Note: Descriptions are shown in the official language in which they were submitted.
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FINE TUNED PROTEGRINS
1. CROSS-REFERENCE TO RELATED APPLICATIONS
This invention was made with funding from NIH Grant No.
~ 5 A122839. The U.S. Government has certain rights in this
invention.
This application is a continuation-in-part of U.S.
Serial No. 08/960,921 filed August 1, 1996, which is a
continuation-in-part of U.S. Serial No. 08/649,811 filed
17 May 1996, which is a continuation-in-part of U.S. Serial
No. 08/562,346 filed 22 November 1995, which is a
continuation-in-part of U.S. Serial No. 08/499,523 filed
7 July 1995, which is a continuation-in-part of U.S. Serial
No. 08/451,832 filed 26 May 1995 which claims priority from
PCT/US94/08305 (WO 95/03325) and which is a continuation-in-
part of U.S. Serial No. 08/243,879 filed 17 May 1994, which
is a continuation-in-part of U.S. Serial No. 08/182,483 filed
13 January 1994, which is a continuation-in-part of U.S.
Serial No. 08/095,769 filed 26 July l9g3, which is a
continuation-in-part of U.S. Serial No. 08/093,926 filed
20 July 1993. Benefit is claimed under 35 U.S.C. 120 with
respect to U.S. Serial Nos. 08/960,921, 08/649,811 and
08/562,346. The contents of these applications are
incorporated herein by reference in their entireties.
2. FIE1D OE THE lNv~NllON
The invention relates to the field of antimicrobial
peptides. In particular, the invention concerns short
peptides, designated "protegrins," that have a wide range of
antimicrobial activities.
3. BACKGROUND OF THE lN v~llON
one of the defense mechanisms against infection by both
~n;~ls and plants is the production of peptides that have
~ 35 antimicrobial and antiviral activity. Various classes of
these peptides have been isolated from tissues of both plants
~ and ~n; ~1 fi. PCT application WO 95/03325 (published
2 February 1995) contains a review of the literature on this
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subject. Such peptides include tachyplesins, which are 17-18
amino acid peptides cont~;ning four invariant cysteines, the
defensins, ~-defensins, and insect defensins, which are
somewhat longer peptides characterized by six invariant
cysteines and antifungal and anti~acterial peptides and
proteins which have been found in plants.
The parent applications provide a new class of
antimicrobial peptides, designated "protegrins,"
representative members of which have been isolated from
porcine leukocytes. The protegrin peptides, which are
generally amphiphilic in nature, exhibit broad spectrum
antimicrobial activity. Thus, these peptides are useful as
antibacterial, anti-fungal and antiviral agents in both
plants and ~n; ~
The isolation of some of the protegrin peptides of the
invention was reported by the present applicants in a paper
by Kokryakov, V.N. et al., 1993, FEBS Lett 337:231-236 (July
issue). A later publication of this group described the
presence of a new protegrin whose sequence, and that of its
precursor, was deduced from its isolated cDNA clone. Zhao,
C. et al., 1994, FEBS Lett 346:285-288. An additional paper
disclosing cationic peptides from porcine neutrophils was
published by Mirgorodskaya, O.A. et al., 1993, FEBS ~ett
330:339-342. Storici, P. et al., 1993, Biochem Bio~hYs Res
Comm 196:1363-1367, report the recovery of a DNA sequence
which encodes a pig leukocyte antimicrobial peptide with a
cathelin-like prosequence. The peptide is reported to be one
of the protegrins disclosed hereinbelow. Additional
publications related to protegrins are Harwig, S.S.L., et
al., 1995, J Pe~tide Sci 3:207; Zhao, C., et al., 1995, FEBS
376:130-134; Zhao, C. et al., 1995, FEBS Lett 368:197-
202; Miyakawa, Y. et al., 1996, Infect Immun 64:926-932;
Yasin, B. et al., 1996, Infect Immun 64:709-713; Qu, X-~ et
al., 1996, Infect Immun 64:1240-1245; Aumelas, A. et al.,
1996, Eur J. Biochem 237:575-583; ~angoni, M.E. et al., 1996,
FEBS Lett 383:93-98; Steinberg et al., 1996, "Protegrins:
Fast Acting Bacterial Peptides," presented at 8th Intl.
SYm~OSiUm on Staphylococci and Sta~hylococcal Infections, Aix
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les Bains, France, June 23-26, 1996; Steinberg et al., 1996,
"Broad Spectrum Antimicrobial Activity of Protegrin
Peptides," presented at 36th Interscience Conference on
Antimicrobial Aaents and Chemotherapy, New Orleans, LA,
September 15-18, 1996; Kung et al., lg96r "Protegrin Protects
Mice From Systemic Infection By Antibiotic-Resistant
Pathogens," presented at 36th Interscience Conference on
Antimicrobial Aqents and Chemotherapy, New Orleans, LA,
September 15-18, 1996; and Steinberg et al., 1996, "In Vitro
Efficacy of Protegrins Against ~elicobacter Pylori,"
presented at 36th Interscience Conference on Antimicrobial
Aqents and Chemotherapy, New Orleans, LA, September 15-18,
1996.
The protegrins have also been found to bind to
endotoxins -- i.e., the lipopolysaccharide (LPS) compositions
derived from Gram-negative bacteria which are believed
responsible for Gram-negative sepsis. The protegrins are
also effective in inhibiting the growth of organisms that are
associated with sexually transmitted diseases such as
C~lamydia trachomatis and Neisseria gonorr~oeae.
The present invention is directed to a new set of
protegrins which offer properties of being rapid acting
microbicides having a broad spectrum of activity with a low
likelihood of resistance. In addition, the class of
protegrins of the present invention offers an additional
opportunity to adjust the spectrum of activity with respect
to the type of microbe or virus most effectively inhibited
and with respect to the conditions under which this
inhibition occurs. The protegrins in this case differ from
those of the parent applications either by deletion of at
least one of the four N-terminal amino acids, or of certain
other designated residues and/or by replacement of certain
amino acids with those of other classes.
4. SUMMARY OF THE INVENTION
In one aspect, the present invention is directed to
antimicrobial peptides contA; n ing about 10-30 amino acid
residues characterized by a "core" structure having two main
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elements: a reverse-turn bracketed by two strands that form
an anti-parallel ~-sheet. Generally, the ~-sheet region of
the molecule is amphiphilic, one surface being net
hydrophobic in character, the other being net hydrophilic in
character. The peptides contain at least one basic amino
acid residue in the reverse-turn region, and have a net
charge of at least +1 at physiological pH. The antimicrobial
peptides may optionally be acylated at the N-terminus and/or
amidated or esterified at the C-terminus, and may contain
zero, one or two disulfide bridges.
In one illustrative embodiment, the invention provides
antimicrobial peptides comprising about 10-30 amino acid
residues and cont~;n;ng the amino acid sequence:
(I) X1~X2~X3~X4~X5~C6~X7~C8~Xg~xlo~xll~xl2~cl3~xl4~cls~xl5~xl7~xls
or a pharamceutically acceptable salt or N-terminal
acylated or C-te~ ; n~ amidated or esterified form thereof,
wherein:
each of C8 and C13 is independently present or not
present, and if present each is independently a cysteine-
like, basic, small, polar/large or hydrophobic;
each of C6 and C1s is independently a cysteine-like,
basic, small, polar/large or hydrophobic amino acid;
each of X1-X5 is independently present or not present,
and if present each is independently a basic, hydrophobic,
polar/large, or small amino acid;
each of X7 and X14 is independently a hydrophobic or a
small amino acid;
each of Xg and X12 is independently present or not
present;
Xg-Xl2 taken together are capable of effecting a reverse-
turn when contained in the amino acid sequence of formula (I)
and at least one of X9-Xl2 must be a basic amino acid;
each of X16-X18 is independently present or not present,
and if present each is independently a basic, hydrophobic,
polar/large or small amino acid;
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and wherein at least about 15% up to about 50% of the
amino acids comprising the antimicrobial peptide are basic
amino acids such that the antimicrobial peptide has a net
charge of at least +1 at physiological pH.
~ 5 The peptides of the invention exhibit broad spectrum
antimicrobial activity, being biocidal against a wide range
of microbial targets, including Gram-positive bacteria, Gram-
negative bacteria, yeast, fungi and protozoa. Accordingly,
the peptides can be used as antimicrobial agents in a wide
variety of applications. For example, the peptides can be
used to preserve or disinfect a variety of materials,
including medical equipment, foodstuffs, cosmetics, contact
lens solutions, medicaments or other nutrient-contAining
materials. The peptides are also useful for the prophylaxis
or treatment of microbial infections or diseases related
thereto in both plants and Ani~
In another aspect, the invention is directed to
recombinant materials useful for the production of certain of
the peptides of the invention as well as plants or animals
modified to contain expression systems for the production of
these peptides.
In another aspect, the invention is directed to
pharmaceutical compositions and to compositions for
application to plants contA;ning the peptides of the
invention as active ingredients or compositions which contain
expression systems for production of the peptides or for }n
situ expression of the nucleotide sequence encoding these
peptides.
In still another aspect, the invention is directed to
methods of preparing the invention peptides synthetically, to
antibodies specific for these peptides, and to the use of the
peptides as preservatives.
In yet another aspect, the present invention is directed
to methods of using the above-described peptides, or~ 35 compositions thereof, to inhibit microbial growth. The
method generally involves contacting a microbe with an
antimicrobially effective amount of one or more of the
peptides or compositions of the invention. In a preferred
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embodiment, a bacteria is contacted with a bactericidally
effective amount of peptide or composition.
In a final aspect, the present invention is directed to
methods of using the above-described peptides, or
compositions thereof, to prevent or treat micro~ial
in~ections or diseases related thereto in both plants and
animals. The method generally involves Al' ; n; stering to a
plant or ~n;~l an effective amount of one or more of the
peptides or compositions of the invention. Such diseases or
infections include eye infections such as con~unctivitis and
keratitis, corneal ulcers, stomach ulcers associated with H.
pylori, sexually transmitted diseases (STDs~, and Gram-
negative sepsis. Clinically relevant infections that can be
treated or prevented ~y the peptides of the invention include
systemic infections caused by multi-drug resistant pathogens
such as vancomycin-resistant Enterococcus faecium,
methicillin-resi~tant Staphylococcus aureus and penicillin-
resistant Streptococcus pneumoniae.
5. BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows the nucleotide se~uence and the deduced
amino acid sequence of the genomic DNA encoding the precursor
protein ~or the antimicrobiai compounds PG-1, PG3, and PG-5.
Figure 2 shows the organization of the protegrin genomic
DNA.
Figures 3a-3c show the antimicrobial activity of
synthetically prepared PG-5 as compared to that of
synthetically prepared PG-1.
Figure 4 is a graphical representation of the effect of
serial transfer into antibiotic-cont~;n;ng media on the
development of drug resistance in methicillin resistant
Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa.
Figure 5 is an illustration of the sequences of
protegrins PG-1 through PG-5; and
Figure 6 is an illustration of a ~-sheet secondary
peptide structure.
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6. ~ETAILED DESCRIPTION OF ~HE lNv~N~ oN
6.1 Definitions
As used herein, the following terms shall have the
following e?n; n~s:
"SecondarY Structure:" As used herein, "secondary
structure" refers to the regular local structure of segments
of polypeptide ~h~in~ including, but not limited to, helices
such as a-helices, extended strands such as ~-strands and
sheets of e~tended strands such as ~-sheets.
lo "Anti-Parallel ~-Sheet:" As used herein "anti-
parallel ~-sheet" refers to a secondary structure of a
polypeptide chain characterized by intermolecular backbone-
backbone hydrogen hon~; ng between anti-parallel peptide
strands. An anti-parallel ~-sheet may optionally contain one
or two interstrand disulfide linkages.
"Amphiphilic Anti-Parallel ~-Sheet:" As used
herein, "amphiphilic anti-parallel ~-sheet" refers to an
anti-parallel ~-sheet wherein one surface has a net
hydrophobic character and another surface has a net
hydrophilic character.
"Reverse-Turn:" As used herein, "reverse-turn"
refers to a characteristic secondary structure that links
adjacent strands of an anti-parallel ~-sheet. Typically, a
"reverse-turn" is a two to four amino acid residue peptide
segment that reverses the direction of a polypeptide chain so
as to allow a single polypeptide chain to adopt an anti-
parallel ~-sheet conformation. Such peptide segments are
well known in the art and include, by way of example and not
limitation, three amino acid residue ~-turns (Rose et al.,
1985, Adv. Protein Chem. 37:1-109; Wilmer-White et al ., 1987,
Trends Biochem. Sci. 12:189-192; Wilmot et al., 1988, J. Mol.
Biol. 203:221-232; S;hAn~A et al ., 1989, J. Mol. Biol.
206:759-777; Tramontano et al., 1989, Proteins: Struct.
Funct. Genet. 6:382-394) and four amino acid residue ~-turns,
as described below.
"~-Turn:" As used herein, "~-turn" refers to a
recognized sub-class of reverse-turns. Typically, a "~-turn"
is a four amino acid residue peptide segment that reverses
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the direction of a polypeptide chain so as to allow a single
polypeptide chain to adopt an anti-parallel ~-sheet secondary
structure. Generally, the two internal amino acid residues
of the ~-turn are not involved in the hydrogen-bonding of the
~-sheet; the two amino acid residues on either side of the
internal residues are included in the hydrogen-bonding of the
~-sheet. The term "~-turn~' expressly includes all types of
peptide ~-turns commonly known in the art including, but not
limited to, type-I, type-II, type-III, type-I', type-~I' and
type-III' ~-turns (see, Rose et al ., 1g85, Adv. Protein Chem.
37:1-109; Wilmer-White et al., 1987, Trends Biochem. Sci.
12:189-192; Wilmot et al ., 1988, J. Mol. Biol. 203:221-232;
S;h~n~ et al., 1989, J. Mol. Biol. 206:759-777; Tramontano
et al., 1989, Proteins: Struct. Funct. Genet. 6:382-394).
"AntimicrobiallY Effective Amount:" As used
herein, "antimicrobially effective amount" refers to an
amount of peptide (or composition thereof) that is biostatic
or biocidal against a target microbe. More specifically, an
antimicrobially effective amount of peptide refers to an
amount of peptide that inhibits the growth of, or is lethal
to, a target microbe.
"TheraPeuticallY Ef~ective Amount" As used herein,
"therapeutically effective amount" refers to an amount of
peptide (or composition thereof) effective to ameliorate the
symptoms of, or ameliorate, treat or prevent microbial
infections or diseases related thereto in both plants and
animals.
"PharmaceuticallY AccePtable Salt:" As used
herein, "pharmaceutically acceptable salt" refers to those
salts which substantially retain the antimicrobial activity
of the free bases and which are obtained by reaction with
inorganic acids.
6.2 DescriPtion of the Preferred Embodiments
The present invention provides protegrin peptides
having antimicrobial activity, compositions comprising the
peptides, methods of using the peptides (or compositions
thereof) to inhibit the growth of or kill a wide variety of
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microbial targets and methods of using the peptides (or
compositions thereo~) to treat or prevent micro~ial
infections and diseases related thereto in both plants and
n t 5~
The peptides of the invention exhibit broad spectrum
antimicrobial activity, being biostatic or biocidal against a
, wide range of microbial targets, including but not limited
to, Gram-positive bacteria such as L. monocytogenes, B.
subtilis, E. faecalis ( including vancomycin-sensitive (VSEF)
and vancomycin-resistant (VREF) strains), E. faecium
(including vancomycin-sensitive (VSEF) and vancomycin-
resistant (VREF) strains), S. aureus (including methicillin-
sensitive (MSSA) and methicillin-resistant (MRSA) strains),
S. epidermis ( including methicillin-sensitive (MSSE) and
methicillin-resistant (MRSE) strains), S. salivarius, C.
minutissium, C. pseudodiptheriae, C. stratium,
Corynebacterium group G1, Coryne~acterium group G2, S.
rne ~niae (including penicillin-resistant (PSRP) strains),
S. mitis and S. sanguis; Gram-negative bacteria including A.
calcoaceticus, E. coli, R. pneumoniae, P. aeruginosa, S.
marcescens, H. influenza, Moraxella sp., N. meningitidis, S.
typhimurium, H. pylori, H. felis, and C. jejuni; as well as
protozoa, yeast and certain strains of viruses and
retroviruses. Significantly, the peptides described herein
are biostatic or biocidal against clinically relevant
pathogens exhibiting multi-drug resistance such as, among
others, v~nc~ ycin-resistant Enterococcus faecium or faecalis
("VR~"), penicillin-resistant Streptococcus pne7~moniae
("PRSP") and methicillin-resistant Staphylococcus aureus
("MRSA").
The peptides of the invention (or compositions thereof)
are useful as biocidal or biostatic agents in a wide variety
of applications. For example, the peptides can be used to
disinfect or preserve a variety of materials including
3~ medical instruments, foodstuffs, medicaments, contact lens
solutions, cosmetics and other nutrient-cont~; n i ng materials.
The peptides of the invention are particularly useful as
bacteriostatic or bactericidal agents against multi-drug-
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resistant pathogens such as VRE, MRSA and MSSE in a variety
of clinical settings.
The peptides of the invention, or compositions thereof,
are also useful for the prophylaxis or treatment of microbial
infections and diseases related thereto in both plants and
animals. Such dis~ include, but are not limited to,
Gram-negative and Gram-positive bacterial infections,
endocarditis, pneumonia and other respiratory infections,
urinary tract infections, systemic candidiasis, oral
mucositis, etc.
The peptides described herein provide significant
advantages over traditional antibiotics and/or other
antimicrobial peptides. For example, as the peptides
described herein are related to antimicrobial peptides found
naturally ~n i ~1 S, it is believed that the relatively high
frequency of resistance observed for traditional antibiotics
will not be observed for the peptides described herein.
A particular advantage of some of the protegrins of the
invention, especially the "mini-protegrin~' form having fewer
than eighteen amino acids, lies in their reduced size. As a
consequence, they are less costly to produce, generally are
expected to provide better distribution in tissue and are
less immunogenic.
6.2.1 The PePtides
Generally, the protegrin peptides of the
invention include the amino acid sequence:
(I) X1_X2_X3_X4_XS-C6-X7-CB-X9-X10-X11-X1Z-C13-X1~-C15-X16 X17 X18
and its defined modified forms. Those peptides which may
coincidentally occur in nature must be in a purified and/or
isolated form or prepared synthetically or recombinantly.
The designation X~ in each case represents an amino acid
at the specified position in the peptide. Similarly, the
designation Cn represents an amino acid at the specified
position and further represents those positions in the amino
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I
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acid sequence of formula (I) which may optiona~ly contain
amino acid residues capable of forming disulfide
interlinkages.
The amino acid residues denoted by Xn or Cn may be the
genetically encoded L-amino acids, naturally occurring non-
genetically encoded L-amino acids, synthetic L-amino acids or
D-enantiomers of all of the above. The amino acid notations
used herein for the twenty genetically encoded L-amino acids
and common non-encoded amino acids are conventional and are
as follows:
Common Amino Acid Abbreviations
One-Letter Common
Amino Acid Symbol Abbreviation
AlAnine A Ala
~rginine R Arg
A~parag$ne N A~n
A~partic acid D A~p
Cy~teine C Cy~
Glutamine Q Gln
Glutamic acLd E Glu
.Glycine G Gly
Hi~tidine H Hi~
I~oleucine I Ile
Leucine L Leu
Ly~ine K Ly~
Meth;o~i no M Met
Phenyl~l~ninO F Phe
Proline P Pro
Serine S Ser
Threonine T Thr
Tryptophan W Trp
Tyrosine Y Tyr
Valine V Val
ornithine O Orn
~ - ~1 Ani no bAla
2 r 3-rl; ~i n~propionic Dpr
acLd
a-aminoiRobutyric acid Aib
N-methylglycine MeGly
(Rarcosine)
Citrulline Cit
t-butyl~l ~ni no t-BuA
t-butylglycine t-BuG
N-methyli~oleucine MeIle
phenylglycine Phg
cyclohexylRl~n;n~ Cha
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O~e-Lottcr Common
Amino Acid Symbol Abbreviation
Norleucine Nle
1-naphthyl~l An i n~ 1-Nal
2-naphthy7~1 ~n i n~ 2-NaL
4-chlorophenyla 1 ~ n ~ n~ Phe(4-Cl)
2-fluorophenylRl~nin~ Phe(2-F)
3-fluo~ophenyl~l ~ni n~ Phe(3-F)
4-fluorophenylF~lAnin~ phe(4-F)
Penicilll i n~ P~n
1,2,3,4-tetrahydro- Tlc
i~oquinoline-3-
carboxylic acid
~-2-thienyla~ n ~ n e Thi
Me~hi n~i n~ sulfoxid~ MSO
Homoarginine Har
N-acetyl ly~ine AcLys
2r4-~i~m; no butyric acid Dbu
p-aminophenyl~l~ n i n~ Phe(pNH2)
N-methylvaline ~eVal
Homocysteine hCys
Homoserine hSer
~-amino hexanoic acid Aha
~-amino valeric acid Ava
2,3-~innbutyric acid Dab
~yd.O~y~ oline Hyp
Parabenzylphenyl~l ~ n; n~ Pba
Homopheny~l A n ~ n~ hPhe
N-methylphenyl~ 1 ~n ~ nF~ MePhe
The compounds of the invention are peptides which are
partially defined in terms of amino acid residues of
designated classes. Amino acid residues can be generally
subclassified into major subclasses as follows:
~ cidic: The residue has a negative charge due to loss
of H~ ion at physiological pH and the residue is attracted by
a~ueous solution so as to seek the surface positions in the
conformation of a peptide in which it is contained when the
peptide is in aqueous medium at physiological pH.
B~sic: The residue has a positive charge due to
association with H~ ion at physiological pH and the residue is
attracted by a~ueous solution so as to seek the surface
positions in the conformation of a peptide in which it is
contained when the peptide is in aqueous medium at
physiological pH.
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H~dro~hobic: The residues are not charged at
physiological pH and the residue is repelled by aqueous
solution so as to seek the inner positions in the
conformation of a peptide in which it is contained when the
peptide is in aqueous medium.
Polar/lar~e: The residues are not charged at
physiological pH, but the residue is not sufficiently
repelled by aqueous solutions so that it would necessarily
seek an inner position in the conformation of the peptide in
which it is contained when the peptide is in aqueous medium.
Depending on the conditions, and on the remaining amino acids
in the sequence, the residue may reside either in the inner
space or at the surface of the protein.
CYsteine-Like: Residues having a side chain capable of
participating in a disulfide linkage. Thus, cysteine-like
amino acids generally have a side chain containing at least
one thiol (SH) group, such as cysteine, homocysteine,
penicillamine, etc., with cysteine being preferred.
Small: Certain neutral amino acids having side ch;~
that are not sufficiently large, even if polar groups are
lacking, to confer hydrophobicity. "Small" amino acids are
those with four carbons or less when at least one polar group
is on the side chain and three carbons or less when not.
The gene-encoded secondary amino acid proline (as well
as proline-like imino acids such as 3-hydroxy proline and 4-
hydroxy proline~ is a special case due to its known effects
on the secondary conformation of peptide ch~;n~, and is not,
therefore, included in a group.
It is understood, of course, that in a statistical
collection of individual residue molecules some molecules
will be charged, and some not, and there will be an
attraction for or repulsion from an aqueous medium to a
greater or lesser extent. To fit the definition of
'Icharged~'' a significant percentage (at least approximately
25%) of the individual molecules are charged at physiological
pH. The degree of attraction or repulsion required for
classification as polar or nonpolar is arbitrary and,
therefore, amino acids specifically contemplated by the
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invention have been classified as one or the other. Most
amino acids not specifically named can be classified on the
basis of known behavior.
Amino acid residues can be further subclassified as
cyclic or noncyclic, and aromatic or nonaromatic, self-
explanatory classifications with respect to the side-chain
substituent yLOU~S of the residues.
Certain commonly encountered amino acids which are not
genetically encoded of which the peptides of the invention
may be composed include, but are not limited to, ~-alanine
(b-Ala) and other omega-amino acids such as 3-aminopropionic
acid, 2,3~ ~;nopropionic acid (Dpr), 4-aminobutyric acid
and so forth; ~-aminoisobutyric acid (Aib); ~-aminohexanoic
acid (Aha); ~-aminovaleric acid (Ava); N-methylglycine or
sarcosine (MeGly); ornithine (Orn); citrulline (Cit);
t-butyl A l~n;ne (t-BuA); t-butylglycine (t-BuG);
N-methylisoleucine (MeIle); phenylglycine (Phg);
cyclohexyl~lAnine (Cha); norleucine (Nle); l-naphthylalanine
(1-Nal); 2-naphthylalanine (2-Nal); 4-chlorophenylalanine
(Phe(4-Cl)); 2-fluorophenyl~l~n;ne (Phe(2-F)); 3-
fluorophenylalanine (Phe(3-F)); 4-fluorophenylalanine (Phe(4-
F)); penicillamine (Pen); 1,2,3,4-tetrahydroiso~uinoline-3-
carboxylic acid (Tic); ~-2-thienylalanine (Thi); methionine
sulfoxide (MSO); homoar~inine (Har); N-acetyl lysine (AcLys);
2,3-di~rinohutyric acid (Dab); 2~4-di~r;nohutyric acid (Dbu);
p-aminophenylalanine (Phe(pNH2)); N-methyl valine (MeVal);
homocysteine (hCys); hydroxyproline (Hyp);
Parabenzylphenylalanine (Pba); HomophenylAl~nine (hPhe); N-
methylphenyl~l~n;~e (MePhe); and homoserine (hSer). These
amino acids also fall conveniently into the categories
defined above.
The classifications of the above-described genetically
encoded and non-encoded amino acids are summarized in Table
1, below. It is to be understood that Table 1 is for
illustrative purposes only and does not purport to be an
exhaustive list of amino acid residues that may comprise the
peptides described herein.
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Table 1
Amino A¢id Classifications
Cla~ification Genetically Fnco~ Non-Genetically
P~.n ~~o~
Hydrophobic Y, V, I, L, M, F, W Phg, 1-Nal, 2-Nal, ThL,
Tic, Phe(4-Cl), Phe(2-
F), Phe(3-F), Phe(4-F),
t-BuA, t-8uG, ~eIle,
Nle, MeVal, Cha, hPhe,
MePhe, Pba
Acidic D, E
BaRic H, K, R Dpr, Orn, hArg, phe(
NH2), Dbu, Da~
PO1artLarge Q, N CLt, AcLy~, MSO
Small G, S, A, ~ bAla, MeGly, ALb, hSer
CyRteine-Like C Pen, hCy~
In the peptides of formula I, the symbol "-" between
amino acid residues Xn and/or Cn generally designates a
backbone interlinkage. Thus, the symbol "-" usually
designates an amide linkage (-C(O)-NH). It is to be
understood, however, that in all of the peptides of the
invention one or more amide linkages may optionally be
replaced with a linkage other than amide. Such linkages
include, but are not limited to, -CH2NH-, -CH2S-, -CH2CH2,
-CH=CH- (cis and trans), -C(O)CH2-, -CH(OH)CH2- and -CH2SO-.
Peptides having such linkages and methods for preparing
such peptides are well-known in the art (see, e.g., Spatola,
1983, Veqa Data ~3) (general review); Spatola, 1983,
"Peptide Backbone Modifications" In: ChemistrY and
BiochemistrY of Amino Acids Pe~tides and Proteins (Weinstein,
ed.), Marcel Dekker, New York, p. 267 (general review);
Morley, 1980, Trends Pharm. Sci. 1:463-468; Hudson et al.,
1979, Int. J. Prot. Res. 14:177-185 (-CH2NH-, -CH2CH2-);
Spatola et al., 1986, Life Sci. 38:1243-1249 (-CH2-S); Hann,
1982, J. Chem. Soc. Perkin Trans. I. 1:307-314 (-CH=CH-, cis
and trans); Almquist et al., 1980, J. Med. Chem. 23:1392-1398
(-COCH2-); Jennings-White et al ., Tetrahedron. Lett. 23:2533
(-COCH2-); European Patent Application EP 45665 (1982)
CA:97:39405 (-CH(OH)CH2-); Holladay et al ., 1983, Tetrahedron
- 15 -
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W O 97/18826 PCTrUS96/18544
~ett. 24:4401-4404 (-C~OH)CH2-); and Hruby, 1982, Life Sci.
31:189-199 (-CH2-S-).
Generally, the peptides of the invention are comprised
of about 10 to 30 amino acid residues. Thus, it is to be
understood that while formula (I) designates eighteen
specified amino acid positions comprising the "core" peptide
structure, the peptides of the invention may contain fewer
than or greater than 18 amino acids without deleteriously
affecting, and in some cases even enhancing, the
antimicrobial or other useful properties of the peptides.
For peptides containing fewer than 18 amino acid residues,
certain specified amino acids are not present within the
peptide sequence, as will be discussed in more detail below.
For peptides containing greater than 18 amino acid residues,
the amino acid sequence shown as formula (I) may contain
extensions at the N- and/or C-terminus of additional amino
acid residues or peptide sequence. It is to ~e understood
that such additional amino acid residues or peptide sequences
are non-interfering in that they will not significantly
deleteriously affect the antimicrobial activity of the
peptide as compared with naturally occurring protegrins.
The peptides of the invention are characterized by a
"core" structure containing two main elements or motifs: a
reverse-turn region bracketed by two strands that form an
anti-parallel ~-sheet. While not intending to be bound by
theory, it is believed that the antimicrobial activity of the
compounds of formula (I) is in part associated with such a
core structure.
The ~-sheet region of the peptides comprises an N-strand
(residues Xl-C~) and a C-strand (residues C13-Xl8). The N-
strand and C-strand are arranged anti-parallel to one another
and are non-covalently linked together via backbone-backbone
hydrogen bonds (for a detailed description of the structure
of ~-sheets the reader is referred to Creighton, 1993,
Protein Structures and Molecular ProPerties, W.H. Freeman and
Co., NY, and references cited therein). While not intending
to be bound by theory, it is believed that the most important
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residues comprising the ~-sheet region are residues X5-C8 and
Cl3--Xl6 -
Preferably, the ~-sheet region of the peptides is
amphiphilic, i.e., one surface of the ~-sheet has a net
hydrophobic character and the other surface has a net
hydrophilic character. ~eferring to the ~-sheet structure
illustrated in Figure 6, the side chA;n~ of L-amino acid
residues adjacent to one another intrastrand-wise (residues
n, n~1, n+2, etc.) point in opposite directions so as to be
positioned on opposite surfaces of the ~-sheet. The side
r.h~;n~ of L-amino acid residues adjacent to one another
interstrand-wise (residues n and c, n~l and c+1, etc.) point
in the same direction so as to be positioned on the same
surface of the ~-sheet. Using this general structural motif
an amphiphilic antiparallel ~-sheet is obtained by selecting
amino acids at each residue position so as to yield a ~-sheet
having hydrophobic side ~~.h;~;n~: positioned on one surface of
the sheet and hydrophilic side chains positioned on the
other.
Of course, it will be appreciated that as the surfaces
of the amphiphilic anti-parallel ~-sheet region need only
have net hydrophobic or net hydrophilic character, each side
chain comprising a particular surface need not be hydrophobic
or hydrophilic. The surfaces may contain side c-h~;n!: that do
not significantly alter the net character of the surface.
For example, both the hydrophobic and hydrophilic surfaces
may contain small amino acid side chains, as these side
c-h~in~: do not significantly contribute to the net character
of the surface.
The ~-sheet region of the peptides of formula I may
contain from one to four cysteine-like amino acids,
designated C~, C8, C13 and Cl5, which may participate in
interstrand disulfide bonds. The peptides of the invention
that contain at least two cysteine-like amino acid residues
may be in straight-chain or cyclic form, depending on the
extent of disulfide bond formation. The cyclic forms are the
t result of the formation of disulfide lin~ages among all or
some of the four invariant cysteine-like amino acids. Cyclic
- 17 -
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W O 97/188Z6 PCT~US96tl8544
forms of the invention include all possible permutations of
disulfide bond formation. The straight-chain forms are
convertible to the cyclic forms, and vice versa. Methods for
forming disulfide bonds to create the cyclic forms are well
known in the art, as are methods to reduce disulfides to form
the linear compounds.
The naturally occurring protegrins (P&-1 through PG-5)
contain two disulfide bonds; one between cysteines C6-Cls and
another between cysteines C8-C13 (Harwig et al., 1995, J.
PePtide Sci. 3:207). Accordingly, in those embodiments
having two disulfide linkages, the C6-Cls, C8-C13 form is
preferred. Such peptides are designated "native" forms.
However, it has been found that forms of the protegrins
containing only one disulfide linkage are active and easily
prepared. Preferred among embodiments having only one
disulfide linkage are those represented by C6-Cls alone and by
C~-C13 alone.
Forms cont~in;ng a C6-C15 disulfide as the only disulfide
linkage are generally designated "bullet" forms of the
protegrins; those wherein the sole disulfide is C8-C13 are
designated the "kite" forms. The bullet and kite forms can
most conveniently be made by replacing each of the cysteine-
like amino acid residues at the positions that are not
involved in a disulfide linkage with amino acids that do not
participate in disulfide bonds, preferably with small amino
acids such as glycine, serine, AlAn;ne or threonine.
Alternatively, Ca and/or C13 may be absent.
As the linearized or "snake" forms of the native
peptides have valuable activities, the peptides of the
invention include linearized forms wherein the sulfhydryl
(SH) groups are chemically stabilized with suitable reagents.
As defined herein, "SH-stabilized" forms of the peptides of
the invention contain sulfhydryl groups that have been
reacted with stAn~rd reagents to prevent reformation of
3~ disulfide linkages or forms wherein the cysteine-like amino
acid residues are replaced by other amino acids as set forth
above. It is preferred that all four cysteine-like amino
acid residues be replaced or SH-stabilized in order to
- 18 -
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W O 97118826 PCTAJS96/18544
in;~; ze the likelihood of the formation of intermolecular
disulfide linkages.
The sulfur atoms involved in an interstrand disulfide
bridge in a ~-sheet are not positioned within the plane~ 5 defined by the interstrand backbone-backbone hydrogen-bonds;
the sulfur atoms are at an angle with respect to the
~-carbons of the bridged amino acid residues so as to be
positioned on a surface of the ~-sheet. Thus, the sulfur
atoms of the disulfide linkages contribute to the net
hydrophilicity of a surface of the ~-sheet. It is to be
understood that in the peptides of formula I a ~-sheet region
defined by the following formula is specifically contemplated
to fall within the definition of amphiphilic antiparallel
sheet as described herein:
ll 6 -X7 -11 8
Cl5--Xl4--Cl3
wherein C6, C8, C13 and C15 are each independently a cysteine-
like amino acid, X7 and Xl4 are each independently a
hydrophobic or small amino acid and ¦¦ is a disulfide bond.
In a particularly preferred embodiment, C6, C8, C13 and C1s are
each cysteine and X7 and X14 are each independently
hydrophobic amino acids.
The ~-sheet secon~y structure illustrated in Figure 6
is composed entirely of L-amino acids. Those having skill in
the art will recognize that substituting an L-amino acid with
its corresponding D-enantiomer at a specific residue position
may disrupt the structural stability or amphiphilicity of
amphiphilic anti-parallel ~-sheet region. The degree to
which any particular enantiomeric substitution disrupts the
structural stability or amphiphilicity depends, in part, on
the size of the amino acid side chain and position of the
residue within the ~-sheet. Preferably, the ~-sheet region
of the peptides of formula I will contain mixtures of L- and
D-amino acids that do not significantly affect the stability
or amphiphilicity of the ~-sheet region as compared to
peptides cont~;ning the corresponding all D- or all
L-enantiomeric forms of the sheet. Enantiomeric
substitutions that do not substantially affect the stability
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or amphiphilicity of the ~-sheet region will be readily
apparent to those having skill in the art.
In a preferred embodiment of the invention, hydrophobic,
basic, polar/large and cysteine-like amino acids comprising
the ~-sheet region are either all L-enantiomers or all
D-enantiomers. Small amino acids comprising the ~-sheet
region may be either L-enantiomers or D-enantiomers.
The reverse-turn region of the peptides of formula I
(residues Xg-XlO-Xll-Xl2 taken together) links the strands of
the anti-parallel ~-sheet. Thus, the reverse-turn region
comprises a structure that reverses the direction of the
polypeptide chain so as to allow a reglon of the peptide to
adopt an anti-parallel ~-sheet secondary structure.
The reverse-turn region of the molecule generally
comprises two, three or four amino acid residues (residue Xg
and/or X12 may be absent). An important feature of the
peptides of the invention is the presence of a positive
charge in the turn region of the molecule. Thus, one of Xg-
X12, and preferably two of Xg-Xl2, must be a basic amino acid.
Such two, three and four amino acid segments capable of
effecting a turn in a peptide are well known and will be
apparent to those of skill in the art.
In a preferred embodiment of the invention, the reverse-
turn is a three amino acid residue ~-turn. Virtually any ~-
turn se~uence known in the art may be used in the peptides of
the invention, including those described, for example, in
Rose et al., 1985, Adv. Protein Chem. 37:1-109; Wilmer-White
e~ al., 1987, Trends Biochem. Sci. 12:189-192; Wilmot et al .,
1988, J. Mol. Biol. 203:221-232; S;h~n~ et al ., 1989, J.
~ol. Biol 206:759-777; and Tramontano et al., 1989, Proteins:
~truct. Funct. Genet. 6:382-394.
In another preferred embodiment the reverse-turn is a
four amino acid residue ~-turn. In such structures, the two
internal amino acid residues of the turn are usually not
involved in the hydrogen-bonding of the anti-~arallel
~-sheet; the two amino acid residues on either side of the
internal residues are usually included in the hydrogen-
bonding of the ~-sheet. While not intending to be bound by
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WO 97/18826 PCT~US96/1854
theory, it is believed that such hydrogen bonding helps
stabilize the ~-sheet region o~ the molecule.
The conformations and sequences o~ many peptide ~-turns
have been well-described in the art and include, by way of~ ~ example and not limitation, type-I, type-I', type-II,
type-II', type-III, type-III', type-IV, type-V, type-V',
type-VIa, type-VIb, type-VII and type-VIII (see, Richardson,
1981, Adv. Protein Chem. 34:167-339; Rose et al., 198~, Adv.
Protein Chem. 37:1-109; Wilmot et al ., 1988, J. Mol. Biol.
203:221-232; S; h~n~ et al ., 1989, J. Mol. Biol. 206:759-777;
Tramontano et al., 1989, Proteins: struct. Funct. Genet.
6:382-394). All of these types of peptide ~-turn structures
and their corresponding sequences, as well as later
discovered peptide ~-turn structures and sequences, are
specifically contemplated by the invention.
The specific conformations of short peptide turns such
as ~-turns depend primarily on the positions o~ certain amino
acid residues in the turn (usually Gly, Asn or Pro).
Generally, the type-I ~-turn is compatible with any amino
acid residue at positions Xg through Xl2, except that Pro
cannot occur at position X11. Gly predominates at position Xl2
and Pro pr~ ;n~tes at position XlO of both type-I and type-
II turns. Asp, Asn, Ser and Cys residues frequently occur at
position Xg, where their side ch~; n~ often hydrogen-bond to
the NH of residue Xll.
In type-II turns, Gly and Asn occur most frequently at
position Xll, as they adopt the required backbone angles most
easily. Ideally, type-I~ turns have Gly at positions XlO and
X1l, and type-II' turns have Gly at position XlO. Type-III
turns generally can have most amino acid residues, but type-
III' turns usually re~uire Gly at positions XlO and Xl~.
Type-VIa and VI~ turns generally have a cis peptide bond
and Pro as an internal residue. For a review of the
different types and se~uences of ~-turns in proteins and
3~ peptides the reader is referred to Wilmot et al.~ 1988, J.
Mol. Biol. 203:221-232.
Preferred ~-turn se~uences are as follows (listed in
order Xg to Xl2): ZZZG; ZZZF; ZZSG; ZZAL; ZGZL; ZFZL; ZPZV;
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ZPZF; ZGZY; IZGZ; LZZF; YZGZ, wherein each Z is independently
an L- or D-enantiomer of R, K, Dbu or Orn.
Additional preferred ~-turns include those wherein XlO
and/or Xl1 are Tic or Hyp, as these residues are known to
effect or induce ~-turn structures in peptides and proteins.
The peptides of the invention are generally basic, i.e.,
they have a net positive charge at physiological pH. While
not int~n~;ng to be bound by theory, it is believed that the
presence of positively charged amino acid residues,
particularly in the turn region of the molecule, is important
for antimicrobial activity.
It is understood that in a statistical collection of
individual amino acid residues in a structure such as a
peptide some of the amino acid residues will be positively
charged, some negatively charged and some uncharged. Thus,
some of the peptides will have a charge and some not. To fit
the definition of "basic," an excess of amino acid residues
in the peptide molecule are positively charged at
physiological pH. Thus, approximately 15% but no more than
up to about 50% of the amino acids must be basic amino acids,
and the compounds must have a net charge of at least +1 at
physiological p~. Preferably, the peptides of the invention
will have a net charge of at least +3 at physiological pH.
For embodiments having as few as 10 amino acids, there
may be only one basic amino acid residue; however, at least
two basic residues, even in this short-chain residue, are
preferred. If the protegrin peptide contains as many as 15
amino acid residues, two basic residues are required. It is
preferred that at least 20~ of the amino acids in the
sequence be basic, with 30% basic amino acids being
particularly preferred.
The amino terminus of the peptides of the invention may
be in the free amino form or may be acylated by a group of
the formula RCO-, wherein R represents a hydrocarbyl group of
1-25C, preferably 1-lOC, more preferably 1-8C. The
hydrocarbyl group can be saturated or unsaturated, straight
chain, branched or cyclic, and is typically, for example,
methyl, ethyl, isopropyl, t-butyl, n-pentyl, cyclohexyl,
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WO 97/18826 PCT~S96/18544
cyclohexene-2-yl, hexene-3-yl, hexyne-4-yl, octyl, decyl,
eicanosyl and the like, with octyl being preferred.
Alternatively, the N-terminus may contain aromatic
groups such as naphthalene, etc. Such groups may be~ 5 conveniently incorporated into the peptides of the invention
by using amino acids such as 1-naphthylalanine or
2-naphthylalanine as the N-terminal amino acid residue.
The N-terminus of the peptides may also be substituted
to use solute-specific transmembrane channels to facilitate
their entry into the bacterial periplasm. For example, the
N-terminus may be conveniently modified with catechol using
catechol-NHS activated ester.
The C-terminus of the peptides of the invention may be
in the form of the underivatized carboxyl group, either as
the free acid or an accepta~le salt, such as the potassium,
sodium, calcium, magnesium, or other salt of an inorganic ion
or of an organic ion such as caffeine. In some embodiments,
it is difficult to make salts since the r- - in~er of the
molecule bears a positive charge which may repel the relevant
cation. The carboxyl terminus may also be derivatized by
formation of an ester with an alcohol of the formula ~OH, or
may be amidated by an amine of the formula NH3, or RNH2, or
R2NH, wherein each R is independently hydrocarbyl of 1-25C as
defined and with preferred embodiments as above. Amidated
forms of the peptides wherein the C-terminus has the formula
CONH2 are preferred.
Addition of lipophilic groups at the C- and/or
N-terminus facilitates the transition of the peptide into the
membrane of the target microbe and penetration into sites of
infection. Choice of optimum substitution is determined by
evaluation with respect to the lipid content of the target
microbe.
Thus, in one illustrative embodiment, the invention
3~ provides antimicrobial peptides comprised of about 10-30
amino acid residues and cont~; n i~g the amino acid sequence:
.r
(I) X1-X2-X3-X4-X5-C6-X,-C8-Xg-Xl0-xll-xl2-cl3-xl4-cl~-xl6-xl7-xl8
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or a pharamceutically acceptable salt or N-terminal
acylated or C-terminal amidated or esterified form thereof,
wherein:
each of C8 and Cl3 is independently present or not
present, and if present each is independently a ~ysteine-
like, basic, small, polar/large or hydrophobic;
each of C6 and Cl5 is independently a cysteine-like,
basic, small, polar/large or hydrophobic amino acid;
each of Xl-X5 is independently present or not present,
and if present each is independently a basic, hydrophobic,
polar/large, or small amino acid;
each of X7 and Xl4 is independently a hydrophobic or a
small amino acid;
each of X9 and Xl2 is independently present or not
present;
Xg-Xl2 taken together are capable of effecting a reverse
turn when contained in the amino acid sequence of formula (I)
and at least one of Xg-Xl2 must be a basic amino acid;
each of Xl6-Xl8 is independently present or not present,
and if present each is independently a basic, hydrophobic,
polar/large or small amino acid;
and wherein at least about 15% up to about 50% of the
amino acids comprising said antimicrobial peptide are basic
amino acids such that said antimicrobial peptide has a net
charge of at least ~1 at physiological pH.
In one class of protegrins described herein, either the
hydrophobic amino acids found in the naturally occurring
protegrins at X5 are replaced with a basic amino acid and/or
at least one of X1-X4 is hydrophobic and/or at least one, and
preferably all four of amino acids X1 and X4 found in the
native forms are deleted; and/or one or more of X5r C8, Xg~
X12, C13 and X16 is absent. By suitable manipulation of these
and other features, the range of conditions under which the
3~ class of protegrins of the present invention are effective
can be varied. Furthermore, the spectrum of microbes against
which they are effective can also be modified.
CA 02238610 1998-0~-22
W ~ 97118826 PCT~US96118~44
In another class, the peptides of the inveniton are
comprised of about 10-14 amino acid residues and each of C8
and C13 is independently present or not present, and if
present each is independently a small, hydrophobic or
polar/large amino acid or cysteine;
each of C6 and C15 is independently a small, hydrophobic
or polar/large amino acid or cysteine;
each of X1-X5 is independently present or not present,
and if present each is independently a basic or small amino
acid and any two of Xl-X5 may be a hydrophobic amino acid;
each of X7 and X14 is independently a hydrophobic amino
acid;
each of X9 and X12 is independently present or not
present, and if present each is independently a basic or
hydrophobic amino acid;
X1~ is a basic, hydrophobic or small amino acid or
proline;
X16 is present or not present, and if present is a basic,
small or hydrophobic amino acid;
each of X17 and X18 is ;~ep~ndently present or not
present, and if present each is independently a basic or
small amino acid.
each of Xl6-Xl8 is independently present or not present,
and if present each is independently a basic, hydrophobic,
polar/large or small amino acid.
In another class, the peptides of the invention are
comprised of about 10-18 amino acid residues and each of CB
and C13 is independently present or not present, and if
present each is independently a small, hydrophobic or
polar/large amino acid or cysteine;
each of C6 and Cl5 is independently a small, hydrophobic
or polar/large amino acid or cysteine;
each of Xl-X4 is independently present or not present,
and if present each is ;n~ep~n~ntly a basic or small amino
acid and any one of Xl-X4 may be a hydrophobic amino acid;
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each of X5 and Xl6 is independently present or not
present, and if present each is idenpendently a hydrophobic
or basic amino acid;
each of X7 and Xl4 is independently a hydrophobic amino
acid;
X9 iS present or not present, and if present is a basic
or hydrophobic amino acid;
X10 is a basic or small amino acid or proline;
Xll is a basic or hydrophobic amino acid;
Xl2 is present or not present, and if present is a
hydrophobic amino acid;
Xl, present or not present, and if present is
independently a small amino acid; and
Xl8 is present or not present, and if present is a basic
amino acid.
The invention peptides can be further illustrated by way
of preferred embodiments. In one preferred embodiment of the
invention, all of the cysteine-like amino acid residues at
po6itions C6, C8, Cl3 and Cl5 are present as are Xg and Xl2.
In another set of preferred embodiments, all of Xl-X4 are
not present. In another set of preferred embodiments, at
least one, and preferably two of Xl-X4 is a hydrophobic amino
acid, preferably I, V, L, Y, F or W.
In another set of preferred embo~; -nts, X9-xl2 contain
at least one hydrophobic amino acid residue, preferably Phe,
Tyr or Trp.
Other preferred embo~i ?nts include those peptides
wherein each of X1 and Xg is independently selected from the
group consisting of R, K, Orn, Dab and Har or hydrophobic;
preferably X1 is R, K, Har and Xg i5 R, K, Har or hydrophobic,
especially I, V, L, W, F or Y. However, each of Xl and Xg may
be absent.
In another class of preferred embodiments, each of X2 and
X3 iS independently selected from the group consisting of G,
A, S, T, I, V, L, F, Y and W; more preferably, X2 and X3 are
G, W, F, Y, L, or V; however, X2 and/or X3 may be absent.
In another set of preferred embodiments, X4 is selected
from the group consisting of R, K, H, Orn, Har, Dab, G, A, S,
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T, F, Y and W; more preferably, X4 iS R, K, Orn, Dab, G or W;
however, X4 may be absent.
In another set of preferred embodiments, each of Xs and
X16 is independently selected from the group consisting of I,
- 5 V, L, Nle, W, Y, and F, preferably I, V, L, W, F and Y.
However, Xs and/or Xl6 may be absent.
In another set of preferred embodiments, each of X7 and
Xl4 is independently selected from the group consisting of I,
V, L, W, Y and F; preferably X7 iS I, F, Y or W and X14 is I,
V, L, W, Y, or F.
In another set of pre~erred embo~; ~nts, X9 iS R, K, H,
Orn, Dab, Har, I, V, L, Nle, W, Y or F, and X12 is I, L, V, W,
F or Y; more preferably and aromatic amino acid such as Y, W,
or F.
In another set of preferred embodiments, X10 is R, Orn,
Dab, G, W or P.
In another set of preferred embodiments, Xll is R, K,
Orn, Dab, G, W or P.
In another set of preferred embodiments, X17 is
preferably absent, but when present, is preferably ~, A, S or
T;
In another set of preferred embodiments, Xl8 is
preferably absent, but when present, is preferably R, K, H,
orn, Dab or Har.
Also preferable when all four amino acids X1-X4 are
present, Xl and X4 are preferably basic and X2 and X3 are small
or hydrophobic. Preferred embodiments of Xl-X4 include R-G-G-
R, R-G-W-R, R-L-L-R and the like.
Preferred embodiments for the basic amino acid to
replace cysteine-like residues are R, K, H, Orn, Dab and Har,
most preferably R, K or orn. Preferred small amino acids to
replace the cysteine-like residues include G, A, S and T,
most preferably A and ~.
Particularly preferred peptides of the invention include
the native, kite, bullet and/or snake forms of the following
peptides:
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W O 97/18826 PCTAUS96/18544
Form-21 R-G-G-R-L-Z-Y-Z-R-R-R-F-Z-V-Z-V
R-G-G-R-R-Z-Y-Z-R-R-R-F-Z-V-Z-V
R-G-G-R-L-Z-Y-Z-R-R-R-F-Z-V-Z-R
R-G-G-R-R-Z-Y-Z-R-R-R-F-Z-V-Z-R
R-Z-Y-Z-R-R-R-F-Z-V-Z-V
L-Z-Y-Z-R-R-R-F-Z-V-Z-R
R-Z-Y-Z-R-R-R-F-Z-V-Z-R
L-Z-Y-Z-R-R-R-F-Z-V-Z-V
Form-22 R-G-G-R-L-Z-Y-Z-R-R-R-F-Z-I-Z-V
R-G-G-R-R-Z-Y-Z-R-R-R-F-Z-I-Z-V
R-G-G-R-L-Z-Y-Z-R-R-R-F-Z-I-Z-R
R-G-G-R-R-Z-Y-Z-R-R-R-F-Z-I-Z-R
R-Z-Y-Z-R-R-R-F-Z-I-Z-V
15 L-Z-Y-Z-R-R-R-F-Z-I-Z-R
R-Z-Y-Z-R-R-R-F-Z-I-Z-R
L-Z-Y-Z-R-R-R-F-Z-I-Z-V
Form-23 R-G-G-G-L-Z-Y-Z-R-R-R-F-Z-V-Z-V
20 R-G-G-G-R-Z-Y-Z-R-R-R-F-Z-V-Z-V
R-G-G-G-L-Z-Y-Z-R-R-R-F-Z-V-Z-R
R-G-G-G-R-Z-Y-Z-R-R-R-F-Z-V-Z-R
R-Z-Y-Z-R-R-R-F-Z-V-Z-V
L-Z-Y-Z-R-R-R-F-Z-V-Z-R
25 R-Z-Y-Z-R-R-R-F-Z-V-Z-R
L-Z-Y-Z-R-R-R-F-Z-V-Z-V
Form-24 R-G-G-R-L-Z-Y-Z-R-G-W-I-Z-F-Z-V
R-G-G-R-R-Z-Y-Z-R-G-W-I-Z-F-Z-V
30 R-G-G-R-L-Z-Y-Z-R-G-W-I-Z-F-Z-R
R-G-G-R-R-Z-Y-Z-R-G-W-I-Z-F-Z-R
R-Z-Y-Z-R-G-W-I-Z-F-Z-V
L-Z-Y-Z-R-G-W-I-Z-F-Z-R
R-Z-Y-Z-R-G-W-I-Z-F-Z-R
35 L-Z-Y-Z-R-G-W-I-Z-F-Z-V
Form-25 R-G-G-R-L-Z-Y-Z-R-P-R-F-Z-V-Z-V
R-G-G-R-R-Z-Y-Z-R-P-R-F-Z-V-Z-V
- 28 -
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R-G-G-R-L-Z-Y-Z-R-P-R-F-Z-V-Z-R
R-G-G-R-R-Z-Y-Z-R-P-R-F-Z-V-Z-R
R-Z-Y-Z-R-P-R-F-Z-V-Z-V
L-Z-Y-Z-R-P-R-F-Z-V-Z-R
R-Z-Y-Z-R-P-R-F-Z-V-Z-R
L-Z-Y-Z-R-P-R-F-Z-V-Z-V
and the N-acylated and/or C-amidated or esterified forms
thereof wherein each Z is independently a hydrophobic, small
or basic amino acid or cysteine, as well as the foregoing
forms wherein X7 is W and/or X12 is W and/or wherein Xlq is W
and/or wherein X16 is W and/or wherein X17 is G and X18 is R;
and/or wherein at least one of X5 ~ Xg ~ X12 and X16 is not
present. In all of these embodiments, Z is preferably S, A,
T or G, most preferably A or T.
In the foregoing peptides, protegrin form 21 consists of
compounds which are characteristic of the present class but
which are otherwise similar to protegrin-l (PG-1); form 22
contains the characteristics of the present class but is
otherwise similar to protegrin-2 (PG-2); forms 23 through 2~
are similarly related to protegrins -3, -4 and -5 (PG-3, PG-4
and PG-5) (see Figure 5).
Another set of preferred compounds includes the native,
bullet, kite and snake forms of the following peptides
(sequences are shown aligned to protegrin PG-1):
CODE S~Q~NCE
PG-1 RGG~LCYCRRRFCVCVGR (SEQ ID NO:1)
WLCFCRRRFCVCV (SEQ ID NO:2)
FLCFCRRRFCVCV (SEQ ID NO:3)
WYCYCRRRFCVCV (SEQ ID NO:4)
WXCYCRRRFCVCV ~X=Cha) (SEQ ID NO:5)
WLCYCRRRF~v~v~K (SEQ ID NO:6)
WXCYCRRRFCVCVGR (X=Cha) (SEQ ID NO:7)
~T.T,RT~YCRRRFCVCVGR (SEQ ID NO:8)
RGGRLCYCRRRFCXCVGR (X=MeVal) (SEQ ID NO:9)
RGVCVCFRRRCYCLW (SEQ ID NO:10)
RGVCVCFRRRCYCLW (SEQ ID NO:11)
- 29 -
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CODE SEQUENCE
VCVCFRRRCYCLW (SEQ ID NO:12)
FCVCFRRRCFCLF (SEQ ID NO:13)
RGVCVCFRRRCYCRGGR (SEQ ID NO:14)
RGVCVCFRRRCYCLRGGR (all D) (SEQ ID NO:15)
5RGVCVCFRRRCYCLW (SEQ ID NO:16)
RGVCVCYRXRCYCLW (X=MeGly) (SEO ID NO:17)
WhCYCRXZYCVCVGR (X=MeGly) (SEQ ID NO:18)
(Z=D-Arg)
RGFCVCFRRVCYCLW (SEQ ID NO:l9)
WLCYCRRRFCVCVGR (SEQ ID NO:20)
10WLCYCRRXFCVCVR (X=D-Arg) (SEQ ID NO:21)
WLCYCKKKFCVCVGK (SEQ ID NO:Z2)
Octyl-WLCYCRRRFCVCVGR (SEQ ID NO:23)
XLCYCRRRFCVCV (X=1-Nal) (SEQ ID NO:24)
WLC RGRF CVR (SEQ ID NO:25)
WLC RGRF CFR (SEQ ID NO:26)
WLCY RR VCVR (SEQ ID NO:27)
WLCYCOOOFCVCV (SEQ ID NO:28)
WLCYCXXXFCVCV (X=Dab) (SEQ ID NO:29)
WLCYCRRRFCVCV (all D) (SEQ ID NO:30)
HWRLCYCRPKFCVCV (SEQ ID NO:31)
KWRLCYCRPKFCVCV (SEQ ID NO:32)
OWRLCYCRPKFCVCV (SEQ ID NO:33)
XWRLCYCRPKFCVCV (X=Dab) (SEQ ID NO:34)
RWHLCYCRPKFCVCV (SEQ ID NO:35)
RWKLCYCRPKFCVCV (SEQ ID NO:36)
RWOLCYCRPKFCVCV (SEQ ID NO:37)
RWXLCYCRPKFCVCV (X=Dab) (SEQ ID NO:38)
WLCYCKXKFCVCVGR (X=Tic) (SEQ ID NO:39)
FCYCKXKFCYCV (X=Hyp) (SEQ I3 NO:40)
WLX~XK~FXVXV (X=hCys) (SEQ ID NO:41)
WOLCYCOXOFCVCVO (X=Tic) (SEQ ID NO:42)
OFCVCVOXOFCVCVO (X=Tic) (SEQ ID NO:43)
OWOLCYCOXOFCVCV (X=Tic) (SEQ ID NO:44)
OFCVCXOLCYCFO (X=Tic) (SEQ ID NO:45)
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CODESEQUENCE
WLCYCKKKFCVCV (SEQ ID NO:46)
OWOLCYCOXOFCVCV (X=~YP) (SEQ ID NO:47)
WLCYCOXOFCVCVO ( X=Pba) (SEQ ID NO:48)
WLCYCOOOFCVCV (all D) (SEQ ID NO:49)
XFCYCLRXFCVCVR (X=D-Arg) (SEQ ID NO:50)
WLCYCRRXFCVCVZX (SEQ ID NO:51)
(X=D-Arg)
(Z=MeG1Y)
PC11LCYCRRRFCVCVGR (SEQ ID NO:52)
PCl2RCYCRRRFCVCV (SEQ ID NO:53)
PC15RGGRLCYCRRRFCVCR ( SEQ ID NO:54)
PC16RCYCRRRFCVCR (SEQ ID NO:55)
PC17LCYCRRRFCVCV (SEQ ID NO:56)
PC18LCYARRRFAVCV (SEQ ID NO:57)
PCl9RCYARRRFAVCR ( SEQ TD NO:58)
PC20LAYCRRRFCVAV ( SEQ ID NO:59)
PC21RAYCRRRFCVAR (SEQ ID NO:60)
PC22RGGRLCY RR VCV (SEQ ID NO:61)
PC31GGRLCYCRRRFCVCV ( SEQ ID NO:62)
PC32RGRLCYCRRRFCVCV ( SEQ ID NO:63)
PC33GRLCYCRRRFCVCV ( SEQ ID NO:64)
PC34RRLCYCRRRFCVCV (SEQ ID NO:65)
PC35RLCYCRRRFCVCV ( SEQ ID NO:66)
PC36RRCYCRRRFCVCV ( SEQ ID NO:67)
PC37CYCRRRFCVCV (SEQ ID NO:68)
PC44RGGRLCYCRRRFCVC (SEQ ID NO:69)
PC47RGGRLCY RRRF VCV ( SEQ ID NO:70)
PC48RGWRLCYCRRRFCVCV (SEQ ID NO:71)
PC37aCYCRRRFCVCVGR (SEQ ID NO:72)
PC45RGGRLCYCRRRFCV ( SEQ ID NO:73)
PC72LCYCRRRFCVC (SEQ ID NO:74)
PC64L~Y'1~KFTVCV (SEQ ID NO:75)
PC64aLTYCRRRFCVTV (SEQ ID NO:76)
PC3laGGRLCYCRRRFCVCVGR ( SEQ ID NO:77)
PC32aRGRLCYCRRRFCVCVGR ( SEQ ID N~:78)
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CODE SEQUENCE
PC33a GRLCYCRRRFCVCVGR (SEQ ID NO:79)
PC34a RRLCYCRRRFCVCVGR (SEQ ID NO:80)
PC35a RLCYCRRRFCVCVGR (SEQ ID NO:81)
PC36a RRCYCRRRFCVCVGR tSEQ ID NO:82)
PC44a RGGRLCYCRRRFCVCR (SEQ ID NO:83)
PC47a RGGRLCY RRRF VCVGR (SEQ ID NO:84)
PC48a RGWRLCYCRRRFCVCVGR (SEQ ID NO:85)
PC54 RGWRLAYCRRRFCVAVGR (SEQ ID NO:86)
PC61 RCYCRRRFCVCV (SEQ ID NO:87)
PC62 LCYCRRRFCVCR (SEQ ID NO:88)
PC63 VCYCFRRFCYCV (SEQ ID NO:89)
PC65 LCYTRPRFTVCV (SEQ ID NO:90)
PC66 LCYTRGRFTVCV (SEQ ID NO:91)
PC67 LCYFRRRFIVCV (SEQ ID NO:92)
~15 PC68 LCYFRPRFIVCV (SEQ ID NO:93)
PC69 LCYTFRPRFVCV (SEQ ID NO:94)
PC70 LCYTFRGRFVCV (SEQ ID NO:95)
PC74 CYCFRRFCVC (SEQ ID NO:96)
PC77 LCYCRRRRCVCV (SEQ ID NO:97)
PC78 LCYCFRRRCVCV (SEQ ID NO:98)
PC79 LCYCRFRRCVCV (SEQ ID NO:99)
PC80 LCYCRRFRCVCV (SEQ ID NO:100)
PC81 LCYCRRFFCVCV (SEQ ID NO:101)
PC82 LCYCRFFRCVCV (SEQ ID NO:102)
PC83 LCYCFFRRCVCV (SEQ ID NO:103)
PC84 LCYCFRRFCVCV (SEQ ID NO:104)
PC85 LCYCFRFRCVCV (SEQ ID NO:105)
PC86 LCYCRFRFCVCV (SEQ ID NO:106)
PC87 LCYCFRFFCVCV (SEQ ID NO:107)
PC88 LCYCFFRFCVCV (SEQ ID NO:108)
PC89 LCYCFFFRCVCV (SEQ ID NO:109)
PC90 LCYCRFFFCVCV (SEQ ID NO:100)
RGGRLCY RR VCVGR (SEQ ID NO:lll)
PC91 YCYCRRRFCVCVGR (SEQ ID NO:112)
- 32 -
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CODESEQUENCE
PC95ICYCRRRFCVCVGR (SEQ ID NO:113)
PC96FCYCRRRFCVCVGR (SEQ ID NO:114)
PC97WCYCRRRFCVCVGR (SEQ ID NO:115)
PC99RCYCRRRFCVCVGR (SEQ ID NO:116)
PC109RLCYTRGRFTVCV (SEQ ID NO:117)
PC110LCYTRGRFTVCVR (SEQ ID NO:118)
PC111RLCYTRGRFTVCVR (SEQ ID NO:119)
PC112LCYCHHHFCVCV (SEQ ID NO:120)
PC113L~Y~l~n~r~l~vCV (SEQ ID NO:121)
RGGLCYCRRRFCVCVGR (SEQ ID NO:122)
RGGRLCYCRRRFCVCVGR (SEQ ID NO:123)
RGGGLCYCRRRFCVCVGR (SEQ ID NO:124)
RGGGLCYCRRGFCVCFGR (SEQ ID NO:12~)
RGGGLCYCRRPFCVCVGR (SEQ ID NO:126)
RGGGLCYCRPRFCVCVGR (SEQ ID NO:127)
RGGRLCYCRXRFCVCVGR (X=MeGly) (SEQ ID NO:128)
RGGLCYCRGRFCVCVGR (SEQ ID NO:129)
RGGRLCYCXGRF~v~vG~ (X=Cit) (SEQ ID NO:130)
XGGRLCYCRGRFCVCVGR (X=Cit) (SEQ ID NO:131)
RGGRVCYCRGRFCVCVGR (SEQ ID NO:132)
RGGGLCYCFPKFCVCVGR (SEQ ID NO:133)
RGWGLCYCRPRF~v~v~K (SEQ ID NO:134)
RGWRLCYCRXRFCVCVGR (X=MeGly) (SEQ ID NO:135)
RGWRLCYCRGRFCVCVGR (SEQ ID NO:136)
RGWRLCYCXPRFCVCVGR (X=Cit) (SEQ ID NO:137)
RWRLCYCRPRFCVCVGR (SEQ ID NO:138)
RGWRLCYCRPRFCVCVGR (SEQ ID NO:139)
RGWRACYCRPRFCACVGR (SEQ ID NO:140)
GWRLCYCRPRFCVCVGR (SEQ ID NO:141)
RWRLCYCKGXFCVCVGR (SEQ ID NO:142)
RGWRLCYCRXRFCVCVGR (X=MeGly) (SEQ ID NO:143)
GGWRLCYCRGRFCVCVGR (SEQ ID NO:144)
RGGWLCYCRGRFCVCVGR (SEQ ID NO:145)
RLLRLCYCRXRFCVCVGR (X--MeGly) (SEQ ID NO:146)
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CODE SEQUENCE
RLLRACYCRXRF~v~v~K (X=MeGly) (SEQ ID NO:147)
RLLRLCYCRRRFCVCVGR (SEQ ID NO:148)
RGLRXCYCRGRFCVCVGR (X=Cha) (SEQ ID NO:149)
RGGRLCYCRXRZCVCWGR (X=MeGly) (SEQ ID NO:15~)
(Z=Cha)
RGGRWCVCRXRZCYCVGR (X=MeGly) (SEQ ID NO:151)
(Z=Cha)
RGLRXCYCRGRFCVCVGR (X=Cha) (SEQ ID NO:152)
RGGRWCVCRGRXCYCVGR (X=Cha) (SEQ ID NO:153)
RGGRLCYCRRRFCXCVGR (X=MeVal) (SEQ ID NO:154)
LCYCRRRFCVCV (SEQ ID NO:155)
LCYCRRCFCVCV (SEQ ID NO:156)
LCYCRRRFCVCF (SEQ ID NO:157)
LCACRRRACVCV (SEQ ID NO:158)
LCYCRXRFCVCV (X=D-Arg) (SEQ ID NO:159)
LCWCRRRFCVCV (SEQ ID NO:160)
WCYCRRRFCVCV (SEQ ID NO:161)
LCYCRRRXCVCV (X-hPhe) (SEQ ID NO:162)
LCYCRRRXCVCV (X=Phe(4-Cl)) (SEQ ID NO:163)
XCYCRRRFCVCV(X=Cha) (SEQ ID NO:164)
LCYCRRRFCXCV (X=D-His) (SEQ ID NO:165)
LCYCRRRXCVCV (X=MeGly) (SEQ ID NO:166)
LCYCRRRXCVCV (X=MePhe) (SEQ ID NO:167)
LCYCRRRFCXCV (X=MeVal) (SEQ ID NO:168)
LCXCRRRXCVCV (X=Cha) (SEQ ID NO:169)
LCGCRRRGCVCV (SEQ ID NO:170)
LCACRGRACVCV (SEQ ID NO:171)
RACYCRPRFCACV (SEQ ID NO:172)
RLCYCRPRFCVCF (SEQ ID NO:173)
RLCYCRPRFCVCV (SEQ ID NO:174)
KLCYCKPKFCVCV (SEQ ID NO:175)
RLCACRGRACVCV (SEQ ID NO:176)
R~CYCRXRFCVCV (X=MeGly) (SEQ ID NO:177)
RXCFCRPRFCVCV (X-Cha) (SEQ ID NO:178)
RWCFCRPRFCVCV (SEQ ID NO:179)
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CODE SEQUENCE
WLCYCRRRFCVCV (SEQ ID NO:180)
WLCFCRRRFCVCV (SEQ ID NO:181)
FLCFCRRRFCVCV (SEQ ID NO:182)
WLCFCRRRXCVCV (X=MePhe) (SEQ ID NO:183)
WYCYCRRRFCVCV (SEQ ID NO:184)
WXCYCRRRFCVCV (X=Cha) (SEQ ID NO:185)
RXCFCRGRZCVCV (X=Cha) (SEQ ID NO:186)
(Z=MePhe)
XLCFCRRRZCVCV (X=Cha) (SEQ ID NO:187)
(Z=MePhe)
RLCYCRPRFCVCVGR (SEQ ID NO:188)
WLCYCRRRFCVCVGR (SEQ ID NO:189)
WXCYCRRRFCVCVGR (X=Cha) (SEQ ID NO:190)
RLCYCRGPFCVCR (SEQ ID NO:191)
RRWCFVCYAGFCYRCR (SEQ ID NO:192)
RRCYCRGRFCGCVGR (SEQ ID NO:193)
RWRCYCGRRFCGCVGR (SEQ ID NO:194)
RARCYCGRRFCGCVGR (SEQ ID NO:195)
GWRCYCRGRFCGC (SEQ ID NO:196)
RGWACYCRGRFCVC (SEQ ID NO:197)
RRCYGRRRFGVCVGR (SEQ ID NO:198)
RGWRLCYGRGRFKVC (SEQ ID NO:199)
RGWRLCYCRGRFCVC (SEQ ID NO:200)
CYCRRRFCVCF (SEQ ID NO:201)
RGWRLCYCRXRFCVC (X--MeGly) (SEQ ID NO:202)
RGWRGCYCRXRFCGC (X=MeGly) (SEQ ID NO:203)
LCYCRPRFCVCVGR (SEQ ID NO:204)
LCYCKPKFCVCVGK (SEQ ID NO:205)
LCYCRGRFCVCVGR (SEQ ID NO:206)
LCYCRPRFCVCVGRGR (SEQ ID NO:207)
RRWCYCRPRFCVC~R (SEQ ID NO:208)
WRLCYCRPRFCVCVGR (SEQ ID NO:209)
GWLCYCRGRFCVCVGR (SEQ ID NO:210)
RWLCYCRGRFCVCVGR (SEQ ID NO:211)
RLLCYCRGRFCVCVGR (SEQ ID NO:212)
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CODE SEQUENCE
RWRLCYCRPRFCVCV (SEQ ID NO:213)
RXRLCYCRZRFCVCV (X=Cha) (SEQ ID NO:214)
(Z=MeGly)
RGWRLCYCRGRXCVCV (X=Cha) (SEQ ID NO:215)
RGGRVCYCRGRFCVCV (SEQ ID NO:216)
LCYCRXRFCVCV (X=D-Ala) (SEQ ID NO:217)
LCYCKPKFCVCV (SEQ ID NO:218)
VCYCRPRFCVCV (SEQ ID NO:219)
LCYCRPRFCVCW (SEQ ID NO:220)
LCYRRPRFRVCV (SEQ ID NO:221)
RGWRLCYCRGRXCVCV (X=Cha) (SEQ ID NO:222)
RXRLCYCRZRFCVCV (X=Cha) (SEQ ID NO:223)
(Z--MeGly)
RXRLCYCRGRFCVCV (X=Cha) (SEQ ID NO:224)
RGGGLCYARGWIAFCVGR (SEQ ID NO:225)
RGGGLCYARGFIAVCFGR (SEQ ID NO:226)
RGGGLCYARPRFAVCVGR (SEQ ID NO:227)
RGGGLCYTRPRFTVCVGR (SEQ ID NO:228)
RGGGLCYARKGFAVCVGR (SEQ ID NO:229)
RGGRLCYARRRFAVCVGR (SEQ ID NO:230)
RGGGLCYKRGFIKVCFGR (SEQ ID NO:231)
RGGGLCYKRGWIKFCVGR (SEQ ID NO:232)
RGGGLCYRLPKFRVCVGR (SEQ ID NO:233)
RGGGLCYRLPGFRVCVGR (SEQ ID NO:234)
RGWRGCYKRGRFKGCVGR (SEQ ID NO:235)
LCYKRGRFKVCV (SEQ ID NO:236)
ICYRPR~v~v~ (SEQ ID NO:Z37)
WLCYCRRRFCVCV (SEQ ID NO:238)
RLCYCRRRFCVCV (SEQ ID NO:240)
~GRVCLRYCRGRFCVRLCFR (SEQ ID NO:241)
RRRLCYCRRRFCVCVGR (SEQ ID NO:242)
and the N-acylated and/or C-amidated or esteri~ied forms
thereof.
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6.2.2 PreParation of the com~ounds
The invention compounds, often designated
herein "protegrins" are essentially peptide backbones which
may be modified at the N- or C-terminus and also may contain~ 5 one or two disulfide linkages. The peptides may first be
synthesized in noncyclized form. These peptides may then be
converted to the cyclic peptides if desired by st~n~Ard
methods of disulfide bond formation. As applied to the
protegrins herein, "cyclic forms" refers to those forms which
contain cyclic portions by virtue of the formation of
disulfide linkages between cysteine-like amino acid residues
in the peptide. If the non-cyclized forms are preferred, it
is preferable to stabilize the sulfhydryl groups for any
peptides of the invention which contain two or more cysteine-
like residues.
St~n~d methods of synthesis of peptides of the sizesdescribed herein are known. Most commonly used currently are
solid phase synthesis techniques; in~e~ automated
equipment for systematically constructing peptide ~~.h;~; n!: can
be purchased. Solution phase synthesis can also be used and
has considerable benefits for large scale production. When
synthesized using these st~n~d techniques, amino acids not
encoded by the gene and D-enantiomers can be employed in the
synthesis. Thus, one very practical way to obtain the
compounds of the invention is to employ these st~n~rd
chemical synthesis techn;ques.
In addition to providing the peptide backbone, the N-
and/or C-terminus can be derivatized, again using
conventional chemical techn;ques. The compounds of the
invention may optionally contain an acyl group at the amino
terminus. Methods for acetylating or, more generally,
acylating, the free amino group at the N-teL ;nn~ are
generally known in the art; in addition, the N-terminal amino
acid may be supplied in the synthesis in acylated form.
3~ At the carboxyl terminus, the carboxyl group may, of
course, be present in the form of a salt; in the case of
pharmaceutical compositions this will be a pharmaceutically
acceptable salt. Suitable salts include those formed with
CA 02238610 1998-0~-22
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inorganic ions such as NH4~, Na+, K~, Mgt~, Ca'~, and the like
as well as salts formed with organic cations such as those of
caffeine and other highly substituted amines. However, when
the compound of formula (I) contains a multiplicity of basic
residues, salt formation may be difficult or impossible. The
carboxyl terminus may also be esterified using alcohols o~
the formula ROH wherein R is hydrocarbyl as defined above~
Similarly, the carboxyl terminus may be amidated so as to
have the formula -CONH2, -CONHR, or -CONR2, wherein each R is
independently hydrocarbyl as herein defined. Techni~ues for
esterification and amidation as well as neutralizing in the
presence of base to form salts are all standard organic
chemical t~çhn;ques.
If the peptides of the invention are prepared under
physiological conditions, the side-chain amino groups of the
basic amino acids will be in the form of the relevant acid
addition salts.
For synthesis of linear peptide with a C-terminal amide,
the peptide sequence is conveniently synthesized on a Fmoc
Rink amide solid support resin (Bachem) using Fmoc chemistry
on an automated ABI g33 peptide synthesizer (ABD, Perkin
Elmer, Foster City, CA) according to the manu~acturer~s
st~n~rd protocols. Cleavage is typically carried out in 10
ml of thioanisole/EDT/TFA (1/1/93 for 2 hours at room
temperature. Crude cleavage product is precipitated with t-
butyl methyl ether, filtered and dried.
Formation of disulfide linkages, if desired, is
conducted in the presence of mild oxidizing agents. Chemical
oxidizing agents may be used, or the compounds may simply be
exposed to the oxygen of the air to effect these linkages.
Various methods are known in the art. Processes use~ul for
disulfide bond formation have been described by Tam, J.P. et
al., Synthesis (1979) 955-957; Stewart, J.M. et al., Solid
Phase PePtide SYnthesis, 2d Ed. Pierce Chemical Company
Rock~ord, IL (1984); Ahmed A.K. et al., J Biol Chem (1975)
250:8477-8482 and Pennington M.W. et al ., Peptides 19gO , E.
Giralt et al., ESCOM Leiden, The Netherlands (1991) 164-166.
An additional alternative is described by Kamber, B. et al .,
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Helv Chim Acta (1980) 63:899-915. A method conducted on
solid supports is described by Albericio, Int ~ Pept Protein
Res (1985) 26:92-97.
A particularly preferred method is solution oxidation
using molecular oxygen. This method has been used by the
inventors herein to refold synthetic PG-1, PG-3 in its amide
or acid forms, enantio PG-1 and the two unidisulfide PG-l
compounds (C6-C1s and C8-C13). Recoveries are as high as 65-
90% .
In this preferred method to form disulfide linkages, the
crude peptide is dissolved in DMSO and added to 20 mM
ammonium acetate buffer, pH 7. The final concentration of the
peptide in the solution is between 1-8 mg/ml, the pH ranges
from 7.0-7.2, and the DMSO concentration ranges from 5-20%.
1~ The peptide solution is stirred overnight at room
temperature.
The pE of the solution is adjusted to pH5 with
concentrated acetic acid and the sample purified on Prep LC.
After loading, the column is washed with 10% acetonitrile/H20
(0.1% TFA) until the W absorbance decreases to the baseline.
The gradient is then started.
Column: Vydac Cat#218TP101522, 2.2 x 25 cm, C18 peptides
& proteins; W ~: 235 nm; Flow Rate: 10 ml/min.
Solvent A is 100% 0.1% TFA/H2O; Solvent B is 100% 0.08%
T~A/ACN. The gradient is as follows.
T (min) %B (l; neA~ gradient)
0 10
18
32
Fractions are analyzed by analytical EPLC and those that
contain the desired peptide are combined. The acetonitrile
is stripped and the resulting aqueous solution lyophilized.
The resulting amide, cont~;n;ng sulfide bonds, is con~irmed
by mass spectrum.
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I~ the peptide backbone is comprised entirely of gene-
encoded amino acids, or if some portion of it is so composed,
the peptide or the relevant portion may also be synthesized
using recombinant DNA techniques. The DNA encoding the
peptides of the invention may itself be synthesized using
commercially available equipment; codon choice can be
integrated into the synthesis depending on the nature of the
host.
Recombinantly produced forms of the protegrins may
require subsequent derivatization to modify the N- and/or
C-teL ;~u~ and, dep~n~i n~ on the isolation procedure, to
effect the formation of disulfide bonds as described
hereinabove. Depending on the host organism used for
recombinant production and the animal source from which the
protein is isolated, some or all of these conversions may
already have been ef~ected.
For recombinant production, the DNA ~coA; ng the
protegrins of the invention is included in an expression
system which places these coding sequences under control of a
suitable promoter and other control sequences compatible with
an intended host cell. Types of host cells available span
almost the entire range of the plant and animal kingdoms.
Thus, the protegrins of the invention could be produced in
bacteria or yeast (to the extent that they can be produced in
2~ a nontoxic or refractile form or utilize resistant strains)
as well as in Ani ~- cells, insect cells and plant cells.
Indeed, modified plant cells can be used to regenerate plants
cont~;ning the relevant expression systems so that the
resulting transgenic plant is capable of self protection vis-
~-vis these infective agents.
The protegrins of the invention can be produced in a
form that will result in their secretion from the host cell
~y fusing to the DNA enco~;ng the protegrin, a ~NA encoding a
suitable signal peptide, or may be produced intracellularly.
They may also be produced as fusion proteins with additional
amino acid se~uence which may or may not need to be
subse~uently removed prior to the use of these compounds as
antimicrobials or antivirals.
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Thus, the protegrins of the invention can be produced in
a variety of modalities including chemical synthesis and
recombinant production or some combination of these
tPchn;ques.
Any members of the protegrin class which occur naturally
are supplied in purified and/or isolated form. By l'purified
and/or isolated" is meant free from the envi.o ~nt in which
the peptide normally occurs (in the case of such naturally
occurring peptides) and in a form where it can be used
practically. Thus, "purified and/or isolated" form means
that the peptide is substantially pure, i.e., more than 90%
pure, preferably more than 95~ pure and more preferably more
than 99~ pure or is in a completely different context such as
that of a pharmaceutical preparation.
6.2.3 Antibodies
Antibodies to the protegrins of the invention may
also be produced using st~n~rd immunological t~chn;ques for
production of polyclonal antisera and, if desired,
immortalizing the antibody-producing cells of the immunized
host for sources of monoclonal antibody production.
Techniques for producing antibodies to any substance of
interest are well known. It may be necessary to enhance the
immunogenicity of the substance, particularly as here, where
the material is only a short peptide, by coupling the hapten
to a carrier. Suitable carriers for this purpose include
substances which do not th~ ;clves produce an immune response
in the mammal to be ~ ; n; ~tered the hapten-carrier
conjugate. C~ ~n carriers used include keyhole limpet
hemocyanin (KLH), diphtheria toxoid, serum albumin, and the
viral coat protein of rotavirus, VP6. Coupling of the hapten
to the carrier is effected by st~ rd t~c-hn; ques such as
contacting the carrier with the peptide in the presence of a
dehydrating agent such as dicyclohexylcarbodiimide or through
the use of linkers such as those available through Pierce
Chemical Company, Chicago, IL.
The protegrins of the invention in immunogenic form are
then injected into a suitable mammalian host and antibody
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titers in the serum are monitored. It should be noted,
however, that some forms of the protegrins require
modification before they are able to raise antibodies, due to
their resistance to antigen processing. For example, the
native form of PG-1, containing two disulfide bridges is
nonimmunogenic when a~in;stered without coupling to a larger
carrier and was a poor immunogen even in the presence of
potent adjuvants and when coupled through glutaraldehyde or
to K~H. Applicants believe this to be due to its resistance
to attack by leukocyte serine proteases (human PMN elastase
and cathepsin G) as well as to attack by an aspartic protease
(pepsin) that resembles several macrophage cathepsins. The
lack of immunogenicity may therefore result from resistance
to processing to a linear form that can fit in the antigen-
presenting pocket of the presenting cell. Immunogenicity of
these forms o~ the protegrins can be enhanced by cleaving the
disulfide bonds. However, the immunogenicity can be enhanced
using the MAPS t~chn;que recently reported by Huang, W et
~1., Mol Immunol ~1994) 15:1191-1199
Po~yclonal antisera may be harvested when titers are
sufficiently high. Alternatively, antibody-producing cells
of the host such as spleen cells or peripheral blood
lymphocytes may be harvested and immortalized. The
immortalized cells are then cloned as individual colonies and
screened for the production of the desired monoclonal
ant; hoAi es.
Reco hinant t~chn;ques are also available for the
production of antibodies, and thus, the antibodies of the
invention include those that can be made by genetic
engineering t~chn;ques. For example, single-chain forms,
such as Fv forms, ch; -~ic antibodies, and antibodies modified
to mimic those of a particular species, such as humans, can
be produced using standard methods. Thus, the antibodies of
the invention can be prepared by isolating or modifying the
genes encoding the desired antibodies and producing these
through expression in recombinant host cells, such as CH0
cells.
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The antibodies of the invention are, of course, useful
in immunoassays ~or determining the amount or presence of the
protegrins. Such assays are essential in quality controlled
production of compositions cont~;ning the protegrins of the
invention. In addition, the antibodies can be used to assess
the efficacy of recombinant production of the protegrins, as
well as scr~;ng expression libraries for the presence of
protegrin encoding genes.
6.2.4 ~omPositions containinq the Proteqrins
and Methods of Use
The protegrins of the invention are effective in
inactivating a wide range of micro~ial and viral targets,
including Gram-positive and Gram-negative bacteria, yeast,
protozoa and certain strains of virus. Because of their
broad spectrum of activities, the protegrins of the invention
can be used as preservatives as well as in treatment and
prophylactic contexts.
They are bactericidal against Gram-positive bacteria
which include major pathogens, such as Staphylococcus aureus,
including MRSA (the methicillin resistant version) and MSSA
(the methicillin-sensitive strain), and Enterococcus faecium
and E. faecalis (including VREF or vancomycin resistant
E. faecium) and VSEF or vancomycin-sensitive E. faecalis).
These are very common pathogens in hospital settings. Other
Gram-positive bacteria which are suitable targets include
Listeria monocytogenes, Streptococcus pneumoniae ( including
PRSP, the penicillin resistant form), S. mitis, S. sanguis,
Staphylococcus epidermis ( including methicillin sensitive
strain MSSE), S. salivarius, Corynebacterium minutissium, C.
pseudodiphtheriae, c. striatum, Corynebacterium groups Gl and
G2, and Bacil l us subtil is . PRSP is also a wide-spread health
hazard.
Among Gram-negative organisms against which the
protegrins are effective are Escherichia coli, Pseudomonas
aeruginosa, ~le~siella pneumoniae, Serratia marcescens,
Haemophilus influenzae, Salmonel7a tyr~i rium, Acinetobacter
calocoaceticus, C. pneumoniae, and Neisseria meningitidus, as
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well as other species including those within the genera
represented above. For example, Neisseria gonorrhoeae is
~ssociated with sexually transmitted diseases (STDs) as is
Chlamydia trachomatis. Also among the Gram-negative
orgAnis~ are the gastric pathogens Hel1cobacter pylori,
H. felis, and Campylo~acter jejuni.
Besides Gram-positive and Gram-negative bacteria, the
protegrins of the invention are also effective against growth
and infection by mycobacteria such as M. tuberculosls and
M. avium (including MAC); fungi, such as Candida albicans and
the related pathogens, C. parapsilosis, C. krusei,
C. tropicalis and C. glabrata, as well as Aspergillus niger.
Among the viruses against which the protegrins are effective
are Herpes simplex I and II and Human immunodeficiency virus
(HIV).
The foregoing is not an exhaustive, but representative
list.
As stated above, the protegrins can also be used in
disinfectant compositions and as preservatives for materials
such as foodstuffs, cosmetics, medicaments, or other
materials contAin;ng nutrients for orgAn;! ~. For use in
such contexts, the protegrins are supplied either as a single
protegrin, in admixture with several other protegrins, or in
admixture with additional antimicrobial agents. In general,
as these are preservatives in this context, they are usually
present in relatively low amounts, of less than 5%, by weight
of the total composition, more preferably less than 1%, still
more preferably less than 0.1%.
The peptides of the invention are also useful as
standards in antimicrobial assays and in assays for
determination of capability of test compounds to bind to
endotoxins such as lipopolysaccharides.
For use as antimicrobials or antivirals for treatment of
animal subjects, the protegrins of the invention can be
formulated as pharmaceutical or veterinary compositions.
Depending on the subject to be treated, the mode of
administration, and the type of treatment desired -- e.g.,
prevention, prophylaxis, therapy; the protegrins are
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formulated in ways consonant with these parameters. A
Sl ~ry of such t~chn;ques is found in Remington's
Pharmaceutical Sciences, latest edition, Mac~ Publishing co.,
Easton, PA.
The protegrins can be used in animal subjects both as
therapeutic and prophylactic treatments; by "treating" an
infection is meant either preventing it from occurring,
ameliorating the symptoms, inhibiting the growth of the
microbe in the subject, and any other negative effect on the
microbe which is beneficial to the subject. Thus, "treating"
or ~treatment~ have both prophylactic and therapeutic
aspects.
The protegrins are particularly attractive as an active
ingredient in pharmaceutical compositions useful in treatment
of sexually transmitted diseases, including those caused by
Chlamydia trachomatis, ~reponema palli~um, Neisseria
gonorrhoeae, Trichomonas vaginalis, Herpes simplex type 2 and
HIV. Topical formulations are preferred and include creams,
salves, oils, powders, gels and the like. Suitable topical
excipients are well known in the art and can be adapted for
particular uses by those of ordinary skill.
In general, for use in therapy or prophylaxis of STDs,
the protegrins of the invention may be used alone or in
combination with other antibiotics such as erythromycin,
tetracycline, macrolides, for example azithromycin and the
cephalosporins. Depending on the mode of administration, the
protegrins will be formulated into suitable compositions to
permit facile delivery to the affected areas. The protegrins
may be used in forms cont~;n;ng one or two disulfide bridges
or may be in linear form. In addition, use of the
enantiomeric forms containing all D-amino acids may confer
advantages such as resistance to those proteases, such as
trypsin and chymotrypsin, to which the protegrins conta;n;ng
L-amino acids are less resistant.
The protegrins of the invention can be a~ ' n; ~tered
singly or as mixtures of several protegrins or in combination
~ with other pharmaceutically active components. The
formulations may be prepared in a manner suitable for
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systemic administration or topical or local aciri n;stration.
Systemic formulations include those designed for injection
(e.g., intramuscular, intravenous, intraperitoneal or
subcutaneous injection~ or may be prepared for transdermal,
transmucosal, or oral a~;n;stration. The formulation will
generally include a diluent as well as, in some cases,
adjuvants, buffers, preservatives and the like. The
protegrins can be administered also in liposomal compositions
or as microemulsions.
If administration is to be oral, the protegrins of the
invention should be protected from degradation in the
digestive tract using a suitable enteric coating. This may
be avoided to some extent by utilizing amino acids in the
D-configuration, thus providing resistance to protease. The
protegrins are relatively acid stable, however, some degree
of enteric coating may still be required.
The protegrins of the invention retain their activity
~gainst microbes in the context of borate solutions that are
commonly used in eye care products. It has also been shown
that when tested for antimicrobial activity against E. coli
in the presence and absence of lysozyme in borate buffered
saline, that the presence of lysozyme ~nhAnced the
effectiveness of PG-3. This effect was more pronounced when
the PG-3 was autoclaved and similar patterns were obtained
for both the ~ree-acid form and the amide. Accordingly, the
protegrins may be used as preservatives in such compositions
or as antimicrobials for treatment of eye infections, such as
conjunctivitis and corneal ulcers.
It is particularly important that the protegrins retain
their activity under physiological conditions including
relatively high salt and in the presence of serum. In
addition, the protegrins are dramatically less cytotoxic with
respect to the cells of higher organisms as compared with
their toxicity to microbes. These pr~perties, described
3~ hereinbelow in the Examples, make them particularly suitable
for in vivo and therapeutic use.
As the examples will show, by appropriately choosing the
member of the protegrin class of the invention, it is
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possible to adap~ the antimicrobial activity to ~; ;7e its
effectiveness with respect to a particular target microbe.
As used herein, "microbe" will be used to include not only
yeast, bacteria, and other unicellular orgAn;~ ~, but also
viruses. For example, PC-l9 is particularly effective, as
co~p~red to PG-l, under certain conditions. The particular
protegrin can also be chosen to be advantageous in a
particular context, such as low salt or physiological salt,
the presence or human serum, or conditions that mimic the
conditions found in blood and tissue fluids.
The protegrins of the invention may also be applied to
plants or to their environment to prevent virus- and microbe-
induced diseases in these plants. Suitable compositions for
this use will typically contain a diluent as well as a
spreading agent or other ancillary agreements beneficial to
the plant or to the environment.
Thus, the protegrins of the invention may be used in any
context wherein an antimicrobial and/or antiviral action is
required. This use may be an entirely in vitro use, or the
peptides may be a~~ ; n; ~tered to orgAn;~ -.
In addition, the antimicrobial or antiviral activity may
be generated in situ by administering an expression system
suitable for the production of the protegrins of the
invention. such expression systems can be supplied to plant
and animal subjects using known t~chn;ques. For example, in
~ , pox-based expression vectors can be used to generate
the peptides in situ. Similarly, plant cells can be
transformed with expression vectors and then regenerated into
whole plants which are capable of their own production of the
peptides.
The protegrins are also capable of inactivating
endotoxins derived from Gram-negative bacteria -- i.e.,
lipopolysaccharides (LPS) -- in stAn~Ard assays.
Accordingly, the protegrins may be used under any
circumstances where inactivation of LPS is desired. One such
situation is in the treatment or amelioration of ~ram-
negative sepsis.
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6.2.5 Conditions Relevant to
Antimicrobial/Antiviral ActivitY
It has been stated above that as used herein
'lantimicrobial" activity refers to inhibition with respect
both to traditional microorganisms and to viruses, although
occasionally, llantimicrobiali' and 'lantiviral'l are both
specifically indicated.
A particularly useful property of the protegrins is
their activity in the presence of serum. Unlike defensins,
protegrins are capable of exerting their antimicrobial
effects in the presence of serum.
Media for testing antimicrobial activity can be designed
to mimic certain specific conditions. The standard buffer
medium, medium A, uses an underlay agar with the following
composition: 0.3 mg/ml of trypticase soy broth powder, 1%
w/v agarose and 10 mM sodium phosphate buffer (final p~ 7.4).
This will be designated either llmedium A" or llst~n~rd in
vitro conditionsll herein.
All of the remaining media contain these same
components. However, in addition:
A second medium contains lOo mM NaCl in order to mimic
the salt levels in blood and tissue fluids. This will be
designated "medium Bll or llsalt mediumll herein.
A third medium is supplemented with 2.5% normal human
serum; however, it is of low ionic strength and thus does not
mimic body fluids. This medium will be designated "medium C"
or 'Iserum-cont~;ning mediumll herein.
A fourth medium contains 80% RPMI-1640, a standard
tissue culture medium which contains the principal ions and
amino acids found in blood and tissue fluids. In addition,
it contains 2.5% normal human serum. This will be designated
'medium Dll or 'Iphysiological mediumll herein.
Other media useful for testing antimicrobial properties
are provided in the Examples.
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6.2.6 Speci~ic Indications
Certain of the protegrins of the invention have
been found to be particularly effective in treating certain
indications. For example, in treating microbial infections
where the infectious agent is Staphylococcus aureus,
particularly methicillin resistant strains, applicants have
found the amides of the formulae:
RGWRLCYCRPRFCVCVGR (SEQ ID NO:139)
GWRLCYCRPRFCVCVGR (SEQ ID NO:141)
XCYCRRRFCVCVGR (X = Cha) (SEQ ID NO:164)
WLCYCRRRFCVCV (SEQ ID NO:180)
to be particularly effective. Also preferred is the free-
acid form of WLCYCRRRFCVCV (SEQ ID NO:180).
In treating Pseudomonas infection, applicants have found
the following amides to be effective:
RGGRLCYCRRRFCVCVGR (SEQ ID NO:1)
RGGRLCYCRPRFCVCVGR (SEQ ID NO:239)
RGGGLCYTRPRFTVCVGR (SEQ ID NO:228)
Also effective is the free-acid form of the peptide
RLCYCRRRFCVCV (SEQ ID NO:66).
Finally, in treating infections caused by H.pylori,
applicants have found the following compounds in the amide
form to be particularly effective:
WLCYCRRRFCVCV (SEQ ID NO:180)
RXCFCRPRFCVCV (X=Cha) (SEQ ID NO:178)
RG&RLCYCRRRFCVCVGR (SEQ ID NO:1)
RGWGLCYCRPRFCVCVGR (SEQ ID NO:134)
RLCYCRPRFCVCVGR (SEQ ID NO:138)
RGWRLCYCRGRFCVCVGR (SEQ ID NO:136)
RGRVCLRYCRGRFCVRLCFR (SEQ ID NO:241)
RGLRXCYCRGRFCVCVGR (X=Cha) (SEQ ID NO:149)
GWRLCYCRPRFCVCVGR (SEQ ID NO:139)
RLCYCRRRFCVCV (SEQ ID NO:66)
WLCYCXXXFCVCV (X=Dab) (SEQ ID NO:29)
OWRLCYCRPKFCVCV (SEQ ID NO:33)
RWOLCYCRPKFCVCV (SEQ ID NO:37)
RRCYCRRRFCVCVGR (SEQ ID NO:82)
XCYCRRRFCVCV (X=Cha) (SEQ ID NO:164)
45 Also effective are the free-acid and enantio (all-D) forms of
the peptide RRRLCYCRRRFCVCVGR (SEQ ID NO:240).
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6.3 ~ffective Dosaqes
The peptides of the invention, or compositions
thereof, will generally be used in an amount effective to
achieve the intended purpose. Of course, it is to be
understood that the amount used will depend on the particular
application.
For example, for use as a disinfectant or preservative,
an antimicrobially effective amount of a peptide, or
composition thereof, is applied or added to the material to
be disinfected or preserved. By antimicrobially effective
amount is meant an amount of peptide or composition that
inhibits the growth of, or is lethal to, a target microbe
population. While the actual antimicrobially effective
amount will depend on a particular application, for use as a
disinfectant or preservative the peptides, or compositions
thereof, are usually added or applied to the material to be
disinfected or preserved in relatively low amounts.
Typically, the peptide comprises less than about 5% by weight
of the disinfectant solution or material to be preserved,
preferably less than about 1% by weight and more preferably
less than about 0.1% by weight. An ordinarily skilled
artisan will be able to determine antimicrobially effective
amounts of particular peptides for particular applications
without undue experimentation using, for example, the in
vltro assays provided in the examples.
For use to treat or prevent microbial infections or
diseases related thereto, the peptides of the invention, or
compositions thereof, are Al' i n; ctered or applied in a
therapeutically effective amount. By therapeutically
effective amount is meant an amount effective top ameliorate
the symptoms of, or ameliorate, treat or prevent microbial
infections or diseases related thereto. Determination of a
therapeutically effective amount is well within the
capabilities of those s~illed in the art, especially in light
of the detailed disclosure provided herein.
As in the case of disinfectants and preservatives, for
topical administration to treat or prevent bacterial, yeast,
fungal or other infections a therapeutically effective dose
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can be determined using, for example, the in vitro assays
provided in the examples. The treatment may be applied while
the infection is visible, or even when it is not visible. An
ordinarily skilled artisan will be able to determine
therapeutically effective amounts to treat topical infections
without undue experimentation.
For systemic a~ ;n;~tration, a therapeutically effective
dose can be estimated initially from in vltro assays. ~or
example, a dose can be formulated in animal models to achieve
a circulating peptide concentration range that includes the
ICso as dete~ ; ne~ in cell culture (i .e ., the concentration of
test compound that is lethal to 50% of a cell culture), the
MIC, as determined in cell culture (i.e., the minimal
inhibitory concentration for growth3 or the IC1oo as
determined in cell culture (i .e., the concentration of
peptide that is lethal to 100% of a cell culture). Such
information can be used to more accurately determine useful
doses in humans.
Initial dosages can also be estimated from ln vivo data,
e.g., ~n; ~1 models, using techn;ques that are well known in
the art. One having ordinary skill in the art could readily
optimize administration to humans based on animal data.
Dosage amount and interval may be adjusted individually
to provide plasma levels of the active peptide which are
sufficient to maintain therapeutic effect. Usual patient
dosages for administration by injection range from about 0.1
to 5 mg/kg/day, preferably from about 0.5 to 1 mg/kg/day.
Therapeutically effective serum levels may be achieved by
A~ ' ; n; stering multiple doses each day.
In cases of local administration or selective uptake,
the effective local concentration of peptide may not be
related to plasma concentration. One having skill in the art
will be able to optimize therapeutically effective local
dosages without undue experimentation.
3S The amount of peptide a inistered will, of course, be
dependent on the subject being treated, on the subject's
- weight, the severity of the affliction, the manner of
administration and the judgment of the prescribing physician.
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The antimicrobial therapy may be repeated intermittently
while lnfections are detectable or even when they are not
detectable. The therapy may be provided alone or in
combination with other drugs, such as for example antibiotics
or ot~er antimicrobial peptides.
6.4 ToxicitY
Preferably, a therapeutically effective dose of the
peptides described herein will provide therapeutic benefit
without causing substantial toxicity.
Toxicity of the peptides described herein can be
determined by standard pharmaceutical procedures in cell
cultures or experimental animals, e.g., by determining the
LDso (the dose lethal to 50% of the population) or the LDlco
(the dose lethal to 100% of the population). The dose ratio
between toxic and therapeutic effect is the therapeutic
index. Compounds which exhibit high therapeutic indices are
preferred. The data obtained from these cell culture assays
and ~n; ~1 studies can be used in formulating a dosage range
that is not toxic for use in human. The dosage of the
peptides described herein lies preferably within a range of
circulating concentrations that include the effective dose
with little or no toxicity. The dosage may vary within this
range depending upon the dosage form employed and the route
of a~ ;nictration utilized. The exact formulation, route of
a~ ;ni~tration and dosage can be chosen by the individual
physician in view of the patient~s condition. ~See, e.g.,
Fingl et al., 1975, In: The Pharmacoloqical Basis of
Thera~eutics, Ch.l, p.1).
SummarY
The protegrins therefore represent a peculiarly useful
class of compounds because of the following properties:
1) They have an antimicrobial effect with respect to a
broad spectrum of target microbial systems, including
viruses, including retroviruses, bacteria, fungi, yeast and
protozoa.
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2) Their antimic~obial activity is effective under
physiological conditions - i.e., physiological saline and in
the presence of serum.
3) They are markedly less toxic to the cells of higher
organisms than to microbes.
4) They can be prepared in non; lnogenic form thus
ext~n~;ng the number of species to which they can be
administered.
5) They can be prepared in forms which are resistant
to certain proteases suggesting they are antimicrobial even
in lysosomes.
6) They can be modified in amino acid sequence so as
to optimize the specificity with respect to target.
7) They can be modified structurally so as to
accommodate the conditions under which antimicrobial activity
is to be exhibited.
The following examples are intended to illustrate but
not to limit the invention.
~m~le 1: Isolation and ActivitY of PG-1, PG-2 and PG-3
As described in PCT/US94/08305, referenced above, three
antimicrobial peptides were isolated from porcine leukocytes
having the amino acid sequences:
PG-1: RGGRLCYCRRRFCVCVGR
PG-2: RGGRLCYCRRRFCICV
PG-3: RGGGLCYCRRRFCVCVGR,
which are amidated at the C-terminus.
These particular protegrins were tested for
antimicrobial activity.
It was shown that PG-1 and PG-3 are more effective
against E. coli ML-35P than human neutrophil peptide ~NP-1
and only slightly less effective than rabbit defensin NP-1.
PG-1 and PG-3 were also effective against Listeria
monocytogenes, strain EGD and against Candida albicans. In
general, these peptides are approximately as effective as
rabbit defensin NP-1 on a weight basis and are more effective
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than HNP-1. In all cases, PG-2 was also effective against
the three organisms tested, but was not as active as the
other two peptides.
PG-1 has also been shown directly to inhibit the growth
of Stap~ylococcus aureus and K. pneumoneae 270. HNP-1 used
as a control was less effective against S. aureus and almost
entirely ineffective against ~. rner7~oneae.
The protegrins have also been tested against various
other organisms and show broad spectrum activity. In
addition to their effectiveness in inhibiting the growth of
or infection by microorganisms associated with STDs, the
protegrins show strong activity against the following
microorganisms in addition to those tested hereinabove:
Pseudomonas aeruginosa, Klebsiella pneumoniae, Salmonella
ty~h;ml~rium, Staphylococcus aureus, Histoplasma capsulatum,
Myobacterium avium-intracellulare, and Mycobacterium
tuberculosis. The protegrins showed only fair activity
against Vlbrio vulnificus and were inactive against Vibrio
c~olerae and Borrelia burgdorferi.
The three protegrins described above retain their
activity in the contexts of a variety of reaction conditions,
including the presence of 100 mM NaCl and the presence of 90
fetal calf serum.
The protegrins have a spectrum of retention of
antimicrobial properties under useful physiological
conditions, including isotonic and borate solutions
appropriate for use in eye care products.
PG-1 and PG-3 retain their activity with respect to
C. albicans and E. coli respectively, in the presence of
100 mM NaCl. Neither NP-1 nor HNP-1 have this property.
Although NP-l and NHP-2 lose their ability to inhibit
C. al~icans in 90% fetal calf serum, inhibition by PG-3 is
retained.
The protegrins show varying patterns of activity when
reaction conditions are altered. Synthetic PG-l was tested
against E. coli ML-3~ (serum sensitive) in underlayered gels
containing only 10 mM sodium phosphate buffer, pH 7.4 and a
1:100 dilution of trypticase soy broth, both in the presence
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and absence of 2.5% normal human serum, which is below the
lytic concentration for this strain of E. coli. In the
presence of serum, the i n; ~1 bactericidal concentration was
reduced from approximately 1.0 ~g/ml to about 0.1 ~g/ml.
This type of effect was not observed either for a linear
fragment of cathepsin G or for the defensin HNP-l.
Similarly, using C. albicans as a target organism,
underlayers were prepared with 10 mM sodium phosphate with
and without 10~ normal human serum. The ~;n; ~1 fungicidal
co~c~ntration fell from about 1.3 ~g/ml in the absence of
serum to 0.14 ~g/ml in its presence. The serum itself at
this concentration did not effect C. albicans. Similar
results were obtained using L. monocytogenes as the target
organism.
The protegrins PG-1 and PG-3 were incubated for 4 hours
at pH 2.0 with 0.5 ~g/ml pepsin and then neutralized. The
residual antimicrobial activity against C. albicans, E. coli
and L. monocytogenes was assessed and found to be fully
retained. Similar experiments show that these compounds are
not degraded by human leukocyte elastase or by human
leukocyte cathepsin G even when exposed to high
concentrations of these enzymes and at a pH of 7.0 - 8.0
favorable for proteolytic activity. In addition, synthetic
PG-3 amide and synthetic PG-3 acid were autoclaved and tested
for antimicrobial activity against E. coli, L. monocytogenes
and C. albicans, ret~;ning full antimicrobial activity in all
cases. It is possible that the stability of these compounds
to protease degradation and to autoclaving is enhanced by the
presence of disulfide bonds.
The protegrins were also tested for their ability to
bind the lipid polysaccharide (LPS) of the Gram-negative
bacterium ~. coli strain 0.55B5.
Both synthetic and native PG-l, PG-2 and PG-3 in the
amidated and nonamidated forms are able to bind LPS at
concentrations as low as 2.5 ~g/0.2 ml. nPG-l and nPG-2 are
effective at somewhat lower concentrations. The protegrins
were substantially more effective than the NP or HNP test
compounds; the most effective among these controls was NP-3a,
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a peptide whose primary sequence most closely resembles that
of the protegrins.
Inhibition of gelation can be overcome by increasing the
concentration of LPS, therefore interaction with LPS is
responsible for the lack of gelation, rather than interfering
with the gelation enzyme cascade.
nPG-1 and nPG-3 were converted to linear form using a
reducing agent to convert the disulfide linkages to
sulfhydryl groups, which were then stabilized by alkylating
with iodoacetamide.
The linearalized forms of the protegrins are equal to
cyclic forms in inhibiting gelation in the endotoxin assay
and in binding to endotoxin.
Both linearalized and cyclic forms of the protegrins
tested continue to show antimicrobial activity, although the
effectiveness of these peptides as antimicrobials depends on
the nature of the target organism and on the test conditions.
The antimicrobial activity of cyclic and linearalized
PG-l and PG-3 in the concentration range 20 ~g/ml-125 ~g/ml
with respect to E. co7i ML-35P was measured with and without
100 mM NaCl. The linear form was slightly more potent in the
presence of buffer alone than was the cyclic form; on the
other hand, the cyclic form was more potent than the linear
form under isotonic conditions.
For L. monocytogenes, both cyclic and linearalized forms
of the protegrins showed strong antimicrobial activity in the
absence of salt and both were approximately e~ually effective
over the concentration range tested (20 ~g/ml-125 ~g/ml).
The cyclic form retained strong antimicrobial activity in
100 mM NaCl with a slightly greater concentration dependence.
Linearalization appeared to lower the activity appreciably
although high concentrations were still able to show an
antimicrobial effect.
A11 forms of these protegrins were effective against
C. albicans in a dose-dependent manner over the above
concentration range when tested in the presence of 10 mM
phosphate buffer alone, although the linearalized peptides
were very slightly less effective. While the cyclized forms
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retained approximately the same level of antimicrobial e~fect
in 100 mM NaCl, the activity of the linearalized forms was
greatly ~; ;n;~h~ so that at concentrations below 100 ~g/ml
of the protegrin, virtually no antimicrobial effect was seen.
At higher concentrations of 130 ~g/ml, a moderate
antimicrobial effect was observed.
Thus, depending on the target microorganism and the
conditions used, both the cyclized and linearalized forms of
the PG-1 and PG-3 have antimicrobial activity.
Contact lens solutions are typically formulated with
borate buffered physiological saline and may or may not
contain EDTA in addition. Protegrins in the form of the
synthetic PG-3 amide and synthetic PG acid were tested
wherein underlay gels contain 25 mM borate buffer, pH 7.4, 1%
(v/v) tryptocase soy broth (0.3 ~g/ml TSB powder) and 1%
agarose. Additions included either 100 mM NaCl, 1 mM EDTA or
a combination thereof. Other test compounds used as controls
were the defensin NP-1 and lysozyme. Dose response curves
were dete ; ne~ .
Although these protegrins are somewhat less active in
25 mM borate buffered saline than in 25 mM phosphate buffer,
the antimicro~ial activity is ~h~n~ by adding
physiological saline and modestly enhanced by 1 mM EDTA.
Tests with C. albicans and with L. monocytogenes
indicate that the protegrins are capable of exerting their
antimicrobial effects under conditions typically associated
with conditions suitable for eye care products.
Bxam~le 2: RecoverY of cDNA Clones
As described in WO 95/03325 referenced above, cDNAs
encoding PG-1, PG-2, PG-3 and PG-4 were prepared from porcine
leukocytes and the DNA sequences encoding these protegrins
are set forth in Figure 7 of the published application.
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Exam~le 3: Recovery of Genomic DNA Encoding
PG-1 PG-3 and PG-5
High molecular weight genomic DNA was purified from pig
white blood cells with the QIAGEN blood DNA kit (QIAGEN,
Chatsworth, CA). To amplify protegrin (PG) genes, PCR as
performed using genomic DNA as a template.
The sense primer (5J-GTCGGAATTCATGGAGAC~A~-AG(A or
G)GCCAG-3') corresponded to the 5' regions of PG cDNAs, of
Example 2 and provided an EcoRI restriction site. The anti-
sense primer ~5'-GTCGTCTAGA~C or G)GTTTCAC~A~-AA~TTATTT-3')
was complementary to 3' ends of PG cDNAs ; ?~;ately
pr~c~;ng their poly(A) tails and provided an XbaI
restriction site. The reaction was carried out in a total
volume of 50 ~1, which contained 200 ng of purified pig
genomic DNA, 25 pmoles of each primer, 1 ~g of 10 mM dNTP, 5
~g of 10X PCR buffer (200 m~ Tris-HCl, 100 mM(NH4)2, 20 mM
MgSO~, 1% Triton X-100, 0.1% BSA), and 2.5 units of cloned Pfu
DNA polymerase (Stratagene, La Jolla, CA). Thirty cycles
were performed, each with 1 min of denaturation at 94~C, 1
min of primer annealing at 55~C, 2 min of primer extension at
72~C, and a final extension step at 72~C for 10 min.
The amplified PCR product was digested with EcoRI and
XbaI, excised from the agarose gel, purified, and ligated
into pBluescript KS+ vector (Stratagene, La Jolla, CA) that
had been digested with EcoRI and XbaI and purified. Both
strands of DNA were sequenced by the dideoxy method using the
Sequenase version 2.0 kit (United States Biochemical,
Cleveland, OH), pBluescript universal primers and specific
oligomer primers based on PG genomic and cDNA sequences.
Computer analysis of the DNA sequences was performed using
the PC-Gene Program (Intelligenetics, Palo Alto, CA).
A PCR product of about 1.85 kb was confirmed as
protegrin-re-ated by hybridization with a protegrin-specific
oligonucleotide probe complementary to nt 403-429 of the
protegrin cDNA seqll~nre~. The PCR product was then subcloned
into pBluescript vector, and recombinant plasmids were
subjected to DNA purification and sequencing. Gene sequences
for three different protegrins were identified PG-~, PG-3 and
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PG-s. The nucleotide sequences and deduced amino acid
se~uences are shown in Figure 1.
Comparison of protegrin cDNAs and genes revealed that
the coding regions of protegrin genes consisted of four
exons, interrupted by three introns (Figure 2). The first
exon contained the 5' noncoding region and codons for the
first 66 amino acids of the protegrin prepropeptide,
including a 29 residue signal peptide and the first 37
cathelin residues. Exons II and III were relatively small,
only 108 and 72 bp respectively, and together cont~;ne~ the
next 60 cathelin residues. The final two cathelin residues
were on Exon IV, and were followed by the protegrin
sequences. The exon-intron splice site sequences are shown
in Table 1, and conform to the consensus rule: all introns
end on an AG doublet, preceded by a T/C rich stretch of 8-12
bases, while all introns start with GT, followed
pred~ ;n~ntly by A/G A/G G sequence.
Table 1
Exon-Intron 8tru¢ture of the PG-1 Gene
Exon Size 5' ~plice donor Intron Size 3' ~plice ~c~ o~
1?~198 AAGGCCgtgagtcg 1 405 tt~c~ ~r-~-
2108 AACGGGgtgaggct 2 152 ccttccagCGGGTG
372 AATGAGgtgagtgg 3 596 ggtcacagGTTGAA
2S 4 313
The highly conserved cathelin region spans exons I-IV
and ~xon IV contains the full sequence of the mature
protegrin peptide followed by an amidation consensus
sequence, a 3~ untranslated region, and the putative
polyadenylation site. The three introns range in size from
152 to 596 bp. If the protegrin genes are representative of
other cathelin-like genes, the third intron o~ cathelin-
associated peptides will be found to separate all but the
last two residues of the highly conserved cathelin region
from the variable antimicrobial peptides encoded in Exon IV.
Such a layout would favor recombination m~rh~ni~m~ involving
association of diverse Exon IVs with the first three exons
specifying cathelin cont~ g prepro-regions.
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The family of naturally occurring protegrins thus
contains at least 5 members. Figure 5 shows a comparison of
the amino acid sequences of the five protegrins found so far
in porcine leukocytes. There is complete homology in
positions 1-3, 5-9, 13 and 15-16.
Homology search of protegrin genes against the
EMBL/GenBank identified no significantly homologous genes.
More specifically, the gene structures and nucleotide
se~uences of protegrins were very different from those of
defensins, which contain three exons in myeloid defensin
genes, and two exons in enteric defensin genes. As expected,
the search yielded the large family of cDNAs corresponding to
cathelin-associated bovine, porcine and rabbit leukocyte
peptides.
To assess protegrin-related genes further, we screened a
porcine genomic library of approximately 2.3 x 105 clones in
EMBL-3 SP6/T7 with the 32P-labeled protegrin cDNA, and
identified 45 hybridizing clones.
A porcine liver genomic library in EMBL3 SP6/T7 phages
was purchased from Clontech (Palo Alto, CA). E. coli strain
K803 was used as a host, and DNA from phage plaques was
transferred onto nylon membranes (DuPont, Boston, MA). The
filters were hybridized with 32P-labeled porcine 691 PG-3
cDNA. The filters were washed several times, finally at 60~C
in O.lx SSC and 0.1% SDS, and exposed to x-ray film with an
intensifying screen at -70~C. Positive clones were subjected
to two additional rounds of pla~ue purification at low
density.
DNA purified from hybridizing clones was digested with
various restriction endonucleases (New ~ngland Biolabs,
Beverly, MA), fractionated on 0.8% agarose gels, and
transferred onto GeneScreen Plus mem~rane (DuPont, Boston,
MA). The hybridization probes were labeled with 32p and
included porcine PG-3 cDNA, and 5~-labeled protegrin-specific
oligonucleotide complementary to nt 403-429 of PG-l, 2 and 3
cDNAs. For the cDNA probe, the hybridization and washing
conditions were carried out as for the library screening.
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For ~he oligonucleotide probe, the membranes were washed at
42~C in O.lx SSC, 0.1% SDS.
Southern blot analysis was carried out with purified DNA
from positive clones by hybridization with protegrin cDNA and
a protegrin specific oligonucleotide complementary to nt 403-
429 of protegrin cDNA sequences. Although all of the clones
hybridized with the complete cDNA probe, on~y about half of
them hybridized with the protegrin-specific probe. A
specific oligonucleotide probe for porcine prophen;n, another
cathelin-associated porcine leukocyte-derived antimicrobial
peptide, hybridized to several of the nonprotegrin clones.
These results confirm a) that the conserved proregion
homologous to cathelin is present within the same gene as the
mature antimicrobial peptides and is not added on by
posttranscriptional events, and b) that the protegrins
account for about half of the cathelin-related genes in the
pig.
A synthetic peptide corresponding to the amino acid
seguence of PG-5 was prepared and tested with respect to
antimicrobial activity against E. coli, L. monocytogenes and
c. albicans. The results were c ,-red to those obtained
with a synthetically prepared PG-1. The results are shown in
Figures 3a-3c. As shown in these graphical representations
of the results, PG-~ has comparable antimicrobial activity to
PG-1 against all three organisms tested.
ExamPle 4: PreParation of EnantioPG-1
Using st~n~rd solid phase t~hn i ~ues, a protegrin
having the amino acid sequence of PG-l, but wherein every
amino acid is in the D form was prepared. This form of
protegrin was tested against E. Co 7 i, L. monocytogenes,
C. al~lcans and other microbes in the absence and presence of
protease and otherwise as described for the radiodiffusion
assay in agarose gels, generally as described by Lehrer, R.I.
et al. J Immunol Met~ ~1991) 137:167-173. The results show
that both native PG-1 and enantio PG-1 in the absence of
protease are equally effective in inhibiting the growth of
E. coli. Neither trypsin nor chymotrypsin inhibits the
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W O 97/18826 PCT~US96/18544
antibacterial effect of enantio PG-1. In the presence of
these proteolytic enzymes, the ability of native PG-1 to
inhibit the growth of L. monocytogenes is adversely affected,
although in the absence of these proteases PG-l is comparably
active to an enantio PG-1.
Using similar techniques, the enantio forms of PG-2,
PG-3, P~-4 and PG-5 are synthesized. Similarly, the enantio
forms of the specific compounds set forth herein are
prepared.
~xamPle 5: Activity of the Protegrins Against
STD Pathoqens
As reported in W0 95/03325, PG-1 was tested against a
variety of organisms which are responsible for the infections
associated with sexually transmitted diseases (STDs). PG-1
was highly active against HIV-1, Chlamydia trachomatis,
Treponema pallidum, Neisseria gonorrhoeae, and was moderately
active against Trlchomonas vagi n~ 7 i.c and Herpes simplex
type 2. PG-1 appeared inactive against Herpes simplex type
1, however, another protegrin, the amide form of
RGGLVYVRGRFCVCVGR, was active against Herpes Simplex virus
type 1.
ExamPle 6: Antiretroviral ActivitY
Both synthetic and native PG-1 and native PG-2 showed
antiviral activity against various strains of HIV using the
method described in Miles, S.A. et al., Blood (1991) 78:3200-
3208, as set forth in W0 95/03325.
The protegrins show similar activity with respect to
other retroviruses.
E~amPle 7: Preparation of Modified Protegrins:
Kite and Bullet Forms
The kite and bullet forms of PG-1 wherein all Z are
~l~n;ne were synthesized using conventional Fmoc chemistry.
The crude synthetic peptide was reduced by adding
dithiothreitol (DTT) equal in weight to the synthetic peptide
which had been dissolved at 10 mg peptide/ml in a solution
cont~;n;ng 6 molar guanidine HCl, 0.5 molar tris buffer, and
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WO 97/18826 PCT/US96/18~;44
2 mM EDTA, pH 8.05 and incubated for two hours at 52~C under
nitrogen. The mixture was passed through a 0.45 ~m filter,
acidified with 1/20 (v/v) glacial acetic acid and subjected
to conventional RP-HPLC purification with a C-18 column.
HPLC-purified, reduced synthetic bullet and kite PG-l were
partially concentrated by vacuum centrifugation in a speed
vac and allowed to fold for 24 hours at room temperature in
ambient air in o.l M Tris pH 7.7 at low concentration (0.1 mg
peptide/ml) to minimize formation of interchain disulfide
disulfides. The mixture was then concentrated and acidified
with HOAC to a final concentration of 5% and subjected to
RP-HPLC purification.
The purity of the final products bullet and kite PG-l
was verified by AU-PAGE, analytical HPLC, and FAB-mass spec.
AU-PAGE showed a single band for the final product in each
case. The observed MH+ mass values were z093 in both cases.
Exam~le 8: Antimicrobial Activity of the Kite
and Bullet Forms
The kite and bullet PG-l compounds prepared in Example 7
were tested for antimicrobial activity using the radial
diffusion assay as published by Lehrer, R.I. et al ., J
T nol Meth (1991) 137:167-173, except that the underlay
agars contained 10 mM sodium phosphate buffer with a final pH
of 7.4. As described in Example 1, 0.3 mg/ml ~rypticase soy
broth powder and 1% agarose were used as well in the underlay
agar. In some cases 100 mM NaCl or RPMI plus 2.5% normal
human serum (NHS) was added to the agar.
In a first set of determinations, the bullet and kite
forms of PG-l were tested for antimicrobial activity against
L. monocytogenes, E. ~aecium (VR) or S. aureus under these
three sets of conditions.
The bullet and kite forms were roughly e~ually effective
against these three bacteria using st~n~rd assay conditions.
When 100 mM NaCl was added to the agar, however, the kite
forms appeared slightly less active than the bullet forms
which appear to have slightly ~h~nced antimicrobial activity
against all three strains except S. aureus under these
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W O 97/18826 PCT~US96/18544
conditions. Similarly, when RPMI plus 2.5% NHS were added,
the bullet forms were again more effective than the kite
forms. The activity of the kite form versus E. faecium was
significantly less under these conditions.
These forms of PG-l were also tested against E. coli,
R. pneumoniae and P. aeruginosa. All three microorgAn; ~mc
were inhibited by both kite and bullet forms under standard
conditions. This antimicrobial activity was maintained also
at 100 mM NaCl and RPMI plus NHS.
ExamPle 9: SYnthesis of the Snake Form of PG-l
The snake form of PG-l wherein all Z are Al~n;ne was
performed using stAn~rd methods by Synpep Inc., Dublin, CA
and the MH+ value in FAB-mass spec was 2031.3 as expected.
The snake form was purified to homogeneity by RP-HPLC.
ExamPle 10: Antimicrobial Activity of Snake PG-l
Snake PG-l was tested with respect to the same six
orgAn; ~r~ and using the same conditions as set forth in
Example 8 with respect to the bullet and kite forms of PG-l.
In this case, the native two-disulfide form of PG-l (native)
was used as a control. While the snake form shows somewhat
superior activity with respect to L. monocytogenes,
E. faecium, and S. aureus under stAn~Ard conditions, it is
notably less effective than the native form in the presence
of either 100 mM NaCl or RPMI plus NHS. The same pattern is
followed when the test orgA~ are E. coli, ~. ~n~t77noniae~
and P. aeruginosa.
ExamPle 11: Preparation of Miniprotegrins and Protegrins
with Enhanced Basicitv
A series of protegrins was prepared, including bullet
and kite forms, wherein one or both of Xs and Xl6 is a basic
as opposed to a hydrophobic amino acid and/or wherein Xl-X4
are absent. The peptides prepared are summarized and
compared to the amino acid sequence of PG-l as shown in Table
2.
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CA 02238610 1998-05-22
W O 9~18826 PCTAJS96/18544
Tnble 2
8eq~ns~R of 8elected M~nirrotegrinS
Code Sequence Ma~
PG-1RGGRLCYCRRR'~v~v~ 2154.04
PC-llLCYCRRR~v~v~K 1730.li
PC-12RCY~KKK~V~ 1773.14
PC-13R~RTCY~K~R~v~v 1943.35
PC-14RGGRLCYCRRRFCICV 1957.38
PC-15RGGRL~Y~K~kr~v~K 2000.41
PC-16RCY~k~v~K 1616.96
PC-17LCY~KRX~v~v 1516.87
PC-18LCYARRRFAVCV 1454.77
PC-l9~ry~RR~AVCR 1554.85
PC-20~AY~Kk~VAV 1454.77
PC-21RAY~KKK~VAR 1554.85
PC-22RGGRLCY RR VCVGR* 1648.97
PC-31aGGRLCY~n~R~v~v~n 1999.40
PC-32aRGRL~Y~KKn ~v~v~ 2098.54
PC-33aGRL~r~nnk~'~v~v~K 1942.35
PC-34aRRLCYCRRR~v~v~K 2041.49
PC-35aRLCY~Knk~'~v~v~K 1885.30
PC-36ak~I~Khh~ ~V~V~K 1928.33
PC-37a ~Y~K~'~V~V~K 1615.95
PC-44a RGGRLCYCRRRFCVCR* 2000.41
PC-45RGGRLCY~nK~v~ 1843.22
PC-46R~RTCY~KKR~V~A 1844.22
PC-47a RGGRLCY RRRF VCVGR 1951.33
PC-48RGWRLCY~nRR~v~v~K 2284.75
The a~teri~k (*) in Table 2 indicate~ C-t~r~in~l free acid; all others
are C-t~rm;n~l amide~.
The protegrin congeners were tested using the radial
diffusion assay described above using mid-log phase
organisms, except that assays with C. albicans used overnight
cultures. As described above, all underlay agars contained
0.3 mg/ml trypticase soy broth powder/ml, 1% w/v agarose and
10 mM sodium phosphate buffer, pH 7.4 and were seeded with 4
x lo6 bacterial or fungal CFU/lOml. The st~n~Ard underlay
agar, medium A, is as described; medium B is identical except
that it also contains 100 mM NaCl; medium C contains the
stAn~Ard medium plus 2.5% normal human serum; and medium D
contains 80% RPMI-1640 plus 2.5% normal human serum.
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The test peptides were dissolved at 500 ~g/ml in 0.01%
acetic acid and serial dilutions were prepared in the same
solvent either as two-fold dilutions or half-log dilutions
(half-log dilutions are, from 500 ~g/ml, 250, 78, 25, 7.8,
2.5, 0.78, 0.25 and 0.078 ~g/ml).
The dilutions were made daily in sufficient amount for
that day's experiments; to perform the tests, 5 ~g volumes of
the solutions were added to prepunched wells ~3 mm diameter)
in the bacterial underlay gels. Overlay gels (2 X
conventional trypticase soy agar) were poured after 3 hours.
Zone sizes were measured to the nearest 0.~ mm after
overnight incubation. Zone sizes in units of clearing (10
units = 1 mm diameter) were graphed against logl0 of the
peptide concentration using the sigma plot program. The
X-intercept corresponds to the ' n; ~1 microbicidal
concentration and was deteL ; ne~ by least mean squares
regression analysis. Only the highest peptide concentration
which showed no clearing was included in the calculation.
The results are provided both in terms o~ ; n; ~1
microbicidal concentrations in ~Lg/ml and relative molar
potency, which corrects for molecular weight.
In the initial experiments, PC-11 and PC-12 were tested
in co ~riSon to PG-1. The results are shown in Tables 3
and 4.
Table 3
Minimal Microbicidal Concentrations (~g/ml)
5Ori~s 1 10 mM Pho~ph~t~ Buff-r 10 mM Buff~r 1 100 mM N~Cl
D~lutlonn
Org~ni~ Prot~grin PC-ll PC-12 Prot~grin PC-ll PC-12
pa-l PG-l
E. col~ ML-35p 5.3 2.3 3.8 2.8 1.3 1.9
. mono., ~CD 5.8 4.8 5.1 3.6 3.7 5.5
C. ~lo~c~nJ 820 6.1 7.1 4.3 7.9 11.6 20.5
S. ~ureu~ 930918-37.0i0.54 3.7 3.3 4.5~0.26 3.3 3.9
MRSA 30371n.t.n.~.n.t. 4.0~0.18 3.2 3.1
MRSA 28841 n.t. n.t. n.t.3.110.03 2.1 2.6
- 66 -
Table 4
Relative Molar Potency (PG-1 = 1.00)
Image
As shown in Table 4, the shortened forms of the
protegrins are slightly more effective against E. Coli and
against S. aureus as compared to PG-1 and comparably
effective against the remaining organisms tested except for
C. albicans where the effectiveness was of the same order of
mangitude.
Tables 5 and 6 show the minimal microbicidal
concentrations and relative molar potency of an embodiment,
PC-15, where X16 is a basic amino acid. PC-13, shown in these
tables, is identical to PG-1 except that it lacks X17 and X18.
As shown in these tables, replacing X16 with a basic amino
acid generally enhances the potency against most organisms,
but alters the response to the addition of salt.
Table 5
Minimal Microbicidal Concentrations (µg/ml)
Image
-67-
Table 5
Minimal Microbicidal Concentrations (µg/ml)
Image
Table 6
Relative Molar Potency PG-1 = 1.00)
Image
Tables 7-10 show results for PC-16 and PC-17, both of
which lack X1-X4; PC-16 also contains basic amino acids at
positions X5 and X16. Tables 8 and 10 relate to minimal
microbicidal concentrations and differ only in that the
dilution method has been altered. In Tables 8 and 9 two-fold
dilutions were used and in Tables 10-11 half-log dilutions
were used. The data in these tables show that the
effectiveness compared to PG-1 varies with the target
organism and with the conditions of testing. As shown in
Table 9, for example, PC-17 (and PC-16) are mostly
comparatively less active than PG-1 in the presence of salt;
as shown in Table 11 the presence of serum is also
problematic, especially for PC-16.
-68-
Table 7
Minimal Microbicidal Concentrations (µg/ml)
Image
Table 8
Relative Molar Potency (PG-1 = 1.00)
Image
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WO 97/18826 PCTAUS96/18544
Tnble 9
~~~n; -1 Micro~c;~l Concentrations (~g/ml)
Series 2 Buffer + 100 mM NaCl RP~I ~ 2.5~ N~S
Dilutions
Organi~m PG-1PC-16 PC-17 PG-1 PC-16 PC-17
E. coli ML-35p 1.40.59* 1.00.36 0.49 0.42
L. mono., EGD 0.7 1.5 0.70.35 0.61 0.42
C. albicans 820 8.6 25.510.5 7.1 24.1* 29.8*
E. faecium 1.210.0 0.630.42 31.1 0.40
VREF, CDC21 1.511.3 1.6 0.5 28.4 0.90
(E. faecalisJ
VREF, 94.132 0.670.44 0.560.37 8.8 0.39
(E. faecium)
P. aeruginosa 1.30.41 1.20.96 8.8 0.80
~5 MR 2330
P. aeruginosa 1.4 1.2 l.l0.81 2.8 0.83
SBI-N
P. aeruginosa 1.30.80 0.60 1.1 8.9 0.88
MR 3007
P. a~ruginosa 1.40.85 0.590.91 3.9 0.81
MR 2133
S. aureus3.3 ~250 3.9 0.440.82* 0.35
930918-3
MRSA 30371 1.5~250 1.60.32 1.0* 0.30
MRSA 28841 1.37.9* 1.6 0.6 9.5 0.20
Tablo 10
Relative Molar Potency (PG-1 = 1.00)
Series 2 Dilutions Buffer + lOO mM RPMI ~ 2.5% N~S
NaCl
organi~m PC-16PC-17 PC-16 PC-17
~. coli ML-35p 1.780.99 0.35 0.60
L. mono., EGD 0.350.70 0.43 0.59
C. albicans 820 0.250.58 0.22 0.17
E. faecium 0.091. 34 0.01 0.74
VREF, CDC21 O.100.66 0.01 0.39
(~. faecali~)
VREF, 94.132 1.140.84 0.03 0.67
(E. faecium)
P. aeruginosa MR 23302.38 0.76 0.08 0.85
P. aeruginosa SBI-N 0.88 O.90 0.22 0.69
P. aeruginos~ MR 30071.22 1.53 0.09 0.88
P. aeruginosa MR 21331.24 1.67 0.18 0.79
s. aureus 930918-3<0.03 0.60 0.40 0.89
MRSA 30371 <0.030.66 0.24 0.75
MRSA 28841 0.120.57 0.05 2.11
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Tables 11-14 show the results ~or the kite forms of the
invention protegrins. Again, it is apparent that the
spectrum of target org~n;~;m~: which are responsive and the
spectrum of conditions under which response is obtained are~ 5 variable. In general, the more cationic form, PC-l9 is less
ef~ective than the unmodified form which merely lacks the
residues Xl-X4. Under physiological conditions, PC-l9 is
substantially inactive.
Table 11
M;n; -1 Microbicidal Concentr~tions (,ug/ml)
8eries 1 10 mM Pho~phate Buffer Buffer + 100 ~M NaCl
Dilution~
organism Pa-1 PC-18 PC-l9 PG-l PC-18 PC-l9
E. faecium7.7 1.7 17.7 4.2 2.9 >250
VREF, CDC21 n.t. n.t. n.t. 4.6 5.3 >250
(E. faecalis)
V~EF 94.132 n.t. n.t. n.t. 3.6 1.4 5.6
(E. faecium)
S. aureus 7.3 3.9 7.3 5.0 2.1 10.4
930918-3
MRSA 30371n.t. n.t. n.t. 4.1 6.5 68.6
MRSA 28841n.t. n.t. n.t. 3.6 3.9 17.4
Table 12
Relative Molar Potency (PG-l - 1.00)
Series 1 Dilutions 10 Mm Phosphate Buff~r + 100 mM NaCl
Buffer
Or~anism PC-18 PC-l9 PC-18 PC-l9
E. faecium 3.06 0.31 0.98 0.01
VREF, CDC21 n.t. n.t. 0.59 0.01
(E. faecalis)
VREF 94.132 n.t. n.t. 1.74 0.46
(E. faecium)
S. aureus 930918-3 1.26 0.72 1.61 0.35
MRSA 30371 n.t. n.t. 0.43 0.04
MRSA 28841 n.t. n.t. 0.62 0.15
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Table 13
Minimal Microbicidal Concentrations (~g/ml)
S~ries 2 10 mM Buffer ~ 100 ~M RPMI + 2.5% N~S
Dilution~ NaCl
Organ sm PG-l PC-lB PC-19 Pa - 1PC-18 PC-l9
E. faecium1. 5 3.2 ~250 2.9 >250
VREF, CDC21 1.4 3.4 >250 10.0 >250
(E. faecalis)
VREF 94.132 0.61 0.50 2.9 0.30 1.2 30.2
(E. faecium)
P. aeruginosa 1.3 1.1 3.0 0.7 3.0 29.2
MR 2330
P. aeru~ino~a 1.3 1.2 2.1 0.85 4.6 ~250
SBI-N
P. aeru~inosa 1.3 1.1 1.1 0.72 3.89 ~250
MR 3007
P. aeruginosa 1. 2 2.4 2.5 O.9 3.9 ~250
MR 2133
Table 14
Relative ~olar Potency ~PG-1 - 1.00~
S~rios 2 Dilutions l0 mM Buff~r + l00 m~ RPMI + 2.5~ N~S
NaCl
Orgnnism PC-18 PC-19 PC-18 PC-l9
E. faecium 0.32 <0.01 0.98 0.01
VREF, CDC21 ~E. faecalis) 0.28 <O.Ol0.03 <0.01
VREF 94.132 (E. faecium) O .81 0.15 0.17 <0.01
P. aeruginosa MR 2330 0.8 0.31 0~16 0.02
P. aeruginosa SBI-N 0.73 0.45 0.12 <0.01
P. aeruginosa MR 30070.80 O.85 0.13 <0.01
P. aeruginosa MR 21330.34 0.35 0.16 <0.01
Tables 15 and 16 provide results obtained with the l~ite
forms of the invention protegrins. The more cationic forms
are generally less effective than PG-1 under conditions of
high salt or serum. However, they are comparably effective
against E. coli.
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Table 15
Minimal Microbicidal Concentrations (~g/ml)
S-ri~n 110 mM Pho~ph~t~ 8u~~~r 1 100 mM N~Cl B~f~er + 2.5% NH8
Dilut~on~ Buff~r
Org~n~m PG-lPC-20 PC-21PG-l PC-20PC-21 PG-l PC-20 PC-21
B. coli ML-35 4.6 0.750.36 2.0 1.9 1.8 0.34 0.27 0.28
L. mono. EGD 4.8 3.31.4 3.421.736.3 1.3 5.5 5.6
C. albica~ 6.1 5.3 8.56.8 67.5~250 8.1 30.1 59.7
820
E. faecium 6.7 6.7 18.03.9 69.0,250
VREF CDC21n.t.n.t. n.t.4.9 ~250~250
(faécali~)
VREF 94.132n.t.n.t.n.t.2.5 3.328.3
(faecium~
S. aureus 10.3 5.8 7.25.1 30.36~.6
93~918-3
MRSA 30371n.t.n.t. n.t.4.2 29.964.6
MRSA 28841n.t.n.t. n.t.3.3 8.4 8.4
Table 16
Rclative Molar Poten¢y (PG-1 = 1.00)
8-r~s 1 D~lutlon~ 10 ~M Pho~ph~t~ Bu~f~r + 100 ~ Bu~f-r t 2.5%
Buff~r ~aCl N~S
Org~ni~P~-20PC-21 PC-20 PC-21PC-20PC-21
E. coli ML-354.149.21 0.71 0.84.500.88
L. mo~o. EGD0.982.47 0.09 0.070.160.17
C. albican~ 8200.78 0.52 0.07c0.020.18 0.10
E. faeci~m 0.680.27 0.03 0.01
VREF, CDC21n.t.n.t. 0.01 0.01
(f~ 7J~)
VREF 94.132n.t.n.t. 0.20 0.06
(~aecium)
S. aureus 930918-3 1.20 1.030.11~0.06
MRSA 30371 n.t.n.t. 0.09 <0.05
MRSA 28841 n.t.n.t. 0.26 0.28
Exam~le 12: Minimal Bioactive Conformation of PG-1
Necessary for Activity Against Neisseria
gonorrhoeae
The susceptibility of N. gonorrhoeae to variants of
protegrin PG-l was determined using the radial diffusion
assay of Lehrer et al., 1991, ~ Immunol Nethods 137:167.
Native protegrin PG-1 was purified for porcine leukocytes by
the method of Kokryakov, V.N. et al. (FEBS Lett (1993)
327:231-236). All other protegrins were prepared
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synthetically with F-moc chemistry and purified by reverse
phase HPLC.
The following N. gonorr~oeae strains were tested: FA19
(sac-l, sac-3; serum-resistant [SacR;), JS-1 (serum-resistant;
sac-1, sac-3; [sacR]) / and F62 (sac-1~, sac-3; serum-sensitive
tsacS]) have been described by Cannon et al. (Infect Immun
(1981) 32:547-552)and Judd, R.C. (Infect Immun (1982) 37:632-
641). Strains FA628 (sac-1~, sac-3) and FA899 (sac-7, sac-3+)
are SacS transformants of FAl9 (Shafer, W.M. et al., In~ect
Immun (1982) 35:764-769). Strains FA5101 and WS1 are pycin-
resistant mutants of strain FA19 that produce a truncated ~OS
(Lucas, C.E. et al., Molec Nicrobiol (1995) 16:1001-lO09).
Bacteria were streaked on NGTM medium, incubated overnight at
37~C in 5% C02/room air and passaged daily. Bacteria from
plates were placed in 25 ml GC broth and incubated at 37~C
with sh~k;ng for 3 hours to obtain mid-log gonococci.
Underlay and overlay gels were prepared as previously
described for the radial diffusion assay of Qu, X.D. et al.
Peptides were dissolved and serially diluted in 0.01% acetic
acid and 5 ml aliquots were tested. Plates were incubated in
a co2 incubator at 37~C for 3 hours before pouring the overlay
gel, to allow peptides to diffuse into the underlays. After
overnight incubation, the minimal inhibitory concentrations
(mg/ml) were determined from the X-intercepts of the curves
as described by Qu et al. Each strain showed protegrin-
sensitivity (Tables 17-19).
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Table 17
Activity Against Neisseria gonorrhoeae
ami~o M~ 1 Inh~b~tory Conc~tratlon
acidll (I g/~nl)
Protcgrin S~, ~n~ n P62 ~8-1 FA19
PG-1 RGGRLCYCRRRF~vCv~K 18 1.3+0.1 1.2~0.1 1.9+0.1
PC-13 RGGRLCYCRRRFCVCV-- ~6 0.6~0.2 1.4+0.04 2.3+0.8
PC-11 ----~CYCRRRF~v~v~K1~ 1.0+0.11.6+0.3 2.8+0.1
PC-17 ----LCYCRRRFCVCV-- 1~ 0.6_0.11.3+0.2 1.7+0.1
PC-37 -----CYCRRRF~v~v~ 13 4.2il.11.8+0.2 10.7+0.5
PC-45 RGGRLCYCRRRFCVC--- 15 1.1+0.3 2.9+0.03 7.6i2.1
PC-71 -----CYCRRRFCVCV-- 11 4.5+0.84.7+1.0 16.1+0.9
PC-72 ----LCYCRRRFCVC--- 11 2.7+0.22.3+0.8 12.8+0.0
PC-73 -----CYCRRRFCVC---1029.8il.5 48.3+22.5 104.4+7.4
PC-8 R~r~T~AY~ ~AVAVGR 18 21.4+5.8 46.0+7.0 431+13
PC-9 ~-GRT~YCRRRFCVAVGR 18 1.0+0.3 1.1+0.2 7.3+2.3
PC-10 RGGRLCYARRRFAVCVGR 18 0.7+0.1 0.8+0.1 1.6+0.O
PC-18 ----LCYARRRFAVCV-- 12 0.7+0.1 2.1~0.4 8.3+2.0
PC-64 ----LCYTRRRFTVCV-- 12 0~7i0-1 1.4$0.2 4.5+0.3
PC-20 ----LAYCRRRFCVAV-- 12 1.1+0.2 8.5+2.5 32.4~5.7
PC-64a ----LTYCRRRFCVTV-- 12 0.7+0.1 0.7+0.1 2.1_0.1
All of the peptides in the table are the C-terminal
amides.
Protegrins PC-13, PC-11 and PC-17 demonstrate that amino
acid residues X1 through X4 and Xl7 through X18 may be deleted
from PG-1 without a loss of inhibitory activity (Table 17).
The effect of the two intramolecular disulfide bonds of
PG-1 on inhibition of N. gonorrhoeae was determined.
Elimination of both disulfide bonds (protegrin PC-8) reduces
the inhibitory effect on all strains tested (Table 17). In
contrast, protegrin PC-10, which lacks the CYs8 Cysl3
disulfide bond has increased activity against strains F62,
JS-1 and FAl9. A variant of PG-1 lacking the CyS6 Cysl5
disulfide bond (PC-9) retains full activity against strains
F62 and JS-l and shows a 3.8-fold reduction of activity
against strain FA1~. Truncated 12-mer variants of PG-l
having a single disulfide bond also show potent inhibitory
activity (see protegrins PC-18, PC-64, PC-20 and PC-64a in
Table 18).
The inhibitory activity of protegrin variants was also
determined for two serum-sensitive strains, FA628 (sac-l+) and
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~A89s (sac-3~), which were derived for ~he serum resistant
FA19 strain. PG-1 showed similar activity against the wild-
type, serum resistant FA19 strain and the serum-sensitive
derivative strains FA628 and FA899 ~Table 18). Serum-
sensitive strains were two to four fold more susceptible toPC-8 than was the serum-resistant strain. Variants PC-9 and
PC-10, which have one disulfide bond, retain strong
inhibitory activity.
Table 18
~fQct of Protegrin~ Against 5e~ S~iti~ N. go~orrhoe~e strain
amino acids ~in; 1 Inhibitory Concentration (~/ml)
Protegrin n FAl9 FA62R FA899
PG-l 18 2.1+0.1 1.6~0.11.6+0.1
PC-8 18420.8+24.2150.9il4.0 263.5+10.3
PC-9 1811.2+1.8 4.0+0.13.9-~0.2
PC-10 18 1.7+0.1 1.2+0.11.3+0.1
Because LOS truncation increases suscepti~ility of
gonococci to antimicrobial proteins of human PMN (Shafer,
W.M. et al., 1986, J Infect Dis 86:910-917), protegrins PG-1,
PC-8, PC-9 and PC-10 were tested against LOS mutants FA5101,
WSl and wild-type strain FA19 (Table l9). LOS truncation
resulted in increased susceptibility of N. gonorrhoeae to the
linearized protegrin PC-8, but had little effect on
susceptibility to PG-l or PC-10.
Table 19
Ef~ect of Protegrins Against N. go~orrhoeae ~05 Mutant~
amino ~cidfi ~~'~i -1 Inhibitory C~nc_~Lldtion (~g/ml)
Protegrin n FAI9 FAS101 WSl
PG-l 18 2.1~0.1 1.5+0.21.4~0.2
PC-8 18420.8+24.223.1+9.329.7il2.8
3~i Pc-s 1811.2+1.8 2.7+0.42.6~:0.7
PC-10 18 1.7+0.1 1.2+0.11.1+0.1
ExamPle 13: Electron Microscopy of N. gonorrhoeae treated
with Proteqrins
The effect of treatment of N. gonorrhoeae with 50 ~g/ml
PG-1 or the truncated derivative PC-17 was determined.
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Transmission electron microscopy of bacteria after 60 minutes
treatment with PG-1 reveals central vacuolation and membrane-
associated, electron-dense structures. S~nn; ng electron
microscopy of bacteria treated with PG-1 or PC-17 revealed
surface lesions approximately 100 nm in diameter.
~ple 14: Effect of Saliva on Antimicrobial Activitv
The radial diffusion assay of Lehrer et al., 1991, J.
Immunol. Meth. 137:167 was used, except that the media in the
underlay agar contained phosphate buffer at 10 mM, pH6.5,
100 mM NaCl, 1% TSB, 1% agarose. The media in the overlay
contain lo mM phosphate buffer, pH 6.5, 100 mM NaClr 2XTSB,
1% agarose. The peptides were diluted from lOX stock made up
in 0.01% acetic acid (AA) either with 10 mM acetate buffer
pH5 or with saliva.
The results are given as the m;~; ~l concentration
required to produce a detectable zone of clearance, or MCZ --
i.e., an extrapolated value to the x-axis when the
concentration of peptide is plotted against the diameter of
the zone. The results are shown in Table 20.
Ta~le 20
Effe¢t of Diluent on MCZ (~g/mL) again~t E. coli 004
8eguonce Acet~te S~llva
bu~r
RGGRLCYCRRRFCVCVGR O. 48 1.14
RGGGLCYARGWIAFCVGR 4.16 38.30
RGGGLCYKR~lK~v~ 2.71 0.56
RGWGLCYCRPRFCVCVGR 0.39 12.60
RGGRLCYCRRRFCVCVGR~ O. 59 1.45
LCYCRRRFCVCF 4.14 6.11
RLCYCRPRFCVCV 3.36 6.83
L~Y~K~V~V~K 2.37 5.68
RLCYCRPRFCVCVGR 1. 60 6.86
RWRLCYCRPRFCVCV 1.04 39.00
RGWRACYCRPRFCACVGR 0. 71 1.84
3~ GWRLCYCRPRFCVCVGR 0.86 47.50
~L~r~ CVCV 13.70 9.76
WLCYCRRRFCVCV~ 5.05 36.00
RL~r~a~F~V~V (X=MeGly) 2.43 2.54
RLCYCRPRFCVCVGR~ 3.65 12.80
RGGGLCYCRPRFCVCVGR~ 3.51 11.90
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Table 20
Effect of Diluent on MCZ (,~g/mI,) against E. eoli 004
8~ ~ Ac--t~t~ 9a~ iva
bu f f -r
RRCYCRRRFCvCVGR 3.02 8.07
Peptides noted with ~ are acid forms; all others are amide forms~
A large number of the peptides tested showed c ~ -rable
or even improved activity in the presence of saliva.
ExamPle 15: Antimicro~ial Activity of
Additional Proteqrins
lo The following example provides assays for measuring the
antimicrobial activity that were used to test additional
peptides of the invention described hereinbelow. The
following reagents, stock solutions and cultures are used in
the assays that follow.
Microorqanisms: l~:seherichia eoli ML-35p and vancomycin-
resistant Enteroeoeeus faeeium (VRE) were obtained from Dr.
Robert Lehrer (UCLA, see also, Lehrer et al., 1988, J.
Immunol. Methods 108:153) and Dr. Gary Schoolnik (Stanford),
respectively. Pseudomonas aeruginosa (ATCC 9027), Candida
albicans (ATCC 1023), and methicillin resistant
Staphyloeoecus aureus (ATCC 33591) were obtained from the
American Type Culture Collection, Rockville, MD.
Microorganisms from other sources, such as, ~or example,
clinical isolates, can be used interchangeably with the
above-described microorganisms in the assays described
herein.
Media and Reaqents:
TrYPticase SoY Aqar (TSA; Becton-Dickinson,
Cockeysville, MD, BBL #4311768): dissolve 40 g in 1 Liter
deionized water, autoclave 121~C, 20 minutes.
TrYPticase SoY Broth (TSB; Becton-Dickinson,
Cockeysville, MD, BBL #4311768): dissolve 30 g in 1 Liter
deionized water, autoclave 121~C, 20 minutes, and store at
room temperature.
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2X TrY~ticase Soy Broth (2X TSB): dissolve 60 g in
1 Liter deionized water, autoclave 121~C, 20 minutes, and
store at room temperature.
Glycerol (20% v/v): mix 20 mL glycerol with 80 mL
deionized water, Filter sterilize with 0.20 ~ filter and
store at room temperature.
Monobasic ~hosPhate buffer (100 mM): dissolve 13.7
g sodium phosphate monobasic ~Fisher #S368-500) in 1 Liter
deionized water. Filter sterilize with 0.20 ~ filter and
store at room temperature.
Dibasic Phosphate buffer (100 mM): dissolve 14.2 g
sodium phosphate dibasic (Fisher #S374-500~ in 1 Liter
deionized water. Filter sterilize with 0.45 ~ filter and
store at room temperature.
Phos~hate-buffered saline (PBS; 10 mM phosphate,
100 mM NaCl, pH 7.4): mix 15 mL dibasic phosphate bu~fer
(100 mM~, 5 mL monobasic phosphate buffer (100 mM), 4 mL NaCl
(5 M~ and 176 mL deionized water. Adjust pH if necessary,
filter sterilize with 0.45 ~ filter and store at room
temperature.
Phos~hate buffer (100 mM, pH 6.5): mix 40 mL
dibasic phosphate buffer (100 mM~ with 160 mL monobasic
phosphate buffer (100 mM). Adjust pH if necessary, filter
sterilize with 0.45 ~ filter and store at room temperature.
Liauid Testinq Medium (LTM~: aseptically combine
the following sterile ingredients: 10 mL Phosphate buffer
(100 mM, pH 6.5~, 1.0 mL TSB, 2 mL NaCl (5 M~ and 87 mL
deionized water. Store at room temperature.
Acetic acid (0.01~ v/v): mix 10 ~L acetic acid with
100 mL sterile deionized water.
Aqarose: mix l g agarose (Sigma #S6013) in 80 mL
deionized water, autoclave 121~C, 20 minutes.
Aqarose Underlay Medium: combine 10 mL Phosphate
buffer (100 mM, pH 6.5), 1.0 mL TSB, 2 mL NaCl ~5 M) and 7 mL
deionized water with 80 mL tempered (50OC) agarose.
2X TSB Aqarose Overla~ Medium: dissolve 60 g TSB
- and 10 g agarose in 1 Liter deionized water, aliguot 100 mL
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W O 97/18826 PCT~US96/18544
per bottle, autoclave 121~C, 20 minutes, and store at room
temperature.
Pre~aration of Microorqanism Slants: Each strain was
cultured on TSA. Isolated colonies were transferred into ~SB
(10 mL in a sterile 50 mL Erlenmeyer flask) using a sterile,
disposable loop and the flask ;n~llh~ted at 37~C (bacteria) or
30~C (yeast) with shaking (200 RPM) for 16-18 hours.
Broth cultures were diluted 1:1 with 20% sterile
glycerol and stored as 1.0 mL aliquots at -80~C. For daily
inocula, liquid was transferred from a thawed vial using a
sterile loop and then spread onto the surface of TSA slants.
The screw capped tubes were incubated overnight and stored at
4OC for up to one month.
PreParation o~ Inoculum:
1. Remove the cap from tube and lightly touch a
sterile loop to the area of heavy growth on the TSA slant.
2. Inoculate 10 mL of TSB (50 mL flask) and
incubate the flask in a sh~k; ng water bath for 18 hours
(overnight) at 37~C (bacteria) or 30OC (yeast) at 200 RPM.
3. In a cuvette, dilute 50 ~L of the overnight
culture 1:20 with TSB and measure the absorbance at 600nm
(A600) using TSB as a reference. The A6~0 of the diluted
culture should be between 0.1-0.4.
4. In a 250 mL Erlenmeyer flask, dilute 50~L of
the overnight culture 1:1000 with TSB (bacteria) or 1:100
with TSB (yeast).
5. Incubate the flask in a shaking water bath at
37~C (bacteria) or 30~C (yeast) at 200 RPM for approximately
2-3 hours until log-phase is reached, i.e. until the A600 of
the culture is between 0.200 and 0.400 without further
dilution.
6. Transfer 25 mL of the log-phase culture to a
sterile centrifuge tube and centrifuge at 2000 rpm and 4~C
for 10 minutes. Decant the supernatant, add 25 mL of sterile
PBS and resuspend the pellet by vortexing.
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7. Centrifuge the suspension at 2000 rpm and 4~C
for 10 minutes. Decant the supernatant and resuspend the
pellet with 5 ml sterile PBS.
8. Measure the A600 of the undiluted suspension.~ 5 If the absorbance is above 0.5, dilute with sterile PBS until
the absorbance is between 0.100 and 0.500.
9. Determine the number colony-forming units per
milliliter suspension (CFUs/mL) by preparing 10-fold serial
dilutions in saline (0.87%) and spre~cl;ng 100 ,uL of the 104-,
105-, and 105-fold dilutions onto TSA plates, one dilution per
plate. Incubate overnight, count the number of colonies and
determine the CFUs/mL (an accurate determination requires
approximately 30-300 colonies per plate).
For the strains reported, the CFUs/mL for a suspension
having an A6~o=0.2 have been determined as reported in the
table below:
CFUs/mL of Suspension (~oo=0.2)
8train CFUs/mh
E. coli 8.0 X 107
P. aeruginosa 7 . 8 x 107
MRSA 2.0 x 107
VRE 3.8 x 107
C. albicans 9 . 7 x 105
PreParation of PePtide Stock Solutions:
1. Weigh approximately 1.0 mg of each peptide to
be tested into a sterile polypropylene cryovial (1.8 mL).
2. Add sufficient acetic acid (0.01%) to make a
stock solution having a concentration of 1280 ~g/mL.
Dispense the stock solution into several vials, lO0 ~L per
vial, and store the aliquots, tightly sealed, at -80~C.
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6.5 Radial Diffusion fMCZ~ Assav
The MCZ assay uses ;n; ~1 amounts of test
materials to determine the sensitivity of microorganisms to
various antimicrobial compounds. Cells are grown to
approximately mid-log phase and resuspended in ;n; ~1
nutrient buffered agarose. Agarose (not agar) is used in
this gel to avoid electrostatic interactions between
antimicrobial peptides and the polyanionic components of
st~n~Ard agar. Peptides diffuse r~ y into the gels from
small wells and the diameter of the zone of growth inhi~ition
is proportional to the concentration of peptide in the
solution (Lehrer et al., 1988, J. Immunol. Methods 108:153;
Lehrer et al., 1991, ~. Immunol. Methods 137:167).
PreParation of MCZ AssaY Plates:
1. For each petri plate to be poured, dispense 10
mL of tempered (50OC) Agarose Underlay Medium into a sterile
polypropylene tube (15 mL). Add 4x105 CFUs of the desired
strain to each tube. Mix well by inverting tube 3 times.
Immediately pour the molten agarose into the petri dishes.
2. After the agarose has solidified, use a
sterile canula (3 mm i.d.) to punch 16 wells (4x4 evenly
spaced grid) into the agarose. Remove the agarose plugs with
a pasteur pipette and trap the agarose in a flas~ with a side
arm port attached to a vacuum.
3. From the peptide stock solution, prepare
serial 2-fold dilutions (from 128 ~g/mL to 0.06 ~g/mL) using
acetic acid (0.01%) as a diluent, or, for peptide
concentrations lower than 50 ~g/mL, sodium acetate (10 mM,
pH 5~ cont~;ning Human Serum Albumin (HSA; 0.1% w/v) as a
diluent.
4. Dispense 5 ~L of each serial dilution into the
agarose wells, one serial dilution per well.
5. Dispense diluents into wells as negative
controls and protegrin-l (U.S. Patent No. 5,464,823; 32
~g/mL, 8 ~g/mL and 2 ~g/mL) into wells as positive controls.
6. Incubate the plates at 37~C (bacteria) or 30~C
(yeast) for 3 hours.
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~. ~ispense 2X TSB Agarose Overlay Medium (10 mL)
onto the surface of each plate, allow the agar to solidify
and incubate plates, inverted, at 37~C (bacteria) or 30~C
(yeast) for 16-18 hours.
8. ~ ;ne the plates and measure (in mm) the
diameter of the zone of growth inhibition (area of clearing
around each well).
9. Plot the diameter of the zone of growth
inhibition (Y-axis) versus the concentration of peptide in
the well (X-axis) and obtain the line of best fit using
l;ne~r regression analysis. The X-intercept of the line of
best fit is the ;ni concentration for zone of growth
inhibition (MCZ) for each peptide concentration.
6.6 Microbroth Dilution (MCB) AssaY
The microbroth dilution method accomodates large
l~l h~rs of samples and is more amenable to automation than
the MCZ assay and the data analysis is direct and simple. A
key step in this assay is combining microorg~n; ~m~ and
peptide in a defined ;n; ~l nutrient buf~er system that
;n; ;zes interference with the peptide's biological
activity. Tn addition, the presence of 0.1% (w/v) human
serum albumin (HSA) or bovine serum albumin (BSA) to the
peptide diluent ; n; ; zes adsorption of peptide to the
container.
Pre~aration of MCB assav Plates:
1. Dispense 100 ~L of log-phase cells in LTM
(MCB-5 is 4x105 CFUs/mL; MCB-3 is 4x103 CFUs/mL) into each
well of a sterile 96-well microtiter plate.
2. From the peptide stock solution, prepare
serial two-fold dilutions (from 1280 ~g/mL to 0.625 ~g/mL)
using acetic acid (0.01%) as a diluent, or, for peptide
concentrations lower than 50 ~g/mL, sodium acetate (10 mM,
pH 5) cont~;n;ng HSA or BSA (0.1%w/v) as a diluent.
3. Dispense triplicate ali~uots (ll ~L) of each
~ serial two-fold dilution into the wells of the microtiter
plate.
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4. Incubate the plate at 37~C (bacteria) or 30~C
(yeast) for 3 hours.
5. Add 100 ~L of 2X TSB to each well, mix, and
incubate at 37~C (bacteria) or 30~C (yeast) for an additional
16-18 hours.
6. F.5r~ ;ne the plates and evaluate each well for
turbidity (cell growth). Often, MRSA will settle out and
form a pellet at the bottom of the well. MRSA can be
evaluated by placing the microtiter plate on a stand and
e~A ;ning the bottom of the well using a tilted mirror.
7. The minimum concentration for inhibition of
growth in broth medium (MCB) is defined as the lowest
conc~ntration of peptide that inhibits all visible growth.
If the MCB values for each of the triplicate samples differ,
the MCB is obtained by averaging the results of the three
samples.
8. The minimum concentration of peptide showing
at least 99.9% biocidal activity (at least a 3 log decrease
from starting inoculum) is determined by incubating a 50 ~L
aliquot from each well on a TSA plate for 24 hours at 37~C
(bacteria) or 30~c (yeast) (for plating, 1.5 mL TSA in each
well of a 24-well plate minimizes cross contamination).
6.7 Modified NCCLS Mirl; Inhibitory
Concentration (MIC) AssaY
The National Committee for Clinical StA~rds
(NCCLS) requires that test compounds be prepared as stock
solutions in Mueller-Hinton Broth ("MH~") at 512 ~gtmL. The
stock solutions are serially diluted (two-fold) in medium and
each serial dilution added 1:1 to medium cont~in;ng lx106
CFU/mL bacteria (National Committee on Clinical Laboratory
Standards, December 19g4, "Performance Standards for
Antimicrobial Susceptibility Testing," NCCLS Document M100-S5
Vol. 14, No. 16; Methods for Dilution Antimicrobial
SuscePtibility Test for Bacteria that Grow AerobicallYl 3d
Ed., Approved Standard M7-A3, National Committee for Clinical
Standards, Villanova, PA).
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It has been found that the peptides of the invention
precipitate in MHB at concentrations greater than 128 ~g/mL.
Thus, following the NCCLS protocol would result in serial
two-fold dilutions cont~;n;ng less peptide than calculated,
- 5 yielding erroneously high MIC values.
To overcome this problem, the following modified NCCLS
assay is the preferred method for determ;ning MICs of the
peptides of the invention. In the method, precipitation is
avoided by preparing concentrated (10X) stock solutions of
lo test peptide in a buffer that is suitable for the peptide and
which does not exhibit deleterious effects on the
microorganisms (0.01% v/v acetic acid, 0.1% w/v HSA)and
diluting the stoc~ 1:10 into MHB cont~;n;ng the
microorganisms.
PreParation of MIC assaY Plates:
1. Prepare a fresh overnight culture of test
organism in Meuller-~inton broth (MHB; ~ecton-Dickinson,
Cockysville, MD, BB2 #11443).
2. Dilute the culture to approximately 4x105
CFUs/mL with fresh MH~ and dispense 100 ~L aliquots into each
well of a sterile 96-well microtiter plate.
3. From the peptide stock solution, prepare
serial two-fold dilutions (from 1280 ~g/mL to 0.625 ~g/mL)
using acetic acid (0.01~) as a diluent, or, for peptide
concentrations lower than 50 ~g/mL, sodium acetate (10 mM,
pH 5) containing Human Serum Albumin (HSA; 0.1%w/v) as a
diluent.
4. Dispense triplicate aliquots (11 ~L) of each
serial dilution into the wells of the microtiter plate.
5. T~cllb~te the plate for 16-18 hours, without
aeration, at 37~C (bacteria) or 30~C (yeast).
6. Examine the plates and evaluate each well for
turbidity (cell growth). Often, MRSA will settle out and
form a pellet at the bottom of the well. MRSA can be
evaluated by placing the microtiter plate on a stand and
m; n; ng the bottom of the well using a tilted mirror.
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7. The minimum inhibitory concentration (MIC) is
defined as the lowest peptide concentration that inhibits all
visible growth. If the MIC values for each of the triplicate
samples differ, the MIC is obt~;ne~ by averaging the results
5 of the three samples.
8. The minimum concentration of peptide showing
100% biocidal activity is determined by incubating a 10 ,uL
aliquot from each well on a TSA plate for 24 hours at 37~C
(bacteria) or 30~C (yeast) (for plating, 1.5 mL TSA in each
10 well of a 24-well plate minimizes cross contA ;nAtion).
6.8 ~inetic Bactericidal AssaY
The following assay is used to determine the rate
at which a peptide kills a target microorganism, as well as
15 to determine if a peptide is bactericidal or bacteriostatic.
AssaY
1. Dispense 200 ,~lL of log-phase cells in LTM (4 x
10~ CFUs/mL) into each well of a 96-well microtiter plate
20 solution.
2. At time T=0 minutes, add 22 ,uL of 1280 ~g/mL
peptide to well Al and mix by triturating 3 times.
3. Wait 30 seconds and add 22 ,uL of a second
concentration of peptide the to the next well (A2) and mix by
25 triturating 3 times.
4. Repeat the process, staggering each peptide
addition by 30 seconds, until all concentrations of peptide
have been added. Typically, 4-fold serial dilutions of stock
peptide (l.e., 1280, 320, 80, 20 and 5 ~g/mL peptide diluted
30 1:10 into each well) produces good comparative ]cill curves.
Add 22 t~L of 0.01% acetic acid to one well as a control.
~ . At time T=15 minutes, mix well Al by
triturating 3 times and transfer 20 ~L to an empty sterile
petri dish (100 mm x 15 nu~).
6. Quickly add 20 mL of tempered (50~C) TSA and
gently swirl plate to mix.
7. ~epeat steps 5-6 until all peptide
concentrations have been plated.
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8. For the control well, dilute the sample l:lOo
with LTM and plate 50 ~L of the dilution to obtain an
accurate determinations of CFUs.
9. After the agar has solidified, invert the
~ 5 plates and incubate at 37~C (bacteria) 30~C (yeast) for 18-24
hours.
10. Repeat steps 5-9 for all peptide
concentrations and control samples at times T=30, 60, 120,
and 240 minutes.
11. Count the n h~r of CFUs per plate and
estimate the reduction in C~Us for each peptide
concentration. In order to assess an effect using this
assay, the peptide must reduce the CFUs by at least one log
(i.e., at least 800 CFUs per plate). Although such numbers
are higher than reco en~ed for accuracy (30-300 CFUs/plate),
log-order changes in recoverable CFUs indicate significant
bacteriocidal efficacy.
12. To obtain comparative kill curves, plot the
log of fractional survival versus peptide concentration.
Table 21 shows the antimicrobial activity of a number of
the compounds of the invention against various target
orgAn;~ ~ using the MCB-3 assay described above (i.e., at 103
CFU. The results are given as a ~; n; ~ l concentration for
inhibition of growth in broth" (MCB) which is the lowest
concentration that results in no visible turbidity. The
~minimal bactericidal concentration~ (MBC) on TSA was
e~uivalent to the MCB. The units of concentration are ~g/ml.
As shown in Table 21, various substitutions can be made in
the peptide chain in order to fine-tune the spectrum of
3~ antimicrobial activity. An asterisk at the C-terminus
indicates that the peptide was supplied in the form of the
free acid; otherwise the amide was used in the assay.
Table 21
E~aluation of ~ntimicrobial Peptides i~ MCB-3 ~g/mh)
Se~cc MRSA Psa VREF ~-n~; ~ E. coli
RGGRLCYCRRRFCVCVGR* 1.5 O.ll 1.2 0.6
RGGGLCYCRRR~v~v~k* 3.29 0.4
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SequenceMRSA Psa VREF ~n~; ~ n E . coli
RGGGLCYCRR~v~n 1.93 0.14 1.62
RGGGLCY~nRP~v~v~n 3.1 0.06 7.69 0.15
RGGGL~_~nPR~v~v~K* 17.7 3.51
RGGRLCY~nkn~v~v~n* (X=MeGly) 5.33 2
RGGRLCYCRXRr~v~v~n (x=MeGly) 4 1.67 0.83
RGGL~Y~n~r~v~v~n10.6 0.83
RGGRLCYCX~Kr~v~v~n (X=CLt)
XGGRLcycRGR~v~v~K (X=Cit)
RG~nv~Y~nGRr'~v~v~n 8
RG~inv~:YI;n~ ~:v~;vGn*
RGGGLCY~K~v~v~k 3.48 1.2 15.96
RGWGLCYCRPR~v~v~k 1.55 0.27O.O9 5.68 O.1
RGWRLCYCR~n~v~v~n* (X=MeGly) 26.7 10.6
RGWRLCYCRGRh~v~v~n 5.3 0.5
R~RT.~Y~Kr~V~V~n (X=Cit)
RWRLCYCRPRFCVCVGR 4.7 3.3 1.7
RGWRLCYCRPR~v~v~n 4.7 5.3 6.1
RGWRACYCRPRFCACVGR 4.7 1.3 2 4.7 0.34
GWRLCYCRPR~v~v~n 4.7 4 2.7 0.86
RWRL~Y~K~v~v~K
RGWRLCY~n~nr~v~v~n (X=MeGly)
GGWRLCYCR~n~v~v~n 16 9.3
RGGWLCYCR~n~v~v~n 32 5.3
RLLRLCY~nkn~v~v~n (X=MeGly) 4 3.3
RLLRACYCRXRFCVCVGR (X=MeGly) 10.6 3
RLLRLCYCRRR~v~v~n
RqT.R~CY~n~n~v~v~n (X=Cha) 6.7 2.3
RGGRLCYCRXRZCVCWGR (X MeGly)
(Z~Cha)
RG~nw~v~n~nZCYCVGR (X=MeGly)
(Z=Cha)
RGLRXCYCRGR~v~v~K* (X=Cha) 16 8
RG~w~v~GRXCYCVGR (xscha)
RGGRL~Y~KkR~CVGR (X MeVal)
LCY~K~V~V 16 4 4 5.9 1.9
LCYCRRRFCVCV* 32 8
LCYCRPCFCVCV 64 5.33.33 >64
L~Y~nRR~v~ 4 2 2 3
T C~ CRRRACVCV >64 13.3
LCYCRXRFCVCV (X=D-Arg) 4 2 1.32 5.72 1.18
L~w~nkk~v~v 5.3 2 4 8.91 O.76
~._Y~nRRFCVCV 5.3 2.7 2 4.7 2.3
L~Y~KkR~v~v (X~hPhe) 8(64)6.7 2.7
L~K~nX~v~v (X=Phe(4-Cl)) 4 2 1.3
XCY~nkk~-~v~v (X=Cha) 2.672.67 1.33
LCYCRRRFCXCV (X=D-Hi~) >6410.7 >64
LCYCRRRXCVCV (x=MeGly) >6413.3 42.7
LCYCRRRXCVCV (X=MePhe)
LCYCRRRFCXCV (X~MeVal)
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WO 97/18826 PCTnUS96/18544
~c~caMRSA P~a VREF ~nn~;~A E. coli
T~X~k~xcvcv (xzCha) 18.7 16 8 3.98 0.37
LCGCRRRGCVCV>32 >16 >128
T~r,R~RACvCV>326.721.3
RLCY~Rkr-~v~v 8 4
RACYCRPRFCACV>642.7
RLCYCR~kr~v~r
RLCYCRPR~v~v5. 32.7 2
KLCY~K~F~v~v 16 22. 67 2.07
Rr~ArRGR~r-VCV~322.710.79.51 3.93
RLCY~nkKr~v~v (X=MeGly) 5.3 2 2 6 2.54
RXCFCR~F~v~v (X~Cha) 2.67 2.67 0.83
RWCFCRPk~v~v 3.3 2 2 5.2 2.2
WLCYCRRRFCVCV5.31.3
WL~r~v~V
FL~r~KKk~v~v
WL~r~KXCVCV (X=MePhe)
WLCYCRRRFCVCV* 8 4 2 7.14 0.99
RLCYCRRRFCVCV* 81.67 2
WYCYCRRRFCVCV*
WXCY~K~R~v~v* (X~Cha)
RXCFCRGRZCVCV (X=Cha)
(Z-MePhe)
XLCFCRRRZCVCV (X=Cha)
(Z~MePhe)
RLCY~P~r~v~v~0.65 0.1 0.89 0.05
RLCY~r~v~v~ 3. 30.71.3 3.3 0.98
RL~Y~nP~r~V~V~* 8 2.7 2 12.1 1.6
WLCY~KR~r~v~v~*
WXCY~nR~v~v~k* (X=Cha)
RLCYCR~r~v~ 16.61.1 7.73
RKW~r'V~YAGFCYRCR64 8 4
RGGRL~Y~kKr~v~ >32 1.6* 2.7
RRCY~KKk~v~v~ >64 1.3 2 8.07
(32)
RRCY~;K iKr CGCVGR
RWRCYC~KkrCGCVGR
RAR~Y~Kkr-CGCVGR
GWRCY~;K~Kr CGC
RGWACY~n~r~vC
RR~;Y~Rr ~. v~v~
RGWRL~_~r~v~
RGWRLCYCRGRFCVC
~Y~nRkr~V~r 53-3 4 3-33 >64
CY~r~v~v~ >64 2 21.3
RGWRLCY~Kk~r~vC (X=MeGly)
RGWRGCY~K~CGC (X=MeGly) >6410.7
LCY~RRr~v~v~ 8 2 5.8
LCY~nP~v~v~ 13.3 4
LCYCK~Kr~v~v~K 64 2
LCYCRGRr~v~v~ 8 2 2 2.14
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S~nc~MRSA P~a VR~EF ~nn~; ~ E. coli
LCYCRPRr~v~v~GR 8 2
R~ ~PKF~v~vK 2.87 O.18 2.98
WRLCYCKPKr~v~v~K 5.3 4 5
GWLCY~K~'~v~v~K 21 16
RWLCYCRGRr~v~v~K 16 5.3
RLLCY~K~K~v~v~x 6.6 1.3
RWRL~Y~n~Xr~v~V 8 4 5
RXRLCYCRZRFCVCV (X=Cha) 16 5.3
(Z~MeGly)
RGWRLCYCRGRXCVCV (X=Cha) 32 13.3
RGGKv~Y~KGRFCVCV 8 2
KG~Kv~YCRGRFCVCV* 64
L~Y~K~K~v~v (X=D-Ala) 32 9.3 3.33 >64
LCY~K~Kr~v~v 64 3 6.7 >64
VCYCR~Kr~v~v 26.7 5.2 5.26
LCYCRPRFCVCW53.3 42.3
LCYRRPRFRVCV >64 4 16 >64
RGWRLCYCR~Kk~v~v* (X=Cha) >32 >16
RXRLCYCRZRFCVCV* (X=Cha) >32 21
(Z=MeGly)
RXRLCYCR~K~v~v (X=Cha)
RGGGTcypT~r-wIAFcvGR 2.1 0.59 32.6 0.81
RGGGLCYARGFIAVCFGR 19 14 65.8 3.27
RGGC~,TCY~RPRFAVCVGR
RGGGL~Y~PR~l~v~K 8.7 0.07 >128 1.53
RGGGLCYARKGFAVCVGR >128 O.Ol >128 Z.65
RGGRLCYARRRFAVCVGR* 0.05 1.6 0.4
R4r-RT~cy~T~RRFAvcvGR O.Ol 3 0.08
Rr~GGT~;yKK~rlKv~r~K 17 O.19 7.73 3.27
RGGr-TCY~R~wlKr~v~K 2.07 0.15 2.72 3.56
RGGGLCYRL~KrKv~v~n 34.770.22 15.09 0.56
RGGGTCY~TP~r-Kv~v~K 30.760.53 31.4 8.95
RGW~G~ K ~(;K ~GCVGR >64 9.3
RGWRGCYKR~KrK~v~K* >32 8*
T~Y~RRRFAVCV >64 2 10.7
L~L~RR~ lV~V >64 4 16
LCYKR~r~v~V
I~rv~v~K >128 6.9 >128 10.78
~ denote~ free acid form; all other~ are amide form.
MRSA i~ methicillin-re~i~tant S. aureus
P~a i~ P. aeruginosa
VREF i3 Vancomycin-re~i~tant E. faecium
C~n~i ~A i5 C. A 7hi~
Tables 22-25 show activity in the kinetic bactericidal
assay described above provided in terms of log reduction in
CFUs for various peptides of the invention against MRSA,
Pseudomonas aeruginosa, and the endogenous flora in saliva.
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Table 22
RQ~IUCtiOn Of MRSA (ATCC 33591) CFUS after
exposure to peptide ~2 ~g/ml) for 15 minutes
in LTN medium at 37~
Sequence Log
reduction
CFUs
RGGRLCYCRRRFCVCVGR 2.44
RGGRLCYCRRRFCVCVGR 1.83
RGGRLCYARRRFAVCVGR* 0.29
RWRLCYCRPRFCVCV 1.41
RGWRLCYCRPRFCVCVGR 2.06
GWRLCYCRPRFCVCVGR >3.19
XCYCRRRFCVCV (X=Cha) 1.32
LCXCRRRXCVCV (X=Cha) >3.19
RLCYCRRRFCVCV* <o,go
RXRLCYCRZRFCVCV (X=Cha) 1.13
(Z=MeGly)
RGLRXCYCRGRFCVCVGR (X=Cha) 2.48
RWLCYCRGRFCVCVGR 1.58
GGWRLCYCRGRFCVCVGR 2.47
RLLRLCYCRXRFCVCVGR (X=MeGly) 1.66
RGGRLCYCRGRFCVCVGR* <0.90
RXRLCYCRZRXCVCWGR* (X=Cha) 1.38
(Z=MeGly)
RGWRLCYCRGRFCVCVGR 2.12
WLCYCRRRFCVCV 3.16
RLLRLCYCRRRFCVCVGR 2.16
RGGRLCYCRRRFCXCVGR (X=MeVal) <0.90
* Peptides noted with * are acid forms; all others are
amide forms.
Initial inoculum approximately 4 x lOs CFUs/ml.
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T~ble 23
Log Redu¢tion of Pse~ n~ R aeruginosa
(Aq~CC 90~7~ CFUs after 15 minutes expos~ure
to peptide (4 ,ug/ml) in LTM medium at 37~
Sequence Log
reduction
CFUs
RGGRLCYCRRRFCVCVGR 3.19
RGGRLCYCRPRFCVCVGR 3.65
RGWGLCYCRPRFCVCVGR <1.2
RGGGLCYTRPRFTVCVGR 2.15
RGGRLCYCRRRFCVCVGR* 3.98
XCYCRRRFCVCV (X=Cha) 2.81
RLCYCRXRFCVCV (X=MeGly) <0.5
RLCYCRPRFCVCVGR* 3.Z9
RXCFCRPRFCVCV (X=Cha) 1.78
RLCYCRRRFCVCV* 3.52
RGLRXCYCRGRFCVCVGR (X=Cha) 2.87
RGGLCYCRGRF~v~v~K 3.61
RLLRLCYCRXRFCVCVGR (X=MeGly) 2.70
RLLRACYCRXRFCVCVGR (X-MeGly) 2.71
RGGRLCYCRGRFCVCVGR* 2.66
RGWRLCYCRGRFCVCVGR 2.54
RGGRVCYCRGRFCVCVGR 2.37
RGGRVCYCRGRFCVCV 2.18
RGGRVCYCRGRFCVCV* 1.55
WLCYCRRRFCVCV 1.27
* Peptide~ noted with * are acid forms; all others are amide forms.
Initial inoculum approximately 4 x 10~ CFUq/ml.
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T~ble 24
Log Reduction of Pse~ aerugi~o~
(ATCC 9027) CFUs after expo~ure to peptide
(0.12 ~g/ml) in LTM medium at 37~
Sequence 15 min 120 min
RGGRLCYCRRRFCVCVGR 3.48 3.68
RGWGLCYCRPRFCVCVGR 3.48' 3.68
RGGGLCYTRPRFTVCVGR 0.75 3.68
RGGGLCYARKGFAVCVGR 1.20 3.68
10GWRLCYCRPRFCVCVGR 2.70 2.21
RGGRLCYCRRRFCVC 2.25 >3.73
LCYCRRRFCVCV 1.35 2.13
Initial inoculum approximately 4 x lOs CFU~/ml.
Table 25
Log Reduction of CFUs endogenous flora in
saliva after 15 minute~ expo~ure to peptide
(320 ~g/ml) at 37~
Sequence Log
reduction
CFUs
RGGRLCYCRRRFCVCVGR 1.80
RGGRLCYCRPRFCVCVGR 2.04
RGGGLCYKRGWIKFCVGR 1.09
RGWGLCYCRPRFCVCVGR 0.53
25RLCYCRPRFCVCVGR 0.53
RGGGLCYTRPRFTVCVGR 1.0 5
RGGRLCYCRRRFCVCVGR* 1.25
LCYCRGRFCVCVGR 1.02
RWRLCYCRPRFCVCV 0.22
30RGWRLCYCRPRFCVCVGR 0.38
RGWRACYCRPRFCACVGR 0.28
GWRLCYCRPRFCVCVGR 0.26
XCYCRRRFCVCV (X=Cha) 0.25
WLCYCRRRFCVCV* 0.16
35RLCYCRXRFCVCV (X=MeGly) 3.43
RLCYCRPRFCVCVGR* 0.66
RGGGLCYCRPRFCVCVGR* 1.51
RXCFCRPRFCVCV (X=Cha) 0.71
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Table 25
Log Reduction of CFUs endogenous flora in
saliva ~fter 15 minutes exposure to peptide
(320 ,ug/ml) at 37~
Sequence Log
reduction
CFUs
RWCFCRPRFCVCV O.52
LCXCRRRXCVCV (X=Cha) 0.23
RGGRLCYCRRRFCVC 0.86
L~Y~l~K~FTVCV 0.64
RRCYCRRRFCVCVGR 0.98
RLCYCRRRFCVCV* 0.21
RXRLCYCRZRFCVCV (X=Cha) <0.6
(X=MeGly)
RGWRLCYCRGRXCVCV (X=Cha) <O.6
RGLRXCYCRGRFCVCVGR (X=Cha) 1.65
RGWRGCYKRGRFKGCVGR <0.97
RGWRGCYCRXRFCGC (X-MeGly) <0.6
RGGLCYCRGRFCVCVGR 2.52
RLLRLCYCRXRFCVCVGR (X--MeGly) 0.65
RLLRACYCRXRFCVCVGR (X=MeGly) 2.11
RGGRLCYCRGRFCVCVGR* 2.16
RGWRLCYCRGRFCVCVGR 1.89
RGGRLCYCRGRFCVCVGR 2.53
RGGRVCYCRGRFCVCVGR 2.37
RG&RVCYCRGRFCVCV 2.07
RGGRVCYCRGRFCVCV* <0.97
WLCYCRRRFCVCV 1.87
25 * Peptide~ noted with * are acid forms; all others are amide forms.
InLtial inoculum appro~; -tely 4 x 107 CFUs/ml saliva. Peptides (3200
~g/ml) are dissolved in 0.01% acetic acid and added as 1/10 volume to
~liva.
Kinetic experiments were also run using Haemophilus
influenzae (ATCC 49247) which does not ordinarily grow on
TSA, but survives several hours in LTM.
Log-phase cells (4.5 x 105 CFUs/ml in LTM) were treated
with peptide and then plated onto Chocolate agar to determine
35 the number of viable CFUs. All peptides tested (acid and
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amide forms of RGGRLCYCRRRFCVCVGR, and amide form o~
RGGLCYCRGRFCVCVGR) were rapidly bactericidal against N.
influenzae. No regrowth was observed at 240 minutes, as seen
previously with P . aeruginosa .
5Finally, the assay described above was conducted using
H. pylori as the target provided the results ~hown in Ta~le
26 .
Table 26
Eff~ct of peptide (32~g/ml) on CFU~ o~ Nelicobacter
pylori ~ATCC 33591) in LTM a~ter 15 minute~ exposure
Sequence Log reduction CFUs
RGGRLCYCRRRFCVCVGR 2.90
RGWGLCYCRPRFCVCVGR 3.43
RLCYCRPRFCVCVGR 1.34
RGGGLCYTRPRFTVCVGR <1.12
RGGGLCYARKGFAVCVGR <1.2
RGGRLCYCRRRFCVCVGR* 3.00
LCYCRGRFCVCVGR <1.12
RGWRACYCRPRFCACVGR <1. 6
XCYCRRRFCVCV (X=Cha) <1.70
WLCYCRRRFCVCV* <1.6
RLCYCRXRFCVCV (X=MeGly) 1.57
RLCYCRPRFCVCVGR* <1.6
RXCFCRPRFCVCV (X=Cha) >4.66
RGGRLCYCRRRFCVC <1.2
L~Y~l~K~FTVCV <1.28
RLCYCRRRFCVCV* <1.2
RXRLCYCRZRFCVCV (X=Cha) 1.97
(Z=MeGly)
RGWRLCYCRGRXCVCV (X=Cha) <1.47
RGLRXCYCRGRFCVCVGR (X=Cha) 3.22
RGGLCYCRGRFCVCVGR 1.57
RLLRLCYCRXRFCVCVGR (X=MeGly) 2.10
RLLRACYCRXRFCVCVGR (X=MeGly) 1.42
RGGRLCYCRGRFCVCVGR* 2.40
RGLRXCYCRGRFCVCVGR* (X=Cha) <1.47
RGWRLCYCRGRFCVCVGR >3.57
RGGRVCYCRGRFCVCV* <1.47
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Sequence Log reduction CFUs
WLCYCR~RFCVCV 3.36
WLCFCRRRFCVCV 1.97
FLCFCRRRFCVCV 1.99
WYCYCRRRFCVCV <1.6
WXCYCRRRFCVCV (X=Cha) <1.6
WLCYCRRRFCVCVGR <1.67
WXCYCRRRFCVCVGR (X=Cha) <1.67
RLLRLCYCRRRFCVCVGR <1.67
RGGRLCYCRRRFCXCVGR (X=MeVal) <1.67
Initial inoculum approximately 4 x 105 CFUs/ml.
Peptides denoted with * are acid forms; all others are amide
forms.
The modified NCCLS assay was also used as described
above, where the ini inhibitory concentration (~g/ml) is
reported in Tables 28 and 29. Peptides were prepared in
O.01% AA with (~HSA) or without (-HSA) human serum albumin at
0.1%. Table 28 shows the results of various peptides tested
20 agsinst various organisms; Table 29 shows the results in the
presence of human serum albumin. Generally, the HSA
decreased the MICs, putatively by preventing absorption to
the plastic surfaces.
Table 28
Minimum Inhibitory Concentration (~g/ml) for
peptides tested in the mod~fied NCC~ a~say
Se~uence P. aeruginosa MRSA V~EF
RGGRLCYCRRRr~v~v~K 8.00 8.00 2.00
30RGWGLCY~KPKr~v~v~K 16.00 16.00
RGGRL~Y~KRKr~v~v~K* 8.00 16.00 2.00
WLCY~K~r~v~v* 32.O 32.00
RGGLCY~K~Kr~v~v~K 4.00 8.00 2.00
Rr~r~R~cycRxRr~v~v~K (X=MeGly) 16.00 16.00
35WLCY~KKKr~v~v 128.00 16.00 3.00
WY~KKRr~v~v 32.0 64.00
WLCY~KkKr~v~v~K >64 64.00
RLLRLCYCRRRr~v~v~K 32.0 32.00
RGGRLCY~K~r~CVGR (X=MeVal) 64.0 >128
* Peptide~ noted with * are acid formq; all other~ are amide formq.
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Peptides were prepared in O.O1~ acetic acid without HSA.
T~ble 29
MICs ~'~g/~l) in Mueller Hinton Media
Organi~ M-d~um RGG~K~K~ ~ V ~ ~ PG-l (~mide) PG-l
(nmide) (acid)
Suppl. - HSA + HSA - HSA + HSA - HSA
Staphylococcus aureus None 4 4 4
MSSA
Staphylococcus aureus None 13.3 2 16 5.3 16
0 MRSA
Enterococcus faecium None 2 0.33 1.3 0.25 2
VREF
- hematin
+ hematin 8
~acillu3 subtilis None o. 7 0.8 0.2
Streptococcus - hematin 2
~n~-r~~niae~
+ hematin 8
Streptococcus - hematin o.12
sali~arius~
viridans group + hematin 0.5
Ps~ aeruginosa None 4 1.33 5.3 0.33 9.3
Klebsiella r~7 iae None 5.3 4 4
Serratia marcescens None 16 16 21
Escherichia coli None 4 0.33 5.3 0.12 4
- hematin 0.5
+ hematin ' 8
~A~ ~r-; 7us influenzae~ - hematin No growth
30+ hematin 8
Acinetobacter None 2 3 4
calocoaceticus
Nei~seria meningitidis~ - hematin 8
+ hematin 32
Candida albicans None 8 4 16 8 16
Table 30 shows the MICs of various peptides against a
variety of microorganisms.
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TablQ 30
MICs of Various PQptia~s
MICs (~g/mL)
~riQu~:Ncri MRSA Psa VREF Can.
R~r~RT~cycnKnr CV~V~ 2 - 40.3 - 1.7 0.13 8, 16
WL~nR~r~CVCV
FLcb~K~b~v~v 5.3 >32 0.5 128
WYCY~KKK~'~V~V* 42.7 32 1 >128
WXCY~nRKr-~v~v* (X=Cha)
WLCY~K~b~v~v~n* 27-32 32-530.25-0.5>128
WXCYCRRRr~v~v~K* (X=Cha)
RLLRCYcnRkrcY~v~n 32 t 32 t
Rr-~RT~cy~KR~c~v~n (X=MeVal) ~128t 64t
R~v~vcrnnnCYCLW
h~v~vc~K~RCYCLW 10. 7 >32 0.5 32
Vcv~rn~CYCLW 16 >32 1 >128
r~vcr~RRCFCLF 16 >32 2 >128
R~v ~v ~ KRR~ y CRGGR 8 8 0.25 16
R~v~v~rnKRCYCLRGGR (all D) 21 8 4 32
R~vcvcrK~CYCLW* 53 >32 2 128
R~v~v~Y~RCYCLW (X=MeGly) 13 32 1 64
WL~rcK~ZY~v~v~K (X=MeGly)
(Z=D-Arg)
k~r~v~rKKv~YcLw >32 128 2 >128
WLCY~KR~r~vcv~n 11 48 0.21 128
WLCYCRR~vcvn (X=D-Arg) 5.3 32 0.25 64
WLCY~KKK~vCVGR 6.7 4.3 0.21 32
Octyl-WLCY~KKKr~v~v~n
XLCYCRRRFCVCV (X=l-Nal) 4 128 0.5 >128
WL~VK* >128 >128 >32 >128
WLCRGRFCFR >128 >128 16 >128
WL~YKKV~VK 64 32 16 16
WL~YCOOOr ~V~v 2 4 0.5 64
WLCY~r-~v~v (X=Dab) 2 2 0.5 32
WLCYcn~n~v~v (all D) 1 8 0.5 128
~wRLcycR~Kr~v~v 13.3 32 1 ~128
RwRLcycnpKr~vcv 1 4 0.5 128
OWRLCYCRPKr~v~v 1 4 0.25 128
XWRLCYCRPKr~cvcv (X~Dbu) 2.67 5 ~ 30.25 107
RwHLcycRpKr~v~v 4.3 13.30.25 ~128
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NICs (~g~mL)
~yU~N~ MRSA Pea VREF Can.
RWKLCYCRPK~vuv 2 13.3 0~5 107
RWOLCYu~EK~v~v 1 2.7 0.25 43
RWXLCYuRPK~v~v (X=Dbu) 2 2 0~5 64
NLCYu~xKr~v~v~ (X=Tic)
5~r~K~K~YCV (X-Hyp)
WL~Y~K~K~v~v (X=hCy8) 16 64
WOLCYCu~Or-~v~vO (X=Tic) 1 2
Or~v~v~OFCVCVO (X=Tic) 16 85
OWOLCYCOXOFCVCV (X=Tic) 4 11
lOO~v~OLCYCFO (X=Tic) 32 >128
NLCYuKKK~v~v 2 5.3
ONOLCYCOXOFCVCV (X=Hyp) 1 2.3
WLCYCOXOFCVCVO (X=Pba)
WLCYCOOOFCVCV (all D)
15XFCYCLR~v~v~* (X-D-Arg) 8 48
WLCYCR~x~-~v~v~* (X=D-Arg) 64 48
(Z=MeGly)
* Peptides denoted wLth * are acid forms; all others are amide forms
MRSA is methicillin-resistant S. aureus
P~a i~ P. aeruginosa
VREF i~ V~nc ycin-resistant E. faecium
Can is C. albicans
t denotes no HSA or BSA in assay
_ 99 _
