Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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CATIONIC, AMPHIPATHIC BETA-SHEET PEPTIDES AND USES THEREOF
This application is a nonprovisional of Serial No.
60/182,495 filed February 15, 2000, which is hereby
incorporated by reference.
Mention of Government Grant
The invention disclosed herein was developed with the
assistance of NIH Grant 1-R-15-AI-47165. The U.S.
Government has certain rights in the invention.
BACKGROUND OF THE INVENTION
Field of the Invention
This invention relates to the use of cationic peptides
whictz can assume an amphipathic beta-sheet secondary
structure for clinical and diagnostic purposes, especially
inhibition of microbial activity.
Description,of the Background Art
We know that diseases caused by bacteria have afflicted
humans since the beginning of recorded history. The Black
Plague devastated the population of Europe in the fourteenth
century. Other major outbreaks of bacterial infection
wreaked havoc on a regular basis through the nineteenth
century. It was not until the mid-1800's that scientists
such as Louis Pasteur first linked microorganisms with human
disease. This breakthrough set the stage for the
identification of therapeutic agents that could selectively
kill them.
In the early 1900's, a compound named Salvarsan, an
arsenic-containing dye, was developed by Paul Erhlich as a
treatment for syphilis. Soon thereafter, in the 1920's,
Alexander Fleming discovered lysozyme (present in human
tears) and penicillin; however, the clinical application of
these discoveries did not materialize until the 1940's. In
the meantime, a new dye called Prontosil was found to cure
Streptococcus infections in mice. This compound, a
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sulfonamide, opened the door to the development of other
antibiotics. In the early 1940's, Howard Floret' and his
coworkers finally purified penicillin. Following the fire
at the Cocoanut Grove in Boston in 1942 (where crude
penicillin used to treat burn victims received tremendous
acclaim), pharmaceutical companies dramatically increased
the production of penicillin, leading to its widespread
availability and use. Penicillin was declared a "miracle
drug". In the late 1940's and early 1950's, oral penicillin
was available over-the-counter without a prescription.
Since most living organisms are very similar at the
molecular level, it is difficult to find substances that are
lethal to certain organisms without being harmful to others.
Antibiotics have proven effective in eliminating or at least
greatly reducing the incidence of many clinical problems
caused by bacteria because these compounds possess the
necessary selectivity to attack bacterial cells while
sparing human cells. These antibiotics rely upon
differences between these cells. For instance, penicillin
is effective because it targets the bacterial cell wall, for
which there is no similar counterpart in human cells.
- Unfortunately, the widespread use of common antibiotics such
as penicillin has selected for resistant strains.that are no
longer susceptible to these agents.
In 1945, Fleming predicted that improper use and
overuse of the new drug would lead to the development of
resistant microorganisms. Penicillin resistance soon
materialized; however, the discovery of new antibiotics
lessened the impact of the resistance problem. Streptomycin
and other aminoglycosides, chloramphenicol, tetracyclines,
cephalosporins, quinolones, semi-synthetic penicillins like
ampicillin and methicillin, and super-potent drugs like
vancomycin enriched the antimicrobial arsenal. In the past
two decades, only a small number of new antimicrobials have
appeared on the market. The array of available drugs seemed
to be sufficiently vast to stifle interest in costly new
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development programs.'
Over the years, however, more and more microorganisms,
exposed to more and more antibiotics, have adapted to these
compounds. Resistance to antimicrobial drugs is now a
worldwide problem. The emergence of methicillin-resistant
Staphylococcus aureus in the 1970's was a major setback in
antibiotic therapy. The prospect of vancomycin-resistant S.
aureus looms on the horizon. Multiply-drug-resistant
strains of Mycobacterium tuberculosis have resulted from
improper or incomplete therapy. Outbreaks of tuberculosis
are now commonplace among indigent populations in cities
throughout the U.S. Since resistance is appearing to even
the most potent antibiotics such as vancorriycin, the
development of new approaches in antimicrobial therapy is
imperative . 1
Some new ideas include limiting the ability of
microorganisms to transfer plasmids containing resistance
genes, reducing the virulence of disease-causing organisms,
and looking for new models of antimicrobial compounds.e-io
The host defense systems of animals are potential
sources of new ideas in antimicrobial therapy. In
particular, small cationic peptides represent a large class
of weaponry used in the protection against-bacterial
infection by animals.li,i2 A diverse collection of host
- defense peptides, discovered in a wide range of species,
shares the common characteristics of a net positive charge
and the ability to form amphipathic structures. Many of
these peptides appear to exert their protective effect by
permeabilizing the membranes of target organisms. The
efficacy of these peptides results from their ability to
- _ disrupt prokaryotic membranes at concentrations that are not
harmful to host membranes. Frog skin is a particularly rich
source of antimicrobial peptides, including magainins and
PGLa.
The discovery of naturally occurring antimicrobial
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peptides opened a new pathway for antibiotic development.z-3
Magainin 1 and mag.ainin 2, first isolated from frog skin in
1987, are representative of th-a class of small linear
cationic peptides that can kill both Gram-positive and Gram-
negative bacteria by increasing the permeability of the
plasma. membrane at concentrations that do not induce
hemolysis.4~5 PGLa, also isolated from frog skin, has
greater antimicrobial activity than magainins while
retaining low hemolytic activity.6 A common feature of these
peptides is their capacity to form an amphipathic a-helix
(with polar and nonpolar groups on opposite faces of the
helix), a structural feature believed to be important in
their function as antimicrobial agents.
These linear cationic peptides, containing 21-23 amino
acid residues, demonstrate broad antimicrobial activity;
however, relatively high concentrations are necessary to
kill most target organisms. It is possible to enhance
antimicrobial activity through simple modifications of the
native peptides. For instance, substituting Ala for Glu-19
in magainin 2 amide substantially increases antimicrobial
activity (47, 52).
Our earliest work involved looking at the role of the
outer membrane and LPS in the interaction between magainin 2
and the Gram-negative cell envelope , is-la, so, s~ Magainin 2
altered the thermotropic properties of the outer membrane-
peptidoglycan complexes from wild-type Salmonella
typhimurium and a series of LPS mutants that display
differential susceptibility to the bactericidal activity of
cationic antibiotics. These results were correlated with the
LPS phosphorylation pattern and charge (characterized by
high-resolution 31P NMR) and outer membrane lipid -
composition, and were compared to the bactericidal
susceptibility. LPS mutants showed a progressive loss of
resistance to killing.by magainin 2 as the length of the LPS
polysaccharide moiety decreased. Disordering of the outer
- membrane lipid fatty acyl chains by magainin 2 depended
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primarily upon the magnitude of LPS charge rather than the
length of the LPS polysaccharide. While disruption of outer
membrane structure most likely is not the primary factor
leading to cell death, the susceptibility of Gram-negative
5 cells to magainin 2 is associated with factors that
facilitate the transport of the peptide across the outer
membrane, such as the magnitude and location of LPS charge,
the concentration of LPS in the outer membrane, outer
membrane molecular architecture, and the presence or absence
of the 0-antigen side chain.
Magainins and PGLa function by binding to bacteria and
inducing leakage. The selectivity for bacteria over
mammalian cells is based at least in part on the presence of
anionic lipids on the outer surface of bacteria, such as
lipopolysaccharide (LPS) in the outer membrane of Gram
negative organisms ls-le and phosphatidylglycerol (PG) and
diphosphatidylglycerol (DPG) in the plasma membrane.l9 In
contrast, the outer surface of mammalian cells is populated
almost exclusively by zwitterionic phospholipids, primarily
phosphatidylcholine (PC) and sphingomyelin. Magainin-
induced leakage in large unilamellar vesicles (LUV) is
generally favored as the ratio of acidic t o neutral lipids
increases. Thus, electrostatic interactions play an
integral role in at least the initial binding process.
Magainins and related peptides can adopt some degree of
a-helical structure in the presence of TFE or when bound to
lipid bilayers. In aqueous solution, however, they possess
no discernable secondary structure. While there is general
agreement that an a-helix is the dominant conformation,
there is debate concerning the level of helicity, ranging
between 60 and 90o.z° In spite of the experimental data,
most often these peptides are considered to be entirely
helical. This point may be important in evaluating models
of how the peptides exert their lytic effects. Another area
of contention is the orientation of the peptides with
respect to the plane of the lipid bilayer. Peptide -
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orientation has been evaluated using solid-state NMR,zI-zz
fluorescence quenching, z3 and oriented circular dichroism
(CD).z4 NMR and fluorescence results show that the peptides
remain associated with the lipid polar groups; however, the
CD study suggests that the hel-ical axis of the peptide
changes from parallel to the lipid bilayer to perpendicular
when the peptide-to-lipid ratio exceeds 1:30.
Much attention has been focused on whether these
peptides form channels or pores in lipid bilayers and
membranes, and if so, the exact nature of these pores. An
early model proposed that the peptides, after binding to the
surface of the membrane, self-associate to form a multimeric
bundle that inserts across the bilayer, with the polar
helical faces lining the cavity of the pore.3~z~ Other more
complex models were developed that include lipid molecules
as part of the pore.z6 More recently, a model invoking a
supramolecular organization of lipids~and peptides forming a
torus was proposed by Matsuzakiz' and Huang.z° If these
models are correct, there are no direct peptide-peptide
interactions involved in pore formation. In fact, the pores
might simply be membrane defects similar to those that would
result from the accumulation of detergent molecules in the
bilayer on the way to formation of micelles and the loss of
bilayer structure. The peptides binding to the interfacial
region of the lipid bilayer may well mimic the action of
detergents by expanding the surface area to induce
sufficient positive curvature to destabilize the bilayer.
Thus far, it appears that microorganisms have not been
able to develop resistance to magainins and related
peptides, even after chronic exposure to sublethal doses.
This is probably due to the fact that the peptides do not
bind to a specific receptor, but instead are attracted to
the anionic membrane surface that would be difficult for the
organism to alter extensively. The most likely resistance
mechanism against these molecules is the presence of
proteases that could degrade the peptides. The emergence of
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proteases might have less impact if it is possible to
identify many diverse sequences that possess potent
antimicrobial activity and good selectivity for bacterial,
cells.
In 1987, Zasloff isolated magainin 1 and magainin 2, a
pair of 23-residue peptides with broad spectrum
antimicrobial activity.4 These peptides kill microorganisms
by increasing the permeability of the bacterial plasma
membrane at concentrations that are not hemolytic. The
sequence of magainin 2 is shown below:
GIGKFLHSAKKFGKAFVGEIMNS (SEQ ID NO:1)
1 5 10 15 20
In water, this peptide has no discernable secondary
structure, but in the presence of trifluoroethanol (TFE), it
is mostly cx-helical 13. Due to the placement of the polar
and nonpolar residues in the amino acid sequence, the
resulting a-helix is reasonably amphipathic.
Magainin 2 amide exhibits only weak potency (i.e., its
minimum inhibitory concentration (MIC) values against most
microorganisms is =256 ug/mL). Deletion of E at position 19
or its replacement by A greatly increases the antimicrobial
activity of the peptide (see Table 2).15 These analogues are
more cationic (+4 vs. +3), but their amphipathic character
as a-helices is virtually unchanged. We have studied the
conformation and activity of the E19A analogue.
MSI-78, with a net charge of +10, is a peptide derived
from the E19 deletion analogue of magainin 2 amide that is
under development as a topical antimicrobia~l agent by
Magainin Pharmaceuticals, Inc.l2
Its sequence is:
MSI-78 GIGKFLKKAKKFGKAFVKILKK- NHz (SEQ ID N0:21)
1 s to is 20
PGLa is a similar peptide also discovered in frog skin.G
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Like magainins, it is a small cationic peptide with the
potential to form an amphipathic a-helix. PGLa is
substantially more active than magainin 1 or 2, while .
maintaining low hemolytic activity. Its amino acid sequence
is:
GMASKAGGIAGKIAKVALKAL- NHZ (SEQ ID N0:2)
i s io is Zo
A more potent analogue was produced by replacing two
glycines with lysines (G1K, G8K). In this analogue, there
are three heptamers with the sequence KXXXKXX. Three new
peptides (each derived from one of the heptamers) were made
as trimeric repeats. The peptide from the middle heptamer,
(KIAGKIA)3-NH2 (SEQ ID N0:4), possessed the most potent
antimicrobial activity, on a par with MSI-78, even though
its net charge is much less (see Table 2).11
Numerous analogues with sequences derived from these
peptides have been prepared and examined. In all cases of
which applicants) are aware, the strategy employed in
enhancing activity involved increasing the amphipathic-a-
helical character of the peptide.
Several other linear amphipathic (3-sheet peptides have
been examined previously. An 18-residue KL repeat was
reported to have no appreciable antimicrobial or hemolytic
activity (52). Peptides containing 6-12 residues with
repeats of either SVKV (SEQ.ID N0:22) or KV were shown to
adopt a (3-sheet structure in the presence of lipid(23).
While some of these peptides could induce leakage in lipid
vesicles, none were antimicrobial below a concentration of
100 ug/mL. The peptide FKVKFKVKVK (SEQ ID N0:23) was able
to inhibit the growth of E. coli, S. aureus, and P.
aeruginosa at concentrations comparable to the peptides in
this study; although the hemolytic activity of this peptide
was not tested (54). FKVKFKVKVK (SEQ ID N0:23) was shown by
C_D to adopt a ~3-sheet structure in the presence of either
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50o TFE or 25 mM sodium dodecyl sulfate.
Recently, a series of (KL)nK-NH2.(for n=7, 15 residues:
SEQ ID N0:24) peptides containing 9 to 15 residues (all
dansylated at the amino terminus) was studied by Castano et
al. (55). The amide I vibrational band in the IR spectra
(either dry, at the air/water interface, or inserted into a
lipid monolayer) of these peptides was very similar to that
of KIGAKIx3 (Fig. 3), centered near 1620 cml. All of the
peptides induced both leakage in lipid vesicles and
hemolysis, with activity increasing as a function of length.
The antimicrobial activity of these peptides was not
examined.
Shai and coworkers studied diastereomeric antimicrobial
peptides based upon 12-mers containing K and L (56, 57) or
derivatives of pardaxin (58). These peptides were derived
from all-L-amino-acid parent compounds that can form
amphipathic a,-helices. Based upon changes in the amide I
infrared band, the conformation of the diastereomeric
peptides was interpreted to be mainly (3-sheet; however, the
appearance of the amide I band. is much different than that
of the peptides in this study or those examined by Castano
et al. (55). Instead of a relatively narrow band near 1620
cml, the diastereomeric peptides gave rise to broad bands
centered between 1640 and 1650 crril. Clearly, the
conformation of these peptides is significantly different
than that of KIGAKI when bound to lipid bilayers.
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SUM~2ARY OF THE INVENTION
The present invention is directed to peptides which
carry a sufficient positive charge to selectively disrupt
microbial but not mammmalian cell membranes, contain enough
5 hydrophobic residues to be able to enter a cell membrane,
preferentially assume a beta sheet structure in a membrane
environment, are substantially amphipathic in that structure
but not in an alpha helical structure, and have
antimicrobial activity.
10 All known naturally occurring linear cationic peptides
adopt an amphipathic a-helical conformation upon binding to
lipids as an initial step in the induction of cell leakage.
We designed an 18-residue peptide, (KIGAKI)3-NHZ (SEQ ID'
N0:8), that has no amphipathic character as an a-helix but
IS can form a highly amphipathic (3-sheet. When bound to
lipids, (KIGAKI)3-NH2 (SEQ ID N0:8) did indeed form (3-sheet
structure, as evidenced by Fourier transform infrared and
circular dichroism spectroscopy. The antimicrobial activity
of this peptide was comparable to that of (KIAGKIA)3-NHz (SEQ
ID N0:4), and better than that of PGLa (SEQ ID N0:2) and
(KLAGLAK)3-NHZ (SEQ ID N0:6), all of which form amphipathic
a-helices when bound to membranes. (KIGAKI)3-NH, (SEQ ID
N0:8) was much less effective at inducing leakage in lipid
vesicles.composed of mixtures of the acidic lipid, -
phosphatidylglycerol and the neutral lipid,
phosphatidylcholine, as compared to the other peptides.
When phosphatidylethanolamine replaced phosphatidylcholine,
however, the lytic potency of PGLa and the a-helical model
- peptides was reduced, while that of (KIGAKI)3-NHZ (SEQ ID
N0:8) was improved. Moreover, fluorescence experiments
using analogs containing a single tryptophan residue showed
that unlike the a-helical peptides, (KIGAKI).3-NHZ (SEQ ID
N0:8) preferentially bound to vesicles containing
phosphatidylethano-lamine instead of phosphatidylcholine,
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suggesting enhanced selectivity between bacterial and
mammalian lipids .
Linear amphipathic ~i-sheet peptides such as (KIGAKI)3-
NHZ (SEQ ID N0:8) may be used as antimicrobial agents.
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BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1. Helical wheel and beta strand diagrams showing the
distribution of amino acid side chains (+ = lysine, white =
glycine, gray = alanine, and black = isoleucine or leucine).
The hydrophobic moment (uH), calculat.ed using the consensus
hydrophobicity scale (15), is noted for each conformation.
(A) (KIAGKIA)3 (SEQ ID N0:4); (B) (KLAGLAK)3 (SEQ ID N0:6);
and (C) (KIGAKI) 3 (SEQ ID N0:8) .
FIG. 2. CD spectra of (KIAGKIA)3 (dashed line), (KLAGLAK)3
(dash-dotted line), and (KIGAKI)3 (solid line) in (A) aqueous
buffer; (B) 50o trifluoroethanol/buffer; and (C) POPG LUV
(lipid-to-peptide ratio = 20).
FIG. 3. FTIR spectra of (KIAGKIA)3 (A), (KLAGLAK)3 (B), and
(KIGAKI)3 (C) in the presence of POPG (lipid-to-peptide ratio
- 20) .
FIG. 4. Percent release of calcein from LUV three minutes
following the addition of PGLa (open bars), (KIAGKIA.)3 (fine
hatched bars), (KLAGLAK)3 (coarse hatched bars), or (KIGAKI);
(cross-hatched). LUV composition: (A) POPG; (B) POPC; and
(C) E. coli polar lipids (67o PE, 23o PG, loo DPG).
FIG. 5. Percent release of calcein from LUV three minutes
following the addition of PGLa (open bars), (KIAGKIA)3 (fine
hatched bars), (KLAGLAK)3 (coarse hatched bars), or (KIGAKI)=
(cross-hatched bars). LUV composition: (A) 1:1 POPC/POPG;
(B) 2:1 POPC/POPG; (C) 3:1 POPC/POPG; (D) 4:1 POPC/POPG; (E)
1:1 POPE/POPG; (F) 2:1 POPE./POPG; (G) 3:1 POPE/POPG; and (H)
4:1 POPE/POPG.
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FIG. 6. Percent release of calcein from LUV three minutes
following the addition of PGLa (open bars), (KIAGKIA)3 (fine
hatched bars), (KLAGLAK)3 (coarse hatched bars), or (KIGAKI)3
(cross-hatched bars). LUV composition: (A) POPC/POPG/DPG
S (6.7/2.3/1); and (B) POPE/POPG/DPG (6.7/2.3/1). The
replacement of PE by PC in the ternary mixture results in an
increase in potency for all peptides except (KIGAKI)3-NH2.
FIG. 7. Shifts in the emission maximum of tryptophan
fluorescence of W-KIAGKIA (SEQ ID N0:5) (A), W-KLAGLAK (SEQ
ID N0:7) (B), and W-KIGAKI (SEQ ID N0:9) (C). The emission
peak positions in aqueous solution were: 356 nm (W-
KIAGKIA); 354 nm (W-KLAGLAK); and 355 nm (W-KIGAKI). The
peptide concentration was 3 ~aM. The lipid-to-peptide ratio
was 20 for measurements in the presence of LUV. The LUV
abbreviations are: PC = POPC; PC/PG = POPC/POPG; PE/PG =
POPE/POPG; PG = POPG; E. coli = E. coli polar lipids;
PE/PG/DPG = POPC/POPG/DPG (6.7/2.3/1); and PC/PG/DPG =
POPC/POPG/DPG (6.7/2.3/1). Errors are less than ~2 nm for
all measurements.
We measured the shift for three peptides (W-KIAGKIA, W-
KLAGLAK, and W-KIGAKI) to estimate binding to LUVs with
different lipid composition at a lipid-to-peptide ratio of
20. For each peptide, the greatest shift occurred with POPG
LUVs. The Trp analogue of (KIAGKIA)3-NHZ had a slightly
higher affinity fo.r LUVs containing POPC vs. POPE. The Trp
analogue of (KLAGLAK)3-NHZ had a much higher affinity for
LUVs containing POPC vs. POPE. In contrast, the Trp
analogue of (KIGAKI)3-NHZ had a higher affinity for LUVs
containing POPE vs. POPC.
Figure 8: Model of Peptide Association and Disruption of the
Lipid Bilayer.
PE promotes negative curvature in the bilayer surface
because its head group is smaller than that of PC. If
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peptide induced disruption is the result of an increase in
positive curvature, than the replacement of PC with PE
should reduce leakage.
Figure 9: Estimate of Peptide Molecules per Bacterium and
Surface Lipid in MIC Assays
Figure 10: Hydrophobic Moments of 18-Mer peptides (A) Alpha-
Helical Conformation, (B) Beta-Sheet Conformation.
Peptides: "1,2-Hex" (KKIGAI)3 (SEQ ID N0:10); "1,3-Hex"
(KIKGAI)3 (SEQ ID N0:11); "1,4-Hex" (KIGKAI)3 (SEQ ID N0:12);
"1,5-Hex" (KIGAKI)3 (SEQ ID N0:8); "1,6-Hex" (KIGAIK)3 (SEQ
ID N0:13); "Short-Hept" (KLAGKLA)zKLAG (SEQ ID N0:14).
Figure 11: Hydrophobic Moments of 21-Mer peptides (A) Alpha-
Helical Conformation, (B) Beta-Sheet Conformation.
Peptides: "1,2-Hept" (KKLAGLA)3 (SEQ ID N0:15); "1,3-Hept"
(KLKAGLA)3 (SEQ ID N0:16); "1,4-Hept" (KLAKGLA)3 (SEQ ID
N0:17); "1,5-Hept" (KLAGKLA)3 (SEQ ID N0:18); '~1,6-Hept"
(KLAGLKA)3 (SEQ ID N0:19); "1,7-Hept" (KLAGLAK)3 (SEQ ID
N0:6); "Long-Hex" (KIGAKI)3 KIG (SEQ ID N0:20).
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DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE
INVENTION
PGLa possesses greater Gram positive antimicrobial
activity than magainin 2 (see Table 2). PGLa is largely a-
5 helical when bound to lipid bilayers and appears to form
pores in membranes. A more potent derivative of PGLa
contains three heptamer repeats of sequence KXXXKXX, where X
represents a nonpolar residue, as shown in Table 1. Cp.
AIAGKIA in PGLa at residue 8-14 of SEQ ID N0:2. A 21-
10 residue amidated peptide containing three heptameric repeats
of KIAGKIA possesses high antimicrobial and relatively low
hemolytic activity. When KIAGKIA1 adopts an a-helical
conformation, the peptide is highly amphipathic. with all six
lysines clustered on the helical face (Fig. 1). Using a
15 consensus hydrophobicity scale, the hydrophobic moment, u~, a
quantitative measure of amphipathicity , for this peptide is
much greater as an a-helix (0.40) as compared to a (3-sheet
(0.16) .
In order to determine whether a highly amphipathic a-
helix is a prerequisite for potent antimicrobial activity,
we synthesized a peptide, KLAGLAK, with a similar amino acid
content but with a heptamer repeat that separates the six
lysines into two groups of three on the helical face,
resulting in a large decrease in uH to 0.25 (see Table 1 and
Fig. 1). Like KIAGKIA, KLAGLAK cannot form a highly
amphipathic (3-sheet structure.
Even though all known naturally occurring linear
antimicrobial peptides have the capability to form at least
a reasonably amphipathic a-helical structure, we designed a
new peptide that can form a highly amphipathic ~3-sheet
rather than a-helix. This 18-residue peptide contains the
hexameric repeat, KIGAKI (Table I). The value of u" as an a-
helix and a (3-sheet is 0 and 0.63, respectively, as shown in
Fig. 1. The three model peptides, KIAGKIA, KLAGLAK, and
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KIGAKI, possess equal charge (+7) and nearly equal mean
hydrophobicity values. We compared the antimicrobial and
hemolytic activity of these peptides, used CD and FTIR
spectroscopy to determine the conformation of the peptides
in solution and when bound to lipid bilayers, and measured
the ability of the peptides to induce leakage in and bind to
LUV of varying lipid composition. Our results show that
KIGAKI does indeed adopt a (3-sheet conformation when bound
to lipids and is comparable in antimicrobial activity to
KIAGKIA and KLAGLAK. KIGAKI appears to possess greater
selectivity for bacterial vs. mammalian lipids as compared
to the a-helical peptides tested.
The appended claims are hereby incorporated by
reference as an enumeration o~f some. of the preferred
embodimens.
Abbreviations
CD circular dichroism
DSC differential scanning calorimetry
FTIR Fourier transform infrared ,
L/P lipid-to-peptide ratio
LUV large unilamellar vesicle
~.z" hydrophobic moment
MIC minimum inhibitory concentration
DiPoPE 1,2-dipalmitoleoylphosphatidylethanolamine _
DPG diphosphatidylglycerol
PC phosphatidylcholine
PE phosphatidylethanolamine
PG phosphatidylglycerol
POPC 1-palmitoyl-2-oleoylphosphatidylcholine
POPE 1-palmitoyl-2-oleoylphosphatidylethanolamine
POPG 1-palmitoyl-2-oleoylphosphatidylglycerol
T" bilayer-to-hexagonal phase transition temperature
TFE trifluoroethanol
LPS lipopolysaccharide
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NMR nuclear magnetic resonance
REDOR .rotational echo double resonance (form of NMR)
CFU colony forming units -
EDTA ethylene diamine tetraacetic acid
PIPES piperazine-N,N~=bis(2-ethanesulfonic~acid)
HEPES N-(2-hydroxyethyl)piperazine-N'(2-ethane sulfonic
acid)
ONGP o-nitrophenyl-~3-D-galactopyranoside
HPLC high pressure liquid chromatography
Cationicity
A "cationic" peptide is one having a net positive
charge. Among the 20 genetically encoded amino acids, only
Arg and Lys have a full positive charge (+1 each) under
normal physiological conditions. His has a partial positive
charge (its pK varies with the environment, but is usually
about 6.8; at pH 7, the charge would be +0.4). In contrast,
Asp and Glu have have full negative charge (-1 each).
With a normal peptide, one terminal is NH2-, with a
charge of +1, and the other is -COON, with a charge of -1,
so the termini balance each other out. However, it is
possible to amidate the second end of the peptide so that it
is -NH2 instead of -COOH.
The peptides of the present invention comprise one or
more positively charged amino acids, so that they have a net
positive charge. The net positive. charge must be sufficient
for the peptide to have some antimicrobial activity.
Preferably, the peptides of the present invention are
cationic peptides with a net charge of at least +4 (like
magainin 2), more preferably at least +5 (like PGLa), still
more preferably at least +6, most preferably at least +7.
If the charge is too high, selectivity_is diminished.
The net-positive charge must not be so high that there is a
complete loss of selectivity between microbial and mammalian
cells. Preferably, the net positive charge is not more than
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+10.
Charge Density
- The "charge density" is the net total charge of the
peptide, divided by the length of the peptide in amino
acids. It is preferable that the charge density as defined
above be in the range- of 0.25 to 0.5. A lower charge
density than 0.25 would imply a longer molecule if the net
charge were held constant, leading possibly to lower yields.
A higher charge density than 0.5 would limit the
10- amphipathicity of the peptide in the beta sheet state (see
below), as it then would not be possible to alternate
hydrophilic (all positively charged residues are
hydrophilic) and hydrophobic residues.
Considerations of amphipathicity alone would point
toward a most preferred charge density of 0.5. However, it
is desirable to saturate the membrane's negative charges
with as great a mass of peptides as possible. Use of a
peptide that is too highly charged could result in a lower
bound mass, through peptide/peptide repulsive effects, or
"overbinding" (a peptide interacting with more membrane
nega-tive charges than is necessary for adequate binding,
thereby denying use of the excess bound negative charges to
another peptide). In the successful (KIGAKI)3 peptide of
the present invention, the. charge density is 0.33.
For peptidomimetics, the charge density may be
expressed as the net charge divided by the length of the
molecule in angstroms. In a beta-sheet, the translation per
residue is 3.2 (parallel) to 3.4 (antiparallel) angstroms.
Hence, the charge density, when expressed in
charge/angstroms, is 0.075 to 0.15.
Hydrophobicity/Hydrophilicity Scales
In order to calculate the total or mean hydrophobicity,
or the amphipathicity, of a peptide,- it is necessary to
select a hydrophobicity scale. The hydrophobicity scale is
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an an attempt to quantify the preference of the amino acids
for polar (esp. aqueous) and nonpolar (esp. lipid)
environments. The quantitative and even qualititative -
differences between the scales is, perhaps, not surprising,
given that it is unrealistic to expect that all aspects of
the interaction of a residue with water, with lipid, and
with other residues in the peptide or protein can be
summarized in a single number.
In a general sense, an amino acid may be considered to
have some hydrophobic characteristics if its value on any
art-accepted scale is of a sign (usually positive) which is
representative, on that scale, of hydrophobicity.
However, to quantify the desired hydrophobicity of a
peptide, it is necessary to pick a particular scale.
Hence, unless otherwise stated, it should be assumed
that any quantitative teaching concerning hydrophobicity is
based on Eisenberg's consensus hydrophobicity scale.
This scale is a simple average of four other scales,
those of von Heijne, Janin, Chothia, and Wolfenden. The von
Heijne scale was theoretical, describing the energetic
effects of the covering of hydrophobic surface area,
hydrogen-bond breakage, and charge neutralization. Janin's
scale is based on the fraction of each type of residue that
is found buried in globular proteins. Chothia looked at the
observed distribution of amino acid side chains between the
surface and t-he interior of proteins. Finally, Wolfenden
tabulated the .Gibbs free energy of transfer from dilute
aqueous solution to the vapor of substances of the class RH,
where R represents an amino acid side chain (e.g., RH for
glycine is H2 ) .
Another experimental scale is that of Nozaki, which is
based on the free energy of transfer of amino acids from
water to ethanol, but which, unfortunately, as originally
published, was incomplete. Segrest and Feldman have
35_ suggested values for some of the omitted residues:
Another consensus scale is that of Kyte and Doolittle,
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which considers both the values from water-to-vapor
transfers and the internal-external distribution of amino
acid residues, adjusted subjectively.
The Argos "membrane-buried preference" scale is derived
5 from the relative frequencies of 1125 amino acids found in
protein segments judged to be within membranes.
The OMH (optimal matching hydrophobicity) scale is
derived from sequence alignments, and assumes that families
of proteins that fold the same way do so because they have
10 the same pattern of residue hydrophobicities along their
amino acid sequences.
The Eisenberg consensus scale has been established only
for the genetically encoded amino acids. For other amino
acids, one must rely on a purely experimental scale, such as
15 that of Wolfenden or Nozaki. Use of the Wolfenden scale is
preferred because it is a component of the Eisenberg
consensus scale.
If a peptide includes amino acids for which there is no
established Eisenberg consensus scale value, the procedure
20 is, for each such amino acid (1) determine its value
experimentally according to an experimental scale, such as
the-Wolfenden scale, and (2) determine its equivalent value
on the Eisenberg consensus scale by finding the least
squares fit between the experimental scale value for the
genetically encoded AAs and the Eisenberg consensus scale
value for those AAs.
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For convenience, the Eisenberg and Wolfenden scales are
set forth below:
Amino Acid Eisenberg Wolfenden.
Hydrophobicity Hydrophobicity
Ile 0.73 2.15.
Phe 0.61 -0.76
Val - 0.54 1.99
Leu 0.53 2.28
Trp 0:37 -5.88
Met 0.26 -1.48
Ala 0.25 1.94
Gly 0.16 2.39
Cys 0.04 -1.24
Tyr 0.02 -6.11
Pro -0.07 --
Thr -0.18 -4.88
Ser -0.26 -5.06
His -0.40 -10.27
Glu -0.62 -10.20
Asn -0.64 -9.68
Gln -0.69 -9.38
Asp -0.72 -10.95
Lys -1.1 ~ -9.52
Arg -1.8 -19.92
Mean Hydrophobicity
Cationic peptides are typical soluble in water.
However, to interact with the bacterial membrane, solubility
in water is not sufficient. The bacterial membrane is
amphipathic in nature, with the hydrophilic moieties on the
outside and the hydrophobic moieties on the inside. For a
cationic peptide to interact with the hydrophobic moieties
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of the membrane, it must have a hydrophobic component.
However,. the positively charged amino acids are highly
hydrophilic. Hence,-the peptides of the present invention
will also include one or more hydrophobic moieties.
~ Preferably, the arithmetic mean hydrophobicity of the
peptide is preferably at least -0.8, more preferably at
least -0.6, with the. individual hydrophobicities being
determined according to Eisenberg's non-normalized consensus
hydrophobicity scale. An (RI)n oligomer will have a mean
hydrophobicity of -0.535 ((-1.8+0.73)/2), a (KI)n oligomer
of -0.185, an (HI)n oligomer of +0.165, an RL oligomer of -
0.435, a (KL)n oligomer of -0.285, etc.
By way of comparison, the mean hydrophobicities are -
0.0357 for magainin 2; -0.1832 for MSI-78; +0.0381 for PGLa.
Peptide length
In order to achieve both the charge density desiderata,
a peptide length of 2*p to 4*p, where p is the number of
positively charged amino acids, is desirable (as it yields
the preferred charge density of 0.5 to 0.25). Since the
net positive charge is preferably at least +4, the preferred
minimum length is at least 8 a.a.'s.
The preferred maximum peptide length is set by
considerations of yield anal cost. Preferably, the peptides
are not more than 50 AA, more preferably not more than 30
AA, still more preferably not more than 20 AA.
The peptides may be composed of 2, 3 or more perfect or
nearly perfect repeats of a 6-8 amino acid repeat sequence,
such as KIGAKI (SEQ ID N0:8, residues 1-6).
Secondary Structure
A peptide may assume a variety of secondary structures,
the most common of which are the (right-handed) alpha-helix
and beta-sheet (strand). While the term beta-strand is
probably more accurate for the short peptides contemplated
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here than is beta-sheet, the literature refers much more
often to beta-sheets than to beta-strands, and so that
convention is followed here.
An alpha helix is stabilized by intramolecular hydrogen
bonding between the amino and carboxyl groups of the peptide
backbone. The structure resembles a coil with 3.6 amino
acid residues per turn,. and a translation of 1.5 angstroms
per residue. Each amino acid side chain, extending out from
the coiled backbone, is offset by 100 degrees from its
nearest neighbors when viewed down the long helical axis.
A beta sheet (strand) consists of an extended peptide
chain that can be stabilized by either intramolecular
(strand or sheet) or intermolecular (sheet only) hydrogen
bonds. In a beta strand, each amino acid side chain
extends in the opposite direction (180 deg. offset) from its
nearest neighbors.
The peptides of the present invention are those which
substantially prefer the beta-sheet structure over the
alpha-helix structure when bound to a lipid bilayer or a
microbial membrane.
More particularly, as measured by circular dichroism
and infrared spectroscopy, when bound to a lipid vesicle
model of a bacterial membrane (the peptide secondary
structure can not be determined when the peptides are bound
to a bacterial membrane, since the latter is already loaded
with protein), they are preferably more than 50%, still more
preferably at least 800, even more preferably at least 90%,
most preferably at least 950, in the beta sheet
conformation, when in a bacterial membrane or in a lipid
vesicle model of a bacterial membrane.
Amphipathicity
An amphipathic peptide is one which,. iri its principal
3D secondary structure conformation, has a hydrophobic face
and a hydrophil-is face. (The terms amphipathic and
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amphiphilic are interchangeable.)
A quantitative value_for the degree of amphipathicity
can be assigned using Eisenberg's consensus hydrophobicity
scale and his definition for mean hydrophobic moment (uY).14
The hydrophobic moment is defined as
N N
sin (fin) ~ 2 +- [~ Hn cos (fin) ] 2 } lie
n=1 n=1
for an amino acid sequence of N residues, where the
individual residue hydrophobicities are Hn for residue n,
and b is the angle (in radians) at which successive side
chains emerge from the backbone when the periodic segment is
viewed down its axis. This angle is 2rt * (100/360) radians
for an alpha helix, and n radians for a beta-sheet.
It can be seen that the hydrophobic moment is the
vector sum of the individual hydrophobicities. The value of
the moment is sensitive to the hydrophobicity scale
employed, and hence the preferred moments set forth below
are to be understood as calculated using the Eisenberg
scale .
According to the Eisenberg scale, for the genetically
encoded amino acids, arginine (-1.8) is the most hydrophilic
amino acid, and isoleucine (0.73) is the most hydrophobic
amino acid. Thus, the most amphipathic peptide
constructible~from the ctenetically encoded amino acids will
be composed just of arginine and isoleucine.
We will use the symbol ~..tHa for the hydrophobic moment
of the peptide in its alpha-helical conformation (the "alpha
moment") , and ~.z"(3 for its moment in its beta-sheet
conformation (the "beta moment").
By way of comparison, for magainin 2, uH has a value of
0.286 as an alpha-helix, and 0.037 as a beta-sheet. For
MSI-78, the moments are 0.449 (alpha) and 0.143 (beta). For
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PGLa, the moments are 0.264 (alpha) and 0.07 (beta).
The peptides of the present invention are preferably
substantially more amphipathic as beta strands than as alpha
helices.
5 Preferably, the alpha moment of the peptides of the
present invention is less than 0..2, more preferably less
than 0.1, still more preferably. less than 0.05.
Preferably, the beta moment of the peptides of the
present invention is greater than 0.2, more preferably
10 greater than 0.4.
Preferably, the beta moment for the peptides of the
present invention is at least 0.2 higher than the alpha
moment.
The difference between the moments is probably more
15 significant than the absolute values, as it is the
difference which is primarily responsible for the
stabilization of the peptide in a membrane or
membranomimetic system into a beta-sheet secondary
structure. For example, in water, or in a TFE/water mixed
20 solvent, the (KIGAKI)3 peptide is mainly alpha-helical in
character. It is only in a lipid bilayer, where the
amphipathic beta-sheet is stabilized relative to the non-
amphipathic alpha-helix,. that the. beta-sheet structure
predominates. The amphipathic beta-sheet aligns with the
25 bilayer so that the hydrophobic.face is on the lipid side of
the bilayer and the hydrophilic face is on the aqueous.side
of the bilayer.
Composition
The peptide of the present invention will necessarily
comprise one or more positively charged amino acids, such as
lysine, arginine, and histidine. Lysine and Arginine 'are
preferred to histidine because they have a full positive
charge. These positively charged amino acids will normally
be in positions which place them in the hydrophilic face of
the desired beta sheet structure. The hydrophilic face may
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also include uncharged/hydrophilic AAs like Ser, neutral AAs
like Gly (which is in our preferred peptide (KIGAKI)3). It
less desirable for it to include negatively charged AAs-
(which lower the net charge), or hydrophobic Aas (of the
latter, those which can form H-bonds are preferred).
The peptides of the present invention also will
.comprise one or more hydrophobic amino acids, such as Leu,
Ile, Val, Met, Phe, Trp, Tyr, or Ala . In -general, for
amino acids which are to be part of the hydrophobic face of
the desired beta-sheet structure, the more hydrophobic the
AA, the better.
Certain amino acids present synthetic difficulties;
these are isoleucine (because of the location of the beta
branch); methionine (because it can oxidize); and Cys
(because of its propensity to form disulfide bonds, which
can cause the peptides to form multimolecular aggregates.
It is noted that the defensins are natural antimicrobial
peptides of about 30 a.a. with an intrinsic beta sheet
structure becasue of three disulfide bonds. However, these
disulfide bonds make it difficult to synthesise the
defensins because of the difficulty of controlling disulfide
bond topology.) However, none of these_AAs are absolutely-
prohibited and Ile, despite its adverse effect on synthetic
yield, remains a preferred amino acid for the hydrophobic
face because of its very high hydrophobicity.
Scientists have determined the propensity of various
amino acids to appear in alpha helices or beta sheets of
proteins. This propensity data is of only marginal value in
the present context, where we are looking at the structure
of small peptides in a lipid bilayer environment. There are
a variety of interactions at work-in the interior of a
protein which are not applicable in our context. Our
peptides are too small to have a stable secondary structure
in solution, so it is expected that the relative
amphipathicity of the peptide in the beta-sheet as opposed
to the alpha-helix state is what will primarily drive which
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27
structure it assumes in the lipid bilayer.
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Nonetheless, in comparing two peptides with similar
relative amphipathicities, these secondary structure
propensities, and especially the relative propensities, of
the individual amino acids may be relevant.
Amino Acid (1) Chou- ~ (2) Chou- Relative
- Fasman (1974) Fasman (1974) Propensity
Alpha-Helix Beta-Sheet (ratio of (1)
Propensity Propensity to (2))
Ala 1.45 0.97 1.494845361
Cys 0.77 1.30 0.592307692
Asp 0.98 0.80 1.225
Glu 1.53 0.26 5.884615385
Phe 1.12 1.28 0.875
Gly 0.53 0.81 0.654320988
His 1.24 0.71 1.746478873
Ile 1.00 1.60 0.625
Lys 1.07 0.74 1.445945946
Leu 1.34 1.22 1.098360656
Met 1.2 1.67 0.718562874
Asn 0.73 0.65 1.123076923
Pro 0.59 0.62 0.951612903
Gln 1.17 1.23 0.951219512
Arg 0.79 0.9 0.877777778
Ser 0.79 0.72 1.097222222
Thr 0.82 1.2 0.683333333
Val 1.14 1.65 0.690909091
Trp 1.14 1.19 0.957983193
Tyr 0.61 1.29 0.472868217
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Similarly, one may take into account the reported
tendencies of homopolymers of oligopeptides to form alpha-
helices or beta-sheets in water. However, this is with the
caveat that we are concerned with the formation of secondary
structure in-a lipid bilayer, rather trian in water. r~elow
are reported helix formation parameters for amino acid
residues; these give the equilibrium constants for initial
helical conformation in a random coil or for adding a
residue of helical conformation to the end of a helix. The
table below is for 18 of the amino acids:
Amino Acid helix initiation helix addition
(x10E4)
Gly 0.1 0.6
Asp- 70 0.66
His+ 0.1 0.68
Ser 0.1 0.77
Asn 0.1 0.79
His 210 ' 0.80
Thr 0.1 0.83
Lys+ 1 0.94
Val 1 0.96
Gln. 33 0.96
Glu- 6 0.96
Tyr 66 0.99
Arg+ 0.1 1.02
Ala 8 1.06
Trp 77 1.08
Phe 18 1.08
Ile 55 1.12
Leu 33 1.14
Met 54 1.17
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Sequence
.The mean hydrophobicity is determined completely by the
AA composition of the peptide. However, the amphipathicity
is a function of both composition and sequence.
5 For a beta-sheet, a perfectly amphipathic sequence has
the structure (wl)n or (lw)~, where w denotes a-hydrophilic
(water-loving) amino acid, 1 a hydrophobic (lipid-loving)
amino acid, and n is a positive integer. In other words,
the maximum amphipathicity is reached when the hydrophobic
10 and hydrophilic amino acids alternate.
If all of the hydrophilic amino acids are positively
charged, this perfectly amphipathic sequence would have a
charge density of 0.5. If only half were positively
charged, and the rest uncharged, this perfectly amphipathic
15 sequence would have a charge density of 0.25.
The peptides of the present invention are preferably
perfectly amphipathic as beta-strands. If they are not
perfectly amphipathic, they are preferably nearly perfectly
amphipathic, i.e., the only departure from perfect
20 amphipathicity is the presence of one or more Gly (a neutral
AA) in one or both faces.
However, it is possible for the. peptide to depart further
from perfect amphipathicity, i.e., have a hydrophobic AA in
the hydrophilic face, or vice versa, although it preferably
25 still satisfies the aforementioned preference concerning the
values of the alpha and beta moments.
For an alpha helix, a perfectly amphipathic 18 a.a.
sequence would be (wwllwwllw llwwllwwl)~, or (llwwllwwl
wwllwwllw)~.
30 If we line up the sequences for a perfectly amphipathic
18 a.a. beta -and a perfectly amphipathic 18 a.a. alpha, we
have
wlwlwlwlwlwlwlwlwl (beta)
wwllwwllwllwwllwwl_(alpha)
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and three permutations thereof.
For an 18 a.a. peptide composed of nine Arg and nine
Ile, the maximum beta moment would be achieved-if the
residues were arranged in the sequence-(RI)9 , and would be
1.27 (This sequence would have an alpha moment of 0.0). The
maximum alpha moment would be for the sequence RRIIRRIIR
IIRRIIRRI, and would be 0.81 (This sequence would have a
beta moment of 0.14).
Increasing or decreasing the length of a peptide which
was 50oR 50oI would not alter the maximum achievable beta
moment, which would be with (RI)n or (IR)n. It would alter
the maximum achievable alpha moment, for example, for 8R/8I,
it would be 0.85, and for lOR/10I, it would be 0.83.
Predicted Helical Character
Agadir is a prediction algorithm based on the
helix/coil transition theory. Agadir predicts the helical
behaviour of monomeric peptides. It only considers short
range interactions.
Unfortunately, the Agadir algorithm is based on the
behavior of peptides in aqueous solution. It does not
consider the role of amphipathicity in stabilizing one
structure over another. Also, it only considers the
helix/coil, and not the beta-strand/coil transition.
Still, for peptides of equal amphipathicity, it may be
of interest to look at the relative helical character
predicted according to the Agadir algorithm. For (RI)9, the
predicted percentage helix was 1.970; for (KIGAKI)3, it was
0.850; for RRIIRRIIRIIRRIIRRI, it was 5.130; for magainin 2,
0.830; MSI-78, 2.180; PGLa, 3.290; all for N-terminal free,
C-terminal amidated peptides under the default conditions of
pH 7.00, Temperature 278, Ionic Strength 0.100. For
melittin, with free N and~C termini, it was 2.040, and, C-
amidated, 2.110.
Preferably, the predicted percentage helical character
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32
of the peptide in aqueous solution, according to the Agadir
algorithm, under its default conditions, for the peptide in
amidated form, is less than 20.
The Agadir algorithm is described in Munoz, V. &
Serrano, L. (1994a)., Elucidating the folding problem of
helical peptides using empirical parameters. Nature: Struct.
Biol...l, 399-409; Munoz, V. & Serrano, L. (1994b).
Elucidating the folding problem of a-helical peptides using
empirical parameters, II. Helix macrodipole effects and
rational modification of the helical content of natural
peptides. J. Mol. Biol 245, 275-296; Munoz, V. & Serrano, L.
(1994c). Elucidating the folding problem of a-helical
peptides using empirical parameters III:Temperature and pH
dependence J. Mol. Biol 245, 297-308; Munoz, V. & Serrano,
TS L. (1997). Development of the Multiple Sequence
Approximation within the Agadir Model of a-Helix Formation.
Comparison with Zimm-Bragg and Lifson-Roig Formalisms.
Biopolymers 4l, 495-509; Lacroix, E., Viguera AR & Serrano,
L. (1998) Elucidating the folding problem of a-helices:
Local motifs, long-range electrostatics, ionic strength
dependence and prediction of NMR parameters. J. Mol. Biol.
284, 173-191; Villegas, V., Viguera, A.R., AvilOs, F.X. &
Serrano, L. (1996). Stabilisation of proteins by rational
design of a-helix stability using helix/coil transition
theory. Folding & Design, 1,29-34; Munoz, V. & Serrano, L.
(1996). Local vs non-local interactions in protein folding
and stability. An experimentalist point of view. Folding &
Design. 1, R71-R77;:~Y~,~-pez-Hernf3ndez, E., Cronet, P.,
Serrano, L. & Munoz, V. (1997). Kinetics of CheY mutants
with enhanced native a-helix propensities. J. Mol. Biol.
266, 610-620. Viguera, A.R., Villegas, V., AvilOs, F.X. &
Serrano, L. (1996). Favourable native-like helical local
interactions can accelerate protein folding. Folding &
Design, 2, 23-33.
The EMBL WWW Gateway to AGADIR is at
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http://www.embl-heidelbera de/Services/serrano/aaadir
agadir-start.html
Should this URL change, the new URL may be identified
by contacting or exploring the root domain, or, if that
fails, by an Internet search on the term "a-gadir".
Amino Acids and Peptides
Amino acids are the basic building blocks with which
peptides and proteins are constructed. Amino acids possess
both an amino group (-NHZ) and a carboxylic acid group (-
COOH). Many amino acids, but not all, have the structure
NHZ-CHR-COOH, where R is hydrogen, or any of a variety of
functional groups.
Twenty amino acids are genetically encoded: Alanine
(A), Arginine (R), Asparagine (N), Aspartic Acid (D),
Cysteine (C), Glutamic Acid (E), Glutamine (Q), Glycine (G),
Histidine (H), Isoleucine (I), Leucine (L), Lysine (K),
Methionine (M), Phenylalanine (F), Proline (P), Serine (S),
Threonine (T), Tryptophan (W), Tyrosine (Y), and Valine (V).
Of these, all save Glycine are optically isomeric, however,
only the L-form is found in humans. Nevertheless, the D-.
forms of these amino acids do have biological significance;
D-Phe, for example, is a known analgesic.
Many other amino acids are also known, including: 2-
Aminoadipic acid; 3-Aminoadipic acid; beta-Aminopropionic
acid; 2-Aminobutyric acid; 4-Aminobutyric acid .(Piperidinic
acid);
6-Aminocaproic acid; 2-Aminoheptanoic acid; 2-
Aminoisobutyric acid, 3-Aminoisobutyric acid; 2-Aminopimelic
acid;
2,4-Diaminobutyric acid; Desmosine; 2,2'-Diaminopimelic
acid; 2,3-Diaminopropionic acid; N-Ethylglycine; N-
Ethylasparagine; Hydroxylysine; allo-Hydroxylysine; 3-
Hydroxyproline;
4-Hydroxyproline; Isodesmosine; allo-Isoleucine; N-
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Methylglycine (Sarcosine); N-Methylisoleucine; N-
Methylv.aline; Norvaline; Norleucine; and Ornithine.
Peptides are constructed by condensation of amino acids
and/or smaller peptides. The amino group of one amino acid
(or peptide) reacts with the carboxylic acid group of a
second amino acid (or peptide) to form a peptide (-NHCO-)
bond, releasing one molecule of water.- Therefore, when an
amino acid is incorporated into a peptide, it should,
technically speaking, be referred to as an amino acid
residue.
A peptide is composed of a plurality of amino acid
residues joined together by peptidyl (-NHCO-) bonds. A
biogenic peptide is a peptide in which the residues are all
genetically encoded amino acid residues; it is not necessary
that the biogenic peptide actually be produced by gene
expression.
The peptides of the present invention include peptides
whose sequences are disclosed in this specification, or
sequences differing from the above solely by no more than
one nonconservative substitution and/or one or more
conservative substitutions, preferably no more than a single
conservative substitution. The substitutions may be of non-
genetically encoded (exotic) amino acids, in which case the
resulting peptide is.nonbiogenic.
A conservative substitution is a substitution of one
amino acid for another of the same exchange group, the
exchange groups being defined as follows
I Gly, Pro, Ser, Ala (Cys) (and any nonbiogenic,
neutral amino acid with a hydrophobicity not
exceeding that of the aforementioned a.a.'s)
II Arg, Lys, His (and any nonbiogeni-c,-positively-
charged amino acids)
III Asp, Glu, Asn, Gln (and any nonbiogenic
negatively-charged amino acids)
IV Leu, Ile, Met, Val (Cys) (and any nonbiogenic,
aliphatic, neutral amino acid with a
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WO 01/60162 PCT/USO1/04822
hydrophobicity too high for I above)
V Phe, Trp, Tyr (and any nonbiogenic, aromatic
neutral amino acid with a hydrophobicity too high
for I above).
5 Note that Cys belongs to both I and IV.
A highly conservative substitution, which is preferred,
is Arg/Lys/His, Asp/Glu, Asn/Gln, Leu/Ile/Met/Val,
Phe/Trp/Tyr, or Gly/Ser/Ala.
Additional peptides witin the present invention may be
10 identified by systematic mutagenesis of the lead peptides,
e.g.
(a) separate synthesis of all possible single
substitution (especially of genetically encoded
AAs) mutants of each lead peptide, and/or
15 (b) simultaneous binomial random alanine-scanning
mutagenesis of each lead peptide, so each amino
acids position may be either the original amino
acid or alanine (alanine being a semi-conservative
substitution for all other amino acids), and/or
20 (c) simultaneous random mutagenesis sampling
conservative substitutions of some or all
positions of each lead peptide, the number of
sequences in total sequences space for a given
experiment being such that any sequence, if
25 active, is within detection limits (typically,
this means not more than about 101° different
sequences).
The mutants are tested for activity, and, if active,
are considered to be within "peptides of the present
30 invention". Even inactive mutants contribute to our
knowledge of structure-activity relationships and thus
assist in the design of peptides, peptoids, and
peptidomimetics.
Preferably, substitutions of exotic amino acids for the
35 original amino acids take the form of
(I) replacement of one or more hyd-rophilic amino
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36 -
acid side chains with another hydrophilic
organic radical, not more than twice the
volume of the origina-1 side chain, or
(II) replacement o.f one or more hydrophobic amino
S acid side chains with ano-ther hydrophobic
organic radical, not more than twice the
volume of the original side chain.
The exotic amino acids may be alpha or non-alpha amino
acids (e. g., beta alanine). They may be alpha amino acids
with 2 R groups on the Ccx, which groups may be the same or
different. They may be dehydro amino acids (HOOC-
C (NHz) =CHR) .
Exotic amino acids of particular interest include those
which differ from a genetically encoded amino acid primarily
by including more or fewer carbons, e.g., 5 or 6 carbon
analogues of Leu, Ile or Val, or analogues of Lys with more
or less than 4 carbons.
For further information on synthesis of peptides
including exotic amino acids, see:
1. Bielfeldt, T., Peters, S., Meldal, M., Bock, K.
and Paulsen, N.A. new strategy for solid-phase synthesis of
O-glycopeptides. Ange-w. Chem. (Engl) 31:857-859, 1992.
2. Gurjar, M.K. and Saha, U.K. Synthesis of the
glycopeptide-O-(3,4-di-O-methyl-2-0-[3,4-di-0-methyl-cx-L-
rhamnopyranosyl]-a-L-rhamnophyranosyl)-L-alanilol: An
unusual part structure in the glycopeptidolipid of
Mycobacterium fortuitum. Tetrahedron 48:4039-4044, 1992.
3. Kessler, H., Wittmann, V., Kock, M. and
Kottenhahn, M. Synthesis of C-glycopeptides via free radical
addition of glycosyl bromides to dehydroalanine derivatives.
Angew. Chem. (Engl.) 31:902-904, 1992.
4. Kraus, J.L. and Attardo, G._Synthesis and
biological activities -of new N-formylated methionyl peptides
containing an a-substituted glycine residue. European
Journal of Medicinal Chemistry 27:19-26, 1992.
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37.
5. Mhaskar, S.Y. Synthesis of N-lauroyl dipeptides
and correlation of their structure with surfactant and
antibacterial properties. J. Am. Oil Chem. Soc.69:647-652,
1992.
6. Moree, W.J., Van der Marel, G.A. and Liskamp,
R.M.J. Synthesis of peptides containing the (3-substituted
aminoethane sulfinamide or sulfonamide transition-state
isostere derived from amino acids. Tetrahedron Lett. 33:69-
6392, 1992.
7. Paquet, A. Further studies on the use of 2,2,2-
trichloroethyl groups for phosphate protection in
phosphoserine peptide synthesis. International Journal of
Peptide and Protein Research 39:82-86, 1992.
8. Sewald, N., Riede, J., Bissinger, P. and Burger,
K. A new convenient synthesis of 2-trifluoromethyl
substituted aspartic acid_and its isopeptides. Part 11.
Journal of the Chemical Society. Perkin Transactions 1
1992:267-274, 1992.
9. Simon, R.J., Kania, R.S., Zuckermann, R.N.,
Huebner, V.D., Jewell, D.A., Banville, S., Ng, S., Wang, L.,
Rosenberg, S., Marlowe, C.K., Spellmeyer, D.C., Tan, R.,
Frankel, A.D., Santi, D.V., Cohen, F.E. and Bartlett, P.A.
Peptoids: A modular approach to drug discovery. Proc.
Natl, Acad. Sci. USA 89:9367-9371, 1992.
10. Tung, C.-H., Zhu, T., Lackland, H. and Stein, S.
An acridine amino acid derivative for use in Fmoc peptide
synthesis. Peptide Research 5:115-118, 1992.
11. Elofsson, M. Building. blocks for glycopeptide
synthesis: Glycosylation of 3-mercaptopropionic acid and
Fmoc amino acids with unprotected carboxyl groups.
Tetrahedron Lett. 32:7-613-7616, 1991.
- 12. McMurray, J.S. Solid phase synthesis of a cyclic
peptide using Fmoc chemistry. Tetrahedron Letters 32:76.79-
7682, 1991.
13. Nunami, K.-I., Yamazaki, T. and Goodman, M. Cyclic
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38
retro-inverso dipeptides with two aromatic side chains. I.
Synthesis. Biopolymers 31:1503-1512, 199.1.
14.. Rovero, P, Synthesis of cyclic peptides on solid
support. Tetrahedron Letters 32:2639-2642, 1.991.
15. Elofsson, M., Walse, B. and Kihlberg, J. Building
blocks for glycopeptide synthesis: Glycosylation of 3-
mercaptopropionic acid and Fmoc amino acids with unprotected
carboxyl groups. Tetrahedron Letter, 32-:7613-7616, 1991.
16. Bielfeldt, T., Peter, S., Meldal, M., Bock, K. and
Paulsen, H. A new strategy for solid-phase synthesis of 0-
glycopeptides. Agnew. Chem (Engl) 31:857-859, 1992.
17. Luning, B., Norberg, T. and Tejbrant, J. Synthesis
of glycosylated amino acids for use in solid phase
glycopeptide synthesis, par 2:N-(9-
fluorenylmethyloxycarbonyl)-3-O-[2,4,6-tri-O-acetyl-cc-D-
sylopyranosyl)-(3-D-glucopyranosyl]-L-serine. J. Carbohydr.
Chem. 11:933-943, 1992.
18. Peters, S., Bielfeldt, T., Meldal, M., Bock, K.
and Paulsen, H. Solid phase peptide synthesis of mucin
glycopeptides. Tetrahedron Lett. 33:6445-6448, 1992.
19. Urge, L., Otvos, L., Jr., Lang, E., Wroblewski,
K., Laczko,I. and Hollosi, M. Fmoc-protected, glyc.osylat.ed
asparagines potentially useful as reagents in the solid-
phase synthesis of N-glycopeptides, Carbohydr. Res. 235:83-
93, 1992.
2b. Gerz, M., Matter, H. and Kessler, H., S-
glycosylated.cyclic peptides, Angew. Chem. (Engl.) 32:269-
271, 1993.
N- and C-terminal modified peptides
In an unmodified peptide, one end of the peptide
terminates in a -NHZ and the other end has a free -COOH.. If
the -COOH is replaced with an -NH2, the peptide is called an
amide. The present invention includes the amide of any
disclosed standard peptide.
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39
In addition, further N- and C-terminal modifications
are contemplated. The NHz- and/or -COOH termini may be
replaced with a group of the form RY-, where R is a
hydrophobic moiety, and Y is a spacer. The resulting
modified peptide should still have a solubility in water of
at least 1 g/L at 20°C. Y may, for example, be -O-, -C(=O)-
or -C(=0)-NH-. R may be a hydrocarbon. If a hydrocarbon,
R may be aliphatic or aromatic, and linear, branched or
cyclic, and may contain alkenyl or alkynyl moieties. It is
preferably alkyl of 2 to 12 carbons.
Modifications of particular interest include N-terminal
alkanoyl modification (linear or branched chains, from 4-
to 12 carbons), cyclic modification (e. g., cyclohexanoyl,
etc.), or aromatic modification (e. g., benzyol, etc.).
If both N- and C-termini are modified, the
modifications may be the same or different. It is noted
that cyclic peptides constitute a special case of N- and C-
terminal modification.
The term "peptide" is understood to include both
modified and unmodified peptides, if not further qualified.
- Cyclic Peptides.
Many naturally occurring peptide are cyclic.
Cyclization is a common mechanism for stabilization of
peptide conformation thereby achieving improved_association
of the peptide with its ligand and hence improved biological
activity. Cyclization is usually achieved by intra-chain
cystine formation, by formation of peptide bond between side
chains or between N- and C- terminals. Cyclization was
usually achieved by peptides in solution, but several
publications have appeared recently that describe
cyclization of_ peptides on beads (see references below).
1. Spatola, A.F., Anwer, M.K. and Rao, M.N. Phase
transfer catalysis in solid phase peptide synthesis.
Preparation of cycle [Xxx-Pro-Gly-Yyy-Pro-Gly] model
peptides and their conformational analysis. Int. J. Pept.
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Protein Res. 40:322-332, 1992.
2. Tromelin, A., Fulachier, M.-H., Mourier, G. and.
Menez, A. Solid phase synthesis of a cyclic peptide derived
from a curaremimetic toxin. Tetrahedron Lett. 33:5197-5200,
5 1992. -
3.. Trzeciak, A. Synthesis of 'head-to-tail' cyclized
peptides on solid supports by Fmoc chemistry. Tetrahedron
Lett. 33:4557-45560, 1992.
4. Wood, S. J. and Wetzel, R. Novel cyclization
10 chemistry especially suited for biologically derived,
unprotected peptides, Int. J. Pept. Protein Res. 39:533-539,
1992.
5. Gilon, C., Halle, D., Chorev, M., Selinger, Z. and
Byk, G. Backbone cyclization: A new method for conferring
15 conformational constraint on peptides. Biopolymers 31:745-
750, 1991.
6. McMurray, J. S. Solid phase synthesis of a cyclic
peptide using Fmoc chemistry. Tetrahedron Letters32:7679-
7682, 1991.
20 7. Rovero, P. Synthesis of cyclic peptides on solid
support. Tetrahedron Letters 32:2639-2642, 1991.
8. Yajima, X. Cyclization on the bead via following
Cys Acm deprotection. Tetrahedron 44:805, 1988.
Peptoid
25 A peptoid is an analogue of a peptide in which one or
more of the peptide bonds (NHCO) are replaced by
pseudopeptide bonds, which may be the same or different.
Such pseudopeptide bonds may be:
Carba ~Y ( CHz-CHZ )
30 Depsi ~ (CO-O)
Hydroxyethylene ~(CHOH-CHZ)
Ketomethylene 1Y (CO-CH2)
Methylene-ocy CHZ-O-
Reduced CHz-NH
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41
Thiomethylene CHZ-S-
Thiopeptide CS-NH
N-modified -NRCO- (where N is cyclic, branched or
linear alkyl of u~p to 12 carbons)
See also
1. Corringer, P.J., Weng, J.H., Ducos, B., Durieux,
C., Boudeau, P., Bohme, A. and Roques, B.P. CCK-B agonist or
antagonist activities of structurally hindered and
peptidase-resistant Boc-CCK9 derivatives. J. Med. Chem.
36:166-172, 1993.
Amino acids reported: aromatic naphthylalaninimide (Nal-
NH2); N-methyl amino acids.
2. Beylin, V.G., Chen, H.G., Dunbar, J., Goel, O.P.,
Harter, W., Marlatt, M. and Topliss, J.G. Cyclic derivatives
of 3,3-diphenylalanine (Dip) (II), novel a-amino acids for
peptides of biological interest. Tetrahedron Lett. 34:953-
956, 1993.
' 3. Garbay-Jaureguiberry, C., Ficheux, D. and Roques,
B.P. Solid phase synthesis of peptides containing the non
gydrolysable analog of (O)phosphotyrosine, p(CHzP03H2)Phe.
Application to the synthesis of 344-357 sequences of the (32
adrenergic receptor. Int. J. Pept. Protein Res. 39:523-527,
1992.
4. Liming, B., Norberg, T. and Tejbrant, J. Synthesis
of glycosylated amino acids for use in solid phase
glycopeptide synthesis, part 2: N-(9-
fluorenylmethyloxycarbonyl)-3-O-[2,4,6-tri-O-acetyl-3-O-
(2,3,4-tri-O-acetyl-cx-D-.xylopyranosyl)-~i-D-glucopyranosylJ-
L]serine. J. Carbohydr. Chem. 11:933-943, 1992.
5. Tung, C.H., Zhu, T., Lackland, H. and Stein, S. An
acridine amino acid derivative for use in Fmoc peptide _
synthesis. Peptide Research 5:115-118, 1992.
6. Eric Frerot, PyBOP and PyBroP: Two reagents for
the difficult coupling of the alpha, alpha-dialkyl amino acid
Aib. Tetrahedron 47:259-270, 1991.
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42
7. Moree, W.J., Van der Marel, G.A. and Liskamp,
R.M.J_. Synthesis of peptides containing the (3-substituted
aminoethane sulfinamide or sulfonamide transition-state
isostere derived from amino acids. Tetrahedron Lett.
33:6389-6392, 1992.
8. Rana, T.M. Synthesis of a metal-binding amino acid
suitable_for solid phase assembly of peptides. Tetrahedron
Lett. 33:4521-4524, 1992.
9. Urge, L., Otvos, L., Jr., Lang, E., Wroblewski, K.,
Laczko, I. and Hollosi, M. Fmoc-protected, glycosylated
asparagines potentially useful as reagents in the solid-
phase synthesis of N-glycopeptides. Carbohydr. Res. 235:83-
93, 1992.
10. Pavone, V., DiBlasio, B., Lombardi, A., Maglio, O.,
Isernia, D., Pedone, C., Benedette, E., Altmann, E. and
Mutter, M. Non coded C"'"-disubstituted amino acids. X-ray
diffraction analysis of a dipeptide containing (S)-a-
methylserine. Int. J. Pept. Protein Res. 41:15-20, 1993
11. Nishino, N., Mihara, H., Kiyota, H., Kobata, K. and
Fujimoto, T. Aminoporphyrinic acid as a new template for
polypeptide design. J. Chem. Soc. Chem. Common. 1993:162-
163, 1993.
12. Sosnovsky, G., Prakash, I. and Rao, N.U.M. In the
search for new anticancer drugs. XXIV: Synthesis and
anticancer activity of amino acids and dipeptides containing
the 2-chloroethyl- and [N'-nitroso]-aminocarbonyl groups.
J. Pharm. Sci. 82:1-10, 1993.
13. Berti, F., Ebert., C. and Gardossi, L. One-step
stereospecific synthesis of cx,(3-dehydroamino acids and
dehydropeptides. Tetrahedron Lett. 33:8145-8148, 1992.
Peptidomimetic
A peptidomimetic is a molecule which mimics the
biological activity of a peptide, by substantially
duplicating the pharmacologically relevant,portion of the
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43
conformation of the peptide, but is not a peptide or peptoid
as defined above. Preferably the peptidomimetic has a
molecular weight of less than 700 daltons.
Designing a peptidomimetic usually proceeds by:
(a) identifying the pharmacophoric groups responsible
for the activity;
(b) determining the spatial arrangements_of the
pharmacophoric groups iri the active conformation
of the peptide; and
(c) selecting a pharmaceutically acceptable template
upon which to mount the pharmacophoric groups in a
manner which allows them to retain their spatial
arrangment in the active conformation of the
peptide.
Step (a) may be carried out by.preparing mutants of the
active peptide and determining the effect of the mutation on
activity. One may also examine the 3D structure of a
complex of the peptide and the receptor for evidence of
interactions, e.g., the fit of a side chain of the peptide
into a cleft of the receptor; potential sites for hydrogen
bonding, etc.).
Step (b) generally involves determining the 3D
structure of the active peptide, in the complex, by NMR
spectroscopy or X-ray diffraction studies. The initial 3D
model may be refined by an energy minimization and molecular
dynamics simulation.
Step (c) may be carried out by reference to a template
database, see Wilson, et al. Tetrahedron, 49:3655-63 (1993).
The templates will typically allow the mounting of 2-8
pharmacophores, and have a relatively rigid structure. For
the latter reason, aromatic structures, such as benzene,
biphenyl, phenanthrene and benzodiazepine, are preferred.
For orthogonal protection techniques, see Tuchscherer, et
al., Tetrahedron, 17:3559-75 (1993).
For more information on peptoids and peptidomimetics,
see USP 5,811,392, USP 5,811,512, USP 5,578,629, USP
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5,817,879, USP 5,817,757, USP 5,811,515.
Analo -ues
Also of interest are analogues of the disclosed
peptides, and other compounds with activity of interest.
Analogues may be identified by assigning a hashed
bitmap structural fingerprint to the compound, based on its
chemical structure, and determining the similarity of that
fingerprint to that of each compound in a broad chemical
database. The fingerprints are determined by the
fingerprinting software commercially distributed for that
purpose by Daylight Chemical Information Systems, Inc.,
according to the-software release current as of January 8,
1999. In essence, this algorithm generates a bit pattern
for each atom, and for its nearest neighbors, with paths up
to 7 bonds long. Each pattern serves as a seed to a
pseudorandom number generator, the output of which is a set
of bits which is logically ored to the developing
fingerprint. The fingerprint may be fixed or variable size.
The database may be SPRESI'95 (InfoChem GmbH), Index
Chemicus (ISI), MedChem (Pomona/Biobyte), World Drug Index
(Derwent)., TSCA93(EPA) May bridge organic chemical catalog'
(Maybridge), Available Chemicals Directory (MDLIS Inc.),
NCI96 (NCI), Asinex catalog of organic compounds (Asinex
Ltd.), or IBIOScreen SC and NP -(Inter BioScreen Ltd.), or an
inhouse database.
A compound is an analogue of a reference compound if it
has a daylight fingerprint with a similarity (Tanamoto
coefficient) of at least 0.85 to the Daylight fingerprint of
the reference compound.
A compound is also an analogue of a reference compound
if it may be conceptually derived from the reference
compound by isosteric replacements.
Homologues are compounds which differ by an increase or
decrease in the number of methylene groups in an alkyl
moiety .
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Classical isosteres are those which meet Erlenmeyer's
definition: "atoms, ions or molecules in which the
peripheral layers of electrons can be considered to be
identical". Classical isosteres include
5 Monovalents Bivalents Trivalents Tetra - Annular
F, OH, NH2, CH3 -O- -N= =C= -CH=CH-
=Si=
Cl, SH, PHz -S- -P= -N+= -S-
Br -S.e- -As- =P+= -O-
10 i -Te- -Sb- =As+= -NH-
-CH= =Sb+=
Nonclassical isosteric pairs include -CO- and -SOZ-, -
COOH and -S03H, -SO2NH2 and -PO (OH) NH2, and -H and -F, -
OC (=O) - and C (=O) O-, -OH and -NH2.
15 Bacteria
The bacteria which are to be inhibited are pathogens of
humans or other animals. They may be obligate or
opportunistic pathogens. They may be gram-negative or gram-
positive bacteria. The gram-negative bacteria include
20 bacteria of the families Pseudomonadacae,
Enterobacteriaceae, Vibrionaceae, Bacteroidaceae,-
Neisseriaceae and Veillonellaceae, and some Bacillaceae, and
the order Chlamydiales. The gram-positive bacteria include
bacteria of the families Micrococcaceae, Streptococcaceae,
25 Peptococcaceae, some Bacillaceae, and Lactobacillaceae, and
the order Rickettsiales. There are also genera of uncertain
affiliation, of which the gram-negative Brucella,
Bordetella, Francisella, Chromobacterium, Haemophilus,
Pasteurella, Actinobacillus, Cardiobacterium,
30 Streptobacillus-, and Calymmatobacterium, and the gram-
positive Listeria Erysipelothrix, and Corynebacterium, are
worthy of note. -
Among the gram-negative bacteria, the
Enterobacteriaceae (Escherichia, Edwardsiella, Citrobacter,
35 Salmonella, Shigella, Klebsiella; Enterobacter, Hafnia,
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Serratia, Proteus, Yersinia, and Erwinia) are of particular
interest.
Other Microbes-
The peptides of the present invention may also be
useful in inhibiting other microbial pathogens, including
algal, fungal and protozooal pathogens.
Other Target Organisms
The peptides of the present invention may also be
useful in inhibiting nonmicrobial pathogens, such as worms
or arthropods, whose membranes are sufficiently different
from mammalian membranes. In addition they may be useful as
spermicides for humans, as the sperm membrane is atypical of
human cell membranes.
Antimicrobial Activity
Preferably, the peptides of the present invention have
an antimicrobial activity at least equal to that of magainin
2 under the same assay conditions.
Patients/Subjects
The terms "patients" and "subjects" are used
interchangeably. The term "animal" includes "humans". The
subject is preferably a mammal, especially of th-a orders
Primata (humans, apes, monkeys), Artiodactyla or
Perissodactyla (esp. cows, pigs, goats, sheep, horses),
Rodenta or Lagomorpha (esp. rats, mice, rabbits, hamsters),
or Carnivora (esp. cats and dogs) or other pet, farm or~
laboratory mammals.. It is especially preferable that the
subject be human, but the subject may be a nonhuman mammal.
Those peptides which are effective in nonhuman mammals
but not humans have the advantage that their animal use does
not endanger human antimicrobial therapy.
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Pharmaceutical Methods and Preparations
The term "protection", as used herein, is intended to
include "prevention," "suppression" and "treatment."
"Prevention" involves administration of the protein prior to
the induction of the disease (or other adverse clinical
condition). "Suppression" involves administration of the
composition prior to the clinical appearance of the disease.
"Treatment" involves administration of the protective
composition after the appearance of the disease.
It will be understood that in human and veterinary
medicine, it is not always possible to distinguish between
"preventing" and "suppressing" since the ultimate inductive
event or events may be unknown, latent, or the patient is
not ascertained until well after the occurrence of the event
or events. Therefore, it is common to use the term
"prophylaxis" as distinct from "treatment" to encompass both
"preventing" and "suppressing" as defined herein. The term
"protection," as used herein, is meant to include
"prophylaxis." It should also be understood that to be
useful, the protection provided need not be absolute,
provided that it is sufficient to carry clinical value. An
agent which provides protection to a lesser degree than do
competitive agents may still be of value if the other agents
are ineffective for a particular individual, if it can be
used in. combination with other agents to enhance the level
of protection, or if it is safer than competitive agents.
The drug may provide a curative effect, an ameliorative
effect, or both.
At least one of the drugs of the present invention may
be administered, by any means that achieve their intended
purpose, to protect a subject against a disease or other
adverse condition. The form of administration may be
systemic or topical. For example, administration of such a
composition may be by various parenteral routes such as
subcutaneous, intravenous, intradermal, intramuscular,
intraperitoneal, intranasal, transdermal, or buccal routes.
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Alternatively, or concurrently, administration may be by the
oral route. Parenteral administration can be by bolus
injection or by gradual perfusion over time.
A typical regimen comprises administration of an
effective amount of the drug, administered over a period
ranging from a single dose, to dosing over a period of
hours, days, weeks, months, or years. The specific amount-
of drug administered can be determined readily for any
particular patient according to recognized procedures and
based on the expertise and experience of the skilled
practitioner. Precise dosing for a patient can be determined
according to routine medical practice.
Prior to use in humans, a drug will first be evaluated
for safety and efficacy in laboratory animals. In human
clinical studies, one would begin with a dose expected to be
safe in humans, based on the preclinical data for the drug
in question, and on customary doses for analogous drugs (if
any). If this dose is effective, the dosage may be
decreased, to determine the minimum effective dose, if
desired. If this dose is ineffective, it will be cautiously
increased, with the patients monitored for signs of side
effects. See, e.g., Berkow et al, eds., The Merck Manual,..
15th edition, Merck and Co., Rahway, N.J., 1987; Goodman et
al., eds., Goodman and Gilman's The Pharmacological Basis of
Therapeutics, 8th edition, Pergamon Press, Inc., Elmsford,
N.Y. , (1990) ; Avery's Drug Treatment: Principles and
Practice of Clinical Pharmacology and Therapeutics, 3rd
edition, ADIS Press, LTD., Williams and Wilkins, Baltimore,
MD. (1987), Ebadi, Pharmacology, Little, Brown and Co.,
Boston, (1985), which references and references cited
therein, are entirely incorporated herein by reference.
The standard dose of a drug will generally be
determined by first administering a trial dose to a test
animal. This trial dose may be determined by a theoretical
calculation (e.g.; one based on the binding affinity of the
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49
drug for a receptor, and the number of receptors in the
body), or by analogy with a related drug for which a safe
- and effective dose is known. The dose is then adjusted
upward if the initial dose is safe but insufficiently
effective, and downward if the initial dose is unsafe.
These adjustments may be arithmetic or logarithmic " and
typically progress from coarse to fine. In this manner, a
range of doses which are reasonably safe and effective in
the test animal is determined.
The initial human dose is then determined on the basis
of the preferred dose in one or more test animal species,
with suitable adjustments for the differences between the
human and the test animal, and usually erring on the side of
safety. If the test animal is a generally accepted model of
the disease in question, there will be known drugs for which
the safe and effective dose in both humans and the test
animal in question are known,~allowing a conversion of the
animal dose to the human dose. If not, the test animal dose
will normally be corrected on the basis of the relative
weight or surface area of the test animal and the human.
This initial human dose is then adjusted in a similar manner
to that described for the test animal.
In one embodiment, the initial toxicological testing
will be in mice and will involve a maximum dose of lg/kg
body weight.
Once a standard dose is determined for the patient
population in the abstract, a clinician may determine what
further adjustment is appropriate for a particular patient.
It is understood that the suitable dosage of a drug of the
present invention will be dependent upon the age, sex,
health, and weight of t-he recipient, kind of concurrent
treatment, if any, frequency of treatment, and the nature of
the effect desired. However, the most preferred dosage can
be tailored to the individual subject, as is understood and
determinable by one of skill in the art, without undue
experimentation. This will typically involve adjustment of
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a standard dose, e.g., reduction of the dose if the patient
has a low body weight.
The total dose required for each treatment may be -
administered -by multiple doses or in a single dose. The
5' total daily dose of a drug for a human adult will typically -
be in the range of 1 pg to 10 g, more typically in the range
of 1 ng to 1 g, still more typically in the range of 1 }.zg to
1 g, most typically in the range of 1 mg to -1 g.
The drug may be administered alone or in conjunction
10 with other therapeutics directed to the disease or directed
to other symptoms thereof. Two drugs are administered in
"conjunction" if their times of administration are
sufficiently close so that (1) one drug alters the
biological response to the other drug, or (2) both drugs
15 have a protective effect on the subject at the same time.
The appropriate dosage form will depend on the disease,
the protein, and the mode of administration; possibilities
include tablets, capsules, lozenges, dental pastes,
suppositories, inhalants, solutions, ointments and
20 parenteral depots. See, e.g., Berker, supra, Goodman,
supra, Avery, supra and Ebadi, supra, which are entirely
incorporated herein by reference, including all references
cited therein.
In the case of peptide drugs, the drug may be
25 administered in the form of an expression vector comprising
a nucleic acid encoding the peptide, such a vector, after in
corporation into the genetic complement of a cell of the
patient, directs synthesis of the peptide. Suitable vectors
include genetically engineered poxviruses (vaccinia),
30 adenoviruses, adeno-associated viruses, herpesviruses and
lentiviruses which are or have been rendered nonpathogenic.
Alternatively, a nonpathogenic bacterium could be
genetically engineered to express the drug: The drug must,
of course, either be secreted, or displayed on the outer
35 membrane of the bacterium (or the coat of a virus) in such a
manner that it can interact with the appropriate receptor.
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The dose of vector will be sufficient to achieve a suitable
expressed and delivered dose .of the peptide drug as ,.
previously discussed. Since vectors replicate, and peptides
are cont-inually manufactured by the transformed cells (at
- least when the corresponding promoter is active), it is
possible to achieve a clinical effect with a relatively
small amount of the vector.
In addition to at least one drug as described herein, a
pharmaceutical composition may contain suitable
pharmaceutically acceptable carriers, such as excipients,
carriers and/or auxiliaries which facilitate processing of
the active compounds into preparations which can be used
pharmaceutically. See, e.g., Berker, supra, Goodman, supra,
Avery, supra and Ebadi, supra, which are entirely
incorporated herein by reference, included all references
cited therein.
The appropriate dosage form depends on the status of
the disease, the composition administered, and the route of
administration. Dosage forms include tablets, capsules,
lozenges, dental pastes, suppositories, inhalants,
solutions, ointments, and parenteral depots. See, e.g.,
B-erker, supra, Goodman, supra, Avery, supra and Ebadi,
supra, which are entirely incorporated herein by reference,
including all references cited therein.
In one embodiment, the drug, is dissolved or suspended
in an aqueous carrier. A variety of aqueous carriers may be
used, e.g., water, buffered water, 0.9o saline, 0.30
glycine, hyaluronic acid and the like. These compositions
may be sterilized by conventional, well known sterilization
techniques, or may be sterile filtered. The resulting
aqueous solutions may be packaged for use as is, or
lyophilized, the 1_yophilized preparation being combined with
a-sterile solution prior to administration. The
compositions may contain pharmaceutically acceptable
auxiliary substances as required to approximate
physiological conditions, such as pH adjusting and buffering
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agents, tonicity adjusting agents, wetting agents and the
like, for example, sodium acetate, sodium lactate, sodium
chloride, potassium chloride, calcium chloride, sorbitan
monolaurate, triethanolamine oleate, etc.
For solid compositions, conventional nontoxic solid-
carriers may be used which include, for example,
pharmaceutical grades of mannitol, lactose, starch,
magnesium stearate, sodium saccharin, talcum, cellulose,
glucose, sucrose, magnesium carbonate, and the like. For
oral administration, a pharmaceutically acceptable nontoxic
composition is formed by incorporating any of the normally
employed excipients, such as those carriers previously
listed, and generally 10-950 of active ingredient, that is,
one or more peptides of the invention, and more preferably
at a concentration of 25o-75o.
For aerosol administration, the drugs are preferably
supplied in finely divided form along with a surfactant and
propellant. Typical percentages of drugs are O.Olo-20o by
weight, preferably 1%-100. The surfactant must, of course,
be nontoxic, and preferably soluble in the propellant.
Representative of such agents are the esters or partial
esters of fatty acids containing from 6 to 22 carbon atoms,.
such as caproic, octanoic, lauric, palmitic, stearic,
linoleic, linolenic, olesteric and oleic acids with an
aliphatic polyhydric alcohol or its cyclic anhydride. Mixed
esters, such as mixed or natural glycerides may be employed.
The surfactant may constitute 0.1%-20o by weight of the
composition, preferably 0.25-50. the balance of the
composition is ordinarily propellant. A carrier can also be
included, as desired, as with, e.g., lecithin for intranasal
delivery.
Specific Diagnostic Uses
The peptides of the present invention are of particular
interest in localizing an infection or detecting sepsis.
Technetium-labeled peptides could be used to specifically
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locate bacteria in vivo using imaging.
Binding Molecule
For the purpose of the discussion of diagnostic methods
and agents which follows, the "bind-ing molecule" may be a
peptide, peptoid or peptidomimetic of the present invention,
or an oligonucleotide of the present invention, which binds
the analyte or a binding partner of the analyte. The
analyte is a target protein.
In Vitro Diagnostic Methods and Reagents
The in vitro assays of the present invention may be
applied to any suitable analyte-containing sample, and may
be qualitative or quantitative in nature. In order to
detect the presence, or measure the amount, of an analyte,
the assay must provide for a signal producing system (SPS)
in which there is a detectable difference in the signal
produced, depending on whether the analyte is present or
absent (or, in a quantitative assay, on the amount of the
analyte). The detectable signal may be one which is
visually detectable, or one detectable only with
instruments. Possible signals include production of colored-
or luminescent products, alteration of the characteristics
(including amplitude or polarization) of absorption or
emission of radiation by an assay component or product, and
precipitation or agglutination of a component or product.
The term "signal" is intended to include the discontinuance
of an existing signal, or a change in the rate of change of
an observable parameter, rather than a change in its
absolute value. The signal may be monitored manually or
automatically.
The component of the signal_producing system which is
most intimately associated with the diagnostic reagent is
called the "label". A_label may be, e.g., a radioisotope, a
fluorophore, an enzyme, a co-enzyme, an enzyme substrate, an
electron-dense compound, an agglutinable particle. One
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diagnostic reagent is a conjugate, direct or indirect, or
covalent or noncovalent, of a label with a binding molecule
of the invention.
The radioactive isotope can be detected by such means
as the use of a gamma counter or a scintillation counter or
by autoradiography. Isotopes which are particularly useful
for the purpose of the present invention are 3H; lzsl ~ lsll
3sS, 14C, and, preferably, lzsI.
It is also possible to label a compound with a
fluorescent compound. When the fluorescently labeled
antibody is exposed to light of the proper wave length, its
presence can then be detected due to fluorescence. Among
the most commonly used fluorescent labelling compounds are
fluorescein isothiocyanate, rhodamine, phycoerythrin,
phycocyanin, allophycocyanin, o-phthaldehyde and
fluorescamine.
Alternatively, fluorescence-emitting metals such as
~zsEu, or others of the lanthanide series, may be attached to
the binding protein using such metal chelating groups as
diethylenetriaminepentaacetic acid (DTPA) of
ethylenediamine-tetraacetic acid (EDTA).
The binding molecules also can be detectably labeled by
coupling to a chemiluminescent compound. The presence of
the chemiluminescent compound is then determined by
detecting the presence of luminescence that arises during
the course of a chemical reaction after a.suitable reactant
is provided. Examples of particularly useful
chemiluminescent labeling compounds are luminol, isolumino,
theromatic acridinium ester, imidazole, acridinium salt and
oxalate ester.
Likewise,- a bioluminescent compound may be used to
label the binding molecule. Bioluminescence is a type of
chemiluminescence found in biological systems in which a
catalytic protein increases the efficiency of the
chemiluminescent reaction. The presence of a bioluminescent
protein is determined by detecting the presence of -
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luminescence. Important bioluminescent compounds for
purposes of labeling are luciferin, luciferase and aequorin.
Enzyme labels, such as horseradish peroxidase and
alkaline phosphatase, are preferred. When an enzyme label
5 is used, the signal producing system must also include a
substrate for the enzyme. If the enzymatic reaction product
is not itself detectable, the SPS will include one or more
additional reactants so that a detectable product appears.
Assays may be divided into two basic types,
10 heterogeneous and homogeneous. In heterogeneous assays, the
interaction between the affinity molecule and the analyte
does not affect the label, hence, to determine the amount or
presence of analyte, bound label must be separated from free
label. In homogeneous assays, the interaction does affect
15 the activity of the label, and therefore analyte levels can
be deduced without the need for a separation step.
In general, a target-binding molecule of the present
invention may be used diagnostically in the same way that a
target-binding antibody is used. Thus, depending on the
20 assay format, it may be used to assay the target, or by
competitive inhibition, other substances which bind the
target. The sample will normally be a biological fluid,
such as blood, urine, lymph, semen, milk, or cerebrospinal
fluid, or a fraction or derivative thereof, or a biological
25 tissue, in the form of, e.g., a tissue section or
homogenate. However, the sample conceivably could be (or
derived from) a food or beverage, a pharmaceutical or
diagnostic composition, soil, or surface or ground water.
If a biological fluid or tissue, it may be taken from a
30 human or other mammal, vertebrate or animal, or from a
plant. The preferred sample is blood, or a fraction or
derivative thereof.
In one embodiment, the binding molecule is
insolubilized by coupling it to a macromolecular support,
35 and target in the sample is allowed to compete with a known
quantity of a labeled or specifically labelable target
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analogue. (The conjugate of the binding molecule to a
macromolecular support.is another diagnostic agent within
the present invention.) The "target analogue" is a molecule
capable of competing with target for binding to the binding
molecule, and the term is intended to include target itself.
It may be labeled already, or it may be labeled subsequently
by specifically binding the label to a moiety
differentiating the target analogue from authentic target.
The solid and liquid phases are separated, and the labeled
target analogue in one phase is quantified. The higher the
level of target analogue in the solid phase, i.e., sticking
to the binding molecule, the lower the level of target
analyte in the sample.
In a "sandwich assay", both an insolubilized target-
binding molecule, and a labeled target-binding molecule are
employed. The target analyte is captured by the
insolubilized target-binding molecule and is tagged by the
labeled target-binding molecule, forming a tertiary complex.
The reagents may be added to the sample in either order, or
simultaneously. The target-binding molecules may be the
same or different, and only one need be a target-binding
molecule according to the present invention (the other may.
be, e.g., an antibody or a specific binding fragment
thereof). The amount of labeled target-binding molecule in
the tertiary complex is directly proportional to the amount
of target analyte in the sample.
The two embodiments described above are both
heterogeneous assays. However, homogeneous assays are
conceivable. The key is that the label be affected by
whether or not the complex is formed.
A label may be conjugated; directly or indirectly
(e. g., through a labeled anti-target-binding molecule
antibody), covalently (e.g., with SPDP) or noncovalently, to
the target-binding molecule, to produce a diagnostic
reagent. Similarly, the target binding molecule may be
conjugated to a solid-phase support to form a solid phase
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("capture") diagnostic reagent. Suitable supports include
glass, polystyrene, polypropylene, polyethylene, dextran,
nylon, amylases, natural and modified celluloses,
polyacrylamides, agaroses, and magnetite. The nature of_the
carrier can be either soluble to some extent or- insoluble
for the purposes of the present invention. The support
material may have virtually any possible structural
configuration so long as the coupled molecule is capable of
binding to its target. Thus the support configuration may
be spherical, as in a bead, or cylindrical, as in the inside
surface of a test tube, or the external surface of a rod.
' Alternatively, the surface may be flat such as a sheet, test
strip, etc.
In Vivo Diagnostic Uses
Analyte-binding molecules can be used for in vivo
imaging.
Radio-labelled binding molecule may be administered to
the human or animal subject. Administration is typically by
injection, e.g., intravenous or arterial or other means of
administration in a quantity sufficient to permit subsequent
dynamic and/or static imaging using suitable radio-detecting'
devices. The preferred dosage is the smallest amount
capable of providing a diagnostically effective image, and
may be determined by means conventional-in the art, using
known radio-imaging agents as a guide.-
Typically, the imaging is carried out on the whole body
of the subject, or on that portion of the body or organ
relevant to the condition or disease under study. The
radio-labelled binding molecule has accumulated. The amount
of radio-labelled binding molecule accumulated at a given
point in time in relevant target organs can then be
quantified.
A particularly suitable radio-detecting device is a
scintillation camera, such as a gamma camera. A
scintillation camera is a stationary device that can be used
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to image distribution of radio-labelled binding molecule.
The detection device in the camera senses the radioactive
decay, the distribution-of which can be recorded. Data
produced by the-imaging system can be digitized. The
digitized information can be analyzed over time
discontinuously or continuously. The digitized data can be
processed to produce images, called frames, of the pattern
of uptake of the radio-labelled binding protein in the
target organ at a discrete point in time. In most
continuous (dynamic) studies, quantitative data is obtained
by observing changes in distributions of radioactive decay
in target organs over time. In other words, a time-activity
analysis of the data will illustrate uptake through
clearance of the radio-labelled binding molecule by the
target organs with time.
Various factors should be taken into consideration in
selecting an appropriate radioisotope. The radioisotope
must be selected with a view to obtaining good quality
resolution upon imaging, should be safe for diagnostic use
in humans and animals, and should preferably have a short
physical half-life so as to decrease the amount of radiation
received-by the body. The_radioisotope used should
preferably be pharmacologically inert, and, in the
quantities administered, should not have any substantial
~ physiological effect.
The binding molecule may be radio-labelled with
different isotopes of iodine, for example 123I, '-'-'I, or 1311
(see for example, U.S. Patent 4,609,725). The extent of
radio-labeling must, however be monitored, since it will
affect the calculations made based on the imaging results
(i.e. a diiodinated binding molecule will result in twice
the radiation count of a similar monoiodinated binding
molecule over the same time frame).
In applications to human subjects, it may be desirable
to use radioisotopes other than lzsl for labelling in order
- to decrease the total dosimetry exposure of the human body
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and to optimize the detectability of the labelled molecule
(though this radioisotope can be used if circumstances
require). Ready availability for clinical use is also a
factor. Accordingly, for human applications, preferred
S radio-labels are for example, 99'"Tc, 6'Ga, 6aGa, 9oY, 111In,
ii3mln ~ i23I ~ iasRe ~ ieaRe or ZilAt .
The radio-labelled binding molecule may be prepared by
various methods. These include radio-halogenation by the
chloramine - T method or the lactoperoxidase method and
subsequent purification by HPLC (high pressure liquid
chromatography), for example as described by J. Gutkowska et
al in "Endocrinology and Metabolism Clinics of America:
(1987) 16 (1):183. Other known method of radio-labelling
can be used, such as IODOBEADS'''".
There are a number of different methods of delivering
the radio-labelled binding molecule to the end-user. It may
be administered by any means that enables the active agent
to reach the agent's site of action in the body of a mammal.
If the molecule is digestible when administered orally,
parenteral administration, e.g., intravenous, subcutaneous,
or intramuscular, would ordinarily be used to optimize
absorption.
Other Uses
The binding molecules of the present invention may also
be used to purify target from a fluid, e.g., blood. For
this purpose, the target-binding molecule is preferably
immobilized on a solid-phase support. Such supports include
those already mentioned as useful in preparing solid phase
diagnostic reagents.
Peptides, in general, can be used as molecular weight
markers for reference in the separation or purification of
peptides by electrophoresis or chromatography. In many
instances, peptides may need to be denatured to serve as
molecular weight markers. A second general utility for
peptides is the use of hydrolyzed peptides as a nutrient
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source. Hydrolyzed peptide are commonly used as a growth
media component for culturing microorganisms, as well as a
food ingredient for human consumption. Enzymatic or acid
hydrolysis is normally carried o.ut either to completion,
5 resulting in free amino acids, or partially,-to generate
both peptides and amino acids. However, unlike acid
hydrolysis, enzymatic hydrolysis (proteolysis) does not
remove non-amino acid functional groups that may be present.
Peptides may also be used to increase the viscosity of a
10 solution.
The peptides of the present invention may be used for
any of the foregoing purposes, as well as for therapeutic
and diagnostic purposes as discussed further earlier in this
specification.
15 Reference Example A
Determination of Peptide Conformation
One method of determining the conformation of a peptide
when bound to lipids is illustrated below, using the A19
analogue of magainin 2 (A1a19-magainin) as an example. We
20 used both Fourier transform infrared_(FTIR). and solid-state
nuclear magnetic resonance (NMR) spectroscopy.
The peptide was synthesized with both 13C and 15N labels
in order to determine conformation through intramolecular
distance measurements using rotational-echo double resonance
25 (REDOR) NMR.32 In these studies, we attempted to (a)
determine whether or not magainins are completely a-helical
when bound to membranes, (b) localize secondary structures
within the molecule, and (c) identify the location of the
peptide. with respect to. the lipid bilayer.zz
30 The amide I band in the infrared spectrum is sensitive
to. peptide conformation. -This band was centered near 1650
cm-1 with a prominent shoulder near 1635 cml. The former
frequency is attributable to a-helical conformation, while
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the latter is generally associated with (3-sheet structure.33
For fully hydrated lipid-peptide mixtures, we estimated that
-the a-helical and ~i-sheet components of the amide I' band
(where amide protons are exchanged with deuterium) account
for 60-75o and 20-35%, respectively, of the total peak area,
depending on the fluidity of the lipids. The level of
hydration of the system is important in determining the
secondary structure of the peptide. In the anhydrous state,
the peptide is almost entirely a-helical. As hydration
occurred (using DZO as the solvent), the helical content
diminished, with a concomitant increase in (3-sheet
structure. Also, the quick disappearance of the amide II
band upon hydration with DZO showed that the peptide
molecules have access to the aqueous surroundings and are
not locked in hydrogen bonds that would impede the exchange
of deuterium for hydrogen along the peptide backbone. Other
investigators also saw a mixture of a-helix and (3-sheet or
turn structures in membrane-bound magainins using CD,19,2s
FTIR, 34 and Raman35 spectroscopy.
In order to confirm the presence of both cc-helical and
(3-sheet structure in A1a19-magainin, we synthesized the
peptide with [ 1-13C] A1a15, and [15N] Alal9. This label-ing
pattern allows for a distance measurement between A1a15 and
A1a19 by using REDOR,to detect dipolar coupling between the
13C and 15N nuclei. If the peptide _adopts an a-helical
conformation, these atoms will be only 4 A apart. The 13C
NMR spectrum showed two peaks at 176.8 and 172.4 ppm for [1-
~3C]Alals, which is consistent with a mixture of a-helix and
~3-sheet conformations. Furthermore, only the peak at 176.8
ppm showed dipolar coupling with 15N in the REDOR experiment,
confirming its assignment as the a-helical component. By
comparing the areas of the two peaks, the proportion of ~a-
helix to (3-sheet structure using solid-state NMR was nearly
identical to the results obtained by FTIR spectroscopy.
Thus, we concluded that while the peptide is primarily a-
helical, there is a substantial amount of (3-sheet structure
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as well.
We also attempted to determine the location of the
peptide in the lipid bilayer using 13C-observe, 31P-dephase
REDOR experiment. If the peptide resides close to the lipid
polar head groups, the distance between-the 13C label in the
peptide and 31P-in the lipid molecules should be short,
resulting in significant dipolar coupling. The results
indicated that most of the peptide must reside near the
surface of the bilayer, although it was not possible to rule
out that some of the peptide might penetrate through the
hydrophobic core of the bilayer in channels or pores.
Example 1
PGLa contains GAIAGKIAK (residues 7-15 of SEQ ID N0:2).
As shown in Table I, the activity of this peptide is very
high and it also possesses low hemolytic activity. This
peptide is capable of forming a highly amphipathic a-helix.
We wanted to test the hypothesis that the ability to form a
highly amphipathic ct-helix 3s a mandatory requirement for
potent antimicrobial activity in these linear cationic
peptides. The placement of lysines at positions 1 and 5 of
a heptamer repeat results in their cluster-ing on the helica l
face of a 21-mer peptide (SEQ ID N0:4). We decided to shift
a lysine from position 5 to 7 of the heptamer. The
resulting peptide, (KLAGLAK)3-NHz (SEQ ID N0:6), has a
considerably reduced hydrophobic moment as an a-helix
because the lysines now are split into two clusters of three
on the helical face. Finally, we designed a third peptide,
(KIGAKI)3-NHZ (SEQ ID N0:8), that would have no propensity to
form an amphipathic helix, but could form a highly
amphipathic (3-sheet. All three of these peptides carry a +7
charge and are nearly equal in overall hydrophobicities.
Fig. 1 shows helical wheel and (3-sheet diagrams for the
peptides, along with the calculated hydrophobic moments for
each of the structures.
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If forming an amphipathic structure is critical to
membrane association, (KIAGKIA)3-NHZ (SEQ ID N0:4) should be
effective primarily as an a-helix, (KLAGLAK)3-NHZ (SEQ ID
N0:6) is only slightly more amphipathic as an a-helix than
- as a (3-sheet, and KIGAKI)3-NHZ (SEQ ID N0:8) is
overwhelmingly amphipathic as a (3-sheet. Our plan was to
compare the antimicrobial and hemolytic activities of the
peptides, their conformations when bound to lipid bilayers,
and their ability to induce leakage from LUV.
EXPERIMENTAL PROCEDURES
Materials. All peptides were synthesized using Fmoc
chemistry on an Advanced Chem Tech model 90 peptide
synthesizer. The crude peptides were purified by reverse
phase HPLC. Purity was checked by reverse phase HPLC,
capillary electrophoresis, and electrospray mass
spectrometry. POPC, POPE, POPG, and E. coli polar lipid
extract were used as supplied from Avanti Polar Lipids, Inc.
E. coli DPG, calcein, TFE, and buffer materials were from
Sigma Chemical Co. Phosphorus content in lipid stock
solutions was determined by a spectrophotometric analysis
(39) .
Antimicrobial and Hemolytic.Assays. Antimicrobial
susceptibility testing against Staphylococcus aureus (ATCC
29213), Escherichia coli (ATCC 25922), and Pseudomonas
aeruginosa (ATCC 27853) was performed using a modification
of the National Committee for Clinical Laboratory Standards
microdilution broth assay (22). Mueller-Hinton broth (BBL)
was used for diluting the peptide stock solution and for
diluting the bacterial inoculum. The inoculum was prepared
from mid-logarithmic phase cultures. Microtiter plate wells
received aliquots-of 100 ~L each of the inoculum and peptide
dilution. The final concentration of peptide solution
ranged-from 0.25 to 256 ~tg/mL in two-fold dilutions. The
final concentration of bacteria in the wells was 5_x 105
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CFU/mL. Peptides were tested in duplicate. In addition to
the test peptide, three standard peptides and a non-treated
growth control were included to validate the assay. The
microtiter plates were incubated overnight at 37°C and the
absorbance was measured at 600 nm. MIC is defined as the
lowest concentration of peptide that completely inhibits
_ growth of the organism. Hemolysis at peptide concentrations
of 500 ~tg/mL was determined using a So suspension of freshly
drawn human erythrocytes, which had been washed twice in
phosphate buffered saline. After incubation at 37°C for 30
min, the suspension was centrifuged at 10,000 x g for 10 min
and the absorbance at 400 nm was measured. Complete
hemolysis was determined by adding 0.2o Triton X-100 in
place of the peptide.
CD Spectroscopy. CD spectra were measured using a
Jasco J-715 spectropolarimeter. Spectra were recorded from
250-190 nm at a sensitivity of 100 mdeg, resolution of 0.1
nm, response of 8 seconds, bandwidth of 1.0 nm, and scan
speed of 20 nm/min, with a single accumulation. The buffer
contained 10 mM potassium phosphate, 150 mM KCl, 1 mM EDTA,
pH 7Ø The peptide concentration in buffer and TFE/buffer
mixtures was 20 ~M. LUV were prepared from aqueous
dispersions of POPG at a concentration of ~lmg/mL in
phosphate buffer. Following five freeze/thaw cycles, the
mixture was extruded ten times through a 0.1-~m-pore
polycarbonate membrane in an Avanti mini-extruder apparatus,
resulting in 100-nm-diameter LUV. The lipid and peptide
concentrations in the cuvet were 100 and 5 ~M, respectively.
FTIR Spectroscopy. Mixtures of POPG (4 pmoles) and
peptide (0.2 umoles) were co-dissolved in 2:1 CHC13/CH30H.
Solvent was removed by evaporation, followed by evacuation
under high vacuum. The mixtures were suspended in D20
buffer (20 mM PIPES, 1 mM EGTA, pD 7.0), isolated by
centrifugation, and placed between CaF2 windows using a 25
~m Teflon_spacer. FTIR spectra were collected using a
Mattson Polaris FTIR spectrometer with a HgCdTe detector. A
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total of 250 interferograms were co-added and Fourier
transformed with triangular apodization to generate
absorbance spectra with 2 cm-1 resolution and data points
encoded every 1 cm 1, with.a signal-to-noise ratio of better
5 than 500(22). -
Peptide-Induced Leakage from Calcein-Loaded LUV. The
ability of the peptides to release calcein (MW 623) from LUV
of varying lipid composition was compared. LUV were
prepared as above, except that the buffer consisted of 50 mM
10 HEPES, 100 mM NaCl, 0.3 mM EDTA, 80 mM calcein, pH 7.4.
Calcein-loaded vesicles were separated from free calcein by
size-exclusion chromatography using a Sephadex G-50 column
and calcein-free buffer. Calcein leakage was monitored
using a Perkin-Elmer LS-500 luminescence spectrometer by
15 measuring the time-dependent increase in fluorescence of
calcein (excitation = 490 nm, emission = 520 nm). LUV
containing 8 nmoles of lipid were added to 1.5 mL of buffer
in a stirred cuvet at 25'C. An aliquot of peptide was added
to achieve the desired lipid-to-peptide ratio (256 to 8).
20 Complete leakage was determined by the addition of 20 laL of
10% Triton X-100. Each value represents at least nine
different measurements using at least three different LUV .
preparations.
Fluorescence Measurement of Peptide Binding to LUV.
25 Peptide binding to LUV was assessed by measuring the shift
in the emission maximum of the tryptophan residue in W-
KIAGKIA, W-KLAGLAK, and W-KIGAKI. LUV were prepared as
described for the leakage experiments except that calcein
was omitted. Fluorescence spectra were collected from 290
30 to 400 nm using an excitation wavelength of 280 nm. The
peptide concentration was kept constant at 3 uM.
DSC. Lipid films were made by_solvent evaporation
under nitrogen of solutions of DiPoPE in chloroform/methanol
(2/l,v/v). Last traces of solvent were removed in vacuum
35 for 2 hours. The films were then hydrated by vortexing at
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room temperature with 20 mM PIPES, 1 mM EDTA, 150 mM NaCl
with _0.002% NaN3, pH 7.4. The final lipid concentration was
3.7 mg/mL. The lipid suspension was degassed under vacuum
before being loaded into a NanoCal high sensitivity scanning
calorimeter (CSC, Provo, UT). A heating scan rate of 0.75°
C/min was generally employed. TN was fitted using parameters
to describe equilibrium with a single van't Hoff enthalpy and
the transition temperature reported as that for the fitted
curve. Data was analysed with the program Origin, version
5Ø
RESULTS
Comparison of Antimicrobial and Hemolytic Activities of
the Peptides. The antimicrobial and hemolytic activities of
the three model peptides are compared to those of magainin 2
amide and PGLa in Table II. KIAGKIAx3, KLAGLAKx3, and
KIGAKIx3 are significantly more potent against all three
microorganisms than either magainin 2 amide or PGLa. At 500
~.tg/mL, all of the peptides tested showed little hemolytic
activity.
Secondary Structure of the Peptides. The conformation of the
peptides was assessed by CD and FTIR spectroscopy. As shown
in Fig. 2, the CD spectra of the three model peptides are
characteristic of a random structure (with a minimum below 200
nm) in buffer (panel A). TFE is often used as a membrane-
mimetic to lower the polarity of the solvent. In 50o TFE
(panel B), the spectra show minima near 208 and 222 nm, which
indicates a-helical content. The amount of helical structure
in KIAGKIAx3 and KLAGLAKx3 is about the same, while the
helical content of KIGAKIx3 is slightly lower under these
conditions. Finally, spectra of the peptides in the presence
of POPG LUV (lipid-to-peptide ratio of 20:1) reveal that both
KIAGKIAx3 and KLAGLAKx3.are mainly a-helical, with the-helical
content of KIAGKIAx3 slightly greater than that of KLAGLAKx3.
The conformation of KIGAKIx3 is dramatically different in the
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presence of POPG vesicles as compared to TFE. This spectrum,
with a single minimum just below 220 nm, suggests ~3-sheet
structure,(20) .
The amide I' vibrational bands of the-three peptides
bound to POPG at a 20:1 molar ratio of lipid to peptide are
shown in Fig. 3. The amide I' band for KIAGKIAx3 is centered
- close to 1650 crril, indicative of primarily a-helical
conformation. In contrast, the band for KLAGLAKx3 is shifted
slightly to lower frequency and is broader, suggesting less a-
helical content, in agreement with the CD data. The amide I'
band for KIGAKIx3 is markedly different, with a maximum below
1620 cm 1 and a small peak near 1680 cm 1, consistent with ~i-
sheet conformation (51).
Peptide-Induced Leakage from Calcein-Loaded LUV. A
comparison of PGLa and the three model peptides in their
ability to release calcein from LUV with varying lipid
composition is shown in Fig. 4. All peptides were able to
induce leakage from anionic LUV composed of POPG (panel A).
The order of potency is: PGLa > KIAGKIAx3 > KLAGLAKx3 >
KIGAKIx3. Thus, PGLa, the peptide with the weakest
antimicrobial activity, is the most potent in inducing calcein
release. In POPC LUV (panel B), the level of calcein release
was.much lower than in POPG LUV; however, it is clear that the
three a-helical peptides (PGLa, KIAGKIAx3, and KLAGLAKx3) were
more active than KIGAKI in permeabilizing POPC vesicles.
In order to better relate the leakage experiments to
bacterial membranes, calcein-loaded LUV composed of E. coli
polar lipids were tested. These results, shown in panel C,
are dramatically different. The order of potency here is:
KIGAKIx3 ~ KIAGKIAx3 > KLAGLAKx3 -- PGLa. The correlation
between the antimicrobial activity and leakage in E. coli LUV
is much better than in POPG or POPC LUV. The composition of
E. coli polar lipids is as follows: 67% PE, 23o PG, and 100
DPG. Therefore, we decided to compare leakage rates in LUV
containing POPG as-the anionic component and either POPC or
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POPE as the neutral component. The ability of the peptides to
increase the permeability of LUV with neutral-to-acidic lipid
ratios of 1:1, 2:1, 3:1, and 4:1 is shown in Fig. 5. The
general trend is a reduction in leakage as the neutral-to-
acidic lipid ratio increases. In POPC/POPG LUV .(panels A-D),
PGLa is the most potent peptides at all ratios, followed
closely. by KIAGKIAx3. The lytic activity of KLAGLAKx3 falls
off dramatically~at higher neutral lipid content. KIGAKIx3 is
the least potent peptide at all ratios. The results change
markedly, however, when POPC is replaced by POPE (panels E-H).
At 2:1 POPE/POPG, leakage rates for the three a-helical
peptides decrease significantly, while the activity of KIGAKI
is slightly enhanced in comparison to 2:1 POPC/POPG. By 4:1
POPE/POPG, the activity of PGLa is quite low even at high
peptide levels. KIAGKIAx3 and KIGAKIx3 are the most potent,
with leakage rates only slightly below those observed in E.
coli LUV.
Since E. coli plasma membrane lipids.contain DPG as a
third major lipid component, LUV were prepared with a ternary
mixture of 23% POPG, 10o DPG, and 67o either POPE or POPC
(Fig. 6). LUV containing POPC as the neutral lipid (panel A)
are highly susceptible to the lytic activity of the a-helica l
peptides but not the (3-sheet peptide. In contrast, the
properties of LUV formed by combining POPE, POPG, and DPG
(panel B) are quite similar to those of LUV made from the E.
coli lipid extract (Fig. 4, C). Only the activity of KIGAKIx3
is enhanced by replacing POPC with POPE in the ternary
mixture.
Fluorescence Emission in Tryptophan-Containing Analogs.
W-KIAGKIA, W-KLAGLAK,- and W-KIGAKI (see Table 1) were
synthesized to assess the interaction of the peptides with LUV
of varying lipid composition. Since each peptide contains a
single tryptophan residue, fluorescence, emission can be used
to monitor binding and local polarity. The antimicrobial
activity and secondary structure of these analogs were similar
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to those of the parent peptides (data not shown). In aqueous
solution, the emission maxima for the three peptides were
nearly identical (354-356 nm). The change in emission maximum
to lower wavelength (blue shift) under different conditions. is
shown in Fig. 7. In 50o TFE, the emission maximum decreased
by 4-5 nm for each peptide. Emission spectra in the presence
of LUV were measured at lipid-to-peptide ratio of 20. The
shift observed in the presence of POPC LUV (<_3 nm) was-smaller
than in 50o TFE for all peptides. The largest blue shifts
were observed in the presence of POPG LUV. For W-KIAGKIA, the
shifts in the presence of POPC/POPG were slightly larger than
POPE/POPG at ratios above 1:1. This enhancement is much
greater for W-KLAGLAK, where the blue shifts with POPC/POPG
LUV are about twice as large as the corresponding POPE/POPG
LUV at neutral-to-acidic ratios >_ 2. The presence of either
POPC or POPE decreased the magnitude of the blue shifts
observed for W-KIGAKI to a much greater degree as compared to
the other peptides; however, the shifts observed with
POPE/POPG LUV were greater than those with POPC/POPG LUV at
all ratios below 4.
The blue shifts observed for W-KIAGKIA in the presence of
LUV containing E. coli lipids or ternary mixtures of
POPC/POPG/DPG or POPE/POPG/DPG were nearly equal. For W-
KLAGLAK, however,-the shift observed in the presence of
POPC/POPG/DPG was greater than with either E. coli lipids or
POPE/POPG/DPG. Only W-KIGAKI showed larger blue shifts in the
presence of E. coli lipids or POPE/POPG/DPG as compared to
POPC/POPG/DPG.
DSC. An indication of the effects of membrane additives
on the curvature properties of membranes may be assessed by
their effect on the bilayer-to-hexagonal phase transition
temperature (TH). Only KLAGLAKx3 caused an appreciable
increase in TH. The change in TH of DiPoPE as a function of
peptide concentration is shown in Table 3.
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DISCUSSION
PGLa and. the three model peptides, KIAGKIAx3, KLAGLAKx3,
and KIGAKIx3, possess no defined secondary structure in
solution, but adopt a conformation that appears to maximize
5 amphipathic character upon interacting with lipid bilayers.
Like PGLa, KIAGKIAx3 and KLAGLAKx3 can form an amphipathic a-
helix at the bil.ayer surface. KIGAKIx3 was designed to mimic
KIAGKIAx3 and KLAGLAKx3 in terms of net charge and
hydrophobicity, but to form an amphipathic ~-sheet instead of
10 an a-helix. In 50o TFE, KIGAKIx3 is mainly helical, but when
bound to LUV, the drive to form an amphipathic structure
dominates and the resulting conformation is ~3-sheet as shown
by CD and FTIR spectroscopy (Figs. 2 and 3). A comparison of
antimicrobial activity (Table 2) shows that KIAGKIAx3 and
15 - KIGAKIx3 are significantly more active than PGLa, with
KLAGLAKs3 only slightly- so. Notably, KIGAKIx3 is the least
hemolytic of the three model peptides, and about the same as
magainin 2 amide and PGLa.
PGLa, KIAGKIAx3, KLAGLAKx3, and particularly KIGAKI were
20 not very effective at inducing leakage in PC LUV, but all were
much more active with PG LUV. The binding of PGLa to
membranes was shown recently to be dominated by electrostatic
and not hydrophobic effects (47). Thus, increased binding
probably accounts for the greater leakage rates observed in
25 POPG vs. POPC LUV. At lower peptide levels, however, PGLa is
more effective than the other peptides at inducing leakage in
LUV containing POPG alone or POPC/POPG mixtures (Fig. 5).
This contrasts with the antimicrobial activities (Table 2)
that show PGLa as the least potent peptide. In LUV.composed
30 of E. coli polar lipids, however, the activity of PGLa is
markedly reduced while that of KIGAKIx3 is enhanced compared
to the other peptides (Fig. 4).
Since PE is the major uncharged polar lipid in E. coli
plasma membranes, we examined the effect of replacing POPC by
35 POPE. In LUV containing equimolar amounts of POPG and neutra?
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lipid, only slight differences were observed. As the
proportion of POPE in the LUV increased, however, the leakage
rates more closely resembled those in E. coli LUV (Fig. 5).
In a comparison of ternary mixtures of either POPE/POPG/DPG or
POPC/POPG/DPG, the presence of PC greatly enhances the
activity of PGLa and.the other a-helical peptides, while
. reducing the activity of KIGAKIx3 (Fig. 6).
One may legitimately question whether the relatively high
level of peptide necessary to induce leakage in LUV composed
of E. coli polar lipid extract and other lipid mixtures with a
high proportion of PE is relevant to the inhibitory effect
upon bacterial growth. We can estimate the number of peptide
molecules per bacterium in the MIC assay. The assay mixture
contains 105 bacteria in a volume of 0.2 mL. The lowest MIC
value reported here is 8 }.zg/mL. With a molecular weight of
2,000, the amount of peptide in the assay (1.6 ug) translates
to >4 x 101" molecules. Thus, there are ~4 x 10~ peptide
molecules per bacterium even at the. lowest MIC value,. How
does this relate to the number of lipid molecules in the
plasma membrane? For a large bacterium of size 2 x 4 ~.un
(i.e., even larger than the bacteria tested here), the surface
area is ~3 x -10' nm2. If the average surface area of a lipid
molecule is estimated to be ~0.7 nmz, then the number of lipid
molecules on the outer surface of the plasma membrane is ~4 x
10'. Therefore, conservatively, there are about 100 peptide
molecules for each lipid molecule on the exterior of the
bacterial plasma membrane. For smaller bacteria or for higher
MIC values, the number of peptides per lipid is proportionally
higher.
30. This does not mean, however, that all of the peptides are
bound to the plasma membrane. Many peptide molecules may be
bound to lipopolysaccharide, peptidoglycan, teichoic acid, or
other components of the cell envelope beyond the plasma
membrane, while other peptides may remain free in solution.
The estimate does point out, however, that a real potential
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exists for a very large number of peptides to interact with
the plasma membrane surface at antimicrobial concentrations.
Further experiments will be necessary to determine the binding
affinity and location of the peptides on intact bacteria.
What is the explanation for the observed differences
between PC and PE in the leakage experiments? One obvious
possibility is that the peptides bind differently to LUV
containing PC or PE as the neutral lipid. We used tryptophan-
containing analogs of the three model peptides to study their
interactions with LUV. A blue shift in the maximum of the
tryptophan emission band results from the decrease in polarity
surrounding the indole side chain as the peptide binds to and
penetrates the bilayer surface. Minimal and maximal blue
shifts were observed in the presence of POPC and POPG LUV,
respectively (Fig. 7). A comparison of LUV with either
POPC/POPG or POPE/POPG reveals that W-KIAGKIA and W-KLAGLAK
showed a greater interaction (i.e., a larger blue shift) with
POPC/POPG while W-KIGAKI interacted more strongly with
POPE/POPG. Thus, binding differences could contribute to the
relative differences in leakage rates in LUV containing POPC
vs. POPE. A second important factor affecting the activity of
the peptides is, once bound,-the precise nature of the
interaction of the peptide with the membrane surface.
The curvature-modulating property (37) of KLAGLAKx3
differs from KIAGKIAx3 and KIGAKIx3 in that KLAGLAKx3 promotes
more positive membrane curvature (Table 3). This property
appears to have consequences for the lipid dependence of the
lytic activity of magainin 2, which was shown to induce
positive curvature to a slightly greater extent than KLAGLAKx3
(38). The curvature effects of these peptides can be
rationalized in terms of their structure. Comparing the two
a-helical model peptides, in KIAGKIAx3 the six lysine residues
are clustered together, while-in KLAGLAKx3 they are separated
by three glycine residues in a helical wheel projection (Fig.
1). Lysines have a special role in the binding of peptides to
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bilayers because of the amphiphilic nature of their side chain
(i.e., four hydrophobic methylene groups between the a-carbon
atom and the side chain amino group) (59). In the case of
KIAGKIAx3, the clustered.lysine residues will allow the
peptide to insert more deeply in the bilayer and thereby
promote less positive curvature (60). In KLAGLAKx3, the two_
gr-oups of lysine-residues are at the interface between the
hydrophobic and hydrophilic sides of the amphipathic helix as
they are in Class A peptides (61), resulting in increased
l0 positive curvature. This difference in insertion might not be
reflected in the fluorescent properties of the tryptophan-
substituted analogs because the W residues should seek a
position close to the interface, regardless of the depth of
insertion of the peptide as a whole (62-64). Also, in W-
KIAGKIA, the substitution is closer to the hydrophilic face as
compared to W-KLAGLAK, where it is near the center of the
hydrophobic face (Fig. 1). Since the (3-sheet peptide,
KIGAKIx3, does not shift TH of DiPoPE, its lytic activity may
not be dependent on the curvature properties of the bilayer
surface.
The toroidal pore mechanism proposed by Matsuzaki (27)
and Huang (65) for the antimicrobial action of magainin-2 is
based upon the induction of sufficient positive curvature to
create supramolecular pores. An alternative ~~carpet" model
was proposed earlier by Shai- and coworkers (39) which also
relies upon changes in bilayer curvature to disrupt the
membrane. Of the peptides examined in this work, only KLAGLAK
resembles magainin 2 in its ability to generate positive
curvature. Both KIAGKIAx3 and KIGAKIx3 have only a negligible
effect on the T" of DiPoPE. If the interactions with DiPoPE
can be generalized to other lipids, then the membrane
disruption caused by KIAGKIAx3 and KIGAKIx3 may well be
different from magainin-like peptides.
We have demonstrated that KIGAKIx3, designed to adopt a
highly amphipathic (3-sheet, possesses -a combination of
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equivalent antimicrobial activity and superior selectivity
compared to the a-helical peptides in this study.
Example 2
In this study, we compared the structure and activity of:
1 5 10 15 20
KIAGKIAx3 K I A G K I A K I A G K I A K I A G K I A-NHZ
(SEQ ID N0:4)
KIGAKIx3 K I G A K I K I G A K I K I G A K I-NHZ
(SEQ ID N0:6)
SH-KIGAKI K I G A K S K H G A K I K I G A K I-NHZ
(SEQ ID N0:25)
KI~-14 K L K L K L K W K L K L K L-NHZ
(SEQ ID N0:26)
KL-18 K L K L K L K W K L K L K L K L K L-NHZ
(SEQ ID N0:27)
KL-14 is a 14-residue KL repeat containing a tryptophan
at position 8 in place of a leucine. KL-18 is the
cor-responding 18-residue peptide. Both of these peptides are
more highly charged but much less in hydrophobic than our
original ~3-sheet peptide, (KIGAKI)3-NH2 (SEQ ID N0:8). Both of
these peptides should be highly amphipathic ~3-sheet peptides.
The conformation of the peptides in aqueous solution, in
50% TFE, and bound to lipid bilayers was determined by FTIR
and CD spectroscopy. The peptides were compared for their
ability to release the fluorescent dye, calcein, from LUVs
composed of either PC, PG, 1:1 PC/PG, 2:1 PC/PG, 3:1 PC/PG,
4:1 PC/PG, 1:1 PE/PG, 2:1 PE/PG, 3:1 PE/PG, 4:1 PE/.;PG, or E
coli polar lipid extract. Interactions with the same set of
LUVs were monitored by the-tryptophan emission specturm of
four of the peptides: KL-14, KL-18, and the analogs W9-KIAGKIA
and We-KIGAKI.. These results then were compared to the
antimicrobial activity of the peptides toward Escherichia
coli, Staphylococcus aureus, and Pseudomonas aeruginosa in
order to assess relationships between peptide structure and
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function.
The data is summarized in Table 4.
Results and Discussion
As shown previously, KIAGKIAx3 and KIGAKIx3 adopt-alpha-
5 helical and beta-sheet conformations, respectively, when bound
to LUVs containing either pure PG or PG mixed with either PC
or PE. KL-14 and KL-18 also adopt beta-sheet conformation
under these conditions. SH-KIGAKI is predominantly beta-sheet
in the presence of PG LUV, but not with either PC/PG or PE/PG
10 LUVs .
KL-18 was most potent in inducing leakage from PC LUVs,
followed by KL-14 and KIAGKIAx3. Both KIGAKI and SH-KIGAKI
showed low lytic activity even at high peptide concentrations.
In PC/PG LUVs, the order of potency was KIAGKIAx3>KL-18>KL-
15 14~KIGAKIx3»SH-KIGAKI. The potency of KIAGKIAx3 was
diminished substantially when PE replaced PC as the neutral
lipid in LUVs. Only KIGAKIx3 was more active with PE/PG vs.
PC/PG LUVs at all molar lipid ratios tested (1:1, 2:1, 3:1,
and 4:1). SH-KIGAKI showed, no ability to induce leakage in
20 any LUVs containing PC/PG or PE/PG. In LUVs composed of E.
coli polar lipids, the order of lytic potency was KL-1>KL-
14>KIGAKIx3>KIAGKIAx3»SH-KIGAKI.
In experiments to estimate peptide binding to LUVs, WB-
KIGAKIx3 differed from W9-KIAGKIA, KL-14, and KL-18, in that it
25 showed much larger blue shifts. in the tryptophan emission
maximum in PE-containing as opposed to PC-containing LUVs.
The low affinity of KIGAKIx3 for PC-containing LUVs is
consistent with the low lytic activity observed in these
vesicles.
30 - These antimicrobial activities of all peptides were
assessed against E. coli, S. aureus, and P. aeruginosa. The
potencies of KIAGKIAx3 and KIGAKIx3 were about equal. Both
KL-14 and KL-18 showed higher MIC values (4-16 ug/mL) than
KIAGKIAx3 or KIGAKIx3.
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Thus, the antimicrobial activity of the KL peptides is
lower than that of both KIAGKIAx3 and KIGAKIx3. The KL
peptides also are much less selective than (KIGAKI)3-NH,.
We conclude that: (1) both amphipathic beta-sheet and
alpha-helical peptides can possess comparable antimicrobial
activity; (2) a relatively small reduction in amphipathic
character (as shown by SH-KIGAKI) can result in a large loss
of activity; (3)~KL-14 and especially KL-18,.which are both
highly effective at inducing leakage in LUVs from E. coli
lipids, are less antimicrobial than KIAGKIA or KIGAKI
(hydrophobicity may be an important factor here); and
(4)KIGAKI is unique among these peptides since binding and
leakage experiments demonstrate it to be highly selective for
membranes containing PE as the neutral lipid instead of PC.~
KIGAKI therefore may have a higher therapeutic index than the
other peptides since PC is a major component in mammalian
plasma membranes. Since KL-14 and KL-18 are quite lytic
toward PC .LUVs, this selectivity clearly is not conferred by
secondary structure alone.
Hypothetical Example 101
We intend- to study two series of peptides of identical
charge and hydrophobicity. Each model peptide will contain
six lysine residues, and will be amidated at the carboxyl
terminus. The first series will consist of five peptides,
each containing a different hexamer sequence of the same set
of amino acids repeated three times (18 residues). These
peptides will possess no potential to form amphipathic a-
helices, but they will have varying capacity to form
amphipathic ~3-sheet structures. A second series will consist
of six peptides, each containing a different heptamer sequence
of the same set of amino acids repeated three times (21
residues). These peptides will have low amphipathic (3-sheet
potential, but will vary considerably in the prospect of
forming amphipathic a-helices.
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These two sets of peptides will enable us to examine a
variety of structure/function relationships. Our experimental
approach will be as follows:
1) Test the antimicrobial and hemolytic activity of each
peptide.
2) Determine the secondary structure of the peptides in
solution and bound to lipids.
3) Measure the ability of the peptides to induce leakage in
LUV of varying lipid composition.
4) Measure the ability of the peptides to induce leakage in
a strain of E. coli with constitutive (3-galactosidase
activity.
5) Determine how many peptide molecules must bind to the
membrane in order to increase permeability in both LUV
and bacteria.
101.1. Peptide Design and Synthesis
As described in the previous section, we have. worked with
two 21-residue peptides containing heptamer repeats and one
18-residue peptide containing a hexamer repeat. We propose
here to create two complete peptide families. All of the
peptides will be amidated at the carboxyl terminus. The first
series will be comprised of 18-residue molecules made up of a
trimeric hexamer repeat in which there are two lysines. One
will be fixed at position 1 and the other will be located at
either position 2, 3, 4, 5, or 6. The other amino acids in
the hexamer repeat - I, G, A, I - will remain constant. The
18-residue family will consist of the following: 1,2-Hex =
(KKIGAI ) 3-NHz; l, 3-Hex = (KIKGAI ) 3-NHz; 1, 4-Hex = (KIGKAI ) 3-NH2;
1, 5-Hex = (KIGAKI) 3-NHZ; and 1, 6-Hex = (KIGAIK) 3-NHZ.
Similarly, the second family will be comprised of 21-residue
molecules made up of a trimeric heptamer repeat in which there
are two lysines. One will be fixed at position 1 and the
other will be located at either position 2, 3, 4, 5, 6, or 7.
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The other amino acids in the heptamer repeat - L, A, G, L, A.-
will remain constant. The 21-residue family will consist of
the following: 1,2-Hept = (KKLAGLA)3-NH2; 1,3-Hept =
(KLKAGLA) 3-NHZ; 1, 4-Hept = (KLAKGLA) 3-NHZ; l, 5-Hept =
(KLAGKLA) 3-NHZ; 1, 6-Hept = (KLAGLKA) 3-NHZ; and 1, 7-Hept =
( KLAGLAK ) 3-NHZ .
All of these peptides are matched both in-terms of net
charge (+7) and mean hydrophobicity (-0.06). The peptides
will differ considerably, however, in their ability to form
amphipathic a-helix or (3-sheet structures. These two
families will form the basis for comparisons in relating
conformation and amphipathicity to activity. In addition, two
more peptides will be made - Long-Hex (a 21-residue version of
1,5-Hex) - (KIGAKI)3KIG-NHZ and Short-Hept (an 18-residue
version of 1,5-Hept) - (KLAGKLA)zKLAG-NH2. Long-Hex and
Short-Hept will have a net charge of +8 and +6, respectively,
while their mean hydrophobicities will be the same as the
other peptides. These peptides should provide some insight
into the effects of length and charge. The amphipathic
potential as both a-helices and (3-sheets of the 18-residue and
21-residue families are shown in Figs. 8 and 9, respectively.
101.2. Antimicrobial and Hemolytic Activity
Antimicrobial-susceptibility testing against Escherichia
coli (ATCC 25922), Staphylococcus aureus (ATCC 29213),
Pseudomonas aeruginosa (ATCC 27853), and Candida albicans
(ATCC 14053) is performed using a modification of the National
Committee for Clinical Laboratory Standards microdilution
broth assay. Mueller-Hinton broth (BBL) is used for diluting
the peptide stock solution and for diluting the bacterial
inoculum. The inoculum is prepared from midlogarithmic phase
cultures at an approximate concentration of 106 CFU/mL.
Microtiter plate wells receive aliquots of 0.1 mL each of the
inoculum and peptide dilution. The final concentration of the
peptide solution ranges from 0.25 to 256 ug/mL in 2-fold
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dilutions. The microtiter plates are incubated overnight at
37°C. Minimum inhibitory concentration (MIC) is defined as
the lowest concentration of peptide that completely inhibits
growth of the organism.
Hemolytic activity is determined by adding a defined
concentration of peptides to a 5% suspension of freshly drawn
human erythrocytes, which had been washed twice in phosphate
buffered saline. After incubation at 37°C for 30 minutes, the
suspension is centrifuged at 10,000 x g for 10 minutes and the
absorbance at 400 nm is measured. Complete hemolysis is
determined by adding 0.2% Triton X-100 in place of the
peptides.
101.3. Peptide Conformation
The conformation of the peptides will be assessed by FTIR
and CD spectroscopy. The data presented in the previous
section demonstrates the utility of these methods for
determining peptide conformation both in solution and when
bound to lipids.
101.3.1. FTIR Spectroscopy. Mixtures of lipid (4 pmoles)
and peptide (varied to achieve the desired peptide-to-lipid
ratio) are codissolved in 2:1 CHC13/CH30H. The solvent is
evaporated and the sample is placed under high vacuum for 3
hours. The mixture is hydrated with D20 buffer (20 mM PIPES, 1
mM EGTA, pD 7.0) and incubated at 50°C for 1 hour. The sample
is isolated by centrifugation and placed between CaF2 windows
separated by a 25 um Teflon spacer. Infrared spectra are
collected using a Mattson Polaris FTIR spectrometer with a
HgCdTe detector. A total of 256 interferograms are co-added
- and Fourier transformed with triangular apodization to
generate absorbance spectra with 2 aril resolution and data
points.encoded every 1 ciril, with a signal-to-noise ratio of
better than 500. Evaluation of secondary structure from the
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amide I' band between 1600 and 1700 crril is performed as
described in the preliminary results section. When necessary,
Fourier deconvolution and curve fitting are used to measure
the subcomponents of the amide I' band.ZZ All of the peptides
5 will be tested in lipid systems consisting of pure PG as well
as PC/PG, PE/PG, and E.coli lipid mixtures at peptide-to-lipid
_ ratios ranging from 1:20 to 1:2.
101.3.2. CD Spectroscopy. CD spectra are measured using a
10 Jasco J-715 spectropolarimeter. Spectra are recorded from
250-190 nm at a sensitivity of 100 mdeg, resolution of 0.1 nm,
response of 8 seconds, bandwidth of 1.0 nm, and scan speed of
20 nm/min, with a single accumulation. For the lowest peptide
concentrations, a response of 16 seconds, scan speed of 10
15 nm/min, and a total of 5 accumulations are used. The buffer
contains 10 mM potassium phosphate, 150 mM KC1, 1 mM EDTA, pH
7Ø The peptide concentration in buffer and TFE/buffer
mixtures is 20 uM. LUV are prepared from aqueous dispersions
of the appropriate lipid systems (containing POPG, POPC/POPG,
20 POPE/POPG, or E. coli lipids) at a concentration of ~lmg/mL in
phosphate buffer. Following 5 freeze/thaw cycles, the mixture
is extruded 10 times through a 0.1-pm-pore polycarbonate
membrane in an Avanti mini-extruder apparatus, resulting in
100-nm-diameter LUV. The lipid concentration is determined
25 by a phosphorus assay.39 The lipid concentration in the cuvet
is kept constant at 100 uM. The peptide concentration is
varied from 1 uM to 50 uM to achieve peptide-to-lipid ratios
ranging from 1:100 to 1:2. The helical content can be
estimated using the method of Luo and Baldwin.4°
101.4. Peptide-Induced Calcein Leakage from LW
The ability of the peptides to release calcein (MW 623)
from LUV of varying lipid composition will be compared. LUV
are prepared as above, except that the buffer consists of 50
mM HEPES, 100 mM NaCl, 0.3 mM EDTA, 80 mM calcein, pH 7.4.
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Calcein-loaded vesicles are separated from free calcein by
size-exclusion chromatography using a Sephadex G-50 column.
Calcein leakage is monitored by measuring the time-dependent
increase in fluorescence of calcein (excitation = 490 nm,
emission = 520 nmj. LUV containing 8 nmoles of lipid are
added to 1.5 mL of buffer in a stirred cuvet at 25°C. An
aliquot of peptide is added to achieve the desired peptide-to-
lipid ratio (1:256 to 1:2). Complete leakage is determined by
the addition of 20 uL of 10% Triton X-100.
101.5. Peptide-Induced Calcein heakage in E. coli
We have obtained a strain of E. coli (ML-35) from Dr.
Renato Gennaro (University of Trieste, Italy). This strain is
lactose permease-deficient and constitutive for cytoplasmic
(3-galactosidase. Using o-nitrophenyl-(3-D-galactopyranoside
(ONPG) as a substrate, it is possible to evaluate peptide-
induced permeabilization of the plasma membrane. In the
absence of peptide, ONPG is impermeable to the cell. The
increase in permeability is monitored colorimetrically by
incubated 106 CFU of bacteria in l0 mM sodium phosphate, 100 ml'~I
NaCl, 1.5 mM ONPG, pH 7.5, in a cuvet at 37°C. Following the
addition of peptide; the entry of ONPG into the cell is
monitored by the increase in absorbance at 405 nm due to the
production of o-nitrophenol.41 We will determine the
concentration of each peptide necessary-to induce leakage and
also examine the effect of changing the number of bacteria in
the assay. This should provide some clue as to the number of
peptides per bacteria that are necessary to induce leakage.
101.6. Peptide Binding to hUV and Bacteria
We will attempt to measure peptide binding to bacteria in
three differentways:
101.6.1. Physical separation of peptides from LW or
bacteria. We have used microfiltration tubes inpreparing
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samples for FTIR spectroscopy. These tubes, with a 0.4 um
nylon filter, can separate particulates (e. g., LUV) from the-
filtrate. By simply measuring the amount of peptide in the
filtrate, it may be possible to determine how much peptide is
bound. There are potential problems with this method,-
however._ Particularly at higher peptide levels, if the LUV
_ are breaking down, the filter may not be able to retain all of
the lipid. An equilibrium dialysis using a low molecular
weight cutoff membrane that allows individual peptides to pass
through but not supramolecular complexes may be an alternative
to microfiltration tubes. A similar approach will be
attempted for bacteria by incubating the cells with peptide
and separating the bacteria from.the solution by
centrifugation. The amount peptide remaining in the
supernatant will be identified using either HPLC, or a peptide-
specific antibody.
101.6.2. Tryptophan emission of fluorescent peptide
analogues. We have made the following fluorescent analogues
of (KIAGKIA) j-NH2, (KLAGLAK) 3-NHZ, and (KIGAKI) 3-NH2.
1 KIAGKIAKWAGKIAKIAGKIA- NHZ (SEQ ID N0:5)
2 KLAGLAKKWAGLAKKLAGLAK- NHz (SEQ ID N0:7)
3 KIGAKIKWGAKIKIGAKI - NHZ (SEQ ID N0:9)
In each case, the tryptophan replacement caused no appreciable
change in antimicrobial or hemolytic activity. The binding or
these peptides to lipid bilayers can be detected by a
significant blue shift in the tryptophan emission maximum. In
water., the emission maximum is near 356 nm. In the presence
of LUV, if the peptide is completely bound, the emission
maximum shifts to near 330 nm as the tryptophan associates
with the lipid bilayer. -
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The antimicrobial activity and secondary structure of
these analogs were- similar to those of the parent peptides.
The change in emission maximum to lower wavelength (blue
shift) under different conditions is shown in Fig. 7. In 500
TFE, the emission maximum decreased by 4-5 nm for each
peptide. Emission spectra in the presence of LUVs were
measured at lipid-to-peptide ration of 20. The shift observed
in the presenced of POPC LUVs (--<3 nm) was smaller than in 50%
TFE for all peptides. The largest blue shifts were observed
in the presence of POPG LUVs. For W-KIAGKIA, the shifts in
the presence of POPC/POPG were slightly larger than POPE/POPG
at ratios above 1:1. This enhancement is much greater for W-
KLAGLAK, where the blue shifts with POPC/POPG LUVs are about
twice as large as the corresponding POPE/POPG LUVs at neutral-
to-acidic ratios >_2. The presence of either POPC or POPE
decreased the magnitude of the blue shifts observed for W-
KIGAKI to a much greater degree as compared to the other
peptides; however, the shifts observed with POPE/POPG LUVs
were greater than those with POPC/POPG LUVs at all ratios
below 4. The blue shifts observed for W-KIAGKIA in the
presence of LUVs containing E. coli lipids or ternary mixtures
of POPC/POPG/DPG or POPE/POPG/DPG-were nearly equal. For W-.
KLAGLAK, however, the shift observed in the presence of
POPC/POPG/DPG was greater than with either E. coli lipids or
POPE/POPG/DPG. Only W-KIGAKI showed larger blue shifts in the
presence of E. coli lipids or POPE/POPG/DPG as compared to
POPC/POPG/DPG. Similar binding experiments may be performed
with the heptamer and hexamer peptide families.
101.6.3. Varying the number of bacteria in MIC assays. As
described above, the number of bacteria used in MIC assays is
105. We can gauge the number of peptide molecules necessary to
cause killing by altering. the number of bacteria in the assay
and observing the effect on the MIC values. For instance, if
the MIC value increases in proportion to the number of
bacteria in the assay, then we can infer that not enough
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peptide molecules remain available in solution to bind to and
kill the cells, given the increased target load. On the other
hand, if no difference is observed in the MIC value as the
number of bacteria in the assay increases, then the
concentration of peptides in solution must remain sufficiently
high to accommodate the increased number of cells. If this is
the case, then the binding affinity at the MIC level rather
than the absolute amount of peptide in the assay is the
limiting factor.
The results of these binding studies should provide some
insight into the actual number of peptide molecules required
to induce leakage in LUV and bacterial cells.
Example 102
We have synthesized the hexamer and heptamer peptide
families described in hypothetical example 101. These
peptides were made with a single tryptophan residue to permit
fluorescence experiments. The sequences are:
Hexamer Family
1,2-Hex
K K I G A I K K W G A J K K I G A I-NHZ (SEQ ID N0:25)
1 5 10 15
1,3-Hex
K I K G A I K W K G A I K I K G A I-NHZ (SEQ ID N0:26)
1,4-Hex
K I G K A I K W G K A I K I G K A I-NHz (SEQ ID N0:27)
1,5-Hex
K I G A K I K W G A K I K I G A K I-NHz (SEQ ID N0:29)
1,6-Hex
K I G A I K K W G A I K K I G A I K-NHZ ( SEQ I D NO : 30 )
Heptamer Family
1,2-Hept
K K L A G L A K K W_ A G L A K K L A G L A-NHZ
1 5 10 15 20
(SEQ ID N0:31)
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1,3-Hept
K L K A G L A K _W K A G L A K L K A G L A-NHZ ( SEQ I D
N0:32) -
1,4-Hept _
5 K L A K G L A K W A- K G L A K L A K G L A-NHz (SEQ ID
N0:33)
1,5-Hept
K L A G K L A K W A G K L A K L A G K L A-NHZ ( SEQ I D
N0:34)
10 1, 6-Hept
K L A G L K A K _W A G L K A K L A G L K A-NHZ ( SEQ I D
N0:35)
1,7-Hept
K L A G L A K K W A G L A K K L A G L A K-NHz (SEQ ID
15 N0:36)
The physical properties and antimicrobial activities of
these peptides are shown in Table 102.
None of the peptides in the hexamer family has the
capacity to form an amphipathic a-helix. Only those peptides
20 that can form a highly amphipathic (3-sheet (i.e., 1,3-Hex and
1,5-Hex) have appreciable antimicrobial activity. For the
heptamer family, the peptides that can adopt an amphipathic a-
helix (1,2-Hept, 1,4-Hept, 1,5-Hept, and 1,7-Hept) all have
relatively low MIC values. 1,6-Hept, which has no amphipathic
25 potential as either an a-helix or ~i-sheet, is devoid of
antimicrobial activity, while 1,3-Hept, with some amphipathic
potential as both an a-helix and (3-sheet, is slightly active.
Example 103
N-Terminal Modified Peptides
3o We have made five derivatives of our (3-sheet peptides
with an octanoyl [CH3-(CHz)6-(C=O)-] group added to the amino
terminus. Four are derivatives of KIGAKI (i-3 repeats) and
one is a derivative of a KL repeat. All of these peptides
contain a single tryptophan for fluorescence studies, but this
35 could be replaced by I or L in any final product. The
sequences are:-
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KIGAKI
K I G A K I K I G A K I K I G A K I-NHZ ( SEQ I D NO : 8 )
Oct-KIGAKI-18A
Oct- K I _G A K I K W G A K I K I G A K I-NHZ ( SEQ ID NO: 9 )
Oct-KIGAKI-18B
Oct- K I G A K I K I G A K I K W G A K I-NHZ ( SEQ I D NO : 37 )
Oct-KIGAKI-12
Oct- K I G A K I K W G A K I-NHZ (SEQ ID N0:38)
Oct-KIGAKI-6
Oct- K W G A K I-NHz (SEQ ID N0:39)
Oct-Beta-11
Oct- K L K W K L K L K L K-NHZ (SEQ ID N0:40)
KL-14
K L K L K L K W K L K L K L-NHZ ( SEQ I D NO : 2 6 )
KL-18
K L K L K L K W K L K L K L K L K L-NHZ ( SEQ I D NO : 27 )
The antimicrobial activity of the octanoylated peptides in
comparison to related peptides is set forth in Table 103.
Adding an octanoyl group to the 18-residue KIGAKI did not
appear to increase potency to a great extent. We have not yet
measured the hemolytic activity of these octanoylated
peptides, nor have we yet tested a 12-residue octanoylated
KIGAKI. A 6-residue octanoylated KIGAKI had no activity.
Oct-Beta-11 is a shortened octanoylated version of KL-14 and
KL-18. This peptide appears to have reasonable antimicrobial
activity, but we have not measured its hemolytic activity and
have not tested whether it possesses the desired selectivity
between bacterial membrane lipids and mammalian membrane
lipids.
Example 104
Application of Combinatorial Chemistry to (3-sheet Framework
We have shown that our original peptide (KIGAKI)3-NH2
(1,5-Hex) and 1,3-hex have antimicrobial-act wity. In an
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effort to increase potency and maintain selectivity, we could
employ a combinatorial approach to scarch for derivatives.
This is a common method that is used in many laboratories
(see, for example, Houghten, R.A.; Pinilla, C.; Blondelle,
S.E.; Appel, J.R.; Dooley, C.T., and Cuervo, J.H. Generation
and use of synthetic peptide combinatorial libraries for basic
research and drug discovery. Nature, 1991, 354:84-86.
Using this method, we could retain the amphipathic (3=
sheet architecture (controlling length and charge) while
searching for favorable amino acid replacements. For example,
we could try the following:
K X G X K X K X G X K X K X G X K X-NHz
1 5 10 15
We could screen a set of 15 hydrophobic amino acids
(these could be natural and/or unnatural) at the positions
marked X in the sequence. Starting at position 2, we would
make 15 separate libraries, each with a unique amino acid at
position 2, and an equimolar mixture of the.l5 amino acids at
the other X positions. These 15 libraries (each containing
millions of different peptides) would then be screened for
antimicrobial and hemolytic activity. The library or
libraries giving the the best results identify candidate amino
acids for position 2. This is then repeated by making 15
libraries each for positions 4, 6, 8, 10, 12, 14, 16 and 18.
After the best amino acids were identified at each position (a
process called positional scanning), then individual peptides
would be made to test whether they are superior to our
original compounds.
It is noted that this specification may, from time to
time,, refer to "KIGAKI", "KIAGKIA", etc. , when the respective
trimers (KIGAKI) 3, (KIAGKIA) 3, etc. , are intended. - The
nomenclature "KIGAKIx3", "KIAGKIAx3", etc. is also used.
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CA 02399668 2002-08-08
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A11 references cited herein, including journal articles
or abstracts, abandoned or pending (whether-or not published)
U.S. or foreign patent applications, issued U.S. or foreign
patents, or any other references, are entirely incorporated by
5 reference herein, including all data, tables, figures, and
text presented in the cited references. Additionally, the
entire contents of the references cited within the-references
cited herein are also entirely incorporated by reference.
Reference to known method steps, conventional methods
10 steps, known methods or conventional methods is not in any way
an admission that any aspect, description or embodiment of the
present invention is disclosed, taught or suggested in the
relevant art.
The foregoing description of the specific embodiments
IS will so fully reveal the general nature of the invention that
others can, by applying knowledge within the skill of the art
(including the contents of the references cited herein),
readily modify and/or adapt for various applications such
specific embodiments, without undue experimentation, without
20 departing from the general concept of the present invention.
Therefore, such adaptations and modifications are intended to
be within the meaning and range of equivalents of the
disclosed embodiments, based on the teaching and guidance
presented herein. It is to be understood that the phraseology
25 or terminology herein is for the purpose of description and
not of limitation, such that the terminology or phraseology of
the present specification is to be interpreted by the skilled
artisan in light of the teachings and guidance presented
herein, in combination with the knowledge of one of ordinary
30 skill in the art.
Any description of a class or range as being useful or
preferred in the-practice of the invention shall be deemed a
description of any subclass or subrange contained therein, as
well as a separate description of each individual member or
35 value in said class or range.
CA 02399668 2002-08-08
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96
Where a set of preferred embodiments are recited for
particular elements of the invention, any combination of a
first set of preferred embodiments for a first element, with a
second set of preferred elements for a second element, shall
also be considered a preferred embodiment, and so forth for
higher combinations including additional elements.
CA 02399668 2002-08-08
WO 01/60162 PCT/USO1/04822
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Table 3. Effect of Peptides on Bilayer-to-Hexagonal Phase
Transition (TH) of DiPoPE
Peptide Linear Regressions
KIAGKIAx3 13265
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