(imidazolinylindole) derivatives with potent 1 antibacterial activity against antibiotic-resistant st

33 Pages·2009·1.35 MB·English

AAC Accepts, published online ahead of print on 27 July 2009
Antimicrob. Agents Chemother. doi:10.1128/AAC.01709-08
Copyright © 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.
1 Novel broad-spectrum bis-(imidazolinylindole) derivatives with potent
2 antibacterial activity against antibiotic-resistant strains
3 Rekha G. Panchal1*, Ricky L. Ulrich1, Douglas Lane2, Michelle M. Butler3, Chad
4 Houseweart3, Timothy Opperman3, John D. Williams3, Norton P. Peet3, Donald T. Moir3,
5 Tam Nguyen2, Rick Gussio4, Terry Bowlin3 and Sina Bavari1*
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8 P1 United States Army Medical Research Institute of Infectious Diseases, 1425 Porter D
9 SPtreet, Fort Detrick, Frederick, MD 21702-5011, USA. o
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11 2Target Structure Based Drug Discovery Group, SAIC-Frederick, Inc., NCI-Frederick, lo
12 FP Prederick, Maryland, 21702-1201, USA. ad
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14 3Microbiotix Inc., One Innovation Dr., Worcester, MA 01605, USA. f
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15 4Target Structure Based Drug Discovery Group, Information Technology Branch,
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16 Developmental Therapeutic Program, National Cancer Institute, Frederick, Maryland, t
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17 21702-1201, USA. p:
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19 Running Title: New chemotype with broad-spectrum antibacterial activity .a
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20 Key words: antibacterial, B. anthracis, broad-spectrum, Gram-positive, Gram-negative, .
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21 antibiotic resistance rg
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23 Abbreviations: CFU, colony-forming unit; MIC, minimum inhibitory concentration; MBC, n

24 minimum bactericidal concentration; MOI, Multiplicity of infection J
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27 Correspondence ry

28 *Rekha G. Panchal or Sina Bavari 7
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29 Telephone: [phone] (R.G.P) or 301-619-4246 (S.B.) 2
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30 Fax: [phone]
31 Email: [email protected] 9

32 [email protected] by
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1 ABSTRACT
2 Given the limited number of structural classes of clinically available antimicrobial
3 drugs, the discovery of antibacterials with novel chemical scaffolds is an important
4 strategy in the development of effective therapeutics for both naturally occurring and
5 engineered resistant strains of pathogenic bacteria. In this study, several diarylamidine D
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6 derivatives were evaluated for their ability to protect macrophages from cell death n
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7 following infection with Bacillus anthracis, a Gram-positive spore-forming bacterium. ad
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8 Four bis-(imidazolinylindole) compounds were identified with potent antibacterial activity f
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9 as measured by the protection of macrophages and by the inhibition of bacterial growth

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10 in vitro. These compounds were effective against a broad range of Gram-positive and p
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11 Gram-negative bacterial species, including several antibiotic resistant strains. Minor a
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12 structural variations among the four compounds correlated with differences in their sm
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13 effects on bacterial macromolecular synthesis and mechanisms of resistance. In vivo r
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14 studies revealed protection by two of the compounds of mice lethally infected with B. n

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15 anthracis, Staphylococcus aureus or Yersinia pestis. Taken together, these results n
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16 indicate that the bis-(imidazolinylindole) compounds represent a new chemotype for the ry

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17 development of therapeutics for both Gram-positive and Gram-negative bacterial 2
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18 species as well as against antibiotic resistant infections. 9

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1 INTRODUCTION
2 Bacillus anthracis is a serious bioterrorism threat because its spores are stable
3 under extreme conditions in the environment, easily cultured and produced, easily
4 distributed by aerosol (in a powder form), and highly fatal via inhalation as dramatically
5 demonstrated in 2001 (4, 16, 17). While B. anthracis cells are sensitive to several D
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6 antibiotics (3, 13), naturally occurring or intentionally engineered drug-resistance is a n
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7 concern. Antibiotic resistance is also a growing problem in the clinic (10), and the ad
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8 recent increased prevalence of community-acquired MRSA has added to the concern f
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9 (11, 12). While effective new agents are in the pipeline, they are all new analogs of

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10 existing classes of antibiotics (22). The development of new antibiotics against p
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11 unexploited targets with novel mechanisms of action is a vital part of the solution to a
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12 these problems because such antibiotics are unlikely to be affected by pre-existing sm
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13 target-based resistance alleles. r
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14 In order to explore potential new chemotypes of antibacterial agents with a n

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15 variety of possible mechanisms of action, we developed a cell-based screen for rescue n
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16 of macrophages from B. anthracis-mediated death and applied it to a focused library ry

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17 containing diarylamidine compounds. This class of compounds has been evaluated 2
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18 previously for antimicrobial properties (2), as well as for antiproteolytic, anticoagulant 9

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19 (23) and antiproliferative activity (5). In our study, four bis-(imidazolinylindole) g
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20 compounds from the diarylamidine library exhibited very potent activity in the s
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21 macrophage rescue screen. Mechanism of action studies indicated that these
22 compounds are neither non-specific inhibitors of macromolecular synthesis nor
23 membrane active but are rapidly bactericidal inhibitors of a broad spectrum of bacterial
3
1 species. Finally, we demonstrate activity of these inhibitors in animal models of
2 infection.
3 MATERIALS AND METHODS
4 Small molecule Library. A focused library containing ~70 compounds sharing a
5 diarylamidine chemical scaffold was obtained from National Cancer Institute and used D
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6 for screening (Supplementary Table 1). n
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7 Bacterial strains. The different bacterial species and strains used in this study include ad
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8 Bacillus anthracis (Sterne), Bacillus anthracis (Ames), Bacillus brevis, Bacillus, f
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9 licheniformis, Bacillus megaterium, Bacillus vollum, Bacillus subtilis, Bacillus pumilus,

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10 Staphylococcus aureus NCTC8325, S. aureus (Smith strain), methicillin-resistant S. p
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11 aureus 1094, Enterococcus faecalis ATCC29212, Mycobacteria smegmatis a
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12 ATCC19420, M. smegmatis ATCC35798, M. smegmatis ATCC700009, Escherichia coli sm
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13 J53, Klebsiella pneumoniae 5657, Pseudomonas aeruginosa PA01, Yersinia pestis r
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14 CO92, Y. pestis KIM (∆pgm pCD1-), Burkholderia mallei ATCC3344, Burkholderia n

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15 pseudomallei DD503, Burkholderia thailandensis and Burkholderia cepacia. n
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16 Cell-based infection assays. J774A.1 macrophages (6 x 105) were infected with B. ry

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17 anthracis Sterne spores at a multiplicity of infection (MOI) of 5, in the presence of 2
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18 DMSO (1%, as control) or test compounds (10 µM). After a 4 hr incubation at 37oC, 9
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19 bacterial growth was inhibited by the addition of antibiotics penicillin (100 IU) and g
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20 streptomycin (100 µg/ml). To determine cell viability, sytox green dye that is st
21 impermeant to live cells was added and incubated for 15 min at 37oC. The cells were
22 centrifuged at 2000 rpm for 2 min and then washed two times with complete medium
23 containing antibiotics. The cells were then analyzed by flow cytometry.
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1 In vitro inhibition of bacterial growth. Minimum inhibitory concentrations (MICs)
2 were determined by the broth microdilution method (20). B. anthracis Sterne spores or
3 bacterial cultures (5 x105 CFU/ml) in log-phase growth were seeded in 96 well plates
4 and treated with DMSO (1%) or compound at concentrations ranging from 0 to 20 µM.
5 Plates were incubated at 370C for 16 to 20 hrs and cell growth determined by D
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6 measuring the absorbance at 600 nm. n
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7 The minimal bactericidal concentration (MBC) was determined by a modification ad
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8 of the MIC method. Bacteria from MIC wells and four dilutions above the MIC were f
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9 diluted and bacteria were plated on sheep blood agar plates. The next day colonies

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10 were counted, and MBC values reflecting a 99.9% reduction in viable counts were p
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11 determined. a
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12 Spore germination assay. Sterne spores (5 x105 CFU/ml) were germinated in Mueller sm
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13 Hinton broth in the presence of DMSO (control) or compounds (1 x MIC). At time r
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14 intervals of 0, 15, and 30 minutes, samples were heated at 70oC for 30 min to kill any n

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15 germinated spores and appropriate dilutions were plated on sheep blood agar plates to n
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16 quantify remaining viable spores. ry

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17 Kinetics of bactericidality. To determine the kinetics of cidality, compounds were 2
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18 diluted in Mueller Hinton broth and tested at concentrations equivalent to 4x their 9

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19 respective MIC values against B. anthracis Sterne spores (5 x105 CFU/ml) or B. g
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20 anthracis Sterne vegetative bacilli or an attenuated Yersinia pestis strain (KIM ∆pgm s
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21 pCD1- ) (1 x106 CFU/ml). The cultures were incubated and sampled at various time
22 points (0, 1, 2, 4, 6 and 24 hr) and diluted appropriately into fresh medium, and then
23 plated onto drug-free agar plates to determine the number of CFU/ml present in the
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1 sample (CFU/ml = number of colonies on the plate multiplied by the dilution factor and
2 adjusted for a volume of 1 ml). An additional experiment was conducted, as described
3 above, using B. subtilis BD54, MBX 1066 and the antibiotic ciprofloxacin, an inhibitor of
4 bacterial DNA replication, at concentrations of 5x their MIC values. The minimum level
5 of detection in these experiments was 50 cfu/mL. The log10 value of CFU/ml was D
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6 plotted versus time. Bactericidal activity is defined as a ≥ 3 log reduction in initial CFU n
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7 count within 24 hours. ad
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8 Determination of Mammalian Cytotoxicity. Cytotoxicity of the compounds was f
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9 measured as described previously, except that HeLa cells were used (9). Cytotoxicity

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10 was quantified as the CC , the concentration of compound that inhibited 50% of p
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11 conversion of MTS to formazan (18). The “selectivity index” is defined as the ratio of a
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12 the mammalian cell cytotoxicity to the MIC value against B. anthracis Ames (i.e. sm
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13 CC /MIC), both measured in the presence of 10% fetal calf serum. The addition of r
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14 10% fetal calf serum had no effect on the MIC values. n

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15 Macromolecular synthesis assays. Compounds were examined at 5x MIC for n
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16 inhibitory effects on bacterial DNA, RNA, protein and cell wall biosynthesis in B. subtilis ry

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17 BD54, in 96-well polystyrene plates. Incorporation of the following radiolabeled 2
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18 precursors into macromolecules was measured -- [methyl-3H] thymidine for DNA, [5-3H] 9

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19 uridine for RNA, L- [4,5-3H] leucine for protein, N-acetyl-D-[1-3H] glucosamine for cell g
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20 wall synthesis. At time zero, compound or pathway-specific control antibiotic (in DMSO) s
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21 at a concentration of 5x its MIC value and radioactive precursors were added to log
22 phase cells. At various times, samples were collected from each 37oC culture,
23 precipitated with an equal volume of ice-cold 20% TCA, collected on 96-well glass fiber
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1 filter plates, dried and counted for radioactivity. Plots of counts (cpm) incorporated vs.
2 incubation time were generated for each compound.
3 Membrane Activity Assays. The effect of compounds on bacterial membrane
4 potential of B. subtilis BD54 was determined using a fluorescent assay essentially as
5 described by Wu and Hancock (25). Briefly, B. subtilis BD54 cultures were grown to D
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6 exponential phase (OD600 = 0.4-0.5) in LB media. Cells were harvested, washed, and n
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7 resuspended in wash buffer (5 mM HEPES pH 7.4, 20 mM glucose) to an OD600 = 0.1. ad
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8 The fluorescent dye DiSC3(5) was added to the cell suspension (final concentration of 1 fr
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9 µM) and incubated at room temperature for 10 min to allow dye uptake. KCl was added

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10 to a final concentration of 100 mM. The cell suspension was transferred to a 96-well p
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11 assay plate (200 µl/well) containing control and test compounds dissolved in DMSO a
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12 (final concentration = 2%). A total of 8 wells were tested for each condition. sm
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13 Fluorescence intensity (RFU) was measured after 5 min using a Molecular Devices r
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14 SpectroMax fluorescence plate reader with an excitation wavelength of 622 nm and n

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15 emission wavelength of 670 nm. Dinitrophenol (DNP), a protonophore, was used at a n
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16 concentration of 200 µg/ml as a positive control. The average RFU and standard ry

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17 deviation for eight assay wells was calculated and is presented in Fig. 4F 2
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18 The effect of the compounds on mammalian cell membrane integrity was 9

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19 determined by measuring the release of lactate dehydrogenase (LDH) from HeLa cells g
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20 treated with a concentration range of compounds. Briefly, HeLa cells were grown to s
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21 confluence in DMEM and were treated for 1 h with 1 % DMSO alone (untreated control),
22 various concentrations of test compounds and a control antibiotic (32x MIC
23 vancomycin). The final concentration of DMSO in all samples was 1%. LDH activity in
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1 the supernatant was measured using the the CytoTox ONETM Homogenous Membrane
2 Integrity Assay Kit (Promega, Madison, WI) according to the manufacturer’s
3 instructions.
4 Selection for resistant mutants. Eight independent cultures of S. aureus NCTC8325
5 were grown in 96 well assay plates in the presence of several concentrations of each of D
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6 the compounds (0.125x- 128x MIC). Cultures were recovered from the well with highest n
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7 compound concentration that exhibited robust growth (>50% of untreated control). This ad
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8 process was repeated for 20 days, and results were displayed as the highest sub-lethal f
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9 compound concentration for each culture for each day. Colonies were isolated from

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10 apparently resistant cultures and confirmed to be resistant by MIC assays. p
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11 Animal studies. Eight to ten week old C57BL/6 mice were used in this study. The a
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12 dosing regimen was based on the MIC values and on initial pilot studies varying dose sm
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13 and administration frequency. For in vivo B. anthracis studies, mice (n=10/group) were r
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14 challenged via intraperitoneal (ip) injection with ~300 CFU of B. anthracis Ames. After 6 n

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15 hours post challenge, mice were treated via ip injection with vehicle control, MBX 1066 n
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16 (5 or 10 mg/kg/injection) or MBX 1090 (0.2, 0.5 or 1.0 mg/kg/injection). Mice were ry

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17 treated every six hours for 5 days and survival monitored for up to 20 days. 2
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18 Compounds in this study were dissolved at a stock concentration of 50 mg/ml in 9

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19 dimethyl sulfoxide (DMSO) and then diluted to an appropriate working concentration in g
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20 5% dextrose in water. s
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21 To determine the protective effect of the compounds during the late stages of
22 infection, treatment with compound MBX 1066 (10 mg/kg/injection) via ip injection was
23 initiated 6, 12, 18 or 24 hours post challenge. Thereafter mice were treated every six
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1 hours for five days and survival was monitored up to 20 days. Compound MBX 1066
2 was dissolved at a stock concentration of 50 mg/ml in DMSO and then diluted to an
3 appropriate working concentration in 5% dextrose in water.
4 To test the efficacy of the compounds in a Y. pestis infection model, C57BL/6
5 mice (n=10/group) were challenged via ip injection with ~ 100 CFU of Y. pestis (strain D
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6 CO92). After 6 hours mice were treated via ip injection with MBX 1066 (5 or 10 n
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7 mg/kg/injection) and treatment continued every six hours for 5 days. Survival of the ad
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8 mice was monitored for 15 days. f
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9 To investigate the efficacy of the compounds administered via a different route

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10 from that of the pathogen challenge, Swiss Webster mice (n=10/group) were challenged p
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11 via ip injection with 8.3 x 108 CFU of S. aureus (Smith strain). After 15 minutes, mice a
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12 were treated via intravenous injection with a single dose of MBX 1066 (10 mg/kg), MBX sm
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13 1090 (10 mg/kg) prepared in 10% dimethyl acetamide/5% dextrose in water, pH 4.0 or r
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14 daptomycin control (10 mg/kg) or vehicle control (10% dimethyl acetamide/5% dextrose n

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15 in water, pH 4.0). Survival was monitored for 48 hr. n
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16 All research was conducted under an approved protocol and in compliance with ry

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17 the Animal Welfare Act and other federal statutes and regulations related to animals 2
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18 and experiments involving animals and adhered to principles stated in the Guide for the 9
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19 Care and Use of Laboratory Animals, National Research Council 1996. The facilities in g
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20 which this research was conducted are fully accredited by the Association for s
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21 Assessment and Accreditation of Laboratory Animal Care International.
22 RESULTS
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1 Small molecules protect macrophages from B. anthracis induced cell-death. B.
2 anthracis, a Gram-positive spore forming bacterium, produces a virulent lethal toxin that
3 causes death of susceptible macrophages (14). A cell-based B. anthracis infection
4 assay was used to test a focused library that contained diarylamidine derivatives (see
5 supplementary Table 1 for chemical structures). Macrophages were infected with B. D
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6 anthracis spores (5 MOI) in the presence of a DMSO control (1 %) or the compounds n
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7 (10 µM) and cell death was monitored by the uptake of membrane impermeable sytox ad
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8 green using flow cytometry. A number of compounds (28% hit rate) protected f
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9 macrophages to varying extents (≥ 50%) from B. anthracis induced cell death.

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10 Representative results for the four most potent compounds (MBX 1066, MBX 1090, p
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11 MBX 1113 and MBX 1128, also known as NSC 317881, NSC 317880, NSC 330687, a
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12 NSC 369718 respectively in supplementary Table 1) are shown in Fig. 1A&B. These sm
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13 potent inhibitors all share a bis-(imidazolinylindole) chemotype; their chemical structures r
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14 are shown in Fig. 1C. The observed cellular protection also suggests that the n

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15 compounds are relatively non-toxic to macrophages at the tested dose. In an n
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16 independent experiment, in the absence of bacteria, only MBX 1128 caused more ry

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17 macrophage death than the DMSO control, and the effect was modest (Fig. 1B). 2
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18 In vitro inhibition of bacterial growth. The protection of macrophages from cell death 9

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19 by the identified compounds suggests that they may be targeting bacterial growth or g
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20 viability, bacterial virulence factors, or host factors vital to the bacteria. The cells s
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21 infected with B. anthracis in the presence of the identified hit compounds did not show
22 any changes in the pH of the medium, and microscopic examination of these samples
23 showed little to no outgrowth of the bacteria. These results suggest that the compounds
10