Tetrazole-based trans-translation inhibitors kill Bacillus anthracis spores to protect host cells

Article abstract of DOI:10.1128/AAC.01199-17

Bacillus anthracis, the causative agent of anthrax, remains a significant threat to humans, including potential use in bioterrorism and biowarfare. The capacity to engineer strains with increased pathogenicity coupled with the ease of disseminating lethal

Full text of DOI:10.1128/AAC.01199-17

AAC Accepted Manuscript Posted Online 31 July 2017
Antimicrob. Agents Chemother. doi:10.1128/AAC.01199-17
Copyright © 2017 American Society for Microbiology. All Rights Reserved.
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Tetrazole-Based trans-Translation Inhibitors Kill Bacillus anthracis
Spores to Protect Host Cells
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John N. Alumasa, Tyler D. P. Goralski, Kenneth C. Keiler#
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Department of Biochemistry & Molecular Biology, The Pennsylvania State University,
University Park, PA, 16802, USA
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Running title: Killing B. anthracis spores to protect host cells
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#Corresponding author: Kenneth C. Keiler, email: kkeiler@psu.edu
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ABSTRACT
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Bacillus anthracis, the causative agent of anthrax, remains a significant threat to
humans, including potential use in bioterrorism and biowarfare. The capacity to
engineer strains with increased pathogenicity coupled with the ease of disseminating
lethal doses of B. anthracis spores makes it necessary to identify chemical agents that
target and kill spores. Here, we demonstrate that a tetrazole-based trans-translation
inhibitor, KKL-55, is bactericidal against vegetative cells of B. anthracis in culture. Using
a fluorescent analog, we show that this class of compounds co-localizes with
developing endospores and bind purified spores in vitro. KKL-55 was effective against
spores at concentrations close to its MIC for vegetative cells. Spore germination was
inhibited at 1.2X MIC and spores were killed at 2X MIC. In contrast, ciprofloxacin killed
germinants at concentrations close to its MIC but did not prevent germination even at
32X MIC. Because toxins are released by germinants, macrophages infected by B.
anthracis spores are killed early in the germination process. At ≥ 2X MIC KKL-55
protected macrophages from death after infection with B. anthracis spores.
Ciprofloxacin required concentrations ≥ 8X MIC to exhibit a similar effect. Taken
together, these data indicate that KKL-55 and related tetrazoles are good lead
candidates for therapeutics targeting B. anthracis spores, and suggest that there is an
early requirement for trans-translation in germinating spores.
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INTRODUCTION
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Bacterial infections continue to pose a significant danger to humans worldwide.
The limited reserve of effective antibiotics calls for continued efforts in the discovery and
development of new antibacterial agents to fight pathogenic species. B. anthracis, the
causative agent of anthrax, is a Gram-positive bacterium that infects both humans and
animals (1). This bacterium is capable of forming dormant spores that are resistant to
radiation and chemical challenges, and are able to survive extreme environmental
conditions for extended periods of time (2,3). Upon encountering favorable nutrient
conditions, the spores germinate and are then able to propagate. The germination and
outgrowth of the bacillus spores into vegetative cells usually occurs within the first 2 – 3
h following exposure to nutrients (4). This transformation enables B. anthracis to
synthesize virulence factors, including the protective antigen (PA), the edema factor
(EF) and lethal factor (LF) (5). While dormant spores are unable to discharge toxins,
release of the LF toxin has been reported to occur in germinants associated with
macrophages within 3 h following infection (6). This finding implies that germinants are
capable of killing host cells.
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Currently, there are limited data on the ability of commonly used antibiotics to
inhibit germination or kill B. anthracis spores (7). Most of the antibiotics used against B.
anthracis infections are believed to target vegetative cells with limited effects on
dormant spores (8,9). Therefore, identification of antibacterial agents that target and kill
spores could prove beneficial in preventing the onset of anthrax in cases of spore
exposure. Anthrax manifests in different forms, of which inhalational anthrax is the most
lethal with 85 – 90% mortality rates in humans if left untreated (10,11). This type of
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infection occurs upon inhalation of spores through the respiratory tract. Inhalational
anthrax lacks initial clinical symptoms but ultimately results in severe respiratory
complications (12). Although cases of natural human inhalational anthrax are rare, B.
anthracis spores pose a threat because of their potential use in bioterrorism or
biowarfare (8,13). This threat is of great concern to the biological defense community
given the potential ease of dissemination of lethal doses of spores to human
populations (14,15). In fact, over the past 20 years B. anthracis has been on the list of
biological agents likely to be weaponized and used in biowarfare (16). The anthrax
attacks of 2001 led to numerous infections and death despite the fact that the
disseminated spores were from a wild-type strain (17,18). The ability to genetically
engineer strains with increased pathogenicity and antibiotic resistance further
compounds this threat (19,20). New therapeutic agents therefore need to be developed,
particularly those with the ability to kill spores. Such drugs could be administered as a
form of prophylactic treatment to exposed populations including first responders and
military personnel prone to encounter spore-contaminated sites.
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Here, we investigate the ability of inhibitors of trans-translation to kill B. anthracis
spores. Trans-translation is a pathway for rescuing ribosomes that are stalled at the 3’
end of an mRNA that has no stop codon (21). These mRNAs (“nonstop” mRNAs) are
abundant in bacteria (21). Trans-translation uses tmRNA, a specialized RNA molecule,
and SmpB, a tmRNA-binding protein, to recognize ribosomes on nonstop mRNAs and
release them using a short reading frame in tmRNA (21). Trans-translation is conserved
throughout bacteria, and is important for maintaining protein synthesis capacity in a
wide range of species (21). Previously, we used a high-throughput screen to identify
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small molecules that inhibit trans-translation (22). KKL-55, one of the compounds
identified in the screen, inhibited trans-translation on nonstop mRNAs in vitro and in
vivo, and had broad-spectrum antibiotic activity (22). Although the molecular target of
KKL-55 is not yet known, in vitro assays showed that KKL-55 did not inhibit normal
translation on mRNAs that have a stop codon, so this compound is a specific inhibitor of
trans-translation (22). The data presented below show that KKL-55 kills both vegetative
cells and spores of B. anthracis, and protects macrophages from killing by B. anthracis
after infection.
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RESULTS
Tetrazole inhibitors interact with B. anthracis endospores
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The tetrazolyl-benzamide KKL-55 (Fig. 1A) was identified using a phenotypic screen for
inhibitors of trans-translation (21). This compound displayed antibacterial activity
against Gram-positive and Gram-negative species including B. anthracis Sterne (22,23).
To further examine the efficacy of KKL-55 against B. anthracis, we first used broth
microdilution assays to confirm that this compound inhibited growth with a minimum
inhibitory concentration (MIC) = 6.6 g/ml and an IC50 = 4.8 g/ml (Fig. 1B & D),
results that were consistent with our previous data (22). Plating assays showed that
KKL-55 was bactericidal at 13.3 g/ml (Fig. 1C). To study the interaction of this class of
compounds with bacterial cells we designed and synthesized a fluorescent derivative,
KKL-182 (Fig. 1A, Scheme 1). Broth microdilution and platting assays with B. anthracis
showed that KKL-182 has a MIC = 6.3 g/ml, an IC50 = 4.3 g/ml, and a MBC = 12.5
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g/ml (Fig. 1D). The similar activities of KKL-55 and KKL-182 suggest that the addition
of the fluorescent moiety did not disrupt the mechanism of action.
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To determine if the tetrazole inhibitors are taken up by B. anthracis, KKL-182 was
added to growing cultures at a concentration below the MIC, and cells were harvested
and washed to remove extracellular KKL-182. Fluorescence microscopy showed that
KKL-182 accumulated within vegetative cells (Fig. 1E). In addition, spots of increased
fluorescence intensity co-localized with developing endospores (Fig. 1E). This
observation prompted us to examine whether KKL-182 would also bind to mature
spores. We induced sporulation of B. anthracis by nutrient starvation and purified the
spores using a Ficoll gradient. The remaining vegetative cells were killed by heat
treatment and the spores were resuspended in medium lacking germination inducers.
Addition of spores to KKL-182 quenched fluorescence in a concentration-dependent
manner (Fig. 1F), suggesting that KKL-182 was interacting with the purified spores.
Fluorescence microscopy confirmed that KKL-182 was co-localizing with spores (Fig.
1F inset). These results cannot distinguish between accumulation of KKL-182 inside
the spores and binding to the surface of the spores, but they do indicate that KKL-55
and related tetrazoles associate with spores in some manner and therefore might limit
or prevent bacterial growth from spores.
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KKL-55 kills B. anthracis spores
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The onset of inhalational anthrax occurs following intake of B. anthracis spores through
the respiratory tract. While in the lungs, spores are phagocytosed by macrophages and
release toxins after germinating, killing the macrophages (24,25). As a result, inhibition
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of spore germination in vivo could prevent toxin-mediated macrophage killing by
germinants and provide additional time for the immune system to eliminate the infection
prior to the onset of disease. Therefore, drugs with activity against both vegetative cells
and spores could be useful for prophylactic and in acute anthrax treatment (26). An in
vitro spore germination model was used to assess whether KKL-55 could kill spores or
early germinants. Germination of purified spores was induced by the addition of a
combination of L-alanine and inosine (27,28) and growth was measured in the presence
or absence of KKL-55. KKL-55 inhibited outgrowth of spores in a dose-dependent
manner with complete inhibition at concentrations ≥ 1.2X MIC (Fig. 2A). The
fluoroquinolone ciprofloxacin (CIP) is one of the antibiotics currently recommended for
treatment of B. anthracis infections (29), but there are limited data on the activity of CIP
against spore germination (7). CIP inhibited growth of B. anthracis in culture with a MIC
= 0.1 g/ml and an IC50 = 0.06 g/ml, in agreement with previous reports (30). Growth
curves of B. anthracis spore cultures in the presence of CIP showed that growth was
arrested at concentrations ≥ MIC (Fig. 2B).
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Micrographs of samples treated with KKL-55 at 1.2X or 2X MIC showed no
vegetative cells present after 24 h, indicating complete inhibition of spore germination
(Fig. 2C). In contrast, the population in samples treated with 2X MIC CIP consisted
predominantly of germinants after 24 h (Fig. 2C). In comparison to ungerminated
spores, germinants are easily identifiable by their larger size (4,31). Samples treated
with 16X MIC CIP contained a mixture of ungerminated spores and germinants at both
the 7 h and 24 h time points (Fig. 2C). These results suggest that at this concentration
CIP did not prevent germination but did prevent outgrowth of germinated spores. Taken
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together, these data show that KKL-55 inhibits spore germination and outgrowth at
concentrations near its MIC, whereas CIP allows germination but effectively prevents
outgrowth of germinants at concentrations up to 16X MIC.
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To determine whether transient exposure to KKL-55 provides lasting inhibition of
germination, spores were incubated with KKL-55, washed with drug-free medium, and
alanine and inosine were added to induce germination. Micrographs of spores pre-
incubated with ≥ 1.2X MIC KKL-55 showed no vegetative cells (Fig. 3A). Cultures of
these pre-treated spores did not increase in optical density and showed a decrease in
CFU/ml (Fig. 3C & E). Control experiments in which spores were pre-treated with no
KKL-55 or with KKL-55 at concentrations < MIC showed production of vegetative cells
and growth of the culture (Fig. 3A). In contrast, spores pre-incubated with CIP at
concentrations up to 32X MIC showed germinants in the micrographs, although there
were no full-size vegetative cells (Fig. 3B). Cultures of spores pre-treated with CIP did
not accumulate optical density and the number of viable cells decreased, indicating that
CIP was killing the cells (Fig. 3D & F). These results show that treatment of B. anthracis
spores with either KKL-55 or CIP inhibits proliferation, even if the drug is not present in
the medium when germination is induced. However, the mechanism of inhibition
appears to be different: CIP allows germination of a large percentage of the spores and
inhibits growth of vegetative cells, whereas KKL-55 prevents germination of spores at a
very early stage.
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KKL-55 protects RAW Macrophages from killing by B. anthracis
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Germination of spores within host cells is characterized by the release of anthrax toxins
(32,33). During the inception of inhalational anthrax, uptake of B. anthracis spores by
macrophages within the lung alveoli leads to germination and release of toxins that kill
the macrophages (34,35). Based on the results demonstrating the capacity of the
tetrazoles to inhibit germination and kill spores in vitro, we assessed the ability of KKL-
55 and CIP to protect macrophages from B. anthracis toxin-induced death during
germination using an ex vivo infection model. The Sterne strain used in these
experiments lacks the capsule but has the capacity to produce toxins, including LF (6).
Heat-treated spores were added to RAW macrophages in the presence or absence of
KKL-55 or CIP. Propidium iodide (PI), which stains membrane-compromised and dead
cells but is impermeable to viable cells, was used to assess killing by B. anthracis.
Addition of KKL-55 at 2X MIC decreased PI staining by 90% relative to untreated
controls, indicating that it was able to protect macrophages from death (Fig. 4A).
Optical density measurements and plating assays confirmed that B. anthracis cells were
killed when KKL-55 was added (Fig. 4B & C). Addition of CIP at 16X MIC to infected
macrophages also killed B. anthracis cells and protected macrophages from B.
anthracis mediated cell death (Fig. 4D-F).
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Having established the activity of KKL-55 and CIP at selected concentrations we
examined the dose-dependent inhibitory activity using the same ex vivo infection model.
Increasing concentrations of either KKL-55 or CIP were added to a typical assay and
the PI fluorescence recorded after 9 h. KKL-55 exhibited a dose-dependent inhibitory
profile limiting toxin-induced macrophage death with IC50 = 5.7 g/ml and completely
inhibiting macrophage necrosis at 4X MIC (Fig. 5A). This trend was consistent with the
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spore germination and outgrowth profile monitored by absorbance which revealed
significant inhibition at concentrations > 0.5X MIC (Fig. 5B). The bactericidal/sporicidal
activity for KKL-55 displayed a trend similar to the in vitro data exhibiting ~ 90%
reduction in the CFUs at 2X MIC and no recovered CFUs at 4X MIC. (Fig. 5C).
Evaluation of any potential toxic effects for KKL-55 against host cells was performed
using an LDH release assay and showed no cytotoxicity at up to 16X MIC (Fig. 5D &
E). This result was in agreement with previous data showing an ED50 > 133 g/ml (20X
MIC) for KKL-55 against HeLa cells (36).
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CIP also displayed a dose-dependent profile preventing macrophage killing by B.
anthracis toxins with IC50 = 0.4 g/ml and providing full protection at concentrations >
8X MIC (Fig. 5F). Surprisingly, no substantial growth was recorded even at 0.5X MIC
based on the absorbance readings (Fig. 5G), unlike results from our in vitro
experiments (Fig. 3D). These data suggest that at CIP concentrations ≤ 8X MIC,
germinants were able to release toxins and kill macrophages, even though these CIP
concentrations prevented bacterial growth. CIP caused 1.5 - 3 log unit reductions in the
CFUs at all the concentrations tested (Fig. 5H). Under these experimental conditions
CIP did not show any significant toxicity at up to 32X MIC, the highest concentration
tested (Fig. 5I).
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DISCUSSION
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The data presented here demonstrate that both KKL-55 and CIP effectively kill
vegetative cells and germinants, but KKL-55 also kills spores. KKL-55 inhibited
germination of spores in vitro at concentrations ≥ 1.2X MIC, whereas spores germinated
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in the presence of CIP up to at least 32X MIC. Even though CIP prevented growth of B.
anthracis vegetative cells and killed early germinants, the sporicidal activity of KKL-55
correlated with increased protection of macrophages from killing by B. anthracis: KKL-
55 protected macrophages infected with spores at concentrations ≥ 2X MIC, but CIP
required a concentration ≥ 8X MIC to provide similar activity. CIP clearly has better
bacterial killing properties than KKL-55, which is a screening hit and has not been
optimized. In units of µg/ml, CIP is 66-fold more active than KKL-55 in preventing
growth of vegetative B. anthracis cells in vitro. However, CIP is only 16-fold more active
than KKL-55 at preventing necrosis of macrophages infected with B. anthracis spores.
These results suggest that optimization of sporicidal activity might produce a compound
that is more active in vivo than optimization of bactericidal activity. Sporicidal agents
such as KKL-55 could also be used to decontaminate areas after dissemination of B.
anthracis spores because KKL-55 inhibited germination in treated spores even after
they were washed with drug-free medium.
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The different relative activities of KKL-55 and CIP in spores and vegetative cells
indicate different requirements for their respective targets. KKL-55 was shown to inhibit
trans-translation, a mechanism used to rescue ribosomes sequestered on mRNAs that
don’t have a stop codon (21,22). trans-Translation is essential in many bacterial
species, presumably because it is required to maintain protein synthesis capacity (21).
Consistent with this hypothesis, species that can survive without trans-translation have
a backup mechanism to rescue ribosomes (21). The sporicidal activity of KKL-55
suggests that trans-translation is required in spores or at the earliest steps of
germination. A recent study has shown that protein synthesis is required for the first
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steps of germination, and that pre-incubation of spores with translation inhibitors prior to
induction prevented germination (4). It is likely that trans-translation is required during
this period to rescue ribosomes and maintain protein synthesis capacity. CIP targets
DNA replication, which is required much later in the spore germination process (4,37).
The delayed requirement for DNA replication likely explains why CIP kills early
germinants but not spores.
234 METHODS
Bacterial strains and culturing conditions. B. anthracis Sterne (34F2) was grown in
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lysogeny broth (LB) or cation modified Mueller Hinton broth at 37 °C. Sporulation was
induced by spreading an overnight culture of B. anthracis onto LB agar plates and
incubating them for 5 days at 37 °C. Microscopy analysis revealed > 95 % spores. The
spores were pooled by adding sterile ice cold deionized water to the plates and
scrapping. This suspension was harvested by centrifugation at 5510 g for 10 min. The
supernatant was aspirated and the spores were resuspended in 500 l ice cold water.
Spores were purified using a Ficoll gradient: respective solutions of Ficoll-400 in water
(20%, 30%, 50% w/v) were sterilized and a gradient prepared with the most
concentrated sample at the bottom. The spore suspension was added gently to the top
layer and then separated from vegetative cells by centrifugation at 3900 g. The
supernatant was discarded and the remaining pellet washed 3 times with sterile ice-cold
water as described above. Finally, the pellet was resuspended in ice cold sterile
deionized water, heat-treated at 65 °C for 30 min, cooled and stored at 4°C. Spore
viability was assessed by heat-treatment followed by serial dilution and plating on solid
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nutrient agar. Spores were cultivated in germination medium (50 mM Tris-Cl [pH 7.5],
10 mM NaCl) and growth induced by adding 40 M L-alanine and 250 M inosine (28).
Prior to each experiment the spore suspension was heat-treated at 65°C for 30 min and
inspected by microscopy to check for the presence of vegetative cells. The spores were
routinely assessed for auto-germination by culturing without the inducers and monitoring
samples by absorbance and microscopy. No germination was detected under these
conditions as previously observed (28).
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Antibacterial assays. MIC and MBC values were determined in accordance with the
Clinical and Laboratory Standards Institute guidelines (38). An overnight culture of B.
anthracis was inoculated into LB to make a 2X working culture (OD600 = 0.004). Serial
dilutions of the test compounds at 2X the desired concentration were prepared in LB in
a 96 well microtiter plate. 25 l diluted 2X working culture was then added to each well
for a final starting OD600 = 0.002. Plates were incubated at 37 °C for 20 h and the MIC
was recorded as the lowest concentration resulting in no visible growth. Absorbance
readings of the plates were recorded using a SpectraMax i3 microplate reader
(Molecular Devices, Sunnyvale, CA) and used to determine the IC50. The MBC was
determined by spotting samples from the wells without visible growth on LB agar plates.
The MBC was recorded as the concentration with ≥ 99 % reduction in the cfu/ml.
Inhibitors were tested in triplicate for each biological repeat.
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Fluorescence microscopy. Samples from growing cultures were transferred to plastic
culture tubes in the presence or absence of KKL-182 at 3.3 g/ml, incubated for an
additional 30 min, harvested by centrifugation at 6,700 g for 10 min, washed once with
drug-free medium and resuspended in ice cold phosphate buffered saline (1.2 g
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Na2HPO4, 0.22 g NaH2PO4, 8.5 g NaCl in 1L pH 7.5). An aliquot was spotted onto an
agararose pad and observed by fluorescence microscopy on a Nikon Eclipse 90i
microscope using a 100X oil emersion objective lens (DAPI emission filter).
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Interaction of spores with KKL-182. KKL-182 (50 g/ml) was added to plastic culture
tubes each containing 0.5 ml of an increasing amount of spores in medium without
germinants. These samples were incubated at 37 °C for 1 h with gentle agitation. 100 l
aliquots were then transferred to a 96 well plate and fluorescence recorded on a
SpectraMax i3 microplate reader at ex 340 nm and em 560 nm.
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Germination assays. Heat-treated spores were re-suspended in germination medium
with and without inducers and the culture was incubated at 37 °C. Germination and
outgrowth were assessed using absorbance readings at 600 nm in conjunction with light
microscopy. Inhibition of spore germination was assayed by incubating spores with and
without the inducers in the presence or absence of an inhibitor. Typically, 1 ml of a re-
suspended spore suspension was transferred into multiple culture tubes with loose caps
to facilitate aeration and drugs were added. Sample tubes were incubated at 37 °C and
inhibition of spore germination was determined by comparing absorbance values and
microscopy images of the drug-treated samples relative to the untreated controls.
Microscopy data were recorded from three independent assays with at least 5 fields
analyzed per sample per assay. On average, a total of 299 – 1153 cells/spores were
counted per sample.
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Spore killing assays. A heat-treated spore suspension was incubated in the presence
or absence of either of KKL-55 or CIP and the absorbance was recorded at 0, 7 and 24
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h. At the 24 h time point, cultures were harvested, washed with drug-free medium and
plated to determine CFUs.
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Ex vivo infection assays. RAW 264.7 macrophages (a gift from Dr. James Drake,
Albany Medical College) were seeded onto 96-well cell culture plates at a density of 1.5
x 105 cells/ml in RPMI-1640 medium (ThermoFisher, USA) with 10% FBS, 1%
glutamine, 1% non-essential amino acids, 1% sodium pyruvate and 1 mM HEPES and
no antibiotic. B. anthracis spores in Hank’s Balanced Salt Solution (HBSS) were diluted
in 1X PBS, and enumerated at various dilutions on LB plates, in order to obtain CFU/ml.
Prior to infection, the spores were heated at 65 °C for 30 min to kill any vegetative cells
that may have germinated. The spores were diluted to the appropriate concentration in
RPMI-1640 medium in order to achieve a multiplicity of infection (M.O.I) of 20:1. KKL-
55 or CIP were assessed at variable concentrations (39). When necessary, DMSO and
propidium iodide (PI) were added to a spore suspension at final concentrations of 0.5%
(v/v) and 13.4 g/ml (20 M) respectively (28). The solutions were then added directly
to the macrophages and the spore infection was synchronized by centrifugation of the
culture plates at 300 g for 10 min. The cells were incubated at 37 °C and 5% CO2 for 9
h, at which point fluorescence (ex 535 nm/em 617 nm) and absorbance (600 nm)
readings were obtained using a SpectraMax i3. Microscopy assays were performed with
0.5 M PI and images collected using an Evos FL Auto Cell Imaging System.
Cytotoxicity assays were performed using RAW 264.7 cells and an LDH release assay
kit (Pierce Biochemicals, USA) according to the manufacturer’s instructions.
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Synthesis and characterization of KKL-182 (3-chloro-N-(1-(2-(5-
(dimethylamino)naphthalene-1-sulfonamido) ethyl)-1H-tetrazol-5-yl)benzamide)
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Materials: All organic solvents and reagents were purchased from Sigma-Aldrich (St.
Louis, MO) unless otherwise indicated. NMR spectra were recorded using a DPX400
NMR spectrophotometer (Bruker) at 400 MHz for 1H-NMR and 100 MHz for 13C-NMR).
Electrospray mass spectroscopy (ESI-MS) for KKL-182 was performed on a 3200
QTRAP mass spectrometer (Applied Biosystems) coupled with a binary HPLC system.
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3-chloro-N-(1-propyl-1H-tetrazol-5-yl)benzamide (3). 3-chlorobenzoic acid (1 g, 6.4
mmol) was added to a flask containing 30 ml DMF followed by DIPEA (4.5 ml, 25.6
mmol) and PyBOP (4 g, 7.7 mmol). The resulting mixture was stirred for an additional
30 min at room temperature. A solution of 5-aminotetrazole (1.1 g, 12.8 mmol) in
DMF/CH2Cl2 (1:1) was slowly added to the flask and the mixture stirred continuously at
room temperature for 12 h. The reaction was terminated and evaporated to dryness
under reduced pressure. 10 ml aq. NaHCO3 (0.5 M) was added to the residue followed
by 30 ml CH2Cl2. The white precipitate was isolated by vacuum filtration and washed 3
times with CH2Cl2 to give the product as a white powder (67 % yield). 1H-NMR (400
MHz, DMSO-d6): 6.45 (bs, 1H) 7.60 (t, J = 8.0 Hz, 1H), 7.73 (d, J = 8.0 Hz, 1H), 8.04
(d, J = 6.9 Hz, 1H) 8.14 (s, 1H). 13C-NMR (100 MHz, DMSO-d6): 127.1, 128.2, 130.7,
132.7, 133.5, 133.8, 150.3, 164.3.
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tert-butyl-2-bromoethylcarbamate (4). Di-tert-butyl dicarbonate (2.8 g, 12.2 mmol)
dissolved in 40 ml CH2Cl2 was added to a solution of 2-bromoethylamine hydrobromide
(5 g, 24.4 mmol) in 20 ml H2O over a period of 10 min. NaOH (2.0 g, 48.8 mmol)
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dissolved in 20 ml H2O was then added the resulting biphasic mixture. The reaction was
stirred vigorously at room temperature for 4 h. The organic phase was then isolated and
washed with H2O and then 0.2 M HCl to a pH of ~ 1, and finally with H2O to a pH = 6-7
for the aqueous phase. The resulting organic phase was dried over anhydrous MgSO4,
filtered and evaporated to dryness in vacuo to give a colorless oil (75 % yield). 1H-NMR
(400 MHz, CDCl3):  1.40 (s, 9H), 3.44 (m, 4H), 5.05 (bs, 1H). 13C-NMR (100 MHz,
CDCl3):  28.7, 33.0, 41.0, 80.1, 156.0.
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N-(1-(2-aminoethyl)-1H-tetrazol-5-yl)-3-chlorobenzamide (5). Compound 3 (1 g, 4.5
mmol), compound 4 (1.5 g, 4.9 mmol) and Et3N (1.36 g, 13.4 mmol) were added to a
flask containing 20 ml DMF. The reaction mixture was refluxed at 150 °C for 12 h.
Volatiles were removed under reduced pressure and the crude sample was dissolved in
CH2Cl2. This solution was extracted with 0.1 M NaHCO3, washed with brine, dried over
anhydrous MgSO4 and concentrated under reduced pressure. The concentrate was
purified over silica gel with 4 % MeOH in CH2Cl2 as the mobile phase. After confirmation
by 1H-NMR the product was subjected to BOC deprotection without further purification.
This intermediate (200 mg, 0.55 mmol) was added to a flask containing a 1:1 solution of
CH2Cl2/TFA (10 ml) at room temperature. This reaction was stirred and allowed to
proceed for 5 h after which the solvent was evaporated in vacuo. The remaining residue
was dissolved in CH2Cl2 and extracted with 0.1 M NaHCO3. The combined organic
phase was evaporated and the resulting crude product purified by flash chromatography
on silica gel. The desired product was eluted using MeOH:CH2Cl2:Et3N (2:98:0.1%) to
give a white solid in 77.6 % yield. 1H-NMR (400 MHz, CDCl3): 2.92 (bs, 2H), 3.62 (m,
2H), 4.69 (m, 2H), 7.48 (m, 2H), 7.11 (d, J = 5.1 Hz, 1H), 7.95 (s, 1H), 9.80 (s, 1H). 13C-
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NMR (100 MHz, CDCl3): 13C-NMR (100 MHz, CDCl3): 47.3, 51.2, 125.4, 126.9, 129.4,
130.6, 132.2, 132.9, 148.7, 159.1.
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3-chloro-N-(1-(2-(5-(dimethylamino)naphthalene-1-sulfonamido)ethyl)-1H-tetrazol-
5-yl)benzamide (KKL-182). To a round bottom flask on ice (0°C) was added compound
3 (200 mg, 0.75 mmol) and Et3N (0.52 ml, 3.8 mmol) dissolved in CH2Cl2. This mixture
was stirred for 5 min and dansyl chloride (224 mg, 0.83 mmol) dissolved in CH2Cl2 was
added dropwise over a period of 15 min. The reaction was left to gradually warm up to
room temperature and stirred continuously for 12 h. A saturated solution of NaHCO3
was added to the resulting product and extracted with CH2Cl2 (3 x 20 ml). The organic
layers were combined, dried over anhydrous MgSO4 and concentrated in vacuo. The
crude product was purified by flash chromatography on silica gel and the product eluted
with CH2Cl2 and a MeOH gradient of 1 – 5% to give 261 mg of the desired product (69.6
% yield). 1H-NMR (400 MHz, CDCl3): 2.90 (s, 6H), 3.47 (t, J = 7.2 Hz, 2H), 4.51 (t, J =
7.2 Hz, 2H), 6.40 (bs, 1H), 7.54 (m, 4H), 8.07 (m, 4H), 10.6 (bs, 1H). 13C-NMR (100
MHz, CDCl3): 41.6, 45.7, 48.3, 115.6, 119.0, 123.4, 123.5, 127.8, 128.8, 129.6, 129.7,
129.8, 130.2, 130.8, 131.2, 131.4, 134.2, 140.2, 152.2, 161.3, 171.4. ESI-MS (+): [M +
H]+ Calc’d for C22H22ClN7O3S m/z = 500.1, observed 500.2.
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ACKNOWLEDGEMENTS
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We thank James R. Drake from the Department of Immunology and Microbial Disease
at the Albany Medical College and Girish S. Kirimanjeswara from the Department of
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Veterinary and Biomedical Sciences at the Pennsylvania State University for providing
RAW 264.7 macrophages.
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FUNDING INFORMATION
This work was supported by NIH grant GM068720 to K.C.K.
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FIGURE LEGENDS
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Figure 1. Tetrazole-based inhibitors kill vegetative B. anthracis cells and bind
spores in vitro. A) Chemical structures of ciprofloxacin and the tetrazole-based
compounds, KKL-55 and KKL-182. B) Growth curves for B. anthracis treated with KKL-
55. C) CFUs determined after 24 h incubation of B. anthracis cultures with KKL-55 (#no
CFUs recovered). Data are presented as the mean from 2 biological replicates (± SD).
D) Representative plots showing growth inhibition by KKL-55 and KKL-182. Data are
plotted as the mean with error bars indicating the standard deviation of 3 biological
replicates. E) Cellular localization of KKL-182 in vegetative B. anthracis cells with no
treatment (top row) or after treatment with 3.3 g/ml (0.5X MIC) KKL-182 (bottom row).
Red arrows show endospores. F) Fluorescence quenching of KKL-182 in the presence
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of B. anthracis spores. Inset: micrograph showing purified spores after incubation with
KKL-182 in vitro. Note: MIC for KKL-55 = 6.6 g/ml and KKL-182 = 6.3 g/ml.
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Figure 2. KKL-55 inhibits B. anthracis spore germination in vitro. A) Inhibition of
spore germination and outgrowth by KKL-55 monitored by absorbance over a 24 h
period in the absence of inhibitor, 1.7 g/ml (0.25X MIC), 8.0 g/ml (1.2X MIC), 13.3
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g/ml (2X MIC) and a control without KKL-55 or the inducers of germination (no
inducers). B) Similar assay as in (A) performed with CIP at 0.05 g/ml (0.5X MIC), 0.2
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g/ml (2X MIC) and 1.6 g/ml (16X MIC). C) Spore germination monitored by light
microscopy in the presence or absence of inducers or inhibitors over a 24 h period.
Representative micrographs from three independent assays are shown. Black arrows
indicate germinated spores and white arrows indicate ungerminated spores.
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Figure 3. KKL-55 kills B. anthracis spores in vitro. Analysis of B. anthracis spore
culture by light microscopy 24 h following induction of germination in the presence or
absence of A) KKL-55 (MIC 6.6 g/ml) and B) CIP (MIC 0.1 g/ml) at the indicated
concentrations. C) Dose-dependent inhibition of germination and outgrowth for purified
B. anthracis spores cultured in the presence or absence of KKL-55 monitored over a 24
h period. D) Similar experiment as in (C) for spores cultured in the presence or absence
of CIP. CFU counts from serially diluted cultures at the 24 h time point in the presence
or absence of the inducers of germination or E) KKL-55 and F) CIP at the indicated
concentrations. Data in C - F show averages from two independent determinations (±
SD). Black arrows indicate germinated spores and white arrows are germinants. (-) is
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the negative control without the inducers of germination or inhibitors. (+) is the positive
control with inducers of germination but no inhibitors present.
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Figure 4. KKL-55 protects RAW macrophages from B. anthracis-induced death.
Macrophages (Mac) cultured in medium containing PI were infected with purified spores
and B. anthracis-mediated death recorded in the presence or absence of the inhibitors
9h after spore treatment. The cultures were then incubated for an additional 15 h and
plated for CFU enumeration. A) Macrophage killing monitored by the increase in PI
fluorescence. Data show control macrophages without spores (SP) ± DMSO or 13.3
g/ml (2X MIC) KKL-55 and spore-treated macrophages ± DMSO or 13.3 g/ml KKL-55.
B) Germination and outgrowth monitored by absorbance at 600 nm ± KKL-55 for
samples in (A) recorded 9 h after spore treatment. C) CFUs determined from spore-
treated macrophage cultures ± 13.3 g/ml KKL-55. D) Macrophage killing monitored in
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the presence of 0.4 g/ml (4X MIC) and 1.6 g/ml (16X MIC) CIP. E) Germination and
outgrowth for samples in (D) recorded 9 h after spore treatment. F) CFUs determined
from spore-treated macrophage cultures ± CIP. G) Micrographs showing the DIC and PI
staining of spore-treated macrophages. B. anthracis macrophage killing is indicated by
the PI fluorescence signal. Data in A - F are averages from two biological replicates
each performed in triplicate wells (± SD). #No recovered CFUs.
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Figure 5. KKL-55 is not cytotoxic to RAW macrophages. A) Dose-dependent
inhibition of the B. anthracis-induced macrophage killing by KKL-55 monitored by PI
fluorescence 9 h after spore treatment. B) Inhibition of the germination and outgrowth of
spores by KKL-55 for samples in (A) monitored by absorbance. C) CFUs determined
from samples of macrophages infected with spores in the presence of varying
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concentrations of KKL-55. D) Cytotoxicity profile for KKL-55 against RAW macrophages
assessed using the LDH release assay 18 h following assay initiation. (DO = 1.6 %
DMSO). E) Micrographs showing macrophages from sample wells in the LDH release
assay after 18 h. F) B. anthracis-induced macrophage killing monitored by PI
fluorescence after 9 h in the presence or absence of CIP at variable concentrations. G)
Inhibition of the germination and outgrowth of spores by CIP monitored by absorbance.
H) CFUs determined from samples of macrophages infected with spores in the
presence of varying concentrations of CIP. I) Cytotoxic profiles for CIP against RAW
macrophages at the highest concentrations tested. Data in A-C, F-H are averages from
two biological replicates each performed in triplicate wells (± SD). The data in (D) and (I)
are averages from three technical replicates (± SD). (-) is the negative control without
the inducers of germination or inhibitors. (+) is the positive control with inducers of
germination but no inhibitors present.
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Scheme 1. Synthetic protocol for KKL-182 i) PyBOP, DIPEA, DMF, rt, 12h; ii) DMF,
150°C, 12h; iii) CH2Cl2:TFA (1:1), rt, 4 h; iv) CH2Cl2, Et3N, 0°C – rt, 12 h.
27