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Antimicrobial agents and chemotherapy 2005 dartois
1. 10.1128/AAC.49.8.3302-3310.2005.
2005, 49(8):3302. DOI:Antimicrob. Agents Chemother.
and Thomas R. Parr Jr.
Granja, Colleen Gritzen, Dana Weinberger, M. Reza Ghadiri
Cabezas, Ellen Chi, Chad Dubbelde, Carrie Dunn, Juan
Véronique Dartois, Jorge Sanchez-Quesada, Edelmira
Synthetic Cyclic Peptides
Systemic Antibacterial Activity of Novel
http://aac.asm.org/content/49/8/3302
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3. In this report, we describe the discovery of novel antibacte-
rial compounds resulting from the combinatorial synthesis of
short cyclic peptides made of alternating D- and L-␣-amino
acids. Six representative compounds were chosen based on
their in vitro potency and their in vivo tolerability and efficacy
in murine models of infection. The present study was con-
ducted to evaluate the in vivo pharmacodynamics and initial
pharmacokinetics of these six cyclic peptides in comparison to
vancomycin and oxacillin.
MATERIALS AND METHODS
Animals. Four-week-old, specific-pathogen-free, ICR female mice (Harlan
Sprague-Dawley, Indianapolis, IN) weighing 21 to 24 g were used for all studies.
The Adaptive Therapeutics Animal Care and Use Committee approved all the
experimental protocols.
Bacterial strains and media. Methicillin-sensitive S. aureus (MSSA) strains
ATCC 25923, 29213, and 13709 (Smith) and MRSA strains ATCC 43300 and
33591 were used for in vitro and in vivo studies. Organisms were grown, subcul-
tured, and quantified in Mueller-Hinton broth (MHB), cation-adjusted MHB
(CAMHB), or tryptic soy broth (TSB) and on tryptic soy agar (TSA) (Difco
Laboratories, Detroit, MI) or blood agar plates (Hardy Diagnostics, Santa
Maria, CA).
Compounds. Vancomycin, oxacillin, and rifampin were obtained from Sigma-
Aldrich (St. Louis, MO) and solubilized in 5% dimethyl sulfoxide (DMSO)–
water for in vitro use or phosphate-buffered saline (PBS) for in vivo use. Peptide
test compounds were purified by high-performance liquid chromatography
(HPLC) using mixtures of trifluoroacetic acid (TFA), water, and acetonitrile as
the solvent system. Purity values of compounds used for in vivo experiments were
between 95% and 98%.
Primary screening of peptide libraries. Primary cyclic peptide libraries were
routinely screened at either 8 g/ml or 16 g/ml for antibacterial activity against
S. aureus ATCC 29213. Nominal 200-g/ml stock solutions of the peptides in 5%
DMSO in water were aliquoted into sterile 96-well flat-bottom polystyrene plates
using a Tomtec Quadra 96 liquid handler. For screening compounds at 8 g/ml,
4 l of 200-g/ml stocks were distributed into plates prefilled with 4 l of 5%
DMSO in water per well. For screening compounds at 16 g/ml, 8 l of the
200-g/ml stocks was distributed into empty plates.
S. aureus was grown in a shaking incubator at 35°C and 200 rpm in 20 ml TSB
using individual colonies retrieved from a fresh overnight TSA-sheep blood
plate. The culture was grown to a density of approximately 1 ϫ 108
cells/ml and
then diluted to 5 ϫ 105
cells/ml in CAMHB. This 5 ϫ 105
-cell/ml dilution was
dispensed into the compound-containing plates (90 l/well) using a Titan Mul-
tidrop 384. Controls on each plate included a blank medium control, a 100%
inhibition control (vancomycin at a final concentration of 4 g/ml), and a 100%
growth control (5% DMSO in water only). After addition of the cell suspension,
plates were sealed and incubated at 35°C for 18 h. The optical density at 595 nm
(OD595) of each well was measured after 18 h using a Tecan Genios spectro-
photometer. OD data were uploaded and processed by an internal database and
expressed as percent inhibition relative to that of the 100% inhibition controls.
MICs and minimum bactericidal concentrations (MBCs). MICs of the various
compounds against a panel of gram-positive pathogens were determined by
standard National Committee for Clinical Laboratory Standards microdilution
methods (26a) with twofold serial dilutions in CAMHB. Each well contained 100
l (50 l inoculum plus 50 l of drug-containing MHB). The final cell density
was 1 ϫ 105
to 5 ϫ 105
/ml. MIC endpoints were determined by visual inspection
after incubation at 35°C for 18 to 24 h. Final results were expressed as means of
two to four determinations. MBCs were defined as the minimum concentration
of agent that brought about Ͼ99.9% killing of the organism.
Antimicrobial killing kinetics. The kinetics of bactericidal activity was deter-
mined by measuring changes in the viable counts of bacteria exposed to various
test compounds. A 20-ml liquid culture of S. aureus ATCC 25923 was grown in
TSB at 35°C for 1 h or until the OD625 reached 0.08 to 0.1 (108
CFU/ml). The
culture was diluted 1:100 in 4 ml (106
CFU/ml) of prewarmed medium containing
the test compound, control antibiotic, or vehicle-only control at a concentration
of four times the MIC and incubated at 35°C. Samples were drawn at 0, 2, 5, 10,
30, 60, and 90 min. Serial dilutions of these samples were plated on CAMHB and
incubated overnight at 35°C. The number of CFU on plates with an appropriate
density was scored the next day.
Resistance development studies. MICs of six peptide compounds (6752, 7251,
1316, 1150, 6756, and 6853), vancomycin, and rifampin were newly determined as
described above using a single culture of S. aureus ATCC 29213 inoculated with
colonies from a fresh streak plate of this strain. The final concentrations of the
serial twofold dilutions of the compounds ranged from 32 g/ml to 0.25 g/ml,
and those of vancomycin and rifampin ranged from 16 g/ml to 0.125 g/ml and
0.25 g/ml to 0.00195 g/ml, respectively, for these MIC experiments.
After this initial MIC experiment, MICs were then determined daily for 15
days for each of the eight compounds using cells from the well in which the
compound concentration was one-half the MIC (1/2 MIC well). For each com-
pound, the 1/2 MIC well from the previous day’s MIC assay plate was resus-
pended and the OD595 of the resuspension was determined. The resuspension
was then diluted to 5 ϫ 105
cells/ml in CAMHB and used to again determine the
MIC of the same compound to which those cells had previously been exposed.
Compound concentration ranges were adjusted during the course of the 15-day
experiment when necessary to keep them useful for MIC determination. All MIC
determinations were done in duplicate.
Aliquots of the 5 ϫ 105
-cell/ml dilutions for each 1/2 MIC well were period-
ically plated on TSA-sheep blood plates and incubated at 35°C for 18 h to verify
the cell densities by colony counts and to visually inspect the phenotype of the
resultant S. aureus colonies.
Hemolysis assay. Serial twofold dilutions of the compounds were prepared in
20% DMSO in water, and 20 l-aliquots were distributed in 96-well plates.
Mouse red blood cells (RBC) were obtained by centrifuging whole blood at 1,000
ϫ g, washed in PBS, and resuspended in PBS containing 10% fetal bovine serum
(FBS) at a final RBC concentration of 5%. The RBC suspension (80 l) was
added to each well, and the plate was incubated at 35°C for 30 min. Following
centrifugation at 1,000 ϫ g, hemolysis was assessed by measuring the OD595 of
the serum layer. Hemolysis is calculated as the percentage of total hemolysis as
defined by hemolysis achieved by 100 g/ml melittin (Sigma Chemical Company,
St. Louis, MO) as the control. Fifty percent hemolysis (HD50) values were
calculated as the compound concentrations required to lyse 50% of the cells.
Cytotoxicity assay. Cellular cytotoxicity was measured by a colorimetric assay that
makes use of the tetrazolium salt MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy-
methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium]. A subconfluent monolayer cul-
ture of rat hepatoma cells (H-4-II-E) (ATCC CRL1548) was trypsinized in 0.25%
trypsin–1 mM EDTA in Hanks balanced salt solution without Ca2ϩ
or Mg2ϩ
. Cells
were collected in complete Eagle’s minimum essential medium containing 10% FBS.
Complete growth medium was Eagle’s minimal essential medium combined with
FBS to a final concentration of 10% and antibiotic-antimycotic liquid so as to contain
100 U/ml penicillin, 100 g/ml streptomycin, and 0.25 g/ml amphotericin B in
0.85% saline. Dilutions of cells were made in complete medium at 5 ϫ 104
cells/ml,
and 90 l of the resultant mixture was aliquoted onto 96-well plates.
Cells were allowed to incubate at 37°C in the presence of 5% CO2, and 10 l
of serial twofold dilutions of compound in 5% DMSO in water was then added
to the 90 l of medium and cells. Thioridazine was included as a positive control.
Post 24-h incubation in the presence of compound, cells were tested with MTS
(25 l each of MTS at 40 mg/ml in 100% DMSO and phenazine methosulfate at
3 mM in PBS per well). Initial readings of the plates were performed at 490 nm
immediately following addition of the reagents. Plates were then allowed to
incubate 3 h more, and an endpoint reading was taken at 490 nm with 5 s of linear
shaking immediately before reading. The net increase between the initial and
final readings was determined for each well. Percent cytotoxicity was calculated
as follows: (control value Ϫ test value) ϫ 100/control value. Fifty percent inhib-
itory concentrations were obtained by using the GraphPad Prizm software (ww-
w.graphpad.com; San Diego, CA).
Tolerance testing. To determine the maximum tolerated dose in the mouse,
compounds were prepared in 5% (wt/vol) dextrose in water and injected intra-
venously (i.v.) starting at 10 mg/kg; an up-down protocol, based on the outcome
obtained with the initial dose of 10 mg/kg, was followed. The following signs were
recorded: reduced motor activity, piloerection, redness in the ear lobe, cyanosis,
protruding eyeballs, slow or labored breathing, loss of response in the rear legs,
convulsions, and death. A score was given based on the intensity and number of
the observed signs listed above as follows: 5, no signs observed; 4, light redness
in the ear lobe; 3, reduced motor activity and redness in the ear lobe; 2, reduced
motor activity, piloerection, and pronounced redness in the ear lobe; 1, protrud-
ing eyeballs, temporary loss of motor activity, piloerection, redness in the ear-
lobes and legs, and labored breathing; 0.5, protruding eyeballs, temporary loss of
motor activity, piloerection, redness in the earlobes and legs, convulsions with
subsequent loss of response in the rear legs, and labored breathing; 0, death.
Neutropenic-mouse thigh model of infection. Mice were rendered neutro-
penic by injecting cyclophosphamide (Henry Schein, Melville, NY) intraperi-
toneally 4 days (150 mg/kg of body weight) and 1 day (100 mg/kg) before
experimental infection. Previous studies have shown that this regimen pro-
duces neutropenia in this model for 5 days (12). Broth cultures of freshly
VOL. 49, 2005 SYSTEMIC ACTIVITY OF NOVEL ANTIMICROBIAL PEPTIDES 3303
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4. plated bacteria were grown overnight in MHB. After a 1:4,000 dilution into
PBS, bacterial counts of the inoculum ranged between 106
and 2 ϫ 106
CFU/ml. Mice were anesthetized briefly with approximately 4% isoflurane
just prior to inoculation. The bacterial suspension (0.1 ml) was injected
intramuscularly into each thigh (approximately 105
CFU/thigh). In all studies,
treatment was initiated 2 h following bacterial inoculation through an i.v.
route. At various time points following treatment, groups of three mice were
humanely sacrificed by CO2 asphyxiation. The thigh muscle mass, including
the bone, was homogenized using a tissue homogenizer (Polytron; Kine-
matica AG) and decimally diluted in iced PBS, and 5-l aliquots of five serial
dilutions were plated on TSA. Following overnight incubation at 35°C, CFU
were enumerated for each thigh and expressed as the log10 CFU/thigh. The
limit of detection was Յ3.3 log CFU/thigh. Efficacy (as presented in Table 2)
was calculated by subtracting the mean log10 CFU/thigh of each group of
mice at a given time point following therapy from the mean log10 CFU/thigh
of untreated control mice taken at the corresponding time point. The post-
antibiotic effect (PAE) was calculated by subtracting the time it took for
organisms to increase 1 log in the thighs of control animals from the time it
took organisms to grow the same amount in treated animals after serum drug
levels fell below the MIC (11): PAE ϭ T Ϫ C, where C is the time for 1 log10
control growth and T is the time for 1 log10 treatment growth after levels have
fallen below the MIC.
Mouse peritonitis model. Type II mucin (Sigma-Aldrich, St. Louis, MO), an
enzyme extract of porcine stomach, was used as an adjuvant for inoculation of
the mice and was prepared as a 7% stock solution in PBS supplemented with 0.2
mM FeNH4–citrate. The bacterial suspensions were diluted 1:10 with ice-cold
mucin solution to give a final mucin concentration of 6.3% and kept on ice prior
to inoculation. Inoculation was performed by intraperitoneal injection of 0.2 ml
of a mucin-bacterial suspension via a 25-gauge syringe. The inoculum contained
3 ϫ 106
CFU/mouse of S. aureus ATCC 13709 (Smith), which causes death of
100% of untreated animals within approximately 18 h following inoculation.
Groups of 10 to 12 mice were treated i.v. with decreasing doses of test and
control compounds at 0 h and 2 h following inoculation. For each experiment, a
group of mice treated with mucin was included as a control for the lethality of the
infection. The endpoint was determined at 48 h. The ED50, the single dose
providing protection to 50% of the mice, was calculated using the Reed and
Muench equation (29).
Pharmacokinetic study. Mice received an 8-mg/kg dose of test compounds
prepared in 5% dextrose via tail vein injection. Groups of three mice were
euthanized by CO2 asphyxiation at different intervals from 2 min to 8 h after
dosing, and blood samples were collected by cardiac puncture. After 15 min at
room temperature, coagulated blood was centrifuged and serum was recovered
and stored frozen at Ϫ20°C. The serum samples were analyzed and quantified for
peptide content by reverse-phase HPLC coupled with mass spectrometry. All
plasma samples were deproteinized with 2 volumes acetonitrile, diluted 1:1 in
running buffer, and injected onto a Waters Atlantics C18 column (2.1 by 150 mm)
maintained at room temperature. The peptide was eluted using a flow rate of 200
l/min with a 1:1 mixture of 0.1% formic acid in water and 50% acetonitrile using
an isocratic gradient. An ABI API 2000 liquid chromatography-tandem mass
spectrometry system was used to quantify the peptide in each serum sample. For
quantitative calibration, standard curves were established using spiked com-
pounds in serum. The limit of detection was between 0.1 and 1.0 g/ml.
Data analysis. One- and two-compartment pharmacokinetic models with bolus
input and first-order elimination from the central compartment were fitted to the
serum concentration-time data using WinNonlin (Pharsight, Mountain View,
CA). The optimal number of exponentials was selected using the Akaike infor-
mation criterion (37). The pharmacokinetic parameters total drug clearance,
volume of the central compartment, volume of distribution at steady state (Vss),
and half-lives at the alpha and beta phases were obtained directly from the
modeling output.
Peptide synthesis: solvents and reagents. Acetonitrile (HPLC grade; Aldrich,
St. Louis, MO), dichloromethane (DCM, HPLC grade; Aldrich), diethyl ether
(American Chemical Society grade; Fisher), isopropanol (American Chemical
Society grade; Fisher), N,N-dimethylformamide (DMF, sequencing grade; Al-
drich), N-methylpyrrolidinone (NMP, peptide synthesis grade; Aldrich), and
N,N-diisopropylethylamine (DIEA, peptide synthesis grade; Aldrich) were ob-
tained from the noted providers and used without further purification. TFA
(reagent grade; Aldrich), diisopropylcarbodiimide (DIC; Aldrich), N-hydroxy-
benzotriazole (anhydrous; Fisher), 1-hydroxy-7azabenzotriazole (Applied Bio-
systems, Foster City, CA), 2-(1-H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate (HBTU; Novabiochem EMD Biosciences, San Diego, CA),
phenylsilane (Aldrich), tetrakistriphenylphosphine palladium (0) [Pd(PPh3)4;
Aldrich], and benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluoro-
phosphate (NovaBiochem) were used as obtained. Trityl chloride polystyrene
macrobeads (500 to 560 m in diameter) were obtained from Peptides Interna-
tional (Louisville, KY). Commercially available amino acids and resins were used
as obtained from Novabiochem, Advanced Chemtech (Louisville, KY), or
NeoMPS (San Diego, CA). The side chain protection groups were as follows for
9-fluorenylmethoxy carbonyl (Fmoc) synthesis: Arg (Pbf), His (Trt), Lys (Boc),
Ser (t-Bu), Thr (t-Bu), Tyr (t-Bu), Asp (Ot-Bu), Glu (Ot-Bu), Asn (Trt), and Gln
(Trt). All other chemicals were used as obtained from Aldrich, Sigma (St. Louis,
MO), or Fisher (Fluka, Acros).
Single-compound-per-bead combinatorial cyclic peptide libraries. The pep-
tides were prepared on 500- to 560-m-diameter macrobeads of trityl chloride
polystyrene-based resin using the split-and-pool approach (17, 24). Coupling of
the first allyl amino acid to the resin was conducted using 1.5 equivalents (eq) of
N-ε-deprotected N-␣-Fmoc–L-Lys–allyl ester and 4 eq DIEA in DCM overnight
(0.2 M Lys). After coupling, the resin was washed with DCM (3 ϫ 15 min), 10%
DIEA, 10% methanol (MeOH), 80% DCM (3 ϫ 15 min), and DCM (3 ϫ 15
min). After drying the beads in vacuo, the resin loading level was calculated by
weight or based on Fmoc release upon treatment with piperidine and monitoring
UV absorbance at 290 nm. Beads were swollen in DMF for at least 30 min prior
to initial Fmoc deprotection. Sequential growing of the peptide chain was ac-
complished via repeated cycles of deprotecting with 25% piperidine in DMF (2
ϫ 15 min); washing with DMF (4 ϫ 15 min); coupling with 4 eq amino acid, 10
eq DIEA, and 4 eq HBTU in DMF (1 ϫ 4 h); and washing with DMF (4 ϫ 15
min). Following the synthesis of the linear peptide, the beads were washed with
DCM. To the resin was added a degassed solution of 0.5 eq Pd(PPh3)4 in 90%
DCM–10% phenylsilane. After shaking under nitrogen for 2 h, the resin was
washed sequentially with DMF, DCM, and DMF. Resin was treated again for 2 h
with 0.5 eq Pd(PPh3)4 in 90% DCM–10% phenylsilane under nitrogen and
washed with a solution of 1% sodium dimethylthiocarbamate in DMF (3 ϫ 15
min) and 1% DIEA in DMF (3 ϫ 15 min). Cyclization was accomplished by
deprotection of the terminal Fmoc using 25% piperidine–DMF (2 ϫ 15 min);
washing with DMF (3 ϫ 15 min), 10% DIEA-DMF (3 ϫ 15 min), and NMP (1
ϫ 15 min); and cyclizing overnight with 3 eq each of HATU, HOAT, and DIEA
in dry NMP (0.2 M HATU). After cyclization, the resin was washed with NMP
(4 ϫ 15 min), DCM (2 ϫ 15 min), and MeOH (1 ϫ 15 min). The beads were
dried in vacuo and arrayed (one bead per well) in 96-well plates (1-ml capacity
per well) analogously to the arraying reported by Clemons et al. (7). To each
well, 95 l of a cleavage cocktail (2.5% triisopropylsilane, 2.5% H2O, 95% TFA)
was added and the hydrolysis was allowed to continue for 2 h. After cleavage,
diethyl ether was added to precipitate the peptide. The plates were centrifuged,
the supernatant was removed, and the volatiles were evaporated. The residue
was dissolved in 5% DMSO in water to prepare nominal 200-g/ml stock solu-
tions (concentration estimate based on average theoretical yields and resin load-
ing) for biological assays and mass spectrometry sequence determination (28).
The final yield per bead was approximately 100 g.
Individual peptide synthesis. Peptides were synthesized using standard solid-
phase Fmoc protocols (36) on the Fmoc–Lys–O-allyl ester-loaded trityl resin with
coupling reaction times ranging from 4 to 12 h either manually (using HBTU as
the coupling reagent, reaction times ranging from 0.5 to 1 h) or in a peptide
synthesizer (Advanced Chemtech APEX 396; DIC–N-hydroxybenzotriazole as
the coupling reagent and employing a double-coupling protocol, 2 ϫ 1 h).
Following synthesis of the linear peptide, the resin was swollen in dry DCM for
20 min. To the resin was added a degassed solution of 0.5 eq Pd(PPh3)4 in 90%
DCM–10% phenylsilane. After shaking under nitrogen for 2 h, the resin was
washed sequentially with DMF, DCM, and DMF. Resin was treated again for 2 h
with 0.5 eq Pd(PPh3)4 in 90% DCM–10% phenylsilane under nitrogen and
washed with a solution of 1% sodium dimethylthiocarbamate in DMF (3 ϫ 15
min) and 1% DIEA in DMF (3 ϫ 15 min). After the final Fmoc deprotection
(25% piperidine in DMF, 2 ϫ 15 min), the resin was washed thoroughly with
DMF (3 ϫ 15 min), 10% DIEA-DMF (3 ϫ 15 min), and 0.8 M LiCl-DMF (3 ϫ
15 min). The resin was treated with 5 eq benzotriazole-1-yl-oxy-tris-pyrrolidino-
phosphonium hexafluorophosphate–5 eq 1-hydroxy-7azabenzotriazole–20 eq
DIEA in 0.8 M LiCl-DMF for at least 12 h. After washing with DMF (3 ϫ 15
min) and DCM (2 ϫ 15 min), followed by MeOH, the peptide was cleaved from
the resin and deprotected by treatment with 95% TFA–2.5% water–2.5% triiso-
propylsilane for 2 h. Peptides were recovered by precipitation with diethyl ether
and purified by HPLC using mixtures of TFA, water, and acetonitrile as eluents.
Mass spectrometric sequence determination of cyclic peptide library mem-
bers. Mass spectrometry was used as previously reported (28) to determine the
likely sequence of cyclic peptide library members. Fidelity for the identification
routinely was Ͼ90%.
3304 DARTOIS ET AL. ANTIMICROB. AGENTS CHEMOTHER.
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5. RESULTS
Library design and screening. Combinatorial libraries of
novel cyclic D,L-␣-peptides were synthesized and screened for
antimicrobial activity. Similar molecules have previously been
shown to form self-assembling supramolecular structures with
bioactivity (15). We generated libraries of both cyclo-D,L-␣-
hexa- and octapeptides designed to determine the effect of the
global amino acid composition of the peptides on their anti-
microbial activity and the optimal ratio of hydrophobic to hy-
drophilic residues for potency and selectivity toward bacterial
cells. Studies were initiated with a hexamer library that was
designed to provide members possessing between two and four
consecutive hydrophilic residues chosen from among six amino
acids having either charged or noncharged side chains (Lys,
Arg, His, Glu, Asn, and Ser) but always having a lysine residue
at position 1 for resin attachment purposes. Positions within
the hydrophobic region were filled with Trp and Leu. The
design of the compounds in the octamer library is analogous,
but the number of consecutive hydrophilic residues ranged
from three to five. A schematic depiction of these original
libraries is given in Fig. 1. In peptide sequences described
throughout this work, the conventional one-letter code for
amino acids is used, except for the unnatural amino acids
ornithine (O) and O-benzyltyrosine (BY); residues having D
stereochemistry are indicated by lowercase letters, and a trail-
ing dash at the end of the peptide sequence indicates that the
peptide is cyclic.
The above-mentioned libraries were screened for antibacte-
rial activity, and the resulting data were used to design subse-
quent libraries in an effort to optimize the biological data and
physical properties (i.e., solubility) of the analyzed members.
Over the full course of our study, approximately 140,000 rep-
resentative compounds from a collection of Ͼ1 ϫ 106
synthe-
sized hexapeptides and octapeptides were assayed for antibac-
terial activity against S. aureus 29213 in primary screens at a
final peptide concentration of either 8 g/ml or 16 g/ml.
Approximately 8,000 of the tested peptides (0.57%) were
found during this screening to inhibit S. aureus growth by 90%
or greater relative to the 100% growth controls under the
specific screening conditions used. These compounds were re-
covered and tested again to confirm antibiotic activity using the
same protocol followed during primary screening. A success
rate of 80 to 90% was routinely obtained upon secondary
screening. Compounds with confirmed activity were then ana-
lyzed by mass spectrometry to determine the sequence of the
cyclic peptide. Compounds for which amino acid sequences
were generated with high confidence were then synthesized on
a larger scale (10 to 100 mg) for further testing.
Identification of lead compounds. After scaled-up synthesis,
compounds were again tested for in vitro antimicrobial activity
against S. aureus. Preliminary in vivo characterization of com-
pounds with confirmed activity led to the identification of sev-
eral compounds which were further investigated (Table 1). The
hexamer peptide l-L-w-H-s-K- (not shown) and the octamer
peptide k-K-h-K-w-L-w-K- (1150) were isolated directly from
the starting combinatorial libraries (Fig. 1). The hexamer pep-
tide properties were optimized by performing point substitu-
tions, resulting in the cyclo-D,L-␣-hexapeptide l-L-w-H-o-K-
(1316). Similarly, octamers with confirmed activity were opti-
mized by amino acid substitution at specific peptide residues to
yield S-w-F-k-T-k-S-k- (6752), S-w-F-k-H-k-S-k- (6756), and
S-w-BY-k-N-k-S-k- (6853). These second-generation com-
pounds have certain improved in vitro or in vivo traits over
those of their parent molecules. Additionally, the sequence
s-W-f-K-t-K-s-K- (7251), the enantiomer of 6752, also was syn-
thesized to investigate whether stereochemistry plays a critical
role in biological activity.
In vitro inhibitory activity. Table 1 lists the MICs of the six
lead compounds against MSSA ATCC 29213 in CAMHB.
MICs were also obtained in CAMHB supplemented with 50%
mouse serum to assess potential protein binding issues. With
the exception of 6853, serum MICs remained within a twofold
dilution of the standard MICs, suggesting only minimum se-
rum protein binding or inactivation. Interestingly, 6752 and its
FIG. 1. Schematic description of hexamer (left) and octamer
(right) libraries. Hydrophilic residue positions are shown in black cir-
cles and hydrophobic positions in gray circles. Positions indicated by
white circles are those that include either hydrophilic or hydrophobic
amino acids.
TABLE 1. Summary of in vitro potency and toxicity and in vivo tolerability of peptide compounds
Compound Sequencea
MIC for
S. aureus 29213
(g/ml)
Serum MIC for
S. aureus 29213b
(g/ml)
MBC for
S. aureus 29213
(g/ml)
HD50
(g/ml)
EC50
d
(g/ml)
Tolerancec
at 8 mg/kg
Max tolerated
dose (mg/kg)
6752 S-w-F-k-T-k-S-k- 8 16 32 Ͼ400 Ͼ100 4 Ͼ25
7251 s-W-f-K-t-K-s-K- 8 16 16 Ͼ400 Ͼ100 4 Ͼ20
6756 S-w-F-k-H-k-S-k- 12 12 16 Ͼ400 Ͼ100 3 Ͼ20
6853 S-w-BY-k-N-k-S-k- 2 8 4 209 63.5 2 20
1316 I-L-w-H-o-K- 2 1 32 190 5.9 1 10
1150 k-K-h-K-w-L-w-K- 2 2 32 101 29.9 0.5 Ͻ10
a
Peptide sequences are written using the conventional one-letter code for amino acids, except for the unnatural amino acids ornithine (O), and O-benzyltyrosine
(BY). Residues having D stereochemistry are in lowercase. A trailing dash at the end of the peptide sequence indicates that the peptide is cyclic.
b
Serum MICs were determined in cation-adjusted MHB in the presence of 50% mouse serum.
c
For an explanation of tolerance scoring, see Materials and Methods.
d
EC50, 50% effective concentration.
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6. enantiomer 7251 exhibited similar potency in vitro. This ob-
servation is consistent with the thought that small antimicro-
bial peptides exert their effect on bacteria via nonchiral inter-
actions with membrane lipids (35), even though enzymatic
inhibitory activities of most small bioactive molecules are en-
antioselective with a highly selective fit for one of the two
enantiomeric forms (9, 10, 34). The MIC90 (the concentration
at which 90% of the isolates tested were inhibited) was deter-
mined for 6752 and 6756 using a panel of 10 strains that
included MRSA strains ATCC 43300 and 33591. The MIC90s
of 6752 and 6756 were 8 and 12 g/ml, respectively, identical to
their corresponding MICs against S. aureus 29213. Bactericidal
activity was assessed by measuring MBCs as shown in Table 1.
Time-kill curves were obtained to study the kinetics of bacte-
ricidal activity. Compounds 6752 and 1150 and vancomycin
were tested against MSSA strain ATCC 25923 at four times the
compound’s MIC. Consistent with their mechanism of action,
both peptides displayed much more rapid bacterial killing than
did vancomycin. Compound 1150 decreased the viable counts
by 100% within 5 min, while 6752 reduced the viable counts by
ϳ3 log units after the same incubation period. On the other
hand, vancomycin caused no change in CFU in this period and
a 1.6-log CFU/ml decrease over an incubation period of 6 h at
four times the MIC.
Attempts to generate spontaneous resistance in vitro. The
ability of S. aureus 29213 to develop resistance to 6752, 7251,
1316, 1150, 6756, and 6853, and to vancomycin and rifampin as
controls, was evaluated by repeated passaging and MIC deter-
mination. S. aureus cells growing in the presence of a com-
pound at half the MIC on a particular day were used the next
day in an MIC assay of that same compound. In this manner,
cells were continually exposed to a single compound at one-
half the MIC while being passaged over 15 days. The MIC of
rifampin increased as much as 512-fold over 15 days—rifampin
is an agent to which resistance is known to arise quite easily by
spontaneous chromosomal point mutations (31). In contrast,
the MICs of 7251, 6853, and 6756 increased only twofold, while
the MIC of 6752 increased fourfold and that of 1150 remained
unchanged. Again, both 6752 and its enantiomer 7251 were
very similar with respect to the capacity of S. aureus to acquire
resistance to each compound. Similarly to the tested peptides,
the MIC of vancomycin under these conditions only increased
twofold; vancomycin is generally regarded as an antibiotic to
which spontaneous resistance development is unlikely to occur
under conditions where horizontal genetic transfer between
species is excluded (22).
To confirm that the observed increases in MICs were due to
adaptive changes in the S. aureus cells and not due to instability
of the anti-infective compounds, MICs of the peptides for the
passaged bacteria were determined at the end of the 15 days in
parallel with S. aureus cells that had not previously been ex-
posed to antibiotics. MICs obtained with fresh S. aureus cells
for all compounds tested were the same as those at the start of
the experiment. This indicated that the compounds were stable
during the period of the experiment and that the cells that had
been continuously exposed to test compounds had acquired
mechanisms allowing for increased resistance (data not
shown). Due to the poor diffusion properties of the peptides in
solid growth medium, we were unable to experimentally per-
form the conventional assay using multiples of the MICs and
detecting spontaneous mutants arising in conventional growth
agar medium. Similar behavior has been observed for orita-
vancin, a new semisynthetic derivative of vancomycin in clinical
development (Thomas R. Parr, unpublished observation).
In vitro toxicity and in vivo tolerability studies. Hemolytic
activity of the peptides was tested using a mouse RBC lysis
assay. As shown in Table 1, 6752, 6756, and 7251 exhibited no
significant hemolytic activity at clinically relevant concentra-
tions, while peptides 6853, 1316, and 1150 had HD50s ranging
between 100 and 200 g/ml, with 1150 being the most hemo-
lytic compound. Melittin was used as a positive control and
showed an HD50 of 2 g/ml under those conditions. Com-
pound cytotoxicity was measured using a dye exclusion
method. Trends in cytotoxicity measured in a rat hepatoma cell
line mirror those obtained in the hemolysis assay, with 6752,
6756, and 7251 having no significant cytotoxicity below 100
g/ml, while 6853, 1150, and 1316 display increasing toxicity in
vitro. Consistent with these results, 6752 and its enantiomer
7251 were well tolerated in vivo in the mouse with no visible
signs of discomfort when administered as a bolus i.v. injection
at 8 mg/kg. The maximum tolerated dose was above 20 mg/kg.
Both 1316 and 1150 were extremely poorly tolerated, with 1150
being tolerated only at doses of 5 mg/kg or less (Table 1).
Thigh model of infection. Mice were made profoundly neu-
tropenic during the course of the thigh model experiments by
administration of cyclophosphamide. Initial inocula were, per
thigh, 5.1 Ϯ 0.1 log10 CFU of S. aureus ATCC 25923. Control
and test compounds were administered i.v. 2 h following thigh
infection, and thigh bacterial counts were determined at reg-
ular intervals following treatment. The organisms expanded 3.1
Ϯ 0.3 log10 CFU/thigh after 10 h in untreated control mice.
The viable recovery of S. aureus from infected thighs following
a single injection of vancomycin, oxacillin, and 6752 is shown in
Fig. 2A. Single 16-mg/kg doses of 6752 and vancomycin pro-
duced a prolonged antibacterial effect against S. aureus, while
oxacillin at 16 mg/kg had a weak bactericidal effect which
lasted for 1 h posttreatment. Escalating doses of 6752 resulted
in dose-dependent killing of S. aureus, with a maximum effect
at single doses exceeding 8 mg/kg (Fig. 2B). In vivo PAEs also
increased with dose and were 3.75 h and Ͼ7 h for 6752 at 8
mg/kg and 16 mg/kg, respectively (see Materials and Methods
for the PAE calculation method used). The in vivo efficacy of
6752 against MRSA 33591 was very similar, with a maximum
bactericidal effect of 2.2 log10 CFU/thigh at 8 h following a
single dose of 8 mg/kg (data not shown).
The efficacy of 7251, 6756, 6853, 1316, and 1150 against
MSSA strain ATCC 25923 in the neutropenic-mouse thigh
model is shown in Table 2. The enantiomer of 6752, 7251
(Table 1), as well as 6756 and 6853, produced a bactericidal
effect similar to that of 6752 at a dose of 8 mg/kg. Overall, the
reduction in bacterial counts produced by the four compounds
was similar to that obtained with vancomycin at the same dose.
In contrast, 1316 and 1150 caused no significant reduction in
the thigh bacterial counts, despite significant potency in vitro.
The latter two compounds were also very poorly tolerated in
the mouse.
Peritonitis model of infection. The in vivo efficacy of 6752,
7251, 6756, 6853, and vancomycin in sepsis studies with MSSA
ATCC 13709 (Smith) is shown in Table 2. All four compounds
exhibited similar 50% protective doses. Following the 24-h
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7. experiment, the spleens and kidneys were recovered from sur-
vivors treated with 6752 (8 mg/kg at 0 h and 2 h following
intraperitoneal infection). Bacterial counts were obtained and
compared to those of untreated animals (Table 3). On average,
mice treated with 6752 showed decreases of 2.9 log10 CFU/
spleen and 3.6 log10 CFU/kidney pair, while vancomycin at the
same dose produced CFU decreases of 3.2 log10 and 3.9 log10
in the spleen and kidneys, respectively. The two peptides which
had no activity against MSSA in the neutropenic-mouse thigh
model, 1316 and 1150, were not tested in the peritonitis model
due to poor systemic tolerance.
In vivo pharmacokinetic studies. The serum concentrations
of 6752 following administration of single i.v. injections of 2, 4,
8, and 16 mg/kg to naive mice are shown in Fig. 3. The decline
in concentration was best described by a two-compartment
model. The values for the pharmacokinetic parameters are
shown in Table 4. In these preliminary experiments, the serum
concentrations showed dose proportionality for doses of up to
8 mg/kg, as indicated by the steady increase in the maximum
concentration of the drug in serum (Cmax) and the area under
the concentration-time curve (AUC). However, the increase in
Cmax and AUC was much less pronounced between 8 and 16
mg/kg. Accordingly, the elimination half-life and Vss remained
within a comparable range at 2, 4, and 8 mg/kg but significantly
increased at 16 mg/kg (Table 4). Figure 3 also shows a more
drastic decrease in the initial serum concentration of 6752 at 16
mg/kg than at lower doses.
Pharmacokinetic profiles were also established for three
other peptides, one showing in vivo efficacy in the thigh model
of infection, namely, 6853, and the two peptides with no ap-
parent efficacy in vivo, 1316 and 1150. Interestingly, the Cmax of
6853 was similar to that of 6752, while both 1316 and 1150
showed significantly lower peak concentrations and AUC val-
ues. Also, 1316 and 1150 displayed increased volumes of dis-
tribution, with 1150 having a 30-fold increased Vss value com-
pared to that of 6752 injected at the same dose. Thus, some
level of correlation appears to exist among Cmax, Vss, and
efficacy in the mouse for all four compounds examined. Spe-
cifically, a high Cmax may correlate with good efficacy in vivo
whereas large volumes of distribution may be indicators of a
lower Cmax and therefore decreased efficacy in infection mod-
els.
DISCUSSION
Until now, application of antimicrobial peptides to the clinic
has been hampered by various barriers, and most therapeutic
peptides are being developed for topical uses only (5, 8, 38),
with the exception of the anionic lipodepsipeptide daptomycin
(21). Daptomycin has been recently introduced into clinical
medicine for the treatment of complicated skin and skin struc-
ture infections caused by susceptible strains of several gram-
positive microorganisms. Though the molecules reported here
and daptomycin both have cell membrane-mediated activity,
there are several differences to be noted. Daptomycin is an
anionic molecule. It has been reported to have cell wall activity
and does not exhibit activity against stationary-phase cells (1).
Furthermore, daptomycin is not recognized to require self-
assembly for its activity. We have obtained the first conclusive
examples of a new class of cationic peptide antimicrobial
agents showing prolonged systemic antimicrobial activity in
infection models, where the molecules are administered i.v. to
treat an infection localized beyond the i.v. compartment.
Positive in vivo efficacy effects in the neutropenic-mouse
thigh model of infection show a more rapid and drastic initial
drop in the bacterial counts for 6752 than for vancomycin and
oxacillin (Fig. 2A), in agreement with the putative bactericidal
mechanism of action of the cyclic peptides. In contrast to the
nearly instantaneous membrane disruption by the cyclic pep-
FIG. 2. (A) Efficacies of vancomycin, oxacillin, and 6752 against S.
aureus ATCC 25923 in the neutropenic-mouse thigh model. Each
curve represents the bacterial titer observed in the thighs versus time.
Treated animals received a 16-mg/kg dose injected i.v. at 2 h postin-
fection. Symbols: Ⅺ, untreated control animals; Œ, 6752; }, oxacillin;
‚, vancomycin. The time above the MIC or the time during which the
serum concentration of each compound was above its corresponding
MIC is indicated in the upper left corner as follows: black rectangle,
oxacillin; hatched rectangle, vancomycin; gray rectangle, 6752.
(B) Dose-response study of 6752 against S. aureus ATCC 25923 in the
neutropenic-mouse thigh model. Each curve represents the bacterial
titer observed in the thighs versus time. Symbols: छ, untreated ani-
mals; Œ, 6752 at 2 mg/kg (i.v.); ‚, 6752 at 4 mg/kg (i.v.); ■, 6752 at 8
mg/kg (i.v.); ᮀ, 6752 at 16 mg/kg (i.v.).
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8. tides, both vancomycin and oxacillin exert their activity by
inhibiting enzymes involved in cell wall synthesis, and their
effect are therefore seen more slowly. In the absence of human
pharmacokinetic and efficacy data for the cyclic peptides, a
16-mg/kg single dose was chosen based on the tolerance data
for 6752 and on the clinical dose recommended for vancomycin
(1 g i.v. once daily for a 70-kg individual, which corresponds to
14.3 mg/kg). The same dose was used for 6752, vancomycin,
and oxacillin in order to compare the intensity and duration of
the bactericidal effect of each compound at similar initial se-
rum concentrations. At 16 mg/kg, 6752 clearly exhibits a more
prolonged antibacterial activity in the thigh model than oxacil-
lin but is similar to vancomycin. In our experiments, the cal-
culated PAEs were Ͼ6 h for vancomycin, in agreement with
values found in the literature (20), and Ͼ7 h for 6752. We
observed a minimal PAE for oxacillin, in contrast with what
has been reported with other, similar, -lactams using the same
model of infection, where the PAE could reach up to 4 h
against S. aureus strains. In the neutropenic-mouse thigh
model, 6752 also demonstrated efficacy against MRSA strain
33591 identical to the activity seen against MSSA strain ATCC
25923.
In vitro, these new peptide molecules displayed fast bacte-
ricidal activity in an enantiomer-independent manner, with
failure of S. aureus to easily develop spontaneous resistance
upon prolonged exposure to the peptides at sublethal concen-
trations. Taken together, our in vitro and in vivo results are
consistent with a previously proposed mechanism of action for
these cyclo-D,L-␣-peptides: insertion into the bacterial mem-
brane with a subsequent increase in membrane permeability,
potential collapse, and fast cell death (3, 6, 15). When tested
against MRSA either in vitro or in vivo, the cyclic peptides had
similar potencies against MSSA and MRSA, in agreement with
the putative mechanism of action of various classes of antimi-
crobial peptides thought to interact with negatively charged
components of the bacterial membrane as a whole, rather than
with individual enzymatic processes (23, 27). This complex set
of interactions between the amino acid side chains and com-
ponents of the bacterial membrane canopy could explain the
failure of S. aureus to easily develop spontaneous resistance
upon prolonged exposure to the peptides at sublethal concen-
trations. The proposed mechanism of action is not likely to be
influenced by individual target alterations, which would not
grossly alter the membrane structure to the extent required for
the appearance of resistant mutants. Furthermore, the unique
abiotic structure of the D,L-cyclic peptides may contribute to a
reduced risk of drug-resistant bacterial emergence. The puta-
tive mode of action is further supported by the MBCs and
MICs being quite similar.
The hypothesis that primary antimicrobial activity occurs at
the membrane level is strengthened by the observation that
enantiomeric cyclopeptides have the same activity in vitro and
show comparable behavior in more complex environments, as
is proven with the results in the murine model experiments
FIG. 3. Mean serum concentration-time profiles of 6752 following
i.v. injection of the compound at 2 mg/kg (‚), 4 mg/kg (Œ), 8 mg/kg
(}), and 16 mg/kg (ᮀ).
TABLE 2. In vivo efficacy of cyclic peptides in the thigh and peritonitis infection models
Compound
Efficacy in the thigh model (mean log CFU reduction Ϯ SD)a
PD50
c
(mg/kg)0.5 h 4 h 8 h
6752 1.1 Ϯ 0.24b
2.2 Ϯ 0.51b
2.4 Ϯ 0.37b
6.7
7251 1.2 Ϯ 0.31b
2.6 Ϯ 0.14b
2.8 Ϯ 0.28b
4.7
6756 1.0 Ϯ 0.32b
2.8 Ϯ 0.44b
3.2 Ϯ 0.25b
5.7
6853 1.2 Ϯ 0.10b
2.5 Ϯ 0.63b
3.0 Ϯ 0.77b
4.0
1316 0.0 Ϯ 0.26 0.0 Ϯ 0.09 0.1 Ϯ 0.05 NDd
1150 0.2 Ϯ 0.60 0.0 Ϯ 0.44 (ϩ) 0.2 Ϯ 0.45e
ND
Vancomycin 0.4 Ϯ 0.14b
1.9 Ϯ 0.31b
2.5 Ϯ 0.62b
1.3
a
All compounds were administered as a single-bolus i.v. injection of 8 mg/kg.
b
P Ͻ 0.001 to 0.01 compared to corresponding parameters for untreated controls (paired t test).
c
PD50, 50% protective dose.
d
ND, not determined.
e
(ϩ), increase in log CFU for treated animals compared to control animals.
TABLE 3. Bacterial counts in the spleens and kidneys of survivors
in the S. aureus peritonitis infection modela
Compound
Log10 CFU in:
Spleen Kidneys
None (untreated) 7.92 Ϯ 0.22 8.13 Ϯ 0.59
6752 5.02 Ϯ 0.24b
4.52 Ϯ 0.67b
Vancomycin 4.71 Ϯ 0.26b
4.07 Ϯ 0.65b
a
Untreated mice were infected with a sublethal dose of S. aureus 13709 of 3
ϫ 105
CFU/mouse.
b
P Ͻ 0.001 to 0.01 compared to corresponding parameters for untreated
controls (paired t test).
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9. with compounds 6752 and 7251. It is unlikely that the critical
mode of action involves interaction with a specific receptor
where changes in stereochemistry would be expected to have
drastic consequences with regard to potency and activity.
Other antimicrobial peptides thought to disrupt bacterial
membranes by a physical mechanism have been shown to be
active regardless of their enantiomeric form (2, 26, 35).
For the peptides described in Table 1, we observed a good
correlation between the toxicity of the cyclic peptides in vitro,
as indicated by the ED50s and HD50s, and their respective
tolerability in vivo. Among the six compounds examined in
detail and presented here, those with low ED50s and/or HD50s
showed very poor acute tolerability in vivo. We have observed
that prototypic cyclic peptides from this structural class, when
prone to amorphous aggregation as measured by in vitro meth-
ods, lead to a terminal blood pressure drop within seconds to
minutes when injected i.v. into instrument-monitored rats
(Neil Granger, Louisiana State University, personal communi-
cation). It must be noted, however, that a correlation between
in vitro cytotoxicity and tolerance in vivo was not always ob-
served for the other cyclic peptides tested. Overall, structure-
activity relationship studies are required to correlate specificity
(or lack thereof) with the primary sequence of the peptides in
order to increase the tolerability levels and thereby the thera-
peutic window of these cyclic-D,L-␣-peptides.
Analysis of our pharmacokinetic data and correlation with
pharmacodynamic properties of the cyclic peptides revealed
the following. Compounds with poor efficacy in vivo, namely,
1150 and 1316, had lower initial Cmaxs than those peptides
which showed good therapeutic activity in vivo. Also, both 1150
and 1316 exhibited a clear increase in their Vsss compared to
the peptides showing in vivo efficacy (Table 4). Interestingly,
those same compounds with high Vsss and lower Cmaxs were
significantly more hemolytic and cytotoxic in vitro and were
also very poorly tolerated systemically. We therefore hypoth-
esize that poor tolerability upon i.v. injection into the mouse
may be due to vascular leakage and partial blood vessel col-
lapse, with a subsequent severe drop in blood pressure and
diffusion of the peptides in deep tissue compartments. Such a
phenomenon could explain the drastic initial decrease in serum
concentrations, the resulting poor efficacy of 1150 and 1316 in
the various infection models, and the lack of correlation be-
tween in vitro potency and positive therapeutic activity for
these compounds. Also, our preliminary pharmacokinetic and
pharmacodynamic results suggest that Cmax could be the phar-
macokinetic index which best correlates with efficacy in vivo,
though further work needs to be done to verify this assump-
tion. Other components of the innate immune system, such as
neutrophil activation to produce superoxide, may also be in-
volved in the positive biological effect of these compounds
(26).
Preliminary studies showed poor bioavailability of these cy-
clic peptides following oral or subcutaneous administration.
Intravenous administration was required for therapeutic effi-
cacy. This is most likely due to the size of these compounds,
with a molecular mass of around 1,000 Da for octamers, and to
their polycationic nature. On the other hand, these alternating
D,L-cyclopeptides are highly resistant to proteolysis and stable
in serum and other biological fluids. This constitutes an im-
portant advantage which distinguishes them from other pep-
tides with therapeutic potential (38). Finally, pharmacokinetic
and pharmacodynamic studies performed with 6752 and 6756
in renally impaired mice indicated that a significant portion of
the injected compounds is cleared renally based on the dra-
matic decrease in the clearance rate and more prolonged in
vivo efficacy seen in the presence of uranyl nitrate (data not
shown).
In this paper, we detail the first conclusive examples of a new
class of cationic peptide antimicrobial agents showing pro-
longed systemic antimicrobial activity in infection models,
where the molecules are administered i.v. to treat an infection
localized beyond the blood compartment. Although additional
structure-activity relationship studies are required to improve
the therapeutic window of this class of antimicrobial peptides,
our data suggest that the amphipathic cyclic D,L-peptides, with
their self-assembling and specific membrane disruption mech-
anism of action, may have potential for systemic administration
and treatment of otherwise antibiotic-resistant infections.
ACKNOWLEDGMENTS
The assistance of Emmett Bond, David Griffith, Shirley Carigo-
Cairel, Dana Johnson, Morag Day, Christian Solem, and Tim Hoffman
is gratefully acknowledged.
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