U.S. patent application number 13/898181 was filed with the patent office on 2014-01-30 for selective poly-n-substituted glycine antibiotics.
The applicant listed for this patent is Annelise E. Barron, Nathaniel P. Chongsiriwatana, Ann M. Czyzewski, Michelle T. Dohm, Tyler M. Miller, James A. Patch, Ronald N. Zuckermann. Invention is credited to Annelise E. Barron, Nathaniel P. Chongsiriwatana, Ann M. Czyzewski, Michelle T. Dohm, Tyler M. Miller, James A. Patch, Ronald N. Zuckermann.
Application Number | 20140031523 13/898181 |
Document ID | / |
Family ID | 40986091 |
Filed Date | 2014-01-30 |
United States Patent
Application |
20140031523 |
Kind Code |
A1 |
Barron; Annelise E. ; et
al. |
January 30, 2014 |
Selective Poly-N-Substituted Glycine Antibiotics
Abstract
Antimicrobial peptoid compounds and related compositions as can
be used against bacteria effectively and selectively.
Inventors: |
Barron; Annelise E.; (Palo
Alto, CA) ; Zuckermann; Ronald N.; (El Cerrito,
CA) ; Czyzewski; Ann M.; (Grayslake, IL) ;
Dohm; Michelle T.; (Palos Park, IL) ; Miller; Tyler
M.; (Englewood, CO) ; Patch; James A.; (San
Francisco, CA) ; Chongsiriwatana; Nathaniel P.;
(Chicago, IL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Barron; Annelise E.
Zuckermann; Ronald N.
Czyzewski; Ann M.
Dohm; Michelle T.
Miller; Tyler M.
Patch; James A.
Chongsiriwatana; Nathaniel P. |
Palo Alto
El Cerrito
Grayslake
Palos Park
Englewood
San Francisco
Chicago |
CA
CA
IL
IL
CO
CA
IL |
US
US
US
US
US
US
US |
|
|
Family ID: |
40986091 |
Appl. No.: |
13/898181 |
Filed: |
May 20, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
12378034 |
Feb 9, 2009 |
8445632 |
|
|
13898181 |
|
|
|
|
61065189 |
Feb 8, 2008 |
|
|
|
Current U.S.
Class: |
530/328 |
Current CPC
Class: |
C07K 7/08 20130101; C07K
5/0815 20130101; Y02A 50/30 20180101; Y02A 50/473 20180101; A61P
31/04 20180101; B01D 71/06 20130101; A61K 38/00 20130101; C07K 7/06
20130101 |
Class at
Publication: |
530/328 |
International
Class: |
C07K 7/06 20060101
C07K007/06 |
Goverment Interests
[0002] This invention was made with government support under Grant
Nos. 1R01 HL67984-01 and AI007266 awarded by the National
Institutes of Health and Contract No. DE-AC02-05CH11231 awarded by
the U.S. Department of Energy. The government has certain rights in
the invention.
Claims
1. A poly-N-substituted glycine antibiotic compound of a formula
##STR00006## wherein A is selected from H and a terminal N-alkyl
substituted glycine residue, where said alkyl substituent is
selected from about C.sub.4 to about C.sub.20 linear, branched and
cyclic alkyl moieties; n is an integer selected from 1-3; B is
selected from NH.sub.2, one and two N-substituted glycine residues,
said N-substituents independently selected from natural
.alpha.-amino acid side chain moieties, isomers and carbon homologs
thereof; X, Y and Z are independently selected from N-substituted
glycine residues, said N-substituents independently selected from
natural .alpha.-amino acid side chain moieties, isomers and carbon
homologs thereof, and proline residues, at least one of said X, Y
and Z residues is N.sub.Lys and at least one said N-substituent is
chiral, said compound of a formula ##STR00007##
Description
[0001] This application is a continuation of and claims priority
benefit from application Ser. No. 12/378,034 filed Feb. 9, 2009 and
issued as U.S. Pat. No. 8,445,632 on May 21, 2013, which claimed
priority benefit from application Ser. No. 61/065,189 filed Feb. 8,
2008--each of which is incorporated herein by reference in its
entirety.
BACKGROUND OF THE INVENTION
[0003] Natural antimicrobial peptides (AMPs) defend a wide array of
organisms against bacterial invaders and show potential as
supplements for or replacements of conventional antibiotics, since
few bacteria have evolved resistance to them. Many AMPs kill
bacteria by permeabilization of the cytoplasmic membrane, causing
depolarization, leakage, and death, whereas others target
additional anionic bacterial constituents (e.g. DNA, RNA, or cell
wall components). Bacterial resistance to AMPs is rare probably
because they have evolved along with the resistance mechanisms
designed to evade them; furthermore, the targets of many AMPs (e.g.
bacterial plasma membranes, anionic intracellular macromolecules)
are sufficiently general that changes to their sequence can be made
that subvert resistance, yet have negligible impact on overall
functionality.
[0004] Although AMPs have been actively studied for decades, they
have yet to see widespread clinical use. This is in part due to the
vulnerability of many peptide therapeutics to rapid in vivo
degradation, which dramatically reduces their bioavailability.
Non-natural mimics of AMPs can circumvent the proteolytic
susceptibility of peptides while retaining their beneficial
features. The short (<40 amino acids), simple structure of AMPs
in the cationic, linear, .alpha.-helical class, which includes the
well-known magainins, are especially amenable to mimicry.
.beta.-peptide mimics of these AMPs have been successfully created
with antibacterial and non-hemolytic in vitro activity.
Poly-N-substituted glycines (peptoids) comprise another class of
peptidomimetics, and are isomers of peptides in that peptoid side
chains are attached to the backbone amide nitrogen rather than to
the .alpha.-carbon. More than any of the other peptidomimetic
systems under study, including .beta.-peptides, .beta.-peptoids,
oligoureas, and oligo(phenylene ethynylene)s, peptoids are
particularly well-suited for AMP mimicry because they are easily
synthesized on solid phase (using conventional peptide synthesis
equipment) with access to diverse sequences at relatively low cost.
By way of an elegant submonomer synthetic method, any chemical
functionality available as a primary amine can be incorporated,
whether it be an analog of a proteinogenic amino acid or a totally
non-natural moiety; thus, peptoids are highly and finely tunable.
Furthermore, they are protease-resistant, and can be designed to
form amphipathic helices that resist thermal and chaotropic
denaturation.
[0005] The poly-N-substituted glycine structure of peptoids
precludes both backbone chirality and intrachain hydrogen bonding;
nevertheless, peptoids can be driven to form stable helical
secondary structures via periodic incorporation of bulky,
.alpha.-chiral side chains. X-ray, NMR, and CD studies of peptoid
oligomers have shown that incorporation of homochiral side chains
can give rise to polyproline type-1-like helices with a periodicity
of .about.3 monomers per turn and a helical pitch of 6.0-6.7 .ANG..
The three-fold periodicity of the peptoid helix facilitates the
design of facially amphipathic structures similar to those formed
by many AMPs; for example, the trimer repeat (X--Y--Z).sub.n forms
a peptoid helix with three faces, composed of X, Y, and Z residues,
respectively.
[0006] Amphipathic secondary structures in which residues are
segregated into hydrophobic and cationic regions are the hallmark
of most AMPs. Regardless of their final target of killing, AMPs
must interact with the bacterial cytoplasmic membrane, and
amphipathicity is integral to such interactions. The cationic
region facilitates electrostatically driven adsorption to anionic
bacterial membranes and imparts some measure of selectivity, since
mammalian cell membranes are largely zwitterionic. The hydrophobic
region provides an additional driving force for incorporation of
the AMP into the lipid bilayer. The precise nature of AMP-membrane
interactions remains controversial and actively debated; a variety
of mechanisms have been proposed, including the carpet,
barrel-stave pore, toroidal pore, and aggregate models.
SUMMARY OF THE INVENTION
[0007] In light of the foregoing, it is an object of the present
invention to provide new poly-N-substituted glycine compounds and
methods for and/or therapies relating to their use as antibiotics,
thereby improving upon the prior art and/or overcoming various
deficiencies or shortcomings thereof. It will be understood by
those skilled in the art that one or more aspects of this invention
can meet certain objectives, while one or more other aspects can
meet certain other objectives. Each objective may not apply
equally, in all its respects, to every aspect of this invention. As
such, the following objects can be viewed in the alternative with
respect to any one aspect of this invention.
[0008] It can be an object of the present invention to provide such
compounds having minimum inhibitory concentrations in the low
micromolar range against both Gram-positive and Gram-negative
bacteria, with lower mammalian cytotoxicities and negligible
hemolysis, at such concentrations, as compared to compounds of the
prior art.
[0009] It can be another object of the present invention to provide
such compounds, variable by residue sequence and/or N-substituent,
so as to affect hydrophobicity and/or amphipathicity and/or to
enhance selectivity.
[0010] It can be another object of the present invention alone or
in conjunction with one or more of the preceding objectives, to
provide a new class of N-alkylated peptoids, providing such
potencies and selectivities at monomer numbers and peptoid lengths
shorter than previously available.
[0011] Other objects, features, benefits and advantages of the
present invention will be apparent from this summary and its
descriptions of certain embodiments, and will be readily apparent
to those skilled in the art having knowledge of various
peptidomimetic compounds and their syntheses. Such objects,
features, benefits and advantages will be apparent from the above
as taken into conjunction with the accompanying examples, data,
figures and all reasonable inferences to be drawn therefrom, alone
or with consideration of the references incorporated herein.
[0012] In part, the present invention can be directed to a
poly-N-substituted glycine antibiotic compound of a formula
##STR00001##
In such a compound, A can be selected from H and a terminal N-alkyl
substituted glycine residue, where such an alkyl substituent can be
selected from about C.sub.4 to about C.sub.20 linear, branched and
cyclic alkyl moieties; n can be an integer selected from 1-3; B can
be selected from NH.sub.2, and one and two N-substituted glycine
residues, such N-substituents as can be independently selected from
.alpha.-amino acid side chain moieties and structural/functional
analogs thereof; and X, Y and Z can also be independently selected
from N-substituted glycine residues, such N-substituents as can be
independently selected from .alpha.-amino acid side chain moieties
and structural/functional analogs thereof and proline residues. As
described elsewhere herein, such X--Y--Z periodicity can provide
such a compound a certain amphipathicity. As would be understood by
those skilled in the art made aware of this invention, such
structural and/or functional analogy can be considered in the
context of any such .alpha.-amino acid side chain, N-substituent
and/or a sequence of such N-substituted glycine residues, such
structure and/or function including but not limited to charge,
chirality, hydrophobicity, amphipathicity, helical structure and
facial organization. Such analogs include, without limitation,
carbon homologs of such side chain--such homologs as would be
understood in the art, including but not limited to plus or minus 1
or 2 or more methylene and/or methyl groups.
[0013] Regardless, in certain embodiments A can be H, and B can be
selected from one or two N-substituted glycine residues, such a
selection as can reduce the hydrophobicity of such a compound, as
compared to compounds of 3-fold periodicity. In certain such
embodiments, X can be an N.sub.Lys residue; n can be 2-3; and B can
be two N-substituted glycine residues. Without limitation, such a
compound can be of a formula
##STR00002##
In various other embodiments, regardless of identity of A, X and B,
at least one of Y and Z can be a proline residue. In certain such
embodiments, X, Y and Z can be proline residues.
[0014] In certain other embodiments, A can be a terminal N-alkyl
substituted glycine residue, with such an alkyl substituent as can
be selected from about C.sub.6 to about C.sub.18 linear alkyl
moieties. Regardless, B can be NH.sub.2, and n can be selected from
1 and 2. In certain such embodiments, A can be a terminal N-alkyl
substituted glycine residue, with an alkyl substituent selected
from about C.sub.6 to about C.sub.18 linear alkyl moieties.
Regardless, B can be an N.sub.Lys residue, and n can be 1.
[0015] In part, this invention can also be directed to a
poly-N-substituted glycine antibiotic compound of a formula
##STR00003##
wherein n can be selected from 2 and 3; and Y, Z, Y' and Z' can be
independently selected from N-substituted glycine residues, where
such substituents can be independently selected from .alpha.-amino
acid side chain moieties and carbon homologs thereof. Such Y' and
Z' residues can be selected to provide such compound reduced
hydrophobicity as compared to a compound of 3-fold periodicity. In
certain such embodiments, at least one of X and Y can be a proline
residue. Regardless, n can be selected from 2 and 3, and Y' can be
an N.sub.Lys residue. In certain such embodiments, one or both X
and Y can be proline residues. Without limitation, such a compound
with reduced hydrophobicity can be of a formula
##STR00004##
[0016] In part, this invention can also be directed to a
poly-N-alkyl substituted glycine antibiotic compound of a
formula
##STR00005##
wherein B can be selected from NH.sub.2 and X'; X, Y, Z and X' can
be independently selected from N-substituted glycine residues,
where such substituents can be independently selected from
.alpha.-amino acid side chain moieties and carbon homologs thereof;
n can be an integer selected from 1 and 2; and R can be an N-alkyl
substituent of such a glycine residue, as can be selected from
about C.sub.4 to about C.sub.20 linear, branched and cyclic alkyl
moieties. In certain embodiments, n can be 2, and B can be
NH.sub.2. In certain other embodiments, n can be 1, and B can be
X'. Accordingly, one or both of X and X' can be N.sub.Lys residues.
Regardless, an alkyl substituent can be selected from about C.sub.6
to about C.sub.18 linear, branched and cyclic alkyl moieties, and X
and X' can be N.sub.Lys residues. Without limitation, such a
compound can be of a formula
H--N.sub.tridec--N.sub.Lys--N.sub.spe--N.sub.spe--N.sub.Lys--NH.sub.2.
[0017] In part, the present invention can be directed to a
poly-N-substituted glycine antibiotic compound comprising an
N-terminus selected from H and an N-alkyl substituted glycine
residue, where such an alkyl substituent can be selected from about
C.sub.4 to about C.sub.20 linear, branched and cyclic alkyl
moieties; a C-terminus selected from NH.sub.2, one and two
N-substituted glycine residues, such N-substituents as can be
independently selected from .alpha.-amino acid side chain moieties
and structural/functional analogs thereof; and 2 to about 15
monomeric residues between the N- and C-termini, each such residue
as can be independently selected from proline residues and
N-substituted glycine residues, said N-substituents independently
selected from .alpha.-amino acid side chain moieties and
structural/functional analogs thereof. As illustrated herein and as
distinguished over the prior art, such monomers can be selected to
provide such a compound a non-periodic sequence of monomers. As
would be understood by those skilled in the art made aware of this
invention, such structural and/or functional analogy can be
considered in the context of any such .alpha.-amino acid side
chain, N-substituent and/or a sequence of such N-substituted
glycine residues, such structure and/or function including but not
limited to charge, chirality, hydrophobicity, amphipathicity,
helical structure and facial organization. Such analogs include,
without limitation, carbon homologs of such side chain--such
homologs as would be understood by those skilled in the art,
including but not limited to plus or minus 1 or 2 or more methylene
and/or methyl groups.
[0018] In certain embodiments, the N-terminus of such a compound
can be H; and the C-terminus can be selected from said one and two
N-substituted glycine residues. Regardless, such a compound can
comprise 2 to about 5 (X--Y--Z) non-periodic trimers. In certain
such embodiments, at least one of X, Y and Z in each of the trimers
can be selected to interrupt 3-fold periodicity. Without
limitation, at least one X in at least one said trimer can be an
N.sub.Lys residue. In certain such embodiments, at least one of Y
and Z in at least one such trimer can be a proline residue. In
other embodiments, the monomeric residues can comprise at least two
non-consecutive of the same or repeat trimers, with at least one
such residue therebetween to interrupt periodicity. In certain such
embodiments, at least one X in at least one such trimer can be an
N.sub.Lys residue, and at least one of Y and Z in at least one said
trimer can be a proline residue.
[0019] In various other non-limiting embodiments, the N-terminus of
such a compound can be an N-alkyl substituted glycine residue, with
an alkyl substituent selected from about C.sub.6 to about C.sub.18
linear alkyl moieties. Regardless, such a compound can comprise 2
to about 5 (X--Y--Z) non-periodic trimers. In certain such
embodiments, at least one of X, Y and Z in each of the trimers can
be selected to interrupt 3-fold periodicity. In certain other
embodiments, the monomeric residues can comprise at least two
non-consecutive of the same or repeat trimers, with at least one
residue therebetween to interrupt periodicity. In certain such
embodiments, at least one X in at least one said trimer can be an
N.sub.Lys residue, and at least one of Y and Z in at least one said
trimer can be a proline residue.
[0020] In part, the present invention can also be directed to one
or more antimicrobial peptoid compositions comprising one or more
of the poly-N-substituted glycine compounds of this invention. Such
compounds as can optionally comprise one or more antimicrobial
peptides and/or peptidomimetic compounds now or hereafter known in
the art. Accordingly, this invention can be directed to a range of
pharmaceutical compositions comprising one or more of the present
polypeptoid/ampetoid compounds, optionally with an antimicrobial
component of the prior art, and a pharmaceutically-acceptable
carrier. Such compositions can be prepared and/or formulated as
would be understood by those skilled in the art made aware of this
invention. Regardless, as illustrated below, any of the present
polypeptoid/ampetoid compounds and/or related compositions can be
used alone or in combination, whether administered together or
sequentially, in conjunction with one or more bacteria or microbial
treatment methodologies. Without limitation, such a method can
comprise providing one or more such poly-N-substituted glycine
compounds and/or related compositions; and administering such
compound(s)/composition(s) and/or contacting bacteria therewith. As
would be understood by those skilled in the art, such
administration can be in vitro or in vivo, using techniques of the
sort described herein or straight-forward modifications thereof,
such modifications as would also be known to those skilled in the
art and made aware of this invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] FIG. 1. Peptoid monomer side chain structures, with full and
shorthand names.
[0022] FIG. 2. Cytotoxicity data for selected peptoids and
comparator peptides against A549 lung epithelial cells.
[0023] FIGS. 3A-B. CD spectra of (A) variants of 1 and (B) variants
of 2 in 10 mM Tris buffer, pH 7.4.
[0024] FIGS. 4A-B. CD spectra of (A) variants of 1 and (B) variants
of 2 in 5 mM POPC/cholesterol (1:1) SUVs suspended in 10 mM Tris
buffer, pH 7.4.
[0025] FIGS. 5A-B. CD spectra of (A) variants of 1 and (B) variants
of 2 in 5 mM POPE/POPG (7:3) SUVs suspended in 10 mM aqueous Tris
buffer, pH 7.4.
[0026] FIG. 6A-C. X-ray reflectivity data (A) and corresponding fit
for DPPG monolayer before (circles; cartoon inset, top (B)) and
after (squares; cartoon inset, bottom (C)) peptoid 1 was injected
into the subphase beneath the monolayer.
[0027] FIG. 7. In vivo efficacy of ampetoids in a mouse peritoneal
injection model. Five or six mice were included in each study group
for each experiment; the bacterial colony counts from each of two
plates per mouse are shown in the dot plot. The horizontal line in
each group represents the geometric mean of the population.
[0028] FIG. 8. A schematic illustration of the sub-monomer
synthetic protocol for polypeptoids. Steps 2 and 3 are simply
repeated for the addition of each monomer unit. Once the full
polypeptoid has been synthesized, it is cleaved off the resin with
trifluoroacetic acid and purified by reversed-phase HPLC.
[0029] FIG. 9: Schematics of ameptoid variants and side chain
structures. By convention, the N-terminus is on the left and the
C-terminus is on the right. Note that the schematics shown here are
for visualization purposes only and not intended to imply the
actual folding behavior of each molecule. Points on the triangular
helicies that have no marker are Nspe monomers.
[0030] FIGS. 10A-C: CD spectra of ampetoid register and sequence
variants. Ampetoid concentrations were 60 .mu.M. (A) CD in 10 mM
Tris buffer (pH 7.4). (B) CD spectra in 10 mM Tris buffer with 5 mM
erythrocyte-mimetic POPC:cholesterol (2:1) SUVs. (C) CD in 10 mM
Tris buffer with 5 mM bacteria-mimetic POPE:PEPG (3:7) SUVs.
[0031] FIGS. 11A-B: CD spectra of net charge variants in (A) 10 mM
Tris buffer and (B) same buffer with 5 mM POPC/cholesterol 2:1
SUVs. Ampetoid concentration is 60 .mu.M.
[0032] FIGS. 12A-C: CD spectra of length variants in (A) 10 mM Tris
buffer and (B) same buffer with 5 mM POPC/cholesterol 2:1 SUVs and
(C) same buffer with 5 mM POPE/POPG 3:7 SUVs. Ampetoid
concentration is 60 .mu.M.
[0033] FIGS. 13A-C: CD spectra of proline variants in (A) 10 mM
Tris buffer and (B) same buffer with 5 mM POPC/cholesterol 2:1 SUVs
and (C) same buffer with 5 mM POPE/POPG 3:7 SUVs. Ampetoid
concentration is 60 .mu.M.
[0034] FIGS. 14A-C: CD spectra of ampetoids containing achiral
monomers in (A) 10 mM Tris buffer and (B) same buffer with 5 mM
POPC/cholesterol 2:1 SUVs and (C) same buffer with 5 mM POPE/POPG
3:7 SUVs. Ampetoid concentration was 60 .mu.M.
[0035] FIGS. 15A-C: CD spectra of ampetoids containing opposite
chirality monomers in (A) 10 mM Tris buffer and (B) same buffer
with 5 mM POPC/cholesterol 2:1 SUVs and (C) same buffer with 5 mM
POPE/POPG 3:7 SUVs. Ampetoid concentration was 60 .mu.M.
[0036] FIGS. 16A-C: CD spectra of ampetoids containing aliphatic
monomers in (A) 10 mM Tris buffer and (B) same buffer with 5 mM
POPC/cholesterol 2:1 SUVs and (C) same buffer with 5 mM POPE/POPG
3:7 SUVs. Ampetoid concentration was 60 .mu.M.
[0037] FIGS. 17A-B: Comparison of antimicrobial/hemolytic activity
profiles for selected ampetoids. Those with the most favorable
profiles appear at the lower right portion of each plot.
DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS
[0038] As illustrated, the present invention provides a class of
helical, cationic, amphipathic, sequence- and/or length-specific
poly-N-substituted glycines (peptoids) with potent and very
selective antibacterial activity. These molecules demonstrate
broad-spectrum antimicrobial activity against a range of pathogens,
and are able to effectively treat a bacterial infection in vivo. It
has been demonstrated that these peptoids are both structurally and
functionally analogous to antimicrobial peptides (AMPs), the
natural molecules. Moreover, by modulating the sequence and side
chain functionality, the activity and selectivity of antimicrobial
peptoids can be tuned. Some compounds have minimum inhibitory
concentrations (MICs) in the low micromolar range against
Gram-positive and Gram-negative bacteria, with low mammalian
cytotoxicity and negligible (<1%) hemolysis at their MICs. These
activities are substantially improved over previous antibacterial
peptoids earlier reported, the best of which were much more
hemolytic toward human red blood cells. This invention also
provides a new class of alkylated antibacterial peptoids, which
retain the antimicrobial potency and selectivity in analogs as
short as 5 monomers in length.
[0039] With reference to examples 1-9 and FIGS. 1-8, below,
peptoids were synthesized, as widely-known in the art, using the
submonomer synthetic method described by Zuckermann et al.,
purified using reverse-phase high performance liquid chromatography
(RP-HPLC), and characterized with electrospray ionization mass
spectrometry (ESI-MS) and circular dichroism (CD) spectroscopy.
(See, Zuckermann, R. N., Kerr, J. M., Kent, S. B. H., & Moos,
W. H. (1992) J. Am. Chem. Soc., 114, 10646-10647, the entirety of
which is incorporated herein by reference.) CD spectrum of
antimicrobial peptoids confirms that they adopt helical structures
in both aqueous buffer and lipid vesicles, such that they possess a
facially amphipathic organization of cationic and hydrophobic
residues. Antibacterial activity was determined according to
Clinical Laboratory Standards Institute (CLSI) protocols for broth
microdilution, and hemolytic activity determined using similar
microdilution methods. The effect of peptoids on cellular metabolic
activity was determined using the colorimetric tetrazolium
salt-based MTS
[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl-
)-2H-tetrazolium]assay also using serial microdilution. These data
demonstrate broad-spectrum activity against a variety of clinically
relevant pathogens, yet the most selective antimicrobial peptoids
do not harm human red blood cells or mammalian fibroblast cells at
concentrations many times their antibacterial concentrations.
Lastly, the ability of two peptoids to treat an infection in vivo
using a murine peritoneal injection model was evaluated. These
results show that peptoids can significantly reduce the bacterial
counts in the mouse peritoneal cavity over a 24 hour exposure
period without mouse fatality.
[0040] Structure-activity studies of antimicrobial peptoids suggest
close analogy between their mechanisms of bacterial killing and
those of AMPs. Numerous studies of AMPs have found that, in
general, increases in hydrophobicity and amphipathicity and
decreases in net charge can bias a peptide toward a less selective
mechanism. The converse is true, in that adding cationic charge,
and decreasing hydrophobicity or amphipathicity can lead to a more
selective peptide. The structure-activity studies conducted show
that peptoids respond similarly to changes in physicochemical
properties. That is, selective peptoids become less selective when
their sequences are altered to make them more hydrophobic, more
amphipathic, or less cationic, and vice versa. Antimicrobial
peptoids are thus functional analogs of AMPs.
[0041] Specular X-ray reflectivity was used to examine
molecular-level interactions of antimicrobial peptoids with lipids.
Just as selective AMPs spend the majority of their time oriented
parallel to the plane of the membrane surface, antimicrobial
peptoids are oriented in anionic DPPG monolayers with 56.degree.
between the surface normal and the long axis of the
peptide--clearly not perpendicular to the membrane, which would be
expected for a transmembrane pore-forming peptide--with clear
interactions between the peptoid and the lipid headgroups. The
similar lipid-bound orientations of AMPs and antimicrobial peptoids
further suggests mechanistic analogy between the two classes of
molecules.
[0042] The present invention, therefore, provides a novel class of
antibiotics which offers a combination of several desirable
properties, including but not limited to: 1. Efficacy in reducing
bacterial counts in a mammalian system over a 24-hour exposure
using an intraperitoneal injection mouse model. 2. low-micromolar
antibacterial activity against a broad spectrum of clinically
relevant pathogens; 3. selectivity that is tunable by molecular
sequence characteristics, whereby mammalian cells are not harmed at
the compounds' MICs; 4. functional analogy to natural antimicrobial
peptides, which implies that, like AMPs, antimicrobial peptoids are
not readily susceptible to the development of bacterial
resistance.
[0043] The role of hydrophobicity is articulated by studies of
antimicrobial lipopeptides, such as polymyxin B and trichogin.
These AMPs are composed of a peptide chain with a fatty acid tail
at the N-terminus. For example, the deacylated version of polymyxin
B is much less bactericidal than the natural lipopeptide. Also,
trichogin analogues with varying lengths of lipids showed greater
activity with longer tails, and analogues with tails shorter than
four carbon atoms were found to be inactive. Attachment of fatty
tails to AMPs that are not normally acylated has also been
attempted, and in some cases, alkylation of inactive cationic
peptides was sufficient to endow the resulting lipopeptides with
antibacterial or antifungal activity. Furthermore, studies by Shai
et al. have found that when varying the length of an alkyl tail, a
threshold of hydrophobicity is reached at which the peptide is no
longer selective. That is, at a point, increasing tail length only
increases hemolytic activity without improving antibacterial
activity.
[0044] As illustrated herein, the in vitro activities of simple
peptoid mimics of AMPs are strikingly similar to those of AMPs
themselves. (See, e.g., Table 1.) Certain antimicrobial peptoids
("ampetoids") exhibit broad-spectrum antimicrobial activity, low
hemolysis, minimal effects on mammalian cellular metabolism, and
efficacy at treating an infection in vivo. Furthermore, conjugation
of fatty tails to peptoids can lead to selective, non-natural
analogs of AMPs as short as five monomers in length that are potent
against both bacteria and fungi. Structure-activity relationships
observed in a library of rationally designed ampetoids are wholly
analogous to those which describe many AMPs. Using synchrotron
radiation to probe the interactions between ampetoids and lipid
layers, AMP-ampetoid analogy was found to extend to molecular-level
interactions.
TABLE-US-00001 TABLE 1 Antibacterial and hemolytic activities of
ampetoids and AMPs. Peptoid monomer abbreviations are explained in
FIG. 2. For minimum inhibitory concentrations (MICs) and hemolytic
doses (HDs) reported as ">x", x = 200 .mu.g/mL-the highest
concentration tested (except for pexiganan, which was tested up to
500 .mu.g/mL). Variant HPLC elution class Shorthand name Sequence
solvent* Basis 1 H-(NLys-Nspe-Nspe).sub.4-NH.sub.2 48% 2
H-(Nlys-Nssb-Nspe).sub.4-NH.sub.2 39% Chirality 1.sub.enantiomer
H-(Nlys-Nrpe-Nrpe).sub.4-NH.sub.2 48% Length 1.sub.6mer
H-(Nlys-Nspe-Nspe).sub.2-NH.sub.2 41% 1.sub.9mer
H-(Nlys-Nspe-Nspe).sub.3-NH.sub.2 46% 1.sub.15mer
H-(Nlys-Nspe-Nspe).sub.5-NH.sub.2 51% Hydro- 2-Nsap.sub.2,5,8,11
H-(Nlys-Nsap-Nspe).sub.4-NH.sub.2 48% phobicity 2-Nsna.sub.6,12
H-(Nlys-Nssb-Nspe-Nlys-Nssb- 47% Nsna).sub.2-NH.sub.2
1-Nsna.sub.6,12 H-(Nlys-Nspe-Nspe-Nlys-Nspe- 53%
Nsna).sub.2-NH.sub.2 1-Nhis.sub.6,12 H-(Nlys-Nspe-Nspe-Nlys-Nspe-
37% Nhis).sub.2-NH.sub.2 1-Pro.sub.6 H-Nlys-Nspe-Nspe-Nlys-Nspe-L-
40% Pro-(Nlys-Nspe-Nspe).sub.2-NH.sub.2 Charge 1-Nglu.sub.4,10
H-(Nlys-Nspe-Nspe-Nglu-Nspe- 60%.sup..dagger. Nspe).sub.2-NH.sub.2
1-Nglu.sub.1,4,7,10 H-(Nglu-Nspe-Nspe).sub.4-NH.sub.2
54%.sup..dagger. Amphi- 1.sub.block
H-(Nlys).sub.4-(Nspe).sub.8-NH.sub.2 54% pathicity 2.sub.scrambled
H-Nlys-Nssb-Nspe-Nssb-Nspe- 42% Nlys-Nspe-Nlys-Nssb-Nssb-
Nspe-Nlys-NH.sub.2 AMPs pexiganan.sup.1 GIGKFLKKAKKFGKAFVKIL 38% KK
(SEQ ID NO: 1)-NH.sub.2 melittin.sup.5 GIGAVLKVLTTGLPALISWIK 54%
RKRQQ (SEQ ID NO: 2)-NH.sub.2 B. subtilis MIC HD.sub.10/HD.sub.50
Selectivity ratio E. coli MIC (.mu.M) (.mu.M) (.mu.M)
(SR).sup..dagger-dbl. 3.5 0.88 21/100 6.0 31 3.9 >120/>120
>39 3.5 0.88 16/86 4.6 27 27 >220/>220 >8.1 9.1 1.2
>150/>150 >16 5.5 1.4 3/19 0.55 7.4 0.95 >120/>120
>16 7.2 0.93 55/>120 7.6 3.3 1.6 4/22 1.2 3.5 6.9
>110/>110 >31 3.1 1.6 63/>110 20 >110 6.9 19/40
<0.17 >219 >219 >110/>110 N/A 6.9 1.7 18/73 2.6 31
15 >120/>120 >39 3.1 1.6 73/>200 24 1.6 0.78 2/6 1.3
*Percent acetonitrile in water, 0.1% (v/v) trifluoroacetic acid
(TFA) at HPLC elution. .sup..dagger.With 10 mM ammonium acetate and
no TFA, pH 7.0. .sup..dagger-dbl.Selectivity ratio, SR =
(HD.sub.10)/(E. coli MIC). .sup..sctn.For concentrations reported
as ">x", x = 200 .mu.g/mL-the highest concentration tested
(except for pexiganan, which was tested up to 500 .mu.g/mL).
Initial antibacterial activity and selectivity screening
[0045] An initial set of 15 ampetoid analogs was synthesized to
determine whether peptoids are affected by structural and sequence
modifications in a manner consistent with AMP activities. The
designs for ampetoids in this library were derived from two
antibacterial and selective amphipathic dodecamers, 1 and 2.
Peptoid 1 [H-(Nlys-Nspe-Nspe).sub.4-NH.sub.2] is composed of 2/3
Nspe, the peptoid analog of phenylalanine, and 1/3 Nlys, the
peptoid analog of lysine (see FIG. 1 for the structures of
representative peptoid monomers and corresponding N-substituents).
Peptoid 2 [H-(Nlys-Nssb-Nspe).sub.4-NH.sub.2] contains 1/3
isoleucine-like Nssb monomers in place of Nspe. The variant
sequences were designed to effect changes in chirality, length,
hydrophobicity, charge, and amphipathicity. All of these compounds
were tested for antibacterial activity against representative BSL1
Gram-negative (E. coli JM 109) and Gram-positive (B. subtilis
BR151) bacterial strains. As an initial measure of selectivity, the
lytic activity of the peptoids was determined against human
erythrocytes. Table 1 summarizes the sequences synthesized, the
solvent composition at RP-HPLC elution as a relative measure of
molecular hydrophobicity, and antibacterial and hemolytic
activities. Ten of the 15 peptoids exhibit low-micromolar MICs
against both E. coli and B. subtilis, demonstrating that
non-natural peptoid oligomers can be as active as AMPs (MICs for
pexiganan--a selective AMP analog of magainin-2- and the bee-venom
AMP melittin are shown in Table 1).
[0046] A selectivity ratio (SR) for each compound was defined, also
shown in Table 1, as the quotient of the 10% hemolytic dose
(HD.sub.10) and the E. coli MIC. Thus, the SR is an estimate of an
ampetoid's tendency to kill bacteria rather than mammalian cells.
Ampetoid 1 has an SR of 6.0, similar to that of pexiganan (SR=5.8).
As expected, melittin (well known to be cytotoxic) has a low SR of
0.16. Most AMPs have antibacterial activities in the low-micromolar
range; since peptoid 1 has MICs in that range, the ampetoid library
was primarily expected to yield variants with increased
selectivity. Indeed, six of 13 variants are more selective than 1
and pexiganan (i.e. SRs>6).
[0047] The biocompatibility of selected oligomers with A549 lung
epithelial cells was evaluated using the MTS
[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl-
)-2H-tetrazolium]assay. FIG. 2 shows that the 10% metabolic
inhibitory dose (ID.sub.10) and 50% inhibitory dose (ID.sub.50) of
these selected peptoids compare very favorably to that of pexiganan
and melittin; ampetoids 1-Pro.sub.6 and 1-Nhis.sub.6,12, with
MICs<7 .mu.M, have ID.sub.10's that are 10 and 20 times that of
pexiganan. Interestingly, the relatively non-selective peptoid
1.sub.15mer exhibits cytotoxicity no worse than pexiganan.
[0048] Structure-activity relationships derived from a library of
ampetoids are discussed below according to the primary
physicochemical parameter that was altered. As in previous studies,
circular dichroism (CD) spectroscopy was used to compare the
helicities of the ampetoids by monitoring the intensity of spectral
extrema, particularly at 190 and 220 nm. CD was performed in 10 mM
aqueous Tris buffer, pH 7.4, alone and in the presence of two types
of small unilamellar vesicles (SUVs) composed of model binary lipid
mixtures: (1) anionic E. coli membrane-mimetic SUVs [POPE/POPG (7:3
mole ratio)], and (2) zwitterionic erythrocyte membrane-mimetic
SUVs [POPC/cholesterol (CH) (1:1 mole ratio)].
[0049] With regard to chirality, using enantiomeric side chains
(Nrpe in place of Nspe; FIG. 1), a left-handed helical analog of
peptoid 1 was created, called 1.sub.enantiomer, as evidenced by its
mirror-image CD spectrum in comparison to that of 1 (FIG. 3A). The
activities and selectivities of 1 and 1.sub.enantiomer are
congruent (Table 1).
[0050] With regard to length, variants of 1 were created, ranging
from 6 to 15 monomers, all with the same three-fold sequence repeat
and 1:3 charge-to-length ratio. The shortened variants of 1,
1.sub.6mer and 1.sub.9mer (approx. 12 .ANG. and 18 .ANG. in length,
respectively), are both active antimicrobials and are more
selective than 1 (Table 1). In contrast, the lengthened variant of
1, 1.sub.15mer (approx. 30 .ANG. in length), is substantially more
hemolytic (SR=0.55) and cytotoxic (FIG. 2) than 1, and slightly
less antibacterial. The helicities of these compounds in buffer are
congruent (data not shown).
[0051] Hydrophobicity was modulated independently of length by
replacing hydrophobic Ile-like Nssb and Phe-like Nspe monomers in 1
and 2 with bulkier and more hydrophobic Nsmb and Nsna, respectively
(FIG. 1). The heightened molecular hydrophobicity of
2-Nsmb.sub.2,5,8,11 and 2-Nsna.sub.6,12 led to increases in both
antibacterial and hemolytic activities (Table 1). 1-Nsna.sub.6,12,
however, shows no enhancement of antibacterial activity relative to
1, but is much more hemolytic (SR=1.2).
[0052] Two variants of 1 with reduced hydrophobicity were also
created. Two evenly spaced hydrophobic Nspe residues in 1 were
replaced by Nhis, an achiral peptoid monomer analog of histidine
(FIG. 1) that is polar yet predominantly uncharged at physiological
pH, yielding 1-Nhis.sub.6,12. This oligomer exhibits substantially
decreased hemolysis (SR>31; Table 1) and decreased cytotoxicity
(FIG. 2), with only a slight reduction in antibacterial activity
against B. subtilis compared to 1. A second reduced-hydrophobicity
variant, 1-Pro.sub.6, was created by replacing the Nspe at position
6 with 1-proline. Although proline is well known to destabilize
peptide .alpha.-helices, 1-Pro.sub.6 and 1 are similarly helical in
buffer (FIG. 3A) since 1-proline is well-accommodated in
right-handed type-1-polyproline-like peptoid helices. 1-Pro.sub.6
(SR=8.9) exhibits less hemolysis and less cytotoxicity than 1, with
similar antibacterial activity (Table 1, FIG. 2).
[0053] With respect to charge, the majority of AMPs are cationic,
and replacement of their basic residues with uncharged or anionic
moieties typically leads to a loss of antibacterial activity. To
investigate this phenomenon in ampetoids, Nlys monomers in 1 were
substituted with glutamate-like Nglu (FIG. 1) to create
1-Nglu.sub.4,10 and 1-Nglu.sub.1,4,7,10. Zwitterionic
1-Nglu.sub.4,10 has significantly reduced activity against B.
subtilis compared to 1 and is inactive against E. coli (Table 1),
likely due to the absence of favorable electrostatic interactions
with anionic bacterial membranes; however, it is quite hemolytic
(SR<0.17). The fully anionic variant, 1-Nglu.sub.1,4,7,10 is
devoid of both antibacterial and hemolytic activity.
[0054] To study amphipathicity, a terminally, rather than facially,
amphipathic isomer of 1 with block-like architecture (1.sub.block)
was created, as well as a scrambled sequence of 2 designed to
preclude global amphipathicity (2.sub.scrambled) (Table 1). The
terminal segregation of cationic and hydrophobic residues in the
1.sub.block sequence ensures a strongly amphipathic structure
independent of the facial organization of residues along a helix.
1.sub.block is slightly less antibacterial and more hemolytic than
1 (SR=2.6), although it has the same monomer composition. The CD
spectra of 2.sub.scrambled in buffer and lipid environments (FIGS.
3B, 4B, 5B) are nearly congruent to those of 2, suggesting
2.sub.scrambled forms a structured helix which, due to its
scrambled sequence, has low global amphipathicity relative to 2.
Peptoid 2.sub.scrambled exhibits antibacterial activity, but no
hemolysis up to 120 .mu.M.
TABLE-US-00002 TABLE 2 Antibacterial, antifungal, and hemolytic
activities of alkylated variants of peptoid 1. See FIG. 1 for a
guide to monomer structures. HPLC PEPTOID SEQUENCE
ELUTION.sup..dagger. 1 H-(Nlys-Nspe-Nspe).sub.4-NH.sub.2 48% C5-1
H-Npent-(Nlys-Nspe-Nspe).sub.4-NH.sub.2 51% C10-1
H-Ndec-(Nlys-Nspe-Nspe).sub.4-NH.sub.2 58% 1.sub.9mer
H-(Nlys-Nspe-Nspe).sub.3-NH.sub.2 46% C5-1.sub.9mer
H-Npent-(Nlys-Nspe-Nspe).sub.3-NH.sub.2 48% C10-1.sub.9mer
H-Ndec-(Nlys-Nspe-Nspe).sub.3-NH.sub.2 59% 1.sub.6mer
H-(Nlys-Nspe-Nspe).sub.2-NH.sub.2 47% C5-1.sub.6mer
H-Npent-(Nlys-Nspe-Nspe).sub.2-NH.sub.2 49% C10-1.sub.6mer
H-Ndec-(Nlys-Nspe-Nspe).sub.2-NH.sub.2 59% C10-1.sub.4mer
H-Ndec-Nlys-Nspe-Nspe-Nlys-NH.sub.2 50% C13-1.sub.4mer
H-Ntridec-Nlys-Nspe-Nspe-Nlys-NH.sub.2 57% Pexiganan
GIGKFLKKAKKFGKAFVKILKK (SEQ ID NO: 1)-NH.sub.2 38% E. coli B.
subtilis C. albicans HD.sub.10/HD.sub.50 MIC (.mu.M) MIC (.mu.M)
MIC (.mu.M) (.mu.M) 6.3 1.6 6.3 18/72 6.3 1.6 6.3 10/40 12.5 1.6
1.6 8/27 25 1.6 15.6 150/>200 16.3 1.0 12.5 65/>200 6.3 3.1
3.1 12/40 27* 27* >100 >220/>220 >200 3.1 100
>200/>200 6.3 1.6 6.25 30/80 >100 6.3 100 >200/>200
12.5 1.6 12.5 70/200 12.5 1.6 50 70/>200 *All assays were
performed in cation-adjusted broth except where denoted by an
asterisk. .sup..dagger.HPLC elution is given as a measure of
hydrophobicity and defined as percent acetonitrile in H.sub.2O (C18
column, conducted in the presence of 0.1% TFA, pH 1).
[0055] In order to test the effects of alkylation on antimicrobial
peptoids, a series of alkylated variants of 1 was synthesized,
systematically varying both the alkyl tail length and the length of
the peptoid chain. In general, the length of the peptoid chain was
decreased in increments 3 monomers (one full helical turn) from the
original sequence of 1. However, based on the report of active
ultrashort AMPs containing two positive charges, an additional Nlys
monomer was retained at the shortest length. Alkyl tails were
incorporated via the submonomer peptoid synthetic protocol (using
the appropriate alkylamine for the substitution step) as the side
chain of the N-terminal peptoid residue. All peptoids were amidated
at the C-terminus. Table 2 lists the shorthand names and sequences
of the alkylated peptoids investigated, as well as the activities
of these alkylated variants. Interestingly, it should be noted that
C13-1.sub.4mer--a compound roughly half the molecular weight of
1--exhibited antibacterial activity comparable to 1 and was in fact
found to be more selective against erythrocytes.
[0056] Additionally, in order to characterize possible antifungal
activity, a library of alkylated compounds was tested against C.
albicans, a representative fungal strain. In several instances,
compounds such as C10.about.1.sub.6mer and C13.about.1.sub.4mer
were found to have potent and selective antifungal activity.
[0057] Whereas the preceding generation of molecules was designed
to elucidate structure function relationships of specific molecular
parameters (chirality, length, hydrophobicity, etc.), another
generation compounds were designed explicitly to explore effects on
selectivity. Antimicrobial activity of these compounds was tested
against bacterial strains E. coli (ATTC 35218) and B. subtilis
(ATTC 6633) in cation-adjusted MHB using the microdilution
protocols previously described. The sequences, antimicrobial
activities, hemolytic activities, and metabolic inhibitory
concentrations against NIH 3T3 mouse fibroblast cells are
summarized in Table 3 and the rationale of their design is
described below.
TABLE-US-00003 TABLE 3 Sequences and activities of
second-generation peptoids designed to explore selectivity. Variant
class Shorthand name Sequence Basis 1
H-(Nlys-Nspe-Nspe).sub.4-NH.sub.2 Register variants 1B
H-(Nspe-Nlys-Nspe).sub.4-NH.sub.2 1C
H-(Nspe-Nspe-Nlys).sub.4-NH.sub.2 Proline variants 1-Pro.sub.3
H-(Nlys-Nspe-L-Pro)-(Nlys-Nspe-Nspe).sub.3-NH.sub.2 1-Pro.sub.6
H-Nlys-Nspe-Nspe-Nlys-Nspe-L-Pro-(Nlys-Nspe-Nspe).sub.2-NH.sub.2
1-Pro.sub.9
H-(Nlys-Nspe-Nspe).sub.2-Nlys-Nspe-L-Pro-Nlys-Nspe-Nspe-NH.sub.2
1-Pro.sub.3,9 H-Nlys-Nspe-L-Pro-Nlys-Nspe-Nspe-Nlys-Nspe-L-Pro-
Nlys-Nspe-Nspe-NH.sub.2 Nhis variants 1-Nhis.sub.6
H-Nlys-Nspe-Nspe-Nlys-Nspe-Nhis-(Nlys-Nspe-Nspe).sub.2-NH.sub.2
1-Nhis.sub.6,12 H-(Nlys-Nspe-Nspe-Nlys-Nspe-Nhis).sub.2-NH.sub.2
1-Nhis.sub.3,6,9,12 H-(Nlys-Nspe-Nhis).sub.3-NH.sub.2 Achiral
variants 1.sub.achiral H-(Nlys-Npm-Npm).sub.4-NH.sub.2
1.sub.achiral-Nspe.sub.12
H-(Nlys-Npm-Npm).sub.3-Nlys-Npm-Nspe-NH.sub.2 1-Npm.sub.2,5,8,11
H-(Nlys-Npm-Nspe).sub.4-NH.sub.2 1-Npm.sub.2,3,8,9
H-(Nlys-Npm-Npm-Nlys-Nspe-Nspe).sub.2-NH.sub.2 Nsdp variants
1-Nsdp.sub.all H-(Nlys-Nsmb-Nsmb).sub.4-NH.sub.2
1-Nsdp.sub.2,5,8,11 H-(Nlys-Nsmb-Nspe).sub.4-NH.sub.2 Charge
1-Nlys.sub.5,11 H-(Nlys-Nspe-Nspe-Nlys-Nlys-Nspe).sub.2-NH.sub.2
distribution 1B.sub.12mer-Nlys.sub.4,10
H-(Nspe-Nlys-Nspe-Nlys-Nlys-Nspe).sub.2-NH.sub.2 variants
1B.sub.15mer-Nlys.sub.4,10
H-(Nspe-Nlys-Nspe-Nlys-Nlys-Nspe).sub.2-Nspe-Nlys-Nspe-NH.sub.2
1B.sub.12mer-Nlys.sub.4,6,10
H-Nspe-Nlys-Nspe-Nlys-Nlys-Nlys-Nspe-Nlys-Nspe-Nlys-
Nlys-Nspe-NH.sub.2 1B.sub.15mer-Nlys.sub.4,6,10
H-Nspe-Nlys-Nspe-Nlys-Nlys-Nlys-Nspe-Nlys-Nspe-Nlys-
Nlys-Nspe-Nspe-Nlys-Nspe-NH.sub.2 AMP comparator pexiganan
GIGKFLKKAKKFGKAFVKILKK (SEQ ID NO: 1)-NH.sub.2 E. coli B. subtilis
HD.sub.10/HD.sub.50 MIC (.mu.M) MIC (.mu.M) (.mu.M)
ID.sub.10ID.sub.50.sup..dagger-dbl. 6.3 1.6 21/100 5.1/1.4 6.3 1.6
40/>100 4.9/2.5 6.3 1.6 25/>100 5.6/2.3 12.5 1.6 74/>200
4.8/12 12.5 1.6 83/>200 8.2/18 12.5 1.6 165/>200 9.8/24 50
1.6 >200/>200 31/71 25 1.6 170/>200 -- >50 1.6
>110/>110 >100/>100 >100 100 >200/>200
72/>100 -- -- >100/>100 5.1/16 25 1.6 164/>200 -- 6.3
1.6 87/>200 3.5/6.8 6.3 1.6 68/>200 -- 50 0.78
>200/>200 6.6/64 12.5 0.78 111/>200 10/20 50 1.6
>100/>100 23/85 50 0.78 >200/>200 -- 50 0.78
>200/>200 -- >100 1.6 >200/>200 -- >100 0.78
>200/>200 -- 12.5 1.6 70/>200 1.9/9
[0058] Without limitation, two molecules in Table 1 found to be
extraordinary selective (1-Pro.sub.6 and 1-Nhis.sub.6,12) were used
to derive several new sequences. Since 1-Pro.sub.6 was found to be
highly antimicrobial but less hemolytic than 1, two isomerically
related compounds (1-Pro.sub.3, 1-Pro.sub.9) were created to
explore the effect of proline residue position on activity and
selectivity. One variant with prolines in both the third and ninth
position (1-Pro.sub.3,9) was also designed to evaluate the effect
of increasing the number of proline residues on the activity
profile. A second pair of molecules (1-Nhis.sub.6 and
1-Nhis.sub.3,6,9,12) was created to explore the effect of
modulating the number of Nhis residues within the sequence.
[0059] The effects of hydrophobicity on activity and selectivity
was further explored with an additional set of molecules described
in Table 3. Several sequences were designed with replacement of
selected Nspe residues by achiral Npm residues (see FIG. 1). Two
sequences contained four Npm residues either aligned on one face
(1-Npm.sub.2,5,8,11) or distributed on two faces
(1-Npm.sub.2,3,8,9) of the peptoid. Additionally, the molecule
(1.sub.achiral-Nspe.sub.12) with all Nspe replaced with Npm except
for the 12.sup.th position was designed to be less hydrophobic, yet
still somewhat helical; it has been shown that inclusion of a
chiral residue at the C-terminus promotes helicity. Lastly, an all
achiral version of 1, 1.sub.achiral, contains all Nspe residues
substituted with Npm.
[0060] The hydrophobic side chain Nsdp, which is isomerically
related to Nsmb (FIG. 1), was incorporated into two additional
sequences aimed at further investigating the use of aliphatic side
chains. 1-Nsdp.sub.all has all Nspe monomers replace with Nsdp, and
1-Nsdp.sub.2,5,8,11 displays four Nsdp monomers on one face.
[0061] A final family of sequences was designed to evaluate the
effect of sequence register, charge distribution, and length on
activity and selectivity. Compounds 1B and 1C are isomers of 1 in
which the sequence register was changed to preclude the presence of
terminal charges (Nspe-Nlys-Nspe) or exhibit a C-terminal charge
(Nspe-Nspe-Nlys), respectively. Several compounds were also created
with increased number of charges (i.e. decreased hydrophobicity)
distributed over multiple faces of the molecule. Compound
I-Nlys.sub.5,11 exhibits the sequence of 1 with the two Nspe
monomers at positions 5 and 11 replaced with Nlys resulting in two
additional charges. Other variants include a 1B register sequence
analogously substituted with Nlys and positions 4 and 10 as both a
12mer (1B.sub.12mer-Nlys.sub.4,10) and a 15mer
(1B.sub.15mer-Nlys.sub.4,10). Similarly, molecules with additional
Nlys monomers distributed on three faces
(1B.sub.12mer-Nlys.sub.4,6,10 and 1B.sub.15mer--Nlys.sub.4,6,10)
were also created.
X-Ray Reflectivity Studies of Ampetoid Orientation in Lipid
Layers
[0062] Liquid (aqueous buffer) surface specular X-ray reflectivity
(XR) studies were conducted using synchrotron radiation to
investigate the membrane orientation and depth of penetration of
ampetoid 1 in a model lipid layer which mimics the outer leaflet of
the cell membrane (FIG. 6). X-rays reflected off of the monolayer
yield an electron density profile perpendicular to the interface,
allowing determination of the layer thickness and the presence and
orientation of added molecules. The experimental data are
represented as a stack of slabs, each with a uniform thickness,
electron density, and interface roughness.
[0063] XR data for a pure DPPG (anionic) film (FIG. 6--circles) fit
well with a two-slab model, yielding a hydrocarbon tail density
(.rho..sub.t/.rho..sub.s) of 0.99 and a hydrocarbon tail slab
thickness (L.sub.1) of 17.9 .ANG., as well as a headgroup electron
density (.rho..sub.h/.rho..sub.s) of 1.54 and headgroup slab
thickness (L.sub.2) of 5.7 .ANG.. These data are in good agreement
with previous DPPG monolayer X-ray work. The XR profile changed
dramatically after peptoid 1 was introduced (FIG. 6--squares), and
fit a four-slab model. According to this fit, the first slab
(.rho..sub.t/.rho..sub.s=0.96, L.sub.1=12.1 .ANG.) corresponds to
the lipid tails without 1, the second slab
(.rho..sub.t+p/.rho..sub.s=1.05, L.sub.2=2.8 .ANG.) to the tails
region with partial insertion of 1, the third slab
(.rho..sub.h+p/.rho..sub.s=1.33, L.sub.2=7.0 .ANG.) to the lipid
headgroup region with 1 fully inserted, and the fourth slab
(.rho..sub.p/.rho..sub.s=1.16, L.sub.2=3.6 .ANG.) to 1 alone,
protruding beyond the DPPG headgroups. This electron density
profile is consistent with insertion of 1 through the lipid
headgroups and partially into the lipid tail region. Furthermore,
assuming that 1 retains its helical structure in model lipid
monolayers, the data suggest that 1 inserts at an angle of
approximately 56.degree. between the interface normal and the long
helical axis of the peptoid.
TABLE-US-00004 TABLE 4 Broad-spectrum antibacterial activity of
pexiganan and selected ampetoids against BSL2 and BSL3 pathogenic
bacteria. MICs (.mu.M) 1.sub.achiral- 1- Ntridec- Strain Gram
pexiganan 1 1-Pro.sub.6 1-Pro.sub.9 1.sub.achiral Nspe.sub.2,5,8,11
Nsmb.sub.2,5,8,11 1.sub.4mer Proteus vulgaris ATCC 49132 - 12.9
17.6 18.2 36.5 18.7 36.3 19.0 38.3 Pseudomonas aeruginosa ATCC
27853 - 1.6 4.4 18.2 18.2 9.4 36.3 9.5 9.6 Proteus mirabilis -
>51.7 >70.4 >72.9 >72.9 >75 >72.6 >76.0 153.3
ATCC 35659 Klebsiella pneumoniae ATCC 33495 - 3.2 8.8 9.1 9.1 4.7
9.1 4.8 9.6 Enterobacter aerogenes ATCC 35029 - 12.9 8.8 72.9 36.5
75.0 36.3 19.0 >153.3 Escherichia coli ATCC 25922 - 3.2 2.2 9.1
4.6 9.4 4.5 4.8 9.6 Serratia marcescens ATCC 13880 - >51.7 70.4
>72.9 >72.9 >75 >72.6 >76.0 153.3 Staphylococcus
aureus ATCC 29213 + 12.9 2.2 4.6 9.1 4.7 4.5 4.8 4.8 Staphylococcus
aureus VAN1* + 6.5 2.2 4.6 4.6 4.7 4.5 4.8 4.8 Staphylococcus
aureus VAN2* + 3.2 2.2 4.6 4.6 4.7 4.5 4.8 4.8 Staphylococcus
aureus NRS100 (COL) + 6.5 2.2 4.6 4.6 4.7 4.5 4.8 4.8
Staphylococcus aureus NRS119 + 25.8 2.2 9.1 9.1 9.4 9.1 9.5 4.8
Staphylococcus aureus NRS120 + 25.8 4.4 9.1 9.1 9.4 9.1 9.5 4.8
Enterococcus faecalis ATCC 29212 + 12.9 2.2 4.6 4.6 9.4 4.5 4.8 9.6
Enterococcus faecalis 99* + 51.7 4.4 36.5 36.5 37.5 36.3 38.0 19.2
Enterococcus faecium 106* + 1.6 2.2 2.3 2.3 2.3 2.3 2.4 4.8
*vancomycin resistant
[0064] XR data for a pure DPPG (anionic) film (FIG. 6A--circles;
cartoon inset, top (B)) fit well with a two-slab model, yielding a
hydrocarbon tail density (.rho..sub.t/.rho..sub.s) of 0.99 and a
hydrocarbon tail slab thickness (L.sub.1) of 17.9 .ANG., as well as
a headgroup electron density (.rho..sub.h/.rho..sub.s) of 1.54 and
headgroup slab thickness (L.sub.2) of 5.7 .ANG.. These data are in
good agreement with previous DPPG monolayer X-ray work. The XR
profile changed dramatically after peptoid 1 was introduced (FIG.
6B--squares; cartoon inset, bottom (C)), and fit a four-slab model.
According to this fit, the first slab
(.rho..sub.t/.rho..sub.s=0.96, L.sub.1=12.1 .ANG.) corresponds to
the lipid tails without 1, the second slab
(.rho..sub.t+p/.rho..sub.s=1.05, L.sub.2=2.8 .ANG.) to the tails
region with partial insertion of 1, the third slab
(.rho..sub.h+p/.rho..sub.s=1.33, L.sub.2=7.0 .ANG.) to the lipid
headgroup region with 1 fully inserted, and the fourth slab
(.rho..sub.p/.rho..sub.s=1.16, L.sub.2=3.6 .ANG.) to 1 alone,
protruding beyond the DPPG headgroups. This electron density
profile is consistent with insertion of 1 through the lipid
headgroups and partially into the lipid tail region. Furthermore,
assuming that 1 retains its helical structure in model lipid
monolayers, the data suggest that 1 inserts at an angle of
approximately 56.degree. between the interface normal and the long
helical axis of the peptoid.
[0065] Using a murine peritoneal injection mouse model, the ability
of peptoids to treat infection was evaluated in vivo. The results
from three separate studies of peptoid 1 and two of peptoid
1-Nhis.sub.6,12 against saline treated controls are shown in FIG.
7. The horizontal line represents the geometric mean of each
population.
[0066] Previous work has shown that peptoids can be used to create
compounds with antimicrobial activities similar to AMPs. In this
study, the facile synthesis and high propensity for helix formation
of peptoids was exploited to create and study a library of ampetoid
variants that suggests functional and mechanistic analogy between
ampetoids and AMPs and that demonstrates ampetoids' potential for
development into clinically useful antibiotics.
[0067] The equivalent activities of 1 and 1.sub.enantiomer
demonstrate that ampetoid mechanism is not dependent on overall
handedness nor on stereospecific interactions with receptors or
enzymes, an attribute which has also been observed for many AMPs.
Evidence that ampetoids interact with and insert into membranes is
provided by X-ray reflectivity studies (FIG. 6). Furthermore, the
depth of insertion of peptoid 1--through the headgroups and
partially into the lipid tails--demonstrates that 1 interacts
simultaneously with hydrophobic and hydrophilic lipid moieties;
thus, as with AMPs, the amphipathic structure of 1 is integral to
its interactions with membranes. The orientation of 1 at an angle
of .about.56.degree. to the interface normal suggests that 1 does
not operate through a barrel-stave mechanism, since that would
require a transmembrane configuration. Although it cannot be
concluded that 1 exhibits identical mechanistic behavior to
natural, .alpha.-helical antimicrobial peptides, these X-ray
results demonstrate ampetoid-lipid interactions consistent with
those seen for AMPs such as pexiganan and LL-37.
[0068] Ultimately, whether ampetoid activity also adheres to trends
relating structure and function was determined in a manner
analogous to AMPs, and physicochemical properties of selective
ampetoids were found consistent with those of selective AMPs and
non-selective peptoids exhibit close similarities to cytotoxic
peptides. Numerous structure-activity studies of a wide variety of
AMPs have delineated the physicochemical characteristics that give
rise to selective antibacterial activity or non-selective
cytotoxicity. Regardless of structural class (i.e. .alpha.-helix,
.beta.-sheet, loop, or extended), non-selective AMPs typically (1)
are very hydrophobic, such that their interactions with membranes
are governed primarily by the hydrophobic effect, and (2) have a
well-defined amphipathic structure. In contrast, the antibacterial
activity of selective AMPs is dependent on (1) high net cationic
charge (although excessive cationic charge can also lead to
hemolytic activity) and (2) only moderate hydrophobicity. Perhaps
counterintuitively, a well-defined amphipathic structure is not
necessary for selective antimicrobial activity; destabilization of
AMP secondary structure often leads to improvements in
selectivity.
[0069] The ampetoids 1-Nglu.sub.4,10, 1.sub.15mer, 1-Nsna.sub.6,12,
and 1.sub.block are all less selective than peptoid 1 (SRs<6.0)
(Table 1). Consistent with properties of non-selective AMPs, these
compounds are all either more hydrophobic (according to RP-HPLC
elution time) and/or less charged (1-Nglu.sub.4,10) than 1.
Furthermore, they are all as (or more) helical than 1 in
erythrocyte-mimetic POPC/CH SUVs (FIG. 4), indicative of their
well-defined membrane-bound amphipathic structures in that lipid
environment.
[0070] In contrast, 1.sub.9mer, 1-Nhis.sub.6,12, and 1-Pro.sub.6
all have antibacterial activities comparable to 1, but enhanced
selectivities (SRs>6.0). These peptoids are all more hydrophilic
than 1, and all have a net charge of at least +3. Thus, ampetoids
are selectively active provided they have a net positive charge and
are sufficiently but not excessively hydrophobic, a phenomenon that
is wholly consistent with observations of selective AMPs.
Additionally, retention of antibacterial activity and enhancement
of selectivity in the shortened 1.sub.9mer is analogous to behavior
observed in studies of truncated AMPs.
[0071] In general, the effect of length on ampetoid potency and
selectivity can largely be attributed to differences in
hydrophobicity (RP-HPLC retention time, Table 1), which increases
proportionally with length. Since the 12mer 1 is more antibacterial
than longer and shorter analogs, these results suggest the
existence of an optimal hydrophobicity at which antibacterial
activity is maximized; added hydrophobicity increases only
hemolytic activity. This conclusion is also supported by the
hydrophobicity variants, since moderately hydrophobic
2-Nsap.sub.2,5,8,11 and 2-Nsna.sub.6,12 are both more antibacterial
and more hemolytic than 2, while the strongly hydrophobic
1-Nsna.sub.6,12 (Table 1) shows enhancement of hemolytic but not
antibacterial activity relative to 1. These results are in
agreement with observations of AMPs and their variants.
[0072] Although they are more hemolytic than 2, peptoids
2-Nsna.sub.6,12 and 2-Nsap.sub.2,5,8,11 are still more selective
(SRs of 7.6 and 16, respectively) than 1, and their physicochemical
properties are consistent with this finding. They are highly
charged (+4), moderately hydrophobic, and exhibit CD spectra
consistent with low helicity (FIG. 3B, 4B, 5B). The CD spectrum of
2-Nsna.sub.6,12 exhibits red-shifted extrema, suggesting a
destabilized secondary structure. Thus, these variants of 2 all
have poorly defined amphipathic structures.
[0073] Indeed, results on the whole suggest that, consistent with
AMPs, a highly amphipathic structure is required for hemolytic
activity, but not for antibacterial activity. CD spectra of
ampetoids in POPE/POPG SUVs (FIG. 5) show that the extent of
helicity in this bacteria-mimetic system is poorly correlated with
antimicrobial activity. 1-Nsna.sub.6,12 for example, is less
helical than 1 (FIG. 5A), but the two compounds have similar
antibacterial potencies (Table 1). In contrast, 2-Nsap.sub.2,5,8,11
is similarly helical to 2 (FIG. 5B), yet 2-Nsmb.sub.2,5,8,11 is
much more antibacterial (Table 1).
[0074] CD spectra in erythrocyte-mimetic POPC/CH SUVs (FIG. 4) do,
however, reveal a correlation between hemolytic activity and
helicity. 1-Nsna.sub.6,12, and 1-Nglu.sub.4,10 are the most
hemolytic ampetoids (Table 1), and are the most helical in POPC/CH
SUVs (FIG. 4A). 1.sub.15mer is also very hemolytic and as helical
as 1 in POPC/CH. Conversely, 1-Nhis.sub.6,12, 2,2-Nsmb.sub.6,12,
and 2.sub.scrambled are less hemolytic (Table 1) and less helical
(FIG. 4) than 1. Thus, for these cationic, facially amphipathic
compounds, it appears that defined helical structure, which leads
to clean segregation between cationic and hydrophobic groups, is
important for hemolytic activity. Further insight is provided by
1.sub.block, which has a well-defined terminally amphipathic
structure independent of its helicity; 1.sub.block is hemolytic
(SR=2.6). Together, these results suggest that helicity is
important only as a means to organize an amphipathic structure,
which facilitates oligomerization and a bias toward a non-selective
mechanism. Based on this conclusion, a helical but poorly
amphipathic compound such as 2.sub.scrambled should be
antimicrobial, but selective; indeed, 2.sub.scrambled kills
bacteria, but exhibits no hemolysis up to 120 .mu.M.
[0075] Results also indicate that hydrophobicity is relevant to
hemolytic activity, and comparison of 1-Pro.sub.6 and 1 establishes
this relationship more concretely. These ampetoids have similar
sequences with the same net charge and yield nearly congruent CD
spectra (FIG. 3A). This suggests that their helical content, and
thus their resultant amphipathicity, are similar. However, the
Nspe.fwdarw.Pro substitution yields a reduction in hydrophobicity,
according to RP-HPLC elution time (Table 1). This single difference
is reflected in the lower hemolytic and cytotoxic activities of
1-Pro.sub.6 compared to 1, and reveals clearly the dependence of
selectivity on hydrophobicity in a case where all other parameters
are held constant.
[0076] The results in Table 3 provide further insight into how
molecular structural features modulate peptoid activity and
selectivity. While antimicrobial activity does not change with
respect to proline position, cytotoxicity is decreased as the
proline is moved closer to the C-terminus. It is possible that
proline provides structural stability at the C-terminus that makes
it less harmful to mammalian cells while not affecting its ability
to kill bacteria. Increasing the number of Nhis residues decreased
both the antimicrobial activity as well as the cytotoxicity.
Interesting, 1-Nhis.sub.6,12, the basis sequence for this family,
was found to be far less active against E. coli ATCC 35218 than the
strain originally tested (JM109).
[0077] The family of compounds containing varying amounts of
achiral residues shows that while the positions of the achiral
residues does not significantly affect antimicrobial activity, it
may have an impact on selectivity (Table 3,1-Npm.sub.2,5,8,11 vs.
1-Npm.sub.2,3,8,9). Interestingly, both of these compounds exhibit
antimicrobial activity equal to that of compound 1, but exhibit
superior selectivity. It also appears that antimicrobial activity
is decreased with increasing achiral residues, an effect that could
be due to the reduced hydrophobicity and decreased organizational
structure of the helix. The sequence containing all Nsdp residues
is relatively inactive, but that containing 4 Nsdp residues along
one face (1-Nsdp.sub.2,5,8,11) exhibits antimicrobial activity.
[0078] Several compounds were made to investigate how increasing
the number of charges and distributing them on multiple faces
effects activity. Two molecules were increased in length in order
to add hydrophobicity to counteract the increased charges.
Interestingly, no molecule in this family was shown to be active
against E. coli, likely due to the decreased hydrophobicity, but
all are very active against B. subtilis. At the same time, all of
them are completely nontoxic to both erythrocytes and mammalian
cells. It is possible that this family acts via a different
mechanism of action than less highly charged molecules.
[0079] The results of the broad spectrum antimicrobial testing and
in vivo efficacy study demonstrate the therapeutic potential of
antimicrobial peptoids. Nearly all seven peptoids tested were more
potent against the nine Gram positive strains (including four
vancomycin-resistant) than pexiganan. Against Gram negative
strains, compounds 1 and 1-Nsdp.sub.2,5,8,11 showed activity
comparable to that of pexiganan.
[0080] The in vivo results shown in FIG. 2 demonstrate that the
bacterial counts from the peritoneal lavage were significantly
reduced upon treatment with peptoid 1 (p<0.05). Additionally, no
mice treated with peptoid 1 died prematurely. Of the eleven mice
treated with peptoid 1-NhiS.sub.6,12 on the other hand, eight died
prematurely during the exposure period. Of the 15 saline treated
controls across all three experiments, only 4 died prematurely.
Also, the average bacterial count was not reduced in comparison to
saline treated controls.
[0081] In summary, the results suggest that antibacterial activity
among AMPs and ampetoids alike is dependent on moderate
hydrophobicity and net cationic charge, while hemolytic activity is
associated primarily with high hydrophobicity and a strongly
amphipathic structure, regardless of helical content. MTS assays
demonstrate a similar trend between hemolytic activity and
cytotoxicity against A549 eukaryotic mammalian cells. The
relationships between structure and function in ampetoids are
empirically analogous to those in AMPs. X-ray reflectivity studies,
which show that ampetoid 1 is membrane-active and adopts a stable
membrane-bound orientation, demonstrate molecular-level analogy
between AMPs and ampetoids.
[0082] Results with alkylated variants demonstrate that alkyl chain
attachment may be used to create very short peptoids that are as
antimicrobially active and non-toxic as longer unalkylated helices.
In particular, both Ndec-1.sub.6mer and Ntridec-1.sub.4mer were
found to be comparable in activity and selectivity to 1. Given that
the lengths of most AMPs range from .about.12-100 amino acids, it
is notable that such low molecular weight (<1 kDa) peptoids
exhibit such potency and selectivity against both bacteria and
fungi. N-terminal attachment of alkyl tails can be a useful motif
for improving the potency and decreasing the molecular weight of
ampetoids. Moreover, alkylation is an effective and tunable
modification that exhibits a clear effect on peptoid function.
[0083] Peptoids have greater potential than peptides to be used as
pharmaceuticals and in biomaterials due to their improved
stability, bioavailability, and highly tunable side chain
chemistry. Since peptides' potential for toxicity is a major
obstacle limiting their clinical use, ampetoids' low cytotoxicity
observed here relative to the AMP pexiganan further emphasizes
their therapeutic potential. The results reported herein will aid
in the rational design and optimization of ampetoids and other
non-natural oligomers as antimicrobials in the future.
[0084] As a continuation of the study described above, various
other ampetoids were created with the goal of more fully exploring
how and to what extent specific structural characteristics
influence selectivity. (See Tables 5-7, FIGS. 9-17 and Examples
10-15.) Additional ampetoids were derived from the previously
reported dodecamer, peptoid 1, which is composed of 1/3 lysine-like
charged monomers (Nlys) and 2/3 phenylalanine-like hydrophobic
monomers (Nspe) arranged in a repeating sequence
H-(Nlys-Nspe-Nspe).sub.4-NH (FIG. 9, Table 5). With reference to
certain compounds previously discussed, additional molecules were
designed to explore the importance of (1) primary sequence, (2)
sequence register, (3) net charge, and (4) charge-to-length ratio
(CTLR) on antimicrobial activities and cell selectivities. The
effects of different hydrophobic moieties were evaluated by
systematically replacing one or more Nspe monomers with other
hydrophobic moieties: (1) L-proline monomers (2) achiral
hydrophobic monomers, (3) opposite chirality hydrophobic monomers
(Nrpe), and (4) aliphatic hydrophobic monomers. Again, as above,
Pexiganan, a widely studied and clinically-relevant AMP analog of
magainin-2, was included in this study as a basis of comparison to
AMPs.
[0085] Schematic structures showing the three-fold periodic
architecture of peptoid 1 (the basis sequence) and ampetoid
variants, including those discussed above, are shown in FIG. 9, to
clarify the design strategy and relationships among ampetoid
variants discussed herein. The three molecular ampetoid faces
discussed throughout this work are depicted as aligned monomers on
the left, back, and right sides, as shown. The full sequence of
each molecule is displayed in Table 5, along with other molecular
properties including molecular weight, net charge, CTLR, and
reversed-phase HPLC (RP-HPLC) solvent composition upon elution, as
a measure of molecular hydrophobicity. In general, as shown above,
the naming convention for ameptoids includes the basis compound (1,
in most cases) followed by a description of how the sequence was
modified compared to the basis compound. For example, compound
1B-Nlys.sub.4,10 describes a variant based on the sequence of 1B in
which Nlys monomers were substituted at positions 4 and 10.
[0086] The activities of all compounds against bacterial strains
and mammalian cells are summarized in Table 6. The potencies of all
compounds were determined against representative Gram-negative (E.
coli ATCC 35218) and Gram-positive (B. subtilis ATCC 6633)
biosafety level 1 (BSL1) organisms. In addition to hemolytic
activity, the conventional measure of toxicity to mammalian cells,
the effect of each ampetoid on NIH 3T3 mouse fibroblast cells was
determined using the MTS tetrazolium salt-based colorimetric assay.
While hemolysis measures the degree to which erythrocytes are
lysed, the MTS assay indirectly quantifies the extent to which
cellular metabolic activity is inhibited.
[0087] Because the range of hemolytic activities displayed by these
molecules is significantly greater than that of inhibitory
activities, hemolysis assays provide a more sensitive gauge of
activity against mammalian cells. Therefore, the selectivity of
each ampetoid for various bacterial strains compared to
erythrocytes is reported as the selectivity ratio (SR), which is
defined as the quotient of the 10% hemolytic dose and the minimum
inhibitory concentration (MIC) for each bacterial strain.
[0088] The ampetoids in this library exhibited antibacterial
potencies against Gram-negative E. coli that ranged from 3.1 .mu.M
to >100 .mu.M. Likewise, hemolytic activity ranged from
HD.sub.10=16 .mu.M to HD.sub.10>200 .mu.M and metabolic
inhibitory activity from ID.sub.50=4.9 .mu.M to ID.sub.50>100
.mu.M. The breadth of activities and selectivities evidenced by
these compounds suggests that this library was well designed to
effect a range of responses for elucidating structure-activity
relationships. Compared to 1 (SR.sub.E. coli=3.3), 17 of the 26
ampetoid variants demonstrate improved selectivity for E. coli
(SR.sub.E. coli=6.8-26). Four ampetoids (1-Npm.sub.2,5,8,11,
1.sub.11mer, 1.sub.ach-Nspe.sub.2, 1.sub.ach-Nspe.sub.12) exhibited
equivalent activity (6.3 .mu.M) and superior selectivity (SR.sub.E.
coli=14-26) compared to pexiganan (SR.sub.E. coli=11). In
comparison to their activities against E. coli, all oligomers were
more potent against B. subtilis, with MICs ranging from 0.78 to 1.6
.mu.M. Corresponding selectivity ratios were as high as
>256.
TABLE-US-00005 HPLC elution Net Compound MW Sequence (% ACN)*
Charge CTLR.sup..sctn. Comparators 1 1819
H-(Nlys-Nspe-Nspe).sub.4-NH.sub.2 65.1 +4 0.33 Pexiganan 2477
GIGKFLKKAKKFGKAFVKILKK (SEQ ID NO: 1)-NH.sub.2 50.2 +9 0.41
Register & 1B 1819 H-(Nspe-Nlys-Nspe).sub.4-NH.sub.2 63.4 +4
0.33 sequence 1C 1819 H-(Nspe-Nspe-Nlys).sub.4-NH.sub.2 64.8 +4
0.33 variants 1scr 1819
H-(Nspe).sub.2-(Nlys-Nspe).sub.3-(Nspe).sub.3-Nlys-NH.sub.2 61.1 +4
0.33 Increased 1-Nlys.sub.5,11 1753
H-(Nlys-Nspe-Nspe-Nlys-Nlys-Nspe).sub.2-NH.sub.2 51.2 +6 0.50
charge 1B-Nlys.sub.4,10 1753
H-(Nspe-Nlys-Nspe-Nlys-Nlys-Nspe).sub.2-NH.sub.2 52.7 +6 0.50
variants 1B-Nlys.sub.4,6,10 1720
H-(Nspe-Nlys).sub.2-Nlys.sub.2-(Nspe-Nlys).sub.2-Nlys-Nspe)-NH.sub.2
45.4 +7 0.58 1B.sub.15mer- 2204
H-(Nspe-Nlys-Nspe-Nlys-Nlys-Nspe).sub.2-Nspe-Nlys- 55.5 +7 0.47
Nlys.sub.4,10 Nspe-NH.sub.2 1B.sub.15mer- 2171
H-(Nspe-Nlys).sub.2-Nlys.sub.2-(Nspe-Nlys).sub.2-Nlys-Nspe.sub.2-
50.8 +8 0.53 Nlys.sub.4,6,10 Nlys-Nspe-NH.sub.2 Length 1.sub.10mer
1497 H-(Nlys-Nspe-Nspe).sub.3-Nlys-NH.sub.2 60.9 +4 0.40 variants
1.sub.11mer 1658 H-(Nlys-Nspe-Nspe).sub.3-Nlys-Nspe-NH.sub.2 63.5
+4 0.36 1.sub.13mer 1948 H-(Nlys-Nspe-Nspe).sub.4-Nlys-NH.sub.2
62.8 +5 0.38 Proline 1-Pro.sub.3 1755
H-Nlys-Nspe-L-Pro-(Nlys-Nspe-Nspe).sub.3-NH.sub.2 63.0 +4 0.33
containing 1-Pro.sub.6 1755
H-Nlys-Nspe.sub.2-Nlys-Nspe-L-Pro-(Nlys-Nspe.sub.2).sub.2-NH.sub.2
62.4 +4 0.33 variants 1-Pro.sub.9 1755
H-(Nlys-Nspe.sub.2).sub.2-Nlys-Nspe-L-Pro-(Nlys-Nspe.sub.2)-NH.sub.2
62.6 +4 0.33 1-Pro.sub.3,9 1691
H-(Nlys-Nspe-L-Pro-Nlys-Nspe-Nspe).sub.2-NH.sub.2 58.1 +4 0.33
Chirality 1-Nrpe.sub.3,6,9,12 1819
H-(Nlys-Nspe-Nrpe).sub.4-NH.sub.2 63.5 +4 0,33 variants
1-Nrpe.sub.2,5,8,11 1819 H-(Nlys-Nrpe-Nspe).sub.4-NH.sub.2 66.4 +4
0.33 1-Nrpe.sub.2,3,5,6 1819
H-(Nlys-Nrpe-Nrpe).sub.2-(Nlys-Nspe-Nspe).sub.2-NH.sub.2 65.0 +4
0.33 Achiral 1.sub.ach 1701 H-(Nlys-Npm-Npm).sub.4-NH.sub.2 59.8 +4
0.33 variants 1.sub.ach-Nspe.sub.2 1721
H-(Nlys-Nspe-Npm)-(Nlys-Npm-Npm).sub.3-NH.sub.2 60.8 +4 0.33
1.sub.ach-Nspe.sub.12 1721
H-(Nlys-Npm-Npm).sub.3-(Nlys-Npm-Nspe)-NH.sub.2 62.0 +4 0.33
1-Npm.sub.2,3,8,9 1763
H-(Nlys-Npm-Npm-Nlys-Nspe-Nspe).sub.2-NH.sub.2 63.3 +4 0.33
1-Npm.sub.2,5,8,11 1763 H-(Nlys-Npm-Nspe).sub.4-NH.sub.2 63.6 +4
0.33 Aliphatic 1-Nsdp.sub.all 1547
H-(Nlys-Nsdp-Nsdp).sub.4-NH.sub.2 63.2 +4 0.33 variants
1-Nsdp.sub.2,3,8,9 1683
H-(Nlys-Nsdp-Nsdp-Nlys-Nspe-Nspe).sub.2-NH.sub.2 64.7 +4 0.33
1-Nsdp.sub.2,5,8,11 1683 H-(Nlys-Nsdp-Nspe).sub.4-NH.sub.2 63.8 +4
0.33
TABLE-US-00006 TABLE 5 Sequence and molecular properties of
ampetoids and comparator peptide pexiganan. See FIG. 9 for the
structures of the peptoid monomers indicated in each sequence. HPLC
elution is reported as the average percentage of acetonitrile in
the solvent mixture upon compound elution for three injections. A
linear acetonitrile/water (0.1% trifluoroacetic acid) gradient of
5%-95% acetonitrile over 45 minutes was run on a C18 column. .sctn.
CTLR stands for charge-to-length ratio, which is defined as the
ratio of the total number of charged monomers to the total number
of monomers in each sequence. Antimicrobial Mammalian Cell Activity
Activity E. coli B. subtilis HD.sub.10/ MIC MIC HD.sub.50 ID.sub.50
Selectivity Ratios* Compound (.mu.M) (.mu.M) (.mu.M) (.mu.M)
SR.sub.E. coli SR.sub.B. subtilis Comparative 1 6.3 1.6 21/ 5.1 3.3
13 Molecules 100 Pexiganan 6.3 1.6 70/ 9 11 44 >200 Register
& 1B 6.3 1.6 55/>100 4.9 8.7 34 sequence 1C 6.3 1.6
25/>100 5.6 4.0 16 variants 1scr 6.3 1.6 64/>200 8.5 10 40
Increased net 1-Nlys.sub.5,11 50 1.6 >100/ 85 >2 >63
charge variants >100 1B-Nlys.sub.4,10 50 0.78 >200/ 83 >4
>256 >200 1B- >100 1.6 >200/ >100 Inactive >125
Nlys.sub.4,6,10 >200 1B.sub.15mer- 50 0.78 >200/ 16 >4
>256 Nlys.sub.4,10 >200 1B.sub.15mer- >100 0.78 >200/
40 Inactive 256 Nlys.sub.4,6,10 >200 Length variants 1.sub.10mer
12.5 0.78 >200/ 54 >16 >256 >200 1.sub.11mer 6.3 0.78
103/>200 11 16 132 1.sub.13mer 3.1 0.78 21/>100 5.6 6.8 27
Proline 1-Pro.sub.3 12.5 1.6 74/>200 12 5.9 46 containing
1-Pro.sub.6 12.5 1.6 83/>200 18 6.6 52 variants 1-Pro.sub.9 12.5
1.6 165/>200 24 13 103 1-Pro.sub.3,9 50 1.6 >200/ 71 4.0 125
>200 Chirality variants 1- 6.3 1.6 16/67 3.8 2.5 10
Nrpe.sub.3,6,9,12 1- 6.3 1.6 22/95 5.2 3.5 14 Nrpe.sub.2,5,8,11
1-Nrpe.sub.2,3,5,6 6.3 0.78 25/96 4.8 4.0 32 Achiral variants
1.sub.ach 12.5 0.78 183/>200 16 15 235 1.sub.ach-Nspe.sub.2 6.3
0.78 160/>200 11 25 205 1.sub.ach-Nspe.sub.12 6.3 1.6
164/>200 15 26 103 1-Npm.sub.2,3,8,9 6.3 1.6 39/>200 15 6.2
24 1- 6.3 1.6 87/>200 6.8 14 54 Npm.sub.2,5,8,11 Aliphatic
variants 1-Nsdp.sub.all 25 0.78 >200/ 64 >8 256 >200 1-
12.5 0.78 77/>200 19 6.2 99 Nsdp.sub.2,3,8,9 1- 12.5 0.78
111/>200 20 9.7 142 Nsdp.sub.2.5.8.11 Table 6: Activity of
ampetoids and pexiganan against bacteria and mammalian cells.
Presented are the minimum inhibitory concentrations (MICs) against
E. coli (ATCC 35218) and B. subtilis (ATCC 6633), 10% and 50%
hemolytic doses (HD), and 50% inhibitory doses (ID) against NIH 3T3
mouse fibroblast cells. *Selectivity ratio (SR) is defined as the
ratio of the 10% hemolytic dose to the MIC for the bacterial strain
of interest.
[0089] The secondary structure of ampetoids was evaluated using
circular dichroism (CD) spectroscopy in 10 mM Tris buffer (pH 7.4)
and the same buffer containing small unilamellar vesicles (SUVs)
comprised of either POPC/cholesterol (2:1 mole ratio) or POPE/POPG
(7:3 mole ratio). Whereas the POPC/cholesterol mixture is a
zwitterionic binary model lipid preparation that mimics the
membrane of erythrocytes, the negatively charged POPE/POPG mixture
mimics the composition of the E. coli outer membrane. A peptoid
composed of aromatic right handed poly-proline-type-1-like helix
exhibits spectral features including a maximum at 192 nm and two
local minima at .about.202 nm and .about.220 nm, respectively.
[0090] Peptoid 1, with a periodic trimer repeat sequence of
Nlys-Nspe-Nspe, is composed of four facially-aligned Nlys monomers
(positions 1, 4, 7, and 10) along the left molecular face and has a
charged N-terminal monomer, as shown in FIG. 9. Two isomeric
variants were made in which the sequence register was modified, a
change that most overtly affects the relative position of monomers
with respect to the terminal positions. Peptoid 1B has a trimer
repeat of Nspe-Nlys-Nspe, exhibits charged monomers along the back
face (positions 2, 5, 8, and 11) and has hydrophobic moieties at
both termini. Ampetoid 1C exhibits a sequence register of
Nspe-Nspe-Nlys, has charged monomers along the right face
(positions 3, 6, 9, and 12), and has a charged monomer at the
C-terminal position (see FIG. 9). The last variant in this family
is a "scrambled" isomer, 1.sub.scr, which was made with a
non-periodic sequence in which the four charged monomers are
distributed across all three molecular faces (FIG. 9).
[0091] The antimicrobial potencies against E. coli (MIC=6.3 .mu.M)
and B. subtilis (MIC=1.6 .mu.M) of all three variants were the same
as for 1, and their toxicities to NIH 3T3 cells were also similar,
with ID.sub.50 values ranging from 4.9-5.6 .mu.M. The hemolytic
activity, however, was reduced for 1.sub.scr (HD.sub.10=64 .mu.M)
and 1B (HD.sub.10=55 .mu.M) compared to 1 (HD.sub.10=21 .mu.M) and
1C (HD.sub.10=25 .mu.M). As a result, the selectivities for
1.sub.scr (SR.sub.E. coli=10) and 1B (SR.sub.E. coli=8.7) were more
favorable in comparison to 1 (SR.sub.E. coli=3.3) and 1C (SR.sub.E.
coli=4.0).
[0092] CD spectroscopy showed that all these variants exhibited
helicity in 10 mM Tris buffer similar to that of 1, with 1.sub.scr
being slightly more helical and 1C, slightly less (FIG. 10A). In
both POPC/cholesterol and POPE/POPG lipids, however, 1B exhibited
significantly increased helical intensity at 220 nm compared to the
other variants (FIGS. 10B, C). By nature of its scrambled sequence
design, the extent of helicity for 1.sub.scr is decoupled from its
degree of amphipathicity; the distribution of charges on all three
molecular faces reduces its amphipathicity regardless of a
three-fold periodic helical architecture. It is possible that the
reduced intramolecular electrostatic repulsion of side chain
moieties along a given molecular face in 1.sub.scr readily
accommodates a more helical secondary structure, despite its
overall reduced amphipathicity. Based on previous studies,
reduction in amphipathicity can improve selectivity without
compromising antimicrobial activity. Indeed, 1.sub.scr exhibits
improved selectivity (SR.sub.E. coli=10) compared to 1 (SR.sub.E.
coli=3.3) with no reduction in antimicrobial activity (MIC.sub.E.
coli=6.3 .mu.M for both molecules).
[0093] A second family of ampetoids was designed to evaluate the
effect of increased charge density on potency and selectivity (FIG.
9). Structure-activity relationships derived from ampetoid variants
with decreased net charge were found to significantly reduce their
selectivity for bacteria, likely due to the less favorable
electrostatic interaction with negatively charged bacterial
membranes. The variants in this family of ampetoids were designed
to explore the effect of increasing net charge and
charge-to-length-ratio (CTLR) on cell selectivity. Ampetoid 1, and
most other variants in this library, have a net charge of +4 and
CTLR of 0.33. These compounds exhibit net charges ranging from +6
to +8, and CTLRs ranging from 0.47-0.58. Additionally, the
hydrophobicities of these compounds (ranging from 45.4% to 55.5%
acetonitrile upon HPLC elution) were all significantly reduced
compared to 1 (65.1%) (Table 9). 1-Nlys.sub.5-11 has a total of six
positive charges with two additional Nlys monomers (compared to 1)
substituted at positions 5 and 11, which are aligned along a back
face of the helix (FIG. 9). 1B-Nlys.sub.4,10 is an isomeric variant
of 1-Nlys.sub.5-11 with the sequence register of 1B, and 1
B-Nlys.sub.4,6,10 similarly has a net charge of +7 with cationic
charge on all three molecular faces. Longer 15mer variants,
1B.sub.15mer--Nlys.sub.4,10 and 1B.sub.15mer--Nlys.sub.4,6,10, were
also made with an additional (Nspe-Nlys-Nspe) turn on the
C-terminus, giving them net charges of +7 and +8, respectively
(FIG. 9).
[0094] All variants in this family were significantly less active
against E. coli (MIC=50->100 .mu.M) and were non-hemolytic
(HD.sub.10>100 .mu.M), a result largely effected by the reduced
hydrophobicity of these variants compared to 1. Because this family
of variants does not exhibit a broad range of selectivities
(SR.sub.E. coli>2 to >4), elucidating a meaningful
relationship between physicochemical properties and selectivity is
difficult. A general trend observed, however, is that variants with
a CTLR of less than .about.0.50, hydrophobicity greater than
.about.50% acetonitrile, and one completely hydrophobic face
(1-Nlys.sub.5,11, 1B-Nlys.sub.4,10, and
1B.sub.15mer--Nlys.sub.4,10) exhibited weak activity (MIC.sub.E.
coli=50 .mu.M). Variants with CTLRs greater than .about.0.50,
hydrophobicity less than .about.50% acetonitrile, and had charges
distributed on all three molecular faces (1B-Nlys.sub.4,6,10 and
1B.sub.15mer--Nlys.sub.4,6,10) were completely inactive (MIC.sub.E.
coli>100 .mu.M). These results are commensurate with structure
activity relationships previously established for both AMPs, and
ampetoids: (1) antimicrobial oligomers must be sufficiently
hydrophobic to be potent against Gram-negative bacteria, and (2)
highly charged and poorly amphipathic structures are generally
selective.
[0095] It is most notable that the reduction in activity against E.
coli exhibited by this family of molecules did not carry over to
their activities against B. subtilis. Despite the marked change in
physicochemical properties (NC as high as +8, hydrophobicity as low
as 45.4% acetonitrile, and CTLR as high as 0.58) exhibited by this
family of molecules compared to 1 (NC=+4, hydrophobicity=65.1%, and
CTLR=0.33), the MICs of these variants against B. subtilis were
very similar to that of 1, ranging from 0.78-1.6 .mu.M. The
resultant selectivity ratios of molecules in this library for B.
subtilis were among the highest of all compounds tested (SR.sub.B.
subtilis>256).
[0096] The CD spectra in FIG. 11 show that, with the exception of
1B-Nlys.sub.4,6,10, all compounds exhibited helicity similar to
that of 1 in 10 mM Tris buffer. In POPC/cholesterol lipids,
however, helicity was inversely related to CTLR, and concomitantly,
achiral monomer content. The effect of increased achiral monomer
content on peptoid CD spectra is demonstrated plainly in the
hydrophobic environment of lipid vesicles; as shown in FIG. 11B,
increased achiral monomer content reduced overall CD signal
intensity, particularly at 220 nm.
[0097] Variants of the dodecameric peptoid 1 that ranged in length
from 10-13 monomers were explored as shown in FIG. 9 and Table 5.
Previous studies that explored ampetoid length variants with a
constant CTLR (1.sub.6mer, 1.sub.9mer, and 1.sub.15mer) showed that
increased length beyond 12 monomers (ampetoid 1) only increased
hemolytic activity without improving antimicrobial potency. The
variants in this study, 1.sub.10mer, 1.sub.11mer, 1, and
1.sub.13mer exhibit small differences in sequence length, but most
notably effect a range of CTLRs (0.33-0.40). This range of CTLR is
significantly lower compared to the range exhibited by charge
density variants (0.47-0.58). The CD spectra shown in FIG. 12
suggest that in both aqueous buffer and lipid environments, all
variants are similarly helical, and therefore exhibit similar
amphipathicities.
[0098] Considering first only those variants with a net charge of
+4 (1.sub.10mer, 1.sub.11mer, 1), both hydrophobicity, and CTLR
scale monotonically with length. Ampetoid 1 (12mer) is the most
hydrophobic (65.1% acetonitrile) and exhibits the lowest CTLR
(0.33), while 1.sub.10mer is the least hydrophobic (60.9%
acetonitrile) and is characterized by the highest CTLR (0.40). The
data in Table 5 show that cell selectivity is directly related to
CTLR and inversely related to hydrophobicity. The slight reduction
in activity of 1.sub.10mer against E. coli (12.5 .mu.M), (a result
of its lowered hydrophobicity), was accompanied by a much larger
improvement in its hemolytic activity (HD.sub.10>200 .mu.M);
1.sub.10mer is therefore the most selective of this group
(SR.sub.E. coli>16). 1.sub.11mer retained antimicrobial activity
equivalent to that of 1 (MIC.sub.E. coli 6.3 .mu.M), but was less
selective than 1.sub.10mer (SR.sub.E. coli=16). Ampetoid 1 was
found to be the least selective (SR.sub.E. coli=3.3). The same
trend was observed for selectivity against B. subtilis (Table
6).
[0099] It is notable that based on the above correlation,
1.sub.13mer (CTLR of 0.38) would be expected to have an improved
selectivity compared to 1.sub.11mer (CTLR=0.36), however this is
not the case. On the contrary, ampetoid 1.sub.11mer (SR.sub.E.
coli=16) is more selective than 1.sub.13mer (SR.sub.E. coli=6.8).
This may be attributable to the fact that while 1.sub.10mer,
1.sub.11mer, and 1 all have a net charge of +4, that of 1.sub.13mer
is increased to +5. As discussed previously, net charge can affect
cell selectivity, particularly against Gram-negative strains.
1.sub.11mer, with a CTLR of 0.36 and net charge of +4 represents
the optimum balance of potent antimicrobial activity and cell
selectivity from this group of molecules. However, the selectivity
ratios of 1.sub.10mer (SR.sub.E. coli>16), 1.sub.11mer
(SR.sub.E. coli=16), and 1.sub.13mer (SR.sub.E. coli=6.8) are all
superior to that of 1 (SR.sub.E. coli=3.3).
[0100] While proline monomers in naturally-occurring AMPs are known
to destabilize .alpha.-helical secondary structure and induce the
formation of helix-bend-helix motifs, here, proline is well
accommodated in the polyproline type-I-like peptoid helical
architecture. Because of proline's reduced hydrophobicity compared
to Nspe, substituting L-proline for a centrally-located hydrophobic
residue in ampetoid 1's sequence (variant called 1-Pro.sub.6) was
found to lower molecular hydrophobicity and improve selectivity. A
family of molecules was designed to evaluate how the relative
position and number of proline monomers affects potency and
selectivity, while maintaining constant CTLR and helicity. Similar
to 1-Pro.sub.6, 1-Pro.sub.3 and 1-Pro.sub.9 have a single proline
monomer substituted into the third and ninth positions of the
ampetoid 1 sequence, respectively. A fourth variant, 1-Pro.sub.3,9
incorporates two substituted proline monomers (FIG. 9 and Table 5).
The relative hydrophobicities of 1-Pro.sub.3, 1-Pro.sub.6, and
1-Pro.sub.9 are all similar (62.4%-63%), and reduced compared to 1
(65.1%); 1-Pro.sub.3,9 was found to be even less hydrophobic
(58.1%). CD spectroscopy in Tris buffer and both zwitterionic and
anionic lipids show that proline-containing ampetoids exhibit a
similar degree of helicity as does 1 (FIG. 13).
[0101] The activity of these variants against E. coli scales with
hydrophobicity; 1-Pro.sub.3, 1-Pro.sub.6, and 1-Pro.sub.9 exhibited
uniformly reduced potencies against E. coli (12.5 .mu.M) compared
to peptoid 1 (6.3 .mu.M). The potency of 1-Pro.sub.3,9 was lessened
further against E. coli (50 .mu.M). Because these variants also
exhibited reduced activity against mammalian cells, selectivity was
improved for all the mono-substituted variants (SR.sub.E.
coli=5.9-13) compared to 1 (SR.sub.E. coli=3.3).
[0102] An intriguing observation regarding the mono-substituted
proline-containing variants is that the relative position of the
proline monomer in the sequence affected cell selectivity.
1-Pro.sub.3, 1-Pro.sub.6, 1-Pro.sub.9 comprise a family of
molecules in which CTLR, net charge, and hydrophobicity are held
constant. Moreover, the degree of amphipathicity among these
variants is similar, based on the similarity of their CD spectra in
both aqueous buffer and lipid environments (FIG. 13). Notably,
however, shifting the proline from the N- to the C-terminal region
resulted in a progressive increase in selectivity against both
erythrocytes and NIH 3T3 cells (Table 6); whereas 1-Pro.sub.3 had
an HD.sub.10=74 .mu.M and ID.sub.50=12 .mu.M, those of 1-Pro.sub.9
were 165 .mu.M and 24 .mu.M, respectively. The resultant
selectivities monotonically increased from 1-Pro.sub.3 (SR.sub.E.
coli=5.9), to 1-Pro.sub.6 (SR.sub.E. coli=6.6) to 1-Pro.sub.9
(SR.sub.E. coli=13). A similar trend was observed for selectivity
ratios against B. subtilis. This suggests that ampetoids may have a
preferred orientation upon interacting with mammalian cells. If,
for example ampetoids interact with mammalian cells preferentially
in the C-terminal region, reducing hydrophobicity specifically in
that portion of the molecule could impair its activity against
mammalian cells and increase selectivity.
[0103] Another strategy for improving selectivity relates to a
family of ampetoids with less hydrophobic, achiral Npm side chains
in place of selected Nspe monomers in ampetoid 1 (FIG. 9 and Table
5). Because molecular chirality of peptoids is derived from the
chirality of the side chains rather than that of the backbone, a
change in the number of chiral monomers is expected to affect the
stability of the secondary structure. This family of molecules,
therefore, was designed to effect a range of decreased
hydrophobicities and helicities compared to 1, independent of any
change in CTLR and net charge constant. 1.sub.achiral has all eight
Nspe monomers replaced with Npm. Two variants, 1.sub.ach-Nspe.sub.2
and 1.sub.ach-Nspe.sub.12 each have only one chiral Nspe monomer at
the second and twelfth positions, respectively. Two other ampetoids
each contain four achiral Npm's, either aligned along the back
molecular face (1-Npm.sub.2,5,8,11), or distributed across both
hydrophobic molecular faces (1-Npm.sub.2,3,8,9) (FIG. 9).
[0104] The hydrophobicities of these compounds ranged from 59.8%
for 1.sub.achiral to 65.1% for peptoid 1 and generally increased
with Nspe content. Moreover, as shown in FIG. 14, the intensity of
the helical signal, which is correlated with amphipathicity,
decreased with Nspe content. The CD spectrum of 1.sub.achiral is
flat, which suggests a lack of stable secondary structure and
reduced molecular amphipathicity, while that of 1 exhibits the most
intense spectral extrema, and correspondingly, the most amphipathic
structure. Interestingly, 1.sub.ach-Nspe.sub.12 showed slightly
more intense CD spectra than isomerically related
1.sub.ach-Nspe.sub.2, an observation that further supports a
previous finding that the C-terminal position plays a particularly
important role in stabilizing peptoid helical structure.sup.57.
[0105] All variants with achiral side chains exhibit activities
similar to peptoid 1 against E. coli (MIC=6.3-12.5 .mu.M) and B.
subtilis (MIC=0.78-1.6 .mu.M), yet have substantially higher
selectivities for bacteria over mammalian cells (SR.sub.E.
coli=6.2-26; SR.sub.B. subtilis=24-235) (Table 6). In general,
selectivity increased with Npm content, a monomer substitution for
Nspe that simultaneously decreases amphipathicity and
hydrophobicity.
[0106] This family of variants provides further insight into how
hydrophobicity and helicity impact potency and selectivity.
Previous studies have shown that variants designed to be less
hydrophobic, (via incorporation of more polar histidine-like side
chains (e.g. 1-Nhis-6,12) or less hydrophobic L-proline monomers
(e.g. 1-Pro.sub.6) improved selectivity, but only at the expense of
reduced antimicrobial activity. Indeed, this observation also held
true for the proline-containing variants reported herein. The
activity and selectivity profiles of four molecules in this family,
however, demonstrate clearly that hydrophobicity can be reduced to
improve selectivity without compromising antimicrobial activity.
Variants 1.sub.ach-Nspe.sub.2, 1.sub.ach-NSpe.sub.12,
1-Npm.sub.2,5,8,11, and 1-Npm 2,3,8,9) demonstrate antimicrobial
activity equivalent to that of 1 (MIC.sub.E. coli=6.3 .mu.M), yet
significantly improved selectivities (SR.sub.E. coli=6.2-26)
compared to 1 (SR.sub.E. coli=3.3). The most overt difference
between using achiral Npm compared to Nhis or L-Pro monomers to
lower hydrophobicity is their effect on helicity. While both
1-Nhis.sub.6,12 and 1-Pro.sub.6 exhibited helicity equivalent to
that of 1, the substitution of Npm monomers resulted in a
progressive decrease in helical stability, as shown in FIG. 14.
This suggests that the reducing amphipathicity (correlated with
helicity) concomitantly with hydrophobicity provides a means of
improving selectivity without compromising antimicrobial
activity.
[0107] Comparison of the isomeric pairs within this group suggest
that the relative position of achiral monomers does not have a
significant effect on selectivity. For example, comparison of
1.sub.ach-Nspe.sub.2 (SR.sub.E. coli=25, ID.sub.50=11 .mu.M) and
1.sub.ach-Nspe.sub.12 (SR.sub.E. coli=26, ID.sub.50=15 .mu.M)
suggests that selectivity against both erythrocytes and NIH 3T3
cells was unaffected by the position of the one chiral Nspe monomer
in the sequence. Comparison of isomeric variants containing equal
numbers of Npm and Nspe monomers in different positions
(1-Npm.sub.2,5,8,11, and 1-Npm.sub.2,3,8,9) suggest that while
1-Npm.sub.2,5,8,11 (SR.sub.E. coli=14) was more selective against
erythrocytes than 1-Npm.sub.2,3,8,9 (SR.sub.E. coli=6.2), the
opposite trend was evident in selectivity against NIH 3T3 cells
(ID.sub.50=6.8 .mu.M and 15 .mu.M, respectively). Taken together,
these results suggest that there is no clear relationship between
the degree of selectivity and relative position of achiral monomers
within the ampetoid sequence.
[0108] 1.sub.enantiomer, a variant of peptoid 1 in which all Nspe
side chains were replaced with enantiomeric Nrpe monomers, has
previously been shown to exhibit left-handed helicity, but
antibacterial activities and cell selectivities congruent to those
of peptoid 1. Because peptoid secondary structure is dictated by
the chirality of its side chains, it is unclear what the resultant
secondary structure (and associated activity/selectivity profiles)
would be in a peptoid that included both enantiomeric side chains.
Moreover, diasteriomeric peptides that contain both D- and L-amino
acids were found to exhibit potent, broad-spectrum antimicrobial
activity and improved selectivity. Here, variants of 1 can contain
equal numbers of Nspe and Nrpe side chains in different
arrangements as shown in FIG. 9 and Table 5. 1-Nrpe.sub.3,6,9,12
and 1-Nrpe.sub.2,5,8,11 are enantiomers that have four
facially-aligned Nspe monomers replaced with enantiomeric Nrpe
monomers. 1-Nrpe.sub.2,3,5,6 has terminally segregated enantiomeric
monomers with Nrpe substitutions at positions 2, 3, 5, and 6 in the
N-terminal portion of the molecule (FIG. 9).
[0109] The antibacterial potencies and cell selectivity profiles of
these variants are very similar to that of peptoid 1, despite their
disparate secondary structures. FIG. 15 shows that in aqueous
buffer as well as both lipid environments, the enantiomeric
molecules, 1-Nrpe.sub.3,6,9,12 and 1-Nrpe.sub.2,5,8,11, yield
mirror image CD spectra. FIG. 15A shows that in buffer,
1-Nrpe.sub.3,6,9,12 and 1-Nrpe.sub.2,5,8,11 appear to adopt helical
secondary structures, with the overall handedness commensurate with
that of the C-terminal monomer. 1-Nrpe.sub.2,3,5,6, also appears to
adopt an overall right-handed spectra, but does not appear to be
strongly helical. In both zwitterionic and anionic lipids, however,
the CD of the facially-aligned 1-Nrpe.sub.3,6,9,12 and
1-Nrpe.sub.2,5,8,11 were markedly altered, suggesting a strong
interaction with lipids and a significant change in secondary
structure (FIGS. 15B and 15C).
[0110] The CD spectacle intensity for all Nrpe containing peptoids
are significantly reduced compared to that of 1, an observation
previously correlated with decreased amphipathicity. However,
because the CD spectra corresponding to variants in cell
membrane-mimetic lipid environments are not typical of a peptoid
helical secondary structure, it is not clear how amphipathicity is
affected in these molecules.
[0111] The effect of side chain chemistry on cell selectivity, was
evaluated with a family of ampetoids that have bulky, hydrophobic,
aliphatic Nsdp side chains in place of some or all of the aromatic
Nspe monomers in ampetoid 1 (FIG. 9 and Table 5). The previously
reported ampetoids containing aliphatic isoleucine-like Nssb side
chains were selective, but exhibited reduced activity, particularly
against Gram-negative bacterial strains. Incorporating the larger
and bulkier aliphatic side chains, 1-methylbutyl glycine (Nsmb),
led to increased antibacterial potency, but also reduced
selectivity. The dipropyl glycine (Nsdp) (FIG. 9) monomer used in
this family of molecules is an isomer of Nsmb and was selected to
evaluate if an aliphatic monomer with branched geometry could
improve potency while maintaining favorable selectivity. All
variants in this group had a CTLR (0.33) and net charge (+4)
equivalent to that of 1.
[0112] 1-Nsdp.sub.all has Nsdp substituted at all eight hydrophobic
monomers in the peptoid 1 sequence. 1-Nsdp.sub.2,5,8,11 and
1-Nsdp.sub.2,3,8,9 are isomers in which half of peptoid 1's Nspe
monomers were replaced with aliphatic Nsdp's. As shown in FIG. 9,
1-Nsdp.sub.2,5,8,11 exhibits four Nsdp monomers aligned along the
back molecular face, while the four Nsdp's included in
1-Nsdp.sub.2,3,8,9 are distributed across both hydrophobic faces.
Whereas 1-Nsdp.sub.2,5,8,11 contains segregated aromatic and
aliphatic faces, 1-Nsdp.sub.2,3,8,9 has a mixture of aromatic and
aliphatic monomers in both hydrophobic faces.
[0113] The CD spectra of right-handed helical peptoids with chiral,
aliphatic side chains have been shown to be distinctly different
from those with aromatic side chains in that the most pronounced
spectral feature is a maximum at 210 nm. Indeed, the spectrum of
1-Nsdp.sub.all exhibits this feature in both aqueous and lipid
environments (FIG. 16). 1-Nsdp.sub.2,5,8,11 and 1-Nsdp.sub.2,3,8,9,
both have spectral characteristics that appear to be a combination
of both the aliphatic and aromatic peptoid helical signals. While
the resultant "combined" CD spectra appear to be less intensely
helical compared to that of 1, it is unclear how the inclusion of
aliphatic and aromatic monomers affected molecular amphipathicity;
both 1-Nsdp.sub.all and 1 exhibit disparate, yet helical CD
spectra.
[0114] The increase in selectivity of 1-Nsdp.sub.all (SR.sub.E.
coli>8) was realized at the cost of significantly reduced
activity against E. coli of (25 .mu.M) in comparison to 1
(SR.sub.E. coli=3.3; MIC.sub.E. coli=6.3 .mu.M). 1-Nsdp.sub.all was
highly potent against B. subtilis (0.78 .mu.M), resulting in a
selectivity ratio of 256 (Table 6).
[0115] The sequences containing equal numbers of aromatic and
aliphatic monomers, 1-Nspd.sub.2,5,8,11 and 1-Nsdp.sub.2,3,8,9
exhibited slightly reduced antimicrobial activity (MIC.sub.E.
coli=12.5 .mu.M) and improved selectivity (SR.sub.E. coli=9.7 and
6.2, respectively) compared to 1 (MIC.sub.E. coli=6.25 .mu.M,
SR.sub.E. coli=3.3). While the slightly reduced hydrophobicity
could be in part responsible for the improved selectivity,
comparison with other variants suggests that the aliphatic side
chain chemistry also plays an important role in its activity
profile. Unlike what has been observed in some sequences that
contain all aromatic hydrophobic monomers, the incorporation of
aliphatic side chains appears to improve selectivity, but only at
the expense of antimicrobial activity. For example variants
1-Npm.sub.2,3,8,9 (63.3% acetonitrile), 1-Npm.sub.2,5,8,11 (63.6%
acetonitrile) and 1.sub.11mer (63.5% acetonitrile) all exhibit
hydrophobicities comparable to that of 1-Nsdp.sub.2,5,8,11 (63.8%
acetonitrile). The balance of the antimicrobial activity and
selectivity profiles of 1-Nsdp.sub.2,5,8,11, however, is less
optimal than for sequences containing only these aromatic side
chains; compared with these three molecules (MIC.sub.E. coli=6.3
.mu.M, SR.sub.E. coli=6.2-16) the antimicrobial activity of
1-Nsdp.sub.2,5,8,11 is reduced (MIC.sub.E. coli=12.5 .mu.M) and
selectivity (SR.sub.E. coli=9.7) comparable. The overall
hydrophobicities of these three molecules (63.2%-64.7%) were
slightly reduced compared to 1 (65.1%). The reduced hydrophobicity
of 1-Nsdp.sub.all (63.2%) could in part be responsible for its
reduced activity, however the lack of aromatic side chains may also
play a role. Variants containing both aliphatic and aromatic side
chains appear to provide a balance of low antimicrobial activity
(MIC.sub.E. coli=12.5 .mu.M) and improved selectivity compared to 1
(SR.sub.E. coli=6.2-12): 1-Nsdp.sub.2,3,8,9 (SR.sub.E.
coli=6.2-12), and 1-Nsdp.sub.2,5,8,11 (SR.sub.E. coli>9.7).
[0116] The antimicrobial activity of selected ampetoids and
comparator peptide pexiganan was tested against 16
clinically-relevant BSL2 bacterial strains. The panel of bacterial
strains included seven Gram-negative species (Proteus vulgaris,
Pseudomonas aeruginosa, Proteus mirabilis, Klebsiella pneumonia,
Enterobacter aerogenes, Escherichia coli, and Serratia marcescens)
and nine strains from three Gram-positive species (Staphylococcus
aureus, Enterococcus faecalis, and Enterococcus faecium). Ampetoids
1,1-Pro.sub.6, 1-Pro.sub.9, 1.sub.achrial, 1-Npm.sub.2,5,8,11, and
1-Nsdp.sub.2,5,8,11 were tested against these organisms. The MICs
(expressed in .mu.g/mL) are shown in Table 7A and corresponding
selectivity ratios (quotient of 10% hemolytic dose and MIC) are
presented in Table 7B.
[0117] The activities of the six peptoids tested were all similar
to that those of pexiganan against P. vulgaris (MIC=32-64
.mu.g/mL), K. pneumoniae (MIC=8-16 .mu.g/mL), and E. coli (4-16
.mu.g/mL). Against P. mirabilis and S. marcescens, pexiganan and
all peptoids tested were inactive (MIC.gtoreq.128). The activities
of ampetoids tested against BSL2 Gram-positive strains, however,
compared very favorably to those of pexiganan. The MIC of pexiganan
against the six strains of S. aureus tested ranged from 8-64
.mu.g/mL, whereas those of all the peptoids tested ranged from 4-16
.mu.g/mL. Interestingly, 1 was uniquely active against both strains
of E. faecalis (MIC=4-8 .mu.g/mL), compared to other ampetoids
(MIC=8-64 .mu.g/mL) and pexiganan (MIC=32-128 .mu.g/mL). All
compounds were equally potent against E. faecium (MIC=4
.mu.g/mL).
TABLE-US-00007 TABLES 7A-B Broad spectrum activity and selectivity
of selected ampetoids and pexiganan. (A) MICs (in .mu.g/mL) of
selected ampetoids against BSL2 microbial strains. (B) Selectivity
ratios are defined as the 10% hemolytic dose divided by the MIC for
the organism of interest. The hemolytic dose (.mu.g/mL) of each
compound (Table 2-2) was multiplied by its molecular weight (Table
2-1) to calculate the selectivity ratio. A Minimum inhibitory
concentration (MIC) (.mu.g/mL) Bacterial organism Pex. 1
1-Pro.sub.6 1-Pro.sub.9 1.sub.achiral 1-Npm.sub.2,5,8,11
1-Nsdp.sub.2,5,8,11 P. vulgaris ATCC 32 32 32 64 32 64 32 49132 P.
aeruginosa ATCC 4 8 32 32 16 64 16 27853* P. mirabilis ATCC >128
>128 >128 >128 >128 >128 >128 35659 K. pneumoniae
8 16 16 16 8 16 8 ATCC 33495 E. aerogenes ATCC 32 16 128 64 128 64
32 35029 E. coli ATCC 25922* 8 4 16 8 16 8 8 S. marcescens ATCC
>128 128 >128 >128 >128 >128 >128 13880 S. aureus
ATCC 29213* 32 4 8 16 8 8 8 VAN1.sup..sctn..dagger. 16 4 8 8 8 8 8
VAN2.sup..sctn..dagger. 8 4 8 8 8 8 8 NRS100 (COL).sup..sctn. 16 4
8 8 8 8 8 NRS119.sup..dagger-dbl. 64 4 16 16 16 16 16
NRS120.sup..dagger-dbl. 64 8 16 16 16 16 16 E. faecalis ATCC 29212
32 4 8 8 16 8 8 99 128 8 64 64 64 64 64 E. faecium 106* 4 4 4 4 4 4
4 B Selectivity ratio (SR) Bacterial organism Pex. 1 1-Pro.sub.6
1-Pro.sub.9 1.sub.achiral 1-Npm.sub.2,5,8,11 1-Nsdp.sub.2,5,8,11 P.
vulgaris ATCC 49132 5.7 1.2 4.6 5.4 9.8 2.4 5.8 P. aeruginosa ATCC
46 4.8 4.6 9.0 19 2.4 12 27853* P. mirabilis ATCC 35659 <1.4
<0.3 <1.1 <2.3 <2.4 <1.2 <1.5 K. pneumoniae ATCC
23 2.4 9.1 18 39 9.6 23 33495 E. aerogenes ATCC 5.7 2.4 1.1 5.4 2.4
2.4 5.8 35029 E. coli ATCC 25922* 23 9.5 9.1 36 19 19 23 S.
marcescens ATCC <1.4 0.3 <1.1 <2.3 2.4 <1.2 <1.5
13880 S. aureus ATCC 29213* 5.7 9.5 18 18 39 19 23
VAN1.sup..sctn..dagger. 11 9.5 18 36 39 19 23
VAN2.sup..sctn..dagger. 23 9.5 18 36 39 19 23 NRS100
(COL).sup..sctn. 11 9.5 18 36 39 19 23 NRS119.sup..dagger-dbl. 2.8
9.5 9.1 18 19 9.6 12 NRS120.sup..dagger-dbl. 2.8 4.8 9.1 18 19 9.6
23 E. faecalis ATCC 29212 5.7 9.5 18 36 19 19 23 99 1.4 4.8 2.3 5.4
4.9 2.4 2.9 E. faecium 106* 46 9.5 36 72 79 38 46 Notes: *indicates
NCCLS recommended standard strain; .sup..sctn.indicates
methicillian-resistant S. aureus (MRSA) strain;
.sup..dagger.indicates vancomycin-resistant strain;
.sup..dagger-dbl.indicates linezolid-resistant strain.
[0118] The selectivity ratios presented in Table 7B show that
against most Gram-negative bacterial species, at least one ampetoid
had greater selectivity compared to pexiganan; the most favorable
selectivity ratio against each Gram-negative species is shown in
boldface type in Table 7B. 1.sub.archiral, 1-Pro.sub.9, and
1-Nsdp.sub.2,5,8,11 were the most selective against selected MDR
bacterial strains. Against Gram-positive strains, ampetoids more
consistently demonstrated improved selectivity compared to
pexiganan or ampetoid 1. Against all S. aureus strains, for
example, the selectivity ratios of pexiganan ranged from 2.8-23,
ampetoid 1 ranged from 4.8-9.5, and those of the panel of more
selective ampetoids ranged from 9.1-39.
[0119] As shown above, ampetoids are a new class of AMP mimics that
have been shown to exhibit potent, broad-spectrum antimicrobial
activity and appear to use mechanisms of action similar to their
natural counterparts. An understanding of factors that influence
cell selectivity can be used in the context of corresponding
pharmaceutical agents. Accordingly, a library of ampetoids was
designed to explore how and to what extent various physicochemical
properties and structural motifs influenced their cell
selectivity.
[0120] To broaden understanding of how ampetoids may be affecting
different types of mammalian cells, the hemolytic dose as well as
the metabolic inhibitory dose was determined against NIH 3T3 mouse
fibroblast cells. While the hemolytic and inhibitory activities
exhibited similar trends for many ampetoids, the hemolytic dose was
consistently higher than the inhibitory dose for the same compound.
This is a trend that has also been reported for pexiganan as well
as other AMP mimics. It is possible that the disparity between the
hemolytic and inhibitory doses is due in part to differences in
membrane composition between the two cell types. The cholesterol
content of erythrocyte membranes, for example is approximately 230
.mu.g/mg protein, whereas NIH 3T3 cholesterol content has been
reported as only 30.5 .mu.g/mg protein. The increased cholesterol
content of erythrocytes may affect the rigidity of the membrane and
offer increased resistance to membrane-active antimicrobial agents
compared to NIH 3T3 cells.
[0121] Another disparity between hemolysis and MTS assays is that
they differ in terms of the measure used to quantify cytotoxicity;
while hemolysis measures the ampetoid dose needed to lyse
erythrocytes, the MTS assay quantifies the dose needed to inhibit
cellular metabolism, measured indirectly by the amount of NADH
produced by the cell population. It is reasonable to expect,
therefore that a dose needed to lyse a cell membrane would not
necessarily be equivalent to the dose required to inhibit cellular
metabolism. Whereas lysis implies membrane disruption activity,
interference with cellular metabolism implies intracellular
targets. While animal testing would be required to determine a true
therapeutic index for these molecules, hemolysis and MTS assay
results together suggest that the relative effect of many ampetoids
against both cell types may be similar.
[0122] This library of ampetoids was designed to include members
that exhibit a variety of structural motifs and possess
physicochemical properties that span a wide range of values.
Different ampetoids demonstrated promising activities against the
various Gram-negative strains, but were most potent against K.
pneumonia (ATCC 33495) and E. coli (ATCC 25922). It is particularly
notable that all 26 ampetoid variants were consistently very potent
against the Gram-positive screening organism, B. subtilis. The
broad-spectrum testing results of selected ampetoids against MDR
strains of S. aureus, E. faecalis, and E. faecium show that
ampetoids are also potent against MRSA (4-8 .mu.M) as well as
vancomycin- and linezolid-resistant organisms (4-8 .mu.M and 4-16
.mu.M, respectively). The corresponding selectivity ratios of
1.sub.achiral (19-39) and 1-Pro.sub.9 (18-36) against MDR S. aureus
strains are particularly favorable compared to those of pexiganan
(2.8-23) or ampetoid 1 (4.8-9.5). The burden of MDR Gram-positive
infections on the healthcare system is significant and only
increasing; these results suggest that ampetoids can be a viable
alternative to conventional therapies to address this unmet
clinical need.
[0123] Structure-activity studies have shown that the antibacterial
activity and selectivity profiles of ampetoids are governed by the
physicochemical properties that, in a similar manner, dictate the
activity and selectivity of AMPs. The structure-activity
relationships gleaned from these studies not only provide further
evidence to re-affirm these findings in ampetoids, but also provide
additional insight into principles that influence how more subtle
changes related to the number, sequence position, arrangement and
chemical structure of specific structural moieties influence
activity and selectivity.
[0124] Potent, but non-selective AMPs and ampetoids tend to be
hydrophobic and adopt well-defined amphipathic structures, while
more selective AMPs and ampetoids are typically highly cationic,
exhibit only moderate hydrophobicity, and are often less
amphipathic. As discussed previously, the activity and selectivity
results of this study, which included several new ampetoid
sequences, re-affirm these general relationships in several ways:
(1) Comparison of the less amphipathic, more selective 1.sub.ser to
1; (2) Comparison of the highly charged, less amphipathic variants
(1-Nlys.sub.5,11, 1B-Nlys.sub.4,10, and
1B.sub.15mer--Nlys.sub.4,10, 1B-Nys.sub.4,6,10 and
1B.sub.15mer--Nlys.sub.4,6,10) compared to 1, (3) Comparison of
less hydrophobic proline-containing variants (1-Pro.sub.3,
1-Pro.sub.6, 1-Pro.sub.9, 1-Pro.sub.3,9) to 1 (4) Comparison among
achiral variants (1.sub.achiral, 1.sub.ach-Nspe.sub.2,
1.sub.ach-Nspe.sub.12, 1-Npm.sub.2,3,8,9, 1-Npm.sub.2,5,8,11),
which exhibit a range of hydrophobicities and amphipathicities that
scale with selectivity. Taking a closer look at more specific
structural characteristics of the ampetoids included in this study,
the influence of subtle molecular changes on activity and
selectivity can be elucidated.
[0125] Comparison among the mono-substituted proline monomers
suggests that the position of monomers along the length of the
molecule and can impact selectivity. The mono-substituted proline
monomers exhibited progressively increased selectivity as the less
hydrophobic proline monomer was moved from the N-terminal toward
the C-terminal region.
[0126] Trends observed in the selectivity profiles of sequence
register variants (1, 1B and 1C) as well as length variants,
1.sub.13mer, suggest that monomer position with respect to the
termini also influences selectivity. All variants are similarly
potent against E. coli: the MIC.sub.E. coli of 1, 1B, and 1C was
6.3 .mu.M, while that of 1.sub.13mer was improved by one dilution
(3.1 .mu.M). While 1 and 1C each have one charged Nlys monomer at
the N and C-termini, respectively, 1B has hydrophobic Nspe monomers
at both termini. Conversely, 1.sub.13mer has charged Nlys monomers
at both terminal positions. Consider these molecules in two groups:
(1) those with both termini charged or hydrophobic (1B and
1.sub.13mer), and (2) those with one charged and one hydrophobic
terminus (1 and 1C). Interestingly, the hydrophobicity of those
with dissimilar termini, 1 (65.1% acetonitrile) and 1C (64.8%
acetonitrile) is greater than that of variants with like monomers
at terminal positions, 1B (63.4% acetonitrile) and 1.sub.13mer
(62.8% acetonitrile). Correspondingly, the selectivity of the less
hydrophobic variants 1B and 1.sub.13mer (SR.sub.E. coli=6.8-8.7,
SR.sub.B. subtilis=27-34) is improved compared to that of 1 and
1C(SR.sub.E. coli=3.3-4.0, SR.sub.B. subtilis=13-16). This suggests
that having similarly charged termini (either both hydrophobic or
both positively charged) reduces molecular hydrophobicity and
results in an improvement in selectivity. This could be related to
a similar phenomenon reported for antimicrobial peptide analogs of
magainins, which found that the relative position of hydrophobic
monomers in the sequence can impact resultant hydrophobicity and
cell selectivity. These results indicate that a strategy for
improving selectivity, while maintaining antimicrobial activity, is
to design the sequence with similarly charged or similarly
hydrophobic terminal monomers.
[0127] As discussed previously, the achiral family of variants
provide evidence that suggests the incorporation of achiral Npm
hydrophobic monomers in place of chiral Nspe monomers is another
means of improving selectivity without compromising antimicrobial
activity. Variants in which as many as seven of the eight Nspe's in
ampetoid 1's structure exhibited equivalent activity and
significantly improved selectivity. It appears that the decrease
amphipathicity that occurs concomitantly with increased Npm content
results in a favorable selectivity profile. It is possible that the
less rigid structure of ampetoids with increased achiral monomer
content is less able to penetrate the rigid cell membranes of
mammalian cells.
[0128] A third way in which this library of compounds was designed
to affect selectivity is through increased CTLR. The increased
charge density variants, with CTLRs in the range of 0.47-0.58
(compared to 0.33 for ampetoid 1), were at best mildly active
against E. coli (MIC=50 to >100 .mu.M). Length variants,
however, which were designed to effect a change in CTLR over a
lower range (0.33-0.40). Of these variants, 1.sub.11mer was found
to exhibit the most optimum balance of CTLR (0.36) and sufficient
hydrophobicity to permeabilize bacterial membranes (63.5%) at a low
minimum inhibitory concentrations (MIC.sub.E. coli=6.3 .mu.M).
[0129] Two other means of reducing hydrophobicity, substituting in
L-proline content or aliphantic Nsdp monomers, are less favorable
because they improve selectivity at the expense of antimicrobial
activity against Gram-negative bacteria. The addition of one
proline monomer (1-Pro.sub.3, 1-Pro.sub.6, and 1-Pro.sub.9,
MIC.sub.E. coli=12.5 .mu.M) and two proline monomers
(1-Pro.sub.3,9, MIC.sub.E. coli=50 .mu.M) progressively decreased
activity compared to that of ampetoid 1 (MIC.sub.E. coli=6.3
.mu.M). Molecules with four (1-Nsdp.sub.2,3,8,9 and
1-Nsdp.sub.2,5,8,11, MIC.sub.E. coli=12.5 .mu.M) and eight
(1-Nsdp.sub.all, MIC.sub.E. coli=25 .mu.M) aliphatic monomers
exhibited a similar trend in reduced antimicrobial activity.
[0130] Another observation is evident from the characterization of
1.sub.achiral, 1-Nrpe.sub.3,6,9,12, and 1-Nrpe.sub.2,5,8,11, which
suggests that ampetoids can exhibit potent antimicrobial activity
without necessarily adopting a stable helical secondary structure.
As the name suggests, 1.sub.achiral is devoid of any chiral
monomers and is thus not optically active; the resultant CD spectra
in aqueous buffer and lipid environments is flat (FIG. 13). While
it is conceivable that 1.sub.achiral could transiently adopt a
helical structure of either handedness, there does not appear to be
external or intrinsic force to stabilize its structure.
1-Nrpe.sub.3,6,9,12, and 1-Nrpe.sub.2,5,8,11, on the other hand,
are enantiomeric molecules that contain equal numbers of Nspe and
Nrpe hydrophobic aromatic side chains. Interestingly, the overall
chirality of these monomers appears to be dictated by the chirality
of the monomer in the 12.sup.th position, a finding commensurate
with the observation that 1.sub.ach-Nspe.sub.12 exhibits a larger
degree of right-handed chirality than does 1.sub.ach-Nspe.sub.2.
This provides further evidence that the C-terminal monomer heavily
influences structural stability. In buffer, both of these variants
produce CD spectra that resemble that of a peptoid
polyproline-type-I-like structure with the extrema normally at 202
nm blue-shifted to approximately 195 nm (FIG. 15). In neutral
POPC/cholesterol lipids, however, the extrema at 220 nm is greatly
diminished, and in POPE/POPG SUVs, this feature is completely
eliminated. This marked change in CD spectra suggests that
1-Nrpe.sub.3,6,9,12, and 1-Nrpe.sub.2,5,8,11 interact strongly with
both of these lipid mixtures such that their overall structure is
significantly altered. Taken together, it is interesting that
1.sub.achiral, which appears to lack a stable secondary structure,
as well as 1-Nrpe.sub.3,6,9,12, and 1-Nrpe.sub.2,5,8,11, which have
a CD spectra in lipids that are distinct from that of a canonical
peptoid polyproline-type-I-like, are all equally potent as ampetoid
1 against E. coli (MIC=6.3 .mu.M). A stable helical secondary
structure does not appear to be necessary for ampetoid
antimicrobial activity. This finding that a stable helical
secondary structure does not appear to be necessary for ampetoid
antimicrobial activity goes beyond previous findings, which
suggested that helicity is important only as a means of organizing
an amphipathic structure.
[0131] A pair of molecules was designed to evaluate if the facial
segregation aliphatic and aromatic hydrophobic monomers impacts
selectivity. While 1-Nsdp.sub.2,5,8,11 has four substituted
aliphatic monomers aligned along molecular faces,
1-Nsdp.sub.2,3,8,9 has a mixture of aliphatic and aromatic
hydrophobic monomers on both faces (FIG. 9). The facially-aligned
isomer 1-Nsdp.sub.2,5,8,11 (SR.sub.E. coli=9.7; SR.sub.B.
subtilis=142) was more selective than its facially-distributed
counterparts (1-Nsdp.sub.2,3,8,9-SR.sub.E. coli=6.2, SR.sub.B.
subtilis=99). While these isomers exhibit similar net charges (both
+4), CTLRs (both 0.33), and hydrophobicities (63.8%-64.7%
acetonitrile), a notable difference between them is that while the
arrangement of hydrophobic monomers on 1-Nsdp.sub.2,5,8,11,
preserved one wholly aromatic face, that of 1-Nsdp.sub.2,3,8,9
exhibits no completely aromatic face. It has been shown previously
that the inclusion of at least one aromatic face increases helical
stability.sup.57; the more intense helicity of 1-Nsdp.sub.2,5,8,11
compared to 1-Nsdp.sub.2,3,8,9 in both zwitterionic and anionic
lipid mixtures supports this observation (FIG. 16). This suggests
that preservation of at least one ampetoid aromatic face may
increase its selectivity independent of changes in other
physicochemical parameters.
[0132] The most promising therapeutic agents exhibit are highly
potent against bacteria and are nontoxic to mammalian cells. This
relationship is depicted graphically in FIG. 17, in which the
hemolytic dose is plotted versus the E. coli (FIG. 17A) and B.
subtilis (FIG. 17B) minimum inhibitory concentrations for selected
peptoids. Peptoid 1 and pexiganan are depicted by red markers for
reference. Those peptoids located in the lower right coordinate
space have the most promising therapeutic potential. Many compounds
reported herein exhibit more favorable activity profiles than
peptoid I and pexiganan. It is most notable that the increased
charge density variants, which were completely non-hemolytic,
demonstrated a marked improvement in activity profile against
Gram-positive B. subtilis.
[0133] As shown, ampetoids are a promising class of AMP mimics that
exhibit potent, broad-spectrum activity, particularly against many
multi-drug resistant Gram-positive organisms. Of the 26 sequences
presented here, 17 demonstrate improved selectivity for E. coli
compared to the basis sequence, 1. The structure-activity
relationships derived from this library of compounds reaffirm and
extend the analogy between the mechanism of action of AMPs and
ampetoids. Selective ampetoids tended to be only moderately
hydrophobic and amphipathic, while non-selective ampetoids were
highly hydrophobic and exhibit more highly amphipathic structures.
The relationships among ampetoid variants in this library also
point to the effects of how more subtle changes in sequence, side
chain chemistry, and monomer position effect selectivity. Three
strategies to improve ampetoid selectivity without compromising
selectivity include (1) Positioning of similarly cationic or
hydrophobic monomers at the sequence terminal positions, (2)
Inclusion of hydrophobic achiral Npm monomers in place of Nspe
monomers (3) Optimizing the CTLR while retaining sufficient
hydrophobicity to permeabilize bacterial cell membranes. Two
approaches, specifically designed to reduce hydrophobicity effected
the desired outcome of improving selectivity, but only at the cost
of reduced antimicrobial activity. This less optimal activity
profile resulted from (1) Substitution of less hydrophobic
L-proline monomers as well as (2) Substitution of aliphatic Nsdp
monomers. Interestingly, while the relative position along the
helix of some monomers played a role in selectivity (e.g.
mono-proline substituted variants), this was not always the case
(e.g. achiral variants). The preservation of at least one aromatic
face may also play a role in increasing selectivity. Lastly, thus
study provides evidence that antimicrobial activity can be
maintained in ampetoids which lack a stable secondary structure
(1.sub.achiral) or appear to adopt a secondary structure different
from that of the canonical peptoid helix (1-Nrpe.sub.2,5,8,11 and
1-Nrpe.sub.3,6,9,12). Because peptoids are sequence-specific
biopolymers that can be made from a diversity of primary amines, it
is conceivable that ampetoid potency and selectivity could be
finely tuned to fight specific, clinically-relevant organisms. The
design heuristics established herein may aid in the design of
potent, yet selective, future generations of ampetoids.
Examples of the Invention
[0134] The following non-limiting examples and data illustrate
various aspects and features relating to the compounds and/or
methods of the present invention, including the preparation of
various helical peptoid compounds, as are available through the
synthetic methodology described herein. In comparison with the
prior art, the present peptoid compounds provide results and data
which are surprising, unexpected and contrary to the prior art.
While the utility of this invention is illustrated through the use
of several compounds, it will be understood by those skilled in the
art that comparable results are obtainable with various other
compounds, peptoid lengths, residue sequences and/or N-pendant side
chains, as are commensurate with the scope of this invention.
Example 1
[0135] Synthesis and purification. Peptoids were synthesized either
using an ABI 433A peptide synthesizer or a parallel synthesis robot
on Rink amide resin according to the submonomer method. (See, e.g.,
Zuckermann, R. N., Kerr, J. M., Kent, S. B. H., & Moos, W. H.
(1992) J. Am. Chem. Soc., 114, 10646-10647.) Briefly, the amide on
the nascent chain is bromoacetylated, followed by S.sub.N2
displacement of bromide by a primary amine to form the side chain.
Peptides were synthesized using standard Fmoc chemistry. Following
synthesis, peptoids and peptides were cleaved and deprotected in
trifluoroacetic acid (TFA):triisopropylsilane:water (95:2.5:2.5 by
vol.) for 10 min. Compounds were purified to >97% homogeneity by
RP-HPLC on a C18 column with a linear acetonitrile/water (0.1% TFA)
gradient. Mass spectrometry was used to confirm the molecular
weight of the purified product.
[0136] The submonomer method is illustrated graphically in FIG. 8.
Each monomer of the growing peptoid polymer is assembled in two
steps, using two readily available submonomeric units. Rink amide
resin is bromoacetylated, using diisopropylcarbodiimide-activated
bromoacetic acid. Next, the bromoacetylated resin undergoes
S.sub.N2 displacement of bromide by a primary amine, which
introduces the desired side chain. Hundreds of potential amine
submonomers and corresponding side chains are commercially or
synthetically available. As a result, (1) the synthesis of peptoids
by the submonomer protocol provides facile access to sequences of
greater chemical diversity than readily obtained via the monomer
approach; and (2) more directly applicable to this invention, the
biomimetic peptoids are limited only by sequence order, length
and/or N-pendant side chain structure sufficient to provide desired
antibacterial activity.
[0137] More specifically, Rink Amide resin
(4-(2',4'-Dimethoxyphenyl-(9-fluorenylmethyl
oxycarbonyl)-aminomethyl)-phenoxy resin, 0.25 mmol; Novabiochem)
can be initially swelled for 30 min in CH.sub.2Cl.sub.2. Following
the resin swelling, the 9-fluorenylmethyloxycarbonyl (Fmoc)
protecting group is removed by treatment with 20% piperidine
solution in 1-methyl-2-pyrrolidone (NMP). The resin-bound
deprotected amine is then bromoacetylated by reaction with 4.2 ml
of 1.2 M bromoacetic acid (50 mmol) in N,N-dimethylformamide (DMF)
and 1.0 ml (11 mmol) neat N,N'-diisopropylcarbodiimide (DIC) for 60
minutes at room temperature with constant mixing. Next, the resin
is rinsed with DMF (3.times.10 ml), followed by NMP rinses
(3.times.10 ml). 6 ml of a 1 M solution (6 mmol) of a primary amine
"submonomer" (see below) in either NMP or CH.sub.2Cl.sub.2 reacted
with the resin-bound bromoacetyl moiety, displacing bromide. A
protected submonomer (N-tert-butoxycarbonyl-1,4-butanediamine) is
synthesized in order to create the N-(4-aminobutyl)glycine residue
(Nlys). The resin is then rinsed again with NMP (3.times.10 ml)
followed by DMF (3.times.10 ml). The product of these two reactions
generates a peptoid "residue", the identity of which depended upon
the submonomer amine employed. Peptoids are elongated by this
submonomer method until the desired chain-length was attained.
[0138] Following synthesis, peptoid oligomers can be cleaved from
the resin, simultaneously removing the tert-butoxycarbonyl (Boc)
protecting group from Nlys residues, by treatment with 10 ml
2,2,2-trifluoroacetic acid (TFA)/triisopropylsilane/H.sub.2O
(95:2.5:2.5 by volume) for 30 min. The cleavage mixture is then
diluted with 25 ml 50% aqueous acetonitrile, frozen, and
lyophilized. Dilution and lyophilization is repeated twice more in
order to remove excess TFA. Subsequent to cleavage, peptoids are
each purified to >97% homogeneity by preparative scale
reversed-phase HPLC using a Waters Prep LC 4000 system, with Waters
2487 dual-wavelength UV detection, and gradient elution (solvent A,
0.1 vol % trifluoroacetic acid (TFA) in water; solvent B, 0.1 vol %
TFA in acetonitrile) through a Vydac (Hysperia, Calif.) 214TP101550
C4 peptide/protein column (10-15 .mu.m, 300 .ANG., 5.times.25 cm).
Following prep HPLC, peptoid purities and crude yields were
determined from analytical scale HPLC, performed with a Waters 2695
Separations Module with a Waters 2487 dual-wavelength UV detector
and gradient elution (solvent A, 0.1 vol % TFA in water; solvent B,
0.1 vol % TFA in acetonitrile) through a Vydac C4 214TP53
peptide/protein column (5 .mu.m, 300 .ANG., 3.2.times.250 mm). The
precise gradient employed for HPLC depended on the identity and
hydrophobicity of the oligomer in question. The composition of the
HPLC solvent at gradient elution is an indication of this
hydrophobicity, and is provided in Table 1. All analytical HPLC was
performed at 0.5 ml/min flow and 58.degree. C. Preparative HPLC was
performed at 50 ml/min flow and room temperature.
[0139] Such synthesis and characterization are also described in
U.S. Pat. No. 6,887,845, the entirety of which is incorporated
herein by reference. As illustrated therein and as would be
understood by those skilled in the art made aware of this
invention, the present N-substituted glycine residues and resulting
peptoid compounds are limited only by synthetic or commercial
availability of the corresponding amine reagents.
Example 2
[0140] SUV preparation. Lipid mixtures, either POPE/POPG (7:3) or
POPC/CH (1:1), were dissolved in chloroform, dried under N.sub.2,
and lyophilized overnight. The resulting lipid film was hydrated
with 10 mM Tris-HCl (pH 7.4) at 40.degree. C. for one hour. The
resulting multilamellar vesicle suspension was vortexed, then
sonicated at 40.degree. C. until the solution clarified to make
SUVs, which were used within 6 hours.
Example 3
[0141] CD spectroscopy. CD measurements were performed on a Jasco
715 spectropolarimeter, using a quartz cylindrical cell (path
length=0.02 cm), with 50 .mu.M peptoid in 10 mM Tris-HCl (pH 7.4)
and 5 mM lipids when SUVs were used. Scans were conducted at 100
nm/min between 185 and 280 nm with 0.2 nm data pitch, 1 nm
bandwidth, 2 s response, 100 mdeg sensitivity, and 40
accumulations.
Example 4
[0142] Antibacterial assays. MICs were determined according to CLSI
M7-A6 protocols in a 96-well microtiter plate. In test wells, 50
.mu.L bacterial inoculum (5.times.10.sup.5 CFU/ml) in
Mueller-Hinton broth (MHB) was added to 50 .mu.L peptoid solution
in MHB (prepared by 1:2 serial dilutions). Positive controls
contained 50 .mu.L inoculum and 50 L MHB without peptoid. The MIC
was defined as the lowest concentration of peptoid that completely
inhibited bacterial growth after incubation at 35.degree. C. for 16
h. MIC values reported were reproducible between three independent
experimental replicates, each consisting of two parallel trials.
Broad-spectrum antimicrobial susceptibility testing was performed
against BSL2 pathogens by Nova Biologicals, Inc. (Conroe,
Tex.).
Example 5
[0143] Antifungal Assays. MICs were determined using the broth
microdilution assay given by CLSI M27-A2 protocols. Candida
albicans (SC5314) was grown on Sabouraud dextrose agar for 24 hours
at 30.degree. C. Cells were suspended in 0.145 M saline, and the
cell concentration was adjusted to 3.times.10.sup.6 cells/mL. After
adjusting the cell concentration, the suspension was diluted 1:1000
with RPMI 1640 (with L-glutamine and without sodium bicarbonate,
Invitrogen) buffered with 0.145 M 3-(N-morpholino) propanesulfonic
acid (MOPS). Two-fold serial dilutions of peptoids, peptides, and
amphotericin B (Calbiochem) were prepared in RPMI 1640 and mixed
with an equal volume of the cell suspension in 96-well plates. The
final testing concentrations for the peptoids and peptides were
0.20 to 100 .mu.M, and concentrations for amphotericin B were 0.031
to 16 .mu.M. Growth controls and sterility controls were also
included. Plates were incubated for 48 hours at 35.degree. C., and
growth of C. albicans was inspected visually to determine the MICs.
The MIC for each compound was defined as the lowest concentration
with no visible fungal growth. Experiments were performed in
duplicate on two separate days.
Example 6
[0144] Hemolysis assays. Erythrocytes were isolated from freshly
drawn, heparanized human blood and resuspended to 20 vol % in PBS
(pH 7.4). In a 96-well microtiter plate, 100 L erythrocyte
suspension was added to 100 .mu.L peptoid solution in PBS (prepared
by 1:2 serial dilutions), or 100 .mu.L PBS in the case of negative
controls. 100% hemolysis wells contained 100 .mu.L blood cell
suspension with 100 .mu.L 0.2 vol % Triton X-100. The plate was
incubated for 1 h at 37.degree. C., then each well was diluted with
150 .mu.L PBS. The plate was then centrifuged at 1200 g for 15 min,
100 .mu.L of the supernatant from each well transferred to a fresh
microtiter plate, and A.sub.350 measured. Percent hemolysis was
determined as (A-A.sub.0)/(A.sub.total-A.sub.0).times.100, where A
is the absorbance of the test well, A.sub.0 the absorbance of the
negative controls, and A.sub.total the absorbance of 100% hemolysis
wells, all at 350 nm.
Example 7
[0145] MTS Assays. A549 carcinoma-derived lung epithelial cells
(ATTC CCL-185) were cultured in Ham's F12K media (ATTC, Manassas,
Va.). A peptoid solution plate (100 .mu.L/well) was prepared by
serial dilution of aqueous peptoid stocks in media. Peptoid
solutions were transferred onto a 96-well plate of day-old cell
monolayers containing 100 .mu.L/well media with .about.5000
cells/well. MTS reagent (Promega Corporation, Madison, Wis.) (40
.mu.L/well) was added to each well and the plate was incubated at
37.degree. C. for 3 h, after which absorbance at 490 nm was
determined. Percent inhibition was determined as [1-(A-A.sub.test
blank)/(A.sub.control-A.sub.blank)].times.100, where A is the
absorbance of the test well and A.sub.control the average
absorbance of wells with cells exposed to media and MTS (no
peptoid). A.sub.test blank (media, MTS, and peptoid) and
A.sub.blank (media and MTS) were background absorbances measured in
the absence of cells. The average of six replicates are reported,
and error bars show one standard deviation.
Example 8
[0146] Specular X-ray reflectivity. XR experiments were carried out
at the 9-ID beamline at the Advanced Photon Source, Argonne
National Laboratory (Argonne, Ill.). The custom-built Langmuir
trough was mounted in a helium-filled, sealed canister and equipped
with a moveable single barrier. The surface pressure was measured
using a Wilhelmy plate. Constant-pressure insertion experiments
were performed at room temperature on Dulbecco's PBS (D-PBS)
subphase. DPPG (Avanti Polar Lipids, Alabaster, Al) was dissolved
to a known concentration in 65/35 (v/v) chloroform/methanol, then
spread at the air-buffer interface using a glass syringe; organic
solvent was allowed to evaporate for 10 minutes. The monolayer was
compressed to the surface pressure thought to occur in cell
membranes, 30 mN/m, and XR was performed on the pure lipid layer.
Then, peptoid 1 dissolved in D-PBS was injected into the subphase
to a total concentration of 6.26 .mu.M (well above the MICs), and
allowed to insert for approximately 45 minutes, after which XR
measurements were again collected. The X-ray reflectivity (XR)
profile was determined by the Fourier transform of the gradient of
the electron density perpendicular to the interface. XR
measurements were carried out over a range of angles corresponding
to q.sub.z values of .about.0-0.6 .ANG..sup.-1, where
q.sub.z=(4.pi./.lamda.)sin(.alpha.), .lamda. is the wavelength, and
.alpha. is the angle.
Example 9
[0147] Murine intraperitoneal infection model. Bacteria was
prepared by inoculating 5 mL of MHB with a single colony of
Staphyloccus aureus (ATCC #25923) from a freshly streaked plate and
grown overnight at 37C. The following morning, the bacteria was
subcultured by diluting 1/3 in MHB and grown for approximately 1.5
hours. The bacteria were then diluted 10-fold in a 5% mucin in PBS
solution and thoroughly mixed. A sample was reserved to later
determine the amount of bacteria the mice received.
[0148] Mice were weighed, marked, and injected I.P. with 200 uL of
S. aureus inoculum. Four hours post infection, mice were treated
I.P. with 4 mg/kg peptoid (.about.100 ug per mouse). The infection
is allowed to proceed overnight. Mice were euthanized by CO.sub.2
asphyxiation after 24 hours, and the peritoneal cavity was exposed
and lavaged with 5 mL PBS. The lavage was mixed and reserved on ice
until plating. The lavages were diluted to 10.sup.-5 (in 1/10
increments) in PBS, and all dilutions were plated onto MH agar, in
duplicate. 50 uL of lavage was spot plated, allowed to dry, and
incubated overnight at 37.degree. C. Colonies were counted the
following day, and the CFU/ml of each sample was calculated. Plates
that had too many colonies to count were assigned an arbitrary
number of 1000 colonies.
Example 10
[0149] Compound synthesis and purification. Peptoids were
synthesized using an ABI 433A peptide synthesizer (Applied
Biosystems, Inc.) on Rink amide MBHA resin (Novabiochem, Inc.)
using the submonomer approach.sup.3. Briefly, bromoacetic acid,
activated by diisopropylcarbodiimide was used to form a
bromocetylated intermediate on the terminal amide group. Bromide
was then substituted with the desired primary amine through
S.sub.N2 displacement to build the peptoid chain. The amines used
in peptoid synthesis include benzylamine, octadecylamine,
(s)-(+)-2-amino-3-methylbutane, (s)-.alpha.-methylbenzylamine,
(r)-(.alpha.-methylbenzylamine (all purchased from Sigma-Aldrich),
and N-tert-butoxycarbonyl-1,4 diaminobutane (Nlys) that was made
using a published procedure.sup.83. Resin-bound peptoids were then
exposed to a mixture of trifluoroacetic acid (TFA):
triisopropylsilane:water (95:2.5:2.5, v:v:v) for ten minutes to
cleave peptoids from the solid phase. Peptoids were purified by
reversed-phase HPLC(RP-HPLC) (Waters Corporation) using a C18
column and a linear acetonitrile/water gradient. A final purity
greater than 97% as measured by analytical RP-HPLC (Waters
Corporation) was achieved, and the identity of each molecule was
checked using electrospray ionization mass spectrometry. All
reagents were purchased from Sigma Aldrich.
Example 11
[0150] Circular dichroism spectroscopy. A Jasco 715
spectropolarimeter was used to perform all CD measurements in a
cylindrical quartz cell with a path length of 0.02 cm. Measurements
were taken over the range of 190 nm to 280 nm at a scanning rate of
100 nm/min. Other parameters include data pitch of 0.2 nm,
bandwidth of 1 nm, response time of 2 seconds, and sensitivity of
100 mdeg. Compound concentration was 60 M in 10 mM Tris buffer (pH
7.4). For samples in the presence of SUVs, the lipid concentration
was 5 mM. 40 accumulations were collected for each sample.
Example 12
[0151] Screening antibacterial assays. MICs were determined in
96-well microtiter plates in accordance with CLSI M7-A6 protocols.
Peptoid solutions with 50 .mu.L total volume were prepared using
2:1 serial dilutions. 50 .mu.L of bacteria inoculum
(1.times.10.sup.6 CFU/mL) prepared in cation-adjusted
Mueller-Hinton broth (CAMHB) was added to test wells. Control wells
contained 100 .mu.L MHB (no growth) or 50 .mu.L inoculums with 50
.mu.L MHB with no peptoid. The MIC was taken as the lowest
concentration of peptoid that completely inhibited bacterial growth
after 16 hours of incubation at 35.degree. C. Reported values were
reproducible over three experiments, each containing two parallel
trials.
Example 13
[0152] Broad-spectrum antibacterial assays. MICs of compounds were
determined by microdilution procedure in Mueller-Hinton broth (MHB)
in accordance with CLSI M7-A6 protocols in a manner similar to that
described for the screen antibacterial assays. Inoculated
microtiter plates were incubated at 35.degree. C. for 24 hours
prior to the result being recorded. Four ATCC strains that were
used as standards are recommended by CLSI: Pseudomonas aeruginosa
ATCC 27853, Escherichia coli ATCC 25922, Staphylococcus aureus ATCC
29213 and Enterococcus faecalis ATCC 29212. Other strains from the
ATCC collection include Proteus vulgaris ATCC 49132, Proteus
mirabilis ATCC 35659, Klebsiella pneumoniae ATCC 33495,
Enterobacter aerogenes ATCC 35029 and Serratia marcescens ATCC
13880. S. aureus NRS100 (COL) is a well characterized
methicillian-resistant S. aureus (MRSA) strain. The strains S.
aureus VAN1 and S. aureus VAN2, vancomycin-resistant MRSA strains
that were isolated in Michigan and Pennsylvania, were the first
vancomycin-resistant strains clinically isolated. S. aureus NRS119
and S. aureus NRS120 are linezolid-resistant isolates from the
Network on Antimicrobial Resistance in S. aureus (NARSA)
collection. E. faecalis 99 and E. faecium 106 are
vancomycin-resistant enterococcal strains.
Example 14
[0153] Hemolysis assays. Erythrocytes were isolated from freshly
drawn, heparanized human blood and resuspended in PBS (pH 7.4) to
make a 20% volume suspension. Peptoid solutions were prepared by
serial dilution (2:1) in a 96-well microtiter plate. For test
wells, 100 .mu.L of erythrocyte suspension was added to 100 .mu.L
of peptoid solution in PBS; PBS without peptoid was used as the
negative control and 0.2 vol % Triton X-100 as the positive control
that indicates 100% hemolysis. After 1 hour incubation at
37.degree. C., each well was diluted with 150 .mu.L PBS. Plates
were then centrifuged at 1,200.times.g for 15 minutes to pellet the
cells. 30 .mu.L of the supernatant from each well were transferred
to the corresponding well of a second 96-well plate that contains
70 .mu.L PBS. Using a plate reader, the absorbance at 350 nm was
measured, and percent hemolysis was defined as
(A-A.sub.0)/(A.sub.total-A.sub.0).times.100, where A is the
absorbance of the test well, A.sub.0 the average absorbance of
negative controls, and A.sub.total the average absorbance of 100%
hemolysis wells.
Example 15
[0154] MTS assays. NIH/3T3 cells (ATCC Corporation) cultured at
37.degree. C. and 5% CO.sub.2 in complete Dulbecco's modified
eagle's media (CDMEM) supplemented with 1% sodium pyruvate, 1%
penicillin-streptomycin, 1.5 g/L NaHCO.sub.3, and 10% fetal bovine
serum. Cells were seeded at a density of 5,000 cells per well for
NIH/3T3 cells in 96-well plates (100 .mu.l total volume). A peptoid
solution plate (100 .mu.L total volume per well) was prepared by
serial dilution of aqueous peptoid stock solution in Hank's
balanced salt solution (HBSS) media. The day-old cell monolayers
were washed with HBSS and media was replaced with 100 .mu.L HBSS.
The contents of the peptoid solution plate were transferred onto
corresponding wells of the cell monolayer plate, and 40 .mu.L MTS
reagent (Promega, Inc.) was added to each well. After incubating
for 3 hours at 37.degree. C., absorbance at 490 nm was determined.
The percentage inhibition was determined as
[1-(A-A.sub.testblank)/(A.sub.control-A.sub.blank)].times.100,
where A is the absorbance of the test well and A.sub.control the
average absorbance of the wells with cells exposed to media and MTS
(no peptoid). A.sub.testblank (media, MTS, and peptoid) and
A.sub.blank (media and MTS) were measured as background absorbances
in the absence of cells. The average of six replicates is
reported.
* * * * *