Augmenting the Activity of Macrolide Adjuvants against Acinetobacter baumannii

Article abstract of DOI:10.1021/acsmedchemlett.0c00276

Approximately 1.7 million Americans develop hospital associated infections each year, resulting in more than 98,000 deaths. One of the main contributors to such infections is the Gram-negative pathogen Acinetobacter baumannii. Recently, it was reported that aryl 2-aminoimidazole (2-AI) compounds potentiate macrolide antibiotics against a highly virulent strain of A. baumannii, AB5075. The two lead compounds in that report increased clarithromycin (CLR) potency against AB5075 by 16-fold, lowering the minimum inhibitory concentration (MIC) from 32 to 2 μg/mL at a concentration of 10 μM. Herein, we report a structure-activity relationship study of a panel of derivatives structurally inspired by the previously reported aryl 2-AI leads. Substitutions around the core phenyl ring yielded a lead that potentiates clarithromycin by 64- and 32-fold against AB5075 at 10 and 7.5 μM, exceeding the dose response of the original lead. Additional probing of the amide linker led to the discovery of two urea containing adjuvants that suppressed clarithromycin resistance in AB5075 by 64- and 128-fold at 7.5 μM. Finally, the originally reported adjuvant was tested for its ability to suppress the evolution of resistance to clarithromycin over the course of nine consecutive days. At 30 μM, the parent compound reduced the CLR MIC from 512 to 2 μg/mL, demonstrating that the original lead remained active against a more CLR resistant strain of AB5075.

Full text of DOI:10.1021/acsmedchemlett.0c00276

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Letter
Augmenting the Activity of Macrolide
Adjuvants Against Acinetobacter baumannii
Veronica B. Hubble, Kyle R. Bartholomew, Alexander W. Weig, Sara L. Brackett, Samantha
Barlock, Anne E. Mattingly, Ansley M. Nemeth, Roberta J. Melander, and Christian Melander
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.0c00276 • Publication Date (Web): 12 Aug 2020
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Augmenting the Activity of Macrolide Adjuvants Against Acinetobacter baumannii
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Authors: Veronica B. Hubble†; Kyle R. Bartholomew‡; Alexander W. Weig†; Sara M.
Brackett†; Samantha L. Barlock†; Anne E. Mattingly†; Ansley M. Nemeth†; Roberta J.
Melander†; and Christian Melander*†
†Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana
46556, United States
‡Department of Chemistry, North Carolina State University, Raleigh, NC, 27695, United States
*Correspondence: Christian Melander, cmelande@nd.edu
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Abstract
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Approximately 1.7 million Americans develop hospital associated
infections each year, resulting in more than 98,000 deaths. One of
the main contributors to such infections is the gram-negative
pathogen Acinetobacter baumannii. Recently, it was reported that
aryl 2-aminoimidazole (2-AI) compounds potentiate macrolide
antibiotics against a highly virulent strain of A. baumannii,
AB5075. The two lead compounds in that report increased
clarithromycin potency against AB5075 by 16-fold, lowering the
minimum inhibitory concentration (MIC) from 32 to 2 µg/mL at a
concentration of 10 µM. Herein, we report a structure-activity
relationship study of a panel of derivatives structurally inspired
by the previously reported aryl 2-AI leads. Substitutions around
the core phenyl ring yielded a lead that potentiates clarithromycin
by 64- and 32-fold against AB5075 at 10 and 7.5 µM, exceeding the
dose response of the original lead. Additional probing of the amide
linker led to the discovery of two urea containing adjuvants that
suppressed clarithromycin resistance in AB5075 by 64- and 128-fold
at 7.5 µM. Finally, the originally reported adjuvant was tested
for its ability to suppress the evolution of resistance to
clarithromycin over the course of nine consecutive days. At 30 µM,
the parent compound reduced the CLR MIC from 512 to 2 µg/mL,
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demonstrating that the original lead was remained active against
a more CLR resistant strain of AB5075.
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Keywords
Acinetobacter baumannii, adjuvant, antibiotic resistance,
macrolide potentiation
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The prevalence of infections that stem from multi-drug
resistant (MDR) bacteria has significantly increased in recent
years, which, in addition to a reduction in the involvement of
pharmaceutical companies in antibiotic development, has severely
limited currently available treatment options.1 Resistance toward
every class of clinically prescribed antibiotics has now been
observed.2 The Centers for Disease Control and Prevention (CDC)
estimates that over 2.8 million people are infected with
antibiotic-resistant bacteria each year, and approximately 35,000
deaths result from these bacterial infections.3 A high number of
these deaths are associated with infections caused by the ESKAPE
pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella
pneumoniae, Acinetobactor baumannii, Pseudomonas aeruginosa,
Enterobacter species), where the ‘ES’ represents gram-positive
species and the ‘KAPE’ pathogens represents gram-negative species.4
Gram-negative pathogens are of particular concern because their
outer membrane (OM) is impermeable to many antibiotics, further
limiting the available antibiotics for treating MDR gram-negative
infections.2
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Despite the target being present in both gram-positive and gram-
negative bacteria, macrolides are generally utilized for the
treatment of gram-positive infections due to low penetration of
the OM. It has been shown, however that if the OM is compromised
then macrolides can become efficacious against gram-negative
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bacteria. For example, A. baumannii strains that lack
lipopolysaccharide (LPS) are highly permeable and are susceptible
to treatment with the macrolide antibiotic azithromycin.5 Compounds
that are able to physically disrupt the outer membrane, such as
pentamidine and polymyxin derivatives, are also able to sensitize
gram-negative bacteria to macrolides.6
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Recently, we reported two small molecules that potentiate the
activity of clarithromycin (CLR) (compounds 1 and 2, Figure 1)
against a strain of A. baumannii (AB5075).7 These compounds, which
we and others refer to as antibiotic adjuvants, are relatively
non-toxic to AB5075 by themselves (MICs of 100 µM); however at 30
M, each compound reduced the minimum inhibitory concentration
(MIC) of CLR by 128-fold, from 32 g/mL to 0.25 g/mL, while at 10
M they reduced the CLR MIC 16-fold to 2 g/mL. Investigations
into the mechanism of action of these molecules revealed that,
unlike other small molecules that compromise the outer membrane
through physical disruption, these compounds antagonized colistin
and altered Lipid A composition in AB5075. This suggests that
alterations in LPS presentation and/or biosynthesis may underpin
these adjuvants mode of action (MoA).7 While research is ongoing
to determine the target for these molecules, we herein report a
structure-activity relationship (SAR) analysis encompassing
structural modifications of the reported lead compounds 1 and 2
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(Figure 1) to further optimize activity in combination with
macrolide antibiotics, specifically CLR which displays the
greatest enhancement in activity of the three macrolide
antibiotics studied (erythromycin, azithromycin, CLR) against
AB5075.7
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To perform analog synthesis, we divided the lead molecules into
three regions: region 1, the aryl tail (boxed green), region 2,
the phenyl core (highlighted purple), and region 3, the amide
linker (highlighted in blue) (Figure 1). First, we elected to
modify the aryl tail of compound 1 in an attempt to delineate the
importance of electron-withdrawing and/or inductive effects. Next,
we introduced various functional groups on the phenyl core while
maintaining the 3.5-dichloro substituent in the aryl tail. After
identifying structural components in each region that augmented
activity, we combined the most effective phenyl core modification
with aryl tails that had shown augmented activity with CLR.
Finally, a brief SAR was performed modifying the original amide
linker.
Figure 1. Initial lead aryl 2-aminoimidazole adjuvants 1 and 2 and
delineation of scaffold regions for SAR examination.7
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Results and Discussion
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We initiated our study by screening additional analogs of 1 and
2 for CLR potentiation against AB5075 (Figure 1). Seventeen of the
25 aryl 2-AI analogs had been previously synthesized and screened
for potentiation of β-lactam antibiotics against gram-negative
bacteria.8 The other analogs were accessed through intermediate 3
by acylation with the appropriate acid chloride, TFA-mediated
removal of the Boc-groups, and finally counter ion exchange. Once
the pilot library was completed, each compound was first analyzed
individually for standalone toxicity against AB5075 by determining
the MIC (Table S1). Of the 25 analogs, one registered an MIC of 25
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M (4n), two returned an MIC of 50 M (4l, 4p), four displayed an
MIC of 100 M (4o, 4r, 4s, and 4v), while the MIC of the rest of
the analogs were ≥ 200 M. To test for potentiation, the MIC of
CLR against AB5075 was determined in the absence or presence of
each compound dosed at 30% their MIC or 30 M, whichever was lower
(Table S1). Each analog was compared to the activity of compounds
1 and 2 at 30 M.
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Scheme 1. Aryl tail modifications of previously reported adjuvants
1 and 2. Reagents and conditions: a) ArCOCl, K3PO4, THF, 25 ºC, 16
h; b) TFA, DCM, 25 ºC, 3 h; c) 6 M HCl, MeOH, 25 ºC, 5 min.8
First, we noted that removal of the halides (4a) abolished
activity (Table S1). We also observed that di-substitution
patterns on the aryl tail were typically more active and achieved
greater CLR potentiation when compared to mono-substituted
compounds. The only mono-substituted compounds that displayed CLR
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potentiation at 30 µM were mono-chlorinated compounds (4d, 4i) and
one mono-brominated compound (4h).
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Compounds 4k-m, which incorporated a 3,5-disubstiution pattern,
allowed a comparison of the effects of replacing Cl/Br with F,
Cl/Br with CF3, and Cl for CH3 (steric isostere). Compound 4k only
exhibited an eight-fold reduction in the MIC of CLR at 30 µM, while
compounds 4l and 4m, also exhibited relatively modest activity,
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returning CLR MICs of 8 and 2 g/mL respectively at 15 µM and 30
µM.
Since chlorination appeared to be a promising halogenation
choice for adjuvant activity, the positioning of the dichloro
substitution pattern was evaluated next. Dichlorination at the
2,3-positions, seen in compound 4q, resulted in only four-fold
potentiation of CLR at 30 µM, while compound 4r in which the chloro
substituents were moved to the 3,4-position displayed a 64-fold
reduction in the MIC of CLR at 30 µM. Compound 4t, which placed
the chloro residues at the 2,4-position, exhibited only an eight-
fold reduction in the MIC of CLR at 30 µM. Analysis of structure-
function of tri- and multi-halogenated patterns on the aryl tail
showed that 4r, 4s, and 4y displayed comparative CLR potentiation
to the original adjuvants 1 and 2 when dosed at 30 µM.
Next, a dose response study was conducted with nine of the most
active compounds (4d, 4i, 4n, 4o, 4p, 4r, 4s, 4u, 4v, and 4y) to
determine CLR potentiation as a function of compound concentration
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(Table 1). At 10 M, 4o, 4p, and 4r showed comparable activity to
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the original leads 1 and 2, returning CLR MICs of 2, 1, and 4 g/mL
respectively. Compound 4y, however, was significantly more active
maintaining a CLR MIC of 0.25 g/mL (128-fold reduction) at 10 M.
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Table 1. Dose-response of active aryl tails for CLR potentiation
against AB5075.
CLR
CLR
MIC
(µg/mL
)
Concentratio
n Tested
(µM)
Concentratio
n Tested
(µM)
Compoun
d
MIC Compoun
(µg/mL
d
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To probe the SAR of region 2, the original 3,5-dichloro aryl
tail was held constant while various functional groups were
introduced on the phenyl core. The synthetic approach to these
analogs is outlined in Scheme 2. Anilines 7a-e were alloc-protected
using allyl chloroformate in a biphasic solvent system under basic
conditions at 0 ºC to afford compounds 8a-e. Compounds 8a-e and
5a-b were then converted into their corresponding acid chlorides
using oxalyl chloride and a catalytic amount of DMF at 0 ºC. Each
acid chloride was reacted with diazomethane at 0 ºC for two hours
followed by addition of hydrobromic acid to generate the
corresponding ⍺-bromo-ketone. The α-bromo-ketones were then
cyclized with Boc-guanidine to deliver the corresponding mono-Boc-
protected 2-AI compounds 9a-e. The alloc protecting groups of 9a-e
were
then
removed
by
treatment
with
tetrakis(triphenylphospine)palladium(0) and sodium borohydride.
Each aniline was then immediately acylated using commercially
available 3,5-dichlorobenzyl chloride under basic conditions,
that, following Boc-deprotections using trifluoroacetic acid and
subsequent conversion to the hydrochloric acid salt, afforded
compounds 10a-e. Intermediates that contained a nitro group as the
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latent aniline were reduced using palladium on carbon and hydrogen
gas. Acylation, Boc-deprotection and ion exchange as above yielded
compounds 6a-b.
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A
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B
C
Scheme 2. Phenyl core modification of compounds 1 and 2. Reagents
and conditions for 2A: a) (COCl)2, DMFcat, DCM, 0 ºC, 2 h; b) CH2N2,
Et2O, DCM, 0 ºC, 1.5 h; c) HBr, 0 ºC, 30 min; d)Boc-guanidine, THF,
56 ºC, 3 h; e) H2, 10% Pd/C, MeOH, 25 ºC, 16 h; f) 3,5-
dichlorobenzoyl chloride, K3PO4, THF, 0 ºC, 16 h; g) TFA, DCM, 25
ºC, 3 h; h) 6 M HCl, MeOH, 25 ºC, 5 min. Reagents and conditions
for 2B: a) allyl chloroformate, NaHCO3, DIPEA, 1,4-dioxane/H2O, 0
ºC, 16 h; b) (COCl)2, DMFcat, DCM, 0 ºC, 2 h; c) CH2N2, Et2O, DCM,
0 ºC, 1.5 h; d) HBr, 0 ºC, 30 min; e) Boc-guanidine, THF, 56 ºC,
3 h; f) Pd(PPh3)4, NaBH4, EtOH, 25 ºC, 4 h; g) 3,5-dichlorobenzoyl
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chloride, K3PO4, THF, 0 ºC, 16 h; h) TFA, DCM, 25 ºC, 3 h; i) 6 M
HCl, MeOH, 25 ºC, 5 min. Difluoro modification on phenyl core of
lead adjuvant 10d. Reagents and conditions for 2C: a) 3,5-
dichlorobenzoyl chloride, K3PO4, THF, 0 ºC, 16 h; b) 5M NaOH,
MeOH/H2O, 25 ºC, 3 h; c) (COCl)2, DMFcat, DCM, 0 ºC, 2 h; d) CH2N2,
Et2O, DCM, 0 ºC, 1.5 h; d) HBr, 0 ºC, 30 min; e) Boc-guanidine,
THF, 56 ºC, 3 h; f) TFA, DCM, 25 ºC, 3 h; i) 6 M HCl, MeOH, 25 ºC,
5 min.
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As detailed above, all compounds were first screened against
AB5075 alone to determine their MICs followed by an initial screen
of each compound at ≤ 30% their MIC in combination with CLR (Table
2). Except for 6a, which returned a standalone MIC of 100 M, all
derivatives were essentially non-toxic showing MIC values >200 M.
When the carbon ortho to the amide linker (R1, Scheme 2) contained
either a methyl or methoxy substituent, similar levels of CLR
potentiation was noted in comparison to compounds 1 and 2. Both
methyl and methoxy compounds, 10a and 6a, displayed a 64-fold
reduction in CLR MIC at 30 µM compared to parent 128-fold activity
at 30 µM. When either Cl or F is placed at this position, activity
decreased significantly, with only a two- and four-fold reduction
in CLR MIC observed for compounds 10b and 10c respectively at 30
µM. We noted a substantially different trend when substituents
were placed meta to the amide linker (R2, Scheme 2). Derivatives
containing a chloro (10e), fluoro (10d), or methyl (6b) potentiated
CLR activity by a similar degree to compound 1, lowering the MIC
of CLR by 64-fold, from 32 to 0.5 µg/mL, when dosed at 30 µM.
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The five analogs (6a, 6b, 10a, 10d, and 10e) showing comparable
activity to 1 were then subjected to a dose response study (Table
2). Compounds 6a, 6b, 10a, and 10e were not as active as 1 at 10
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M, effecting an MIC reduction of two-, four-, two-, and four-fold
respectively. The fluoro analog 10d, however, surpassed the
activity of 1 and displayed a 64-fold reduction of the CLR MIC at
10 µM.7
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Table 2. CLR potentiation data against AB5075 using phenyl core
derivatives 6a-b and 10a-e.
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Compound Concentration CLR MIC Fold
MIC (µM) Tested (µM) (µg/mL) Reduction
Compound R1
R2
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OCH3
H
CH3
H
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H
>200
>200
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CH3
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Cl
F
H
H
>200
>200
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4
0.25
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4
128
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10d
10e
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H
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>200
>200
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Given the activity of the mono-fluoro compound (10d), the
difluoro analogue 13, was synthesized to probe if either symmetry
or additional electronic effects would further modulate activity
(Scheme 2C). Interestingly, compound 13 was essentially inactive
at 10 µM, displaying only a two-fold reduction in the MIC of CLR.
At 20 and 30 µM, compound 13 lowered the MIC of CLR to 8 and 4
µg/mL, respectively.
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As the mono-fluoro analog 10d was significantly less toxic to
AB5075 in comparison to 1 and 2, we probed whether this type of
modification could attenuate the standalone toxicity of some of
the initial tail derivatives we studied.7 To explore this, we chose
some of the aryl tails that exhibited the ability to potentiate
CLR yet were also more toxic, registering standalone MIC values
(i.e. no antibiotic added) of ≤ 100 µM. We postulated that the
addition of a fluoro substituent would reduce the overall toxicity
of these adjuvants while either retaining or improving activity
with CLR against AB5075.
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B
Scheme 3. Evaluation of active aryl tails in combination with
fluorinated phenyl core. Reagents and conditions: Scheme A
protocol: a) ArCOCl, K3PO4, THF, 25 ºC, 16 h; b) TFA, DCM, 25 ºC,
3 h; c) 6 M HCl, MeOH, 25 ºC, 5 min. Scheme B protocol: a) ArCOCl,
K3PO4, THF, 25 ºC, 16 h; b) 5 M NaOH, MeOH/H2O, 25 ºC, 3 h; c)
(COCl)2, DMFcat, DCM, 0 ºC, 2 h; d) CH2N2, Et2O, DCM, 0 ºC, 1.5 h;
d) HBr, 0 ºC, 30 min; e) Boc-guanidine, THF, 56 ºC, 3 h; f) TFA,
DCM, 25 ºC, 3 h; i) 6 M HCl, MeOH, 25 ºC, 5 min.
The aryl tail from compound 2 in Figure 1 was chosen first for
further analog synthesis, and was accessed by coupling 3,5-
dibromobenzoyl chloride to mono-Boc-protected 2-AI aniline 14.
After TFA-mediated Boc-deprotection followed by counter ion
exchange, the fluorinated analog of 2 (15a) was screened for
standalone toxicity and CLR potentiation (Scheme 3). Fluorination
did indeed attenuate toxicity as 15a returned an MIC of >200 M
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(compound 2 has a standalone MIC of 100 M); however, it also
compromised adjuvant activity. At 30 µM, compound 15a only reduced
the CLR MIC 16-fold against AB5075 (Table 3), while compound 2
effected a 128-fold reduction at the same concentration.
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The 3,4-dichlorobenzoyl tail 15b showed both a reduction in the
standalone toxicity and improvement in CLR potentiation over the
original motif 4r. Compound 15b afforded a standalone MIC of >200
µM (vs. 100 M for 4r) and delivered a 32-fold reduction in CLR at
a concentration of 7.5 M, exceeding the activity of the parent
compound 4r and as well as 10d. When the activity of 17a and 17b
were directly compared to their non-fluorinated variants 4n and
4o, we noted that standalone toxicity was essentially unchanged
(two-fold changes are not considered to be significant in a
standard MIC assay). The ability of 17a to potentiate CLR activity
at 7.5 µM was severely compromised in comparison to compound 4n,
showing an insignificant two-fold reduction in the MIC of CLR
compared to a 16-fold reduction at 7.5 µM for 4n. The adjuvant
activity of compound 17b was identical to compound 4o (Table S1,
1, and 3).
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Table 3. CLR potentiation data for compounds 15a-b, 17a-b, and
20a-c against AB5075.
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Compound Concentration CLR MIC Fold
Compound
MIC (µM) Tested (µM) (µg/mL) Reduction
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≤0.0625
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4
In a previous report from our lab9, we described related aryl
2-AI analogs that inhibit E. coli biofilm formation. Within that
report, compound 4k was determined to be the lead biofilm
inhibitor, indicating that biofilm inhibition activity was greater
when the amide linker of the 3,5-difluoro aryl tail was para in
relation to the 2-AI head group opposed to meta (18) or ortho (19)
(Figure 2).9 We screened these same compounds for CLR potentiation
against AB5075 and observed a similar trend, with the para amide
linker 4k (highest adjuvant activity) > meta amide linker 18 >
ortho amide linker 19 (lowest adjuvant activity), when dosed at 30
µM (Figure 2). Based on this we maintained the para position and
explored diversifying the linker itself instead. We focused on
incorporating a urea due to past reports showing that urea linkers
are active in the context of antibiotic potentiation against gram-
negative species, including A. baumannii.10-11
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Figure 2. Previously reported 3,5-difluoro aryl 2-AI analogs with
varying amide linker connectivity to the phenyl core.9
Three of the most active aryl tails from the initial screenings,
the aryl tails seen in compounds 1, 2, and 4r, were utilized for
the synthesis of para urea derivatives (Scheme 4). Compounds 20a-c
were synthesized by converting the 2-AI aniline intermediate 3
into its corresponding isocyanate using sodium carbonate and
triphosgene in a biphasic solvent system at room temperature. Each
isocyanate was then reacted with the appropriate halogenated
aniline derivative to deliver the tri-Boc protected 2-AI
intermediates. Finally, each urea derivative was exposed to
trifluoroacetic acid to remove the Boc protecting groups and
converted to the hydrochloric acid salt for biological testing
(Scheme 4).
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Scheme 4. Linker modification of active CLR adjuvants 1, 2, and
4r. Reagents and conditions: a) Na2CO3, H2O, DCM, then triphosgene,
25 ºC, 2 h; c) ArNH2, DCM, 25 ºC, 1 h; d) TFA, DCM, 25 ºC, 3 h; e)
6 M HCl, MeOH, 25 ºC, 5 min.
Modification of the amide linker to a urea led to an increase
in adjuvant activity, yet at the cost of increasing their
standalone toxicity toward AB5075 (Table 3). All three urea analogs
20a-c potentiate CLR ≥ 16-fold at concentrations as low as 7.5 µM,
with 20a showing the greatest CLR potentiation of 128-fold and
becoming our lead urea derivative (Table 3). However, compounds
20a and 20b displayed higher toxicity, returning MIC values of 50
and 25 µM, respectively. The least toxic urea was compound 20c,
which displayed a standalone MIC of 100 µM. Compound 20c had
superior adjuvant activity at 30 µM of all of the screened
compounds, including the amide linkers, lowering the MIC of CLR to
≤ 0.0625 µg/mL (≥ 512-fold, lowest concentration tested).
Interestingly, when compound 20c was dosed at 7.5 µM, its adjuvant
activity was comparable to the most active amide linker 15b
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activity, lowering the MIC of CLR by 16-fold from 32 to 2 µg/mL
(Table 3).
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In our initial report describing the macrolide potentiation
activity of adjuvants 1 and 2, we examined their activity against
a panel of A. baumannii isolates that encompasses all major and
most minor clinically relevant clades.7, 12-13 Both adjuvants
suppressed macrolide resistance in 23 different AB strains at 30
µM, lowering CLR MIC values to ≤ 0.125 to 1 µg/mL.7 Since the amide
linker compound 15b and the urea analog 20a displayed greater CLR
potentiation than compounds 1 and 2 in AB5075, we chose to screen
the same 23 isolates at lower concentrations with compounds 15b
and 20a (Table S3). To begin, compounds 15b and 20a were screened
individually to determine their standalone MIC values to ensure
that toxicity is avoided during antibiotic potentiation screening
(Table S2). Compound 15b returned MIC values of 50 µM in three of
the AB isolates, though it was relatively non-toxic (≥100 µM) in
the remaining 20 strains; therefore, compound 15b was tested at 10
µM when combined with CLR against all 23 strains. Compound 20a
displayed MIC values ranging from 25 to 100 µM, thus was tested at
7.5 µM for all of the 23 AB isolates to avoid potential toxic
effects. Compound 15b at 10 µM lowered the MIC of CLR in all 23 AB
isolates, with reductions ranging from eight- to 32-fold (Table
S3). Compound 20a at 7.5 µM exhibited even greater CLR
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potentiation, lowering the MIC of CLR between 16- and 128-fold in
all 23 AB isolates (Table S3).
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We previously reported that compounds 1 and 2 antagonize the
activity of the polymyxin colistin against AB5075, effecting an
increase in colistin MIC from 1 to 4 µg/mL at 30 µM.7 The mechanism
of action of colistin is well-documented, and it is known that
binding to LPS plays a significant role in polymyxin activity.5 In
addition, a genome sequencing study of some colistin resistant
strains of A. baumannii, revealed that loss of LPS imparts
increased colistin resistance.5 It was posited that adjuvants 1
and 2 could be interfering with LPS production or assembly and
thus affecting the ability of colistin to bind to its target.
Therefore, we wanted to evaluate the effect of these second-
generation adjuvants at their active concentrations on colistin
activity against AB5075 (Table S4). Some compounds that display
moderate CLR potentiation at 30 µM, such as compounds 4h, 4m, or
4u, did not potentiate nor antagonize colistin activity when dosed
at 30 µM (Table S4). However, the most active compounds from the
CLR potentiation screenings did increase the MIC of colistin by
two- to eight-fold when dosed at their active concentrations
against AB5075 (Table S4), as seen with adjuvants 1 and 2.
Finally, parent adjuvant 1 was evaluated for its ability to
suppress the evolution of CLR resistance in AB5075 in comparison
to that occurring upon exposure to CLR alone over the course of
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nine consecutive days. Evaluation of resistance evolution was
conducted by serially passaging AB5075 in the presence of CLR with
and without adjuvant 1 and determining CLR MICs of the resultant
bacterial populations. When exposed to CLR alone, after day 2 of
the evolution assay, the MIC of CLR increased from 32 µg/mL to ≥
128 µg/mL in AB5075 (Table S5). By day 6 of serial exposure, the
MIC of CLR had risen to 512 µg/mL, and the MIC of CLR remained
approximately 512 µg/mL throughout the remaining three days of the
assay (Table S5). We then tested whether adjuvant 1 could reduce
the MIC of this mutant. At 30 µM, compound 1 reduced the CLR MIC
from 512 to 2 µg/mL, demonstrating that the adjuvant was still
active against a more CLR resistant strain.
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The evolution assay was next conducted via serial passaging of
AB5075 in the presence of CLR and 30 µM of adjuvant 1. We observed
that adjuvant 1 suppressed the increase in macrolide resistance in
AB5075 (Table S6) throughout the entire nine-day course. On day 2
and day 9 of passaging, the CLR MIC remained unchanged at 32 µg/mL,
and the combination of adjuvant 1 at 30 µM lowered the CLR MIC
128-fold to 0.25 µg/mL, identical to what we observe for the
activity of 1 against the parent AB5075 (Table S6).
In summary, we report optimized adjuvants within the aryl 2-AI
class for macrolide potentiation against a virulent strain of gram-
negative bacteria, AB5075 (Figure 3). Compounds 4o, 4p, 4r, and
17b retained significant adjuvant activity at 10 µM, lowering the
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MIC of CLR 16-, 32-, eight-, and 16-fold, respectively, which was
comparable to the 16-fold reduction at 10 µM for original adjuvants
1 and 2.7 Adjuvants that surpassed the activity of 1 and 2 were
the amide-linked derivatives 4y, 10d, 15b as well as urea analogs
20a-c. At 10 µM, compound 4y reduced the MIC of CLR by 128-fold,
where compounds 10d and 15b reduced the MIC of CLR by 64-fold at
10 µM. However, compounds 10d and 15b both surpassed compound 4y’s
activity at 7.5 µM, lowering the MIC of CLR by eight- and 32-fold,
respectively.
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Figure 3. Structure-activity relationship trends observed and
representative clarithromycin potentiation results against AB5075.
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The urea analog 20c, exhibited comparable adjuvant activity as
amide compounds 10d and 15b at 7.5 µM, lowering the MIC of CLR by
16-fold. Interestingly, compound 20c was able to lower the MIC of
CLR to ≤ 0.0625 µg/mL when dosed at higher concentrations (≥ 20
µM), which surpassed the fold reductions seen thus far with CLR.
Urea derivatives 20a and 20b were more toxic, displaying standalone
MIC values of 50 and 25 µM; however, they maintained the highest
adjuvant activity at 7.5 µM out of all of the screened compounds.
Compound 20a lowered the MIC of CLR 128-fold from 32 to 0.25 µg/mL
at 7.5 µM, and compound 20b lowered the MIC of CLR 64-fold to 0.5
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g/mL at 7.5 µM.
Lead adjuvants 15b and 20a potentiated CLR activity against 23
additional clinically relevant A. baumannii strains at 10 and 7.5
µM, respectively. An evolution assay indicated that original
adjuvant 1 suppresses the acquisition of CLR resistance in AB5075.
Overall, these compounds represent valuable tools in ongoing
mechanism of action studies, as well as future murine model studies
to determine whether these compounds can potentiate CLR in vivo.
ACKNOWLEDGMENTS
The authors would like to thank the National Institutes of
Health (GM055769 and AI136904) for funding.
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ABBREVIATIONS
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2-AI, 2-aminoimidazole; CLR, clarithromycin; CLSI, Clinical and
Laboratory Standards Institute; HCAI, hospital care-associated
infection; MDR, multi-drug resistance; MIC, minimum inhibitory
concentration; OM, outer membrane; SAR, structure-activity
relationship.
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NOTES
The authors declare the following competing interest(s): Dr.
Melander is a co-founder and a broad of directors member of Agile
Sciences, a biotechnology compound that is seeking to
commercialize the antibiofilm and antibiotic sensitization
activity of 2-aminoimidazoles.
SUPPORTING INFORMATION
1
13
Biology Experimental, Chemistry Experimental, H and C Nuclear
Magnetic Resonance Spectra, Bibliography.
REFERENCES
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