
930/19 (20130101) |
Current International
Class: |
C07K
14/435 (20060101); C07K 14/46 (20060101); A61K
38/00 (20060101); C07K 007/08 (); C07K
007/10 () |
Field of
Search: |
;530/324,325,326,317,842,827,326,842 ;514/21,12,13,21 |
References Cited
[Referenced By]
U.S. Patent Documents
|
|
|
4195018 |
March 1980 |
Takita et al. |
4409210 |
October 1983 |
Kawaguchi et al. |
|
Other References
Williams, Chemical & Engineering News, Oct. 5, 1987. .
Goldsmith, Arch Dermatol, 123:1087-1088, 1987. .
Zasloff Proc. Natl. Acad. Sci. USA 84:5449-5453, 1987. .
Giovannini et al., Biochem. J., 243:113-120, 1987. .
Csordas et al., Toxicon &103-108, 1969. .
Eisenberg, Ann Rev. Biochem. 53:595-623, 1984. .
Kaiser et al., Proc. Natl. Acad. Sci. USA 80:1137-1143, 1983. .
Sures et al., Proc. Natl. Acad. Sco. USA 81:380-384, 1984. .
CRC Handbook of Chemotherapeutic Agents vol. I, pp. 178-185,
"Peptide Antibiotics". .
Kempf et al., J. Biol. Chem. 257:2469-2476, 1982. .
Steiner et al., Nature 292:246-248, 1981. .
Hoffmann et al., Embo, J. 2:711-714, 1983. .
Andreu et al., Biochem. 149:531-535, 1985. .
Merrifield et al., Biochem. 21:5020-5031, 1982. .
Gibson et al., J. Biol. Chem. 261:5341-5349, 1986. .
Richter et al., Peptides 6:17-21, 1985..
|
Primary Examiner: Phillips; Delbert R.
Assistant Examiner: Wessendorf; T. D.
Attorney, Agent or Firm: Holman & Stern, Chartered
Claims
What is claimed is:
1. An antibiotic having an amino acid sequence of:
(NH.sub.2)GIGKFLHSAGKFGKAFVGEIMKS(OH).
2. An antibiotic having an amino acid sequence of:
(NH.sub.2)GIGKFLHSAKKFGKAFVGEIMNS(OH).
3. An antibiotic having an amino acid sequence of:
(NH.sub.2)GIGKFLHSAKKFGKAFVGEIMN(OH).
4. An antibiotic having an amino acid sequence of:
(NH.sub.2)IGKFLHSAKKFGKAFVGEIMNS(OH).
5. An antibiotic having an amino acid sequence of:
(NH.sub.2)GKFLHSAKKFGKAFVGEIMNS(OH).
6. An antibiotic having an amino acid sequence of:
(NH.sub.2)KFLHSAKKFGKAFVGEIMNS(OH).
7. An antibiotic having an amino acid sequence of:
(NH.sub.2)FLHSAKKFGKAFVGEIMNS(OH).
8. An antibiotic having an amino acid sequence of:
(NH.sub.2)LHSAKKFGKAFVGEIMNS(OH).
Description
BACKGROUND OF THE INVENTION
The present invention relates generally to antimicrobial compounds.
More particularly, the present invention is related to a new class
of polypeptides, designated herein "Magainins", which have a broad
range of antimicrobial activity. It is noted that the "Magainin"
family of compounds having the properties as described herein have
never heretofore been discovered or known.
SUMMARY OF THE INVENTION
It is, therefore, an object of the present invention to provide a
novel family of compounds, termed "Magainins."
It is a further object of the present invention to provide a novel
class of antibiotics providing effective defense against infection
by such organisms as gram-positive and gram-negative bacteria,
fungi, protozoan species and the like.
Various other objects and advantages will become apparent from the
Detailed Description of the Invention.
DETAILED DESCRIPTION OF THE INVENTION
The above and various other objects and advantages of the present
invention are achieved by Magainins which are defined as a class of
polypeptides having a molecular weight of about 2500 or less, being
water soluble at a concentration of greater than 5 mg per ml at
neutral pH or in an aqueous solution of physiologic ionic strength,
being non-cytolytic to animal cells including red blood cells,
being amphiphilic and having a broad antibiotic spectrum at
physiologic ionic strength and pH. Such polypeptides include
Magainin I, Magainin II, Magainin III and analogs or derivatives
thereof.
Table I shows the amino acid sequence (single letter code) of
certain Magainin polypeptides of the present invention.
TABLE I
Primary Sequence of Magainin Polypeptides
Magainin I: (NH.sub.2)GIGKFLHSAGKFGKAFVGEIMKS(OH)
Magainin II: (NH.sub.2)GIGKFLHSAKKFGKAFVGEIMNS(OH)
Magainin III: (NH.sub.2)GIGKFLHSAKKFGKAFVGEIMN(OH)
Unless defined otherwise, all technical and scientific terms used
herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, the preferred methods and materials are now described.
All publications mentioned hereunder are incorporated herein by
reference.
Of course, having described the amino acid sequence of the
Magainins, these polypeptides can be routinely synthesized by
standard techniques well known in the art, such as by commercially
available peptide synthesizers and the like. Such standard
techniques of poplypeptide synthesis can be found described in such
publications as Merrifield, J. Chem. Soc. 85: 2149-2154, 1963;
Hunkapillar et al, Nature 310: 105-111, 1984.
It is noted, however, that the amino acid sequences of Magainins
listed in Table I show only essential portions required for
antibiotic activity. In otherwords, the polypeptides are not
limited to the sequences shown in Table I, but must have, at least
in part or in whole the amino acid sequence shown in Tables I and
II. Of course, various analogs and derivatives of Magainins can be
easily predicted, generated and/or prepared by such standard and
common methods as NMR (Nuclear magnetic resonance), computer
modeling and the like and all such analogs or derivatives which are
equivalent in structure and function to the Magainins as defined
herein are encompassed within the scope of the disclosure contained
herein. Some derivatives of Magainin II and their antimicrobial
activities, for instance, are shown in Table II. It is clear from
the results presented in Table II that the Magainins could be
manipulated, for example progressively made shorter at the amino
terminus, and still retain antimicrobial potency.
Table III shows the antimicrobial spectrum of the synthetic
Magainin peptides of the present invention.
TABLE II
__________________________________________________________________________
Antimicrobial Activity of Truncated Synthetic Derivatives of
Magainin II Zone of Inhibition on Lawn of E. coli Y1088 Generated
by 50 .mu.g of Derivative (Single Letter Code) the Peptide (mm)
__________________________________________________________________________
Magainin II(a) (NH.sub.2)IGKFLHSAKKFGKAFVGEIMNS(OH) 10 Magainin
II(b) (NH.sub.2)GKFLHSAKKFGKAFVGEIMNS(OH) 10 Magainin II(c)
(NH.sub.2)KFLHSAKKFGKAFVGEIMNS(OH) 10 Magainin II(d)
(NH.sub.2)FLHSAKKFGKAFVGEIMNS(OH) 2 Magainin II(e)
(NH.sub.2)LHSAKKFGKAFVGEIMNS(OH) 1 Magainin II(f)
(NH.sub.2)HSAKKFGKAFVGEIMNS(OH) 0
__________________________________________________________________________
TABLE III ______________________________________ Antimicrobial
Spectrum of Synthetic Magainin Peptides Organism(ATCC) Magainin I
Magainin II Magainin III ______________________________________
(Minimal Inhibitory Concentration (.mu.g/ml) Eschericia coli D31
1-20 1-10 1-10 Acinetobacter 7-35 1-7 7-35 caloaceticus(19606)
Shigella sonnei 7-35 1-7 7-35 (25931) Enterobacter sonnei 7-35 1-7
7-35 (23355) Eschericia coli 7-35 1-7 7-35 (25922) Streptococcus
7-35 1-7 7-35 pyogenes (19615) Shigella flexneri 35-70 7-35 7-35
(12022) Citrobacter freundii >100 7-35 7-35 (8090) Enterobacter
>100 7-35 >100 aerogenes (13048) Klebisiella >100 7-35
>100 pneumonia (13883) Staphyloccus >100 7-35 35-70
epidermidis (1228) Streptococcus >100 35-70 >100 faecalis
(19433) Pseudomonas >100 35-70 35-70 aeruginosa (27853)
Salmonella >100 35-70 35-70 typhimurium (14028) Staphylococcus
aureus >100 35-70 >100 (25923) Candida albicans >100 35-70
>100 (14053) Proteus vulgaris >100 >100 >100 (13315)
Serratia marcescens >100 >100 >100 (8100)
______________________________________
TABLE IV ______________________________________ Antiprotozoan
Activity of Synthetic Magainin Peptides Organism Magainin I
Magainin II Magainin III ______________________________________
(Concentration required for osmotic lysis (.mu.g/ml)) Paramecium
caudatum 5 5 5 Amoeba proteus 5 5 5 Euglena gracilis 5 5 5
______________________________________
Table III also lists the range of minimal inhibitory concentrations
for various organisms assayed in trypticase soy broth. These
determinations were made following standard techniques well known
in the art and described in such publications as, DIFCO Manual,
10th edition, 1984, pages 78-80.
Table IV lists the approximate concentration of Magainins which
induces physical lysis of several representative protozoan species.
These determinations were made by visual assessment of the treated
organisms by light microscopy. Of course, the optimal effective
antimicrobial concentration of a particular Magainin can be easily
determined in a routine manner without undue expermentation by
techniques well known to one of ordinary skill in the art.
It is clear from the results presented in Tables III and IV that
the Magainin polypeptides of the present invention have a broad
range of potent antibiotic activity against a plurality of
microorganisms including gram-positive and gram-negative bacteria,
fungi, protozoa and the like. Hence it is apparent that the
Magainin antibiotics of the present invention allow a method of
treating or controlling microbial infection caused by those
organisms which are sensitive to Magainin. Such treatment comprises
administering to a host or tissue susceptible to or afflicted with
microbial infection an antimicrobial amount of Magainin.
Clearly, due to their antibiotic properties, the Magainins of the
present invention can also be used as preservatives or sterilants
of materials susceptible to microbial contamination.
Of course, pharmaceutical compositions comprising Magainin
polypeptides of the present invention as an active ingredient in an
amount sufficient to produce antibiotic effect in suseptible host
or tissue and a pharmaceutically acceptable, non-toxic sterile
carrier can be easily prepared based on the data provided herein.
Such carriers could be fillers, non-toxic buffers, physiological
saline solution and the like. The preparation can be used topically
or systemically and may be in any suitable form such as liquid,
solid or semi-solid, which include injectable solutions, tablets,
ointments, lotions, pastes, capsules and the like. Of course, the
Magainins may also be administered in combination with other
adjuvants, protease inhibitors, or compatible drugs where such
combination is seen desirable or advantageous in controlling the
infection caused by harmful microorganisms including protozoa,
viruses and the like.
It is understood that the examples and embodiments described herein
are for illustrative purposes only and that various modifications
or changes in light thereof will be suggested to persons skilled in
the art and are to be included within the spirit and purview of
this application and the scope of the appended claims.
* * * * *