An FtsZ-targeting prodrug with oral antistaphylococcal efficacy in vivo

Article abstract of DOI:10.1128/AAC.01016-13

The bacterial cell division protein FtsZ represents a novel antibiotic target that has yet to be exploited clinically. The benzamide PC190723 was among the first FtsZ-targeting compounds to exhibit in vivo efficacy in a murine infection model system. Despite its initial promise, the poor formulation properties of the compound have limited its potential for clinical development. We describe here the development of an N-Mannich base derivative of PC190723 with enhanced drug-like properties and oral in vivo efficacy. The N-Mannich base derivative (TXY436) is~100-fold more soluble than PC190723 in an acidic aqueous vehicle (10 mMcitrate, pH 2.6) suitable for oral in vivo administration. At physiological pH (7.4), TXY436 acts as a prodrug, converting to PC190723 with a conversion half-life of 18.2±1.6 min. Pharmacokinetic analysis following intravenous administration of TXY436 into mice yielded elimination half-lives of 0.26 and 0.96 h for the TXY436 prodrug and its PC190723 product, respectively. In addition, TXY436 was found to be orally bioavailable and associated with significant extravascular distribution. Using a mouse model of systemic infection with methicillin-sensitive Staphylococcus aureus or methicillin-resistant S. aureus, we show that TXY436 is efficacious in vivo upon oral administration. In contrast, the oral administration of PC190723 was not efficacious. Mammalian cytotoxicity studies of TXY436 using Vero cells revealed an absence of toxicity up to compound concentrations at least 64 times greater than those associated with antistaphylococcal activity. These collective properties make TXY436 a worthy candidate for further investigation as a clinically useful agent for the treatment of staphylococcal infections. Copyright

Full text of DOI:10.1128/AAC.01016-13

An FtsZ-Targeting Prodrug with Oral
Antistaphylococcal Efficacy In Vivo
Malvika Kaul, Lilly Mark, Yongzheng Zhang, Ajit K. Parhi,
Edmond J. LaVoie and Daniel S. Pilch
Antimicrob. Agents Chemother. 2013, 57(12):5860. DOI:
10.1128/AAC.01016-13.
Published Ahead of Print 16 September 2013.
Updated information and services can be found at:
http://aac.asm.org/content/57/12/5860
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An FtsZ-Targeting Prodrug with Oral Antistaphylococcal Efficacy In
Vivo
Malvika Kaul,a Lilly Mark,b Yongzheng Zhang,b Ajit K. Parhi,b Edmond J. LaVoie,c Daniel S. Pilcha
Department of Pharmacology, Rutgers Robert Wood Johnson Medical School, Piscataway, New Jersey, USAa; Taxis Pharmaceuticals, Inc., North Brunswick, New Jersey,
USAb; Department of Medicinal Chemistry, Ernest Mario School of Pharmacy, Rutgers-The State University of New Jersey, Piscataway, New Jersey, USAc
The bacterial cell division protein FtsZ represents a novel antibiotic target that has yet to be exploited clinically. The benzamide
PC190723 was among the first FtsZ-targeting compounds to exhibit in vivo efficacy in a murine infection model system. Despite
its initial promise, the poor formulation properties of the compound have limited its potential for clinical development. We de-
scribe here the development of an N-Mannich base derivative of PC190723 with enhanced drug-like properties and oral in vivo
efficacy. The N-Mannich base derivative (TXY436) is ϳ100-fold more soluble than PC190723 in an acidic aqueous vehicle (10
mM citrate, pH 2.6) suitable for oral in vivo administration. At physiological pH (7.4), TXY436 acts as a prodrug, converting to
PC190723 with a conversion half-life of 18.2 ؎ 1.6 min. Pharmacokinetic analysis following intravenous administration of
TXY436 into mice yielded elimination half-lives of 0.26 and 0.96 h for the TXY436 prodrug and its PC190723 product, respec-
tively. In addition, TXY436 was found to be orally bioavailable and associated with significant extravascular distribution. Using
a mouse model of systemic infection with methicillin-sensitive Staphylococcus aureus or methicillin-resistant S. aureus, we show
that TXY436 is efficacious in vivo upon oral administration. In contrast, the oral administration of PC190723 was not effica-
cious. Mammalian cytotoxicity studies of TXY436 using Vero cells revealed an absence of toxicity up to compound concentra-
tions at least 64 times greater than those associated with antistaphylococcal activity. These collective properties make TXY436 a
worthy candidate for further investigation as a clinically useful agent for the treatment of staphylococcal infections.
ntibiotics in current clinical use are directed at a limited num- tion. We show that TXY436 acts as a prodrug at physiological pH,
A
ber of targets (e.g., the cell wall, protein synthesis, and DNA converting to PC190723. Unlike PC190723, the TXY436 prodrug
synthesis), many of which are associated with known resistance exhibits antistaphylococcal efficacy in vivo after oral administra-
pathways. As a result, there is a need for new antibiotics with novel tion in a murine model of systemic infection. In addition, TXY436
mechanisms of action that can help address the growing resistance is minimally toxic to mammalian cells at concentrations up to 64
times those associated with antistaphylococcal activity. These
properties make TXY436 an attractive lead for future clinical de-
velopment.
problem. The bacterial protein FtsZ represents a novel drug target
that has yet to be exploited clinically. FtsZ is a widely conserved
bacterial cytoskeletal protein that plays a critical role in cell divi-
sion (1). Using GTP as a cofactor, FtsZ self-assembles into poly-
mers at the bacterial membrane, forming a Z-ring at the midcell
site of division (1–5). The Z-ring serves as a scaffold for recruit-
ment of other key protein constituents of the bacterial divisome
(1–4). The essential role that FtsZ plays in cell division makes it an
appealing new antibiotic target (6–26).
MATERIALS AND METHODS
Bacterial strains. S. aureus 8325-4 was a gift from Glenn W. Kaatz (John
D. Dingell VA Medical Center, Detroit, MI), S. aureus Mu3 was a gift from
George M. Eliopoulos (Beth Israel Deaconess Medical Center, Boston,
MA), and Bacillus subtilis FG347 was a gift from Richard Losick (Harvard
The substituted benzamide derivative PC190723 (see structure University, Boston, MA). All other bacterial strains were obtained from
the American Type Culture Collection (ATCC).
in Fig. 1) was among the first FtsZ-targeting agents to be identified
(6, 7). It has been shown to bind FtsZ and stimulate the GTP-
dependent polymerization of the protein, which, in turn, leads to
the formation of stable FtsZ polymers that cannot recapitulate the
function of the Z-ring (13, 27). As a result, bacterial cell division is
inhibited, eventually leading to cell death. PC190723 is associated
with potent and specific activity against Staphylococcus sp., including
multidrug-resistantstrainsofmethicillin-resistantStaphylococcusau-
reus (MRSA) (7, 12). It has also been reported to exhibit in vivo activ-
ity in a mouse peritonitis model of S. aureus infection (7).
General chemistry methods and synthesis of PC190723 and
TXY436. All reactions were done under nitrogen atmosphere. Reaction
monitoring and follow-up were done using aluminum backed Silica G
TLC plates with UV254 (Sorbent Technologies), visualizing with UV
light. Flash column chromatography was done on a Combi Flash Rf Tele-
dyne ISCO. The 1H (300 MHz) and 13C (75 MHz) nuclear magnetic
resonance (NMR) spectra were acquired on a Varian Unity Inova (300
MHz) multinuclear NMR spectrometer. The data are expressed in parts
per million relative to the residual nondeuterated solvent signals, spin
Although initially promising, the hydrophobic nature of
PC190723 (calculated logP [ClogP] ϭ 2.64) makes it difficult to
formulate in vehicles suitable for in vivo treatment, a property that
has limited the potential clinical utility of the compound. We de-
scribe here an N-Mannich base derivative of PC190723 (TXY436;
see structure in Fig. 1) that is substantially more polar when pro-
tonated under acidic conditions (ClogP ϭ Ϫ0.51), and formulates
easily in acidic aqueous vehicles suitable for in vivo administra-
Received 13 May 2013 Returned for modification 20 June 2013
Accepted 6 September 2013
Published ahead of print 16 September 2013
Address correspondence to Daniel S. Pilch, pilchds@rwjms.rutgers.edu.
Copyright © 2013, American Society for Microbiology. All Rights Reserved.
doi:10.1128/AAC.01016-13
5860 aac.asm.org
Antimicrobial Agents and Chemotherapy p. 5860–5869
December 2013 Volume 57 Number 12

FtsZ-Targeting Prodrug with Oral In Vivo Efficacy
Reverse-phase HPLC. For all HPLC measurements, a reverse-phase
SPHER-100 C18 column (Princeton Chromatography, Inc.) was used on a
Shimadzu LC-20AT liquid chromatograph equipped with a Shimadzu
SPD-20AV UV/VIS detector (set at 296 nm). The column size was 150
mm by 4.6 mm, with particle and pore sizes of 5 ␮m and 100 Å, respec-
tively. A 20-␮l sample of each experimental solution was injected, and a
flow rate of 1 ml/min was applied, along with a gradient of 10 to 90%
acetonitrile (containing 0.1% [vol/vol] trifluoroacetic acid) and water in
the mobile phase. The total run time was 20 min, with the sampling fre-
quency and response time being 2 Hz and 1 s, respectively. Under these
conditions, TXY436 eluted ϳ9.8 min after sample injection, and
PC190723 eluted ϳ11.7 min after sample injection. Peak areas were de-
termined using the Shimadzu EZStart 7.4 SP3 software package.
MIC assays. MIC assays were conducted in accordance with Clinical
and Laboratory Standards Institute (CLSI) guidelines for broth microdi-
lution (29). Briefly, log-phase bacteria were added to 96-well microtiter
plates (at 5 ϫ 105 CFU/ml) containing 2-fold serial dilutions of com-
pound or comparator drug in cation-adjusted Mueller-Hinton (CAMH)
broth, with each compound concentration being present in duplicate. The
final volume in each well was 0.1 ml, and the microtiter plates were incu-
bated aerobically for 24 h at 37°C. Bacterial growth was then monitored by
measuring the optical density at 600 nm using a VersaMax plate reader
(Molecular Devices, Inc.), with the MIC being defined as the lowest com-
pound concentration at which growth was Ն90% inhibited. The follow-
ing S. aureus strains were included in these assays: 8325-4 (methicillin-
susceptible S. aureus [MSSA]), ATCC 49951 (mucoid MSSA), ATCC
19636 (MSSA), ATCC 29213 (MSSA), ATCC 33591 (MRSA), ATCC
43300 (MRSA), and Mu3 (MRSA). As recommended by CLSI (29), the
CAMH broth was supplemented with 2% NaCl in all of the MRSA exper-
iments so as to maintain selection pressure for MRSA. When present,
filtered mouse serum (Lampire Biological Laboratories, Inc.) was used at
50% (vol/vol).
FIG 1 Chemical structures of PC190723 and TXY436. TXY436 is depicted
with the Mannich base functionality in its protonated (cationic) form. The
indicated ClogP values were calculated using the weighted method (VG ϭ
KLOP ϭ PHYS ϭ 1) in the Marvin 5.12 Software Suite (ChemAxon, Ltd.),
with ClϪ and Naϩ/Kϩ concentrations being set at 0.1 mol/dm3.
multiplicities are given as s (singlet), d (doublet), m (multiplet), and bs
(broad singlet), and coupling constants (J) are reported in Hertz (Hz).
Melting points were determined using a Mel-Temp II apparatus (Labora-
tory Devices, Inc.) and are uncorrected.
PC190723 was synthesized as previously described (28). TXY436 was
synthesized as follows. In a sealed tube, a mixture of PC190723 (200 mg,
0.56 mmol), formaldehyde (37%, 0.3 ml, 3.7 mmol), and dimethylamine
(2 M in tetrahydrofuran [THF], 1.9 ml, 3.70 mmol) in H2O/THF (8/4 ml)
was heated at 65°C for 2 h. After cooling to room temperature, the reac-
tion mixture was diluted with ethyl acetate, washed with brine, dried over
Na2SO4, and concentrated to afford a brown solid. Subsequent purifica-
tion by column chromatography afforded the pure product TXY436 as a
light brown solid (154 mg, 67% yield): melting point, 152 to 154°C; 1H
NMR (CDCl3, 300 MHz); ␦: 8.57 (d, J ϭ 2.1 Hz, 1H), 8.24 (d, J ϭ 2.1 Hz,
1H), 7.17 to 7.09 (m, 1H), 6.92-6.86 (m, 1H), 6.20 (bs, 1H), 5.48 (s, 2H),
4.30 (d, J ϭ 6.0 Hz, 2H), 2.37 (s, 6H). HRMS calculated for
C17H15ClF2N4O2S (M ϩ H)ϩ 413.0645. Found 413.0656.
MBC assays. Minimum bactericidal concentration (MBC) assays
were conducted in accordance with CLSI guidelines (29). Broth microdi-
lution assays were conducted as described in the preceding section. After
the 24-h incubation period, aliquots from the microtiter wells were plated
onto tryptic soy agar (TSA). The colonies that grew after 24 h of incuba-
tion were counted using an Acolyte colony counter (Synbiosis, Inc.), with
MBC being defined as the lowest compound concentration resulting in a
Ն3-log reduction in the number of CFU.
Time-kill assays. Exponentially growing S. aureus 8325-4 bacteria
were diluted in CAMH broth to a final count of 105 to 106 CFU/ml. The
colony count at time zero was verified by plating serial dilutions of the
culture in duplicate on TSA plates. The initial culture was aliquoted into
tubes, each containing either a compound or comparator drug at final
concentrations ranging from 0ϫ to 8ϫ the MIC. An equivalent volume of
DMSO was added to the vehicle control tube. The cultures were then
incubated at 37°C with shaking. The CFU/ml in each culture was deter-
mined over time by withdrawing samples at time points raging from 2 to
24 h and plating appropriate serial dilutions on to TSA plates. To avoid
the possibility of carryover effects in instances when no dilutions were
made of the cultures, the samples were centrifuged at 16,000 ϫ g, the
supernatant was removed, and the bacterial pellet was resuspended in
compound-free medium before plating. All TSA plates were incubated
at 37°C, and the CFU/ml at each time point determined by counting
colonies after 24 h.
Phase-contrast microscopy. Log-phase S. aureus 8325-4 cells were
cultured in CAMH broth at 37°C for 4 h in the presence of DMSO (solvent
control), PC190723 at 3 ␮g/ml, or TXY436 at 3 ␮g/ml. A 1-ml sample was
withdrawn from each bacterial culture, and centrifuged at 16,000 ϫ g for
3 min at room temperature. The supernatant was removed, and the bac-
terial pellet was washed with 1 ml of PBS. The final bacterial pellet was
then resuspended in 50 ␮l of PBS, with 5 ␮l of the resulting bacterial
Comparator antibiotics and S. aureus FtsZ. Vancomycin-HCl,
erythromycin, and oxacillin (sodium salt) were obtained from Sigma-
Aldrich Co. S. aureus FtsZ was expressed in Escherichia coli and purified as
described previously (21).
Compound solubility studies. The maximum solubilities of PC190723
and TXY436 in 10 mM citrate (pH 2.6) or phosphate-buffered saline
(PBS; pH 7.4) were determined at 25°C. In these assays, the compounds
were combined with vehicle at weight/volume (wt/vol) ratios above their
solubility limit, and the amount of dissolved compound quantified. The
wt/vol ratios targeted in the PBS vehicle were 0.2 mg/ml for both com-
pounds, while the wt/vol ratios targeted in the citrate vehicle were 0.2
mg/ml for PC190723 and 2.5 mg/ml for TXY436. Each solubility assess-
ment was conducted in duplicate. All samples were vortexed for 3 min to
afford maximal compound dissolution and then centrifuged at 16,000 ϫ
g for 1 min to pellet out any unsolubilized compound. The concentration
of compound in each of the resulting supernatants was then quantified by
using reverse-phase high-performance liquid chromatography (HPLC) as
described below and generating standard curves of peak area versus com-
pound concentration (in citrate or PBS vehicle).
Compound stability studies. Experimental solutions of TXY436 or
PC190723 at concentrations of either 10 or 20 ␮g/ml were prepared from
4-mg/ml dimethyl sulfoxide (DMSO) stock solutions in either 10 mM
citrate (pH 2.6) or PBS (pH 7.4) vehicle. The relative amount of TXY436
and PC190723 in each experimental solution was then assessed as a func- suspension being transferred onto a microscope slide, together with 5 ␮l
tion of time at 25°C using reverse-phase HPLC as described below.
of 1% molten agarose (made in PBS). A coverslip was applied, and the
December 2013 Volume 57 Number 12
aac.asm.org 5861

Kaul et al.
slide was visualized with a Zeiss Axioplan 2 microscope equipped with a ducted in full compliance with the standards established by the U.S. Na-
Plan-Apochromat ϫ100 objective lens (NA ϭ 1.40). Images were cap- tional Research Council’s Guide for the Care and Use of Laboratory An-
imals and were approved by the Institutional Animal Care and Use
Committee of Rutgers University. Groups of six female Swiss-Webster
mice with an average weight of 25 g were infected intraperitoneally with a
lethal inoculum of either MSSA (500 ␮l of saline containing 8 ϫ 106
CFU/ml and 1.5% [wt/vol] porcine mucin [Sigma-Aldrich Co.]) or
MRSA (500 ␮l of saline containing 108 CFU/ml and 5% [wt/vol] porcine
mucin).
The mice were fasted overnight prior to their use in the studies. All
compound and vehicle administrations were performed p.o. by gavage,
with the vehicle being 10 mM citrate (pH 2.6). Both TXY436 and
PC190723 were formulated at 2.0 mg/ml, with the resulting TXY436 for-
mulation being a solution and the resulting PC190723 formulation being
a suspension.
tured with a Zeiss Axiocam HR camera by using the OpenLab software
package.
Fluorescence microscopy. The impact of TXY436 and PC190723 on
FtsZ Z-ring formation in B. subtilis FG347 bacteria was assessed in a man-
ner similar to that described previously (30). Specifically, exponentially
growing FG347 bacteria were cultured in CAMH broth for 1 h at 37°C in
the presence of DMSO (solvent control), PC190723 at 3 ␮g/ml, or
TXY436 at 3 ␮g/ml. The expression of green fluorescent protein (GFP)-
conjugated ZapA was then induced by the addition of xylose to a final
concentration of 0.25% (wt/vol), and the cells were incubated for 1 addi-
tional hour at 37°C. The bacterial cultures were then treated and visual-
ized as described above, with the additional incorporation of a standard
GFP filter set.
Four experimental groups of MSSA-infected mice were treated as fol-
lows: group 1, untreated; group 2, vehicle only; group 3, TXY436 at 128
mg/kg (in four divided doses of 32 mg/kg); and group 4, PC190723 at 128
mg/kg (in four divided doses 32 mg/kg). The first dose of compound was
administered 10 min after infection, with subsequent doses being admin-
istered at 12-min intervals thereafter. In the MRSA studies, three experi-
mental groups of infected mice were treated as follows: group 1, untreat-
ed; group 2, vehicle only; and group 3, TXY436 at 192 mg/kg (in six
divided doses of 32 mg/kg). In these studies, the first dose of compound
was administered 1 h after infection, with subsequent doses being admin-
istered at 12-min intervals thereafter. The dosing volume for all of the p.o.
administrations was 16 ml/kg.
An additional group of six infected mice was also included as a positive
control group in both the MSSA and the MRSA studies. This group was
treated with a single i.v. administration of vancomycin (formulated in
saline) at a dose of 16 mg/kg in the MSSA studies and 24 mg/kg in the
MRSA studies. The vancomycin was administered 10 min after MSSA
infection, while being administered 1 h after MRSA infection. The dosing
volume for the i.v. vancomycin control groups was 8 ml/kg.
The body temperatures of all mice were monitored for a period of 5
days after infection. Body temperatures were recorded at the Xiphoid
process using a noninvasive infrared thermometer (Braintree Scientific,
Inc.). Infected mice with body temperatures of Յ28.9°C were viewed as
being unable to recover from the infection (32) and were euthanized.
MTT cytotoxicity assay. The cytotoxicity of TXY436 versus Vero cells
(African green monkey kidney epithelial cells; ATCC) was assessed using
a 72-h continuous 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) assay as described previously (33).
Frequency of resistance assay. The frequency of resistance (FOR) to
TXY436 was assayed using a large inoculum approach. In this approach,
TSA plates containing TXY436 at concentrations ranging from 4ϫ to 32ϫ
the MIC were prepared. A large inoculum of ϳ3 ϫ 109 CFU/ml of S.
aureus 8325-4 was spread onto each plate. The colony count of the inoc-
ulum was verified by plating serial dilutions of the culture onto nonselec-
tive TSA plates. All plates were incubated at 37°C overnight and examined
after 24 h. Mutational frequency was calculated from the ratio of the
number of colonies observed on the selective plates to the total number of
plated bacteria (31).
FtsZ polymerization assay. Polymerization of S. aureus FtsZ was
monitored using a microtiter plate-based spectrophotometric assay in
which changes in FtsZ polymerization are reflected by corresponding
changes in absorbance at 340 nm (A340). PC190723 or TXY436 (at con-
centrations ranging from 0 to 10 ␮g/ml) were combined with 5 ␮M FtsZ
in 100 ␮l of reaction solution. Reaction solutions contained 50 mM Tris-
HCl (pH 7.4), 50 mM KCl, and 10 mM magnesium acetate. Reactions
were assembled in half-volume, flat-bottom, 96-well microtiter plates and
initiated by the addition of 4 mM GTP. Polymerization was continuously
monitored at 25°C by measuring A340 in a VersaMax plate reader over a
time period of 60 min.
Pharmacokinetic studies. Pharmacokinetic experiments were con-
ducted by SAI Life Sciences, Ltd. (Pune, India) in accordance with guide-
lines established by the Indian Committee for the Purpose of Control and
Supervision of Experiments on Animals (CPCSEA). Approval by the In-
stitutional Animal Ethics Committee (IAEC) was obtained prior to initi-
ation of the studies. Healthy male BALB/c mice (8 to 12 weeks old and
weighing between 25 and 35 g) were obtained from ACTREC (Mumbai,
India). Twelve mice were divided into two groups of six mice each, with
three mice being housed per cage. Food and water were provided to the
mice ad libitum. The first group of mice received a single intravenous (i.v.)
dose of TXY436 at 5 mg/kg by tail vein injection. The second group re-
ceived single peroral (p.o.) dose of TXY436 at 10 mg/kg by gavage. In both
cases, the compound was formulated in 10 mM citrate vehicle (pH 2.6) at
a concentration of 0.85 mg/ml. Blood samples (ϳ60 ␮l) were collected
from the retro-orbital plexus at 0.08, 0.25, 0.5, 1, 4, and 8 h after the i.v.
dose and 0.25, 0.5, 1, 4, and 8 h after the p.o. dose. The blood samples were
collected from a set of three mice at each time point and placed in micro-
centrifuge tubes containing a 20% K2EDTA solution as an anticoagulant.
The blood samples were then centrifuged at 4,000 rpm for 10 min at 4°C to
separate the plasma. The plasma samples were stored at Ϫ80°C prior to
their bioanalysis. Plasma concentrations of TXY436 and PC190723 were
quantified by LC-MS/MS, with the lower limit of quantitation (LLOQ)
being 20.2 ng/ml for both compounds. The time-dependent plasma con-
centration data were analyzed by using the sparse sampling mode in the
noncompartmental analysis (NCA) tool of the Phoenix WinNonlin ver-
sion 6.3 software package to yield relevant pharmacokinetic parameters.
In vivo efficacy studies. Antistaphylococcal efficacy in vivo was as-
sessed in a mouse peritonitis model of systemic infection with S. aureus
RESULTS AND DISCUSSION
TXY436 is associated with a 100-fold enhanced solubility rela-
tive to PC190723 in an acidic aqueous vehicle (10 mM citrate,
pH 2.6) suitable for in vivo drug administration. We sought to
determine whether the N-Mannich base functionality on TXY436
afforded the compound enhanced aqueous solubility relative
to PC190723. To this end, we used reverse-phase HPLC to
determine the maximal solubilities of TXY436 and PC190723
in two different aqueous vehicles with differing values of pH:
10 mM citrate (pH 2.6) and PBS (pH 7.4). The maximal solu-
bilities derived from these studies are listed in Table 1. The
solubility of TXY436 is ϳ2.8-fold greater than that of
PC190723 in PBS at pH 7.4 (66.8 Ϯ 15.7 ␮g/ml for TXY436
versus 23.9 Ϯ 4.0 ␮g/ml for PC190723), while being ϳ100-fold
greater in citrate at pH 2.6 (2,290 Ϯ 199 ␮g/ml for TXY436
versus 22.5 Ϯ 1.6 ␮g/ml for PC190723). Thus, the N-Mannich
base functionality affords TXY436 increased aqueous solubility
relative to PC190723, with the degree of solubility enhance-
ment being significantly greater at acidic pH (2.6) than at phys-
ATCC 19636 (MSSA) or ATCC 43300 (MRSA). These studies were con- iological pH (7.4). Importantly, the solubility of TXY436 in the
5862 aac.asm.org
Antimicrobial Agents and Chemotherapy

FtsZ-Targeting Prodrug with Oral In Vivo Efficacy
TABLE 1 Solubilities of TXY436 and PC190723 in 10 mM citrate and
PBS at 25°Ca
Mean solubility (␮g/ml) Ϯ SD
Compound
Citrate (pH 2.6)
PBS (pH 7.4)
TXY436
PC190723
2,290 Ϯ 199
22.5 Ϯ 1.6
66.8 Ϯ 15.7
23.9 Ϯ 4.0
a Each solubility value reflects the average of two independent determinations.
acidic citrate vehicle (2,290 ␮g/ml) is sufficient in magnitude
for in vivo efficacy studies of TXY436 using this vehicle.
TXY436 is chemically stable in the acidic citrate vehicle. We
next sought to assess the chemical stability of TXY436 in the acidic
citrate vehicle over time using reverse-phase HPLC. Figure 2
shows the HPLC chromatogram of TXY436 at 10 ␮g/ml after 24 h
of incubation in the acidic citrate vehicle at 25°C, with the corre-
sponding chromatogram of PC190723 at 10 ␮g/ml also shown for
comparative purposes. Under the conditions used in these HPLC
measurements (detailed in Materials and Methods), TXY436
elutes at ϳ9.8 min after injection, while PC190723 elutes at ϳ11.7
min. The shorter retention time of TXY436 relative to PC190723
is consistent with its correspondingly decreased hydrophobicity
(ClogP ϭ Ϫ0.51 for TXY436 and ϩ2.64 for PC190723). Note that
the chromatographic profile of TXY436 remained essentially un-
changed, even after 72 h of incubation (data not shown), indicat-
ing that TXY436 is chemically stable over this period of time in the
acidic citrate vehicle.
Although stable at pH 2.6, TXY436 converts to PC190723 at
pH 7.4 with a half-life (t1/2) of 18.2 ؎ 1.6 min at 25°C. N-Man-
nich base derivatives of drugs are susceptible to pH-dependent
chemical hydrolysis, which has been used as a platform for making
prodrugs with improved formulation and delivery properties (34,
35). Rolitetracyclin (transcycline) is an example of a clinically used
N-Mannich base prodrug of an antibiotic (tetracycline) (35). One
of the by-products of N-Mannich base hydrolysis is formaldehyde
(34, 35). Although the release of formaldehyde after N-Mannich
base prodrug hydrolysis raised concerns early on regarding poten-
tial toxicity, these concerns have been allayed by studies demon-
FIG 3 (A) Reverse-phase HPLC chromatograms of TXY436 at 20 ␮g/ml after
the indicated times of incubation in PBS (pH 7.4) at 25°C. For comparative
purposes, the corresponding chromatogram of PC190723 at 20 ␮g/ml after 60
min of incubation in PBS is also presented. The solid arrow indicates the peak
corresponding to TXY436, while the dashed arrow indicates the peak corre-
sponding to PC190723. (B) Plot of ln(peak area) versus time for the peak
corresponding to TXY436. The indicated half-life (t1/2) for the conversion of
TXY436 to PC190723 was determined using the relationship, t1/2 ϭ Ϫ0.693/m,
where m is the slope derived from the linear least-squares fit of the experimen-
tal data points (as indicated by the solid line). The uncertainty in t1/2 reflects
the maximal error in m propagated through the above relationship.
strating that the level of formaldehyde exposure is well below the
toxic threshold (36).
If TXY436 were susceptible to pH-dependent chemical hydro-
lysis like other N-Mannich base derivatives, one of the expected
products would be PC190723. We used reverse-phase HPLC to
probe for the potential conversion of TXY436 to PC190723 in
solution at the physiological pH of 7.4. To this end, we monitored
the chemical stability of TXY436 over time in PBS (pH 7.4) at
25°C. Figure 3A shows the HPLC chromatograms of TXY436 at 20
␮g/ml after incubation in PBS for various times over a period of 60
FIG 2 Reverse-phase HPLC chromatograms of TXY436 and PC190723 after
incubation for 24 h in 10 mM citrate (pH 2.6) at 25°C. The concentration of
each compound was 10 ␮g/ml.
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Kaul et al.
TABLE 2 Antibacterial activities of TXY436 and PC190723 against
TABLE 3 Comparison of MICs and MBCs for TXY436 and PC190723
MSSA and MRSAa
against MSSA and MRSAa
MIC (␮g/ml)
Strain and compound
MIC (␮g/ml)
MBC (␮g/ml)
MBC/MIC
8325-4 (MSSA)
TXY436
PC190723
Vancomycin
Erythromycin
Strain
TXY436 PC190723 Vancomycin Oxacillin
0.5
0.5
1.0
0.125
1.0
1.0
2.0
32
2
2
2
256
8325-4 (MSSA)
0.5
1.0
0.5
1.0
0.5
1.0
0.5
0.5
0.5
0.5
0.5
1.0
1.0
1.0
1.0
2.0
2.0
2.0
0.125
0.25
0.125
0.25
ATCC 29213 (MSSA)
ATCC 19636 (MSSA)
ATCC 49951 (MSSA)
ATCC 33591 (MRSA) 0.5
ATCC 43300 (MRSA) 0.5
Ͼ64
Ͼ64
Ͼ64
ATCC 19636 (MSSA)
TXY436
PC190723
Vancomycin
Erythromycin
0.5
0.5
1.0
0.5
0.5
0.5
1.0
Ͼ64
1
1
1
Mu3 (MRSA)
0.5
a Vancomycin and oxacillin are included as control antistaphylococcal agents. Mu3 is a
clinical MRSA isolate also identified as being hetero-GISA (hGISA) (43).
Ͼ128
ATCC 33591 (MRSA)
TXY436
PC190723
Vancomycin
Erythromycin
0.5
0.5
2.0
Ͼ64
1.0
1.0
4.0
NA
2
2
2
NA
min. The chromatogram of PC190723 at 20 ␮g/ml after 60 min of
incubation in PBS is also shown as a reference. Note that over this
time period, TXY436 becomes almost fully converted to
PC190723. Thus, in essence, TXY436 acts as a stable acid-soluble
prodrug that converts to PC190723 under physiological pH condi-
tions. A linear regression analysis of the time-dependent decrease in
the peak areas of TXY436 indicates that the TXY436-to-PC109723
conversion follows first-order kinetics, while also yielding a half-life
(t1/2) for the conversion of 18.2 Ϯ 1.6 min (Fig. 3B).
ATCC 43300 (MRSA)
TXY436
PC190723
Vancomycin
Erythromycin
0.5
0.5
2.0
Ͼ64
1.0
1.0
4.0
NA
2
2
2
NA
a Vancomycin and erythromycin are included as control bactericidal and bacteriostatic
agents, respectively. NA, not applicable.
The TXY436 prodrug retains the antistaphylococcal and
FtsZ-targeting activities of the PC190723 product. For an N-
Mannich base prodrug such as TXY436 to be active in vivo, it must
maintain the same biological activities in vitro as the product
(PC190723) itself. We therefore compared TXY436 and Both MRSA strains were resistant to erythromycin, precluding
PC190723 with regard to antistaphylococcal activity (as indicated MBC determinations against these strains. Significantly, TXY436
by MIC, MBC, and kinetics of kill), as well as FtsZ-targeting ac- and PC190723 exhibited MBC/MIC ratios of 1 to 2 against all of
tivity (as reflected by the impact on bacterial morphology, FtsZ the MSSA and MRSA strains, indicative of bactericidal antistaphy-
polymerization, and Z-ring formation).
lococcal behavior.
Antistaphylococcal activity of TXY436. Our initial studies
In addition to assessing the bactericidal activities of TXY436
compared the activities of TXY436 and PC190723 against four and PC190723 against S. aureus with regard to MBC/MIC ratios,
different MSSA and three different MRSA strains, with the result- we also probed for bactericidal activity using a time-kill assay at
ing MICs being summarized in Table 2. The MICs of TXY436 were compound concentrations of 1ϫ, 2ϫ, 4ϫ, and 8ϫ MIC. Figure 4
similar, if not identical, to those of PC190723 against all of the shows representative time-kill curves, with vancomycin being in-
MSSA and MRSA strains examined, with these MICs ranging cluded as a comparator control agent. Inspection of these time-kill
from 0.5 to 1.0 ␮g/ml. With regard to the three MRSA strains used data reveals that both TXY436 and PC109723 produce Ͼ3 logs of
in the studies, the activities of TXY436 were comparable to the kill at concentrations of 2ϫ the MIC or greater. Significantly, this
activity of the prototypical anti-MRSA drug vancomycin (MIC ϭ observation is consistent with the bactericidal behavior revealed
2.0 ␮g/ml), which was included as a comparator antibiotic. Oxa- by our MBC/MIC analysis described above. It is also of interest to
cillin (also included as a control agent) was predictably inactive note that at concentrations of Ն2ϫ the MIC, the killing kinetics of
versus the MRSA strains (MIC Ͼ 64 ␮g/ml).
TXY436 and PC190723 are more rapid than those of vancomycin.
PC190723 has been shown to be associated with a frequency of
We next evaluated the antistaphylococcal activities of TXY436
and PC190723 with regard to their bactericidal behavior. As a first resistance (FOR) in S. aureus of approximately 3 ϫ 10Ϫ8 (7, 37).
step toward this goal, we determined the MBC values of TXY436 We sought to determine the corresponding FOR to the TXY436
and PC190723 against two of the MSSA strains (8325-4 and ATCC prodrug in S. aureus using the large inoculum approach described
19636) and two of the MRSA strains (ATCC 33591 and 43300). in Materials and Methods. These studies yielded an FOR to
The bactericidal antistaphylococcal drug vancomycin and the TXY436 of (2.0 Ϯ 0.7) ϫ 10Ϫ8, in agreement with the previously
bacteriostatic drug erythromycin were used as comparator con- reported value for PC190723.
trols in these determinations. In accordance with CLSI standards,
FtsZ-targeting activity of TXY436. One of the hallmarks of
an MBC/MIC ratio of 1 to 2 is considered indicative of bactericidal FtsZ-targeting compounds that disrupt bacterial cell division is
behavior (29). In contrast, an MBC/MIC ratio Ն8 is viewed as the induction of an enlarged morphology in treated bacteria (7,
being indicative of bacteriostatic behavior. As expected, the con- 12, 27, 37). Therefore, in our initial studies probing the FtsZ-
trol bactericidal agent vancomycin yielded MBC/MIC ratios of 1 targeting activity of TXY436, we determined the impact of the
to 2 against all of the MSSA and MRSA strains examined (Table 3). compound on the morphology of S. aureus bacteria using phase-
Also, as expected, the control bacteriostatic drug erythromycin contrast microscopy. Control DMSO-treated S. aureus bacteria
yielded MBC/MIC ratios of Ͼ128 against the two MSSA strains. are ϳ1 ␮m in diameter, in agreement with previously reported
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FtsZ-Targeting Prodrug with Oral In Vivo Efficacy
purified S. aureus FtsZ in vitro. For this assay, we used a microtiter
plate-based spectrophotometric approach in which FtsZ poly-
merization is detected in solution by a time-dependent increase in
solution absorbance at 340 nm (A340). Figure 6A shows the time-
dependent A340 profiles of S. aureus FtsZ in the absence or pres-
ence of TXY436 at concentrations ranging from 1 to 10 ␮g/ml.
The corresponding results for PC190723 are also included for
comparative purposes (Fig. 6B). Note that TXY436 stimulates
FtsZ polymerization to a similar extent as PC190723, with the
magnitudes of these stimulatory effects also being similarly de-
pendent on compound concentration. Vancomycin was included
as a non-FtsZ-targeting control drug in these assays. As expected,
vancomycin did not impact the polymerization of S. aureus FtsZ
to any significant degree.
In addition to investigating the impact of TXY436 on the func-
tion of purified FtsZ in vitro, we also sought to examine the impact
of the compound on FtsZ function in live bacteria. To this end, we
monitored the impact of the compound on the formation of FtsZ
Z-rings using a strain of B. subtilis (FG347) that inducibly ex-
presses GFP-tagged ZapA, a known protein marker for FtsZ Z-
rings (30). We treated the FG347 bacteria with DMSO (solvent
control), TXY436, or PC190723 and examined the bacteria using
fluorescence microscopy. In the absence of compound, fluores-
cent foci corresponding to Z-rings are evident at midcell (high-
lighted by the arrows in Fig. 7A). In contrast, the TXY436-treated
bacteria lack these midcell foci, instead exhibiting punctate fluo-
rescent foci distributed throughout each cell (Fig. 7B). Recall that
TXY436 acts as an enhancer of FtsZ polymerization. It is therefore
likely that the punctate fluorescent foci distributed throughout
the TXY436-treated bacteria reflect multiple nonfunctional FtsZ
polymeric structures distinct from Z-rings. This type of behavior
is characteristic of agents such as PC190723 that stabilize FtsZ
polymers (Fig. 7C) (6, 7). In addition, the TXY436-treated bacte-
ria, like the PC190723-treated bacteria, are significantly more
elongated (filamentous) relative to the control DMSO-treated
bacteria, a morphological change typical of rod-shaped bacteria in
which cell division has been inhibited through disruption of FtsZ
function (6, 7, 30, 40–42).
Taken together, the MIC/MBC, time-kill, microscopy, and FtsZ
polymerization results described in the preceding sections indicate
that the TXY436 prodrug maintains all the antistaphylococcal and
FtsZ-targeting activities of the PC190723 product itself.
TXY436retainsantistaphylococcalactivityinthepresenceof
mouse serum. An important criterion for a compound with re-
gard to its potential for exhibiting antibacterial efficacy in vivo is
for it to retain antibacterial activity in the presence of serum. In
recognition of this important property, we assessed the impact of
50% (vol/vol) mouse serum on the activity of TXY436 against S.
FIG 4 Time-kill curves for S. aureus 8325-4 (MSSA). Each data point reflects
the average of two independent measurements, with the error bars reflecting
the standard deviation from the mean. Bacteria were treated with DMSO ve-
hicle only (no drug), TXY436 (A), PC190723 (B), or vancomycin (C). All
agents were used at concentrations ranging from 1ϫ to 8ϫ the MIC.
diameters characteristic of S. aureus (38) (Fig. 5A). In contrast, the aureus. As highlighted in Table 4, the presence of mouse serum
TXY436-treated bacteria exhibited 2- to 3-fold-enlarged pheno- resulted in a 4-fold reduction in the antistaphylococcal potency of
types, with diameters ranging from 2 to 3 ␮m (Fig. 5B). TXY436 (with the MIC increasing from 0.5 ␮g/ml in the absence of
PC190723-treated bacteria exhibited a similar behavior (Fig. 5C). serum to 2.0 ␮g/ml in the presence of serum). A similar reduction in
Thus, TXY436 is able to induce the type of morphological change potency was observed for PC190723 itself. Note that the MIC of
in S. aureus that is a hallmark of FtsZ-directed inhibitors of cell TXY436 in the presence of serum is identical to the corresponding
division such as PC190723 (6, 7).
Stimulation of FtsZ polymerization dynamics and stabilization retain antistaphylococcal activity in the presence of serum.
of nonfunctional FtsZ polymeric structures are thought to under- Pharmacokinetic analysis of the TXY436 prodrug and its
MIC for the control drug vancomycin. Thus, TXY436 is still able to
lie the antibacterial activities of FtsZ-targeting agents such as PC190723 product after both intravenous and oral administra-
PC190723 (7, 13, 27, 37, 39). We probed for the propensity of tion of TXY436 to mice reveals the prodrug product to be 73%
TXY436 to exert these types of effects on the polymerization of orally bioavailable. As a prelude to conducting in vivo efficacy
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Kaul et al.
FIG 5 Phase-contrast micrographs of S. aureus 8325-4 bacteria cultured for 4 h in the presence of DMSO (solvent control) (A), TXY436 at 3 ␮g/ml (B), or
PC190723 at 3 ␮g/ml (C).
experiments, we sought to evaluate the plasma pharmacokinetics
of the TXY436 prodrug and its PC190723 product after both i.v.
and p.o. administration of TXY436 to mice. In these studies, a
single i.v. dose of 5 mg/kg or p.o. dose of 10 mg/kg was adminis-
tered to mice, and the plasma concentrations of the TXY436 pro-
drug and the PC190723 product were measured at time points
ranging from 0.08 to 8 h using LC-MS. For both the i.v. and the
p.o. studies, 10 mM citrate (pH 2.6) was used as the vehicle. The
plasma concentration of TXY436 was detectable and quantifiable
up to 1 h after i.v. administration and up to 2 h after p.o. admin-
istration (Fig. 8A). The plasma concentration of PC190723 was
detectable and quantifiable up to 4 h after i.v. administration and
up to 8 h after p.o. administration (Fig. 8B).
Pharmacokinetic analyses of the time-dependent plasma con-
centration profiles for TXY436 and PC190723 with the NCA mod-
ule of the Phoenix WinNonlin version 6.3 software package
yielded the pharmacokinetic parameters listed in Table 5. Analysis
of the TXY436 plasma concentrations after i.v. administration
yielded an elimination half-life (t1/2) of 0.26 h and a high total
body clearance (CL) of 312 ml/min·kg, ϳ3.5-fold that of normal
liver blood flow in mice (90 ml/min·kg). The volume of distribu-
tion at steady state (Vss) was found to be 5.77 liters/kg. Signifi-
cantly, this value of Vss is ϳ8.2-fold greater than that of normal
body water (0.7 liters/kg), indicating substantive extravascular
distribution of the compound. After p.o. administration of
TXY436, the peak plasma concentration was achieved within 30
min (tmax ϭ 0.5 h). A comparison of the areas under the curves up
to the last detectable concentrations of TXY436 (AUClast) after
both p.o. and i.v. administration yields an absolute oral bioavail-
ability (%F) for the prodrug itself of 33% after normalization for
the dose.
Analysis of the PC190723 plasma concentrations after i.v. ad-
ministration of TXY436 yielded an ϳ4-fold-longer t1/2 (0.96 h)
FIG 6 Concentration dependence of the impact of TXY436 (A) or PC190723
than that of TXY436 and an ϳ3.6-fold-lower CL of 86.0 ml/min-
(B) on the polymerization of S. aureus FtsZ, as determined by monitoring
time-dependent changes in absorbance at 340 nm (A340) at 25°C. The time-
dependent A340 polymerization profiles were acquired in the presence of
kg, a value that is comparable to normal liver blood flow. The Vss
of PC190723 (5.39 liters/kg) was similar to that of TXY436, indic-
ative a similar degree of extravascular distribution. After p.o. ad-
ministration of TXY436, the peak plasma concentration of
PC190723 was achieved within 30 min (tmax ϭ 0.5 h). A compar-
ison of the AUClast values of PC190723 after both p.o. and i.v.
administration of TXY436 yields an absolute oral bioavailability
DMSO (solvent control) or the indicated concentrations of compound (rang-
ing from 1 to 10 ␮g/ml). The polymerization profile acquired in the presence
of vancomycin at 10 ␮g/ml is also included as a negative (non-FtsZ-targeting)
control. Experimental conditions for the polymerization studies were 5 ␮M
FtsZ, 50 mM Tris-HCl (pH 7.4), 50 mM KCl, and 10 mM magnesium acetate.
Polymerization reactions were initiated by the addition of 4 mM GTP at the
for the prodrug product of 73% after normalization for dose. times indicated by the arrows.
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FtsZ-Targeting Prodrug with Oral In Vivo Efficacy
FIG 7 Fluorescence micrographs of B. subtilis FG347 bacteria that express a GFP-tagged, Z-ring marker protein, ZapA. The bacteria were cultured for 1 h in the
presence of DMSO (solvent control) (A), TXY436 at 3 ␮g/ml (B), or PC190723 at 3 ␮g/ml (C), at which point GFP-ZapA expression was induced by the addition
of xylose. The bacteria were incubated for an additional hour at 37°C and then visualized by fluorescence microscopy. The arrows in panel A highlight FtsZ
Z-rings at midcell.
Thus, unlike many clinical MRSA drugs, including vancomycin, infection. Similarly, none of the mice treated with citrate vehicle
daptomycin, and the streptogramins, TXY436 is orally bioavail- alone survived beyond 2 days postinfection (Fig. 9B). However, in
able.
the TXY436-treated group, 5 of 6 mice (83%) survived for 3 days
TXY436 is orally efficacious in vivo against both MSSA and postinfection, and 4 of 6 mice (67%) survived the infection en-
MRSA systemic infections. Armed with the pharmacokinetic re-
sults described above, we next evaluated the oral in vivo efficacy of
TXY436 using a mouse peritonitis model of systemic infection
with both MSSA and MRSA. In the MSSA studies, four groups of
six female Swiss Webster mice were inoculated intraperitoneally
with a lethal inoculum of S. aureus ATCC 19636. The four groups
of infected mice were treated as follows: group 1, untreated; group
2, treated p.o. with 10 mM citrate vehicle alone; group 3, treated
p.o. with TXY436 at 128 mg/kg administered in four divided dos-
es; and group 4, treated p.o. with PC190723 at 128 mg/kg admin-
istered as a suspension in four divided doses. All untreated mice
died within 1 day of infection. In addition, none of the mice
treated with citrate vehicle alone or with PC190723 survived be-
yond 1 day postinfection (Fig. 9A). Thus, oral administration of
PC190723 itself was not efficacious in vivo. It is likely that the lack
of oral efficacy associated with PC190723 is related to the com-
pound being administered as a suspension (due to its poor solu-
bility), which may, in turn, limit the oral bioavailability of the
compound. In striking contrast to PC190723, oral administration
of TXY436 proved to be highly efficacious, with 100% of the
TXY436-treated mice surviving the infection.
In our MRSA studies, three groups of six mice were inoculated
intraperitoneally with a lethal inoculum of MRSA ATCC 43300.
The three groups of infected mice were treated as follows: group 1,
untreated; group 2, treated p.o. with 10 mM citrate vehicle alone;
and group 3, treated p.o. with TXY436 at 192 mg/kg administered
in six divided doses. All untreated mice died within 2 days of
TABLE 4 Impact of mouse serum on the antistaphylococcal activities of
TXY436 and PC190723
S. aureus 8325-4 MIC (␮g/ml)
Compound
No serum
50% mouse serum
TXY436
PC190723
Vancomycin
0.5
0.5
1.0
2.0
2.0
2.0
FIG 8 Time-dependent plasma concentrations of TXY436 (A) and PC190723
(B) after a single i.v. administration of TXY436 at 5 mg/kg (Œ) or a single p.o.
administration of TXY436 at 10 mg/kg (Œ) to male BALB/c mice.
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Kaul et al.
TABLE 5 Pharmacokinetic parameters of the TXY436 prodrug and the PC190723 product following a single intravenous or peroral administration
of TXY436 to male BALB/c micea
Compound
TXY436
Route
Dose (mg/kg)
t
max (h)
C0b (ng/liter)
AUClast (ng/liter·h)
t
1/2 (h)
CL (ml/min·kg)
V
ss (liters/kg)
%Fc
i.v.
p.o.
5
10
NA
0.50
878
178
252
164
0.26
NA
312
NA
5.77
NA
NA
33
PC190723
i.v.
p.o.
NA
NA
NA
0.50
978
343
923
1340
0.96
NA
86.0
NA
5.39
NA
NA
73
a Parameters were calculated by analysis of the plasma concentration versus time plots shown in Fig. 8 using the sparse sampling mode in the noncompartmental analysis (NCA)
module of the Phoenix WinNonlin version 6.3 software package. NA, not applicable.; i.v., intravenous; p.o., peroral.
b An extrapolated concentration was used for the i.v. group.
c The AUClast value was used to calculate the %F value.
tirely. Thus, TXY436 is not only orally efficacious against systemic cytotoxicity, if any, of TXY436 against Vero African green monkey
MSSA infection but also against systemic MRSA infection.
kidney epithelial cells. TXY436 was found to be minimally toxic to
In both the MSSA and the MRSA studies, an additional positive Vero cells, with a 50% inhibitory concentration (IC50) of 64 ␮g/
control group of six infected mice was also included. This group ml. Significantly, this IC50 is 64ϫ to 128ϫ the antistaphylococcal
was treated i.v. with vancomycin at doses of 16 mg/kg in the MSSA MICs of TXY436 (which range from 0.5 to 1.0 ␮g/ml). Albeit
studies and 24 mg/kg in the MRSA studies. As anticipated, all derived from a single mammalian cell line, this differential sug-
vancomycin-treated mice survived their infections.
gests that TXY436 is associated with a significant therapeutic win-
TXY436 is minimally toxic to mammalian cells. An impor- dow. Future studies with additional mammalian and human cell
tant criterion for the potential clinical utility of an antibacterial lines will be directed toward assessing the general applicability of
agent is minimal toxicity against mammalian cells. In this connec- this therapeutic window.
tion, we used a 3-day tetrazole (MTT)-based assay to assess the
In conclusion, TXY436 is a prodrug of the FtsZ-targeting benz-
amide PC190273 with enhanced formulation properties as well as
oral efficacy in vivo against both MSSA and MRSA infections.
These characteristics, coupled with a minimal potential for toxic-
ity to mammalian cells, make TXY436 a worthy candidate for
further development as a clinically useful agent to treat staphylo-
coccal infections.
ACKNOWLEDGMENTS
This study was supported by research agreements between TAXIS Phar-
maceuticals, Inc., and both Rutgers Robert Wood Johnson Medical
School (D.S.P.) and Rutgers Ernest Mario School of Pharmacy (E.J.L.).
We are indebted to Nancy Connell (Rutgers New Jersey Medical
School, Newark, NJ) for assistance with the mammalian cytotoxicity stud-
ies. We are also indebted to Glenn W. Kaatz (John D. Dingell VA Medical
Center, Detroit, MI) for providing the S. aureus 8325-4 strain and to
Richard Losick (Harvard University, Boston, MA) for providing the B.
subtilis FG347 strain.
REFERENCES
1. Bi EF, Lutkenhaus J. 1991. FtsZ ring structure associated with division in
Escherichia coli. Nature 354:161–164.
2. Errington J, Daniel RA, Scheffers DJ. 2003. Cytokinesis in bacteria.
Microbiol. Mol. Biol. Rev. 67:52–65.
3. Adams DW, Errington J. 2009. Bacterial cell division: assembly, mainte-
nance, and disassembly of the Z ring. Nat. Rev. Microbiol. 7:642–653.
4. Kirkpatrick CL, Viollier PH. 2011. New(s) to the (Z-)ring. Curr. Opin.
Microbiol. 14:691–697.
5. Erickson HP, Anderson DE, Osawa M. 2010. FtsZ in bacterial cytokine-
sis: cytoskeleton and force generator all in one. Microbiol. Mol. Biol. Rev.
74:504–528.
6. Stokes NR, Sievers J, Barker S, Bennett JM, Brown DR, Collins I,
Errington VM, Foulger D, Hall M, Halsey R, Johnson H, Rose V,
Thomaides HB, Haydon DJ, Czaplewski LG, Errington J. 2005. Novel
inhibitors of bacterial cytokinesis identified by a cell-based antibiotic
screening assay. J. Biol. Chem. 280:39709–39715.
7. Haydon DJ, Stokes NR, Ure R, Galbraith G, Bennett JM, Brown DR,
Baker PJ, Barynin VV, Rice DW, Sedelnikova SE, Heal JR, Sheridan JM,
Aiwale ST, Chauhan PK, Srivastava A, Taneja A, Collins I, Errington J,
Czaplewski LG. 2008. An inhibitor of FtsZ with potent and selective
antistaphylococcal activity. Science 321:1673–1675.
FIG 9 Oral (p.o.) in vivo efficacy of TXY436 in a mouse peritonitis model of
systemic infection with MSSA 8325-4 (A) or MRSA ATCC 43300 (B). In the
MSSA experiments, TXY436 and PC190723 were administered at 128 mg/kg in
four divided doses. In the MRSA experiments, TXY436 was administered at
192 mg/kg in six divided doses. The vehicle was 10 mM citrate (pH 2.6) in all
experiments.
5868 aac.asm.org
Antimicrobial Agents and Chemotherapy

FtsZ-Targeting Prodrug with Oral In Vivo Efficacy
8. Huang Q, Kirikae F, Kirikae T, Pepe A, Amin A, Respicio L, Slayden
RA, Tonge PJ, Ojima I. 2006. Targeting FtsZ for antituberculosis drug
discovery: noncytotoxic taxanes as novel antituberculosis agents. J. Med.
Chem. 49:463–466.
9. Czaplewski LG, Collins I, Boyd EA, Brown D, East SP, Gardiner M,
Fletcher R, Haydon DJ, Henstock V, Ingram P, Jones C, Noula C,
Kennison L, Rockley C, Rose V, Thomaides-Brears HB, Ure R, Whit-
taker M, Stokes NR. 2009. Antibacterial alkoxybenzamide inhibitors of
the essential bacterial cell division protein FtsZ. Bioorg. Med. Chem. Lett.
19:524–527.
10. Schaffner-Barbero C, Martin-Fontecha M, Chacón P, Andreu JM. 2012.
Targeting the assembly of bacterial cell division protein FtsZ with small
molecules. ACS Chem. Biol. 7:269–277.
11. Ma S. 2012. The development of FtsZ inhibitors as potential antibacterial
agents. ChemMedChem 7:1161–1172.
12. Haydon DJ, Bennett JM, Brown D, Collins I, Galbraith G, Lancett P,
Macdonald R, Stokes NR, Chauhan PK, Sutariya JK, Nayal N, Srivas-
tava A, Beanland J, Hall R, Henstock V, Noula C, Rockley C,
Czaplewski L. 2010. Creating an antibacterial with in vivo efficacy: syn-
thesis and characterization of potent inhibitors of the bacterial cell divi-
sion protein FtsZ with improved pharmaceutical properties. J. Med.
Chem. 53:3927–3936.
13. Andreu JM, Schaffner-Barbero C, Huecas S, Alonso D, Lopez-
Rodriguez ML, Ruiz-Avila LB, Núñez-Ramírez R, Llorca O, Martín-
Galiano AJ. 2010. The antibacterial cell division inhibitor PC190723 is an
FtsZ polymer-stabilizing agent that induces filament assembly and con-
densation. J. Biol. Chem. 285:14239–14246.
14. Lock RL, Harry EJ. 2008. Cell-division inhibitors: new insights for future
antibiotics. Nat. Rev. Drug Discov. 7:324–338.
15. Boberek JM, Stach J, Good L. 2010. Genetic evidence for inhibition of
bacterial division protein FtsZ by berberine. PLoS One 5:e13745. doi:10
.1371/journal.pone.0013745.
16. Awasthi D, Kumar K, Ojima I. 2011. Therapeutic potential of FtsZ
inhibition: a patent perspective. Expert Opin. Ther. Patents 21:657–679.
17. Huang Q, Tonge PJ, Slayden RA, Kirikae T, Ojima I. 2007. FtsZ: a novel
target for tuberculosis drug discovery. Curr. Top. Med. Chem. 7:527–543.
18. Kumar K, Awasthi D, Berger WT, Tonge PJ, Slayden RA, Ojima I. 2010.
Discovery of anti-TB agents that target the cell-division protein FtsZ. Fu-
ture Med. Chem. 2:1305–1323.
19. Kumar K, Awasthi D, Lee SY, Zanardi I, Ruzsicska B, Knudson S,
Tonge PJ, Slayden RA, Ojima I. 2011. Novel trisubstituted benzimida-
zoles, targeting Mtb FtsZ, as a new class of antitubercular agents. J. Med.
Chem. 54:374–381.
20. Kapoor S, Panda D. 2009. Targeting FtsZ for antibacterial therapy: a
promising avenue. Expert Opin. Ther. Targets 13:1037–1051.
21. Kaul M, Parhi AK, Zhang Y, LaVoie EJ, Tuske S, Arnold E, Kerrigan JE,
Pilch DS. 2012. A bactericidal guanidinomethyl biaryl that alters the dy-
namics of bacterial FtsZ polymerization. J. Med. Chem. 55:10160–10176.
22. Kelley C, Zhang Y, Parhi A, Kaul M, Pilch DS, LaVoie EJ. 2012.
3-Phenyl substituted 6,7-dimethoxyisoquinoline derivatives as FtsZ-
targeting antibacterial agents. Bioorg. Med. Chem. 20:7012–7029.
23. Kelley C, Lu S, Parhi A, Kaul M, Pilch DS, LaVoie EJ. 2013. Antimi-
crobial activity of various 4- and 5-substituted 1-phenylnaphthalenes.
Eur. J. Med. Chem. 60:395–409.
Yadav A, Srivastava A, Haydon DJ. 2013. An improved small-molecule
inhibitor of FtsZ with superior in vitro potency, drug-like properties, and
in vivo efficacy. Antimicrob. Agents Chemother. 57:317–325.
27. Adams DW, Wu LJ, Czaplewski LG, Errington J. 2011. Multiple effects
of benzamide antibiotics on FtsZ function. Mol. Microbiol. 80:68–84.
28. Sorto NA, Olmstead MM, Shaw JT. 2010. Practical synthesis of
PC190723, an inhibitor of the bacterial cell division protein FtsZ. J. Org.
Chem. 75:7946–7949.
29. CLSI. 2009. Methods for dilution antimicrobial susceptibility tests for
bacteria that grow aerobically; approved standard, 8th ed. Document
M07-A8. Clinical and Laboratory Standards Institute, Wayne, PA.
30. Gueiros-Filho FJ, Losick R. 2002. A widely conserved bacterial cell divi-
sion protein that promotes assembly of the tubulin-like protein FtsZ.
Genes Dev. 16:2544–2556.
31. O’Neill AJ, Cove JH, Chopra I. 2001. Mutation frequencies for resistance
to fusidic acid and rifampicin in Staphylococcus aureus. J. Antimicrob.
Chemother. 47:647–650.
32. Stiles BG, Campbell YG, Castle RM, Grove SA. 1999. Correlation of
temperature and toxicity in murine studies of staphylococcal enterotoxins
and toxic shock syndrome toxin 1. Infect. Immun. 67:1521–1525.
33. Ekins S, Reynolds RC, Kim H, Koo M-S, Ekonomidis M, Talaue M,
Paget SD, Woolhiser LK, Lenaerts AJ, Bunin BA, Connell N, Freundlich
JS. 2013. Bayesian models leveraging bioactivity and cytotoxicity informa-
tion for drug discovery. Chem. Biol. 20:370–378.
34. Bundgaard H, Johansen M. 1980. Prodrugs as drug delivery systems. IV.
N-Mannich bases as potential novel prodrugs for amides, ureides, amines,
and other NH-acidic compounds. J. Pharm. Sci. 69:44–46.
35. Huttunen KM, Rautio J. 2011. Prodrugs: an efficient way to breach
delivery and targeting barriers. Curr. Top. Med. Chem. 11:2265–2287.
36. Dhareshwar SS, Stella VJ. 2008. Your prodrug releases formaldehyde:
should you be concerned? No! J. Pharm. Sci. 97:4184–4193.
37. Tan CM, Therien AG, Lu J, Lee SH, Caron A, Gill CJ, Lebeau-Jacob C,
Benton-Perdomo L, Monteiro JM, Pereira PM, Elsen NL, Wu J, Des-
champs K, Petcu M, Wong S, Daigneault E, Kramer S, Liang L, Maxwell
E, Claveau D, Vaillancourt J, Skorey K, Tam J, Wang H, Meredith TC,
Sillaots S, Wang-Jarantow L, Ramtohul Y, Langlois E, Landry F, Reid
JC, Parthasarathy G, Sharma S, Baryshnikova A, Lumb KJ, Pinho MG,
Soisson SM, Roemer T. 2012. Restoring methicillin-resistant Staphylo-
coccus aureus susceptibility to ␤-lactam antibiotics. Sci. Transl. Med.
4:126ra135.
38. Jorge AM, Hoiczyk E, Gomes JP, Pinho MG. 2011. EzrA contributes to
the regulation of cell size in Staphylococcus aureus. PLoS One 6:e27542.
doi:10.1371/journal.pone.0027542.
39. Kaul M, Zhang Y, Parhi AK, LaVoie EJ, Tuske S, Arnold E, Kerrigan JE,
Pilch DS. 2013. Enterococcal and streptococcal resistance to PC190723
and related compounds: molecular insights from a FtsZ mutational anal-
ysis. Biochimie 95:1880–1887.
40. Haeusser DP, Garza AC, Buscher AZ, Levin PA. 2007. The division
inhibitor EzrA contains a seven-residue patch required for maintaining
the dynamic nature of the medial FtsZ ring. J. Bacteriol. 189:9001–9010.
41. Haeusser DP, Schwartz RL, Smith AM, Oates ME, Levin PA. 2004. EzrA
prevents aberrant cell division by modulating assembly of the cytoskeletal
protein FtsZ. Mol. Microbiol. 52:801–814.
42. Ma X, Ehrhardt DW, Margolin W. 1996. Colocalization of cell division
proteins FtsZ and FtsA to cytoskeletal structures in living Escherichia coli
cells by using green fluorescent protein. Proc. Natl. Acad. Sci. U. S. A.
93:12998–13003.
43. Hiramatsu K, Aritaka N, Hanaki H, Kawasaki S, Hosoda Y, Hori S,
Fukuchi Y, Kobayashi I. 1997. Dissemination in Japanese hospitals of
strains of Staphylococcus aureus heterogeneously resistant to vancomycin.
Lancet 350:1670–1673.
24. Parhi A, Lu S, Kelley C, Kaul M, Pilch DS, LaVoie EJ. 2012. Antibac-
terial activity of substituted dibenzo[a,g]quinolizin-7-ium derivatives.
Bioorg. Med. Chem. Lett. 22:6962–6966.
25. Zhang Y, Giurleo D, Parhi A, Kaul M, Pilch DS, LaVoie EJ. 2013.
Substituted 1,6-diphenylnaphthalenes as FtsZ-targeting antibacterial
agents. Bioorg. Med. Chem. Lett. 23:2001–2006.
26. Stokes NR, Baker N, Bennett JM, Berry J, Collins I, Czaplewski LG,
Logan A, Macdonald R, Macleod L, Peasley H, Mitchell JP, Nayal N,
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