U.S. patent application number 16/869745 was filed with the patent office on 2020-08-20 for compounds for inhibiting bacterial growth via phosphatidylglycerol binding.
The applicant listed for this patent is Wichita State University. Invention is credited to Dennis H. Burns, Douglas S. English.
Application Number | 20200261415 16/869745 |
Document ID | 20200261415 / US20200261415 |
Family ID | 1000004812893 |
Filed Date | 2020-08-20 |
Patent Application | download [pdf] |
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United States Patent
Application |
20200261415 |
Kind Code |
A1 |
Burns; Dennis H. ; et
al. |
August 20, 2020 |
COMPOUNDS FOR INHIBITING BACTERIAL GROWTH VIA PHOSPHATIDYLGLYCEROL
BINDING
Abstract
Antibacterial small molecule compounds, termed liptins, bind to
phosphatidylglycerol in bacterial plasma membranes. The small
molecule compounds comprise a three-dimensional complementary
binding pocket for phosphatidylglycerol, disrupting membrane
function in a bacteriostatic or bactericidal manner. Methods of
inhibiting bacterial growth and/or treating Gram-positive or
Gram-negative bacterial infection using such compounds are also
disclosed.
Inventors: |
Burns; Dennis H.; (Wichita,
KS) ; English; Douglas S.; (Derby, KS) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Wichita State University |
Wichita |
KS |
US |
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|
Family ID: |
1000004812893 |
Appl. No.: |
16/869745 |
Filed: |
May 8, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16048009 |
Jul 27, 2018 |
10668048 |
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16869745 |
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PCT/US2017/015428 |
Jan 27, 2017 |
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16048009 |
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62289027 |
Jan 29, 2016 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07D 487/22 20130101;
C07D 321/00 20130101; A01N 43/90 20130101; A61P 31/04 20180101;
A61K 31/395 20130101; A61K 31/407 20130101; A61K 31/357 20130101;
A01N 43/72 20130101; A01N 43/22 20130101; C07D 273/08 20130101 |
International
Class: |
A61K 31/407 20060101
A61K031/407; C07D 487/22 20060101 C07D487/22; A01N 43/90 20060101
A01N043/90; C07D 273/08 20060101 C07D273/08; C07D 321/00 20060101
C07D321/00; A01N 43/72 20060101 A01N043/72; A01N 43/22 20060101
A01N043/22; A61P 31/04 20060101 A61P031/04; A61K 31/357 20060101
A61K031/357; A61K 31/395 20060101 A61K031/395 |
Goverment Interests
FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was made with government support under # P20
GM103418 awarded by the National Institutes of Health. The United
States government has certain rights in the invention.
Claims
1. An antibacterial small molecule compound that binds to
phosphatidylglycerol in bacterial plasma membranes, said small
molecule comprising a central scaffold and a plurality of
functional groups cooperatively forming a three-dimensional
complementary binding pocket for said phosphatidylglycerol, wherein
said compound is selected from the group consisting of:
##STR00003## combinations thereof, and substituted forms thereof,
where: M is 3, 4, or 5; N is 1, 2, 3, or 4; each R.sup.b is
--CH.sub.2CH.sub.2OH, --CH(OH)CH.sub.2OH, or
--CH.sub.2CO.sub.2NHOH; each R.sup.c is --CONHPh,
(2H).sup.++X.sup.-, or --C(.dbd.NH.sup.+)NHR', where R' is an alkyl
(e.g., C.sub.1-C.sub.8 alkyl) or aryl group; each R.sup.d is an
--COHNalkyl (preferably C3-C8 alkyl),
--CONHCH.sub.2CH.sub.2OCH.sub.2C.sub.6H.sub.5,
--CONHCH.sub.2CH.sub.2OH,
--CONH(CH.sub.2CH.sub.2O).sub.3CH.sub.2CH.sub.3,
--CONHCH.sub.2CH.sub.2CH.sub.3, --CH.sub.2NH.sub.3.sup.+X.sup.-,
--CH.sub.2NH.sub.2.sup.+CH.sub.2C.sub.6H.sub.5,
--CH.sub.2NH.sub.2.sup.+CH.sub.2CH.sub.2CH.sub.3,
--CH.sub.2NH.sub.2.sup.+CH.sub.2(CH.sub.2).sub.4CH.sub.3,
--CH.sub.2CH.sub.2(OCH.sub.2CH.sub.2)yCH.sub.3, --CH.sub.2Ph, or
--CH.sub.2R'', where R'' is 2-(aminomethyl)-5-methylphenol or
5-(aminomethyl)-2-methylphenol; and X-- is any anionic counter
ion.
2. The compound of claim 1, wherein said compound is
bacteriostatic.
3. The compound of claim 1, wherein said compound is
bactericidal.
4. An antibacterial composition comprising a bacteriostatic or
bactericidal amount of an antibacterial small molecule compound
according to claim 1 dispersed in a pharmaceutically-acceptable
carrier.
5. The composition of claim 4, wherein said carrier is selected
from the group consisting of saline, buffered saline, sterile
water, aqueous dextrose solutions, aqueous glycerol solutions,
ethanol, allantoic fluid, oil-in-water emulsion, water-in-oil
emulsions, dimethyl sulfoxide, petroleum jelly, cocoa butter,
cottonseed oil, olive oil, sodium pyruvate, vitamin E, white
petrolatum, white wax, stearyl alcohol, cholesterol, mineral oil,
ceryl ester wax, sodium lauryl sulfate, propylene glycol,
polyethylene glycol, and mixtures thereof.
6. The composition of claim 4, said composition being substantially
free of antibiotics and/or antimicrobial peptides.
7. The composition of claim 4, said composition consisting
essentially of said small molecule compound and said carrier.
8. The composition of claim 4, said compound has a minimum
inhibitory concentration (MIC) of from about 1 to about 4
.mu.M.
9. A method of inhibiting bacterial growth, said method comprising
contacting bacteria with an antibacterial small molecule compound
that binds to phosphatidylglycerol in bacterial plasma membranes,
said small molecule comprising a central scaffold and a plurality
of functional groups, at least one functional group for binding to
an anion, and at least one functional group for glycerol binding,
said scaffold and said functional groups cooperatively forming a
three-dimensional complementary binding pocket for said
phosphatidylglycerol.
10. The method of claim 9, wherein said compound is selected from
the group consisting of: ##STR00004## combinations thereof, and
substituted forms thereof, where: X.sup.- is any anionic counter
ion; m is 3, 4, or 5; n is 3, 4, 5, or 6; M is 3, 4, or 5; N is 1,
2, 3, or 4; each R.sup.a is --(CH.sub.2).sub.2CONHC.sub.6H.sub.13,
--CH.sub.2CONHC.sub.6H.sub.13,
--(CH.sub.2).sub.2CONH(CH.sub.2).sub.yNHCOCH.sub.3,
--(CH.sub.2).sub.2CONH(CH.sub.2).sub.yNHCONH(CH.sub.2).sub.yCH.sub.3,
--(CH.sub.2).sub.2CONHOH, or --NHNHCONH(CH.sub.2).sub.yCH.sub.3,
where each y is 1, 2, 3, or 4; each R.sup.b is
--CH.sub.2CH.sub.2OH, --CH(OH)CH.sub.2OH, or
--CH.sub.2CO.sub.2NHOH; each R.sup.c is --CONHPh,
(2H).sup.++X.sup.-, or --C(.dbd.NH.sup.+)NHR', where R' is an alkyl
(e.g., C.sub.1-C.sub.8 alkyl) or aryl group; and each R.sup.d is an
--COHNalkyl (preferably C3-C8 alkyl),
--CONHCH.sub.2CH.sub.2OCH.sub.2C.sub.6H.sub.5,
--CONHCH.sub.2CH.sub.2OH,
--CONH(CH.sub.2CH.sub.2O).sub.3CH.sub.2CH.sub.3,
--CONHCH.sub.2CH.sub.2CH.sub.3, --CH.sub.2NH.sub.3.sup.+X.sup.-,
--CH.sub.2NH.sub.2.sup.+CH.sub.2C.sub.6H.sub.5,
--CH.sub.2NH.sub.2.sup.+CH.sub.2CH.sub.2CH.sub.3,
--CH.sub.2NH.sub.2.sup.+CH.sub.2(CH.sub.2).sub.4CH.sub.3,
--CH.sub.2CH.sub.2(OCH.sub.2CH.sub.2).sub.yCH.sub.3, --CH.sub.2Ph,
or --CH.sub.2R'', where R'' is 2-(aminomethyl)-5-methylphenol or
5-(aminomethyl)-2-methylphenol.
11. The method of claim 9, wherein said antibacterial small
molecule compound is dispersed in a carrier for administration or
application.
12. The method of claim 9, wherein said compound is fluorescent,
said method further comprising monitoring changes in fluorescence
of said compound after said contacting.
13. A method of treating bacterial infection in a subject suffering
from an infected area, said method contacting said infected area
with therapeutically-effective amount of an antibacterial small
molecule compound that binds to phosphatidylglycerol in bacterial
plasma membranes, said small molecule comprising a central scaffold
and a plurality of functional groups, at least one functional group
for binding to an anion, and at least one functional group for
glycerol binding, said scaffold and said functional groups
cooperatively forming a three-dimensional complementary binding
pocket for said phosphatidylglycerol.
14. The method of claim 13, wherein said compound is selected from
the group consisting of: ##STR00005## combinations thereof, and
substituted forms thereof, where: X.sup.- is any anionic counter
ion; m is 3, 4, or 5; n is 3, 4, 5, or 6; M is 3, 4, or 5; N is 1,
2, 3, or 4; each R.sup.a is --(CH.sub.2).sub.2CONHC.sub.6Ho,
--CH.sub.2CONHC.sub.6H.sub.13,
--(CH.sub.2).sub.2CONH(CH.sub.2).sub.yNHCOCH.sub.3,
--(CH.sub.2).sub.2CONH(CH.sub.2).sub.yNHCONH(CH.sub.2).sub.yCH.sub.3,
--(CH.sub.2).sub.2CONHOH, or --NHNHCONH(CH.sub.2).sub.yCH.sub.3,
where each y is 1, 2, 3, or 4; each R.sup.b is
--CH.sub.2CH.sub.2OH, --CH(OH)CH.sub.2OH, or
--CH.sub.2CO.sub.2NHOH; each R.sup.c is --CONHPh,
(2H).sup.++X.sup.-, or --C(.dbd.NH.sup.+)NHR', where R' is an alkyl
(e.g., C.sub.1-C.sub.8 alkyl) or aryl group; and each R.sup.d is an
--COHNalkyl (preferably C3-C8 alkyl),
--CONHCH.sub.2CH.sub.2OCH.sub.2C.sub.6H.sub.5,
--CONHCH.sub.2CH.sub.2OH,
--CONH(CH.sub.2CH.sub.2O).sub.3CH.sub.2CH.sub.3,
--CONHCH.sub.2CH.sub.2CH.sub.3, --CH.sub.2NH.sub.3.sup.+X.sup.-,
--CH.sub.2NH.sub.2.sup.+CH.sub.2C.sub.6H.sub.5,
--CH.sub.2NH.sub.2.sup.+CH.sub.2CH.sub.2CH.sub.3,
--CH.sub.2NH.sub.2.sup.+CH.sub.2(CH.sub.2).sub.4CH.sub.3,
--CH.sub.2CH.sub.2(OCH.sub.2CH.sub.2)yCH.sub.3, --CH.sub.2Ph, or
--CH.sub.2R'', where R'' is 2-(aminomethyl)-5-methylphenol or
5-(aminomethyl)-2-methylphenol.
15. The method of claim 13, wherein said contacting comprises
directly applying said antibacterial small molecule compound to
said infected area of said subject.
16. The method of claim 13, wherein said contacting comprises
systemically administering said antibacterial small molecule
compound to said subject.
17. The method of claim 13, wherein said bacterial infection is
caused by Gram-negative or Gram-positive bacteria.
18. The method of claim 17, wherein said bacteria is selected from
the group consisting of Acinetobacter baumannii, Escherichia coli,
Staphylococcus aureus, Mycobacterium smegmatis, Enterococcus
faecalis, Streptococcus pneumoniae, Streptococcus pyogenes,
Streptococcus mitis, Streptococcus mutans, Streptococcus bovis,
Klebsiella pneumoniae, Pseudomonas aeruginosa, Enterococcus
faecium, Staphylococcus epidermidis, Staphylococcus haemolyticus,
Salmonella typhimurium, Bacillus subtilis, Neisseria meningitides,
Neisseria gonorrhoeae, and Haemophilus influenza.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a divisional of U.S. Ser. No.
16/048,009, filed Jul. 27, 2018, which is a continuation-in-part of
International Patent Application No. PCT/US2017/015428, filed Jan.
27, 2017, which claims the priority benefit of U.S. Provisional
Patent Application Ser. No. 62/289,027, filed Jan. 29, 2016,
entitled PHOSPHATIDYLGLYCEROL RECEPTORS FOR TREATING BACTERIAL
INFECTIONS, each of which is incorporated by reference in its
entirety herein.
BACKGROUND OF THE INVENTION
Field of the Invention
[0003] The present invention relates to small molecules that bind
to phosphatidylglycerol for treating bacterial infections.
Description of Related Art
[0004] Although antibiotics still remain the first line of defense
against pathogenic bacteria, there has been an emergence of many
strains of multidrug-resistance (MDR) bacteria. The emerging crisis
of bacterial antibiotic resistance is considered to be epidemic.
Besides the well-known cases of Gram-positive MRSA and VRSA (S.
aureus), there is a threat of truly untreatable infections by MDR
and pan-drug resistant (PDR) Gram-negative bacteria. Pathogenic
strains of Acinetobacter baumannii, Escherichia coli, Klebsiella
pneumonia, and Pseudomonas aeruginosa are now resistant to some
(MDR) or all (PDR) antibiotics commonly used to treat these
Gram-negative bacteria, such as penicillins, cephalosporins,
carbapenems, monobactams quinolones, aminoglycosides, tetracyclines
and polymyxins. Extensively drug-resistant strains (XDR) of
Mycobacterium tuberculosis and carbapenem-resistant (CRE) strains
of Klebsiella pneumoniae are up and coming threats with high
mortality rates of those infected. These MDR pathogens (commonly
called superbugs) are a threat to US public health and national
security. Indeed, infectious diseases remain the second-leading
cause of death worldwide and the third-leading cause of death in
the United States. Every year over 23,000 Americans die of
nosocomial infections caused by antibiotic resistant bacteria (and
with untold billions of dollars added to health care costs). To
make matters worse, during this same time period there has been a
continuous decrease in the number of newly approved antimicrobial
agents for use in the United States, a situation that has become a
cause of grave concern to the medical community.
[0005] Conventional antibiotics work by disrupting a specific cell
target (e.g., cell wall synthesis, protein or DNA synthesis), and
bacteria have evolved myriad ways to by-pass the antibiotic's
single target, leading to resistance. Due to the rapid rise in
bacterial resistance for extant antibiotics, there has been a
growing interest in the use of antimicrobial peptides (AMPs) or
their mimics as potential antibiotics. Cationic AMPs are able to
permeabilize the bacterial cell wall by binding to the negative
lipopolysaccharide of Gram-negative bacteria or the teichoic acids
and peptidoglycan layer of Gram-positive bacteria via
"self-promoted uptake." The outer leaflet of prokaryotic cell inner
membrane contain an abundant supply of acidic (anionic)
phospholipids, such as phosphatidylglycerol (PG), whereas the outer
leaflets of eukaryotic cell membranes are almost exclusively
composed of zwitterionic phospholipids. Antimicrobial cationic
peptides utilize this difference in lipid head structure to bind to
anionic phospholipids of the bacterial membrane. AMP-membrane
complex formation is followed by insertion of its hydrophobic
portion into the membrane, leading to eventual disruption of the
membrane and to cell death. Bacteria have been exposed to such
peptides since the dawn of multicellular organisms, yet have shown
only limited resistance, likely because targeting a membrane causes
a broad range of molecular consequences, which are difficult to
overcome by evolving a specific resistance mechanism.
[0006] AMPs kill bacteria by disrupting membrane structure. FIG. 1
shows the barrel-stave, carpet and toroidal pore models used to
describe the way cationic peptides disrupt the bacterial membrane.
More recent studies have shown that the activity of AMPs was not
limited to perforation of bacterial membranes. They can also
inhibit cellular processes such as DNA/RNA synthesis, protein
synthesis, cell division, cell wall synthesis and protein folding,
by translocating across the bacterial plasma membrane. The
barrel-stave model describes the formation of antimicrobial peptide
dimers and multimers after the binding of the peptides to the
negatively charged bacterial membrane. This assembly of AMPs
penetrates the membrane with their hydrophobic part facing the
lipid bilayer and the hydrophilic components forming the internal
lumen pores. The assembled peptide molecules inside the pore have a
barrel like structure. In the carpet model, the peptides cover the
surface of the outer membrane of the bilayer and destroy it with
concomitant pore formation. The toroidal pore formation involves
the fixation of the inner and outer lipid bilayer by the AMPs
[0007] Unfortunately, there are many impediments to the use of
AMP's as antibiotics, such as high production costs, low
bioavailability, degradation by serum proteases and reduced
activity by serum salts. Perhaps most importantly, due to limited
membrane selectivity, AMPs or their mimics can exhibit host
toxicity (systemically, AMPs are contained in, and brought to the
site of infection by, cells such as macrophages and neutrophils).
Very few AMPs have shown success in clinical trials (not due to
lack of activity but for inability to demonstrate an advantage over
existing antibiotics), and those cationic antimicrobial peptides
presently licensed are for topical use only. These disadvantages
have shifted focus from the use of AMPs or their mimics as
antimicrobial agents that can be used systemically.
SUMMARY OF THE INVENTION
[0008] Described herein are synthetic small molecule compounds,
termed "liptins," which are defined herein to refer to small
molecules that bind to the PG head group in solution, in synthetic
lipid membranes, or in bacterial membranes (plasma or outer) via
multifunctional groups correctly aligned with both the phosphate
anion portion and glycerol hydroxyl portion of PG. In
supramolecular chemistry vernacular, the liptin is the anion
receptor and the lipid headgroup the ligand. The liptin compounds
comprise a scaffold comprising functional groups (aka binding
units) arranged to present a three-dimensional binding pocket for
PG. More specifically, the liptin scaffold is structured such that
it will place binding units in proper orientation to interact with
both the phosphate anion portion and glycerol hydroxyl portion of
PG head group. This requires correct alignment of binding units
along an 8-10 angstrom head group length. Note that the PG head
group is positioned orthogonal to the fatty acid hydrocarbon tails.
Use of hydrogen bonds in binding units provides for fine control
over directionality of interactions within the liptin-PG complex.
This enhances the liptin's binding affinity and selectivity for PG.
Accordingly, the liptins comprise functional groups capable of
hydrogen bonding with neutral (glycerol hydroxyls) and/or charged
oxygens (phosphate anion portion) in PG.
[0009] As such certain embodiments of the invention are concerned
with antibacterial small molecule compounds that bind to PG in
bacterial plasma membranes (see FIG. 1B). These small molecule
compounds generally comprise a central scaffold and a plurality of
functional groups, at least one functional group for binding to an
anion, and at least one functional group for glycerol binding. As
such, the scaffold and functional groups cooperatively form a
three-dimensional complementary binding pocket for PG.
[0010] For example, liptin structures preferably comprise at least
two sets of binding units, with one set designed to bind to the
anion portion of the PG head group and one set that binds with the
neutral glycerol hydroxyls. Usually this means the stronger of the
two hydrogen-bonding binding units will interact with the anionic
phosphate head group portion (more effective stabilization of
charge), especially if the liptin scaffold has positively charged
ammonium groups (Coulombic interactions). Thus, the liptins present
complementary and multifunctional binding pockets for the PG
lipid's head group aligning correctly with the phosphate anion
portion and (neutral) glycerol head group of PG. For example, the
working examples demonstrate that the four pickets on meso-phenyl
rings of a porphyrin scaffold, or on the ortho-para substituents on
a linked bis-phenol scaffold, when appropriately functionalized,
formed a complementary binding pocket to the PG lipid's
multifunctional head group. Thus, the liptins are the first
target-based designed small molecule capable of tight binding to a
lipid anionic head group at a membrane interface using non-covalent
interactions. The liptin design results in high binding affinity,
unlike AMPs that use non-specific Coulombic interactions to bind to
bacterial plasma membranes. Also unlike AMPs that permeate the
bacterial membrane, certain liptins according to the invention
appear to stay strongly bound to the lipid head group.
[0011] The liptins disrupt various physiochemical properties of
plasma membranes. For example, upon bonding, the liptin 3e-PG
complex dramatically alters the effective lipid head group size and
charge from mono-anionic to tetra-cationic. This, in turn, alters
the physical chemistry and disrupts the homeostasis of the
lipidome, with lowered cell viability appearing to be attributable
to depolarization and/or loss of bacterial plasma membrane
function.
[0012] Also described herein are antibacterial compositions
comprising a bacteriostatic or bactericidal amount of an
antibacterial small molecule compound according to embodiments
herein, dispersed in a pharmaceutically-acceptable carrier.
[0013] These compounds and compositions are useful in inhibiting
bacterial growth, killing bacteria, as well as in treating
bacterial infection in a subject suffering from an infected
area.
[0014] The methods generally comprise contacting the infected area
or the bacteria with a therapeutically-effective amount of an
antibacterial small molecule compound according to embodiments
described herein. Thus, described herein is a new therapeutic
modality to treat bacterial infections, and would save lives for
those with infections, particularly those that are resistant to
conventional antibiotics. Due to the general nature of the membrane
damage caused by liptins, the rate of resistance occurrence is
expected to be low. Liptins have a high potential to be developed
into highly efficient antimicrobials of broad spectrum.
[0015] Further, fluorescent liptins that bind the PG head group and
alter membrane properties have utility as molecular tools to study
the dynamics of lipid-lipid and lipid-protein interactions
essential for bacterial membrane integrity and function. This could
help identify the roles of lipid-lipid and lipid-protein
interactions in bacterial membrane integrity and function,
interactions that are difficult to target with current tools. As
importantly, the development of supramolecular recognition
principles for targeting many kinds of membrane lipids would
provide a new set of biophysical tools to enhance basic
understanding of heterogeneous membrane function.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1A is a representation of three models of pore
formation by AMP molecules Brogden, K. A., Antimicrobial Peptides:
Pore formers or metabolic inhibitors in bacteria? Nat. Rev.
Microbiol., 2005. Vol. 3: p. 238-250;
[0017] FIG. 1B is a cartoon illustration of the mechanism of action
of the liptins on PG;
[0018] FIG. 2A is an illustration of representative families of
multifunctional small molecules (liptins) that have been
synthesized and which bind selectively to PG;
[0019] FIG. 2B is an illustration of additional proposed families
of multifunctional small molecules (liptins) for selective binding
to PG;
[0020] FIG. 3 is a Molecular Dynamic simulation with a
porphyrin-based molecule that binds selectively to PG involving a
heterogeneous bilayer lipid patch;
[0021] FIG. 4 is a reaction Scheme 1 for synthesis of liptins with
bis-phenol-based scaffolds;
[0022] FIG. 5 is a reaction Scheme 2 for synthesis of a second
class of liptins with bis-phenol-based scaffolds;
[0023] FIG. 6 is a reaction Scheme 3 for synthesis of liptins with
porphyrin-based scaffolds;
[0024] FIG. 7 is a reaction Scheme 4 a second class of liptins with
porphyrin-based scaffolds;
[0025] FIG. 8 is a reaction Scheme 5 of a third class of liptins
with porphyrin-based scaffolds;
[0026] FIG. 9 is a representation of autocorrelation decays for
liptin 3e membrane-bound to PG in (A) liposomes and (C) surfactant
vesicles containing varying amounts of PG, with binding constants
for each in (B) and (D), respectively.
[0027] FIG. 10A is a graph from the initial efflux experiments in
synthetic lipid vesicles showing a comparison of efflux data for
carboxyfluorescein dye from vesicles with pure PG in the presence
and absence of liptin 3e. Additional efflux experiments with
liposomes containing a 20/80 mixture of PG and
phosphatidylethanolamine to better simulate a bacterial membrane
are shown in FIGS. 19A-F with liptins 3e-h and 1h-k;
[0028] FIG. 10B is a graph of the results with liptin 3e used in
cell lysis studies;
[0029] FIG. 11A is a photograph of MIC experiments with E. coli
cultures;
[0030] FIG. 11B is a photograph of MIC experiments with E. coli
cultures;
[0031] FIG. 11 C is a photograph of MIC experiments with S. aureus
cultures;
[0032] FIG. 11 D is a photograph of MIC experiments with S.
faecalis cultures;
[0033] FIG. 12A is a graph of growth inhibition of liptin 3e on E.
coli (MC4100);
[0034] FIG. 12B is a graph of the UV/Visible spectrum of bacterial
solution and liptin 3e after 12 hours;
[0035] FIG. 13A is a graph showing data of long term growth
experiments with S. aureus inoculated with various concentrations
of liptin 3e;
[0036] FIG. 13B is a graph showing data of long term growth
experiments with E. coli inoculated with various concentrations of
liptin 3e;
[0037] FIG. 14 shows photographs of S. aureus plated on LB plates
in the presence of 1 .mu.M 3e (top left plate) or 5 .mu.M 3e (top
right plate), with the control (no liptin) in the bottom panel;
[0038] FIG. 15A is a graph of data from depolarization experiments
measuring fluorescence of 3,3' diethylthiodicarbodyanine iodide in
S. aureus incubated in the presence of a known antimicrobial
(CSA-25) at varying concentrations;
[0039] FIG. 15B is a graph of data from depolarization experiments
measuring fluorescence of 3,3' diethylthiodicarbodyanine iodide in
S. aureus incubated in the presence of an ammonium-picket porphyrin
at varying concentrations;
[0040] FIG. 15C is a graph of data from depolarization experiments
measuring fluorescence of 3,3' diethylthiodicarbodyanine iodide in
S. aureus incubated in the presence of a combination of
ammonium-picket porphyrin and CSA-25 at varying concentrations
[0041] FIG. 16 is an image of a gel electrophoresis of the
periplasmic and cytosolic proteins of E. coli bacteria (with and
without IPTG induction of the overexpression of PapD);
[0042] FIG. 17 is a graph of the toxicity of 3e when incubated with
hepatocytes for 9-12 hours at varying concentrations;
[0043] FIG. 18 is a graph demonstrating the lack of toxicity of
liptin 3e to eukaryotic erythrocytes as assessed by UV/Visible
spectroscopy;
[0044] FIG. 19A is an efflux graph of liptin 3e with a PG liposome
showing carboxy-fluorescein dye leakage;
[0045] FIG. 19B is an efflux graph of liptins 3f-h with a PG
liposome showing carboxy-fluorescein dye leakage;
[0046] FIG. 19C is an efflux graph of liptin 1j with a PG liposome
showing carboxy-fluorescein dye leakage;
[0047] FIG. 19D is an efflux graph of liptin 1i with a PG liposome
showing carboxy-fluorescein dye leakage;
[0048] FIG. 19E is an efflux graph of liptin 1l with a PG liposome
showing carboxy-fluorescein dye leakage;
[0049] FIG. 19F is an efflux graph of liptin 1k with a PG liposome
showing carboxy-fluorescein dye leakage;
[0050] FIG. 20A is a growth curve in BHI culture for E. coli with
one inoculation of liptin 3e;
[0051] FIG. 20B is a growth curve in BHI culture for E. Faecium
with one inoculation of liptin 3e;
[0052] FIG. 20C is a growth curve in BHI culture for M. smegmatis
with one inoculation of liptin 3e;
[0053] FIG. 20D is a growth curve in BHI culture for
Methicillin-resistant S. aureus (MRSA) with one inoculation of
liptin 3e;
[0054] FIG. 20E is a growth curve in BHI culture for S. aureus with
one inoculation of liptin 3e;
[0055] FIG. 20F is a growth curve in BHI culture for K. pneumoniae
with one inoculation of liptin 3e;
[0056] FIG. 20G is a growth curve in BHI culture for A. baumannii
with one inoculation of liptin 3e;
[0057] FIG. 20H is a growth curve in BHI culture for P. aeruginosa
with one inoculation of liptin 3e;
[0058] FIG. 21 is a graph that shows the reduction of growth rate
of E. coli and S. aureus with one inoculation of liptin 3e in a
concentration-dependent manner;
[0059] FIG. 22 is a graph that shows the decrease in culture
density of E. coli and S. aureus with one inoculation of liptin 3e
in a concentration-dependent manner;
[0060] FIG. 23A is a graph depicting the plasma membrane
depolarization in E. coli caused by liptins 1h and 1k in a
concentration-dependent manner;
[0061] FIG. 23B is a graph depicting the plasma membrane
depolarization in S. aureus caused by liptins 1h and 1k in a
concentration-dependent manner;
[0062] FIG. 24 shows scanning electron micrographs (SEM) of (A)
treated and (B) untreated E. coli cells with 5 .mu.M liptin 1k,
evidencing a clear stunting of growth but no holes in the outer
membrane;
[0063] FIG. 25 shows scanning electron micrographs (SEM) of (A)
treated and (B) untreated MRSA cells with 5 .mu.M liptin 1k,
evidencing a wrinkling or non-uniformity of the outer membrane of
the MRSA exposed to liptin 1k;
[0064] FIG. 26 depicts the molecular dynamics simulation of how
liptin 1h binds to PG in a membrane; and
[0065] FIG. 27 details toxicity studies of liptins 1h and 1k with
eukaryotic cell lines HeLa and A459.
DETAILED DESCRIPTION
[0066] The present invention is concerned with new therapeutic
modalities for treating bacterial infection and represents a new
approach to antimicrobials. The major phospholipid components of
both Gram-negative and Gram-positive bacterial membranes are
anionic cardiolipin (CL) and phosphatidylglycerol (PG) and
zwitterionic phosphatidylethanolamines (PE), while the outer
leaflets of eukaryotic cell membranes are almost exclusively
composed of zwitterionic phospholipids. In prokaryotes, the
relative abundance of PE and PG can vary between species and with
life-cycle and environmental factors, but PG is always present in
significant quantities and makes up approximately 20-30% of the
lipid content in E. coli, 12% in B. subtilis and up to 50-60% in
Staphylococcus aureus and Streptococcus pneumonia. Recent work
indicates that lipid homeostasis appears crucial for specific
protein placement within cytosolic membrane hyperstructures and
hence for cellular processes in the cell life cycle. Thus,
disrupting the lipidome may well disrupt several important cellular
processes.
[0067] The present invention is broadly concerned with the new
class of small molecule compounds referred to herein as liptins,
having antibacterial (aka bacteriostatic, and/or bactericidal)
activity against Gram-negative and Gram-positive bacteria. In this
disclosure, the term "small molecule" refers to synthetic compounds
in which the molecular weight does not exceed 2000 grams per mole.
These liptins are at least bacteriostatic, and in some cases can be
bactericidal. The term "bacteriostatic" as used herein means that
the liptin at least stops or slows down bacterial reproduction
(biostatic), while not necessarily killing the bacteria. In other
words, when the liptin is removed, the bacteria may resume growth
and/or proliferation. In some cases, at higher concentrations
(>10 .mu.M), the liptin is preferably bactericidal, killing the
bacteria. At lower concentrations (10 .mu.M or less), the liptins
can be used in methods of inhibiting bacterial growth, and in some
cases as part of treating bacterial infection.
[0068] These liptins are small molecule compounds that bind anionic
PG lipids in bacterial plasma membranes, disrupting the membrane,
and resulting in inhibition of bacterial cell growth, or bacterial
cell death. As described herein, the liptins have a
three-dimensional complementary binding pocket for the PG head
group, and thus a binding affinity and selectivity for bacterial
PG. Thus, the liptins comprise H-bonding functional groups that can
bind to an anion, in this case, the phosphate anion portion of the
PG headgroup. There can be more than one of these groups on a
liptin, and liptins can be acyclic or cyclic. In one or more
embodiments, the liptin is based upon one of two scaffolds: a
porphyrin ring; or a macrocyclic system comprising two linked
phenol rings, which preferably contain one or more ammonium groups
for positively-charged hydrogen bonding. There are a number of
different donor groups for anions, with the list expanding greatly
in recent years. Classic H-bonding donor groups also include,
without limitation:
##STR00001##
This list is not exhaustive, but provides good examples.
[0069] Advantageously, the small molecules also bind to the
glycerol portion of the PG headgroup found in the bacterial plasma
membrane. Exemplary functional groups for glycerol binding include,
without limitation groups that are both H-bond donors and H-bond
acceptors with hydroxyl groups for formation of neutral H-bonds
(e.g., --OH, --CONH.sub.2, --CONHOH, --NHCONHR'--CO.sub.2H). Also,
ammonium groups may form charged hydrogen-bonds with the oxygen
atom in the glycerol headgroup. Thus, overall the small molecule
structure can be neutral or charged.
[0070] Exemplary compounds for use as liptins include:
##STR00002##
and combinations thereof, where: [0071] X.sup.- is PF.sub.6.sup.-,
CF.sub.3CO.sub.2.sup.-, or any other anionic counter ion, such as
halogen anions (e.g., Cl.sup.- or Br), phosphate anions (e.g.,
H.sub.2PO.sub.4.sup.-), organophosphate anions (e.g.,
ReHPO.sub.3.sup.-), sulfate anions (e.g., HSO.sub.4),
organosulfonate anions (e.g., ReSO.sub.3), or other carboxylate
anions (e.g., CH.sub.3CO.sub.2 or ReCO.sub.2.sup.-), where each Re
is an alkyl or aryl group.
[0072] m is 3, 4, or 5; [0073] n is 3, 4, 5, or 6; [0074] M is 3,
4, or 5; [0075] N is 1, 2, 3, or 4; [0076] each R.sup.a is
--(CH.sub.2).sub.2CONHC.sub.6H.sub.13,
--CH.sub.2CONHC.sub.6H.sub.13,
--(CH.sub.2).sub.2CONH(CH.sub.2).sub.yNHCOCH.sub.3,
--(CH.sub.2).sub.2CONH(CH.sub.2).sub.yNHCONH(CH.sub.2).sub.yCH.sub.3,
--(CH.sub.2).sub.2CONHOH, or NHNHCONH(CH.sub.2).sub.yCH.sub.3,
where each y is 1, 2, 3, or 4; [0077] each R.sup.b is
--CH.sub.2CH.sub.2OH, --CH(OH)CH.sub.2OH, or
--CH.sub.2CO.sub.2NHOH; [0078] each R.sup.c is --CONHPh,
(2H).sup.++X.sup.-, or --C(.dbd.NH.sup.+)NHR', where R' is an alkyl
(e.g., C.sub.1-C.sub.8 alkyl) or aryl group; and [0079] each
R.sup.d is --COHN-alkyl (preferably C3-C8 alkyl),
--CONHCH.sub.2CH.sub.2OCH.sub.2C.sub.6H.sub.5,
--CONHCH.sub.2CH.sub.2OH,
--CONH(CH.sub.2CH.sub.2O).sub.3CH.sub.2CH.sub.3,
--CONHCH.sub.2CH.sub.2CH.sub.3, --CH.sub.2NH.sub.3.sup.+X.sup.-,
--CH.sub.2NH.sub.2.sup.+CH.sub.2C.sub.6H.sub.5,
--CH.sub.2NH.sub.2.sup.+CH.sub.2CH.sub.2CH.sub.3,
--CH.sub.2NH.sub.2.sup.+CH.sub.2(CH.sub.2).sub.4CH.sub.3,
--CH.sub.2CH.sub.2(OCH.sub.2CH.sub.2)yCH.sub.3, --CH.sub.2Ph, or
--CH.sub.2R'', where R'' is 2-(aminomethyl)-5-methylphenol or
5-(aminomethyl)-2-methylphenol, wherein any of the foregoing alkyl
or aryl groups may be substituted or unsubstituted.
[0080] Thus, the liptins for use in the invention contain within
their complementary binding pocket multiple hydrogen bonding
functional groups able to align correctly and specifically with the
PG lipid's phosphate anion portion and the glycerol hydroxyl
groups. These liptins bind to the PG displayed on the surface of
bacterial plasma membranes with high affinity and selectivity, and
the resulting liptin-PG complex formation has been shown to inhibit
bacterial growth in both Gram-negative and Gram-positive bacteria.
This result is most likely from the depolarization of the plasma
membrane upon liptin complexation with PG. A synthetic advantage to
the liptin porphyrin structure is the inherent symmetry of the four
pickets that are capable of alignment with the two different sets
of lipid functional groups. NMR studies have been used to determine
binding motifs within the liptin-PG complex. Using the
thermodynamics of binding (determined from NMR and ITC techniques)
associated with particular liptin-PG complex structures enables us
to iteratively change liptin design to enhance liptin affinity for
PG.
[0081] Compositions according to the invention comprise a
bacteriostatic or bactericidal amount of the liptin dispersed in a
pharmaceutically-acceptable carrier. In general, the compositions
may comprise from about 0.1 to about 95% of the liptin, based upon
the total weight of the composition taken as 100%, and preferably
from about 0.5 to about 50% of the liptin. The amounts will depend
upon the desired use, the particular agent used, and the particular
carrier(s) selected. The term carrier is used herein to refer to a
base, diluent, excipient, vehicle, or the like, in which the liptin
may be dispersed for administration or application. Suitable
carriers will be pharmaceutically acceptable. As used herein, the
term "pharmaceutically acceptable" means not biologically or
otherwise undesirable, in that it can be administered to a subject
without excessive toxicity, irritation, or allergic response, and
does not cause unacceptable biological effects or interact in a
deleterious manner with any of the other components of the
composition in which it is contained.
[0082] A pharmaceutically-acceptable carrier would naturally be
selected to minimize any degradation of the liptin or other agents
and to minimize any adverse side effects in the subject, as would
be well known to one of skill in the art.
Pharmaceutically-acceptable ingredients include those acceptable
for veterinary use as well as human pharmaceutical use, and will
depend on the route of administration. Exemplary carriers include
aqueous solutions such as normal (n.) saline (.about.0.9% NaCl),
phosphate buffered saline (PBS), sterile water/distilled autoclaved
water (DAW), aqueous dextrose solutions, aqueous glycerol
solutions, ethanol, normal allantoic fluid, various oil-in-water or
water-in-oil emulsions, dimethyl sulfoxide (DMSO), petroleum jelly,
cocoa butter, cottonseed oil, olive oil, sodium pyruvate, vitamin
E, white petrolatum, white wax, stearyl alcohol, cholesterol,
mineral oil, ceryl ester wax, sodium lauryl sulfate, propylene
glycol, polyethylene glycol, and the like.
[0083] Other ingredients may be included in the composition, such
as adjuvants, other active agents, preservatives, buffering agents,
salts, other pharmaceutically-acceptable ingredients, including
residual amounts of ingredients used in pharmaceutical
manufacturing.
[0084] In some embodiments, the bacteriostatic compositions of the
invention consist essentially or even consist of the liptin
dispersed in a carrier. In some embodiments, the compositions are
substantially free of additional bactericidal or bacteriostatic
agents, where the term "substantially free" means having no
significant amount of that component purposefully added to the
composition to import a certain characteristic (as contrasted with
intentional ingredients listed above), it being understood that
trace amounts of incidental elements and/or impurities may
sometimes find their way into a desired end product (e.g., due to
contamination from incidental additives or through contact with
certain processing and/or holding equipment). In some embodiments,
the compositions are substantially free of antibiotics,
antimicrobial peptides, alkaline earth metals, and the like. As
liptins are synthetic molecules, such ingredients if present,
preferably represent no more than 0.05%, preferably less than
0.005%, and more preferably less than about 0.001% by weight of the
composition taken as 100% by weight in total.
[0085] Methods of inhibiting bacterial growth are also described
herein. The methods generally comprise contacting bacteria with the
liptin. In one or more embodiments, the liptin is in a composition,
which is then contacted with the bacteria. In one or more
embodiments, the liptin is administered or applied to a subject
suffering from a bacterial infection. Thus, methods of treating
bacterial infection in a subject suffering from a bacterial
infection are also described herein. The methods generally comprise
contacting an infected area of the subject with a liptin described
herein. In one or more embodiments, the liptin is in a composition,
which is then contacted with the area. An "infected area" of the
subject, as used here, may refer to a defined site of local
infection, such as a wound, but is also used to refer to a systemic
infection characterized by the presence of pathogenic
microorganisms or their components in the blood or tissues and
organs other than primary infected area of the subject. Regardless,
the infection is due to Gram-negative or Gram-positive bacteria.
Thus, in one or more embodiments, methods of the invention comprise
directly and/or topically applying a liptin to an infected area of
the subject. In one or more embodiments, methods of the invention
comprise systemically administering a liptin to the subject, for
example, remote from the infected area, such as by introducing a
liptin into the blood circulation system of the subject.
[0086] Various routes of administration can be used depending upon
the particular carrier and other ingredients used. For example,
topical administration may involve rubbing, dabbing, or otherwise
applying liptins to the infected area, followed by an appropriate
dressing, gauze, bandage, or other covering, if desired. The
liptins can also be injected intramuscularly, subcutaneously,
intradermally, or intravenously using a needle and syringe, or a
needleless injection device. The liptins can also be administered
mucosally, such as intranasal administration. Oral administration
is also contemplated, provided that the liptins are formulated
appropriately for passage through the gastrointestinal system
(e.g., in an enteric-coated dosage form). In some embodiments, the
methods described herein are useful for reducing the effects,
severity, or morbidity of bacterial infection, as described
herein.
[0087] Methods of the invention will utilize a therapeutically
effective amount of liptin in inhibiting bacterial growth or
treating bacterial infection. As used here, a "therapeutically
effective" amount refers to the amount that will elicit the
biological or medical response of a tissue, system, or subject that
is being sought by a researcher or clinician, and in particular
elicit some desired inhibitory effect as against the bacterial
infection by reducing bacterial growth and/or killing bacteria.
Thus, a therapeutically amount of liptin includes bacteriostatic as
well as bactericidal amounts of liptin. One of skill in the art
recognizes that an amount may be considered therapeutically
"effective" even if the condition is not totally eradicated or
prevented, but it or its symptoms and/or effects are improved or
alleviated partially in the subject.
[0088] In some embodiments, the liptins are provided in unit dosage
form in a suitable container. The term "unit dosage form" refers to
a physically discrete unit suitable as a unitary dosage for use.
Each unit dosage form may contain a predetermined amount of liptin
(and/or other active agents) in the carrier calculated to produce
the desired effect. In other embodiments, the liptins can be
provided separate from the carrier (e.g., in its own vial, ampule,
sachet, or other suitable container) for on-site mixing before
administration to a subject. A kit comprising the liptins is also
disclosed herein. The kit further comprises instructions for
administering the liptins to a subject.
[0089] Our studies with Gram-negative and Gram-positive bacteria
and studies of protein overexpression in E. coli suggest
liptin-complex formation changes the plasma membrane physical
properties. Liptin binding interrupts lipid head group
interactions, altering membrane fluidity, or disrupting lipid
microdomains and therefore protein localization and function.
Additionally, liptin-PG complex formation has been shown to
depolarize the plasma membrane, which will lead to the observed
inhibition of bacterial replication and growth.
[0090] The liptins used in the invention have broad spectrum action
against both Gram-negative and Gram-positive bacteria. A key
advantage of liptins in this class is sparing of the endogenous
microbiome. In some embodiments, these liptins exhibit
bacteriostatic effects that the immune system capitalizes on
through normal clearance methods. Unlike invading pathogens,
endogenous flora are protected from immune responses due to
peripheral tolerance mechanisms. Therefore, even as these liptins
do impede bacterial growth universally, immune cells will not clear
endogenous organisms.
[0091] In one or more embodiments, the liptin has a minimum
inhibitory concentration (MIC) of from about 1 to about 4
preferably between 1 and 4 .mu.M. It will be appreciated that the
MIC of the liptins are stated in micromolar (.mu.M), because the
liptin molecule is relatively large and weighs nearly twice as much
as most conventional antibiotics. Accordingly, the lowest
concentration of liptin that inhibits growth of bacteria is more
precisely provided as a unit of concentration in the micromolar
range. The liptins would be effective against one or more of the
following pathogenic microbes: Acinetobacter baumannii, Escherichia
coli, Staphylococcus aureus (including MRSA), Enterococcus
faecalis, Mycobacterium smegmatis, Streptococcus pneumoniae,
Streptococcus pyogenes, Streptococcus mitis, Streptococcus mutans,
Streptococcus bovis, Klebsiella pneumoniae, Pseudomonas aeruginosa,
Enterococcus faecium, Staphylococcus epidermidis, Staphylococcus
haemolyticus, Salmonella typhimurium, Bacillus subtilis, Neisseria
meningitides, Neisseria gonorrhoeae, Haemophilus influenzae, and
the like.
[0092] The invention provides a significant advancement in the art,
of a completely new therapeutic modality to treat bacterial
infections, whereby formation of the liptin-PG complex at the
plasma membrane by itself disrupts bacterial cellular processes
inhibiting bacterial growth and/or causing bacterial cell death,
without the need for any adjuvants, or other antibiotic agents.
Liptin-PG complex formation, by changing membrane properties,
causes some or perhaps many membrane proteins, such as transporter
proteins, to function less efficiently or perhaps not at all, or
keep membrane protein complexes necessary for function from
forming, or inhibit transporter functions. For example, any
deleterious effects on a bacteria's SecYEG or TAT secretory systems
(both composed of multi-protein assemblies), ion channels or
bacterial two component systems, all found in the cytosolic
membrane, could inhibit growth, replication, interfere with
bacterial virulence mechanisms (i.e., prevent pilus or biofilm
formation, or slow or stop the production of bacterial toxins and
effectors), and in general lower the bacteria's vitality. The
action of the liptins on fundamental features, such as bacterial
membrane function prevents selection pressure for the emergence of
resistant strains. In a broader sense, the liptins can also be used
to study the organizing principles in membrane heterogeneity, and
the interactions of bacterial membrane protein hyperstructures and
lipid microdomain structures.
[0093] As noted, the liptin/PG binding significantly perturbs
lipid-lipid interactions by altering the PG head group charge and
size and leads to measurable changes in membrane physical
properties (such as the fluidity and viscoelasticity of the
membrane) and, ultimately, the viability of cell membrane function.
FIG. 3 shows Molecular Dynamic simulations with a porphyrin-based
liptin (3e, see FIG. 2A) involving a heterogeneous bilayer lipid
patch in explicit water solvent illustrates how our small molecule
liptin caps the lipid head group.
[0094] Specifically, the bound PG lipid is illustrated as a
space-filling representation, while other PG and PE lipids are in
stick representation. Liptin 3e is shown in a stick representation
in both FIGS. 3A and 3B. All water molecules have been removed for
clarity.
[0095] Additional advantages of the various embodiments of the
invention will be apparent to those skilled in the art upon review
of the disclosure herein and the working examples below. It will be
appreciated that the various embodiments described herein are not
necessarily mutually exclusive unless otherwise indicated herein.
For example, a feature described or depicted in one embodiment may
also be included in other embodiments, but is not necessarily
included. Thus, the present invention encompasses a variety of
combinations and/or integrations of the specific embodiments
described herein.
[0096] As used herein, the phrase "and/or," when used in a list of
two or more items, means that any one of the listed items can be
employed by itself or any combination of two or more of the listed
items can be employed. For example, if a composition is described
as containing or excluding components A, B, and/or C, the
composition can contain or exclude A alone; B alone; C alone; A and
B in combination; A and C in combination; B and C in combination;
or A, B, and C in combination.
[0097] The present description also uses numerical ranges to
quantify certain parameters relating to various embodiments of the
invention. It should be understood that when numerical ranges are
provided, such ranges are to be construed as providing literal
support for claim limitations that only recite the lower value of
the range as well as claim limitations that only recite the upper
value of the range. For example, a disclosed numerical range of
about 10 to about 100 provides literal support for a claim reciting
"greater than about 10" (with no upper bounds) and a claim reciting
"less than about 100" (with no lower bounds).
EXAMPLES
[0098] The following examples set forth methods in accordance with
the invention. It is to be understood, however, that these examples
are provided by way of illustration and nothing therein should be
taken as a limitation upon the overall scope of the invention. It
is important to note that with these following examples we show 1)
liptins are the first target-based designed small molecule capable
of tight binding to a lipid anionic head group at a membrane
interface using non-covalent interactions, and 2) completely
different structures (3e and 1h-k), that only have in common the
target PG lipid head group, show similar in vitro effects via a new
mechanism of action. The data establishes a proof of concept that
liptin-PG complex formation (by itself) results in highly
bacteriostatic and/or bactericidal action by disrupting plasma
membrane homeostasis, notwithstanding differences in the particular
liptin structures.
[0099] The first two examples clarify the structural requirements
for a liptin binding pocket that exhibits multifunctional
complementarity for the phosphatidylglycerol (PG) head group.
Notably, the outer leaflet of prokaryotic plasma membrane contains
an abundant supply of the anionic lipid PG, while the outer
leaflets of eukaryotic cell membranes are almost exclusively
composed of zwitterionic phospholipids. Thus, a liptin that binds
to PG will selectively target the bacterial plasma membrane. Three
families of liptins were prepared as presented in Example 1.
Example 1 illustrates the synthesis and characterization of new
liptins which can increase the binding affinity and selectivity for
phosphatidylglycerol. The structures of the liptin-PG complexes
were studied in solution, with mixed-lipid liposomes and synthetic
vesicles, and with efflux studies to determine membrane leakage
with liposomes, as presented in Example 2. Importantly, the studies
of a porphyrin liptin at a membrane interface (Example 2) of a
synthetic vesicle doped with PG show similarities to the liptin's
solution binding motif and high selectivity of binding to PG
because of binding pocket complementarity. Example 3 details how
short-term and long-term bacterial growth and viability is affected
by liptin 3e when it is introduced to both Gram-negative and
Gram-positive bacterial strains, and the determination of MICs for
Gram-negative and Gram-positive bacteria. Example 3 shows that
liptin 3e, simply by binding to PG in the plasma membrane,
depolarizes both the S. aureus and E. coli plasma membrane. Example
3 details the in vitro studies that measure the effects on
development of bacterial resistance to a porphyrin picket PG liptin
with both Gram-negative and Gram-positive bacterial strains by
serial passage studies. Example 4 is an examination of toxicity of
liptin 3e on eukaryotic hepatic cells. Example 5 is an initial
examination of toxicity of liptin 3e on erythrocytes. Example 6
provides the additional characterization of liptins 3e-h and 1h-k.
Included is an examination of how the binding of liptins 3e-h and
1h-k to PG liposomes affects membrane permeability via efflux
experiments with carboxy-fluorescein dye leakage. Included are
additional bacterial growth curves of Gram-negative and
Gram-positive bacteria in BHI enriched medium with one inoculation
of liptin 3e. Included are minimum inhibition concentrations and
minimum bactericidal concentrations of liptins 1h-k in Muller
Hinton culture with Gram-negative and Gram-positive bacteria.
Included are Live Dead staining results of liptins 1h and 1k with
Gram-negative and Gram-positive bacteria. Included are plasma
membrane depolarization studies with liptins 1h and 1k with E. coli
and S. aureus. Included are scanning electron micrographs of E.
coli and MRSA showing the comparison between treated and untreated
bacterial cells with liptin 1k. Included is a molecular dynamics
study of liptin 1h and a PG lipid patch showing the binding motif
of the liptin 1k to PG in a membrane. Example 7 shows a toxicity
study of eukaryotic HeLs (vaginal carcinoma) and A549 (lung
epithelial carcinoma) cells with liptins 1h and 1k.
Example 1
[0100] In a project designed to develop targeting systems for
bacterial membranes, we have generated novel families of small
molecules (termed liptins) that are the first to show good
complementarity with the PG head group (Koralegedara et al., J.
Org. Chem. 2011, 76, 1930-1933; Alliband et al., J. Org. Chem.
2013, 78, 356-362; Alliband et al., Org. Biomol. Chem. 2015, 13,
502-512). Specifically, we have synthesized liptin's binding pocket
with suitably spaced functionality to bind and correctly align with
two different sets of lipid functional groups, the phosphate anion
portion and the neutral glycerol hydroxyl groups. We demonstrated
that the four pickets on meso-phenyl rings of a porphyrin scaffold,
or the ortho-para substituents on two types of linked bis-phenol
scaffolds, when appropriately functionalized, formed a
complementary binding pocket to the PG lipid's multifunctional head
group. Thus, the liptins are the first target-based designed small
molecules capable of tight binding to a lipid anionic head group at
a membrane interface using non-covalent interactions.
[0101] (1.1) Liptin Syntheses.
[0102] The preparations of liptins 1a-g and 3a-e (FIG. 2A) has been
described in previous work. The detailed syntheses of 1h-k, 2a-d,
and 3f-h (FIG. 2A) are presented below. Additional compounds have
been contemplated, as illustrated in FIG. 2B. All liptins are
furnished with binding-pocket (positively charged) ammonium and
(neutral) amide or hydroxyl hydrogen-bonding groups able to align
and bind with the phosphate anion portion and neutral glycerol
portion of the PG head group, respectively. Molecular dynamics
simulations using a lipid patch illustrated (FIG. 3) that the 3e-PG
complex configuration at the membrane interface has its pickets
pointing towards the membrane. This indicates that elaboration of
all or some of the porphyrin pickets, as seen in 3f-h with groups
able to insert into the membrane, will increase the complexation
entropy via loss of solvent molecules and reduce complexation
enthalpy via favorable interaction with lipid tails.
[0103] (1.1.1) Preparation of Bis-Phenol Liptins 1h-1k and Proposed
Elaborations.
[0104] The binding of liptins 1a-g, R.dbd.CH.sub.3, to PG in
solution has been described, as part of previous work directed
towards developing PG liptins as targeting moieties. Since then, we
have replaced the liptin's methyl para-substituent with acetic or
propanoic amide units for binding to the glycerol hydroxyl groups.
Modeling indicates this is the correct length for the amides to
hydrogen bond with the PG hydroxyl groups while the ammonium groups
hydrogen bond to the phosphate anion portion. We have synthesized
bis-phenols 1a-g (and will prepare additional bis-phenols 6) by
starting with a phenol ring that contains the commercially
available para-acetic or -propanoic ester (Scheme 1, FIG. 4).
Coupling the di-ester 4 with a primary amine (compounds 5) followed
by protonation furnished charged liptins 1a-g (or will furnish 6)
able to H-bond with glycerol hydroxyl groups. Amide formation via
the methyl ester proved to be straightforward using La(Tf).sub.3 as
catalyst. Bis-amidation could also be accomplished by
de-esterification followed by amidation of the di-acid with a
primary amine using well known coupling reagents. Additionally, we
can prepare other functionalized para-R groups with commercially
available N-alkylhydrazinecarboxamides coupled to the bis-acid with
TBTU to furnish liptins with semicarbazide urea functional groups
to bind to the PG glycerol groups. Or, using commercially available
w-aminoalkylamides or w-aminoalkylureas we can prepare the
para-amide with additional amide or urea groups to interact with
neighboring lipid head groups once the liptin forms a complex with
membrane-bound PG.
[0105] (1.1.2) Preparation of Bis-Phenol Liptins 2a-2d and Proposed
Elaborations.
[0106] Liptins 2a-2d (FIG. 2A) have been prepared and
characterized, but not published. However, example syntheses of
several precursors required for the preparation of liptins 2a-2d,
via precursors 7-8, utilizing soluble copper catalyst developed in
our laboratory to couple the two aromatic rings and to elaborate
the ring structure is based upon our previous experience, as is our
methodology to efficiently ureidoalkylate the bis-phenol
intermediates (FIG. 5). Prior research has shown that urea liptins
2a or 2b exhibited no significant binding to PG, whereas 2c
exhibited a rather modest binding constant for PG
(K.sub.a.ltoreq.10.sup.2 M.sup.-1) in DMF solution. In contrast,
inorganic phosphate anion bound more tightly to 2c
(K.sub.a=10.sup.3 M.sup.-1), indicating the anion binding unit with
one methylene unit linking the two phenolic oxygens was a
complementary fit for phosphate anion. By adding two additional
methylene groups to the bridge linking the two rings [i.e., now
(CH.sub.2).sub.5, 2d], the size of the pocket better accommodates
the PG head group, leading to increased liptin 2c-PG binding with a
K.sub.a.apprxeq.500 M.sup.-1 in DMF. To further increase binding
affinity, we changed the 3-hydroxypropane para-substituent into a
2,3-propane diol para-substituent (seen in 8 prepared from 7, FIG.
5). This increased the number of possible interactions between the
liptin and the PG glycerol hydroxyls and reduced possible
conformational constraints in the binding pocket when only two
hydroxyl groups are present. FIG. 5 shows the synthetic pathway to
prepare 2d with a ring linkage of five methylene units (same
pathway can be used for four or six methylene units). Importantly,
we changed the phosphate anion-binding urea groups to the stronger
hydrogen-binding ammonium groups as shown in FIG. 5. Elaboration of
2d can be accomplished from reductive amination of an aldehyde with
the ortho-methylamines to form 9. In this way hydrogen-bonding
groups can be added to interact with neighboring lipid head groups.
A second route would replace carbamate formation (during nitrile
reduction) with amide formation (10) followed by mild, selective
hydrosilation-deoxygenation of the amide to furnish 9.
Additionally, the ortho-methylamines can be transformed into
multiple, stronger hydrogen-bonding groups, such as guanidinium
functional groups.
[0107] (1.1.3) Preparation of Elaborated Liptins 3f-h and Proposed
Elaborations. Modification of Porphyrin Pickets with Membrane
Insertion Units.
[0108] The structure of the entire picket in porphyrin liptins 3a
and 3c was found to influence the enthalpy and entropy of lipid
binding, suggesting that PG liptins elaborated with groups able to
insert into membranes to interact with the lipid (and not just the
lipid headgroup) would increase the liptin's overall membrane
affinity and selectivity with the goal of lowering MIC's as
determined from bacterial experiments. The synthetic manipulations
employed allow us to determine experimentally how iterative changes
in the lipid-liptin complex structure affect the K.sub.a, enthalpy
and entropy of non-covalent interactions (.sup.1H NMR, FCS and ITC
experiments).
[0109] A homologous series of alkyl additions to the pickets (above
the ammonium groups) is intended to provide stabilizing van der
Waals' interactions between the insertion unit and lipid tail,
along with different insertion depth of picket structures within
the membrane. Polyether insertion units allow for dipole-dipole and
hydrophobic interaction, and aromatic insertion units with
appropriately substituted ring substituents allow for hydrogen
bonding interactions with the fatty acid ester groups. It may prove
preferable to elaborate only one or two (cis) pickets as membrane
insertion units to allow the same glycine ammonium binding motif
for PG seen in 3e while still providing membrane insertion
units.
[0110] Three potential routes to prepare porphyrins with extended
pickets (3f-g are proposed) are presented in FIG. 6). Route 1
involves reductive amination of the uncharged glycine picket with
an aldehyde. Route 2 involves amidation of the meso-phenylamines
via addition of chloroacetyl chloride followed by the S.sub.N2
addition of amines to the resultant alkyl chloride (examples of
alkyl, polyether, or benzyl amines are shown in Scheme 1). Route 2
is capable of furnishing a more diverse set of pickets, and
modeling has shown that the use of the aryl amines shown in Scheme
1 would position their phenolic OH groups to allow for hydrogen
bonding with the lipid's fatty acid ester groups. At the present
time, porphyrins 3f-h have been furnished using Route 2. Route 3
involves the transformation of the meso-phenylamines into
phenylisocyanates, followed by the addition of a substituted
hydrazine (prepared from an aldehyde and hydrazine to form the
hydrazone followed by reductive amination or with commercially
available hydrazines) to furnish a semicarbazide group whose
nitrogen can be charged (FIG. 6). The urea portion of the
semicarbazide would provide additional strong hydrogen-bonding
sites for the glycerol hydroxyl groups. Porphyrin 3e that contains
TFA counterions is quite water soluble, and we expect similar
results with modified porphyrins.
[0111] (1.1.4) Elaboration of Porphyrins's Meso-Phenyl Rings:
Liptin-PG Complex Interactions with Neighboring Lipid Head
Groups.
[0112] A second method to increase overall membrane affinity is to
prepare liptin porphyrins that not only bind to a specific PG
lipid, but also engage in secondary interactions with neighboring
PG or phosphatidylethanolamine (PE) lipids found in the bacterial
plasma membrane. Modification of para-positions on the porphyrin's
meso-phenyl rings with hydroxylamine or amide groups would provide
additional binding units for neighboring lipid head groups via 1)
H-bonding to adjacent lipid anionic phosphate groups, or 2) head
group interaction with adjacent PE ammonium hydrogens or PG
hydroxyl groups. Two possible para-substituents are hydroxamic acid
or bis-amide groups that able to hydrogen bond to neighboring lipid
head groups, whose synthetic pathway is shown in FIG. 7
(benzaldehyde precursor 11 and porphyrin 12). Use of the Adler
method to furnish porphyrin A (FIG. 6) could lead to lower yields,
and a Lindsey approach reacting 5-aryldipyrromethanes with
substituted aromatic nitrobenzaldehydes, or via N-tosyl imines, may
prove a longer but more fruitful approach. Also, polar
meso-substituents will provide water solubility to the liptins,
perhaps important if the extended pickets add hydrophobicity. As
with liptins in general, such multidentate liptins will change the
viscoelasticity of the plasma membrane and thereby interfere with
lipidome homeostasis and perhaps interfere with membrane protein
dynamics and function.
[0113] (1.1.5) Multiple Porphyrin Arrays.
[0114] If results from the above experiments suggest that higher
affinity and selectivity for PG liptins is desirable (to lower
bacterial MIC), then we can undertake the synthesis of liptins with
two or more PG binding units. Assuming the correct linker size,
complex stability should be increased due to the well-known chelate
effect. Synthetic routes to multiporphyrin arrays have been well
established. While there are no reports of (covalently bound)
multiple picket porphyrin arrays, we will prepare picket porphryins
with the proper functionality to furnish a picket porphyrin dimer,
trimer, or larger array via covalent assembly. The linker coupling
the two porphyrins will need to be 8-10 angstroms or longer in
length to allow for two bound PG lipids to reside side-by-side in
the membrane. Coupling of the two porphyrin rings after the pickets
have been prepared will avoid potential problems with the formation
of an all a-atropisomer of multiple meso-phenylamines and the
preparation in one reaction of multiple pickets. The preparation of
a dimer will be `proof of concept` that the picket dimer can be
synthesized from porphyrin 16 using standard Sonogashira coupling
in the preparation of multiple porphyrin arrays. Note that
depending on the synthetic pathway, porphyrins can be prepared with
one, two (5,15: trans), or with four bromophenyl groups (porphyrins
17,18) for linking via Sonogarshira coupling of alkynes to the
bromophenyl position (FIG. 8).
[0115] Synthetic lipid vesicles with varying ratios of anionic and
zwitterionic lipids will be utilized in binding studies with these
elaborated porphyrin liptins to examine the affect porphyrin arrays
have on lipid organization and membrane structure. In vesicles,
these effects can be measured with differential scanning
calorimetry and fluorescence polarization.
Example 2
[0116] (2.1) Determination of Liptin-PG Binding in Solution.
[0117] The lipid binding ability of liptins 1-3 (FIG. 2A) have been
investigated in solution using isothermal titration calorimetry
(ITC) and/or .sup.1H NMR. Stoichiometry of binding was accomplished
with Job plots using .sup.1H NMR. Table 1 details liptin-PG
association constants, and ITC data shows both the enthalpy and
entropy associated with binding, for the TBA phosphatidylglycerol
anion with a 1:1 stoichiometry of binding. The use of organic
solvents allowed the determination of binding motif using .sup.1H
NMR. Spectroscopic studies showed that liptins with the
preorganized (ortho-ring substituted) ammonium or urea groups found
in compounds 1 or 2, or the four ammonium or urea pickets on the
porphyrin ring as in 3, bound to the PG lipid's phosphate anion
group via hydrogen bonds. Additionally, published spectroscopic
data showed that the PG's glycerol hydroxyl group hydrogen-bonded
with 3e's amide group, while unpublished data showed PG's glycerol
hydroxyl group interacted with 2d's bis-hydroxyl functional
groups.
TABLE-US-00001 TABLE 1 PG Liptin 1b 1e 2c 2d 3a 3c 3e K.sub.a
(M.sup.-1) 2 .times. 10.sup.2 2.5 .times. 10.sup.2 4.2 .times.
10.sup.2 1.7 .times. 10.sup.4 2.1 .times. 10.sup.3 3.7 .times.
10.sup.3 2.8 .times. 10.sup.3 (.+-.6 .times. 10.sup.1) (.+-.4
.times. 10.sup.1) (.+-.6 .times. 10.sup.1) (.+-.1.4 .times.
10.sup.3) (.+-.1 .times. 10.sup.2) (.+-.4.5 .times. 10.sup.2)
(.+-.1.0 .times. 10.sup.3) .DELTA.H (kcal -1.4 -2.8 -1.2 0.7
mol.sup.-1) (.+-.0.02) (.+-.0.07) (.+-.0.04) (.+-.0.09) .DELTA.S
e.u. 8 6 12 18 Results from ITC (errors in parentheses) for
liptin-lipid complexes that exhibit 1:1 binding stoichiometry (1b,
1e, 2d from NMR titrations). Liptins 1b, 1e, 2c, 2d, 3a, 3c in
DMF/5% CHCl.sub.3; 3e in 50% DMSO, 45% CHCl.sub.3, 5%
CH.sub.3OH.
[0118] To examine in detail the structural requirements needed for
1's scaffold to bind the phosphate anion portion of PG, we prepared
a family of small molecules whose linkages between the two phenolic
oxygens and two benzyl amines were of different lengths. In this
way small molecule PG-liptins were constructed to support various
binding motifs and maximize the entropic contribution to
complexation. Because both the lipid head group and liptin were
charged and studies were conducted in polar solvents, it was
expected that complex formation would be entropy driven. As
reported, liptins 1b and 1e both bound to PG, with association
constants of 2-2.5.times.10.sup.2 M.sup.-1, and using ITC (results
not shown) we determined that complex formation was indeed entropy
driven.
[0119] The scaffolding in liptin 2 contained either neutral (urea,
2c) or charged (ammonium, 2d) groups to bind with the phosphate
anion portion in PG, and hydroxyl groups on the para substituents
to bind the glycerol hydroxyl groups. Liptin 2d, with the charged
ammonium groups and four para-hydroxyl groups bound strongly to PG
in 1:1 binding stoichiometry. Its association constant of
approximately 2.times.10.sup.4 M.sup.-1 is almost two orders of
magnitude stronger than the binding constant of 2c, showing that
charged ammonium hydrogens bonds and two sets of bis-hydroxyl
groups were important to efficient binding of PG in solution.
[0120] .sup.1H NMR and isothermal titration calorimetry (ITC) were
used to determine liptin 3's PG binding stoichiometry, liptin-lipid
complex structure, binding constant, and associated thermodynamic
properties of complexation in solution. Thermodynamic properties
determined from ITC showed that liptin 3e-PG complex formation was
entropy driven, while PG-binding for the neutral liptins 3a and 3c
were driven by both enthalpy and entropy. All three exhibited
association constants of 2-4.times.10.sup.3 M.sup.-1 in organic
solution. .sup.1H NMR spectroscopy detailed the binding motif of
liptins 3a, 3c, and 3e, and showed that the PG headgroup was
positioned just above the porphyrin ring with the liptin's urea or
ammonium pickets aligned correctly to bind both the phosphate anion
portion. In porphyrins 3a and 3c the urea groups interacted with
the hydroxyl portion of PG, while in 3e the amide groups interacted
with the hydroxyl portion. Data for new liptins 1h, 1i, and 3f are
shown in Table 2. The increase in K.sub.a from 1b to 1h is greater
than one order of magnitude. We infer that 1h's para-amide
functional group (as opposed to a non-hydrogen bonding para-methyl
group in 1b) is hydrogen-bound to the PG hydroxyl group and results
in a more thermodynamically stable complex. The association
constants determined from ITC or NMR titrations for liptin 1h were
very similar. However, the two liptins 1b and 1h were measured in
different solvent systems due to different solubility requirements,
which may also contribute to the large difference observed in
association constants. The lower association constant for liptin 3f
compared to liptin 3e may be due to steric bulk of the pickets
which may make it more difficult for the PG head group to fully
access the binding pocket. .sup.1H NMR spectroscopy shows that the
picket's amide groups of 3f are not involved in hydrogen bonding to
the PG's glycerol group, and that the PG head group lies farther
above the ring than in the 3e-PG complex.
TABLE-US-00002 TABLE 2 PG Liptin 1h 1i 3f K.sub.a (M.sup.-1) 4.6
.times. 10.sup.3 1.5 .times. 10.sup.3 2.8 .times. 10.sup.3 (.+-.3.8
.times. 10.sup.2) (.+-.4.3 .times. 10.sup.2) (.+-.1.0 .times.
10.sup.3) .DELTA.H (kcal mol.sup.-1) 0.6 3.7 (.+-.0.03) (.+-.2.1)
.DELTA.S e.u. 19 27 Results from ITC or NMR titrations (errors in
parentheses) for liptin-lipid complexes that exhibit 1:1 binding
stoichiometry for PG (3f from NMR titrations). Liptins 1h and 1i
40% DMSO, 60% CHCl.sub.3, 3f in 20% DMSO, 80% CHCl.sub.3.
[0121] (2.2) Determination of Liptin 3e Affinity and Selectivity of
Binding PG at the Membrane Interface.
[0122] The porphyrin 3e was used to examine the liptin's affinity
and selectivity for PG when PG was present as part of a membrane
bilayer. In this example, fluorescence correlation spectroscopy
(FCS) was used to measure the fraction of liptin bound to
surfactant vesicles or liposomes containing a known amount of PG.
This approach is fast, reliable and accurate, and is possible when
the liptin is a stable fluorophore, as in the case of 3e.
[0123] FCS results are evaluated by treating the time dependent
fluorescence intensity acquired from a dilute solution of the
liptin using a focused laser beam as the excitation source.
Temporal fluctuations in the fluorescence intensity are a result of
the diffusion of the liptin through the focused laser beam. When
the liptin binds to a vesicle, its apparent diffusion coefficient
decreases by two orders of magnitude. The data is analyzed using a
correlation analysis that results in an autocorrelation decay,
G(t), and presented in FIG. 9.
[0124] FIGS. panels 9A and 9C contain examples of autocorrelation
decays for liptin 3e in with liposomes (FIG. 9A) and surfactant
vesicles (FIG. 9C) containing varying amounts of PG. Specifically,
FIG. 9 illustrates an autocorrelation analysis of liptin 3e binding
to PG-doped membranes of liposomes and positively-charged
surfactant vesicles. A) Autocorrelation decays for liptin with
varying percentages of PG in PE liposome. B) Binding constant
values as a function of PS and PG in PE liposome. C)
Autocorrelation decays for liptin with varying percentages of PG in
positively-charged surfactant vesicles. D) Binding constant of
liptin with varying percentages of PG in positively-charged
surfactant vesicles. In both examples, a slowly-decaying component
becomes more prevalent in G(t) as the amount of PG present in the
bilayer increases. The slowly decaying component arises from the
slower diffusion of 3e, when bound to vesicles. Note that the decay
of G(t) consists of a single fast component when the samples
contain 0% PG. For a quantitative treatment, the autocorrelation
decays are fit to a function that gives the binding fraction of the
liptin in solution. In FIGS. 9A and 9C, the markers are the
experimental data and the solid lines are the fits that determine
the fraction of liptin bound, f. This quantity is then used to
calculate the binding constant:
K = [ bound receptor ] [ free receptor ] [ free PG ] = f ( 1 - f )
( [ PG ] 2 - [ R ] f ) ##EQU00001##
where [R] is the total concentration of liptin, and it has been
assumed that half of the PG is located on the membrane inner
leaflet and thus not available for binding. Binding constants are
given in FIGS. 4B and 4D. Lipid vesicles containing 10-30% PG all
exhibited a K.sub.a=10.sup.4 for 3e.
[0125] Our FCS studies revealed the following important
discoveries: [0126] 1) PG binding affinities at the membrane
interface are similar to those found for 3e in solution, on the
order of 10.sup.4M.sup.-1. [0127] 2) Binding is specific for PG and
is not attributed merely to pure Coulombic interactions; since no
binding is observed in vesicles that contain anionic headgroups
that differ from PG. Negligible binding of the liptin was observed
when the surfactant vesicle contained no PG and the only anionic
component was sodium dodecylbenzenesulfonate (SDBS), see FIG. 9D.
When the vesicle contained as little as 1% of PG and 35% SDBS, the
binding constant increased from negligible to
4.7.times.10.sup.4M.sup.-1 [0128] 3) Analysis of binding constants
for 3e with lipid vesicles formed from binary mixtures of either PG
or phosphatidylserine (PS) with phosphatidylethanolamine (PE)
showed that binding is selective for PG over the PS (also anionic).
Note that 30% PG: 70% PE is an approximation of an E. coli membrane
composition.
[0129] (2.3) Physiochemical Effect of 3e Complexation with PG in
Synthetic Lipid Vesicle (FIG. 10A).
[0130] Liptin/bilayer interactions were evaluated using
conventional efflux measurements. These experiments make use of the
self-quenching fluorescence of carboxyfluorescein. At relatively
high concentrations (ca. 50 mM) the fluorescence from
carboxyfluorescein is diminished by approximately 70% due to
intermolecular interactions.
[0131] To perform efflux experiments vesicles were prepared by
rehydrating a film of pure DPPG with a 50 mM solution of
carboxyfluorescein in buffer. The solution was then extruded
through a polycarbonate membrane with 200 nm pore size. The
solution was extruded repeatedly for a series of seven passes after
which the resulting vesicle-containing solution was purified on a
size exclusion chromatography column packed with Sephadex50. After
chromatography, the solution consists of vesicles filled with 50 mM
dye solution suspended in dye-free buffer solution. Under these
conditions, as the dye leaves the vesicles by efflux, fluorescence
intensity increases. Monitoring the fluorescence intensity over
time provides a measure of the rate at which dye crosses the
membrane and an indication of the "leakiness" of the vesicle
bilayer. Efflux can be expressed as % Efflux by the following
expression:
% Efflux = F ( t ) - F O F .infin. - F O ##EQU00002##
where F.sub.0 is the fluorescence intensity corresponding to the
initial sample when efflux first begins and F.sub..infin. is the
intensity after efflux is complete and is determined by rupturing
the vesicles by the addition of a small amount of concentrated
detergent. F(t) is the fluorescence intensity measured as the
efflux occurs.
[0132] FIG. 10A shows a comparison of efflux data for
carboxyfluorescein from vesicles prepared with pure PG in the
presence and absence of the liptin. At room temperature, there is
no measurable efflux from the vesicles during the course of the
three hour experiment. This changes dramatically when liptin 3e is
added and efflux is 5% complete after three hours. The large
increase in efflux rate indicates that liptin binding at the
interface increases the permeability of the membrane.
Example 3
[0133] (3.1) Bacterial Experiments: Determination of Minimum
Inhibitory Concentration (MIC).
[0134] The uptake of phosphatidylglycerol liptin 3e by E. coli
(Gram-negative bacillus), or Staphylococcus aureus or Enterococcus
faecalis (both Gram-positive cocci) inhibits bacterial growth. We
have reported that the 3e is able to penetrate both Gram-negative
and Gram-positive bacterial walls and reach the plasma membrane.
FIG. 10B shows the absorbance (420 nm) values of supernatants of
various trials from cell lysis experiments with E. coli and
Bacillus thuringiensis after incubating the bacterial solutions
with liptin (10 or 25 .mu.M) for 1 h. This previous work
demonstrates that the liptin (previously referred to as a
"receptor") binds to bacterial membrane components.
[0135] Here, we have determined the MIC of liptin 3e to be less
than 2 .mu.M. We assessed the MIC for E. coli and growth only in
seen in control and 1 .mu.M tubes, establishing an MIC between 1-2
.mu.M. We also assessed the MIC for Staph aureus and growth was not
observed in any tube except control, establishing an MIC at or
below 1 .mu.M.
[0136] Experimental for MIC with Liptin:
[0137] An E. Coli (JMR 223-MC4100) plate was grown up overnight in
LB agar at 37.degree. C. A good colony was selected and added to 5
mL LB growth media. This was incubated at 37.degree. C. with
shaking at 250 rpm until the solution reaches an OD of at least 0.5
or higher at 600 nm. A final solution was diluted 500.times. to an
OD of 0.001, correlating to a CFU of 1.times.10.sup.6. 5 ml
solutions of the diluted bacterial broth were measured out. Liptins
were added to make solutions of 20 .mu.M, 15 .mu.M, 10 .mu.M, 5
.mu.M, 4 .mu.M, 3 .mu.M, 2 .mu.M, and 1 .mu.M (This means adding
0.2 mL, 0.15 mL, 0.1 mL, 0.05 mL, 0.04 mL, 0.03 mL, 0.02 mL, 0.01
mL of the 500 .mu.M solution). Cultures were allowed to incubate
overnight at 37.degree. C. with shaking at 250 rpm. The following
day, cultures were analyzed to evaluate the presence or absence of
bacterial growth by spectrophotometry at 600 nm (0.66 mg of liptin
in 0.95 mL of water; 0.15 OD Diluted 150.times. to OD of 0.001).
Growth was observed in the control and 1 .mu.M cultures. Growth was
not observed in the 2 .mu.M and up solutions. See FIGS. 11A-11B for
a visual image of the E. coli cultures.
[0138] Staph aureus (33186) cultures were prepared and analyzed as
above. A good colony was selected and added to 5 mL LB growth
media, incubated at 37.degree. C./250 rpm until the solution
reached an OD of at least 0.5 or higher at 600 nm. The final
solutions were diluted 500.times. to an OD of 0.001. This
correlates to a CFU of 1.times.10.sup.6. The liptin solution was
made by adding 0.68 mg of liptin in 1 ml of water to yield 500
.mu.M solution. The control showed significant growth overnight.
(OD600=0.63), However, none of the solutions with the liptin showed
any growth indicating an MIC for liptin 3e of less than 1 .mu.M.
See FIG. 11C for a visual image of the S. aureus cultures.
[0139] Strep faecalis (aka Enterococcus faecalis 29213) cultures
were prepared the same way as S. aureus, and analyzed as above. The
control and 1 microM solutions showed significant growth. The OD600
of the control was 0.72. None of the others showed any growth. See
photos for visual representation. See FIG. 11D for a visual image
of the S. fuecalis cultures.
[0140] (3.2) Bacterial Experiments: Growth Experiments:
[0141] We further examined the effect of 3e on bacterial growth and
3e stability overnight.
[0142] (3.2.1) Overnight Study.
[0143] A one-time addition of a 10 .mu.M solution of liptin 3e to
an E. coli (MC4100) bacterial culture (growth phase, 37.degree. C.,
OD=0.2) in LB broth completely stops bacterial replication within
1-2 hours (stops at OD=0.35) for 12 hours while the UV/Visible
spectrum of membrane-bound 3e shows no change over the same time
period (OD vs time in minutes). Specifically, FIG. 12A illustrates
the growth inhibition of liptin 3e on E. coli (MC4100),
demonstrating complete stoppage of E. coli replication at 10 .mu.M.
The UV/Visible spectrum of bacterial solution and liptin after 12
hours is shown in FIG. 12B. This spectrum is identical to the
spectrum of just liptin in water illustrating that the liptin is
not degraded or metabolized when exposed to bacteria over long
periods of time.
[0144] (3.2.2) Long Term Study in BH Culture.
[0145] Staphylococcus aureus str. ATCC 29213 was grown as
shake-tubes in brain-heart infusion medium supplemented with liptin
and culture densities were measured by turbidity (FIG. 13A). At 0.5
.mu.M 3e, there was an 8-h lag phase before culture density began
to increase, which was not seen without addition of liptin 3e.
Further, the growth rate was substantially lower over the next 12 h
as culture density approached that of controls. Lag phases were on
the order of days to weeks at liptin concentrations of >1.5
.mu.M, and slow recovery followed. While growth was seen at 10
.mu.M, the density of cultures after two weeks were much lower than
controls. Growth of Escherichia coli str. MC4100 under similar
conditions was substantially inhibited by liptin, but somewhat less
so than Staphylococcus. Long lag phases of 8 h were observed at 1.5
.mu.M liptin, as shown in FIG. 13B. Lag phases greater than 24 h
were observed at 10 .mu.M liptin with E. coli and culture densities
were approaching those of controls within a week. Our novel
antibacterial compounds attack plasma membranes through a less
specific mode of action, which may avoid the pitfalls that lead to
antibiotic resistance.
[0146] (3.3) Bacterial Experiments: Bactericidal Experiments:
[0147] MBC results for E. coli (MC4100) and E. fuecalis (29213)
were similar, in that low or no growth was only seen in LB plates
grown from LB cultures that contained much more than four times the
MIC concentrations. Cultures (5 mL) starting at 0.001 OD were grown
overnight at 37.degree. C. with different concentrations of liptin,
and then a sample of culture was added to an LB plate and then it
was incubated for 16 hours at 37.degree. C. However, results for S.
aureus (33186) showed strong bactericidal effects at 5 .mu.M
concentration of 3e. The LB plates shown in FIG. 14 for S. aureus
are representative: top left plate, 1 .mu.M 3e; top right, 5 .mu.M
3e; bottom, control (left plate, 2 uL of the LB culture was put in
1 mL of LB growth media to dilute it, then this was placed onto an
LB plate; right plate, 1 mL of the culture was directly plated onto
a LB plate). All experiments were done in triplicate, including
control. MIC of S. aureus previously determined to be 1 .mu.M or
less.
[0148] (3.4) Bacterial Experiments: Resistance Experiments:
[0149] Triplicate serial passage experiments were undertaken with
E. coli (MC4100), S. aureus (33186) and E. feucalis (29213). In
brief, after the growing bacterial culture exhibited an OD between
0.5-1.0, a small amount was removed and added to new LB culture
broth for a starting OD around 0.001. In each experiment the
concentration of 3e was kept at 0.5 MIC determined for each
bacterial strain in LB culture at 37.degree. C. Preliminary
experimental data shows that although liptin-PG complex formation
initially stunts bacterial growth, at 0.5 MIC it is not
bactericidal. Based on the OD change from bacterial growth during
each experiment, we estimate that over the 15 serial passages a
total of 80-100 generations of bacteria were grown. MICs (not
shown) determined from bacterial cultures taken the end of the 15
serial passages were no different than initial MICs.
[0150] (3.5) Bacterial Experiments: 3e S. aureus Causes Plasma
Membrane Depolarization Upon Complexation with PG:
[0151] The graph in FIGS. 15A-15C represents the depolarization of
S. aureus plasma membrane measured by an increase in the
fluorescence of 3,3-diethylthiodicabodyanine iodide. The dye, which
has direct access to the Gram-positive plasma membrane, becomes
highly fluorescent in membranes where polarization is lost. Liptin
3e was added after 1.5 min at concentrations shown, and
fluorescence increased in a concentration-dependent manner. The
membrane effects were measured against the known
membrane-disruptive agent, cationic steroid antimicrobial-25
(CSA-25), one of a group of cholic acid-derived antimicrobial also
known as ceragenins. This assay measured the effects of the
Ammonium-picket porphyrin alone those of CSA-25 alone or a
combination of Ammonium-picket porphyrin and CSA-25.
[0152] Experimental:
[0153] Bacterial cultures were grown overnight in TSB at 37.degree.
C. Cells were harvested by centrifugation and washed in a buffer
containing 250 mM sucrose, 5 mM MgSO4, and 10 mM potassium
phosphate (pH 7.0). After three washings, pellets were re-suspended
in the same buffer. Fractions from each cell suspension were
diluted in the same buffer in a cuvette to an optical density
(A600) of 0.085 along with the dye DiS-C2(5) at a concentration of
1 M. The dye was allowed to incorporate for 7 min at room
temperature, followed by 7 min at 37.degree. C., which gave a
stable baseline. An excitation wavelength of 600 nm and an emission
wavelength of 660 nm were used to monitor depolarization. Samples
were stirred during the experiment at a constant temperature of
37.degree. C. Fluorescence measurements were taken at 30-s
intervals before and after addition of ceragenins. FIG. 15A
illustrates the depolarization of S. aureus measured by an increase
in fluorescence of 3,3' diethylthiodicarbodyanine iodide
(DiS-C2(5)). CSA-25 was added at 90 s at the following
concentration: 0.69 .mu.M, 1.3 .mu.M, 2 .mu.M, and 2.6 .mu.M.
[0154] FIG. 15B illustrates the depolarization of S. aureus
measured by an increase in fluorescence of 3,3'
diethylthiodicarbodyanine iodide. Ammonium-picket porphyrin was
added at 90 s at the following concentration: 0.69 .mu.M, 1.3
.mu.M, 2 .mu.M, and 2.6 .mu.M.
[0155] FIG. 15C illustrates the depolarization of S. aureus
measured by an increase in fluorescence of 3,3'
diethylthiodicarbodyanine iodide. CSA-25 and Ammonium-picket
porphyrin was added at 90 s at the following concentration: 0.69
.mu.M, 1.3 .mu.M, 2 .mu.M, and 2.6 .mu.M.
[0156] (3.6) Bacterial Experiments: Membrane Depolarization in E.
coli.
[0157] The E. coli strain MC4100 will produce the pili chaperone
PapD, a periplasmic protein, when induced by IPTG. When bacterial
cultures were incubated with both IPTG and 5 .mu.M 3e no PapD was
found in either the periplasmic space or cytosol. Gel
electrophoresis results shown in FIG. 16. The left panel represents
the periplasmic fraction with lanes running left to right: ladder,
NR, C, R, all with no IPTG; NR+IPTG, C+IPTG, R+IPTG (negative
control was porphyrin A) or in the cytosol represented in the right
panel with lanes running from left to right: NR, C, R, all with no
IPTG; ladder, NR+IPTG, C+IPTG, R+IPTG (negative control was
porphyrin A). (NR=no liptin, C=control, R=liptin). IPTG gets into
the cytoplasm most efficiently via uphil symport via the plasma
membrane protein lac permease when the plasma membrane is fully
polarized. Thus, the lack of transport which stopped over
expression of PapD strongly suggests that the plasma membrane has
been depolarized and the pmf shut down.
[0158] On the other hand, with E. coli BL 21 (Tuner pLysS)
tetracycline-induced protein YqhD (a cytosolic alcohol
dehydrogenase) was produced whether or not 3e was present in the
bacterial culture (gel not shown). Although different strains
(liptin 3e was shown to pass through the cell wall in both strains)
and proteins, the larger difference between the two gel
electrophoresis experiments is that IPTG requires lac permease to
efficiently transverse the plasma membrane, where the inducer
tetracycline will permeate the membrane by itself. Since it would
not be expected that inhibition of lac permease alone would inhibit
bacterial growth (as many other food sources are available in the
broth), las permease function in general was deleteriously
affected, most likely by pmf disruption. When 3e was incubated with
the bacteria at 10 .mu.M most protein synthesis was effectively
disrupted (not shown).
Example 4
Hepatocyte Toxicity
[0159] Toxicity of compounds to eukaryotic cells was assessed by
MTT assay of cultured HepG2 human hepatocyte cells grown in media
supplemented with 10% fetal bovine serum and antibiotics.
[0160] Cell Culture:
[0161] Human liver HepG2 cells were cultured in 100 mm.sup.2 Falcon
tissue culture plates in DMEM with high glucose (4500 mg/L)
supplemented with 10% fetal bovine serum, 50 .mu.g/ml streptomycin
and 50 IU/ml penicillin at 37.degree. C. and 5% CO.sub.2. Cells
were cultured to about 70-80% confluence, and then seeded into 96
well plates depending and grown to 70-80% confluence for 2-3 days
unless otherwise stated.
[0162] Measurement of Cell Viability:
[0163] Cell viability was determined by MTT
[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)]
assay (Denizot & Lang 1986). Briefly, cells were seeded on
96-well plates and allowed to grow until 70-80% confluence. Prior
to the experiment, medium was replaced with KRB containing a
desired concentration of the toxin or other reagents and incubated
for 9 h to 12 h depending on the experiment at 37.degree. C. After
the incubation, 10 .mu.L of 5 mg/mL MTT solution was added to each
well and was incubated for 2 h at 37.degree. C. The resulting
formazan was solubilized by the addition of 210 .mu.L detergent
solution (50% DMF, 20% SDS) followed by incubation for 4 h at
37.degree. C. and was quantified based on the difference in the
absorbance at 570 nm and 650 nm (Mosmann 1983). Results are
expressed as viability of toxin treated cells with respect to
control cells which were treated under the same conditions except
in the absence of the toxin. Reference: Kadigamuwa C C, Le V Q
Wimalasena K (2015) "2,2'- and 4,4'-Cyanines are Transporter
Independent in vitro Dopaminergic Toxins with the Specificity of
Toxicity similar to MPP.sup.+" J. Neurochemistry, 135, 755-767.
FIG. 17 illustrates the toxicity of liptin 3e when incubated with
Hepatocytes for 9-12 hours. These data demonstrate the lack of
toxicity of liptin 3e down to 0.5 .mu.M.
Example 5
Erythrocyte Toxicity
[0164] Toxicity of liptin 3e to eukaryotic erythrocytes was
assessed by UV/Visible spectroscopy, whereby the change in
absorbance at 414 nm was used to quantify the release of
hemoglobin, where 100% hemolysis was measured by adding 1% Triton
X-100 detergent. Red blood cells (RBCs) were suspended in a buffer
of 25 mM TRIS and 1.5 mM dihydrogenphosphate anion (serum inorganic
phosphate levels). To the RBCs samples was added differing
concentration of 3e--2.5 .mu.M, 5 .mu.M, 7.5 .mu.M, and 10 .mu.M,
as well as control containing no 3e, and after 30 minutes
incubation and gentle shaking at 37.degree. C. spectra were
obtained. FIG. 18 shows that at or near 3e's MIC concentrations of
2.5-5 .mu.M, there was minimal 4.5-11% damage to red blood cells,
compared to the absorbance of 100% hemolysis and release of
hemoglobin minus the control release.
Example 6
Additional Characterization of Liptins 1h-k and 3e-h
[0165] Efflux Experiments with Liposomes Containing PG and
Phosphatidylethanolamine (PA)
[0166] Liptin 3e causes membrane leakage when it binds to PG in a
membrane, as evidenced by the efflux of fluorescent
carboxy-fluorescein from model lipid vesicles of 80% PE/20% PG (the
approximate lipid content models E. coli's plasma membrane), shown
in FIG. 19A. In the graph, the circles represent the data for 2.5
mM liptin 3e, while the square are the control without liptin 3e.
Not shown is the effect of the bee AMP melittin, which forms pores
in membranes at 2.5 .mu.M concentrations. Upon addition of melittin
within a few minutes of addition the efflux is 100%. This strongly
suggest that the liptins are not forming pores, but rather making
the membrane more permeable to dye leakage.
[0167] Liptins 3f-h also cause membrane leakage when they bind to
PG in a membrane, as evidenced by the efflux of fluorescent
carboxy-fluorescein from model lipid vesicles of 80% PE/20% PG (the
lipid content models E. coli's plasma membrane), shown in FIG. 19B.
The circle bottom line data set is the control without liptin. The
triangle second to bottom line data set shows data for 1.5 .mu.M
liptin 3f. The diamond second to top line data set shows the data
for 1.5 liptin 3h. The square top line data set shows data for 1.5
.mu.M liptin 3g. Y-axis: % Efflux; X-axis: Time in minutes.
[0168] While the efflux caused by liptin 3e at 2.5 .mu.M
concentration resulted in approximately 5-10% dye leakage after 90
minutes, the porphyrins with extended pickets above the ammonium
functionality, either propyl (3g) or hexyl (3h) groups, cause
60-80% dye leakage quickly after addition of the liptin to the
liposomes at 1.5 .mu.M concentration. Dynamic light scattering of
the treated liposomes show that they are still extant, and leakage
is not a result of dissolution of the synthetic vesicle. This
greater efflux activity may well translate to lower MICs of these
two compounds relative to liptin 3e, and may well change the liptin
activity from bacteriostatic to bactericidal. The benzyl extension
of the porphyrin's pickets above the ammonium functional groups
does not lead to dye leakage, perhaps because it is unable to bind
to the model membrane due to steric constraints of the larger
pickets.
[0169] Liptins 1h-k causes membrane leakage as evidenced by the
efflux of fluorescent carboxy-fluorescein from model lipid vesicles
of 80% PE/20% PG (the lipid content models E. coli's plasma
membrane), as shown in FIGS. 19C-F. In all graphs the positive
control bee AMP melittin, which forms pores in membranes at 2.5
.mu.M concentrations, results in 100% loss of dye as shown in
graphs below (efflux shown on Y-axis on right side). Liptins 1h-k
when bound to model membrane cause dye leakage of 10-20% over
ninety minutes. This strongly suggest that the liptins, unlike
melittin, are not forming pores, but rather making the membrane
more permeable to dye leakage.
New Growth Curves for Several Bacteria in BHI
(Brain-Heart-Infusion) Culture Media Show Liptin 3e to be Highly
Bacteriostatic.
[0170] Growth curves were generated from liquid shake-flask
cultures using absorbance measurements at 600 nm at different
concentrations of liptin 3e. The results are shown in FIGS.
20A-20H. Uninoculated control flasks were used as blanks.
Absorbance values of 0.1 OD unit was considered reasonable
threshold for positive growth. The use of the nutrient-rich BHI
culture allowed us to measure the effects of liptin 3e on bacterial
growth rates over long periods of time, which showed that liptin 3e
is more bacteriostatic with Gram-positive than Gram-negative
bacteria. Liptin exposure appears to stop bacterial growth, as
evidenced by long lag phases, which increase with liptin
concentration. In most cases, cultures recover, but growth rates
and maximum cell densities are decreased with liptin treatment.
Gram-positive, Gram-negative, and Mycobacteria were all inhibited
by liptin 3e with one inoculation, demonstrating the liptin's
highly bacteriostatic activity.
Liptin 3e Reduces Bacterial Growth Rates.
[0171] Bacterial growth rates were reduced by liptin 3e treatment
in a concentration-dependent manner. The data is shown in FIG. 21.
Rates were determined as the slope of the log phase of the growth
curve, after the lag phase. An exponential relationship exists with
marginally significant differences (p<0.1).
Liptin 3e Decreases Maximum Culture Density.
[0172] The highest culture densities observed were marginally
significantly correlated (p<0.1) to liptin 3e concentration.
Maximum culture densities represent the carrying capacity of a
culture flask. As shown in FIG. 22, cells grow less robustly while
using energy to resist environmental insult (liptin), and hence
reach lower final culture densities before the medium is
expended.
Minimum Inhibitory Concentration (MIC) Determinations in Muller
Hinton Culture Media and Growth Curves for Bacteria in BHI
(Brain-Heart-Infusion) Culture Media Show that Minor Structural
Changes in Liptins 1h-k Modulates their Antibacterial Activity.
[0173] MICs were determined in cation-adjusted Muller Hinton
culture. Standard CLSI protocol for serial dilutions of liptin
concentrations were used to determine the MIC, starting at 0.01 OD.
Two different experimental cultures were used and each data point
measured in triplicate. The liptin concentrations that show no
growth after 24 hours are shown in the table below.
TABLE-US-00003 TABLE 3 MIC (.mu.M) Liptin Liptin Liptin Liptin
Bacteria 1j 1i 1h 1k Escherichia coli 3.5 3 2 1.5 Staphylococcus
aureus 4 3 2.5 2 MRSA 4.5 3.5 3 2.5 Mycobacterium smegmatis 3.5 3
2.5 2 Pseudomonas aeruginosa 14 12 9 8 Klebsiella pneumoniae 3.5 4
3 2.5 Acinetobacter baumannii 4 3.5 2.5 2
[0174] Growth curves in BHI culture were generated (not shown) from
liquid shake-flask cultures using absorbance measurements at 600 nm
at different concentrations of liptins 1h-k. Uninoculated control
flasks were used as blanks. Absorbance values of 0.1 OD unit was
considered reasonable threshold for positive growth. The use of the
nutrient-rich BHI culture allowed us to measure the effects of the
small structural differences in liptins 1h-k on bacterial growth
rates over long periods of time. The growth curves showed that the
liptins are bactericidal, and that liptins 1h and 1k were the most
potent with Gram-positive and Gram-negative bacteria. Liptin
exposure appears to stop bacterial growth, as evidenced by long lag
phases, which increase with liptin concentration, and growth rates
and maximum cell densities are decreased with liptin treatment.
Most Gram-positive and Gram-negative bacteria were all killed or
greatly slowed in growth by liptins 1h and 1k with one inoculation
in BHI culture, demonstrating the liptin's highly bactericidal
activity. Liptins 1h-k showed high bacteriostatic effects with
Pseudomonas aeruginosa in BHI culture but not bactericidal
effects.
Minimum Bactericidal Concentration (MBC) Determination.
[0175] Minimum bactericidal concentration (MBC) were determined in
cation-adjusted Muller Hinton culture. Standard CLSI protocol for
serial dilutions of liptin concentrations were used to determine
the MBC, starting at 0.01 OD. Two different experimental cultures
were used and each data point measured in triplicate. P. aeruginosa
did not exhibit MBC below 20 .mu.M liptin concentration.
[0176] The MBC for liptins 1h-k was determined. The results are
shown below.
TABLE-US-00004 TABLE 4 MBC (.mu.M) Liptin Liptin Liptin Liptin
Bacteria 1j 1i 1h 1k Escherichia coli 15 14 9 7.5 Staphylococcus
aureus 15.5 10.5 11 9.5 MRSA 15 11.5 10 11.5 Mycobacterium
smegmatis 12 10.5 11.5 9.5 Pseudomonas aeruginosa n/a n/a n/a n/a
Klebsiella pneumoniae 13 12 8.5 9.5 Acinetobacter baumannii 12.5
11.5 8.5 7.5
As can be seen from the data in the table above, the MBCs for these
liptins are roughly 4-5 times the concentrations of their
respective MICs, and are therefore mostly bactericidal. Live-Dead
Stains for Bacteria Inoculated with Liptins 1h and 1k.
[0177] Molecular Probes BacLight.COPYRGT. kits containing the SYTO
9 green-fluorescent dye and propidium iodide red-fluorescent dye
were used to detect live or dead bacteria after ninety minute
exposure of bacteria to liptins 1h or 1k. Bacteria in Muller Hinton
culture were grown to an OD=0.3, were inoculated with liptins 1h
and 1k and after ninety minutes stained with a mixture of the above
dyes and affixed to a slide to be examined under a light microscope
(40.times.) to determine the number of live and dead cells (green
colored cells considered alive and red-colored cells considered
dead). Data was calculated based upon an average of 5 different
plate counts from each slide, and is based on a percent of total
bacteria counted (live and dead). Control sample contains no
liptin. The results presented demonstrate the potent bactericidal
efficacy of liptins after bacteria have been exposed to either 5 or
10 .mu.M liptin for only 90 minutes after one inoculation.
TABLE-US-00005 TABLE 5 Live-Dead Stains Liptin 1h Liptin 1k Control
Bacteria Live Dead Live Dead Live Dead Escherichia coli 35% 75% 35%
65% 100% 0 (10 .mu.M) Staphylococcus aureus 61% 39% 58% 42% 97% 3%
(5 .mu.M) MRSA (5 .mu.M) 28% 72% 30% 70% 98% 2% MRSA (10 .mu.M) 15%
84% 0 100% Mycobacterium 7% 93% 0 100% 100% 0 smegmatis (5 .mu.M)
Klebsiella 33% 67% 0 100% 86% 14% pneumoniae (5 .mu.M) Klebsiella 0
100% na na -- -- pneumoniae (10 .mu.M) Acinetobacter 12% 88% 3% 97%
95% 5% baumannii (5 .mu.M)
Plasma Membrane Depolarization as Mechanism of Bactericidal Action
of Liptins 1h and 1k.
[0178] The Molecular Probes BacLight.RTM. Bacterial Membrane
Potential kit was used to detect changes in the polarization of the
bacterial plasma membrane upon inoculation of bacteria with varying
concentrations of liptins 1h or 1k. The dye
3,3-diethyloxacarbocyanine iodide exhibits either green or red
fluorescence depending on the membrane potential, and the graph is
a ratio of red (high membrane potential) to green (low membrane
potential) fluorescence. CCCP (carbonyl cyanide
3-chlorophenylhydrazone) is a proton ionophore that destroys plasma
membrane potential and as such is a positive control. The results
in FIGS. 23A and 23B show that both a Gram-positive and
Gram-negative bacteria exhibit large membrane depolarization when
the bacteria are exposed to one half than the liptins' MICs, and
the loss of membrane potential is concentration dependent. With
plasma membrane depolarization the bacterial cell loses its ability
to synthesize ATP and proteins. Additionally loss of membrane
potential stops the ability for cell replication due to an
inability for proper placement of divisome proteins in the plasma
membrane. While this may be a result of plasma membrane cation
leakage, as stated above the liptins do not appear to make pores in
the membrane. Thus, it is possible that the liptin's mechanism of
action is a result of their deleterious effect on the functioning
of electron transport proteins found in the plasma membrane.
Scanning Electron Microscopy.
[0179] FIG. 24 shows a comparison of scanning electron micrographs
of E. coli (A) treated or (B) untreated with 5 .mu.M of liptin 1k.
The images are to the same scale. There is a clear stunting of
growth in the E. coli that was exposed to the liptin. There also
seems to be a wrinkling of the outer membrane of the E. coli
exposed to liptin 1k as opposed to a smoother outer membrane in the
unexposed bacterial cell. Importantly, no hole or apparent
destruction of the outer membrane is seen in the liptin-exposed
bacterial cell.
[0180] FIG. 25 shows a comparison of SEM images of MRSA (A) treated
or (B) untreated with 5 .mu.M of liptin 1k. The images are to the
same scale. There appears to be a wrinkling or non-uniformity of
the outer membrane of the MRSA exposed to liptin 1k as opposed to a
smoother outer membrane in the unexposed bacterial cell.
Importantly, no hole or apparent destruction of the outer membrane
is seen in the liptin-exposed bacterial cells. The liptin treated
MRSA cells also appear to be somewhat desiccated compared to those
that are untreated.
Molecular Dynamics (MD) of Liptin 1h Binding to Two PG Lipid Head
Groups in Lipid Patch
[0181] FIG. 26 shows an MD simulation of the binding of the liptin
1h to two PG head groups, showing multiple hydrogen bonding
interactions between the lipid head group and liptin. Only two
lipids, hydrogen-bound water molecules, and liptin 1h shown-patch
lipids and other waters removed for purposes of clarity. The
simulation was run with six liptin 1h molecules (10 mM) in aqueous
solution above an equilibrated lipid membrane of 80:20 DPPE:DPPG
(DP=Dipalmitoyl) with 52 DPPE and 13 DPPG per side in explicit
water. Binding occurs within 20 ns, with no input to steer liptin
toward PG lipid head group. Unlike liptin 3e, which binds to one PG
lipid head group, the liptin 1h is seen interaction with two PG
lipid head groups. One head group exhibits hydrogen bonding to the
liptin's para amide functionality as well as the ammonium hydrogen
via an interceding water molecule, both bound to the lipid's
phosphorus negatively charged oxygen. The other ammonium hydrogen
is also hydrogen bound to the second PG lipid head group via an
interceding water molecule to the phosphorus oxygen that is
negatively charged.
Example 7
Initial Toxicity Study
[0182] Toxicity Studies of Human HeLa (Vaginal Carcinoma) and A549
(Lung Carcinoma Epithelial) Cells with Liptins 1h and 1k.
[0183] Eukaryote cell toxicity was determined using a Pierce LDH
assay kit including the Chemical Compound-Mediated Cytotoxicity
assay. This kit measures cell death by measuring the amount of
lactate dehydrogenase released from cells. Lactate dehydrogenase
(LDH) is a cytosolic enzyme present in many different types of
cells. When the plasma membrane is damaged, LDH is released into
cell culture media. The released LDH can be quantified by a coupled
enzymatic reaction. First, LDH catalyzes the conversion of lactate
to pyruvate via reduction of NAD+ to NADH. Second, diaphorase uses
NADH to reduce a tetrazolium salt (INT) to a red formazan product.
Therefore, the level of formazan formation is directly proportional
to the amount of released LDH in the medium.
[0184] The results are shown in FIG. 27. With both cell lines the
toxicity associated with liptin 1h was substantially greater than
that observed with liptin 1k. For example, with HeLa cells there is
no cell toxicity observed up to about 15 .mu.M with liptin 1k,
where there is large toxicity with liptin 1h at that concentration.
With the A549 lung epithelial cells the toxicity near 15 .mu.M
liptin 1k is around 8%, whereas with liptin 1h the cytotoxicity of
the lung cells was over 25%. The MICs of 1k determined for the 6
bacteria stated above was measured at 1.5-2.5 .mu.M liptin, thus
there is no measured cytotoxicity observed in the two cells lines
between 5-10 times the concentrations of 1k's MICs.
* * * * *