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Antimicrobial Agents and Chemotherapy, August 1999, p. 1982-1987, Vol. 43, No. 8
0066-4804/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Inhibitors of DNA Polymerase III as Novel
Antimicrobial Agents against Gram-Positive Eubacteria
Paul M.
Tarantino Jr.,
Chengxin
Zhi,
George E.
Wright, and
Neal C.
Brown*
Department of Pharmacology and Molecular
Toxicology, University of Massachusetts Medical School, Worcester,
Massachusetts 01655
Received 14 December 1998/Returned for modification 22 March
1999/Accepted 2 June 1999
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ABSTRACT |
6-Anilinouracils are selective inhibitors of DNA polymerase III,
the enzyme required for the replication of chromosomal DNA in
gram-positive bacteria (N. C. Brown, L. W. Dudycz, and
G. E. Wright, Drugs Exp. Clin. Res. 12:555-564, 1986). A new
class of 6-anilinouracils based on N-3 alkyl substitution of the uracil ring was synthesized and analyzed for activity as inhibitors of the
gram-positive bacterial DNA polymerase III and the growth of
gram-positive bacterial pathogens. Favorable in vitro properties of
N-3-alkyl derivatives prompted the synthesis of derivatives in which
the R group at N-3 was replaced with more-hydrophilic methoxyalkyl and
hydroxyalkyl groups. These hydroxyalkyl and methoxyalkyl derivatives
displayed Ki values in the range from 0.4 to
2.8 µM against relevant gram-positive bacterial DNA polymerase IIIs
and antimicrobial activity with MICs in the range from 0.5 to 15 µg/ml against a broad spectrum of gram-positive bacteria, including methicillin-resistant staphylococci and vancomycin-resistant
enterococci. Two of these hydrophilic derivatives displayed protective
activity in a simple mouse model of lethal staphylococcal infection.
| |
INTRODUCTION |
The escalating incidence of
infection with multiple-antibiotic-resistant forms of low G+C content
gram-positive bacteria is a major and rapidly growing clinical problem
(20). In an effort to address this problem, we have sought
to identify a new gram-positive-bacterium-specific antimicrobial target
and to develop selective "bullets" to hit it. The target we have
selected is DNA polymerase III (pol III), the product of the
polC gene (10, 13, 15, 16).
We have targeted the polC-specific pol III for three
reasons. First, it is absolutely essential for the replication of the host chromosome of the low G+C content gram-positive bacteria; when its
action is blocked, chromosomal DNA fails to replicate and the bacterial
host dies (5, 6, 10, 15, 25). Second, this pol III is a
target whose essential structure is strongly conserved in a broad
group of relevant low G+C content gram-positive pathogens, including
Staphylococcus, Streptococcus,
Enterococcus, and Mycoplasma (2, 13).
Third, the active-site domain of this enzyme incorporates a unique
receptor which renders it specifically susceptible to the small
molecule inhibitors of the 6-anilinouracil (AU) class (see references
6 and 7 and structure shown in Fig. 1A, below). It is this AU class of pol III-specific
inhibitors which we seek to develop as
gram-positive-bacterium-selective antimicrobials. The results
described below summarize the progress we have made in this effort.
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MATERIALS AND METHODS |
Determination of water solubility.
A known mass of pure,
dried compound was dissolved in dilute NaOH at pH 12.0. The solution
was then neutralized to pH 7.0 with 0.1 N HCl for determination of the
extinction coefficient at the absorbance maximum. To determine water
solubility, an excess of each compound was stirred in deionized water
(pH 7.0) at 25°C for 1 h. After removal of undissolved compound
by filtration, the absorbance of the solution at the absorbance maximum
was determined and used to calculate the concentration of the compound
remaining in solution. Each reported value is the mean of three
independent experiments.
Bacterial strains and media.
Bacillus subtilis was the
standard penicillin-sensitive laboratory strain, BD54 (16).
Enterococcus faecalis, Enterococcus faecium, and
Staphylococcus aureus were clinically derived strains which
were typed and kindly provided by Gary Doern, University of
Massachusetts Medical Center. Unless noted otherwise, bacteria were
grown in Luria broth medium consisting of 0.5% yeast extract (Difco),
1% tryptone (Bacto-tryptone; Difco) and 0.5% NaCl in deionized water.
Inhibitors.
N-3-substituted AUs were synthesized, purified,
and analyzed as described in reference 19.
6-(p-Hydroxyphenylazo)uracil (HPUra) and other AUs were
synthesized and purified as described (26).
Enzymes.
The pol IIIs of B. subtilis and S. aureus were homogeneous recombinant proteins expressed and
prepared as described (12, 18). E. faecalis pol
III was fraction V purified from ATCC 8043 as described by Barnes and
Brown (1). The dnaE-encoded E. coli pol III was the recombinant form of the reconstituted core
enzyme (alpha-epsilon-theta [14]) and was kindly
provided by Charles McHenry, University of Colorado, Denver.
Immunopurified calf thymus DNA pol alpha was kindly provided by Ulrich
Hübscher of the University of Zürich-Irchel, Zürich, Switzerland.
Determination of MIC.
Log-phase bacterial cultures were
diluted to a concentration of ~105 CFU per ml in Luria
broth, and samples of this suspension were distributed to the wells of
a 48-well microassay plate. Each compound was dissolved in
dimethylsulfoxide (DMSO) and added to one well at a concentration of
200 µM. The contents of this well were then serially diluted, twofold
each time, through seven wells, to produce a series of eight wells
containing 0.5 ml of suspension and compound at the following
successive concentrations: 200, 100, 50, 25, 12.5, 6.25, 3.125, and
1.5625 µM. All wells, including controls incubated in the absence of
inhibitors, contained 1% DMSO. DMSO at this concentration did not
significantly affect the growth of any of the test organisms. Plates
were incubated for 24 h at 37°C and read by visual inspection of
the wells. MIC was defined as the lowest concentration of inhibitor at
which bacterial growth was not visually apparent. Reported values are
the median values for three experiments. MICs were converted from
micromolar concentrations to micrograms per milliliter for tabulation.
Bactericidal activity.
Each inhibitor was dissolved in
sterile DMSO and diluted 100-fold into Mueller-Hinton broth (MHB;
Difco) containing log-phase methicillin-sensitive S. aureus
(MSSA) (Smith strain) at a concentration of ~106 CFU per
ml. The control culture contained only 1% DMSO. The cultures were
incubated at 37°C, and at intervals during a 24-h period, samples
were removed, diluted extensively in sterile MHB, and plated on Luria
broth-based agar plates to determine the numbers of CFU.
DNA polymerase activity.
DNA polymerases were assayed by
using activated calf thymus DNA and the basic method described in
reference 1. Briefly, enzyme was added to buffered
solution containing Mg2+, dithiothreitol, glycerol,
saturating concentrations of activated calf thymus DNA, dATP, dCTP,
dTTP, and [3H]dTTP, and appropriate concentrations of
inhibitor included as a dilution of a stock solution in DMSO. Assay
mixtures were incubated at 30°C for 10 min, reactions were stopped
with cold trichloroacetic acid, and mixtures were filtered to capture
the cold acid-insoluble 3H-labeled DNA. The filters were
washed in turn with cold trichloroacetic acid and ethanol and then
dried. The incorporated radioactivity was measured by scintillation
counting of the dried filters as described (1). Apparent
inhibitor constants (Kis) of the AUs were
determined directly by truncated assay in the absence of dGTP as
described by Wright and Brown (23). Each reported value is
the average for three independent experiments. The average standard
deviation for the values presented was ±17.4%.
Assay for protection against lethal staphylococcal infection in
vivo.
This assay was performed by the Pharmacology Services
Laboratory of MDS Panlabs, Inc. Female Swiss-Webster mice (20 g) were infected with a single intraperitoneal injection of MSSA (ATCC Smith
strain; 0.5 ml in physiological saline; 4 × 107
CFU/mouse), and 15 min thereafter, mice in each group of ten infected
animals were injected intraperitoneally once with one of the following
solutions: (i) 0.1 ml of physiological saline (vancomycin vehicle
control); (ii) 0.1 ml of vancomycin (4 mg/ml in physiological saline;
dose, 20 mg/kg of body weight); (iii) 0.1 ml of AU vehicle control
(10% DMSO-90% peanut oil); or (iv) 0.1 ml of 10% DMSO-90% peanut
oil containing the relevant N-3-substituted AU at a concentration
sufficient to give a dose of either 5 or 10 mg/kg. Following injection,
each group of ten animals was monitored for survival over a 3-day period.
Assay of DNA and RNA synthesis in B. subtilis
BD54.
At time zero [3H]adenine (7 × 105 cpm/pmol; final concentration, 0.1 mM) and inhibitor or
inhibitor diluent were added to a log-phase culture growing at 37°C
in a minimal medium (8) with a doubling time of 35 min.
After 30 min, duplicate 1-ml samples were removed to determine
incorporation into alkali-soluble (RNA) and alkali-stable (DNA)
fractions of cold trichloroacetic acid-insoluble material as described
previously (4).
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RESULTS AND DISCUSSION |
Summary of the mechanism of action of AUs on the gram-positive
bacterial pol III.
Although simple molecules, the AUs have two
distinct domains essential for their inhibitory action. These are
illustrated in Fig. 1A. One, the
so-called base-pairing domain, is comprised of three uracil-specific
components
the ring 1-NH, the 2-keto, and the 6-NH components. The
second, enzyme-specific, domain is contributed by an appropriately
substituted aryl group at N-6. Although formally pyrimidines, the AUs
derive their capacity to inhibit pol III by mimicking the guanine
component of the purine 2'-deoxyribonucleoside triphosphate dGTP. As
shown in Fig. 1B, the AUs derive this property from the capacity of the
unconventional base-pairing domain to specifically form three hydrogen
bonds with the pyrimidine base cytosine. Fig. 1C summarizes how the base-pairing and enzyme-specific domains of the AU molecule cooperate to inhibit pol III as it tries to elongate the primer terminus past a
cytosine residue in the DNA template. The base-pairing domain of the
inhibitor forms hydrogen bonds with the cytosine, and simultaneously,
the enzyme-specific aryl moiety binds to the enzyme's unique aryl
receptor. As a result, the inhibitor sequesters the enzyme with high
affinity into a nonproductive ternary complex with template-primer
(5, 9, 10).
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FIG. 1.
Structures and mechanisms of action of AUs which
selectively inhibit the gram-positive pol III. (A) Relevant domains of
the AU molecule. (B) Equivalence of the base-pairing domains of the
guanine (left) and AU (right) molecules. (C) The AU molecule inhibits
its pol III target by sequestering it into an inactive DNA-drug-protein
complex. PUR, purine; PYR, pyrimidine. (D) Structures of two relevant
AUs, EMAU and TMAU.
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Effect of alkyl substitution at N-3 on antibacterial and anti-pol
III activities of AUs.
Among the AUs which we have
synthesized (5, 7, 23-26), two of the most potent,
6-(3-ethyl-4-methylanilino)uracil (EMAU) and
6-([3,4-trimethylene]anilino)uracil (TMAU), contain
alkyl groups in the 3 and 4 positions of the anilino ring (Fig. 1D
shows structures). Although TMAU and EMAU were potent inhibitors of the
gram-positive bacterial pol III (26), they showed weak
antibacterial activity, yielding MICs of 9 to 18 µg/ml (Table
1). In an attempt to improve their
antibacterial activities, we developed structure-activity relationships
involving substituents at ring nitrogen 3 (see numbering in Fig. 1B and
D), the only position in the AU molecule where nondestructive
substitution is feasible (21).
The first structure-activity relationship to exploit N-3
alkylation was performed on the TMAU platform. It had two
components,
one of which assessed antimicrobial potency and the other
of which
assessed potency for enzyme inhibition. The former utilized
several
strains of relevant gram-positive bacteria, while the latter
utilized
purified pol IIIs derived from three relevant
gram-positive organisms
B. subtilis,
S. aureus,
and
E. faecalis.
Table
1 summarizes the relative antibacterial activities (i.e., MICs)
of a series of N-3-substituted TMAUs. The MIC results
show that N-3
alkylation with up to three carbons generally improved
potency for all
organisms relative to that of the unsubstituted
TMAU. The ethyl and
allyl groups increased potency, but larger
groups, such as the butyl
group, did not. Derivatives with the
largest substituents, hexyl and
benzyl, had considerably weaker
activities than
TMAU.
Table
2 summarizes the effect of N-3
alkylation of TMAU on its potency against the three isolated pol IIIs.
The results,
displayed as apparent inhibitor constants
(
Kis [
23]), indicate
that
potency increased with increasing size of the N-3 alkyl chain,
reached
a maximum (>10-fold) with addition of the butyl substituent,
and
diminished with addition of the larger, hexyl group. Addition
of
the benzyl group had a decidedly negative effect on anti-pol
activity,
reducing it 15- to 18-fold relative to that for the
unsubstituted
TMAU.
To determine whether the effects of N-3 substitution on TMAU were
applicable more generally to another active AU platform,
we synthesized
and tested the ethyl and allyl derivatives of EMAU.
The results are
summarized in the bottom three rows of Tables
1 and
2.
With respect to antibacterial activity (Table
1), ethyl or allyl
substitution of EMAU had essentially the same effect as
it had on TMAU,
i.e., it increased potency against all organisms
except
E. faecium. The effect of ethyl or allyl substitution was
most
dramatic in the case of
B. subtilis, increasing potency
approximately
20-fold. In all cases, the N-3 ethyl form of EMAU
(Et-EMAU) displayed
slightly greater potency than the allyl derivative.
For the two
strains of methicillin-resistant
S. aureus
(MRSA) both the N-3
allyl form of EMAU and Et-EMAU displayed potency
less than twofold
greater than that of the unsubstituted EMAU
molecule. For the
other strains, N-3 alkyl substitution increased
potency from 2-
to 18-fold.
Both N-3 substituents significantly increased the anti-pol III potency
of EMAU (Table
2), and with the exception of
E. faecalis pol
III, the orders of relative potency were qualitatively the
same as
those for the ethyl and allyl derivatives of TMAU (Table
2). In the
case of
E. faecalis pol III, the respective potencies
of the
ethyl and allyl forms did not demonstrate a specific rank
order. As
anticipated, N-3 substitution also did not apparently
change the
selectivity of the molecule (
5) for low G+C content
gram-positive bacteria as evidenced by the lack of significant
inhibitory effect of high concentrations on the growth of the
gram-negative organism
Escherichia coli (Table
1, right
column).
In sum, the above results indicated that N-3 substitution on the AU
platform can lead to increases in both the antibacterial
and anti-pol
III activities of these gram-positive-bacterium-selective
inhibitors.
This effect was apparently maximal with N-3 ethyl
and allyl
substitutions. As expected for compounds which putatively
hit a
novel target, the N-3 alkyl-substituted AUs were essentially
equivalent
with respect to potency against antibiotic-sensitive
and
antibiotic-resistant strains of relevant gram-positive
organisms.
Effects of hydrophilic N-3 substitution of EMAU.
Given their
strong anti-pol III actions and their favorable in vitro antimicrobial
potencies, we attempted to assess Et-EMAU and Et-TMAU for their
capacities to prevent gram-positive bacterial infection in mice and
thus "prove principle" in vivo. However, their marginal aqueous
solubilities (3 µM for Et-TMAU and 6.3 µM for Et-EMAU; determined
as described in the Materials and Methods section) severely restricted
their bioavailabilities following either oral or parenteral
administration and therefore precluded their use for this purpose. To
improve water solubility and bioavailability, we focused on the EMAU
platform and proceeded to synthesize two N-3 hydroxyalkyl forms, i.e.,
the 2-hydroxyethyl derivative, HE-EMAU, and the 3-hydroxypropyl
derivative, HP-EMAU (19). As expected, the water
solubilities of both agents were increased significantly compared to
that of Et-EMAU. The aqueous solubility of HE-EMAU was 32 µM, and
that of HP-EMAU was 98 µM. Even the respective methoxyalkyl
intermediates from which HE-EMAU and HP-EMAU were synthesized were more
water soluble than Et-EMAU (the water solubility of the
2-methoxyethyl intermediate [ME-EMAU] was 23 µM, and that of the 3-methoxypropyl intermediate [MP-EMAU] was 22 µM).
Table
3 summarizes the effects of the N-3
hydroxyalkyl and methoxyalkyl substitutions on the antimicrobial
activity of the
EMAU platform. These substituents all enhanced
antibiotic activity
relative to that of EMAU; their MICs, with the
exceptions of those
for vancomycin-resistant
E. faecalis
(VRE; Table
1) and MSSA
(Table
1), equalled or were better than those
of Et-EMAU. Significantly,
the propyl derivatives, MP-EMAU and HP-EMAU,
were as active against
the MRSA and VRE strains as they were against
the corresponding
antibiotic-sensitive strains, and like the
alkyl-substituted derivatives,
none of these more-hydrophilic compounds
was active against
E. coli at ~60 µg/ml, the highest
concentration tested (right column,
Table
3).
The anti-polymerase activities of the methoxyalkyl- and hydroxyalkyl
EMAUs are summarized in Table
4. These
substituents
did not significantly change either the potency of the
EMAU derivative
or its selectivity for the gram-positive bacterial pol
III. All
three gram-positive bacterial pol IIIs were clearly sensitive
to the four compounds, with
S. aureus pol III slightly less
sensitive
than the enzymes of
B. subtilis and
E. faecalis. In contrast,
none of the four agents displayed
significant inhibitory activity
against either the
dnaE-specific gram-negative bacterial (
E. coli)
pol III or the mammalian (calf thymus) replicative enzyme, pol
alpha.
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TABLE 4.
Inhibitor constants (Kis) of N-3
hydroxyalkyl and methoxyalkyl EMAU derivatives against
relevant DNA polymerases
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Bactericidal properties of the methoxyalkyl and hydroxyalkyl
EMAUs.
Our earlier studies of the effects of HPUra, the prototype
for the AUs, indicated that it was bactericidal for E. faecalis (6). To determine if the N-3-substituted
hydroxyalkyl and methoxyalkyl EMAUs also were bactericidal,
time-kill studies were conducted with the MSSA Smith strain which was
to be used (see below) to examine their abilities to protect against in
vivo infection. The results are summarized in Fig.
2. As shown in panel A, the ethyl
derivatives, HE-EMAU and ME-EMAU, were bactericidal at 4× MIC, while
at MIC both of them permitted some cell replication. The two propyl
compounds, HP-EMAU and MP-EMAU (Fig. 2, panel B), were clearly
bactericidal at 4× MIC, while at MIC they were, at best,
bacteriostatic.
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FIG. 2.
Bactericidal activities of N-3 methoxyalkyl- and
hydroxyalkyl-EMAUs against S. aureus (Smith strain).
Each of the four agents was tested, as indicated, at its MIC and at
four times (4×) its MIC. The MICs of the agents for this strain were
as follows: HE-EMAU, 8 mg/ml; ME-EMAU, 16 mg/ml; HP-EMAU, 8 mg/ml; and
MP-EMAU, 8 mg/ml. (A) Activities of HE-EMAU and ME-EMAU. (B) Activities
of HP-EMAU and MP-EMAU.
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Preservation of other key properties of HPUra.
In addition to
its bactericidal effect, HPUra has three well-documented properties
which characterize its antibacterial action (5, 6, 9, 10, 17,
25). The first is its spectrum. HPUra is active against only low
G+C content gram-positive eubacteria and Mycoplasmatales
(3) and thus has no significant effect on the growth of
gram-negative eubacteria, mammalian cells, or high G+C content
gram-positive eubacteria such as Streptomyces and
Mycobacterium (4). The second distinct property
of HPUra is its selectivity for replicative DNA synthesis.
Administration of the agent to an exponentially growing culture of a
sensitive organism at concentrations up to 20× MIC completely inhibits
the synthesis of DNA with little or no effect on the synthesis of RNA
or other macromolecules (5, 6, 8). The third important characteristic of HPUra is its target within the DNA replication machinery. It is absolutely specific for the polC-encoded
pol III enzyme that is unique to low G+C content gram-positive
eubacteria (10, 11, 13, 25). The active form of HPUra has no
inhibitory effect on a wide variety of prokaryotic and eukaryotic
polymerasesincluding the mammalian pol alpha and the pol IIIs of
gram-negative and high G+C content gram-positive bacteria (4-6,
10, 11).
Considering their potential for further development as antibiotics (see
below), we sought to determine whether the latest
generation of soluble
EMAUs (i.e., HE-EMAU, ME-EMAU, HP-EMAU,
and MP-EMAU) faithfully
retained the HPUra-like specificity for
DNA replication and pol
III target. We assessed their pol III
specificities by exploiting
E. coli pol III and calf thymus DNA
pol alpha as relevant
control enzymes. As the results shown in
the rightmost columns of Table
4 indicate, the four compounds
displayed HPUra-like specificity; none
of them displayed significant
activity against either of these test
enzymes.
We next sought to determine if the N-3 hydroxyalkyl- and N-3
methoxyalkyl-EMAUs also conserve HPUra's specificity for DNA
replication in the intact cell. To this end, we exploited an
experimental
approach that we used previously to assess the pol III and
DNA
replication specificities of
N2-(3,4-dichlorobenzyl)guanine, an even more
distant guanine derivative
of the HPUra pharmacophore (
4).
The experiment compared the
effect of each agent at 100 µM with that
of 100 µM HPUra on the
incorporation of [
3H]adenine
into RNA and DNA (see the Materials and Methods section
for
experimental details). The results, expressed as percent
inhibition
of
3H incorporation into DNA:percent inhibition
of
3H incorporation into RNA, were as follows: HPUra
control, 88%:6%;
HE-EMAU, 85%:5%; ME-EMAU, 88%:5%; HP-EMAU,
86%:8%; and MP-EMAU,
89%:4%. In sum, these results strongly suggest
that the four hydrophilic
N-3-substituted EMAUs and the HPUra molecule
from which they evolved
share the same level of target selectivity in
vivo in the intact
cell.
Effects of MP-EMAU and HP-EMAU on murine staphylococcal
infection.
Given their relatively high aqueous solubilities and
the potency of their bactericidal effects against the Smith strain of S. aureus (Fig. 2B), we selected the two propyl derivatives,
MP-EMAU and HP-EMAU, for testing in a simple in vivo lethal infection model employing the same organism (details of model appear in the
Materials and Methods section). Specifically, mice were infected by
intraperitoneal injection of a fixed, lethal dose of bacteria, and 15 min thereafter, groups of 10 mice each were injected by the same route
with (i) the agent to be tested, (ii) vehicle, or (iii) vancomycin as a
positive control agent of known efficacy. Thereafter, each treatment
group was monitored over a 3-day period. The results, expressed as the
number of mice surviving at 3 days after infection, are summarized in
Table 5.
As expected, all of the animals that received 20 mg of vancomycin per
kg survived throughout the 3-day period, while 9 of
the 10 control mice
that received only vehicle died. Similarly,
all animals treated with
HP-EMAU at a dose of 10 mg/kg survived,
and 7 of 10 animals
treated with half that dose (5 mg/kg) survived.
After they
were treated with MP-EMAU at 10 mg/kg, only 5 of 10
animals survived,
and for those treated at 5 mg/kg, survival (20%)
was barely
distinguishable from that seen for animals in the vehicle
(control)
group (10%).
What is the significance of this in vivo protective effect of HP-EMAU
and MP-EMAU? This so-called "furry test tube" infection
model in
which animals are infected and treated in the same, intraperitoneal,
compartment, is one of the simplest infection models available
for use
in antibiotic testing. We selected this specific model
in an effort
simply to "prove principle" of antibiotic potential,
reasoning that
any candidate agent which failed to display efficacy
in this system was
unlikely to show efficacy in a more valid,
pharmacokinetically complex
model exploiting infection at one
site and administration of agent at a
different enteral or parenteral
site. These preliminary results
strongly suggest that the N-3-substituted
AUs, indeed, have
considerable potential as antiinfectives
a potential
which we intend
to probe further with experiments employing more
relevant infection
models. To achieve this goal we presently are
evaluating, in mice, the
basic pharmacokinetics of HP-EMAU to
permit its rational parenteral and
oral application, and we are
continuing to devise even-more-soluble and
-potent AU derivatives
through further manipulation of both the ring
N-3 substituent
of the base-pairing domain and substituents of the aryl
ring.
Potential of pol III as an antibiotic target.
Like DNA
topoisomerase, the target of the highly effective fluoroquinolones, pol
III is an essential, rate-limiting component of the bacterial DNA
replication machinery (10, 15, 22). Given the essential,
replication-specific function of pol III and the results presented
above for the AU class of pol III-specific inhibitors, we propose that
the development of pol III-specific antibiotics is feasible. We are
presently pursuing this proposition with two approaches. The first,
which is noted above, is to continue pursuit of the anti-infective
potential of our pol III-specific AU platform compounds. The
second is to identify and characterize novel pol III-targeted molecules
by exploiting high-throughput screening of other, unrelated
compounds for activity in vitro against both the
polC-specific pol III of the low G+C content gram-positive
organisms and the dnaE-specific pol III of relevant gram-negative pathogens.
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ACKNOWLEDGMENT |
This work was supported by STTR phase I grant AI41260 from the
National Institutes of Health.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Dept. of
Pharmacology and Molecular Toxicology, University of
Massachusetts Medical School, Worcester, MA 01655. Phone: (508)
856-2152. Fax: (508) 856-5080. E-mail:
neal.brown{at}ummed.edu.
Present address: GLSynthesis, Inc., 222 Maple Ave.,
Shrewsbury, MA 01545.
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Antimicrobial Agents and Chemotherapy, August 1999, p. 1982-1987, Vol. 43, No. 8
0066-4804/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
