Date post: | 06-Jul-2018 |
Category: |
Documents |
Upload: | nguyenduong |
View: | 214 times |
Download: | 0 times |
1
In Vitro Evaluation of CBR-2092, a Novel Rifamycin-Quinolone Hybrid Antibiotic: 1
Studies of the Mode of Action in Staphylococcus aureus 2
3
Gregory T. Robertson, Eric J. Bonventre†, Timothy B. Doyle
‡, Qun Du, Leonard 4
Duncan‡, Timothy W. Morris
#, Eric D. Roche
§, Dalai Yan
¶ and A. Simon Lynch* 5
6
Cumbre Pharmaceuticals Inc., 1502 Viceroy Drive, Dallas, Texas 75235-2304. 7
8
*Corresponding author: 9
A. Simon Lynch 10
Cumbre Pharmaceuticals Inc. 11
1502 Viceroy Drive, Dallas, TX 75235-2304 12
Phone: (214) 631-4700 ext. 7510. 13
Fax: (214) 631-4710 14
E-mail: [email protected] / [email protected] 15
16
Present addresses: Department of Pharmacology, University of Texas Southwestern 17
Medical Center, Dallas, TX 75390†, Vertex Pharmaceuticals Inc., Coralville, IA 52241
‡, 18
Bausch & Lomb Inc., Rochester, NY 14609#, Healthpoint Ltd., Fort Worth, TX 76107
§, 19
Department of Microbiology & Immunology, Indiana University School of Medicine, 20
Indianapolis, IN 46202¶. 21
Running title: Mode-of-Action of CBR-2092 in S. aureus 22
Keywords: Rifampin, quinolone, hybrid antibiotic, RNA polymerase, 23
DNA topoisomerase24
ACCEPTED
Copyright © 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Antimicrob. Agents Chemother. doi:10.1128/AAC.01649-07 AAC Accepts, published online ahead of print on 28 April 2008
on July 15, 2018 by guesthttp://aac.asm
.org/D
ownloaded from
2
ABSTRACT 25
Rifamycins have proven efficacy in the treatment of persistent bacterial 26
infections. However, the frequency with which bacteria develop resistance to 27
rifamycin agents restricts their clinical use to antibiotic combination regimens. In a 28
program directed toward the synthesis of rifamycins with a lower propensity to 29
elicit resistance development, a series of compounds were prepared that covalently 30
combine rifamycin and quinolone pharmacophores to form stable hybrid 31
antibacterial agents. Herein we describe mode-of-action studies in Staphylococcus 32
aureus for CBR-2092, a novel hybrid that combines the rifamycin SV and 4H-4-oxo-33
quinolizine pharmacophores. In biochemical studies, CBR-2092 exhibits rifampin-34
like potency as an inhibitor of RNA polymerase and is an equipotent inhibitor of 35
DNA gyrase and DNA topoisomerase IV with retention of activity against a 36
prevalent quinolone-resistant variant. Macromolecular biosynthesis studies confirm 37
that CBR-2092 has rifampin-like effects on RNA synthesis in rifampin-susceptible 38
strains and quinolone-like effects on DNA synthesis in rifampin-resistant strains. 39
Studies of mutant strains that exhibit reduced susceptibility to CBR-2092 further 40
substantiate RNA polymerase as the primary cellular target of CBR-2092 with DNA 41
gyrase and DNA topoisomerase IV as secondary and tertiary targets, respectively, in 42
strains exhibiting pre-existing rifampin-resistance. In contrast to quinolone 43
comparator agents, no strains with altered susceptibility to CBR-2092 were found to 44
exhibit changes consistent with altered efflux properties. The combined data 45
indicate that CBR-2092 may have potential utility in monotherapy for the treatment 46
of persistent S. aureus infections. 47
ACCEPTED
on July 15, 2018 by guesthttp://aac.asm
.org/D
ownloaded from
3
INTRODUCTION 48
Antibiotics of the rifamycin class exhibit potent antimicrobial activity against an 49
array of gram-positive bacteria including the Mycobacteria, and have been used globally 50
for the treatment of tuberculosis (TB). Rifamycins, however, exert their antibacterial 51
activity as inhibitors of a single enzyme target - DNA-dependent RNA polymerase - and 52
a variety of single point mutations in the rpoB gene (encoding the β subunit of the 53
enzyme) give rise to strains that exhibit highly elevated MICs (7, 12). This resistance 54
development liability limits the approved use of rifamycin class agents to combination 55
regimens. 56
Rifamycins, alone or in combination, exhibit activity against susceptible bacteria 57
propagated in the biofilm state, including data from in vitro biofilm assays (3, 28, 32, 37, 58
41) and animal models of biofilm-associated infections (6, 19, 20, 37). In addition, 59
clinical studies of rifampin in combination with fluoroquinolones (24, 36, 42, 43), 60
vancomycin (21, 38), fusidic acid (38) and amoxicillin (38) have led to the adoption of 61
specific rifampin-containing regimens as standard therapies for the treatment of biofilm-62
associated infections of indwelling medical devices (5). 63
The efficacy of the rifamycins in the treatment of biofilm-associated infections and 64
other persistent or latent infections that are often recalcitrant to other antibiotics is likely 65
explained by two distinct features. First, at the mechanistic level, the transcription 66
process is thought to be essential for the establishment and maintenance of bacteria in 67
alternate survival modes including biofilms and metabolically quiescent states typical of 68
latent cell populations. Second, at the physicochemical level, rifamycin agents exhibit 69
ACCEPTED
on July 15, 2018 by guesthttp://aac.asm
.org/D
ownloaded from
4
excellent tissue distribution (1), efficiently penetrate biofilms formed in vitro (41), and 70
exhibit good activity against a number of obligate or facultative intracellular pathogens. 71
In a program directed toward the synthesis of rifamycin derivatives with improved 72
resistance development properties, a series of compounds was prepared in which 73
rifamycin and quinolone pharmacophores are covalently combined. The design of these 74
rifamycin-quinolone hybrids was such that they act as stable, dual-pharmacophore agents 75
and therein not active as pro-drugs. This strategy should ensure matched 76
pharmacokinetics, pharmacodynamics and tissue distribution of the composite 77
pharmacophores. In CBR-2092, the rifamycin SV pharmacophore is combined with a 78
quinolone pharmacophore derived from the 4H-4-oxo-quinolizine (or ‘2-pyridone’) 79
subfamily of fluoroquinolones that exhibit balanced (equipotent) activity against both 80
DNA gyrase and DNA topoisomerase IV and retain activity against ciprofloxacin-81
resistant strains (26). In an accompanying article (30), we describe the results of 82
microbiology studies to characterize the in vitro profile of activity of CBR-2092 against 83
staphylococci and streptococci. Herein we describe the results of biochemical, cell 84
biology and genetic studies undertaken to characterize the mode-of-action of CBR-2092 85
in a primary target pathogen, Staphylococcus aureus. 86
(Portions of this work were previously presented [47th
Intersci. Conf. Antimicrob. 87
Agents and Chemother., abstr. F1-2101, 2007].) 88
89
MATERIALS AND METHODS 90
Antimicrobial Agents. CBR-2092 and ABT-719 (A-86719.0) were synthesized at 91
Cumbre Pharmaceuticals Inc. Rifampin, ethidium bromide and reserpine were purchased 92
ACCEPTED
on July 15, 2018 by guesthttp://aac.asm
.org/D
ownloaded from
5
from Sigma-Aldrich (St. Louis, MO). Ciprofloxacin, levofloxacin, gatifloxacin and 93
nadifloxacin were purchased from LKT Laboratories (St. Paul, MN). 94
Bacterial strains. Table 1 shows relevant details of a series of derivatives of S. 95
aureus ATCC 29213 (CB190) or RN4220 (CB1244) that exhibit stable resistance to 96
agents of the rifamycin and/or quinolone classes that were isolated and characterized as 97
described below. 98
Isolation and characterization of resistant mutants. Single-step selections of 99
antibiotic resistant mutants were undertaken by standard agar-based methods. In all 100
cases, mutants were purified through drug-free passage and the initial antibiotic 101
resistance phenotype then verified to ensure that stable, true-breeding mutants had been 102
obtained. Step-wise passage for multi-step resistance selection was undertaken in glass 103
tubes with MHII medium plus or minus 0.002% (vol:vol) Polysorbate-80 (P-80) and were 104
inoculated with 106 CFU/mL at sub-MIC doses of test agents; see legends to Tables 5A 105
and 5B for details. Cultures were incubated with shaking at 37°C for 20-24 hours. 106
Thereafter, the cell inoculum was prepared from the highest consecutive drug 107
concentration which had supported growth to an absorbance equivalent to or greater than 108
≈108 CFU per mL. Daily passages were performed until compound solubility issues 109
limited further elaboration of individual step-selection studies, or it was apparent that a 110
terminal resistance endpoint had been attained in specific selections. Genotypic analysis 111
of strains exhibiting stable resistance phenotypes was undertaken by standard methods 112
employing PCR amplification and DNA sequencing of target loci. Assessment of the 113
contribution of mutations in efflux pathways towards altered antibiotic-resistance 114
phenotypes was undertaken by determining altered sensitivity to the unrelated efflux 115
ACCEPTED
on July 15, 2018 by guesthttp://aac.asm
.org/D
ownloaded from
6
substrate ethidium bromide and/or whether quinolone susceptibility was impacted by 116
reserpine. 117
Biochemical studies. Recombinant forms of S. aureus σA RNA polymerase 118
holoenzyme and a rifampin-resistant RpoB (H481Y) variant were prepared as previously 119
described (27). Inhibition of transcription of S. aureus σA RNA polymerase holoenzymes 120
was measured in single-round G-less cassette assays that employed the pGL2B-T7A1-G-121
less template (27). Quantitative gel electrophoresis of a single 272 nucleotide G-less 122
RNA product species was used to determine the minimal concentration necessary to 123
inhibit 50% (IC50) of the holoenzyme-specific RNA product formed in the absence of test 124
agents. Derivatives of the pET28b vector (Novagen Inc.) were constructed to express 125
recombinant forms of the S. aureus GyrA or GyrA(S84L) and ParC or ParC(S80F) 126
subunits bearing amino-terminal oligo-histidine6 and T7-Tag epitopes and carboxyl-127
terminal FLAG epitope tags. The previously described vectors pTrcHisB-SA-GyrB and 128
pTrcHisA-SA-GrlB (18) were used for expression of recombinant variants of the S. 129
aureus GyrB and ParE (GrlB) proteins, respectively, bearing amino-terminal oligo-130
histidine6 affinity tags. In all cases, the recombinant subunits were purified from E. coli 131
Rosetta cells (Novagen Inc.) by immobilized metal affinity chromatography (IMAC). 132
Peak fractions eluted from the IMAC resin via imidazole gradient were identified by 133
SDS-PAGE, pooled and further purified by combination of size-exclusion 134
chromatography, affinity chromatography and/or dialysis. DNA topoisomerase 135
holoenzymes were then reconstituted through combination of the purified subunits at 136
ratios optimized for specific activity to yield either wild-type or quinolone-resistant forms 137
of DNA gyrase and DNA topoisomerase IV. For the studies described herein, DNA 138
ACCEPTED
on July 15, 2018 by guesthttp://aac.asm
.org/D
ownloaded from
7
gyrase preparations exhibited specific activity in the range of 200 [GyrA (S84L):GyrB] to 139
250 [GyrA:GyrB] units per µg, wherein 1 unit is equivalent to the minimal enzyme 140
necessary to effect the supercoiling of 150 ng of relaxed pBR322 DNA in 1 hr at 37ºC. 141
Wild-type [ParC:ParE] and mutant [ParC (S80F):ParE] preparations of DNA 142
topoisomerase IV enzymes exhibited specific activity of 25-30 units per µg (wherein 1 143
unit is equivalent to the minimal enzyme necessary to effect the relaxation of 150 ng of 144
supercoiled pUC19 DNA in 1 hr at 37ºC). Assays to measure the inhibition of the in 145
vitro activity of Type II topoisomerases employed relaxed (DNA gyrase) or negatively 146
supercoiled (DNA topoisomerase IV) forms of covalently closed DNAs (ccDNAs) as 147
substrates. Assays of DNA gyrase were undertaken in 50mM Tris-HCl (pH 7.5), 50mM 148
potassium glutamate, 5mM MgCl2, 5mM DTT, 1mg/mL acetylated BSA, 1.5mM ATP, 149
15 µg/mL relaxed pBR322 DNA and 100 µg/mL E. coli tRNA for one hour at 37ºC. 150
Assays of DNA topoisomerase IV were undertaken in 50mM Tris-HCl (pH 7.5), 50mM 151
potassium glutamate, 5mM MgCl2, 5mM DTT, 1mg/mL acetylated BSA, 1.5mM ATP, 152
15 µg/mL supercoiled pUC19 DNA and 100 µg/mL tRNA for one hour at 37ºC. In all 153
cases, reactions were terminated by the addition of sodium dodecyl sulfate (SDS) and 154
proteinase K. Quantitative gel electrophoresis of the linear DNA products species was 155
employed to determine the minimal concentration of inhibitor necessary to induce 50% 156
cleavage over background of a ccDNA substrate (CC50) with mean values calculated 157
from ≥ 2 independent assays. 158
Macromolecular biosynthesis assays. Effects of test agents on macromolecular 159
biosynthesis in S. aureus were determined using assays wherein the incorporation of 160
specific radiolabeled precursors was measured in a time-course fashion. The radiolabels 161
ACCEPTED
on July 15, 2018 by guesthttp://aac.asm
.org/D
ownloaded from
8
employed were [Methyl-3H]-Thymidine (Perkin Elmer # NET-027) for DNA synthesis, 162
[5,6-3H]-Uridine (Perkin Elmer # NET-367) for RNA synthesis, L-[3,4,5-3H]-Leucine 163
(Perkin Elmer # NET-460) for protein syntheis and [2,3-3H]-D-Alanine (American 164
Radiolabeled Chemicals # ART-179) for cell wall synthesis. At each time point, the ratio 165
of radiolabel incorporated in the presence of test agent relative to that of the DMSO-166
treated control culture was used to determine the percent incorporation. Radiolabel 167
incorporated at time zero (T0) was set to 100%. 168
Antimicrobial susceptibility testing. Determination of MICs was done in 169
accordance with Clinical Laboratory Standards Institute (CLSI) methodology by either 170
the broth microdilution or agar dilution methods using cation adjusted Mueller Hinton 171
(MHII) as a base medium (10). Unless otherwise indicated, microdilution broth assays 172
employed MHII medium supplemented with 0.002% (vol:vol) P-80. 173
174
RESULTS 175
Structure of CBR-2092 and related agents. In a program directed toward the 176
synthesis and evaluation of rifamycin-based hybrid antibiotics, a series of compounds 177
was prepared in which rifamycin and quinolone pharmacophore were covalently joined. 178
In total, approximately 300 rifamycin-quinolone hybrids were synthesized in an effort 179
that entailed combination of different rifamycin backbone scaffolds with quinolones 180
representative of various sub-series, including experimental 4th
generation quinolone 181
pharmacophores. Structure-Activity-Relationships (SARs) derived from data generated 182
in a range of assays described herein revealed that the potency of the quinolone entity 183
was a critical parameter in terms of the retention of antimicrobial activity of rifamycin-184
ACCEPTED
on July 15, 2018 by guesthttp://aac.asm
.org/D
ownloaded from
9
quinolone hybrid agents against strains exhibiting rifampin-resistance. Of the 4th
185
generation quinolone pharmacophores tested, members of the experimental 4H-4-186
oxoquinolizine subfamily (25) proved one of the most promising. In CBR-2092 (Fig. 1), 187
the rifamycin SV pharmacophore is combined via a chiral linking group with a quinolone 188
entity from the 4H-4-oxoquinolizine subfamily (25); for comparison, also shown are the 189
structures of rifampin, ciprofloxacin and a representative 4H-4-oxoquinolizine (ABT-190
719) that were employed herein as comparator agents. ABT-719 was studied as a 191
comparator as the most extensively characterized member of the 4H-4-oxoquinolizine 192
series and exhibits equipotent (balanced) biochemical activity against DNA gyrase and 193
DNA topoisomerase IV enzymes, potent antimicrobial activity against both Gram-194
positive and Gram-negative pathogens including ciprofloxacin-resistant isolates of S. 195
aureus, and is efficacious in rodent infection models (26). 196
Biochemical studies of CBR-2092 and comparator agents. As shown in Table 2, 197
CBR-2092 retains rifampin-like potency against wild-type S. aureus RNAP with an IC50 198
of 0.034µM – approximately two-fold less active than rifampin (0.015µM). In contrast, 199
both rifampin and CBR-2092 exhibit no detectable activity (IC50’s of >25 µM) against a 200
mutant form of the RNAP enzyme bearing a high-level rifampin-resistant RpoB (H481Y) 201
subunit. These combined data suggest that appendage of the 4H-4-oxoquinolizine 202
pharmacophore via the 3’-position of the rifamycin SV scaffold does not significantly 203
impact the ability of CBR-2092 to interact with RNA polymerase in a manner similar to 204
that of rifampin. 205
As also shown in Table 2, biochemical studies indicate that CBR-2092 exhibits 206
equipotent (balanced) activity against wild-type S. aureus DNA topoisomerase IV and 207
ACCEPTED
on July 15, 2018 by guesthttp://aac.asm
.org/D
ownloaded from
10
DNA gyrase enzymes with 50% cleavage concentrations (CC50’s) of 1.7 and 1.5 µM, 208
respectively, that are in the same potency range as ciprofloxacin and gatifloxacin and 209
correspond to a CC50 ratio for DNA gyrase/DNA topoisomerase IV of 0.9. The apparent 210
target balance of CBR-2092 for wild-type topoisomerase IV and DNA gyrase is 211
improved over that of ciprofloxacin (CC50 ratio of 11.5) and gatifloxacin (CC50 ratio of 212
3.2) and is similar to that of the ABT-719 compound for which we observed a CC50 ratio 213
of 1.5 and for which a value of 2.1 has previously been reported (33). However, in 214
contrast to all of the other fluoroquinolone comparators tested, CBR-2092 maintains its 215
activity against a prevalent fluoroquinolone-resistant variant of DNA topoisomerase IV 216
with CC50 values of 2.7 and 1.7 µM determined for ParC (S80F) and wild-type variants, 217
respectively. These data yield a CC50 ratio for CBR-2092 for DNA topo IV ParC 218
(S80F)/DNA topo IV (ParCWT
) of 1.6 that is improved over ciprofloxacin (77.5), 219
gatifloxacin (59.1) and ABT-719 (18.3). Retention of the activity of CBR-2092 against 220
the ParC (S80F) variant of DNA topoisomerase IV was considered a key attribute of the 221
quinolone-driven activity of the molecule as this mutation most commonly underlies 222
target mediated resistance in ciprofloxacin-resistant isolates of S. aureus. 223
Activity of CBR-2092 against S. aureus strains exhibiting rifampin and/or 224
quinolone resistance. Table 3 shows that activity of CBR-2092 and comparator agents 225
against an otherwise-isogenic set of derivatives of S. aureus CB190 (ATCC 29213) that 226
bear all possible combinations of a high-level rifampin-resistance mutation rpoB 227
(H481Y) and the prevalent quinolone-resistance mutations parC (S80F) and gyrA 228
(S84L). For CBR-2092, MIC endpoints are shown here and elsewhere from assays 229
conducted in both the agar dilution and broth microdilution formats, with the latter 230
ACCEPTED
on July 15, 2018 by guesthttp://aac.asm
.org/D
ownloaded from
11
conducted using MHII medium supplemented with 0.002% (vol:vol) Polysorbate-80 (P-231
80). As observed with a number of other semi-synthetic antibiotics, CBR-2092 exhibits 232
apparent high avidity for plastic and glass materials. In the broth microdilution format, 233
P-80 minimizes surface loss of CBR-2092 to the plastic surface of the assay plates 234
(Robertson, Du & Lynch, unpublished) and improves the concordance of MIC endpoints 235
with those determined by the agar dilution method (wherein it is assumed that such 236
compound loss is minimized by slower diffusion through the agar matrix). 237
As anticipated from past studies, the wild-type S. aureus strain CB190 (ATCC 29213) 238
strain and its rifamycin-resistant derivative CB370 [rpoB (H481Y)] were observed to 239
exhibit marked difference in their susceptibility to rifampin with the MIC against CB370 240
(>250 µg/mL) >31,250 times higher than determined for CB190 (0.008 µg/mL). In 241
contrast, the difference in MICs observed for these two strains with CBR-2092 is 242
markedly smaller, with the microbroth MIC against CB370 (0.12 µg/mL) only some 8 243
times higher than determined for CB190 (ATCC 29213) (0.015 µg/mL). Similarly, as 244
anticipated from past studies, the wild-type S. aureus strain CB190 (ATCC 29213) and 245
its fluoroquinolone-resistant derivative CB814 [gyrA (S84L), parC (S80F)] were 246
observed to exhibit marked differences in their susceptibility to fluoroquinolone agents. 247
For instance, the ciprofloxacin MIC against CB814 (16 µg/mL) is observed to be ~67 248
times higher than determined for CB190 (ATCC 29213) (0.25 µg/mL). In contrast, no 249
difference in potency is observed with CBR-2092 against these two strains with 250
microbroth MICs of 0.015 µg/mL observed for both CB814 and CB190. These 251
combined data indicate that the primary antimicrobial activity of CBR-2092 is conferred 252
by its rifamycin pharmacophore. 253
ACCEPTED
on July 15, 2018 by guesthttp://aac.asm
.org/D
ownloaded from
12
These data are also informative about the nature of the fluoroquinolone activity of the 254
tested agents and, in particular, in delineating the relative contributions of cellular 255
inhibition of each of the Type II DNA topoisomerase targets. Ciprofloxacin shows the 256
anticipated pattern of activity against this genetically-defined strain panel with 8-fold 257
differences in MICs observed between otherwise isogenic strains bearing either the gyrA 258
(S84L) or parC (S80F) resistance determinants. Analysis of the MICs determined for 259
CBR-2092 against the four rifamycin-resistant strains in the panel suggest that CBR-2092 260
is a relatively well balanced inhibitor of both Type II DNA topoisomerase targets in S. 261
aureus with a possible slight preference for DNA gyrase. The mechanistic basis 262
underlying the antimicrobial activity that CBR-2092 exhibits against the CB815 [rpoB 263
(H481Y), gyrA (S84L), parC (S80F)] strain cannot be directly determined from these 264
data. However, as CBR-2092 exhibits near equivalent activity in vitro as an inhibitor of 265
both wild-type and fluoroquinolone-resistant forms of S. aureus DNA topoisomerase IV 266
(Table 2), it seems reasonable to assume that the activity of CBR-2092 against strain 267
CB815 is mediated via residual effects on DNA topoisomerase IV. 268
Effects of CBR-2092 and comparator agents on macromolecular biosynthesis. 269
The effects of rifampin, ciprofloxacin and CBR-2092 on the four macromolecular 270
biosynthesis pathways studied in CB190 (ATCC 29213) are shown in Figure 2. As 271
anticipated, rifampin has a primary effect on de novo RNA synthesis with a delayed, 272
secondary effect on protein synthesis and tertiary and less significant overall effects on 273
cell wall and DNA synthesis. Similarly, consistent with past studies, ciprofloxacin has a 274
primary effect on de novo DNA synthesis with minimal effects on the other pathways 275
studied. In contrast, CBR-2092 exhibits intermediary effects that are somewhat distinct 276
ACCEPTED
on July 15, 2018 by guesthttp://aac.asm
.org/D
ownloaded from
13
from either rifampin or ciprofloxacin. Like rifampin, CBR-2092 has a primary effect on 277
de novo RNA synthesis with delayed, secondary effects on protein and cell wall 278
synthesis. However, CBR-2092 also appears to have a secondary effect on DNA 279
synthesis that is more pronounced than the effect observed with rifampin and likely 280
reflects the secondary mode of action conferred by the quinolone pharmacophore. 281
Fig. 2 also shows data from equivalent studies undertaken in the rifampin-resistant 282
strain CB370 [rpoB (H481Y)]. As expected, rifampin has no significant effects at the 283
concentration tested (equivalent to 4× MIC for CB190 (ATCC 29213)) on any of the four 284
macromolecular biosynthesis pathways studied while ciprofloxacin continues to exhibit a 285
primary effect on de novo DNA synthesis. In contrast, CBR-2092 now appears to have a 286
primary effect on de novo DNA synthesis with minimal effects on the other pathways 287
studied. Also shown in Fig. 2 are the effects of the same compounds on macromolecular 288
biosynthesis in the quinolone-resistant strain CB814 [gyrA (S84L), parC (S80F)]. As 289
expected, ciprofloxacin has no apparent effects on de novo DNA synthesis or other 290
pathways while rifampin exhibits an activity profile that is essentially similar to that 291
observed with the CB190 (ATCC 29213) strain. CBR-2092 also exhibits effects on the 292
four macromolecular pathways in CB814 that are similar to those observed in the CB190 293
(ATCC 29213) strain with a primary effect on RNA synthesis and a somewhat 294
diminished effect on DNA synthesis that more closely resembles that of rifampin. 295
Finally, at the concentrations tested here, neither rifampin, nor CBR-2092 nor 296
ciprofloxacin had discernible effects on the macromolecular pathways studied when 297
tested in the CB815 [rpoB (H481Y), gyrA (S84L), parC (S80F)] strain (data not shown). 298
ACCEPTED
on July 15, 2018 by guesthttp://aac.asm
.org/D
ownloaded from
14
Single-step resistance studies with CBR-2092. To further characterize the mode of 299
action of CBR-2092 in S. aureus, we undertook a series of single-step genetic selections 300
wherein spontaneous mutants that exhibit reduced susceptibility to CBR-2092 were 301
isolated and chacterized. As summarized in Table 4A, a series of agar based selections 302
undertaken with the rifampin-sensitive CB190 (ATCC 29213) strain and CBR-2092 in 303
the concentration range 0.08 to 0.12 µg/mL (5- to 8-fold MIC) gave rise to 28 304
independently isolated “first step” mutants that exhibit elevated MICs for CBR-2092 and 305
rifampin in the ranges 0.12 to 0.5 µg/mL and 0.25 to > 64 µg/mL, respectively, but 306
exhibited no apparent change in their susceptibility to ciprofloxacin. The recovery of 307
such first step resistance mutations at the indicated concentrations, but not at higher drug 308
concentrations, is wholly consistent with the anticipated low spontaneous resistance 309
potential of CBR-2092 at drug concentrations above the mutant prevention concentration 310
where the secondary antimicrobial activity of CBR-2092 exerts its effects; see 311
accompanying article (30). Genotypic characterization of these strains revealed a series 312
of mutations corresponding to amino acid substitution or insertion mutations that span 313
residues Gln468 to His481 in the RpoB subunit of RNA polymerase and therein overlap 314
with Cluster I of the previously characterized rifampin-resistance determining region 315
(RRDR) of S. aureus and other pathogens. These genetic data are again wholly 316
consistent with the notion that the primary antimicrobial activity of CBR-2092 in wild-317
type S. aureus is mediated by its rifamycin pharmacophore. 318
As shown in Table 4B, a series of agar-based selections undertaken with the rifampin-319
resistant strains CB370 [rpoB (H481Y)] or CB1522 [rpoB (H481Y)] and CBR-2092 in 320
the concentration range 0.1 to 0.25 µg/mL (1- to 2-fold MIC) gave rise to a series of 9 321
ACCEPTED
on July 15, 2018 by guesthttp://aac.asm
.org/D
ownloaded from
15
independently isolated mutants that exhibit elevated MICs for CBR-2092 in the range 0.5 322
to 2 µg/mL. Genotypic characterization of these strains revealed a series of mutations 323
corresponding to amino acid substitutions in the gyrA gene that with one exception [gyrA 324
(S84L)] lie distal to the canonical quinolone-resistance determining region (QRDR) of 325
the S. aureus gyrA gene. Further characterization of these strains revealed minimal (1- to 326
2-fold) shifts in MICs for a variety of fluoroquinolone agents (data not shown). In 327
contrast, selections undertaken with CB370 [rpoB (H481Y)] and ABT-719 (0.05 µg/mL) 328
or gatifloxacin (0.2 µg/mL) gave rise to a series of independently isolated mutants that 329
exhibited 4 to 8 fold shifts in ciprofloxacin MICs and were found to all possess 330
previously described QRDR mutations in either the parC or parE loci, encoding the 331
subunits of DNA topoisomerase IV (data not shown). These combined data are 332
consistent with the notion that, in contrast to gatifloxacin and ABT-719, the preferred 333
DNA topoisomerase target of CBR-2092 in S. aureus is DNA gyrase. 334
As is also shown in Table 4B, a series of selections undertaken with strains CB809 335
[rpoB (H481Y), parC (S80F)], CB812 [rpoB (H481Y), gyrA (S84L)] and CB815 [rpoB 336
(H481Y), gyrA (S84L), parC (S80F)] and CBR-2092 in the concentration range 0.2 to 337
0.8 µg/mL (1- to 8-fold MIC) gave rise to a series of independently isolated mutants that 338
exhibited elevated MICs for CBR-2092 in the range 1 to >16 µg/mL. Genotypic 339
characterization of these strains revealed a series of mutations corresponding to amino 340
acid substitutions in parE, parC and gyrA including both previously described QRDR 341
mutations and a further series of novel mutations. Importantly, when strains that possess 342
both the rpoB (H481Y) and gyrA (S84L) mutations were employed, CBR-2092 selections 343
resulted exclusively in mutations in the ParC or ParE subunits of DNA topoisomerase IV 344
ACCEPTED
on July 15, 2018 by guesthttp://aac.asm
.org/D
ownloaded from
16
including mutations outside of the previously described QRDR for DNA topoisomerase 345
IV in S. aureus. These data further substantiate DNA topoisomerase IV as the secondary 346
DNA topoisomerase target of CBR-2092 in S. aureus. 347
Finally, to determine the potential contribution of efflux class mutations in CBR-348
2092-selected mutants, MICs for the efflux substrate ciprofloxacin were determined in 349
the presence and absence of the efflux pump inhibitor reserpine. In no case was a 350
significant (>2-fold) shift in ciprofloxacin MIC observed in the presence of reserpine 351
(data not shown). Likewise, no significant shifts in MIC values were apparent for any 352
CBR-2092-selected mutants when tested against minocycline, a structurally unrelated 353
antibiotic that is subject to additional efflux pathways in S. aureus (data not shown). 354
These data suggest that efflux mechanisms do not contribute in mutational mechanisms 355
associated with first- or second-step resistance to CBR-2092 in S. aureus. 356
Multi-step passage selections undertaken with CBR-2092 and comparator agents 357
with CB190 (ATCC 29213). To delineate the genetic mechanism(s) by which a wild-358
type, drug-naïve strain of S. aureus [CB190 (ATCC 29213)] may develop higher level 359
resistance to CBR-2092, we also undertook studies involving the step-wise enrichment of 360
mutants with reduced susceptibility through serial passage in MHII broth medium 361
starting from a sub-MIC concentration range. Parallel studies were also undertaken with 362
rifampin, ciprofloxacin, and ABT-719. Table 5A shows the characterization of 363
intermediary and/or terminal isolates from each study. 364
Consistent with past literature precedents, step selections undertaken with the 365
comparator agents over the course of 2-15 days resulted in the isolation of strains that 366
exhibit resistance to benchmark agents of the parental classes that is elevated > 31,250-367
ACCEPTED
on July 15, 2018 by guesthttp://aac.asm
.org/D
ownloaded from
17
fold (rifampin, 2 days), > 500-fold (ciprofloxacin, 15 days) and > 250-fold (ABT-719, 15 368
days; data not shown) relative to those of the parental CB190 (ATCC 29213) strain. 369
Further, in the selections with fluoroquinolone agents, it is apparent that efflux 370
mechanisms contributed to the development of stable resistance to the test agents as 371
ethidium bromide MICs were elevated 16-fold (see Table 5A). 372
Step selections undertaken with CBR-2092 over the course of 26 days of serial, step-373
wise passage enrichment resulted in the isolation of a strain (CB1884) that exhibits 374
resistance elevated ~4,000-fold or 500-fold over the parent strain [CB190 (ATCC 375
29213)] as determined by broth microdilution or agar dilution MIC assays, respectively. 376
Table 5A includes a summary of the phenotypic and genotypic characterization of stable, 377
true-breeding mutants derived from days 2, 5, 10, 15 and 26 of the CBR-2092 step-378
selection study. 379
On day 2 of the CBR-2092 step-selection study, a mutant derivative (CB1880) was 380
isolated that had highly elevated resistance to rifampin and genotypic analysis revealed 381
an rpoB (R484H) mutation in the canonical RRDR of rpoB. A secondary mutational 382
event was apparent on day 5 as MICs for CBR-2092 are further elevated 4- to 8-fold and 383
genotypic analysis of a purified day 5 isolate (CB1881) revealed a deletion mutation in 384
the gyrA gene [gyrA (∆L520)] corresponding to the leucine residue at position 520 of 385
GyrA. Interestingly, this residue lies outside of the known QRDR of gyrA and is not 386
associated with a significant change in the MIC for ciprofloxacin (Table 5A). 387
A tertiary mutational event was apparent on Day 10 of the CBR-2092 step-selection 388
study with a further 2-fold shift in the MICs determined for CBR-2092. Genotypic 389
analysis of a purified day 10 isolate (CB1882) revealed a duplication mutation in the 390
ACCEPTED
on July 15, 2018 by guesthttp://aac.asm
.org/D
ownloaded from
18
parC gene [parC (R236dupl)] corresponding to a tandem duplication of the arginine 391
residue at position 236 of the ParC subunit of DNA topoisomerase IV. Similarly, this 392
residue also lies outside of the canonical QRDR of parC and is not associated with a 393
significant change in the MIC for ciprofloxacin. A further mutational event was apparent 394
on day 15 with CBR-2092 MICs elevated a further 2- to 8-fold. Genotypic analysis of a 395
purified day 15 isolate (CB1883) revealed a substitution mutation in the gyrA gene [gyrA 396
(S84L)] corresponding to a serine to leucine substitution at residue 84 of GyrA subunit 397
and corresponds to a prevalent QRDR mutation in fluoroquinolone-resistant S. aureus 398
isolates. A final mutational event was apparent on day 26 as the CBR-2092 MICs are 399
elevated a further 4- to 8-fold and genotypic analysis of a purified day 26 isolate 400
(CB1884) revealed a substitution mutation in the parC gene [parC (H103Y)] 401
corresponding to a histidine to tyrosine substitution at residue 103 of the ParC subunit of 402
DNA gyrase. While apparently atypical in fluoroquinolone-resistant clinical isolates, this 403
mutation has previously been reported in laboratory studies of resistance development to 404
non-fluorinated quinolones (31) and lies within the canonical QRDR of parC in S. 405
aureus. Hence, as expected, an 8-fold shift in the ciprofloxacin MIC is observed between 406
the day-15 (CB1883) and day-26 (CB1884) isolates. 407
During the course of the CBR-2092 step-selection study it was apparent that the 408
mutant derivatives that were isolated and characterized exhibited slower growth in vitro 409
than the parental strain. Table 5A also shows the doubling times for each characterized 410
isolate when growing in MHII broth medium at 37ºC with good aeration. Interestingly, 411
the doubling time of the terminal day-26 isolate (CB1884), which bears five mutations 412
[rpoB (R484H), gyrA (∆L520), gyrA (S84L) parC (R236dupl) & parC (H103Y)], is 65 413
ACCEPTED
on July 15, 2018 by guesthttp://aac.asm
.org/D
ownloaded from
19
minutes and therein significantly longer than that of the parent CB190 (ATCC 29213) 414
strain (38 minutes). The significance of these findings with regard to the in vivo fitness 415
or pathogenicity of strains exhibiting decreased susceptibility to CBR-2092 remains to be 416
determined. 417
Finally, in contrast to the fluoroquinolone comparator agents employed, it does not 418
appear that the mutational activation of efflux systems is a contributory factor in the 419
stepwise development of CBR-2092 resistance. In the case of ciprofloxacin and ABT-420
719, the terminal day-15 isolates exhibit MICs for ethidium bromide of 64 µg/mL that are 421
16-fold elevated over that of the parent strain (CB190 (ATCC 29213), 4 µg/mL). In 422
contrast, no decrease in susceptibility to ethidium bromide is apparent for CBR-2092 423
selectants through the day-26 terminal endpoint. 424
Multi-step passage selections undertaken with strains with pre-existing 425
rifamycin and/or quinolone resistance mutations. In a follow-up study, the day-26 426
terminal isolate from the CBR-2092 step-selection study (CB1884) was used as the 427
starting strain for a step-selection undertaken with ABT-719 in MHII medium. As shown 428
in Table 5B, after 20 days of step-wise passage in increasing concentrations of ABT-719, 429
a strain (CB1887) was isolated that has broth microdilution MICs for CBR-2092 and 430
ciprofloxacin of > 250 and 64 µg/mL, respectively. Genotypic analysis of strain 431
CB1887 confirmed that each of the five mutations previously characterized in strain 432
CB1884 had been stably maintained and also revealed two further substitution mutations 433
in parC [parC (E84G) and (S80Y)] corresponding to glutamate to glycine and serine to 434
tyrosine substitutions at residues 84 and 80 of the ParC subunit of DNA topoisomerase 435
IV, respectively, and a further substitution mutation in gyrA [gyrA (E88K)] 436
ACCEPTED
on July 15, 2018 by guesthttp://aac.asm
.org/D
ownloaded from
20
corresponding to a glutamate to lysine substitution at residue 88 of the GyrA subunit of 437
DNA gyrase. These combined data are consistent with the notion that the mode of action 438
of CBR-2092 in the concentration ranges employed herein - up to 250 and 8 µg/mL in the 439
broth and agar conditions, respectively - can be accounted for by combined effects on the 440
cellular function of RNA polymerase, DNA gyrase and DNA topoisomerase IV. 441
A final series of step-selections were undertaken with CBR-2092 in studies 442
employing starting strains that bear pre-existing rifampin- and/or quinolone-resistance 443
mutations. In these studies, MHII medium was supplemented with 0.002% (vol:vol) P-444
80 to enable selections involving higher concentrations of CBR-2092. Table 5B also 445
shows a summary of the phenotypic and genotypic characterization of stable, true-446
breeding mutants derived from the terminal isolates resulting from each of these step-447
selections. In toto, the combined data from this last set of genetic studies further 448
substantiate conclusions from previously described genetic selections including the 449
notion that CBR-2092 acting as a quinolone (in rifamycin-resistant strains) elicits 450
resistance mutations in DNA gyrase and DNA topoisomerases IV that are atypical of 451
traditional quinolones. In addition, the selection of secondary mutations in both GyrA 452
and ParC subunits in each of two independent selections undertaken with a rifampin-453
resistant strain (CB815) bearing pre-existing canonical fluoroquinolone-resistance alleles 454
[gyrA (S84L) and parC (S80F)] supports the notion that CBR-2092 exhibits residual 455
activity against the prevalent fluoroquinolone-resistant variants and is consistent with the 456
afore mentioned biochemical data. Finally, it is noted again that the terminal isolates 457
resulting from all CBR-2092 step-selections exhibit no change with regard to their 458
susceptibility to ethidium bromide suggesting that the mutational activation of efflux 459
ACCEPTED
on July 15, 2018 by guesthttp://aac.asm
.org/D
ownloaded from
21
systems is not a contributory factor in genotypic adaptations associated with decreased 460
CBR-2092 susceptibility in vitro. 461
462
DISCUSSION 463
Examination of rifampin-RNAP co-crystal structures suggest that the tight rifampin 464
binding interaction is mediated by key hydrogen bonds formed by hydroxyl groups at C-465
1, C-8, C-21 and C-23 as well as the carbonyl oxygen of the C-25 acetoxy group (Figure 466
1), while the C3-appended piperazine functionality of rifampin is spatially oriented away 467
from the RNAP binding surface and appears to be solvent accessible (4, 9). As all of the 468
chemical features of the rifampin pharmacophore identified as critical elements in RNAP 469
interaction are preserved in CBR-2092, and the quinolone moiety is appended to the C3 470
position via a secondary hydrazone functionality identical to that of rifampin, it is 471
perhaps not surprising that CBR-2092 exhibits in vitro potency as an RNAP inhibitor that 472
is nearly equivalent to that of rifampin. Preliminary evidence for distinct features of the 473
interaction of CBR-2092 with RNA polymerase is suggested by the retention of activity 474
of CBR-2092 against derivatives of a high-level quinolone resistant strain [CB1623, gyrA 475
(S84L), parC (S80F), parE (D434V), norAUP
] bearing intermediary level rifampin-476
resistance alleles including rpoB (H481N), rpoB (S464P) or rpoB (I527F) (Du, Duncan, 477
Robertson & Lynch, unpublished observations). 478
The activity of CBR-2092 against rifampin-resistant strains, combined with the range 479
of gyrA, parE and parC mutations resulting from genetic selections undertaken with 480
CBR-2092 in strains with pre-existing high-level rifampin resistance, suggest that CBR-481
2092 exhibits secondary activity against S. aureus via its quinolone pharmacophore and 482
ACCEPTED
on July 15, 2018 by guesthttp://aac.asm
.org/D
ownloaded from
22
that DNA gyrase is the preferred topoisomerase target in vivo. In this regard, it is 483
interesting to note that other hybrid quinolone antibiotics, including quinolone dimers 484
(14, 22, 23, 40), sulfonamide-quinolones (2, 29) and oxazolidinone-quinolone hybrids 485
(13, 15, 16), similarly appear to preferentially target DNA gyrase in gram-positive 486
bacteria. These data could be interpreted as indicating that the quinolone-binding pocket 487
of DNA gyrase in ternary complexes may be more accommodating of compounds that in-488
effect bear large, bulky substitutions extending from position 7 of the classical 4-489
quinolone core nucleus. 490
DNA gyrase and DNA topoisomerase IV mutations characterized in strains resulting 491
from genetic selections employing CBR-2092 include an array of mutational changes that 492
have not previously been reported in studies of traditional quinolone agents and as such 493
lie outside of the classical quinolone-resistance determining regions. The selection of 494
such mutants could reflect distinct aspects of the binding interaction between CBR-2092 495
and the DNA topoisomerase target proteins. Alternately, these atypical mutations may 496
simply attenuate the activity of the enzyme such as to alter the sensitivity of the mutant 497
toposiomerase to inhibition by CBR-2092. In this regard, it is of interest to note that past 498
studies of laboratory isolated quinolone-resistant mutants of S. aureus have similarly 499
revealed mutations outside of the classical QRDRs and have been found to either reduce 500
the expression of DNA topoisomerase IV (17) or yield enzymes with apparent reduced 501
catalytic function (X. Zhang and D. Hooper, unpublished observations cited in (17)). 502
Finally, the mutational activation of efflux systems has been shown to be a common 503
mechanism that contributes to the development of fluoroquinolone resistance in a variety 504
of human pathogens. Studies of S. aureus suggest that up to ~50% of fluoroquinolone-505
ACCEPTED
on July 15, 2018 by guesthttp://aac.asm
.org/D
ownloaded from
23
resistant strains of clinical origin exhibit an enhanced quinolone efflux phenotype (11, 34, 506
35, 39) and that mutations affecting the expression of two specific efflux pumps - NorA, 507
a member of the major facilitator superfamily (MFS) class and MepA, a multidrug and 508
toxic extrusion (MATE) family member - are most commonly found. However, in 509
contrast to most fluoroquinolones, including the comparator agents studied herein, the 510
mutational activation of efflux systems does not appear to be a contributory factor in the 511
development of resistance to CBR-2092. Further, in an accompanying article (30), we 512
show through the use of a panel of genetically-defined mutant strains of S. aureus that the 513
increased expression of either norA or mepA has no discernable impact on the in vitro 514
activity of CBR-2092. It is of interest to note that quinolone dimer agents linked via the 515
C7 position also appear to exhibit a lower propensity for quinolone efflux in S. aureus 516
(14, 22, 23). In addition, the improved antimicrobial activity of other quinolone hybrid 517
agents versus gram-positive cocci when compared to parent agents (or cocktails thereof) 518
may also be explained by circumvention of intrinsic or mutationally-activated efflux 519
systems (8, 13, 15, 16). One possible explanation for this apparent commonality among 520
quinolone hybrid antibiotics (including CBR-2092) is that the MFS and MATE class 521
pumps involved in quinolone efflux in gram-positive cocci exhibit relatively narrow 522
substrate specificities when compared to the resistance-nodulation-division (RND) efflux 523
systems principally involved in quinolone efflux in gram-negative pathogens. In light of 524
the increasing prevalence of efflux-mediated resistance traits in gram-positive cocci, this 525
feature may confer a selective advantage of CBR-2092 over rifamycin-fluoroquinolone 526
cocktail combinations with regard to its activity against fluoroquinolone-resistant clinical 527
isolates. 528
ACCEPTED
on July 15, 2018 by guesthttp://aac.asm
.org/D
ownloaded from
24
529
530
ACKNOWLEDGEMENTS 531
The authors wish to thank David Hooper for provision of plasmids used for the 532
inducible over-expression of S. aureus GyrB and ParE subunits in E. coli, Ian Chopra for 533
providing strain RN4220 and Douglas Beeman and Katrina Chapo for experimental 534
contributions in the early stages of the work. The authors also acknowledge past 535
contributors to the rifamycin-quinolone program at Cumbre Pharmaceuticals including 536
Donghui Bao, Keith Combrink, Jing Li, Zhenkun Ma and Paul Renick and the 537
contributions of current colleagues Charles Ding, Steve Madden and William Weiss. 538
539
REFERENCES 540
1. Acocella, G. 1983. Pharmacokinetics and metabolism of rifampin in humans. 541
Rev. Infect. Dis. 5:S428-32. 542
2. Alovero, F. L., X.-S. Pan, J. E. Morris, R. H. Manzo, and L. M. Fisher. 2000. 543
Engineering the Specificity of Antibacterial Fluoroquinolones: 544
Benzenesulfonamide Modifications at C-7 of Ciprofloxacin Change Its Primary 545
Target in Streptococcus pneumoniae from Topoisomerase IV to Gyrase. 546
Antimicrob. Agents Chemother. 44:320-325. 547
3. Amorena, B., E. Gracia, M. Monzon, J. Leiva, C. Oteiza, M. Perez, J. L. 548
Alabart, and J. Hernandez-Yago. 1999. Antibiotic susceptibility assay for 549
Staphylococcus aureus in biofilms developed in vitro. J Antimicrob Chemother 550
44:43-55. 551
ACCEPTED
on July 15, 2018 by guesthttp://aac.asm
.org/D
ownloaded from
25
4. Artsimovitch, I., M. N. Vassylyeva, D. Svetlov, V. Svetlov, A. Perederina, N. 552
Igarashi, N. Matsugaki, S. Wakatsuki, T. T. H., and D. G. Vassylyev. 2005. 553
Allosteric modulation of the RNA polymerase catalytic reaction is an essential 554
component of transcription control by rifamycins. Cell 122:351-63. 555
5. Baddour, L. M., W. R. Wilson, A. S. Bayer, V. G. Fowler, Jr., A. F. Bolger, 556
M. E. Levison, P. Ferrieri, M. A. Gerber, L. Y. Tani, M. H. Gewitz, D. C. 557
Tong, J. M. Steckelberg, R. S. Baltimore, S. T. Shulman, J. C. Burns, D. A. 558
Falace, J. W. Newburger, T. J. Pallasch, M. Takahashi, and K. A. Taubert. 559
2005. Infective Endocarditis: Diagnosis, Antimicrobial Therapy, and Management 560
of Complications: A Statement for Healthcare Professionals From the Committee 561
on Rheumatic Fever, Endocarditis, and Kawasaki Disease, Council on 562
Cardiovascular Disease in the Young, and the Councils on Clinical Cardiology, 563
Stroke, and Cardiovascular Surgery and Anesthesia, American Heart Association: 564
Endorsed by the Infectious Diseases Society of America. Circulation 111:e394-565
434. 566
6. Blaser, J., P. Vergeres, A. Widmer, and W. Zimmerli. 1995. In vivo 567
verification of in vitro model of antibiotic treatment of device-related infection. 568
Antimicrob. Agents Chemother. 39:1134-1139. 569
7. Bryskier, A. 2005. Ansamycins. Antimicrobial Agents: Antibacterials and 570
Antifungals. ASM Press, Washington, DC. 571
8. Butler, M. M., W. A. LaMarr, K. A. Foster, M. H. Barnes, D. J. Skow, P. T. 572
Lyden, L. M. Kustigian, C. Zhi, N. C. Brown, G. E. Wright, and T. L. 573
Bowlin. 2007. Antibacterial Activity and Mechanism of Action of a Novel 574
ACCEPTED
on July 15, 2018 by guesthttp://aac.asm
.org/D
ownloaded from
26
Anilinouracil-Fluoroquinolone Hybrid Compound. Antimicrob. Agents 575
Chemother. 51:119-127. 576
9. Campbell, E. A., N. Korzheva, A. Mustaev, K. Murakami, S. Nair, A. 577
Goldfarb, and S. A. Darst. 2001. Structural mechanism for rifampicin inhibition 578
of bacterial RNA polymerase. Cell Microbiol 104:901-12. 579
10. Clinical and Laboratory Standards Institute. 2006. Methods for Dilution 580
Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically-Seventh 581
Addition; Approved Standard- Seventh Edition CLSI Document M7-A7., vol.26 582
no. 2. Clinical and Laboratory Standards Institute. 583
11. DeMarco, C. E., L. A. Cushing, E. Frempong-Manso, S. M. Seo, T. A. A. 584
Jaravaza, and G. W. Kaatz. 2007. Efflux-Related Resistance to Norfloxacin, 585
Dyes, and Biocides in Bloodstream Isolates of Staphylococcus aureus. 586
Antimicrob. Agents Chemother. 51:3235-3239. 587
12. Floss, H. G., and T.-W. Yu. 2005. Rifamycin - Mode of Action, Resistance and 588
Biosynthesis. Chem. Rev. 105:621-32. 589
13. Gordeev, M. F., C. Hackbarth, M. R. Barbachyn, L. S. Banitt, J. R. Gage, G. 590
W. Luehr, M. Gomez, J. Trias, S. E. Morin, G. E. Zurenko, C. N. Parker, J. 591
M. Evans, R. J. White, and D. V. Patel. 2003. Novel oxazolidinone-quinolone 592
hybrid antimicrobials. Bioorg. Med. Chem. Lett. 13:4213-6. 593
14. Gould, K. A., X.-S. Pan, R. J. Kerns, and L. M. Fisher. 2004. Ciprofloxacin 594
Dimers Target Gyrase in Streptococcus pneumoniae. Antimicrob. Agents 595
Chemother. 48:2108-2115. 596
ACCEPTED
on July 15, 2018 by guesthttp://aac.asm
.org/D
ownloaded from
27
15. Hubschwerlen, C., J. L. Specklin, D. K. Baeschlin, Y. Borer, S. Haefeli, C. 597
Sigwalt, S. Schroeder, and H. H. Locher. 2003. Structure-activity relationship 598
in the oxazolidinone-quinolone hybrid series: influence of the central spacer on 599
the antibacterial activity and the mode of action. Bioorg. Med. Chem. Lett. 600
13:4229-33. 601
16. Hubschwerlen, C., J. L. Specklin, C. Sigwalt, S. Schroeder, and H. H. 602
Locher. 2003. Design, synthesis and biological evaluation of oxazolidinone-603
quinolone hybrids. Bioorg. Med. Chem. 11:2313-9. 604
17. Ince, D., and D. C. Hooper. 2003. Quinolone Resistance Due to Reduced Target 605
Enzyme Expression. J. Bacteriol. 185:6883-6892. 606
18. Ince, D., X. Zhang, L. C. Silver, and D. C. Hooper. 2002. Dual Targeting of 607
DNA Gyrase and Topoisomerase IV: Target Interactions of Garenoxacin (BMS-608
284756, T-3811ME), a New Desfluoroquinolone. Antimicrob. Agents Chemother. 609
46:3370-3380. 610
19. Kadurugamuwa, J. L., L. V. Sin, J. Yu, K. P. Francis, R. Kimura, T. 611
Purchio, and P. R. Contag. 2003. Rapid Direct Method for Monitoring 612
Antibiotics in a Mouse Model of Bacterial Biofilm Infection. Antimicrob. Agents 613
Chemother. 47:3130-3137. 614
20. Kadurugamuwa, J. L., L. V. Sin, J. Yu, K. P. Francis, T. F. Purchio, and P. 615
R. Contag. 2004. Noninvasive Optical Imaging Method To Evaluate 616
Postantibiotic Effects on Biofilm Infection In Vivo. Antimicrob. Agents 617
Chemother. 48:2283-2287. 618
ACCEPTED
on July 15, 2018 by guesthttp://aac.asm
.org/D
ownloaded from
28
21. Karchmer, A. W. 2000. Infections of Prosthetic Heart Valves. Infections 619
Associated with Indwelling Devices (3rd edition) Edited by Waldvogel and Bisno 620
(ASM Press):pp. 145-172. 621
22. Kerns, R. J., M. J. Rybak, G. W. Kaatz, F. Vaka, R. Cha, R. G. Grucz, and 622
V. U. Diwadkar. 2003. Structural features of piperazinyl-linked ciprofloxacin 623
dimers required for activity against drug-resistant strains of Staphylococcus 624
aureus. Bioorg. Med. Chem. Lett. 13:2109-12. 625
23. Kerns, R. J., M. J. Rybak, G. W. Kaatz, F. Vaka, R. Cha, R. G. Grucz, V. U. 626
Diwadkar, and T. D. Ward. 2003. Piperazinyl-linked fluoroquinolone dimers 627
possessing potent antibacterial activity against drug-resistant strains of 628
Staphylococcus aureus. Bioorg. Med. Chem. Lett. 13:1745-9. 629
24. Konig, D. P., J. M. Schierholz, U. Munnich, and J. Rutt. 2001. Treatment of 630
staphylococcal implant infection with rifampicin-ciprofloxacin in stable implants. 631
Arch Orthop Trauma Surg 121:297-9. 632
25. Li, Q., D. T. Chu, A. Claiborne, C. S. Cooper, C. M. Lee, K. Raye, K. B. 633
Berst, P. Donner, W. Wang, L. Hasvold, A. Fung, Z. Ma, M. Tufano, R. 634
Flamm, L. L. Shen, J. Baranowski, A. Nilius, J. Alder, J. Meulbroek, K. 635
Marsh, D. Crowell, Y. Hui, L. Seif, L. M. Melcher, R. Henry, S. Spanton, R. 636
Faghih, L. L. Klein, S. K. Tanaka, and J. J. Plattner. 1996. Synthesis and 637
structure-activity relationships of 2-pyridones: a novel series of potent DNA 638
gyrase inhibitors as antibacterial agents. J. Med. Chem. 39:3070-88. 639
26. Li, Q., L. A. Mitscher, and L. L. Shen. 2000. The 2-pyridone antibacterial 640
agents: bacterial topoisomerase inhibitors. Med. Res. Rev. 20:231-93. 641
ACCEPTED
on July 15, 2018 by guesthttp://aac.asm
.org/D
ownloaded from
29
27. Lynch, A. S., and Q. Du. 2007. Methods to Identify and Characterize Inhibitors 642
of Bacterial RNA Polymerase. Published in ‘New Antibiotic Targets’; Molecular 643
Medicine series published by Humana Press Inc (Totowa, NJ). 142:37-52. 644
28. Monzon, M., C. Oteiza, J. Leiva, and B. Amorena. 2001. Synergy of different 645
antibiotic combinations in biofilms of Staphylococcus epidermidis. J Antimicrob 646
Chemother 48:793-801. 647
29. Pan, X.-S., P. J. Hamlyn, R. Talens-Visconti, F. L. Alovero, R. H. Manzo, and 648
L. M. Fisher. 2002. Small-Colony Mutants of Staphylococcus aureus Allow 649
Selection of Gyrase-Mediated Resistance to Dual-Target Fluoroquinolones. 650
Antimicrob. Agents Chemother. 46:2498-2506. 651
30. Robertson, G. T., E. J. Bonventre, T. B. Doyle, Q. Du, L. Duncan, T. W. 652
Morris, E. D. Roche, D. Yan, and A. S. Lynch. 2008. In Vitro Evaluation of 653
CBR-2092, a Novel Rifamycin-Quinolone Hybrid Antibiotic: Microbiology 654
Profiling Studies Undertaken in Staphylococci and Streptococci. Antimicrob. 655
Agents Chemother. Submitted. 656
31. Roychoudhury, S., T. L. Twinem, K. M. Makin, M. A. Nienaber, C. Li, T. W. 657
Morris, B. Ledoussal, and C. E. Catrenich. 2001. Staphylococcus aureus 658
Mutants Isolated via Exposure to Nonfluorinated Quinolones: Detection of 659
Known and Unique Mutations. Antimicrob. Agents Chemother. 45:3422-3426. 660
32. Saginur, R., M. Stdenis, W. Ferris, S. D. Aaron, F. Chan, C. Lee, and K. 661
Ramotar. 2006. Multiple combination bactericidal testing of staphylococcal 662
biofilms from implant-associated infections. Antimicrob. Agents Chemother. 663
50:55-61. 664
ACCEPTED
on July 15, 2018 by guesthttp://aac.asm
.org/D
ownloaded from
30
33. Saiki, A. Y., L. L. Shen, C. M. Chen, J. Baranowski, and C. G. Lerner. 1999. 665
DNA cleavage activities of Staphylococcus aureus gyrase and topoisomerase IV 666
stimulated by quinolones and 2-pyridones. Antimicrob Agents Chemother 667
43:1574-7. 668
34. Schmitz, F., A. Fluit, M. Luckefahr, B. Engler, B. Hofmann, J. Verhoef, H. 669
Heinz, U. Hadding, and M. Jones. 1998. The effect of reserpine, an inhibitor of 670
multidrug efflux pumps, on the in-vitro activities of ciprofloxacin, sparfloxacin 671
and moxifloxacin against clinical isolates of Staphylococcus aureus. J. 672
Antimicrob. Chemother. 42:807-810. 673
35. Schmitz, F. J., A. C. Fluit, S. Brisse, J. Verhoef, K. Kohrer, and D. Milatovic. 674
1999. Molecular epidemiology of quinolone resistance and comparative in vitro 675
activities of new quinolones against European Staphylococcus aureus isolates. 676
FEMS Immunol. Med. Microbiol. 26:281-7. 677
36. Schrenzel, J., S. Harbarth, G. Schockmel, D. Genne, T. Bregenzer, U. 678
Flueckiger, C. Petignat, F. Jacobs, P. Francioli, W. Zimmerli, D. P. Lew, and 679
S. S. S. Group. 2004. A randomized clinical trial to compare fleroxacin-680
rifampicin with flucloxacillin or vancomycin for the treatment of staphylococcal 681
infection. Clin. Infect. Dis. 39:1285-92. 682
37. Schwank, S., Z. Rajacic, W. Zimmerli, and J. Blaser. 1998. Impact of Bacterial 683
Biofilm Formation on In Vitro and In Vivo Activities of Antibiotics. Antimicrob. 684
Agents Chemother. 42:895-898. 685
ACCEPTED
on July 15, 2018 by guesthttp://aac.asm
.org/D
ownloaded from
31
38. Stein, A., M. Drancourt, and D. Raoult. 2000. Management of Infected 686
Orthopedic Implants. Infections Associated with Indwelling Devices (3rd edition) 687
Edited by Waldvogel and Bisno (ASM Press). 688
39. Tanaka, M., T. Wang, Y. Onodera, Y. Uchida, and K. Sato. 2000. Mechanism 689
of quinolone resistance in Staphylococcus aureus. J. Infect. Chemother. 6:131-9. 690
40. Zhao, X., B. Quinn, R. Kerns, and K. Drlica. 2006. Bactericidal activity and 691
target preference of a piperazinyl-cross-linked ciprofloxacin dimer with 692
Staphylococcus aureus and Escherichia coli. J. Antimicrob. Chemother. 58:1283-693
1286. 694
41. Zheng, Z., and P. S. Stewart. 2002. Penetration of rifampin through 695
Staphylococcus epidermidis biofilms. Antimicrob Agents Chemother 46:900-3. 696
42. Zimmerli, W., A. Trampuz, and P. E. Ochsner. 2004. Prosthetic-joint 697
infections. N. Engl. J. Med. 351:1645-54. 698
43. Zimmerli, W., A. F. Widmer, M. Blatter, R. Frei, and P. E. Ochsner. 1998. 699
Role of rifampin for treatment of orthopedic implant-related staphylococcal 700
infections: a randomized controlled trial. Foreign-Body Infection (FBI) Study 701
Group. JAMA. 279:1537-41. 702
703
ACCEPTED
on July 15, 2018 by guesthttp://aac.asm
.org/D
ownloaded from
32
704
TABLE 1. Bacterial strains used in this study. 705
Strain Relevant Genotype Relevant Characteristics Source or
Reference
CB190 Wild-type ATCC 29213 Parent strain for isogenic strain panel ATCC
CB370 rpoB (H481Y) Spontaneous rifampin-resistant variant of CB190 This work
CB808 parC (S80F) Spontaneous ciprofloxacin-resistant variant of CB190 This work
CB809 rpoB (H481Y), parC (S80F) Spontaneous ciprofloxacin-resistant variant of CB370 This work
CB811 gyrA (S84L) Spontaneous nadifloxacin-resistant variant of CB190 This work
CB812 rpoB (H481Y), gyrA (S84L) Spontaneous nadifloxacin-resistant variant of CB370 This work
CB814 parC (S80F), gyrA (S84L) Spontaneous higher level ciprofloxacin-resistant variant of CB808 This work
CB815 rpoB (H481Y), parC (S80F), gyrA (S84L) Spontaneous higher level ciprofloxacin-resistant variant of CB809 This work
CB1244 Wild-type RN4220 Parent strain: rsbU, agr, restriction minus, methylation
plus laboratory strain I. Chopra
CB1522 rpoB (H481Y) Spontaneous rifampin-resistant variant of CB1244 This work
706
ACCEPTED on July 15, 2018 by guest
http://aac.asm.org/
Dow
nloaded from
33
TABLE 2. Effects of CBR-2092 and comparator agents on the in vitro activity of target enzymes from S. aureus. 707
σA
RNA Polymerase
(IC50 in µM) Type II DNA Topoisomerases (CC50 in µM, or ratios of CC50 values)
DNA Topoisomerase IV DNA Gyrase CC50 Ratios Test Agent
Rif-S
(RpoBWT
)
Rif-R
(RpoBH481Y
) ParCWT
ParCS80F
GyrAWT
GyrAS84L
GyrA
WT
/ ParCWT
ParCS80F
/ ParCWT
CBR-2092 0.034 > 25 1.7 2.7 1.5 >150 0.9 1.6
Rifampin 0.015 > 25 nr nr nr nr nr nr
Ciprofloxacin nr nr 0.4 31 4.6 > 150 11.5 77.5
Gatifloxacin nr nr 0.22 13 0.7 18 3.2 59.1
ABT-719 nr nr 0.06 1.1 0.09 0.9 1.5 18.3
708
Abbreviations: nr, not relevant. 709 ACCEPTED on July 15, 2018 by guest
http://aac.asm.org/
Dow
nloaded from
34
TABLE 3. Antibacterial activity of CBR-2092 against isogenic Staphylococcus aureus strains bearing combinations of rifamycin- or 710
quinolone resistance mutations. 711
MIC (µg/mL) for Strain, Genotype
CB190, CB811, CB808, CB814, CB370, CB812, CB809, CB815,
Test Agent
(condition)a
WT gyrAS84L
parCS80F
gyrA
S84L
parCS80F
rpoB
H481Y
rpoBH481Y
gyrAS84L
rpoBH481Y
parCS80F
rpoBH481Y
gyrAS84L
parCS80F
CBR-2092 (Agar) 0.008 0.008 0.008 0.008 0.06 0.12 0.06 0.25
CBR-2092 0.015 0.015 0.015 0.015 0.12 0.50 0.25 2
Rifampin 0.008 0.008 0.008 0.008 > 250 > 250 > 250 > 250
Ciprofloxacin 0.25 0.25 2 16 0.25 0.25 2 16
Levofloxacin 0.25 0.25 1 8 0.25 0.25 1 8
Gatifloxacin 0.06 0.12 0.25 8 0.06 0.12 0.25 8
ABT-719 0.015 0.03 0.03 0.50 0.015 0.03 0.03 0.25
712
aMinimum inhibitory concentrations (MICs) in µg/mL were determined by the broth microdilution method in the presence of 0.002 % 713
(v:v) polysorbate-80 unless otherwise indicated. 714
ACCEPTED on July 15, 2018 by guest
http://aac.asm.org/
Dow
nloaded from
35
TABLE 4A. Characterization of CBR-2092 single-step mutants derived from a rifampin-sensitive strain. 715
Microbroth MIC (µg/mL) Mutation Identified
in rpoB
Number of
Independent
Isolations Rifampin CBR-2092 Ciprofloxacin
WT Parent nr 0.008 0.015 0.25
H481Y 12 >64 0.12 0.25
Q468L 1 >64 0.25 0.25
Q468K 1 >64 0.50 0.25
H481D 1 >64 0.25 0.25
<H472> (insertion) 1 32 0.12 0.25
D471Y 2 16 0.25 0.50
<E473> (insertion) 1 8 0.25 0.25
H481N 1 2 0.25 0.25
N474K 2 2 0.25 0.25
A477V 2 1 0.12 0.25
D471G 4 0.25 0.25 0.25
716
ACCEPTED on July 15, 2018 by guest
http://aac.asm.org/
Dow
nloaded from
36
TABLE 4B. Characterization of CBR-2092 single-step mutants derived from rifampin-resistant strains. 717
Strain
Parent
Strain Parent Strain Genotype
Selection
Conc.
(µg/mL)
Ending Strain Genotype
CBR-2092
Microbroth
MIC (µg/mL)
CB1522 CB1244 rpoBH481Y
na na 0.25
CB1523 CB1522 rpoBH481Y
0.25 rpoBH481Y
gyrAA26V
1
CB1524 CB1522 rpoBH481Y
0.25 rpoBH481Y
gyrAG773V
2
CB1416 CB370 rpoBH481Y
0.1 rpoBH481Y
gyrAL795S
0.5
CB1417 CB370 rpoBH481Y
0.1 rpoBH481Y
gyrAG532V
0.5
CB1418 CB370 rpoBH481Y
0.1 rpoBH481Y
gyrAD705N
0.5
CB1419 CB370 rpoBH481Y
0.1 rpoBH481Y
gyrAG572D
1
CB1420 CB370 rpoBH481Y
0.1 rpoBH481Y
gyrAG584V
0.5
CB1326 CB370 rpoBH481Y
0.2 rpoBH481Y
gyrAS784F
0.5
CB1327 CB370 rpoBH481Y
0.2 rpoBH481Y
gyrAS84L
0.5
CB1421 CB809 rpoBH481Y
parCS80F
0.18 rpoBH481Y
parCS80F
gyrAS84L
1
CB1430 CB812 rpoBH481Y
gyrAS84L
0.18 rpoBH481Y
parEE474K
gyrAS84L
16
CB1333 CB815 rpoBH481Y
parCS80F
gyrAS84L
0.8 rpoBH481Y
parCS80F
parEL517F
gyrAS84L
16
CB1334 CB815 rpoBH481Y
parCS80F
gyrAS84L
0.8 rpoBH481Y
parCS80F, Q27H
gyrAS84L
8
CB1335 CB815 rpoBH481Y
parCS80F
gyrAS84L
0.8 rpoBH481Y
parCS80F
parEE474Q
gyrAS84L
> 16
CB1353 CB815 rpoBH481Y
parCS80F
gyrAS84L
0.53 rpoBH481Y
parCS80F
parEV458G
gyrAS84L
16
CB1354 CB815 rpoBH481Y
parCS80F
gyrAS84L
0.53 rpoBH481Y
parCS80F
parER455H
gyrAS84L
16
718
Abbreviations: na, not applicable. 719
ACCEPTED on July 15, 2018 by guest
http://aac.asm.org/
Dow
nloaded from
37
TABLE 5A. Characterization of mutants from step-selections undertaken with wild-type strains. 720
MIC in µg/mL for:
CBR-2092 Strain
Selection
Agenta
Dayb Mutations Identified
Broth Agar Rifampin Cipro EtBr
Doubling
Time
(mins)c
CB190 CBR-2092 na Wild-Type 0.015 0.008 0.008 0.25 4 38
CB1880 CBR-2092 2 rpoBR484H
0.12 0.03 > 250 0.25 4 43
CB1881 CBR-2092 5 rpoBR484H
gyrA∆L520
0.5 0.25 > 250 0.5 2 41
CB1882 CBR-2092 10 rpoBR484H
gyrA∆L520
parCR236dupl.
1 0.5 > 250 1 2 45
CB1883 CBR-2092 15 rpoBR484H
gyrA∆L520, S84L
parCR236dupl.
8 1 > 250 1 2 47
CB1884 CBR-2092 26 rpoBR484H
gyrA∆L520, S84L
parCR236dupl., H103Y
64 4 > 250 8 2 65
CB1871 Rifampin 2 rpoBH481Y
0.12 nd > 250 0.25 8 nd
CB1875 Ciprofloxacin 15 gyrAS84L
parCR298K, <LNVIKEE>461
0.008 nd 0.016 > 128 64 nd
CB1891 ABT-719 15 gyrAS84L
parCE84K
parEP587S, F638V
0.03 nd 0.008 > 64 64 nd
721
aStep-selections were undertaken in MHII broth medium without P-80 supplementation. 722
bThe day on which the indicated mutant strain was purified. 723
cThe time necessary for the OD600 of a logarithmic culture propagated at 37ºC in MHII broth medium to double. 724
Abbreviations: na, not applicable; Cipro, ciprofloxacin; EtBr, Ethidium Bromide.725
ACCEPTED on July 15, 2018 by guest
http://aac.asm.org/
Dow
nloaded from
38
TABLE 5B. Characterization of mutants from step-selections undertaken with strains exhibiting resistance to rifamycin and/or 726
quinolone agents. 727
MIC in µg/mL for:
CBR-2092 Strain Selectiona Relevant genotype
Broth Agar Rifampin Cipro
b EtBr
c
CB1884 Parent rpoBR484H
gyrA∆L520, S84L
parC<R236>, H103Y
64 4 > 250 8 2
CB1887 CB1884 plus 20-days
selection with ABT-719 rpoB
R484H gyrA
∆L520, S84L, E88K parC
<R236>, H103Y, S80Y, E84G > 250 > 8 > 250 64 nd
CB370 Parent rpoBH481Y
0.12 0.03 > 250 0.25 4
CB1947 CB370 plus 20 days
selection with CBR-2092 rpoB
H481Y gyrA
S84L, ∆E697 parC
G167V 4 1 > 250 2 4
CB814 Parent rpoBWT
gyrAS84L
parCS80F
0.015 0.06 0.008 16 4
CB1952 CB814 plus 21 days
selection with CBR-2092 rpoB
S486L gyrA
S84L, E88Q, G532S parC
S80F, E84L 31 4 > 250 64 4
CB815 Parent rpoBH481Y
gyrAS84L
parCS80F
1 0.25 > 125 16 4
CB1953 CB815 plus 7 days
selection with CBR-2092 rpoB
H481Y gyrA
S84L, V598I parC
S80F, R570H 16 2 > 125 64 4
CB1954 CB815 plus 7 days
selection with CBR-2092 rpoB
H481Y gyrA
S84L, ∆K809 parC
S80F, A523D 16 2 > 125 64 4
728
ACCEPTED on July 15, 2018 by guest
http://aac.asm.org/
Dow
nloaded from
39
aStep-selections were undertaken in MHII broth medium with (CBR-2092) or without (ABT-719) supplementation with 0.002% (v/v) 729
polysorbate-80. 730
bCipro, ciprofloxacin. 731
cEtBr, ethidium bromide.732
ACCEPTED on July 15, 2018 by guest
http://aac.asm.org/
Dow
nloaded from
40
FIGURE 1. 733
N
N
F
O
H2N
ABT-719
7
8
O
O
OH
OHOH
NH
O
O
OHOH
O
O
O
N
N
N N
N
F
O
O
O
OH
OHOH
NH
O
O
OHOH
O
O
O
N
N
N
CBR-2092
Rifampin
NN
HN
F
O O
OH
Ciprofloxacin
6
7
O
OH
O
OH
3
3
18
2123
25
734
735
FIG. 1. Chemical structures of CBR-2092 and related molecules. 736
ACCEPTED
on July 15, 2018 by guesthttp://aac.asm
.org/D
ownloaded from
41
FIGURE 2. 737
CB190 (Wild-type) 738
739
740
741
742
743
CB370 (Rifamycin-resistant, rpoBH481Y
) 744
745
746
747
748
749
CB814 (Quinolone-resistant, gyrAS84L
plus parCS80F
) 750
751
752
753
754
755
FIG. 2. Effects of CBR-2092 and comparator agents on de novo macromolecular 756
biosynthesis in S. aureus. The radiolabels employed were [Methyl-3H]-Thymidine 757
(closed circles) for DNA synthesis, [5,6-3H]-Uridine (open circles) for RNA synthesis, 758
L-[3,4,5-3H]-Leucine (closed triangles, dashed line) for protein syntheis and [2,3-3H]-D-759
0 50 100 150 200
0
50
100
150
Rifampin(0.032 µg/ml)
Time (min)
0 50 100 150 200
0
50
100
150
CBR-2092(0.5 µg/ml)
Time (min)
0 50 100 150 200
0
50
100
150
DNA
Cell Wall
Protein
RNA
Ciprofloxacin(2 µg/ml)
Time (min)
Lab
el I
nco
rpo
rati
on
%
(CP
Md
rug/C
PM
DM
SO)
0 50 100 150 200
0
50
100
150
Rifampin(0.032 µg/ml)
Time (min)
0 50 100 150 200
0
50
100
150
CBR-2092(0.5 µg/ml)
Time (min)
0 50 100 150 200
0
50
100
150
DNA
Cell Wall
Protein
RNA
Ciprofloxacin(2 µg/ml)
Time (min)
Lab
el I
nco
rpo
rati
on
%
(CP
Md
rug/C
PM
DM
SO)
Lab
el I
nco
rpo
rati
on
%
(CP
Md
rug/C
PM
DM
SO)
0 50 100 150 200
0
50
100
150
Rifampin(0.032 µg/ml)
Time (min)
0 50 100 150 200
0
50
100
150
CBR-2092(0.5 µg/ml)
Time (min)
0 50 100 150 200
0
50
100
150
DNA
Cell Wall
Protein
RNA
Ciprofloxacin(2 µg/ml)
Time (min)
Lab
el I
nco
rpo
rati
on
%
(CP
Md
rug/C
PM
DM
SO)
0 50 100 150 200
0
50
100
150
Rifampin(0.032 µg/ml)
Time (min)
0 50 100 150 200
0
50
100
150
CBR-2092(0.5 µg/ml)
Time (min)
0 50 100 150 200
0
50
100
150
DNA
Cell Wall
Protein
RNA
Ciprofloxacin(2 µg/ml)
Time (min)
0 50 100 150 200
0
50
100
150
Rifampin(0.032 µg/ml)
Time (min)
0 50 100 150 200
0
50
100
150
CBR-2092(0.5 µg/ml)
Time (min)
0 50 100 150 200
0
50
100
150
DNA
Cell Wall
Protein
RNA
Ciprofloxacin(2 µg/ml)
Time (min)
Lab
el I
nco
rpo
rati
on
%
(CP
Mdru
g/C
PM
DM
SO)
0 50 100 150 200
0
50
100
150
Rifampin(0.032 µg/ml)
Time (min)
0 50 100 150 200
0
50
100
150
CBR-2092(0.5 µg/ml)
Time (min)
0 50 100 150 200
0
50
100
150
DNA
Cell Wall
Protein
RNA
Ciprofloxacin(2 µg/ml)
Time (min)
Lab
el I
nco
rpo
rati
on
%
(CP
Mdru
g/C
PM
DM
SO)ACCEPTED
on July 15, 2018 by guesthttp://aac.asm
.org/D
ownloaded from
42
Alanine (open triangles, dashed line) for cell wall synthesis. At each time point, the ratio 760
of radiolabel incorporated in the presence of test agent relative to that of the DMSO-761
treated control culture was used to determine the percent incorporation. Radiolabel 762
incorporated at time zero (T0) was set to 100%. 763
ACCEPTED
on July 15, 2018 by guesthttp://aac.asm
.org/D
ownloaded from
N
N
F
O
H2N
ABT-719
7
8
O
O
OH
OHOH
NH
O
O
OHOH
O
O
O
N
N
N N
N
F
O
O
O
OH
OHOH
NH
O
O
OHOH
O
O
O
N
N
N
CBR-2092
Rifampin
NN
HN
F
O O
OH
Ciprofloxacin
6
7
O
OH
O
OH
3
3
18
2123
25
AAC01649-07 v2: FIGURE 1
ACCEPTED on July 15, 2018 by guest
http://aac.asm.org/
Dow
nloaded from
CB190 (Wild-type)
CB370 (Rifamycin-resistant, rpoBH481Y)
CB814 (Quinolone-resistant, gyrAS84L plus parCS80F)
0 50 100 150 200
0
50
100
150
Rifampin(0.032 µµµµg/mL)
Time (min)
0 50 100 150 200
0
50
100
150
CBR-2092(0.5 µµµµg/mL)
Time (min)
0 50 100 150 200
0
50
100
150DNA
Cell Wall
Protein
RNA
Ciprofloxacin(2 µµµµg/mL)
Time (min)
Lab
el
Inc
orp
ora
tio
n %
(C
PM
dru
g/C
PM
DM
SO)
0 50 100 150 200
0
50
100
150
Rifampin(0.032 µµµµg/mL)
Time (min)
0 50 100 150 200
0
50
100
150
CBR-2092(0.5 µµµµg/mL)
Time (min)
0 50 100 150 200
0
50
100
150DNA
Cell Wall
Protein
RNA
Ciprofloxacin(2 µµµµg/mL)
Time (min)
Lab
el
Inc
orp
ora
tio
n %
(C
PM
dru
g/C
PM
DM
SO)
La
bel In
co
rpo
rati
on
%
(CP
Md
rug/C
PM
DM
SO)
0 50 100 150 200
0
50
100
150
Rifampin(0.032 µµµµg/mL)
Time (min)
0 50 100 150 200
0
50
100
150
CBR-2092(0.5 µµµµg/mL)
Time (min)
0 50 100 150 200
0
50
100
150DNA
Cell Wall
Protein
RNA
Ciprofloxacin(2 µµµµg/mL)
Time (min)
La
bel In
co
rpo
rati
on
%
(CP
Md
rug/C
PM
DM
SO)
0 50 100 150 200
0
50
100
150
Rifampin(0.032 µµµµg/mL)
Time (min)
0 50 100 150 200
0
50
100
150
CBR-2092(0.5 µµµµg/mL)
Time (min)
0 50 100 150 200
0
50
100
150DNA
Cell Wall
Protein
RNA
Ciprofloxacin(2 µµµµg/mL)
Time (min)
0 50 100 150 200
0
50
100
150
Rifampin(0.032 µµµµg/mL)
Time (min)
0 50 100 150 200
0
50
100
150
CBR-2092(0.5 µµµµg/mL)
Time (min)
0 50 100 150 200
0
50
100
150DNA
Cell Wall
Protein
RNA
Ciprofloxacin(2 µµµµg/mL)
Time (min)
Lab
el In
co
rpo
rati
on
%
(CP
Md
rug/C
PM
DM
SO)
0 50 100 150 200
0
50
100
150
Rifampin(0.032 µµµµg/mL)
Time (min)
0 50 100 150 200
0
50
100
150
CBR-2092(0.5 µµµµg/mL)
Time (min)
0 50 100 150 200
0
50
100
150DNA
Cell Wall
Protein
RNA
Ciprofloxacin(2 µµµµg/mL)
Time (min)
Lab
el In
co
rpo
rati
on
%
(CP
Md
rug/C
PM
DM
SO)
AAC01649-07 v2: FIGURE 2
ACCEPTED on July 15, 2018 by guest
http://aac.asm.org/
Dow
nloaded from