Scope and limitations of the designer proline-richantibacterial peptide dimer, A3-APO, alone or insynergy with conventional antibiotics

Marco Cassone a, Paraskevi Vogiatzi a, Raffaele La Montagna a, Vanessa De Olivier Inacio b,Predrag Cudic b, John D. Wade c, Laszlo Otvos Jr.a,*aSbarro Institute for Cancer Research and Molecular Medicine, Temple University, Philadelphia, PA 19122, United StatesbDepartment of Chemistry and Biochemistry, Florida Atlantic University, Boca Raton, FL 33431, United StatescHoward Florey Institute and Department of Chemistry, University of Melbourne, Victoria 3010, Australia

p e p t i d e s 2 9 ( 2 0 0 8 ) 1 8 7 8 – 1 8 8 6

a r t i c l e i n f o

Article history:

Received 30 June 2008

Received in revised form

22 July 2008

Accepted 22 July 2008

Published on line 5 August 2008

Keywords:

A3-APO

Synergy

Antimicrobial peptide

DnaK

a b s t r a c t

The proline-rich antimicrobial peptide dimer, A3-APO, was designed based on a statistical

analysis of native antibacterial peptide and protein sequences. Analysis of a series of

structural analogs failed to identify any single or multiple amino acid modification or

architectural changes that would significantly improve its potential as a clinical therapeutic.

However, a single chain Chex1-Arg20 version, a natural in vivo metabolite, showed a 2 to 8-

fold increase in activity against test Enterobacteriaceae strains. In addition to bacterial species

close to Escherichia coli in phylogeny, A3-APO analogs were able to effectively kill Pseudomonas

aeruginosa and Staphylococcus saprophyticus. Antibacterial efficacy analysis together with

biochemical experiments provided further evidence for a multiple mode of action of A3-

APO that includes binding and inhibition of the bacterial heat shock protein DnaK. Through

inactivating of resistance enzymes, A3-APO was able to recover the lost activity of con-

ventional antibiotics including chloramphenicol, b-lactams, sulfonamides or trimethoprim

against multidrug resistant strains with partial or full synergy. However, the synergy

appeared to be individual strain and small molecule drug combination-dependent.

# 2008 Elsevier Inc. All rights reserved.

avai lable at www.sc iencedi rec t .com

journal homepage: www.e lsev ier .com/ locate /pept ides

1. Introduction

The antimicrobial drug industry is not keeping in pace with

the overwhelming appearance and circulation of pathogens

causing known or novel infectious syndromes. Perhaps the

most worrisome event is the worldwide spread of antibiotic

resistance in hospitals and the community alike [17,28].

Without discounting the efficacy of preventive measures,

there is an impelling need to develop new antimicrobial

molecules, and to use antimicrobial combinations capable of

exerting synergistic activities. Antimicrobial peptides, which

* Corresponding author at: BioLife Sciences Building, 1900 North 12th Sfax: +1 215 204 4021.

E-mail address: otvos@temple.edu (L. Otvos Jr.).

0196-9781/$ – see front matter # 2008 Elsevier Inc. All rights reserveddoi:10.1016/j.peptides.2008.07.016

are largely diffused in nature, are a promising emerging class

of anti-infective drugs. Among those, proline-rich peptides are

unique since they have a very specific mechanism of action,

allowing structure–activity relationships (SAR) studies [21]. In

general, antimicrobial peptides carry little potential to induce

resistance [11], however their systemic use is frequently

hampered by unacceptable pharmacokinetic parameters and

low safety margins [5].

Peptide A3-APO is a flagship representative of a new class of

synthetic peptide antimicrobials derived from natural insect

products [22]. A3-APO selectively binds to the multihelical lid

treet, Philadelphia, PA 19122, United States. Tel.: +1 215 204 4020;

.

p e p t i d e s 2 9 ( 2 0 0 8 ) 1 8 7 8 – 1 8 8 6 1879

region of the 70-kDa bacterial heat shock protein DnaK [21] and

inhibits DnaK functions including protein refolding [15]. A

combination of DnaK binding and bacterial membrane disin-

tegration leads to effective killing of genetically related

Enterobacteriaceae such as Escherichia coli, Klebsiella pneumoniae,

Salmonella typhimurium and Haemophilus influenzae [14]. The

membrane-onlyactivitiesof theproline-rich dimersare enough

to combat Pseudomonas aeruginosa strains, albeit at higher

concentrations than needed to kill bacteria with complemen-

tary DnaK sequences [4]. The specific role of several specific

residues as well as some structural aspects of this peptide

warrant further investigation, and this was the first aim of the

current study.

The Asp2-Lys3 dipeptide and Tyr6-Leu-Pro-Arg-Pro10

pentapeptide fragments are crucial for antibacterial activity

[17]. A combination of protein folding and membrane stability

assays indicate that while the role of the positively charged

Lys3 is likely to initiate attachment to the negatively charged

bacterial membrane surface, the prolines serve as structural

constrains, and Asp2, Tyr6, Leu7 and Arg9 are involved in

binding to the DnaK protein of E. coli and related bacteria [4].

These seven residues are conserved in the designer dimer A3-

APO and are positioned as Asp4-Lys5 and Tyr9-Leu-Pro-Arg-

Pro13. The two extra N-terminal residues and the C-terminal

half of the peptide are thought to partially destroy bacterial

membranes and drive the DnaK-inactivating pharmacophore

into cells [22]. SAR of the designer dimer family indicate that in

two of three inactive analogs, the Tyr-Leu-Pro-Arg-Pro

pentapeptide is altered; in one Ile is substituted for Leu, and

in another Lys is substituted for Arg [23]. This suggests that

killing of susceptible strains requires the intact DnaK-binding

domain. However, one of the inactive analogs featuring an

Arg! Lys change and additional modifications at the carboxy-

terminus, does bind a fluorescein-labeled E. coliDnaK D-E helix

preparation suggesting that perhaps alterations in the C-

terminal delivery unit (compared to A3-APO) are responsible

for the lack of antibacterial activity. We wanted to test this

hypothesis experimentally. Examination of the C-terminal

delivery halves indicate that the active peptides are compact

and lack residues that do not promote entry into cells.

Positively charged residues, especially Arg [25] together with

Pro are known to facilitate peptide entry into cells [9]. Yet, the

frequent appearance of His (a residue both positively charged

and hydrophobic) in native proline-rich antibacterial

sequences such as apidaecins, warrant the synthesis and

testing of a few His-analogs in the place of Arg. While the

function of residues N-terminal to the YLPRP active site other

than the Asp-Lys dipeptide fragment is unclear [4], it is

tempting to speculate that the improved antibacterial activity

of A3-APO compared to some analogs is due to its cell-entry

focused N-terminal heptapeptide fragment. In support,

another pyrrhocoricin-apidaecin construct, based on apidae-

cin 1a without the N-terminal Lys-Val dipeptide fragment fails

to kill the test bacterial strains. The argument presented above

questions the need for the near-C-terminal Val, the only

residue remaining in A3-APO without DnaK-binding or cell-

penetrating functions. Likewise, we are not fully convinced

that the N-terminal Chex residue or the C-terminal Dab are the

best amino acid mimics, although they show favorable

efficacy and toxicity properties. Additionally, we not pre-

viously considered reversing the N-terminal DnaK-binding—

C-terminal delivery architecture or using single chain (non-

dimeric) peptides but with the sequence optimizations

included.

The second aim of this study was to test the potential of A3-

APO as a synergic agent in combination with antimicrobials

currently on the market and to which resistance is increas-

ingly observed. In a preliminary report we documented how

preincubation of b-lactamase-expressing E. coli strains with

the designer antimicrobial peptide dimer, A3-APO, is able to

reinstall the lost activity of amoxicillin against these b-lactam

resistant bacteria [20]. The inhibition of protein refolding

mediated by A3-APO through DnaK binding is the basis for a

possible restoration of the efficacy of antibiotics that are

inactivated by specific enzymes. Since inhibition of DnaK

impairs the function of many enzymes, A3-APO could be the

first molecule able to be synergic in a broad range of

combinations, and also to be effective just when it is most

needed and other combinations fail, i.e. in the presence of

highly active, antibiotic-cleaving or diverting enzymes

responsible for high-level resistance. Encouraged by the

success of our preliminary results with E. coli and b-lactam

antibiotics, in the current study we expanded the investiga-

tions to other Enterobacteriaceae and different small molecule

antibiotics that are impaired by increasingly diffuse resis-

tance-causing enzymes.

2. Materials and methods

2.1. Peptide synthesis

The peptides were synthesized by solid-phase methods.

Peptide chain assembly was carried out on a CEM Liberty

microwave-assisted automated synthesizer using TentaGel S-

Ram-Fmoc resin with an initial load of 0.3 mmol/g (Advanced

ChemTech). Standard Fmoc-chemistry was used throughout

[10] with a 4-fold molar excess of the acylating amino acids.

Non-natural amino acids were coupled manually to ensure

completion. The peptides were cleaved from the solid support

with trifluoroacetic acid (TFA) in the presence of thioanisole

(5%), and water (5%) as scavengers. After cleavage, the

peptides were purified by reversed-phase high performance

liquid chromatography (RP-HPLC). The final products were

characterized by RP-HPLC and matrix-assisted laser deso-

rption/ionization mass spectroscopy (MALDI-MS). Mass spec-

tra (PerSeptive Biosystems, Voyager DE instrument) identified

correct and highly purified samples.

2.2. Bacterial strains

For the study we used bacteria belonging to the species E. coli

(HK101, HK179, HK131, BF1023, SOTE40, 5770, S5081, S4362,

SEQ102, and 045-849), K. pneumoniae (HK123, HK186, HK127,

012-3132, MH4, RP1 and K6), S. typhimurium (ATCC 14028, S5

and G10215), P. aeruginosa (ATCC 39329 and 10), H. influenzae

(ATCC 49247 and R387), Haemophilus ducreyi 51620, Bulkholderia

cepacia 8Q, Proteus vulgaris ATCC 6896, Proteus mirabilis ATCC

7002, Staphylococcus aureus 27660, Staphylococcus saprophyticus

ATCC 15305, and Enterococcus faecalis JWL. Each strain was

p e p t i d e s 2 9 ( 2 0 0 8 ) 1 8 7 8 – 1 8 8 61880

grown to mid-exponential phase in an incubator at 37 8C and

5% CO2, and in the case of E. coli, the flasks were agitated at

200 rpm. For Haemophilus strains, hemin and nicotinamide

adenine dinucleotide (NAD) were added at the concentration

of 15 mg/L each. All assays involving live bacteria were

performed in a BSL2 environment.

2.3. Minimal inhibitory concentration (MIC)determination assays

MICs were determined in duplicate by a liquid growth

inhibition microdilution assay in sterile polypropylene 96-

well plates (Nunc F96 microtiter plates), with a final volume

of 100 mL [1]. The cell concentrations were estimated by

measuring the ultraviolet absorbance at 595 nm and apply-

ing the formula CFU/mL = A595 (3.8 � 108), where CFU is the

number of colony-forming units. Briefly, 50 mL of a suspen-

sion of midlogarithmic phase bacterial cultures diluted to

5 � 105 cfu/mL in 1/4 strength Mueller–Hinton broth was

added to 50 mL of 2-fold serially diluted peptides, dissolved in

the same medium. For S. saprophyticus, additional MICs were

acquired in full strength broth. The final peptide concentra-

tions ranged between 0.12 and 128 mg/mL, (approximately

0.03–32 mM for the full-length peptides and 0.06–64 mM for

the two single chain peptides (A3 single chain and A3 Inverse

single chain; Table 1). For each strain a well with no peptide

was included as growth control, and for each test a row of

medium-only wells was included as a sterility control. The

plates were then incubated at 37 8C, 5% CO2 for 16–20 h

without shaking, and growth inhibition was measured by

recording the absorbance at 595 nm using a microplate

reader. MICs were identified as the lowest antimicrobial

concentrations where the 595 nm absorbance value did not

exceed that of the medium only.

2.4. Synergism assays

Two different assays were employed to study the presence of

synergism between A3-APO and conventional antibiotics in

Table 1 – Sequences of synthetic peptides used in this study

A3-APO (H-Chex-Arg-Pro-Asp-Lys-Pro-Arg

Glu4A3 (H-Chex-Arg-Pro-Glu-Lys-Pro-Arg-

Arg5A3 (H-Chex-Arg-Pro-Asp-Arg-Pro-Arg

Gly11A3 (H-Chex-Arg-Pro-Asp-Lys-Pro-Arg

Lys12A3 (H-Chex-Arg-Pro-Asp-Lys-Pro-Arg

A3-desVal (H-Chex-Arg-Pro-Asp-Lys-Pro-Arg

Arg2A4 (H-Pip-Arg-Pro-Glu-Arg-Pro-Arg-Pr

His2A4 (H-Pip-His-Pro-Glu-Arg-Pro-Arg-Pro

A3 Inverse (H-Chex-Arg-Pro-Arg-Pro-Pro-Arg-

A3 Inverse single chain H-Chex-Arg-Pro-Arg-Pro-Pro-Arg-P

A3 Single chain H-Chex-Arg-Pro-Asp-Lys-Pro-Arg-

Chex-pyrr-Dap dimer (H-Chex-Asp-Lys-Gly-Ser-Tyr-Leu

Residues in italics indicate the amino acid changes compared to the seq

bacteria harboring antibiotic-disrupting or antibiotic-insensi-

tive enzymes. With the first method, bacteria were preincu-

bated with A3-APO in full-strength Mueller–Hinton broth at

concentrations of 1/2 or 1/4 of its MIC and the conventional

antibiotics were serially diluted in the entire concentration

range [11]. Preincubated bacteria where then added and the

OD at 600 nm was measured after 16–20 h. The second method

we used was the conventional checkerboard assay [8]. Bacteria

grown to mid-logarithmic phase in 1/4 strength Mueller–

Hinton broth were preincubated with serially diluted con-

centrations of A3-APO and the conventional antibiotics were

added in a similar 2-fold dilution pattern. The occurrence and

extent of synergy were calculated based on the sum of the MIC

ratios for each compound alone and in the presence of the

other at half MIC (FIC score). Synergy was defined by a FIC

score below 0.5. The small molecule antibiotics included

amoxicillin, chloramphenicol, sulfamethoxazole or the

trimethoprim/sulfamethoxazole (TMP/SXT) combination as

resistance to all these is manifested through the action of

specific enzymes. Those E. coli, S. typhimurium and K.

pneumoniae strains were selected for which the involvement

of enzymatic activity was known or suggested by the

resistance profile. A few tests were also undertaken on

bacteria lacking specific antibiotic-resistance enzymes, in

which synergy was not expected.

2.5. b-galactosidase assay

The inhibition of the enzyme-refolding functions of DnaK by

peptide A3-APO was confirmed by measuring the b-

galactosidase activity of live cells. To this end, a modified

ortho-nitrophenol (ONP)-substrate method was employed

according to manufacturer’s instructions (b-galactosidase

assay, Stratagene, Cedar Creek, TX). The test strain here

was K. pneumoniae K6 harvested after 1 h of exposure to

A3-APO alone, chloramphenicol or TMP/SXT alone or the

small molecule antibiotics together with peptide A3-APO.

All the antibiotics were used at a concentration 1/4 of

their MIC.

-Pro-Tyr-Leu-Pro-Arg-Pro-Arg-Pro-Pro-Arg-Pro-Val-Arg)2-Dab

Pro-Tyr-Leu-Pro-Arg-Pro-Arg-Pro-Pro-Arg-Pro-Val-Arg)2-Dab-NH2

-Pro-Tyr-Leu-Pro-Arg-Pro-Arg-Pro-Pro-Arg-Pro-Val-Arg)2-Dab-NH2

-Pro-Tyr-Leu-Gly-Arg-Pro-Arg-Pro-Pro-Arg-Pro-Val-Arg)2-Dab-NH2

-Pro-Tyr-Leu-Pro-Lys-Pro-Arg-Pro-Pro-Arg-Pro-Val-Arg)2-Dab-NH2

-Pro-Tyr-Leu-Pro-Arg-Pro-Arg-Pro-Pro-Arg-Pro-Arg)2-Dab

o-Tyr-Leu-Pro-Lys-Pro-Arg-Pro-Pro-Arg-Pro-Arg)2-Dab-NH2

-Tyr-Leu-Pro-Lys-Pro-Arg-Pro-Pro-Arg-Pro-Arg)2-Dab-NH2

Pro-Val-Arg)2-Dab-Asp-Lys-Pro-Arg-Pro-Tyr-Leu-Pro-Arg-Pro-NH2

ro-Val-Arg-Dab-Asp-Lys-Pro-Arg-Pro-Tyr-Leu-Pro-Arg-Pro-NH2

Pro-Tyr-Leu-Pro-Arg-Pro-Arg-Pro-Pro-Arg-Pro-Val-Arg-NH2

-Pro-Arg-Pro-Thr-Pro-Pro-Arg-Pro-Ile-Tyr-Asn-Arg)2-Dap(DapAc)-NH2

uence of A3-APO in the non-architecture-modified analogs.

p e p t i d e s 2 9 ( 2 0 0 8 ) 1 8 7 8 – 1 8 8 6 1881

2.6. DnaK binding assay

To identify which A3-APO analogs bind DnaK, a dot-blot assay

was used. Ten micrograms of each peptide was immobilized

on a nitrocellulose membrane, which was blocked by incuba-

tion in phosphate-buffered saline (PBS) - 0.1% Tween20

mixture with 5% bovine serum albumin (BSA) for 2 h at room

temperature. Fifty micrograms of DnaK (Stressgen, Victoria,

British Columbia) were added in 5 mL PBS - 0.1% Tween20 for

1 h. Bound DnaK protein was detected after overnight

incubation with a bacterial DnaK-specific mouse monoclonal

antibody diluted 1/10,000 (clone 8E2/2, Stressgen) at 4 8C

overnight, followed by development of the membrane with

horseradish peroxidase (HRP)-conjugated anti-mouse IgG

antibody (dilution 1:20,000, Pierce, Rockford, Illinois).

3. Results

3.1. Analog and assay design

The new analogs (Table 1) include single residue conservative

changes in the active sites (Glu4A3, Arg5A3, Gly11A3,

Lys12A3), elimination of the valine (A3-desVal), multiple

amino acid substitutions including pipecolic acid at the amino

terminus (Arg2A4, His2A4) and a single chain analog (A3 Single

chain). In the last set of new derivatives we included a reverse

design with a single or dual N-terminal membrane penetrating

and a single C-terminal pharmacophore orientation (A3

Inverse single chain, A3 Inverse). This design places Pro to

the C-terminus. The antimicrobial efficacies of these analogs

were compared with the parent peptide A3-APO and an earlier

dimer that shows less attractive antibacterial properties [6].

A3-APO kills bacteria in full-strength Mueller–Hinton broth,

Table 2 – Minimal inhibitory concentrations of peptide A3-APOthe structural analogues A3 single chain and A3-desVal againinhibition assay in quarter strength Mueller–Hinton broth at 3

Bacterial strain

A3-APO Arg5A3 Glu4A3

E. coli HK101 2 2 2

E. coli HK179 2 2 2

E. coli SOTE40 8 8 8

E. coli 5770 2 2 2

E. coli HK131 4 4 8

E. coli SEQ102 2 2 2

E. coli 045-849 4 2 4

K. pneumoniae HK 123 4 4 8

K. pneumoniae HK 186 8 4 8

K. pneumoniae HK 127 8 8 8

K. pneumoniae 012-3132 2 2 2

K. pneumoniae K6 4 4 8

S. typhimurium ATCC 14028 8 4 8

S. typhimurium S5 8 8 8

S. typhimurium G10215 4 4 4

P. aeruginosa ATCC 39329 8 4 8

P. aeruginosa 10 8 8 8

S. saprophyticus ATCC 15305 2 2 2

but earlier analogs do not [22]. To be able to discriminate

between the efficacies of the new peptides and draw

conclusions on the relative efficacy of analogs, in this report

all antimicrobial assays were conducted in 1/4 strength

Mueller–Hinton broth.

3.2. Antibacterial activity and DnaK binding of peptidescontaining single residue conservative changes

Peptide Arg5A3 has a conservative mutation at the hypothe-

tical membrane-active residue Lys, Gly11A3 features a non-

charged substitution at the DnaK-binding domain and Glu4A3

as well as Lys12A3 involve conservative and charged sub-

stitutions in the fragments interacting with DnaK (Table 1). As

Table 2 shows, A3-APO is indeed a close-to-optimal dimer in

killing Enterobacteriaceae. All but one (Gly11A3) of the new

derivatives with charged substitutions were about equally

potent as the parent designer peptide. Glu4A3 and Lys12A3

showed a slightly decreased activity in three strains each, but

still within the expected experimental variability range.

Arg5A3 exhibited marginally improved activity against 3 of

the 15 E. coli, K. pneumoniae, and S. typhimurium strains studied.

Although this minor improvement can be expected due to the

stronger positive charge of Arg compared to Lys and the

generally observable superiority of Arg in cell and nuclear

penetration-enhancement [2], the MIC differences were in the

experimental error range, so no clear-cut conclusion can be

drawn. Since the Glu4A3 and Lys12A3 analogs showed no

major loss in the antibacterial activity, it is likely that Asp4 and

Arg12 in A3-APO interacts with DnaK through ionic forces. In

contrast, a major drop in the antibacterial activity was

observed for Gly11A3 that features a Pro! Gly substitution,

in spite of both residues being reverse-turn formers [13]. This

derivative reproducibly killed all 15 E. coli, K. pneumoniae, and S.

, of its conservative amino acid replacement analogues andst selected strains measured in an overnight liquid growth7 8C

MIC (mg/mL)

Lys12A3 Gly11A3 A3 single chain A3-desVal

2 4 0.5 2

2 4 1 2

8 32 2 2

2 4 0.5 1

8 32 1 4

2 4 1 2

4 8 2 2

8 32 2 16

8 32 0.25 4

8 32 0.25 2

2 8 0.25 2

4 16 2 4

8 16 1 4

8 16 0.5 4

4 8 0.25 2

4 8 32 8

4 8 32 4

2 2 4 2

Fig. 1 – Binding of A3-APO, conservative change analogs

and control peptides to recombinant Escherichia coli DnaK.

The peptides were dried on a nitrocellulose sheet and

bound DnaK was developed with an N-terminal specific

antibody. The peptides used are from top to bottom—left

column: G11A3, K12A3, R5A3, E4A3; right column: A3-

APO, magainin II, scrambled Rb2 fragment, blank.

p e p t i d e s 2 9 ( 2 0 0 8 ) 1 8 7 8 – 1 8 8 61882

typhimurium strains 2 to 8-fold less efficiently than the other 4

analogs.

To confirm the specificity of the above mentioned

substitutions to DnaK interaction, we investigated whether

the conservative changes affect efficacy against bacteria

whose DnaK is not a known target of native pyrrhocoricin

or other proline-rich peptides. Here the test strains included P.

aeruginosa, B. cepacia, Proteus vulgaris, P. mirabilis, H. influenzae

and H. ducreyi as Gram-negatives as well as S. aureus, S.

saprophyticus and E. faecalis as Gram-positives. Since in these

experiments DnaK-binding did not play a role, all five

peptides, including the Enterobacteriaceae less active Gly11A3,

showed almost identical antibacterial efficacies. Among the

named species, P. aeruginosa and S. saprophiticus were

susceptible to the peptides (Table 2), while the others were

not (MIC > 32 mg/mL) (data not shown). We documented

earlier that modified proline-rich peptides kill P. aeruginosa

through membrane disintegration [4], while a study is

undergoing to characterize the mechanism of action towards

S. saprophyticus.

To correlate the retention or loss of antibacterial activity

with DnaK interactions, we studied the binding of A3-APO and

the single residue conservative change derivatives to DnaK

protein on dot-blot. The negative control peptides were the

only-membrane active antibacterial peptide magainin II and a

scrambled Rb2 protein fragment that similarly to the

antibacterial peptides of this study is positively charged and

of the same overall length [2]. The results fully supported our

hypothesis: peptides K12A3, R5A3, G11A3 and E4A3 bound to

DnaK just like A3-APO did. Magainin II and the scrambled Rb2

fragment did not bind at all (Fig. 1). Although dot-blot is a

rather qualitative assay, the less active analog G11A3

exhibited significantly weaker DnaK binding. These assays

provided further proof for the notion that DnaK inhibition is

one of the mechanisms by which the proline-rich native

peptides and designer analogs kill bacteria [22].

3.3. Antimicrobial activity of A3-APO derivativesfeaturing structural modifications

This group of analogs included the valine-eliminated deriva-

tive, the single-chain Chex1-Arg20 analog, the reversed DnaK-

binding—delivery unit architecture in single chain and

dimeric version and two multiply modified A3-APO analogs.

These latter contained the Glu4, Arg5, Lys12 alterations that

were previously shown not to negatively influence the

antibacterial efficacy, and pipecolic acid instead of 1-amino-

cyclohexane carboxylic acid at the N-terminus. One of them

also contained a histidine for an N-terminal arginine. Chex is a

cyclic valine mimic and was introduced into the early proline-

rich dimers to replace the native Val and stabilize the peptides

from premature aminopeptidase cleavage in body fluids [6]. In

pipecolic acid the free amino group required for maximum

activity [14] is placed inside the hydrocarbon ring, endowing

the secondary amine with a stronger positive charge than the

primary amine in Chex. Clearly, the lack of a primary amine

deteriorated the antimicrobial activity. The Pip-containing

peptides were also inactive (MIC > 32 mg/mL) against both E.

coli SEQ102 and S. typhimurium S5, strains that are efficiently

killed by A3-APO and the other primary amine-containing

analogs (Table 2). Likewise, the N-terminal DnaK-binding and

C-terminal membrane penetrating-unit architecture cannot

be changed. The inverse derivatives A3 inverse and A3 inverse

single chain remained completely inactive (MIC > 32) against

both E. coli and K. pneumoniae, the two most sensitive bacterial

species to native proline-rich peptides and their designer

analogs. Arg2A4 and His2A4 exhibited MIC values of 32 and

>64 against E. coli SEQ102, and >64 for K. pneumoniae K6 and S.

typhimurium S5.

Against expectations, the elimination of the C-terminal

valine did not influence the antimicrobial efficacy in most of

the cases (Table 2). A3-desVal showed a comparable, and in

several cases slightly better activity in all but one the tested

strains. This observation confirms that Arg and Pro are indeed

the responsible residues for cellular penetration. A3-desVal

and A3-APO were 2–4 times more active than the first

generation Chex-pyrr-Dap dimer against the strains sensitive

to this control peptide; the Chex-pyrr-Dap dimer control was

inactive against representative E. coli, K. pneumoniae and S.

typhimurium strains that are killed by A3-APO by a median MIC

of 4 mg/mL.

Even more surprisingly, the most active peptide of this

study was the Chex1-Arg20 A3 single chain analog. This

peptide consistently exhibited lower MIC values compared to

A3-APO against the test phylogenetically related E. coli, K.

pneumoniae and S. typhimurium strains. The A3 Single chain

peptide is the most active antimicrobial peptide against these

Enterobacteriaceae we developed during our antibacterial

peptide drug development program. Interestingly, the oppo-

site was true for the Pseudomonas and S. saprophyticus strains,

which are killed by A3-APO but display a DnaK sequence that

is significantly different from that of E. coli, Klebsiella and

Salmonella. Since staphylococci are halophiles, for S. saprophy-

ticus we also run the MICs of A3-APO and the A3 Single chain

peptide in full-strength broth, to have a comparison under

p e p t i d e s 2 9 ( 2 0 0 8 ) 1 8 7 8 – 1 8 8 6 1883

conditions allowing the fastest possible growth of the bacteria.

In this case, the difference between the MIC for A3-APO (8 mg/

mL) and the A3 Single chain (>64 mg/mL) was even more

pronounced. The data obtained for Pseudomonas and S.

saprophyticus show that while the dimeric and presumably

increasingly membrane-penetrating A3-APO retains activity

on some bacteria independently of DnaK specificity, this is not

the case for the otherwise more active, but less membrane

destroying A3 Single chain derivative.

3.4. Synergy with small molecule antibiotics

We tested the synergistic activity of A3-APO in combination

with amoxicillin, chloramphenicol, sulfamethoxazole or TMP/

SXT on various E. coli, S. typhimurium, K. pneumoniae and H.

influenzae strains. The culprit resistance-carrying enzymes for

these bacteria are b-lactamase (penicillins and cephalospor-

ins), chloramphenicol acetyltransferase, dihydrofolate reduc-

tase (TMP), and tetrahydropteroic synthetase (sulfonamides).

We performed two sets of assay involving the same strains

and antibiotics but using two different techniques.

In the first set of assays, we expanded the same assay design

as previously described [20] that is we used A3-APO in fixed

concentration at 1/2 or 1/4 of its MIC, and the small molecule

antibiotics in the full concentration range from 0.5 to 64 mg/mL.

During these experiments, preincubation of bacteria with A3-

APO lowered the MIC of amoxicillin against H. influenzae R387,

but did not help the b-lactam againstK. pneumoniaeK6 or RP1, or

S. typhimurium G10215. Likewise, preincubation with A3-APO

could not restore any efficacy of sulfonamide against E. coli

S5081 or TMP against E. coli S4362. Partial synergy was observed

for chloramphenicolagainstH. influenzaeR387. In this series, the

best synergism was observed with chloramphenicol and K.

pneumoniaeK6 (Fig. 2). The single A3-APO concentration synergy

assays were run in full-strength Mueller–Hinton broth. During

these conditions, the MIC of A3-APO against K. pneumoniae

without any other antibiotic present was 16 mg/mL. Addition of

Fig. 2 – Killing of Klebsiella pneumoniae K6 by antibiotics as repr

assay. Peptide A3-APO or chloramphenicol were added to bacter

64 mg/mL concentration range. Synergy assays were run on the

concentration (1/4 of its MIC) 1 h prior to small antibiotic additi

chloramphenicol varied between 0.5 and 64 mg/mL. The horizon

4 mg/mL A3-APO only. Synergy can be detected above 16 mg/mL

4 mg/mL A3-APO to the serially diluted chloramphenicol

preparation significantly inhibited the bacterial growth com-

pared to either chloramphenicol or 4 mg/mL A3-APO alone,

allowing a significant reduction of the bacterial load already at

8 mg/mL chloramphenicol concentration, and totally inhibiting

growth at 32 mg/mL (Fig. 2).

The second set of assays was performed using the checker-

board method [8], which has a better sensitivity to determine

the presence of full synergism, and can be performed using a

broader range of antibiotic and peptide concentrations. Byusing

this assay, we could confirm the full synergy, presented in the

previous publication [20], between peptide A3-APO and amox-

icillin for E. coli SEQ102 and BF1023. We also confirmed the

synergy with chloramphenicol against K. pneumoniae K6 (Fig. 3,

top left panel) as presented in the paragraph above. Additional

complete synergistic effects of the peptide were observed with

chloramphenicol againstE. coliS5081 (Fig. 3, bottom right panel),

with sulfamethoxazole against S. typhimurium G10215 (Fig. 3,

bottom left panel) and with TMP/SMX againstE. coliS5081 (Fig. 3,

top right panel), aswell aswith SXT againstK.pneumoniaeK6and

with chloramphenicol against E. coli SEQ102. An additive effect

(limited cooperation not implying synergy) was detected

against K. pneumoniae RP1 and S. typhimurium G10215, both with

chloramphenicol. However A3-APO produced antagonism with

amoxicillin against K. pneumoniae RP1 and S. typhimurium

G10215, in full support of the assays using fixed A3-APO

concentrations. In further support of the single concentration

results, the checkerboard assay was unable to detect A3-APO

synergy with TMP against E. coli S4362. Additional antagonism

was observed with amoxicillin against K. pneumoniae MH4 and

addition with chloramphenicol against this strain.

3.5. Correlation of synergy with inhibition of DnaKfunctions

As shown above, A3-APO could successfully attack more than

one resistance mechanism of the multidrug-resistant strain K.

esented by a broth microdilution liquid growth inhibition

ial cultures grown in Mueller–Hinton broth alone in the 0.5–

same plates, where peptide A3-APO was added at 4 mg/mL

on. In the synergy lanes the concentration of

tal line indicates the bacterial growth in the presence of

chloramphenicol concentration.

Fig. 3 – Checkerboard synergy assay between peptide A3-APO and small molecule antibiotics. The concentration of both A3-

APO and chloramphenicol (top left and bottom right), sulfamethoxazole (bottom left) or the trimethoprim/sulfamethoxazole

combination (top right) was varied in both directions. The assays were run in 1/4 strength Mueller–Hinton broth. The letter

G in the boxes indicates of bacterial growth. Empty boxes indicate complete lack of bacterial growth. The FICI indices are

calculated below each panel. In all four cases full synergy was observed.

p e p t i d e s 2 9 ( 2 0 0 8 ) 1 8 7 8 – 1 8 8 61884

pneumoniae K6. We wondered whether A3-APO maintained its

DnaK inhibitory properties in the presence of small molecule

antibiotics with which it was found to be synergistic. To

answer this question, we used the b-galactosidase assay. b-

galactosidase is a substrate of DnaK in bacteria, where DnaK

is needed for the a-complementation of b-galactosidase,

processes required for cell growth [18]. While the native

antibacterial peptide pyrrhocoricin largely eliminates the b-

galactosidase activity of E. coli, the inactive D-analog has no

such an effect [14]. Thus the b-galactosidase activity of live

bacterial cells can be construed as a direct measure of

functional intracellular DnaK. During the course of our

current studies, we measured the b-galactosidase activity

of K. pneumoniae K6 without any drug added, with A3-APO,

chloramphenicol or TMP/SXT added alone, as well as upon

incubation of mixtures of A3-APO and chloramphenicol or

TMP/SXT. The antibiotics were added at 1/4 concentrations of

their MIC to allow the cells to grow to some degree and

produce the enzyme. As expected, treatment with A3-APO

drastically reduced the b-galactosidase activity of K. pneumo-

niae K6 (Fig. 4). Such reduction could not be observed upon

incubation of the cells with either of the small molecule

antibiotics alone, indicating that these antimicrobials did not

influence protein folding. However, the b-galactosidase

activity was returned to minimal when the A3-APO/small

molecule antibiotic mixtures were applied (Fig. 4).

4. Discussion

The A3-APO peptide dimer is the result of a statistical/

combinatorial design process that took into consideration all

proline-rich native antimicrobial peptide sequences with

additional input from SAR studies from earlier drug develop-

ment studies [22]. Peptide A3-APO and its major in vivo

metabolite kill fluoroquinolone, b-lactam, sulfonamide, tri-

methoprim (TMP) resistant bacteria at low concentrations in

vitro and protect mice from systemic E. coli bacteremia as

equally well as imipenem [19]. As it exhibits an in vitro

therapeutic index of over 100 [22], naturally, the peptide is

currently under consideration for development as a clinical

therapeutic. However, before major efforts and funds are

committed to the industrial development process, the scope

and limitations of the drug as a single antibiotic and in

combination have to be characterized. One of the major

objectives of the current study was to identify amino acid or

structural alterations that would further improve the in vitro

efficacy. While none of the single amino acid modifications

yielded any peptide dimer that would be more appropriate for

Fig. 4 – Inhibition of b-galactosidase activity of live K.

pneumoniae K6 cells. The enzymatic activity of the culture

was measured as described in Section 2. While A3-APO

eliminates the enzymatic activity either alone or in

combination with small molecule antibiotics, neither

chloramphenicol (CHF) nor the trimethoprim/

sulfamethoxazole combination (TMP/SXT) alone

influences the cells’ ability to produce active enzyme.

p e p t i d e s 2 9 ( 2 0 0 8 ) 1 8 7 8 – 1 8 8 6 1885

clinical development than A3-APO, some interesting data

emerged from the panel of structurally modified analogues.

Quite surprisingly, one half of A3-APO, the Chex1-Arg20 A3

Single chain derivative showed an overall improved activity

spectrum against test Enterobacteriaceae. The dimeric structure

was earlier identified as necessary in native antimicrobial

peptides for maximum interaction with bacterial membranes,

with the non-natural diaminobutyric acid scaffold providing

stability against carboxypeptidase cleavage [6]. Apparently,

after primary sequence optimization from native peptides to

the designer A3-APO, the dimeric construct is no longer

necessary, at least for efficacy. Notably, we recently deter-

mined that Chex1-Arg20 fragment is one of the major in vivo

metabolites of peptide A3-APO [19]. In addition to the

microbiological improvements, the shorter version is more

stable in blood ex vivo than the prodrug and is easier to mass-

produce.

It is an intriguing question why peptide A3-APO and its

conservative change analogs killed S. saprophyticus. The

antibacterial activity spectrum of native proline-rich peptides

such as drosocin, correlate with the E. coli similarity of the

DnaK D-E helix sequences of sensitive bacteria [3]. The

sequence of S. saprophyticus DnaK D-E helix region [14] has

low similarity to that of E. coli (48%), just like the non-sensitive

S. aureus [21]. Nevertheless, a few Gram-positive strains are

killed by the native peptides pyrrhocoricin and drosocin at

moderate concentrations, including Bacillus megaterium (44%

homology to E. coli DnaK D-E helix) and Micrococcus luteus [12].

A3-APO showed a broader spectrum of activity compared to

the A3 Single chain peptide, which seems to be restricted to

the specific DnaK sequence of E. coli and other Enterobacter-

iaceae whose DnaKs display a high similarity to E. coli. As

shown in Table 2, while the A3 Single chain peptide was

superior to A3-APO in E. coli, Klebsiella and Salmonella, the

opposite was true for Pseudomonas and S. saprophyticus. From

our data it can be thus hypothesized that while the A3 Single

chain Chex1-Arg20 derivative is able to penetrate the bacterial

membrane and specifically bind DnaK, the dimer A3-APO is

able not only to penetrate, but also to damage the membrane

to such an extent that it retains killing activity even towards

some strains in which the DnaK binding does not play a role.

On the other hand, the inhibition of DnaK may be more

effective in the A3 Single chain peptide, potentially because of

steric restrictions towards the dimer.

S. saprophyticus is the leading pathogen in cystitis in young

women [24]. Regardless of the mode of action, the known

efficacy of proline-rich peptide dimers in animal models of

urinary tract infections (UTI) [7], offers A3-APO or its dimeric

analogs as viable prospective alternatives to current anti-

microbials for the treatment of UTI in a clinical setting.

In view of A3-APO’s potential in inhibiting antibiotic-

inactivating enzymes such as b-lactamases [11], we also

studied the synergistic activity of A3-APO and a series of

antibiotics against which the resistance mechanism involves

production of drug-inactivating enzymes. Synergy between

two antibiotics is defined by a 10-fold decrease of viable

bacterial count using the antibiotics in combination as

opposed to each drug alone [16]. This effect provides an

additional therapeutic choice by allowing the use of conven-

tional antibiotics against multi-drug resistant bacteria, for

which there may currently be no other drug available [27]. The

classical synergic combinations, such as aminoglycoside

antibiotics with b-lactams [26], are however ineffective when

resistance is due to the presence of a modified target or

antibiotic-cleaving enzymes.

We could show A3-APO synergy with each of the tested

antibiotics and against all four bacterial species when the

bacteria were preincubated with the antibacterial peptide

prior to addition of the small molecules to inhibit protein

folding. Importantly, in some multi-resistant strains we were

able to retrieve multiple synergic effects. The synergy with

small molecule antibiotics was a generally observable phe-

nomenon, although the effect is individual strain and small

molecule drug combination-dependent. A possible explana-

tion may reside in the different structural stability of different

enzymes, so that the protein refolding activity of DnaK may be

less important for some enzymes. Also, resistance factors

other than properly folded enzymes or compositional differ-

ences among different strains of the same species may be

responsible for maintaining the resistant phenotype. To verify

our results, we also evaluated some antibiotic combinations

for which synergy was not implied due to the absence of a

functional antibiotic-inactivating enzyme. No synergism was

detected in any of those cases.

Acknowledgement

This work was supported in part by the Sbarro Health Research

Organization.

r e f e r e n c e s

[1] Amsterdam D. Susceptibility testing of antimicrobials inliquid media. In: Lorian V, editor. Antibiotics in laboratory

p e p t i d e s 2 9 ( 2 0 0 8 ) 1 8 7 8 – 1 8 8 61886

medicine. Philadelphia, PA: Lippincott Williams andWilkins; 1996. p. 52–111.

[2] Bagella L, Sun A, Tonini T, Abbadessa G, Cottone G, PaggiGM, et al. A small molecule based on the pRb2/p130 spacerdomain leads to inhibition of cdk2 activity, cell cycle arrestand tumor growth reduction in vivo. Oncogene2007;26:1829–39.

[3] Bikker FJ, Kaman-van Zanten WE, de Vries-van de Ruit AM,Voskamp-Visser I, van Hooft PA, Mars-Groenendijk RH,et al. Evaluation of the antibacterial spectrum of drosocinanalogues. Chem Biol Drug Des 2006;68:148–53.

[4] Bower MA, Cudic M, Campbell W, Wade JD, Otvos Jr L.Walking the fine line between intracellular and membraneactivities of antibacterial peptides. Lett Pept Sci2004;10:463–73.

[5] Bush K, Macielag M, Weidner-Wells M. Taking inventory:antibacterial agents currently at or beyond Phase I. CurrOpin Microbiol 2004;7:466–76.

[6] Cudic M, Condie BA, Weiner DJ, Lysenko ES, Xiang ZQ,Insug O, et al. Development of novel antibacterial peptidesthat kill resistant isolates. Peptides 2002;23:2071–83.

[7] Cudic M, Lockatell CV, Johnson DE, Otvos Jr L. In vitro and invivo activity of a designed antibacterial peptide analogagainst uropathogens. Peptides 2003;24:807–20.

[8] Eliopoulos GM, Moellering RC. Antimicrobial combinations.In: Lorian V, editor. Antibiotics in laboratory medicine. 3rded., Baltimore, MD: Lippincott Williams & Wilkins; 1991. p.432–92.

[9] Fernandez-Carneado J, Kogan MJ, Castel S, Giralt E.Potential peptide carriers: amphipathic proline-richpeptides derived from the N-terminal domain of gamma-zein. Angew Chem Int Ed Engl 2004;43:1811–4.

[10] Fields GB, Noble RL. Solid-phase peptide synthesis using 9-fluorenylmethoxycarbonyl amino acids. Int J Pept ProteinRes 1990;35:161–214.

[11] Ge Y, MacDonald DL, Holroyd KJ, Thornsberry C, Wexler H,Zasloff M. In vitro properties of pexiganan, an analogof magainin. Antimicrob Agents Chemother 1999;43:782–8.

[12] Hoffmann R, Bulet P, Urge L, Otvos Jr L. Range of activityand metabolic stability of synthetic antibacterialglycopeptides from insects. Biochim Biophys Acta1999;1426:459–67.

[13] Hollosi M, Kawai M, Fasman GD. Studies on proline-containing tetrapeptide models of beta-turns. Biopolymers1985;24:211–42.

[14] Kragol G, Hoffmann R, Chattergoon MA, Lovas S, Cudic M,Bulet P, et al. Identification of crucial residues for theantibacterial activity of the proline-rich peptide,pyrrhocoricin. Eur J Biochem 2002;269:4226–37.

[15] Kragol G, Lovas S, Varadi G, Condie BA, Hoffmann R, OtvosJr L. The antibacterial peptide pyrrhocoricin inhibits theATPase actions of DnaK and prevents chaperone-assistedprotein folding. Biochemistry 2001;40:3016–26.

[16] Leng B, Meyers BR, Hirschman SZ, Keusch GT.Susceptibilities of gram-negative bacteria to combinationsof antimicrobial agents in vitro. Antimicrob AgentsChemother 1975;8:164–71.

[17] Levy SB, Marshall B. Antibacterial resistance worldwide:causes, challenges and responses. Nat Med 2004;10:S122–129.

[18] Lopez Ferreira N, Alix JH. The DnaK chaperone is necessaryfor a-complementation of b-galactosidase in Escherichia coli.J Bacteriol 2002;184:7047–54.

[19] Noto PB, Abbadessa G, Cassone M, Mateo GD, Agelan A,Wade JD, et al. Alternative stabilities of a proline-richantibacterial peptide in vitro and in vivo. Protein Sci2008;17:1249–55.

[20] Otvos Jr L, de Olivier Inacio V, Wade JD, Cudic P. Priorantibacterial peptide-mediated inhibition of protein foldingin bacteria mutes resistance enzymes. Antimicrob AgentsChemother 2006;50:3146–9.

[21] Otvos Jr L, de Olivier Inacio V, Rogers ME, Consolvo PJ,Condie BA, Lovas S, et al. Interaction between heat shockproteins and antimicrobial peptides. Biochemistry2000;39:14150–9.

[22] Otvos Jr L, Snyder C, Condie BA, Bulet P, Wade JD. Chimericantimicrobial peptides exhibit multiple modes of action. IntJ Pept Res Ther 2005;11:29–42.

[23] Otvos Jr L, Wade JD, Lin F, Condie BA, Hanrieder J,Hoffmann R. Designer antibacterial peptides killfluoroquinolone-resistant clinical isolates. J Med Chem2005;48:5349–59.

[24] Raz R, Colodner R, Kunin CM. Who are you, Staphylococcussaprophyticus? Clin Infect Dis 2005;40:896–8.

[25] Rothbard JB, Kreider E, VanDeusen CL, Wright L, Wylie BL,Wender PA. Arginine-rich molecular transporters for drugdelivery: role of backbone spacing in cellular uptake. J MedChem 2002;45:3612–8.

[26] Serra P, Brandimarte C, Martino P, Carlone S, Giunchi G.Synergistic treatment of enterococcal endocarditis: in vitroand in vivo studies. Arch Intern Med 1977;137:1562–7.

[27] Tarasi A, Cassone M, Monaco M, Tarasi D, Pompeo ME,Venditti M. Activity of moxifloxacin in combination withvancomycin or teicoplanin against Staphylococcus aureusisolated from device-associated infections unresponsive toglycopeptide therapy. J Chemother 2003;15:239–43.

[28] Weber JT, Courvalin P. An emptying quiver: antimicrobialdrugs and resistance. Emerg Infect Dis 2005;11:791–3.