Potentiation of Francisella resistance to conventional antibiotics through small molecule adjuvants

Article abstract of DOI:10.1039/c5md00353a

A screen of 20 compounds identified small molecule adjuvants capable of potentiating antibiotic activity against Francisella philomiragia. Analogue synthesis of an initial hit compound led to the discovery of a potentially new class of small molecule adjuvants containing an indole core. The lead compound was able to lower the MIC of colistin by 32-fold against intrinsically resistant F. philomiragia.

Full text of DOI:10.1039/c5md00353a

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CONCISE ARTICLE
Potentiation of Francisella resistance to
conventional antibiotics through small molecule
Cite this: DOI: 10.1039/c5md00353a
adjuvants†
a
a
b
c
Matthew D. Stephens, Veroncia B. Hubble, Robert K. Ernst, Monique L. van Hoek,
a
d
a
Roberta J. Melander, John Cavanagh and Christian Melander*
Received 18th August 2015,
Accepted 18th September 2015
A screen of 20 compounds identified small molecule adjuvants capable of potentiating antibiotic activity
against Francisella philomiragia. Analogue synthesis of an initial hit compound led to the discovery of a
potentially new class of small molecule adjuvants containing an indole core. The lead compound was able
to lower the MIC of colistin by 32-fold against intrinsically resistant F. philomiragia.
DOI: 10.1039/c5md00353a
www.rsc.org/medchemcomm
9
–13
antibiotic activity against a wide variety of bacteria.
We
Introduction
completed a screen of randomly chosen molecules from our
in house library and identified four compounds, all of which,
did not contain a 2-AI or 2-ABI core, but another nitrogen
containing heterocycle (indole, indoline, or oxazoline) (Fig. 1)
that were capable of potentiating antibiotic response in
F. philomiragia. A structure activity relationship (SAR) study
of compound 4 was carried out to discover potentially more
potent leads and to better understand the structural require-
ments for activity. Herein, we report the discovery of the first
example of a potentially new class of lead compounds
containing an indole core capable of breaking F. philomiragia
resistance to the antibiotic colistin.
Francisella are small, facultative Gram-negative bacteria that
are responsible for zoonotic disease in which humans are
infected, usually from bites or contact with infected blood.
1
The most common vectors of disease are ticks and wild rab-
2
bits. Francisella can also be a highly infectious aerosol and
3
has the potential to be weaponized. Encompassed under the
Francisella genus are two species of consequence: Francisella
philomiragia and Francisella tularensis. Very resilient, the bac-
teria are capable of surviving multiple weeks in the environ-
ment and have been found in water sources around the
4
world. Although, not as virulent and somewhat rare in
5
humans, F. philomiragia is a potential model organism to
study Francisella. Human cases of F. philomiragia are often
Results and discussion
Adjuvant screen for MIC suppression of antibiotics against
associated with individuals with weakened immune systems
6
or who have suffered from a near drowning experience.
F. philomiragia
F. philomiragia is also the causative agent of the disease
7
francisellosis, most common among various species of fish.
An initial pilot screen of 20 compounds from our in-house
1
4–18
Francisellosis outbreaks have been responsible for mortality
small molecule library
was conducted by first determining
8
rates of up to 95% within farmed fish.
Our laboratory is interested in using small molecule adju-
vants to potentiate antibiotic activity. We have shown that
2
-aminoimidazoles (2-AIs) are capable of potentiating
a
Department of Chemistry, North Carolina State University, Raleigh, North
Carolina 27695, USA. E-mail: ccmeland@ncsu.edu
b
Department of Microbial Pathogenesis, University of Maryland – Baltimore,
Baltimore, MD 21201, USA
c
School of Systems Biology & National Center for Biodefense and Infectious
Diseases, George Mason University, Manassas, VA 20110, USA
d
Department of Molecular and Structural Biochemistry, North Carolina State
University, Raleigh, North Carolina 27695, USA
†
Electronic supplementary information (ESI) available. See DOI: 10.1039/
c5md00353a
Fig. 1 Lead structures identified from initial screening.
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the intrinsic antibiotic activity of each molecule by
establishing its minimum inhibitory concentration (MIC)
against F. philomiragia (Fig. S1, ESI†). Following our lab's pre-
Library synthesis for structure activity relationship (SAR)
study
Based on the results of our screen, availability of starting
materials and cost of antibiotics, we opted to perform ana-
logue synthesis of the indole 4. We were able to rapidly
assemble an 11-membered analogue library of 4 (ESI† Table
S1). The core is structurally similar to tryptamine with the
only difference being a shorter 2-carbon linker. Direct reac-
tion of tryptamine and derivatives with a variety of acylating
agents in the presence of TEA gave a library of various struc-
tural motifs. Testing revealed that shortening the three-
carbon linker to two carbons (5) drastically increased
9
viously reported screening protocol, the MIC of candidate
conventional antibiotics were then determined in the absence
or presence of each compound at 25% of the compound's
MIC. Previous studies from our group has established that
molecules from our internal library had little (if any) effect
on bacterial growth at 25% MIC, allowing us to study non-
microbicidal repotentiation of the conventional antibiotics
under study. Streptomycin, an aminoglycoside, was chosen as
the initial antibiotic to screen for potential adjuvants as it is
considered to be the drug of choice for tularemia treat-
potency (Scheme 1), lowering the MIC of colistin 32-fold to
1
9
ment. Initial testing with streptomycin revealed of the 20
−1
8
μg mL . The amide appears to be necessary as any
compounds screened only compound 1 showed activity, a
change in functionality: amine, ester, or sulfonamide,
results in complete loss in activity (ESI† Table S1). Alkyl-
ation or acylation of the indole nitrogen-1 position, also
caused complete loss in MIC suppression. Changes in the
alkyl tail length from butyl also gave disappointing results,
as methyl, ethyl, and propyl derivatives had no impact on
the antibiotic MIC.
−
1
reduction in MIC of four-fold to 1 μg mL (Table 1). Genta-
micin, another aminoglycoside, has also been shown to be
an effective treatment option for patients suffering from tula-
2
0
remia.
Screening the same library for gentamicin
repotentiation gave the same results for streptomycin, with
−
1
compound 1 lowering the MIC four-fold from 1 μg mL to
−
1
0
.125 μg mL .
Another class of antibiotics tested were macrolides, specif-
Time kill curve of compound 5
ically azithromycin. Azithromycin is an attractive option for
treatment because of its ability to concentrate within macro-
phages, where Francisella replicates at intracellular levels
In order to determine whether compound 5 was acting
through a toxic or non-toxic mechanism, bacterial growth
was measured as a function of time. F. philomiragia was
grown with/without compound (50 μM) being present. Bacte-
rial growth was checked at 8, 12, 16, 20, and 24 h time points
2
1
that are even greater than serum levels. Upon screening our
library, compound 1 exerted no change in azithromycin activ-
ity. Another compound (2) displayed an eight-fold reduction
−
1
−1
in MIC, from 4 μg mL to 0.5 μg mL ). Compound 2 is of
interest because unlike previously reported small molecule
adjuvants from our lab it is neither a 2-AI nor 2-ABI, but
rather contains an indoline core.
(
Fig. 2). Based on the analysis of the time kill curve, we
observe minor growth delay at earlier time points; however
growth is identical by 16 hours.
Our screen was further expanded to include a phosphonic
acid antibiotic, FR900098. FR900098 has been shown to be
effective at inhibiting Francisella sub-species. Compound 3
MIC determination in Francisella novicida
2
2
With lead compounds in hand, we then wanted to determine
whether compounds retained activity against F. novicida, a
sub-species of F. tularensis. As before, the MICs of the active
compounds (1, 2, and 5) were determined and then antibiotic
MICs were established in the absence or presence of each
compound at 25% MIC. Compound 1 showed no change in
MIC for any of the antibiotics tested (Table 2). The indole
compound 5 displayed a two-fold reduction in MIC for colis-
displayed a significant decrease in MIC of 16-fold from 1024
−
1
−1
μg mL to <64 μg mL .
Finally, we used colistin, a polymyxin antibiotic that is
known to be ineffective at treating Francisella infections due
2
3
to intrinsic resistance. The MIC of colistin alone was 256
−
1
μg mL . Library screening revealed an indole containing
compound (4) that was also absent of a 2-AI or 2-ABI motif.
The indole 4 exhibited a four-fold MIC reduction for colistin
−
1
−1
tin from 1024 μg mL to 512 μg mL . The concentration of
5 was increased by 10 μM increments from 50 μM to 100 μM;
−
1
to 64 μg mL .
Table 1 MIC data for the initial lead compounds against F. philomiragia. ND = not determined
MIC
(μM)
Concentration
tested (μM)
Streptomycin
Gentamicin
Azithromycin
FR900098
Colistin
(μg mL )
−
1
−1
−1
−1
−1
Compound
(μg mL
)
(μg mL
)
(μg mL
)
(μg mL
)
—
1
2
3
4
—
—
50
50
50
50
50
4
1
4
8
4
4
0.5
0.125
0.5
1
0.25
0.25
4
4
0.5
2
2
1024
1024
1024
<64
ND
256
32
256
512
64
>200
>200
200
>200
>200
5
0.5
1024
8
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Synthesis
General procedure for amide, ester, and sulfonamide cou-
pling. In a flame dried round bottom under N atmosphere
2
was added tryptamine (300 mg, 1.87 mmol), TEA (777 μL,
5.61 mmol), and anhydrous DCM (10 mL). The reaction mix-
ture was cooled to 0 °C and 4-butylbenzoyl chloride (314 μL,
1.68 mmol) was added dropwise. The reaction mixture was
stirred until completion as determined by TLC. The reaction
mixture was then washed with 1 N HCl (1 × 5 mL), brine (1 ×
Scheme 1 Synthesis of the most active compound (5). Reagents and
conditions: (a) 4-butylbenzoyl chloride, TEA and DCM.
5
2 4
mL) and the organic layer was dried over Na SO , then fil-
tered. The solvent was removed in vacuo and the residue was
purified by flash chromatography using 4 : 1 Hex/EtOAc to 2 :
1
Hex/EtOAc to give a white solid (538 mg, 93% yield).
Biological
Bacteria, media, and antibiotics. Francisella philomiragia
was obtained from the American Type Culture Collection
ATCC), Manassas, VA, as ATCC # (25015) and Francisella
(
novicida was obtained from BEI Resources as BEI # (U112). F.
philomiragia and F. novicida were grown in cation adjusted
Mueller Hinton broth (CAMHB) supplemented with 2% Iso-
VitaleX (BD #211876) enrichment or tryptic soy broth
supplemented with 0.1% cysteine (TSB-C) at 37 °C with shak-
ing, or on cysteine heart agar supplemented with 5% rabbits
blood (Hemostat; DRB050) at 37 °C. The bacteria were incu-
bated at 37 °C for 24 h. Antibiotics were purchased from
Sigma-Aldrich, St. Louis, MO, USA.
Fig. 2 Time kill curve of compound 5. Blue diamonds represent
bacterial control. Red squares represent bacteria and compound 5
(
50 μM).
however the increased concentration of 5 was unable to
potentiate colistin in F. novicida further. Compound 2 was
the only adjuvant tested to show any significant activity,
Minimum inhibitory concentration (MIC) assay. One day
5
−1
cultures (24 h) were subcultured to 5 × 10 CFU mL in
CAMHB supplemented with 2% IsoVitaleX (F. philomiragia) or
TSB-C (F. novicida). Aliquots (1 mL) of the subcultured media
were added to small culture tubes. Compound from a 100 mM
stock solution in DMSO was added to the small culture tube
to give the desired starting concentration (200 μM or 2–2048
−
1
reducing the MIC of azithromycin 16-fold fro 2 μg mL to
−
1
0
.125 μg mL .
Experimental section
Materials and methods
−
1
μg mL ). Rows 2–12 of a 96-well microtiter plate were filled
with 100 μL per well of the bacterial subculture. The wells of
the first row were filled with 200 μL each of the compound-
containing sample. Row 1 wells were mixed a minimum of five
times, followed by a 100 μL aliquot to row 2. The process was
repeated until the final row, in which the last 100 μL aliquot
was discarded. The 96-well plate was then sealed with a plastic
lid and incubated under stationary conditions at 37 °C for 48
h. The compound MIC was recorded as the lowest concentra-
tion at which no bacterial growth was observed.
All reagents and solvents for synthesis were obtained from
Sigma-Aldrich, St. Louis, MO, USA. Triethylamine was dried
by refluxing CaH , followed by distillation and storage over 4
2
Å molecular sieves. Deuterated solvents were acquired from
Cambridge Isotope Laboratories (CIL). Purification was
1
performed using 60 mesh standard silica from Sorbtech. H
1
3
NMR (300 MHz) and C NMR (100 MHz) spectra were
recorded at 25 °C on Varian Mercury spectrometers. Chemi-
cal shifts (δ) are given in ppm relative to TMS as an internal
standard or the respective NMR solvent. Mass spectra were
recorded on Thermo Fisher Scientific Exactive Plus MS (ESI).
Antibiotic resensitization assay. One day cultures (24 h)
5
−1
were subcultured to
5
×
10 CFU mL
in CAMHB
supplemented with 2% IsoVitaleX (F. philomiragia) or TSB-C
Table 2 MIC data for active compounds against F. novicida
−1
−1
−1
Compound
MIC (μM)
Concentration tested (μM)
Gentamicin (μg mL
)
Azithromycin (μg mL
)
Colistin (μg mL )
—
1
2
—
—
50
50
50
1
1
4
4
2
2
1024
1024
1024
512
>200
>200
>200
0.125
1
5
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(F. novicida). Aliquots (5 mL) of subcultured media were Notes and references
added to large culture tubes. Compound from a 100 mM
stock solution in DMSO was added to the large culture tubes
to give the concentration to be tested (25% of the MIC value).
One culture tube had nothing added and served as the con-
trol. Aliquots (1 mL) were taken from the large culture tubes
and added to small culture tubes. Antibiotic was added to
the small culture tubes, including the control, to give the
desired starting concentration (2–2048 μg mL ). Rows 2–12
of a 96-well microtiter plate were filled with 100 μL per well
of the bacterial subculture with or without compound. The
wells of the first row were filled with 200 μL each of the anti-
biotic dosed samples. Row 1 wells were mixed a minimum of
five times, followed by a 100 μL aliquot to the subsequent
row. The process was repeated until the final row, in which
the last 100 μL aliquot was discarded. The 96-well plate was
then sealed with a plastic lid and incubated under stationary
conditions at 37 °C for 48 h. The compound MIC was
recorded as the lowest concentration at which no bacterial
growth was observed.
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Conclusions
In summary, a pilot library screen resulted in the identifica-
tion of several unique small molecule adjuvants, capable of
potentiating various classes of antibiotics against F.
philomiragia. Analogue synthesis of one lead (4) gave rise to
compound 5, which displayed enhanced activity, culminating
in a 32-fold MIC reduction to 8 μg mL for colistin. Bacterial
growth over time was measured to elucidate whether com-
pound 5 was bactericidal. The CFUs measured for compound
containing samples, correlate strongly with the control provid-
ing evidence 5 is acting through a non-toxic mechanism. This
is the first example of a small molecule adjuvant able to
potentiate F. philomiragia resistance to colistin, and 5 may
represent the basis of a new class of small molecule adjuvants.
Further exploration of this unique class of compound is cur-
rently underway in attempts to break F. novicida resistance.
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