Using Small-Molecule Adjuvants to Repurpose Azithromycin for Use against Pseudomonas aeruginosa

Article abstract of DOI:10.1021/acsinfecdis.8b00288

A major contributor to fatalities in cystic fibrosis (CF) patients stems from infection with opportunistic bacterium Pseudomonas aeruginosa. As a result of the CF patient's vulnerability to bacterial infections, one of the main treatment focuses is antibiotic therapy. However, the highly adaptive nature of P. aeruginosa, in addition to the intrinsic resistance to many antibiotics exhibited by most Gram-negative bacteria, means that multi-drug-resistant (MDR) strains are increasingly prevalent. This makes the eradication of pseudomonal lung infections nearly impossible once the infection becomes chronic. New methods to treat pseudomonal infections are greatly needed in order to eradicate MDR bacteria found within the respiratory tract, and ultimately better the quality of life for CF patients. Herein, we describe a novel approach to combatting pseudomonal infections through the use of bis-2-aminoimidazole adjuvants that can potentiate the activity of a macrolide antibiotic commonly prescribed to CF patients as an anti-inflammatory agent. Our lead bis-2-AI exhibits a 1024-fold reduction in the minimum inhibitory concentration of azithromycin in vitro and displays activity in a Galleria mellonella model of infection.

Full text of DOI:10.1021/acsinfecdis.8b00288

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Article
Using Small Molecule Adjuvants to Repurpose
Azithromycin for Use Against Pseudomonas aeruginosa
Veronica Hubble, Brittany A Hubbard, Bradley Minrovic, Roberta Melander, and Christian Melander
ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.8b00288 • Publication Date (Web): 16 Nov 2018
Downloaded from http://pubs.acs.org on November 19, 2018
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Using Small Molecule Adjuvants to Repurpose Azithromycin for Use Against Pseudomonas
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aeruginosa
Authors: Veronica B. Hubble†, Brittany A. Hubbard‡, Bradley M. Minrovic†, Roberta J.
Melander†, and Christian Melander*,†
†Department of Chemistry and Biochemistry, University of Notre Dame, 236 Cavanaugh Dr.,
Notre Dame, Indiana 46556, United States
‡Department of Chemistry, North Carolina State University, 2520 Yarbrough Dr., Raleigh, NC
27607, United States
*Correspondence: Christian Melander, cmelande@nd.edu
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A major contributor to fatality in cystic fibrosis (CF) patients stems from infection with
the opportunistic bacterium Pseudomonas aeruginosa. As a result of the CF patient’s
vulnerability to bacterial infections, one of the main treatment focuses is antibiotic therapy.
However, the highly adaptive nature of P. aeruginosa, in addition to the intrinsic resistance to
many antibiotics exhibited by most Gram-negative bacteria, means that multi-drug resistant
(MDR) strains are increasingly prevalent. This makes eradication of pseudomonal lung
infections nearly impossible once the infection becomes chronic. New methods to treat
pseudomonal infections are greatly needed in order to eradicate MDR bacteria found within the
respiratory tract and ultimately better the quality of life for CF patients. Herein, we describe a
novel approach for combatting pseudomonal infections through the use of bis-2-aminoimidazole
adjuvants that can potentiate the activity of a macrolide antibiotic commonly prescribed to CF
patients as an anti-inflammatory agent. Our lead bis-2-AI exhibits a 1024-fold reduction in the
minimum inhibitory concentration of azithromycin in vitro and displays activity in a Galleria
mellonella model of infection.
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Keywords
Antibiotic resistance, antibiotic adjuvant, cystic fibrosis, Pseudomonas aeruginosa
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Cystic fibrosis (CF) is a life-threatening genetic disease inherited by approximately one
in 2,000 children annually within the American Caucasian population alone.1 The Cystic Fibrosis
Foundation estimates that approximately 70,000 people suffer with CF worldwide.2 CF is
defined by a defect in the cystic fibrosis transmembrane conductance regulator (CFTR) gene,
which causes over-accumulation of thick, sticky mucus that coats the lungs and intestines while
acting as an ideal breeding ground for bacterial infections.3 Pulmonary failure as a result of
prolonged inflammation is directly associated with the presence of Pseudomonas aeruginosa and
remains the main cause of fatality in CF patients.4 The ongoing persistence of P. aeruginosa
within the CF community is due in part to its highly adaptive defense mechanisms and the lack
of novel, effective antimicrobial drugs to treat multidrug-resistant (MDR) pseudomonal strains.
The average age at which a CF patient acquires a P. aeruginosa infection is approximately 16
years, and once physicians observe a change in pseudomonal cells from non-mucoid to mucoid,
life expectancy of each patient is soon established, with the average being 37 years.1-2
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Treating MDR P. aeruginosa is challenging and requires aggressive action, typically the
administration of a combination of two or three antimicrobials. Some of the commonly used
antipseudomonal
drugs
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all of these antibiotic classes have been found in numerous strains of MDR P. aeruginosa.6 This
is especially troubling in the case of colistin, as it is a last resort antibiotic prescribed to CF
patients in Europe. Colistin, however, has not been approved as a CF treatment option within the
United States because of its reported nephro- and neurotoxicity.7-8 Novel treatment approaches to
combat pseudomonal infections are clearly needed.
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In addition to antibiotic therapy, anti-inflammatory drugs are administered to CF patients
as a life-long regimen in attempts to increase patient comfort and expand life expectancy.9
Azithromycin is a macrolide antibiotic that has demonstrated the ability to reduce tissue
inflammation in several lung-associated diseases and is currently prescribed to CF patients as the
main anti-inflammatory drug.9-12 Outside of CF treatment, azithromycin is used as an antibiotic
to treat infections caused by Gram-positive pathogens, such as Staphylococcus aureus.13
However, like all macrolide antibiotics, azithromycin does not possess the ability to combat P.
aeruginosa infections due the intrinsic resistance imparted by both the outer membrane and
efflux pump systems.14
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Previously, we have demonstrated that select members of our in-house marine alkaloid
derived library possess adjuvant activity and can break acquired resistance towards ceftazidime
and meropenem in planktonic MDR P. aeruginosa.15 Other groups have shown that cationic
peptides enhance the activity of azithromycin against Gram-negative pathogens in a synergistic
manner through perturbing the outer membrane and thus allowing antibiotics like azithromycin
entry into the cell.16-17 Given azithromycin’s established use in CF therapy, we wondered
whether we could identify an adjuvant from our in-house library of nitrogen-dense heterocycles
that would break Pseudomonas aeruginosa’s intrinsic resistance to azithromycin and in the
process allow us to repurpose azithromycin for antibacterial use against this pathogen. Herein,
we describe the identification of non-microbicidal bis-2-aminoimidazoles (bis-2-AIs) 1 and 2
that potentiate azithromycin activity in highly resistant strains of P. aeruginosa. Furthermore, we
demonstrate the requirement for the bis-2-AI moiety compared to a mono-2-AI analog thorough
structure-activity relationship (SAR) studies. After identifying a lead bis-2-AI adjuvant, we
sought to identify the potential mechanism of action by studying its effect on bacterial membrane
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integrity and efflux. Next, we evaluated and confirmed in vivo activity of a combination of bis-2-
AI 2 and azithromycin in a Galleria mellonella model of infection. We then demonstrated that
bis-2-AI 2 is able to sensitize PAO1 to an additional macrolide antibiotic, clarithromycin, as well
as other classes of antibiotics. Finally, we established the ability of adjuvant 2 to potentiate
azithromycin activity against several additional clinically relevant P. aeruginosa strains isolated
from CF patients.
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Figure 1. Initial library bis-2-AIs screened for enhancement of azithromycin activity against a
highly resistant strain of P. aeruginosa.
Results and Discussion
This study was initiated by screening a structurally diverse subset of compounds from our
internal library for the ability to lower the minimum inhibitory concentration (MIC) of
azithromycin against the P. aeruginosa laboratory strain PAO1, which exhibits high levels of
resistance to azithromycin (MIC 256 µg/mL) (see Supporting Information for a representative
list of compounds screened). From this screen, compound 1, which possesses two 2-AI groups,
lowered the MIC of azithromycin by eight-fold, from 256 to 32 µg/mL at 60 µM. Prompted by
this result, structurally similar compounds 2 and 3 were screened against PAO1 (also at 60 µM).
Bis-2-AI 2 exhibited potent adjuvant activity, lowering the MIC of azithromycin 1024-fold from
256 to 0.25 µg/mL. Interestingly, bis-2-AI 3 did not sensitize PAO1 to azithromycin, inspiring us
to explore the structure-activity relationship (SAR) of these bis-2-AI analogs.
Using lead adjuvant 2 as an inspiration for SAR studies, a pilot library was synthesized
for the direct comparison of structure and biological activity (Schemes 1 and 2). Beginning with
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amino acid methyl esters 4 and 5, 2-AI analogs 6 and 7 were synthesized using a previously
published procedure, which utilized standard Akabori reduction conditions followed by the
condensation of cyanamide in a temperature and pH controlled environment (Scheme 1A).18 The
pH of the cyanamide condensation reaction was previously determined to be of importance by
Lancini and Lazzari19 to avoid the production of urea in highly acidic environments or undesired
aminoketone formations in neutral or alkaline environments. For SAR purposes, 2-AI derivatives
10 and 13 were accessed using commercially available carboxylic acid 8 or acid chloride 11.
After conversion of compound 8 into its 6-azido acid chloride congener, this and 11 were
subjected to reaction with diazomethane followed by a HBr workup to produce the
corresponding α-bromoketones (Scheme 1B and 1C). These intermediates were subsequently
cyclized with Boc-guanidine to produce compounds 9 and 12. Upon reduction of the azide
moiety of compound 9 and deprotection of the 2-AIs, synthesis of compounds 10 and 13 was
completed by conversion into their corresponding HCl salts for biological testing.
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Scheme 1. Synthesis of Core 2-AI Scaffold
Bis-2-AI derivatives were then accessed via an oxidative dimerization route developed by
the Horne group.20 Compounds 14 and 15 were previously prepared in our group.21 Treatment of
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each 2-AI compound with 1 eq. of N-chlorosuccinimide under acidic conditions followed by
reaction with commercially available 2-AI sulfate generated bis-2-AIs 1, 2, and 16-19 as well as
tricyclic compounds 20-21 as low yielding byproducts (Scheme 2).
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Scheme 2. Synthesis of Bis-2-AI Analogs
The MIC of each bis-2-AI was first determined and all active adjuvants returned MICs
≥200 µM, with the exception of compound 19, which exhibited an MIC of 100 µM (select data
summarized in Table 1). The MIC of azithromycin in the presence of each compound at 60 µM
(30 µM for compound 19) was next determined. The mono-2-AI intermediates, 6, 7, 10, and 13-
14, did not enhance azithromycin activity, suggesting the importance of a second 2-AI functional
group for activity (Table S2). In addition, tricyclic compounds 20-21 containing two 2-AI heads
tethered by a 7-membered ring also lacked the ability to potentiate azithromycin, implying the
significance of the two 2-AI heads being in close proximity to one another, while retaining
conformational flexibility (Table S2).
Considering the highly polar nature of active bis-2-AI analogs, we evaluated the
biological importance of polarity by synthesizing and screening bis-2-AIs 17-19, which possess
aliphatic tails (4, 13 or 15 carbons, respectively) lacking a positively charged amine in this
region of the molecule. All analogues failed to sensitize PAO1 to azithromycin; however, bis-2-
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AI 19 did exhibit greater toxicity against PAO1 alone. The fact that adjuvants 17-19 failed to
sensitize PAO1 to azithromycin led to the conclusion that the presence of a free amine in the
distal tail region of the molecule is crucial for maintaining activity (Table S2).
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Finally, to delineate the ideal length of the aliphatic tail for amine-containing bis-2-AIs,
the synthesis of bis-2-AI 16 was carried out. Bis-2-AI 16 did modestly potentiate azithromycin
activity by 16-fold, similar to that of bis-2-AI 1 (Table 1). With this knowledge, it was revealed
that compound 2, which contained a four-carbon alkyl chain, remained the most active
compound in these studies. To continue investigation of lead bis-2-AI 2 activity, a dose response
study was performed against PAO1 over a wide range of concentrations (Table 2). This revealed
substantial adjuvant activity at 40 µM, where there was a 256-fold reduction in the MIC of
azithromycin, before a drop-off in activity was observed at lower adjuvant concentrations.
Table 1. Azithromycin potentiation data for bis-2-AI derivatives against PAO1
Compound
Concentration
Tested (µM)
Azithromycin
Compound
MIC (µM)
Fold
Reduction
Compound
MIC a
(µg/mL)
1
>200
200
60
60
60
60
60
30
30
60
30
32
0.25
128
16
8
1024
2
2
3
>200
>200
>200
>200
100
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19
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16
2
128
256
128
128
256
0
2
>200
>200
2
0
aAzithromycin MIC alone is 256 µg/mL.
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Table 2. Dose-response of compound 2 against PAO1
Compound
Azithromycin
Fold
Compound
Concentration
Tested (µM)
MIC (µg/mL) Reduction
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0
256
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2
1
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1024
1024
0.25
0.25
Next, we wanted to probe the mechanistic basis by which compound 2 is able to sensitize
PAO1 toward azithromycin. Gram-negative species are far more challenging to combat than
Gram-positive species, predominantly due to the protection imparted in the form of the outer
membrane.22 Therefore, we first investigated whether compound 2 was acting by increasing the
permeability of the bacterial cell membrane, using a commercially available LIVE/DEADTM
BacLightTM Bacterial Viability Kit. The BacLight assay quantifies the fluorescence of two DNA-
binding stains to measure the ratio of intact/damaged cells. Lead compound 2 was tested in
addition to both compounds 1 and 3 for comparison. PAO1 cells were treated with each
compound at 60 µM for one hour, after which cells were collected, washed and fluorescence was
determined. The degree of membrane permeability imparted by compound 1 was not as
substantial as that of compound 2, while compound 3 had negligible effect, indicating a
correlation between membrane permeability and adjuvant activity. Exposure to compound 2
resulted in an increase in permeability of 50% compared to untreated cells, while exposure to
compound 3 only resulted in a 1% increase in membrane permeability (Table S3). Furthermore,
compound 1, which effected a more modest reduction in the MIC of azithromycin than
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compound 2, caused a 31% increase in membrane permeability in PAO1 (Table S3). These
results suggested that increasing cell membrane permeability is correlated to adjuvant activity,
and may play a role in the mechanism by which bis-2-AI 2 enhances azithromycin activity.
Bis-2-AI adjuvants, 1 and 2, were also screened for potentiation of azithromycin against
another important Gram-negative pathogen, Klebsiella pneumoniae. Both bis-2-AI adjuvants 1
and 2 did not potentiate azithromycin activity against K. pneumoniae ATCC BAA-2146,
effecting only a two-fold or less reduction in azithromycin MIC (Table S4). Compound 3 was
likewise tested for azithromycin potentiation activity against K. pneumoniae BAA-2146 and
resulted in merely a two-fold MIC reduction (Table S4). In order to further probe the role of
disruption of the integrity of the cell membrane in bis-2-AI activity, a BacLight assay was
performed using K. pneumoniae BAA-2146, which revealed a 5% increase in cell membrane
permeability after treatment with compound 2, considerably less than the 50% increase observed
for PAO1 at the same concentration (Table S5). This further indicates that an increase in cell
membrane permeability has a correlation with adjuvant activity, although it is not yet known
whether this is direct or indirect.
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Many bacterial species, including P. aeruginosa23 depend on biological metals obtained
from the human host to maintain metabolism and cell growth, as well as contribute to bacterial
virulence.24 As divalent metal ions are also important for eukaryotic cell growth and metabolism,
one approach implemented by the host during infection is to sequester free metals from the
environment using host proteins, such as transferrin, in order to reduce metal availability for
invading pathogens.25 Therefore, metal deprivation, also known as ‘nutritional immunity’, is a
crucial defense mechanism used by the innate immune system to essentially starve microbial
pathogens.26 P. aeruginosa utilizes several strategies to circumvent nutritional immunity, such as
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pirating metal ions from host resources (e.g. transferrin) using siderophores, or scavenging free
metal ions from the environment and transporting it into the cell via several well-studied
mechanisms.24 Previous studies within our lab have shown that 2-aminobenzimidazole (2-ABI)
small molecule adjuvants that inhibit and disperse Gram-positive biofilms, do so via a zinc-
dependent mechanism.27-28 When analyzing the structure of these bis-2-AIs, we recognized that
these molecules could be acting as metal chelators. We therefore measured the effect of
increasing concentrations of iron and other commonly encountered divalent metal cations upon
the ability of compound 2 (at 60 µM) to potentiate azithromycin against PAO1 (Table 3).
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Table 3. Metal doping effect on the ability of compound 2 to potentiate azithromycin
activity against PAO1
Azithromycin MIC (µg/mL)b-c + 2d
Conc. of
FeSO4•7H2O
ZnCl2
≤0.25
≤0.25
≤0.25
0.5
CaCl2
0.12
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0.25
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CuCl2
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MnCl2
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Metal Tested (µM)a
0
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128
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aEach divalent metal exhibited a MIC >200 µM when dosed alone.
bAzithromycin MIC was 256 µg/mL against untreated PAO1.
cAzithromycin MIC was ≥128 µg/mL when combined with each divalent metal at 200 µM.
dCompound 2 was tested at 60 µM.
Increasing concentrations of Fe(II) or Mn(II) both suppressed the ability of compound 2
to potentiate azithromycin. When supplemented with 200 µM FeSO4•7H2O, the activity of
compound 2 was negligible, and supplementation with 200 µM of MnCl2 also caused a reduction
in activity (16-fold reduction in azithromycin MIC compared to 1024-fold). However, doping
with Zn(II), Ca(II), Cu(II), or Mg(II) did not affect the activity of adjuvant 2. Prompted by the
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effects of iron and manganese on bis-2-AI activity, we conducted additional studies to evaluate
the mechanism of action of bis-2-AI 2 against PAO1.
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To further probe the mechanism of action of bis-2-AI 2, and the effect that metal ions
elicited on activity, we wanted to analyze compound 2’s ability to potentiate azithromycin in a
chelating-competitive environment by co-dosing with ethylenediaminetetraacetic acid (EDTA).
Many studies have shown EDTA can displace divalent cation cross-bridges necessary for P.
aeruginosa membrane integrity, and it has been used in combination with antimicrobials to
combat Gram-negative bacterial pathogens.29-30 EDTA chelates positively charged ions, thus co-
dosing with EDTA would sequester any Fe(II) from the media and allow observation of the
activity compound 2 in the absence of Fe(II). A dose-dependent study was performed against
PAO1 in which the ability of compound 2 to potentiate azithromycin was measured in increasing
concentrations of EDTA (Table S6). The supplementation of EDTA at high concentrations (200
µM) did not exhibit any biological activity on PAO1 when dosed alone, nor did it alter the MIC
of azithromycin against PAO1 (Table S6). Furthermore, the triple-dosing of EDTA at
increasingly high concentrations (up to 200 µM), adjuvant 2, and azithromycin did not alter the
previously observed adjuvant activity of 2, with a 1024-fold reduction in azithromycin MIC
noted.
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To further validate that bis-2-AI 2 adjuvant activity is not due to metal-chelation, the
azithromycin potentiation assay was performed in minimal salts media (M9 media). Cation-
adjusted Mueller-Hinton broth (CAMHB) is typically the medium used for MIC determination
against P. aeruginosa according to the Clinical and Laboratory Standards Institute (CLSI).31 It
has been reported that divalent ions can affect the mode of action of several antimicrobial agents
and stabilization of cell walls; therefore, the supplementation of ions in CAMHB has been the
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standardized medium for accurate antimicrobial results against PAO1.32 However, we wanted to
observe activity of adjuvant 2 in iron-deficient media, such as M9. We first established the MICs
of azithromycin and compound 2 against PAO1 in M9 media (Table S7). As expected, the MICs
of azithromycin and compound 2 were consistent with their MICs against PAO1 in CAMHB,
being 256 µg/mL and 200 µM respectively, suggesting that the potency of azithromycin against
PAO1 is not affected when in an iron-deprived environment, which is in agreement with the
EDTA experiment. Likewise, compound 2 exhibits comparable potentiation of azithromycin
against PAO1 in M9 as in CAMHB media, lowering the MIC 512-fold from 256 to 0.5 µg/mL.
Prompted by the effects of high concentration of both Fe(II) and Mn(II) on potentiation
activity, the effect of compound 2 upon PAO1 membrane integrity in the presence of 200 µM of
Fe(II) and Mn(II) was evaluated again using the BacLight assay. We first analyzed the effect of
Fe(II) and Mn(II) on PAO1 membrane integrity before combining metal and adjuvant against
PAO1, and observed that neither metal ion affected permeability when administered alone.
(Table S8). The presence of Fe(II) at 200 µM significantly reduced the effect of compound 2 (at
60 µM) upon PAO1 membrane permeability, resulting in only a 14% increase in membrane
permeability compared 50% in the absence of Fe(II) (Table S8). Furthermore, the presence of
Mn(II) at 200 µM also reduced the increase in PAO1 membrane permeability effected by
compound 2, to just 8%.
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Next, we wanted to exclude the possibility that bis-2-AI 2 acts through a non-specific
aggregation-based (i.e. PAINS) mechanism.33 To address this issue, we conducted the antibiotic
potentiation assay in the presence of a detergent, Tween 80. Bis-2-AI 2 adjuvant activity was
unaffected in the presence of 0.01% Tween 80, indicating that adjuvant activity is not driven by
compound aggregation (Table S9).
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To analyze eukaryotic toxicity, we measured the hemolytic activity of bis-2-AI
compounds 2 and 3 upon defibrinated sheep’s blood.34 Both highly active compound 2 and
inactive bis-2-AI 3 exhibited less than 3% hemolysis at concentrations up to 500 µM, a
concentration well above the adjuvant activity values (Table S10). We also analyzed toxicity of
our lead bis-2-AI 2 in a whole organism study using Galleria mellonella, and observed 100%
worm survival after 6 days following dosing with compound 2 at a high concentration of 400
mg/kg (Table S16).34-35 Both the hemolysis assay and Galleria studies suggest that bis-2-AI 2
does not exhibit significant eukaryotic toxicity.
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Given the favorable eukaryotic toxicity profile, we next explored the ability of adjuvant 2
to potentiate azithromycin against PAO1 in G. mellonella model of infection, which has been
shown to be predictive of activity in murine studies.35 We first identified meropenem as a
positive control for PAO1 infections in the worm model. In G. mellonella, 30 minutes after
worms were inoculated with 105 CFU of PAO1, a single dose of meropenem at 5 mg/kg resulted
in a 100% survival rate after 6 days (Table S17). We then attempted to determine the dosage of
azithromycin necessary for minimal protection against infection; however, azithromycin dosed at
concentrations up to 100 mg/kg provided no survival (Table S12 and S13). Since azithromycin is
not used as an antibiotic for P. aeruginosa, and we did not want to surpass toxic levels, we
decided to continue with combinatorial treatments by modeling our treatment dosage off
previously published work, and treat worms with azithromycin at 50 mg/kg.35-37 Treatment of
infected worms with bis-2-AI 2 at 50 mg/kg provided no improvement in survival rates
compared to untreated, infected worms (Table S14). However, a single dose of compound 2 at 50
mg/kg in combination with 50 mg/kg azithromycin improved the survival rate to 43% (Figure 2;
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Table S15). This survival rate is equivalent to treatment of infected worms with clavulanic
acid/penicillin, one of the few clinically approved antibiotic/adjuvant combinations.36
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100
90
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Bacteria Only
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0
Azm 50 mg/kg +
Compound 2 50 mg/kg
Azm 50 mg/kg
Compound 2 50 mg/kg
Meropenem 5 mg/kg
0
1
2
3
4
5
6
Time (Days)
Figure 2. Percent survival of G. mellonella after antibiotic-adjuvant, antibiotic, or DMSO
(control, i.e. bacteria only) dosage followed with an inoculation of PAO1
Following promising model organism results for lead bis-2-AI 2, its potential as an
adjuvant for additional antibiotics other than azithromycin was investigated against PAO1 (Table
4). Compound 2 (at 60 µM) was able to substantially sensitize PAO1 to another macrolide
antibiotic, clarithromycin, decreasing the MIC 128-fold from 256 µg/mL to 2 µg/mL. Bis-2-AI 2
was also able to sensitize PAO1 to rifampin, an antibiotic prescribed to patients with the lung-
associated disease tuberculosis.38 Additionally, adjuvant 2 potentiated the activity of the ß-lactam
antibiotic doripenem, lowering the MIC 32-fold. The activity of colistin, another antibiotic that
possesses activity against P. aeruginosa, was not enhanced by bis-2-AI 2. It is interesting that
bis-2-AI 2 did not sensitize P. aeruginosa to colistin, which elicits its antibacterial activity by
binding to the outer cell membrane of Gram-negative bacteria and promoting its own uptake,6
yet was able to sensitize PAO1 to doripenem and rifampin, both of which must diffuse across the
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cell membrane to produce their antibiotic effect on the bacterium.38-39 The presence or lack of
adjuvant activity of compound 2 across this range of classes of antibiotics correlates to the data
obtained in the BacLight analysis, indicating that compound 2 is affecting the outer membrane of
the bacterium.
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Table 4. Additional antibiotic potentiation of bis-2-AI 2 against PAO1
Azithromycin MIC (µg/mL)
Antibiotic
Azithromycin MIC (µg/mL)
Fold Reduction
+ 2a
Clarithromycin
Doripenem
Rifampin
256
2
32
2
2
0.06
0.5
1
128
32
64
2
Colistin
aCompound 2 was tested at 60 µM.
Finally, we evaluated the adjuvant potential of bis-2-AI 2 upon azithromycin activity
against a panel of pseudomonal strains that are clinically relevant to CF, by obtaining six isolates
from CF patients, three of which are mucoidal and three non-mucoidal isolates (Table 5). Bis-2-
AI 2 caused a 512-, 32-, and 128-fold reduction in the azithromycin MIC against the three non-
mucoid strains CEC 45, 57, and 102, respectively. A similar pattern of activity was observed in
two of the three mucoid strains, where 32- and 16-fold reductions in MIC were observed against
CEC 103 and 111, respectively. A substantial increase in adjuvant activity was revealed against
the third mucoid strain, CEC 70, for which a 4267-fold reduction in azithromycin MIC was
recorded. These results confirmed adjuvant 2’s viability in a range of P. aeruginosa strains that
are found among CF patients.
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Table 5. Azithromycin potentiation data for bis-2-AI 2 against cystic fibrosis strains
Cell
CF
Compound 2
Azithromycin
Azithromycin MIC
Fold
Type
Strainsa
MIC (µM)
MICb (µg/mL)
(µg/mL) + 2
Reduction
7
8
9
CEC 45
CEC 57
CEC 102
CEC 70
>200
200
>200
200
>200
50
256
256
256
256
128
128
0.5
8
2
512
32
128
4267
32
Non-
mucoid
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0.06
4
Mucoid CEC 103
CEC 111
8c
16
aCystic fibrosis strains (three non-mucoid strains CEC 45, 57, 102 and three mucoid strains CEC
70, 103, 111) were obtained from Robert K. Ernst, in the Department of Microbial
Pathogenesis at University of Maryland-Baltimore.
bAll MIC values are in µg/mL, compound 2 tested at 60 µM for all strains except for CEC 111.
cCompound 2 tested at 15 µM.
In conclusion, there is no question that MDR P. aeruginosa infections have devastated
the quality of life and diminished the life expectancy for CF patients. Novel approaches to
eradicate pseudomonal infections are greatly needed. We report a novel adjuvant containing a
bis-2-AI moiety that potentiates the activity of a non-pseudomonal antibiotic, azithromycin,
against a highly intrinsically resistant strain of P. aeruginosa, PAO1, as well as six CF clinical
pseudomonal isolates. SAR studies revealed bis-2-AI 2 as the lead adjuvant, which enhances
azithromycin activity by 1024-fold against PAO1, without displaying signs of hemolytic activity
or G. mellonella toxicity well above the active concentration. Bis-2-AI 2 sensitized PAO1 to
additional antibiotics including another macrolide antibiotic, clarithromycin, a ß-lactam
antibiotic doripenem, as well as rifampin, but did not potentiate the activity of the membrane
active antibiotic colistin. Interestingly, all of the antimicrobials that were potentiated by bis-2-AI
2 are known to require penetration through the outer membrane in order to be effective. We
hypothesize that the ability of compound 2 to enhance macrolide activity in P. aeruginosa is
somehow related to its effect on membrane integrity, as seen in the BacLight assay; however,
further mechanistic studies for compound 2 are currently ongoing to address whether this is due
to a direct interaction with the membrane or an indirect effect due to altered cell membrane
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structure/presentation. Activity was impacted by both Fe and Mn ions; however, the activity of
these compounds does not appear to be driven by chelation. Lastly, the non-toxic and non-
microbicidal bis-2-AI 2 was able to potentiate azithromycin in a model organism, exhibiting a
43% survival rate for worms inoculated with PAO1, similar to that reported for clavulanic
acid/penicillin.36 As the G. mellonella model has inherent limitation, specifically it is limited to a
single dose, we expect that moving to a more clinically relevant model where multiple doses are
given will return significantly greater survival.
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Experimental
All reagents used for chemical synthesis were purchased from commercially available sources
and used without further purification. Flash chromatography was performed using 60 Å mesh
standard grade silica gel from Sorbetch. NMR solvents were obtained from Cambridge Isotope
1
Labs and used as is. All H NMR (300, 400, or 600 MHz) were recorded at 25°C on Varian
Mercury spectrometers or a (700 MHz) Bruker Avance spectrometer. All 13C NMR (100, 125, or
175 MHz) spectra were recorded at 25°C on Varian Mercury spectrometers. Chemical shifts ()
are given in parts per million (ppm) relative to the respective NMR solvent; coupling constants
(J) are in hertz (Hz). Abbreviations used are s, singlet; bs, broad singlet; d, doublet; dd, doublet
of doublets; t, triplet; dt, doublet of triplets; m, multiplet. All high resolution mass spectrometry
measurements were made in the Molecular Education, Technology, and Research Innovation
Center (METRIC) at NC State University. The purities of the tested compounds were all verified
to be >95% by LC-MS analysis on a Shimadzu LC-MS 2020 with Kinetex, 2.6 mm, C18 50 ×
2.10 mm.
General Procedure for Akabori Reduction and Cyanamide Cyclization (6 and 7). The
commercially available amino acid methyl ester derivatives 4 or 5 (54 mmol, 1 eq) were
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dissolved in deionized water (125 mL) and 250 g of 5% sodium amalgam were added over one
hour. The pH was maintained at 1.5 by the addition of concentrated HCl and the temperature was
kept at 0°C using an ice bath. The reaction mixture was stirred at 0°C for an additional 30
minutes whereupon the mercury was separated. To the resulting solution was added cyanamide
(540 mmol, 10 eq) and the pH was adjusted to 4.3 by addition of 1M NaOH. The solution was
heated to 95°C and stirred for 3.5 h. and then evaporated to dryness under reduced pressure. The
residue was washed in Et2O to remove the unreacted cyanamide, then dissolved in a minimal
amount of MeOH and decanted to remove NaCl, then concentrated to under reduced pressure.
The residue was purified by flash chromatography (25% – 100% MeOH sat. with NH3 in DCM).
Purified product was dissolved in 6 M HCl in MeOH (10 mL) and solvent was removed to yield
6 or 7 as their HCl salt.
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General Procedure for Oxidative Heterodimerization (1-2 and 16-21). NCS (2 mmol, 1 eq) was
added to a solution of the corresponding 2-AI derivative (2 mmol, 1 eq) in methanesulfonic acid
(8 mL) at room temperature. After 10 min, commercially purchased 2-AI sulfate (2 mmol, 1 eq)
was added, and the resulting solution was stirred for 48 h. The reaction mixture was then diluted
with acetone and decanted (200 mL × 2). The resulting residue was purified using flash
chromatography (25% - 100% MeOH sat. with NH3 in DCM) and the purified product was
dissolved in 6 M HCl in MeOH and solvent was removed to yield the corresponding bis-2-AI 1,
2, 16-21 as their HCl salt.
General Procedure for Boc-guanidine Cyclization (9 and 12). The corresponding α-bromoketone
(2 mmol, 1 eq) and Boc-guanidine (6 mmol, 3 eq) were dissolved in anhydrous DMF (20 mL)
and stirred for 48 h. The reaction mixture was diluted with deionized water and the desired
product was extracted with EtOAc (50 mL × 3). The organic fractions were combined and
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washed with brine (10 mL × 2), dried with MgSO4, and concentrated under reduced pressure.
The residue was purified by flash chromatography (5% – 100% EtOAc/hexanes) to afford
desired compounds 9 or 12.
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General Procedure for Boc-deprotection (10 and 13). The corresponding Boc-protected 2-AI
(1.3 mmol, 1 eq) was dissolved in DCM (1 mL) and TFA (2 mL) was added dropwise into the
reaction vessel. The reaction was stirred for 3 h, and then the solvent was removed under reduced
pressure. The resulting TFA salt was dissolved in 6 M HCl in MeOH (10 mL) and solvent was
removed to yield 10 or 13 as their HCl salt.
Previously Reported Compounds18, 40, 41
4-(3-aminopropyl)-1H-imidazol-2-amine (6): Compound 6 was prepared following a previously
published protocol, using the general procedure for the Akabori reduction and cyanamide
cyclization to obtain compound 6 as a white solid in 41% yield. Spectral data for compound 6
was consistent with known values.41 1H-NMR (300 MHz, CD3OD)  6.61 (s, 1H), 2.98 (t, J = 7.8
Hz, 2H), 2.64 (t, J = 7.6 Hz, 2H), 2.06 – 1.90 (m, 2H).
4-(4-aminobutyl)-1H-imidazol-2-amine (7): Compound 7 was prepared following a previously
published protocol, using the general procedure for the Akabori reduction and cyanamide
cyclization was followed to afford compound 7 as an orange solid in 18% yield. Spectral data for
compound 7 was consistent with known values.18 1H NMR (400 MHz, CD3OD) δ 6.57 (s, 1H),
2.98 (t, J = 7.3 Hz, 2H), 2.57 (t, J = 6.6 Hz, 2H), 1.76 – 1.67 (m, 4H).
tert-butyl 2-amino-4-(5-azidopentyl)-1H-imidazole-1-carboxylate (9): Compound 9 was prepared
following a previously published protocol, using the general procedure for Boc-guanidine
cyclization was followed to afford compound 9 as an orange solid in 33% yield. Spectral data for
compound 9 was consistent with known values.40 1H NMR (300 MHz, CDCl3) δ 6.50 (s, 1H),
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5.63 (bs, 2H), 3.26 (t, J = 6.9 Hz, 2H), 2.36 (t, J = 7.3 Hz, 2H), 1.65 – 1.55 (m, 13H), 1.45 – 1.36
(m, 2H).
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9
Novel Compound Characterization
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tert-butyl 2-amino-4-butyl-1H-imidazole-1-carboxylate (12): The commercially available acid
chloride 11 (17 mmol, 1 eq) was dissolved in DCM (10 mL) and added dropwise to a solution of
CH2N2 (51 mmol, 3 eq) generated with Diazald®/KOH in Et2O (100 mL) and cooled to 0°C. The
reaction mixture was stirred for 1 h at 0°C, whereupon concentrated HBr (4 mL) was added
dropwise. The reaction mixture was stirred an additional 30 min at 0°C, then quenched with sat.
aq. NaHCO3 and the organic material was extracted with Et2O (100 mL × 2). The organic
fractions were combined and washed with brine (20 mL × 2), dried with MgSO4, and then
concentrated under reduced pressure. The residue was purified by flash chromatography (5%
EtOAc/hexanes) to afford its corresponding α-bromoketone, which was then used in the general
procedure for Boc-guanidine cyclization to afford compound 12 as an orange solid in 47% yield.
1H NMR (300 MHz, CD3OD) δ 6.54 (s, 1H), 2.33 (t, J = 7.9 Hz, 2H), 1.60 (s, 9H), 1.53 – 1.49
13
(m, 2H), 1.39 – 1.32 (m, 2H), 0.93 (t, J = 7.3 Hz, 3H) ppm; C-NMR (125 MHz, CD3OD) 
149.3, 138.2, 105.9, 84.6, 30.3, 27.1, 26.7, 22.0, 12.8 ppm; HRMS (ESI) calculated for
C12H21N3O2 [M+H]+ 240.1707, found 240.1700.
4-butyl-1H-imidazol-2-amine (13): Using general procedure for Boc deprotection, compound 13
1
was obtained as a white solid in 100% yield. H NMR (300 MHz, CD3OD) δ 6.48 (s, 1H), 2.49
(t, J = 7.6 Hz, 2H), 1.65 – 1.51 (m, 2H), 1.45 – 1.30 (m, 2H), 0.95 (t, J = 7.3 Hz, 3H) ppm; 13C-
NMR (75 MHz, CD3OD)  147.0, 127.8, 108.1, 30.0, 23.7, 21.7, 12.6 ppm; HRMS (ESI)
calculated for C7H13N3 [M+H]+ 140.1182, found 140.1179.
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4-(5-aminopentyl)-1H-imidazol-2-amine (10): Using general procedure for Boc deprotection was
followed to obtain compound 10 as an orange solid in 94% yield. 1H NMR (600 MHz, CD3OD)
δ 6.46 (s, 1H), 2.87 (t, J = 7.6 Hz, 2H), 2.47 (t, J = 7.6 Hz, 2H), 1.68 – 1.55 (m, 4H), 1.39 (p, J =
13
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7.8 Hz, 2H) ppm; C NMR (150 MHz, CD3OD) δ 148.5, 128.7, 109.8, 40.6, 28.7, 28.2, 26.7,
25.2 ppm; HRMS (ESI) calcd for C8H16N4 [M+H]+ 169.1448, found 169.1446.
5-(3-amino-1-(2-amino-1H-imidazol-4-yl)propyl)-1H-imidazol-2-amine (1): Using general
procedure for oxidative heterodimerization, compound 1 was obtained as an orange solid in 5%
yield. 1H NMR (300 MHz, D2O) δ 6.75 (s, 2H), 4.10 (t, J = 7.7 Hz, 1H), 3.01 (t, J = 8.1 Hz, 2H),
2.27 (q, J = 7.9 Hz, 2H) ppm; 13C NMR (125 MHz, D2O) δ 147.6, 125.0, 110.9, 37.2, 30.2, 28.8
ppm; HRMS (ESI) calcd for C9H15N7 [M+H]+ 222.1462, found 222.1459.
5-(4-amino-1-(2-amino-1H-imidazol-4-yl)butyl)-1H-imidazol-2-amine (2): Using general
procedure for oxidative heterodimerization, compound 2 was obtained as an orange solid in 5%
yield. 1H NMR (400 MHz, D2O) δ 6.39 (s, 2H), 3.57 (t, J = 7.7 Hz, 1H), 2.60 (t, J = 7.2 Hz, 2H),
13
1.79 (q, J = 8.0 Hz, 2H), 1.38 (p, J = 7.4 Hz, 2H) ppm; C NMR (125 MHz, D2O) δ 147.4,
126.0, 110.6, 39.0, 32.1, 28.1, 24.5 ppm; HRMS (ESI) calcd for C10H17N7 [M+H]+ 236.1618,
found 236.1616.
5-(5-amino-1-(2-amino-1H-imidazol-4-yl)pentyl)-1H-imidazol-2-amine (16): Using general
procedure for oxidative heterodimerization, compound 16 was obtained as an orange solid in 5%
yield. 1H NMR (300 MHz, D2O) δ 6.46 (s, 2H), 3.64 (t, J = 7.8 Hz, 1H), 2.92 (t, J = 7.4 Hz, 2H),
13
1.85 (q, J = 7.7 Hz, 2H), 1.70 – 1.56 (m, 2H), 1.39 – 1.24 (m, 2H) ppm; C NMR (175 MHz,
D2O) 147.1, 126.3, 110.2, 39.2, 33.1, 30.4, 26.8, 23.2 ppm; HRMS (ESI) calcd for C11H19N7
[M+H]+ 250.1775, found 250.1774.
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5-(1-(2-amino-1H-imidazol-4-yl)butyl)-1H-imidazol-2-amine (17): Using general procedure for
oxidative heterodimerization, compound 17 was obtained as a brown solid in 7% yield. 1H NMR
(400 MHz, CD3OD) δ 6.70 (s, 2H), 3.91 (t, J = 7.6 Hz, 1H), 1.91 (q, J = 7.7 Hz, 2H), 1.41 – 1.31
13
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(m, 2H), 0.98 (t, J = 7.3 Hz, 3H) ppm; C NMR (125 MHz, CD3OD) δ 149.1, 128.3, 111.4,
35.0, 33.8, 21.3, 13.9 ppm; HRMS (ESI) calcd for C10H16N6 [M+H]+ 221.1509, found 221.1506.
5-(1-(2-amino-1H-imidazol-4-yl)tridecyl)-1H-imidazol-2-amine (18): Using general procedure
1
for oxidative heterodimerization, compound 18 was obtained as a brown solid in 10% yield. H
NMR (400 MHz, CD3OD) δ 6.70 (s, 2H), 3.89 (t, J = 7.7 Hz, 1H), 1.93 (q, J = 7.4 Hz, 2H), 1.34
13
– 1.27 (m, 20H), 0.89 (t, J = 6.9 Hz, 3H) ppm; C NMR (125 MHz, CD3OD) δ 149.0, 128.3,
111.4, 34.1, 33.1, 33.0, 30.8, 30.8, 30.7, 30.7, 30.6, 30.5, 30.3, 28.2, 23.7, 14.5 ppm; HRMS
(ESI) calcd for C19H34N6 [M+H]+ 347.2918, found 347.2915.
5-(1-(2-amino-1H-imidazol-4-yl)pentadecyl)-1H-imidazol-2-amine
(19):
Using
general
procedure for oxidative heterodimerization, compound 19 was obtained as a brown solid in 6%
yield. 1H NMR (700 MHz, CD3OD) δ 6.61 (s, 2H), 3.79 (t, J = 7.7 Hz, 1H), 1.87 – 1.78 (m, 2H),
13
1.53 – 0.93 (m, 24H), 0.82 (t, J = 7.0 Hz, 3H) ppm; C NMR (175 MHz, CD3OD) δ 149.7,
128.9, 111.8, 40.0, 40.0, 34.5, 33.6, 33.4, 31.3, 31.2, 31.2, 31.2, 31.1, 31.0, 30.7, 28.7, 24.2, 14.9
ppm; HRMS (ESI) calcd for C21H38N6 [M+H]+ 375.3231, found 375.3234.
8-ethyl-4-propyl-3H-cyclohepta[1,2-d:4,5-d']diimidazole-2,6-diamine (20): Using general
procedure for oxidative heterodimerization, compound 20 was obtained as a brown solid in 4%
yield. 1H NMR (300 MHz, CD3OD) δ 7.87 (s, 1H), 3.41 – 3.33 (m, 2H), 3.19 (q, J = 7.5 Hz, 2H),
1.82 – 1.69 (m, 2H), 1.38 (t, J = 7.5 Hz, 3H), 1.11 (t, J = 7.3 Hz, 3H) ppm; 13C NMR (175 MHz,
CD3OD) δ 162.8, 161.6, 150.1, 148.3, 147.1, 147.0, 139.5, 130.0, 121.8, 32.6, 29.0, 22.5, 13.9,
13.2 ppm; HRMS (ESI) calcd for C14H18N6 [M+H]+ 271.1666, found 271.1663.
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4-dodecyl-8-undecyl-3H-cyclohepta[1,2-d:4,5-d']diimidazole-2,6-diamine (21): Using general
procedure for oxidative heterodimerization, compound 21 was obtained as a brown solid in 4%
yield. 1H NMR (300 MHz, CD3OD) δ 7.84 (s, 1H), 3.38 (t, J = 8.2 Hz, 2H), 3.13 (t, J = 7.8 Hz,
13
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2H), 1.77 – 1.66 (m, 4H), 1.26 – 1.16 (m, 34H), 0.87 (t, J = 5.9 Hz, 6H) ppm; C NMR (175
MHz, CD3OD) 180.2, 173.3, 171.2, 162.2, 160.9, 149.2, 141.1, 137.5, 130.6, 125.7, 122.5, 75.2,
50.8, 48.1, 35.9, 31.8, 31.7, 30.9, 30.2, 29.8, 29.6, 29.5, 29.4, 29.4, 29.4, 29.3, 29.3, 29.3, 29.2,
29.1, 22.3, 13.0 ppm; HRMS (ESI) calcd for C32H54N6 [M+H]+ 523.4483, found 523.4486.
Bacterial Strains and Antimicrobial Agents. The bacterial strains used in this study included
seven Pseudomonas aeruginosa and one Klebsiella pneumoniae. Cystic fibrosis clinical isolates
(three non-mucoid strains CEC 45, 57, 102 and three mucoid strains CEC 70, 103, 111) were
obtained from Robert K. Ernst, in the Department of Microbial Pathogenesis at University of
Maryland-Baltimore. All strains were maintained and subcultured in cation-adjusted Mueller-
Hinton broth (CAMHB) in liquid culture or on lysogeny broth (LB) plates until utilized in
various assays outlined below. Antibiotics were purchased from Sigma Aldrich or TCI America.
All assays were run in duplicate and repeated at least two separate times. All compounds were
dissolved as their HCl salts in molecular biology grade DMSO as 10 or 100 mM stock solutions
and stored at - 20 °C.
Bacterial Membrane Permeabilization Assay. The BacLight assay (Invitrogen) was used to
assess membrane permeability. PAO1 was grown overnight in CAMHB at 37 °C with shaking.
The culture was diluted 1:40 in CAMHB and grown to an optical density at 600 nm (OD600) of
~1.0 (~4 h of growth). The cultures were centrifuged at 10,000 g for 15 min, the cell pellet was
washed once with sterile water, resuspended at 2 times the original volume, and aliquoted, and
adjuvants were added. Suspensions were incubated at 37 °C with shaking for 1 h and then
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centrifuged at 10000g for 10 min, washed once with sterile water, and resuspended in water. A
1:1 mixture of SYTO-9 and propidium iodide was added to the suspension (3 μL/mL) and mixed
well. One hundred microliters of the suspension was added to each well of a 96-well plate, and
the plates were incubated in the dark for 15 min at room temperature. Green fluorescence
(SYTO-9) was read at 530 nm, and red fluorescence (propidium iodide) was read at 645 nm
(excitation wavelength, 485 nm). The ratio of green to red fluorescence was expressed as a
percentage of the control.
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Bacterial Membrane Permeabilization Assay with Divalent Cation Metals. The procedure
for ‘bacterial membrane permeabilization assay’ was followed, with the addition of spiking 200
µM FeSO4·7H2O or MnCl2 into a suspension of bacterial solution containing test compound
before incubating at 37 °C with shaking for 1 h.
Broth microdilution method for determination of minimum inhibitory concentrations. Day
cultures (6 h) of each bacterial strain in cation adjusted Mueller Hinton II broth (CAMHB, Fisher
Scientific U.S.) were subcultured to 5×105 CFU/mL in CAMHB. Aliquots (1 mL) were placed
in culture tubes and compound was added from 100 or 10 mM stock samples in DMSO, such
that compound concentration equaled highest concentration tested (200 μM). Samples were then
aliquoted (200 μL) into the first wells of a 96- well plate, with all remaining wells being filled
with 100 μL of initial bacterial subculture. Row one wells were mixed five times, before 100 μL
was transferred to row two. Row two was then mixed five times, and 100 μL was transferred to
row three. This process was repeated until the final row had been mixed, this served to serially
dilute the compound. Plates were then covered with GLAD Press n’ Seal and incubated under
stationary conditions at 37 °C for 16 h. MIC values were then recorded as the lowest
concentration at which no bacterial growth was observed.
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Broth microdilution method for antibiotic potentiation. Day cultures (6 h) of bacteria in
CAMHB were subcultured to 5x105 CFU/mL in CAMHB. Aliquots (4 mL) were placed in
culture tubes, and dosed with compound from 100 or 10 mM stock samples to give the desired
concentration of the compound to be tested against the particular bacterial strain; this insured
non-toxic DMSO concentrations ≤0.3% in each well. 1 mL of the resulting solution was placed
in a separate culture, and dosed with antibiotic at the highest concentration to be tested. Bacteria
treated with antibiotic alone served as the control. Row one of a 96 well plate was filled with 200
μL of the antibiotic/2-AI solution, and rows 2-12 were filled with 100 μL each of the remaining
4 mL of bacterial subculture containing adjuvant at the desired concentration except for the
control lane which contained only bacterial subculture. Row one was then mixed five times, and
100 μL was transferred to row two, which was then mixed five times before being transferred to
row three. This process was repeated until all rows had been mixed, except for row twelve,
which would have only compound, to serve as a control. The 96-well plate was then covered in
Glad Press n’ Seal and incubated under stationary conditions at 37 °C for 16 h. MIC values were
determined as the lowest concentration at which no bacterial growth was observed, fold
reductions were determined by comparison to control lane.
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Broth microdilution method for antibiotic potentiation with detergent. Day cultures (6 h) of
bacteria in CAMHB were subcultured to 5x105 CFU/mL in CAMHB and then TweenTM 80
Surfact-AmpsTM Detergent Solution (Fisher Scientific, U.S.) was added to make a 0.01%
containing bacterial solution. Then procedure for ‘broth microdilution method for antibiotic
potentiation’ was followed starting with making 4 mL aliquots from TweenTM 80 bacterial
solution.
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Dose-response of Adjuvant Assay. The procedure for ‘broth microdilution method for
antibiotic potentiation’ was followed using 4 mL aliquots of adjuvant at various concentrations:
0, 15, 30, 35, 40, 50, and 60 µM.
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EDTA Assay. The procedure for ‘broth microdilution method for antibiotic potentiation’ was
followed, with the addition of doping the 4 mL aliquots containing adjuvant with either 25, 50,
100, or 200 µM of a prepared EDTA stock. Bacterial solutions lacking adjuvant were used as
controls to examine effect of 200 µM EDTA on P. aeruginosa growth. Row 1 of a 96-well plate
was filled with 200 μL of the antibiotic/adjuvant/EDTA solution, and rows 2−8 were filled with
100 μL each of the remaining bacterial subculture dosed with adjuvant/EDTA. Serial dilution of
rows followed as previously stated. Plates were covered with GLAD Press n’ Seal and incubated
under stationary conditions at 37 °C. After 16 h, MIC values were determined as the lowest
concentration at which no bacterial growth was observed, fold reductions were determined by
comparison to control lanes.
Galleria mellonella Assay. G. mellonella larvae (Speedy Worm, Alexandria, MN) were used
within 10 days of shipment from the vendor. After reception of worms, larvae were kept in the
dark at room temperature for at least 24 h before infection. Larvae weighing between 200 to 300
mg were used in the survival assay. Using a 10 µL glass syringe (Hamilton, Reno, NV) fitted
with a 30 G needle (Exel International, St. Petersburg, Fl), a 5 µL solution of the desired
compound(s) and concentration(s) were injected into the last left proleg. Azithromycin was
dosed at 1 mg/kg dissolved in DI water, 2-AIs at 50 mg/kg dissolved in DMSO, meropenem at
30 mg/kg dissolved in DMSO, and DMSO was injected for “bacteria only” as a control. For
bacterial injections, 50 µL from an overnight culture of P. aeruginosa (PAO1) in Miller LB
broth (Fisher Scientific U.S.) was subcultured into 5 mL Miller LB broth and incubated for an
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additional 3 h before use. Then, 2.5 h after the first injection, a second 5 µL injection containing
105 CFU of PAO1 in phosphate buffer solution (Fisher Scientific U.S.) was injected into the
second to last left proleg. Injected worms were left at room temperature in the dark while being
assessed at 24 h intervals over 6 days. Larvae were considered dead if they did not respond to
physical stimuli. Experiment was repeated 3 times using 10 larvae per experimental group. No
ethics approval was needed for G. mellonella.
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Hemolysis Assay. Hemolysis assays were performed on mechanically difibrinated sheep blood
(Hemostat Laboratories: DSB100). Difibrinated blood (1.5 mL) was placed into a
microcentrifuge tube and centrifuged for 10 min at 10,000 rpm. The supernatant was then
removed, and then the cells were resuspended in 1 mL of phosphate-buffered saline (PBS). The
suspension was centrifuged, the supernatant was removed, and cells were resuspended two
additional times. The final cell suspension was then diluted 10-fold. Test compound was added
to aliquots of the 10-fold suspension dilution of blood. PBS was used as a negative control and a
zero hemolysis marker. Triton × (a 1% sample) was used as a positive control serving as the
100% lysis marker. Samples were then placed in an incubator at 37 °C while being shaken at 200
rpm for 1 h. After 1 h, the samples were centrifuged for 10 min at 10,000 rpm. The resulting
supernatant was diluted by a factor of 40 in distilled water. The absorbance of the supernatant
was then measured with a UV spectrometer at a 540 nm wavelength.
Minimal Salts Assay. The procedure for ‘broth microdilution method for antibiotic potentiation’
was followed, with the exception of culturing P. aeruginosa in LB broth for 6 h, then
subculturing in minimal salts media (M9) instead of CAMHB.
Procedure to Determine the Mitigating Effects of Divalent Cation Metals on P. aeruginosa.
The procedure for ‘broth microdilution method for antibiotic potentiation’ was followed, with
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the addition of doping the 4 mL aliquots containing adjuvant with either 25, 50, 100, or 200 µM
of divalent cation metal (FeSO4·7H2O, ZnCl2, CaCl2, CuCl2, MgCl2, or MnCl2). Controls in
which no adjuvant was added were used to examine effects of 200 µM metal on P. aeruginosa
growth.
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Abbreviations
2-ABI, 2-aminobenzimidazole; 2-AI, 2-aminoimidazole; azm, azithromycin; CF, cystic fibrosis;
CFTR, cystic fibrosis transmembrane conductance regulator; CLSI, Clinical and Laboratory
Standards Institute; MDR, multi-drug resistance; MIC, minimum inhibitory concentration; PI,
propidium iodide; r.t., room temperature; SAR, structure activity relationship.
Acknowledgments
The authors would like to thank the National Institutes of Health (GM055769 and AI136904) for
support, the Ernst lab (University of Maryland, Baltimore) for providing CF isolates, and the
Manoil lab for providing PAO1.
Supporting Information
Representative list of compounds screened; azithromycin potentiation data against PAO1; PAO1
membrane permeabilization data using BacLight assay; mitigating effects of EDTA with
compound 2; azithromycin MIC potentiation of compound 2 in minimal salts media; PAO1
membrane permeabilization data with divalent cation metals; azithromycin potentiation data
against KP2146; KP2146 membrane permeabilization data using BacLight assay; detergent
Tween 80 results; hemolysis results; Galleria mellonella results; references for initial compound
screen; representative 1H and 13C Nuclear Magnetic Resonance spectra
References
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