Small Molecule Inhibitors of the BfrB-Bfd Interaction Decrease Pseudomonas aeruginosa Fitness and Potentiate Fluoroquinolone Activity

Article abstract of DOI:10.1021/jacs.9b00394

The iron storage protein bacterioferritin (BfrB) is central to bacterial iron homeostasis. The mobilization of iron from BfrB, which requires binding by a cognate ferredoxin (Bfd), is essential to the regulation of cytosolic iron levels in P. aeruginosa. This paper describes the structure-guided development of small molecule inhibitors of the BfrB-Bfd protein-protein interaction. The process was initiated by screening a fragment library and followed by obtaining the structure of a fragment hit bound to BfrB. The structural insights were used to develop a series of 4-(benzylamino)- A nd 4-((3-phenylpropyl)amino)-isoindoline-1,3-dione analogs that selectively bind BfrB at the Bfd binding site. Challenging P. aeruginosa cells with the 4-substituted isoindoline analogs revealed a dose-dependent growth phenotype. Further investigation determined that the analogs elicit a pyoverdin hyperproduction phenotype that is consistent with blockade of the BfrB-Bfd interaction and ensuing irreversible accumulation of iron in BfrB, with concomitant depletion of iron in the cytosol. The irreversible accumulation of iron in BfrB prompted by the 4-substituted isoindoline analogs was confirmed by visualization of BfrB-iron in P. aeruginosa cell lysates separated on native PAGE gels and stained for iron with Ferene S. Challenging P. aeruginosa cultures with a combination of commercial fluoroquinolone and our isoindoline analogs results in significantly lower cell survival relative to treatment with either antibiotic or analog alone. Collectively, these findings furnish proof of concept for the usefulness of small molecule probes designed to dysregulate bacterial iron homeostasis by targeting a protein-protein interaction pivotal for iron storage in the bacterial cell.

Full text of DOI:10.1021/jacs.9b00394

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Article
Small molecule inhibitors of the BfrB-Bfd interaction decrease
Pseudomonas aeruginosa fitness and potentiate fluoroquinolone activity
Achala Punchi Hewage, Huili Yao, Baskar Nammalwar, Krishna Kumar
Gnanasekaran, Scott Lovell, Richard A Bunce, Kate Eshelman, Sahishna Phaniraj,
Molly Lee, Blake R. Peterson, Kevin P. Battaile, Allen B Reitz, and Mario Rivera
J. Am. Chem. Soc., Just Accepted Manuscript • Publication Date (Web): 30 Apr 2019
Downloaded from http://pubs.acs.org on April 30, 2019
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Small molecule inhibitors of the BfrB-Bfd interaction
decrease Pseudomonas aeruginosa fitness and
potentiate fluoroquinolone activity
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Achala N. D. Punchi Hewage, a§ Huili Yao, b§ Baskar Nammalwar, c¥ Krishna Kumar Gnanasekaran,
c¥ Scott Lovell, d Richard A. Bunce, c Kate Eshelman, a ¥ Sahishna M. Phaniraj, e Molly M. Lee, e ¥ Blake R.
Peterson, e Kevin P. Battaile, f Allen B. Reitz, g and Mario Rivera* b
aDepartment of Chemistry, University of Kansas, 2030 Becker Dr. Lawrence, KS, 66047
bDepartment of Chemistry, Louisiana State University, 229A Choppin Hall, Baton Rouge, LA 70803.
cDepartment of Chemistry, Oklahoma State University, Stillwater, OK 74078.
dProtein Structure Laboratory, University of Kansas, 2034 Becker Drive, Lawrence, KS 66047
eDepartment of Medicinal Chemistry, University of Kansas, 2034 Becker Drive, Lawrence, KS 66047
fIMCA-CAT, Hauptman Woodward Medical Research Institute, 9700 South Cass Avenue, Building 435A,
Argonne, IL 60439
gFox Chase Chemical Diversity Center, Inc. 3805 Old Easton Road, Doylestown, PA 18902.
¥Present address: Baskar Nammalwar: 10522 Parkdale Ave., San Diego, CA 92126; Krishna K.
Gnanasekaran: 4591 Gatineau Ave., Mississauga, Ontario, Canada L4Z 2R9; Kate Eshelman: 10 Waterview
Blvd., Parsippany, NJ 07054; Molly M. Lee: National Cancer Institute, Building 10, Hatfield CRC,
Bethesda, MD 20892.
§These authors contributed equally.
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*Corresponding author: Mario Rivera, Department of Chemistry, Louisiana State University, 229A
Choppin Hall, Baton Rouge, LA 70803. Telephone: 225-578-1527; Fax: 225-578-3458; Email:
mrivera@lsu.edu
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ORCID: 0000-0002-5692-5497
Funding Sources
This study was supported by a grant from the National Institutes of Health to M.R. (AI125529). M.R. also
thanks the National Science Foundation for support (MCB1615767). Use of the University of Kansas
Protein Structure Laboratory was supported by a grant by the National Institute of General Medical Sciences
(P30 GM110761) at the National Institutes of Health.
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ABSTRACT
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The iron storage protein bacterioferritin (BfrB) is central to bacterial iron homeostasis. The
mobilization of iron from BfrB, which requires binding by a cognate ferredoxin (Bfd), is essential to the
regulation of cytosolic iron levels in P. aeruginosa. This paper describes the structure-guided development
of small molecule inhibitors of the BfrB-Bfd protein-protein interaction. The process was initiated by
screening a fragment library and followed by obtaining the structure of a fragment hit bound to BfrB. The
structural insights were used to develop a series of 4-(benzylamino)- and 4-((3-phenylpropyl)amino)-
isoindoline-1,3-dione analogs that selectively bind BfrB at the Bfd binding site. Challenging P. aeruginosa
cells with the 4-substituted isoindoline analogs revealed a dose-dependent growth phenotype. Further
investigation determined that the analogues elicit a pyoverdin hyperproduction phenotype that is consistent
with blockade of the BfrB-Bfd interaction and ensuing irreversible accumulation of iron in BfrB, with
concomitant depletion of iron in the cytosol. The irreversible accumulation of iron in BfrB prompted by the
4-substituted isoindoline analogs was confirmed by visualization of BfrB-iron in P. aeruginosa cell lysates
separated on native PAGE gels and stained for iron with Ferene S. Challenging P. aeruginosa cultures with
a combination of commercial fluoroquinolone and our isoindoline analogs results in significantly lower cell
survival relative to treatment with either antibiotic or analog alone. Collectively, these findings furnish
proof of concept for the usefulness of small molecule probes designed to dysregulate bacterial iron
homeostasis by targeting a protein-protein interaction pivotal for iron storage in the bacterial cell.
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INTRODUCTION
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Antibiotic resistant infections are a worldwide threat to public health. The challenge posed by the
emergence of antibiotic resistant strains is compounded by slow to nearly stalled development of new
antibiotics and validation of new targets.1-3 Hence, antibiotic resistant infections have the potential to
undermine many achievements in modern medicine, such as organ transplantation, major surgery and
cancer chemotherapy. The World Health Organization (WHO) published a priority list for research and
development of new antibiotics to combat multi-drug resistant bacteria, and assigned critical priority to the
Gram-negative carbapenem-resistant Acinetobacter baumanii and Pseudomonas aeruginosa, and third-
generation cephalosporin resistant Enterobacteriaceae.4 P. aeruginosa is one of the leading Gram-negative
pathogens associated with hospital infections due to their propensity to colonize urinary catheters and
endotracheal tubes,5-6 and accelerate lung function decay that lowers the survival of cystic fibrosis patients.7-
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Responding to this call requires vibrant research and continued investment in the early stages of drug
development, in order to ensure a pipeline of novel ideas and approaches.5 In this context, strategies that
interfere with bacterial iron acquisition and homeostasis are regarded as having potential as new therapeutic
interventions.9-13 Iron is essential for bacteria because of its involvement in multiple metabolic processes,
including respiration and fundamental enzymatic reactions.14 Pathogenic bacteria must obtain iron from the
host, but host nutritional immunity maintains extremely low concentrations of free iron, thus denying the
essential nutrient to invading pathogens.15-18 In addition, the very low solubility of the ferric ion (Fe3+)
severely limits its bioavailability, and the reactivity of the soluble ferrous iron (Fe2+) toward hydrogen
peroxide and oxygen induces oxidative stress. Consequently, the processes of bacterial iron homeostasis
(acquisition, storage and utilization) are highly regulated to ensure sufficiency for metabolic needs while
preventing iron-induced toxicity.19-20 Herein, we describe a new approach to dysregulate iron homeostasis
in P. aeruginosa which utilizes small molecule probes designed to block the interaction between the iron
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storage protein bacterioferritin B (BfrB) and its cognate partner, the bacterioferritin-associated ferredoxin
(Bfd).
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Bacteria store iron reserves in bacterial ferritin (Ftn) and in bacterioferritin (Bfr).21-23 The roughly
spherical and hollow structures of Bfr and bacterial Ftn, which are formed from 24 identical subunits, have
an outer diameter of ~120 Å, an inner diameter of ~80 Å, and an interior cavity that can store up to ~3,000
iron ions in the form of a Fe3+ mineral (Figure 1A). Bfrs, which exist only in bacteria, bind 12 heme groups
buried under the external protein surface, with the heme propionates protruding into the interior cavity.21-22
Despite sharing a nearly identical subunit fold and quaternary structures, the eukaryotic Ftns and the Bfrs
share less than 20% sequence similarity, which results in divergent subunit packing, 24-mer dynamics and
function.23-26 Although in P. aeruginosa the ftnA and bfrB genes encode a bacterial ferritin (FtnA) and a
bacterioferritin (BfrB), respectively,27-28 BfrB functions as the main iron storage protein.19 Importantly, the
mobilization of iron stored in BfrB requires specific interactions with Bfd.19, 23, 29 A crystal structure of the
BfrB-Bfd complex revealed that up to 12 Bfd molecules can bind at identical sites on the BfrB surface, at
the interface of subunit dimers, above a heme molecule (Figure 1B).30 Characterization of the complex in
solution showed that the 12 Bfd binding sites are equivalent and independent, and that Bfd binds to BfrB
with a Kd of approximately 3 µM.31 These investigations also revealed that M1, Y2 and L5 in Bfd form a
continuous set of interactions with L68 and E81 in BfrB, which contribute significantly to the stabilization
of the BfrB-Bfd complex (Figure 1C). In agreement, the Kd values for the association between Bfd and the
L68A or E81A mutants of BfrB are approximately 100-fold larger, and the association between Bfd and
the BfrB L68A/E81A double mutant is undetectable.31
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The repercussions of blocking the BfrB-Bfd interaction on P. aeruginosa iron metabolism have been
investigated by deleting the bfd gene. These investigations, which showed an irreversible accumulation of
Fe3+ in BfrB with concomitant iron deprivation in the cytosol, established the BfrB-Bfd interaction as a
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Figure 1. Structure and function of BfrB, Bfd and the BfrB-Bfd complex. (A) BfrB is a nearly spherical
molecule assembled from 24 identical subunits and 12 hemes. The 24-mer assembly harbors a hollow cavity
approximately 80 Å in diameter where iron is stored in the form of a Fe3+ mineral. (B) Each heme molecule
(red) is buried at the interface of a subunit dimer (green and gray), with the heme propionates protruding
into the interior cavity. Each molecule of Bfd (cyan) binds BfrB at the subunit dimer interface to facilitate
electron flow from the [2Fe-2S] cluster (orange and yellow spheres) in Bfd to the Fe3+ mineral in the interior
cavity of BfrB through a heme, thus promoting the mobilization of Fe2+. (C) Zoomed-in view of the BfrB-
Bfd protein-protein interface, depicting the proximity of the [2Fe-2S] cluster in Bfd (orange and yellow) to
the BfrB surface, as well as the burial of key Bfd residues (Y2 and L5) on pockets formed at the BfrB
surface by residues L68 and E81.
novel target to rationally induce iron homeostasis dysregulation in bacteria.19 Consequently, it is important
to discover small molecule inhibitors of the BfrB-Bfd interaction, which can be used as chemical probes to
study bacterial iron homeostasis and uncover additional vulnerabilities in the bacterial cell exposed by iron
metabolism dysregulation. Chemical probes are a powerful complement to the utilization of genetic
techniques because they offer dose-dependent, selective and temporal control over target proteins, which
can be utilized in combination with other synergistic or antagonistic probes.32-33 Herein we present the
results from a structure-guided program aimed at the development of small molecules designed to inhibit
the BfrB-Bfd interaction in P. aeruginosa. These novel probes are capable of penetrating the bacterial cells,
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where they inhibit the mobilization of iron from BfrB and elicit perturbations in iron homeostasis that
decrease bacterial fitness, and also potentiate the bactericidal activity of fluoroquinolone antibiotics.
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EXPERIMENTAL
Chemicals, strains, and growth media
Chemicals were purchased from Fisher Scientific unless otherwise stated. Pseudomonas aeruginosa
(PAO1) was purchased from the University of Washington Genome Center. The PAO1-derived strain with
an unmarked, in-frame deletion of the bfrB gene had been prepared previously.19 P. aeruginosa clinical
isolates (MR3B and MR60) were obtained from Seattle Children’s Research Foundation.# All strains were
kept on Pseudomonas Isolation Agar (PIA) (BD Biosciences, CA). M63 media was prepared as previously
reported,34 with a small modification. It contained per liter: 2 g of (NH4)2SO4, 13.6 g of KH2PO4 (Sigma
Aldrich), 2 g of glucose, 4 g of citric acid, 0.25 g of tryptophan (Acros organics), 5 g of non-technical grade
casamino acids (BD scientific) and 0.24 g of MgSO4 (Alfa Aesar), and the pH was adjusted to 7.0 with
KOH. The M63 media also contained 0.1% (w/v) of hypermellose (HPMC, Sigma Aldrich) to prevent
aggregation of the analogs in aqueous solution.35 Colorimetric analysis showed that the M63 media contains
2 µM Fe. When necessary, the M63 media was supplemented with additional iron by addition of a small
volume of 1 mM (NH4)2Fe(SO4)2 (pH ~ 2.0) to give the desired final iron concentration.
Fragment library screening using saturation transfer difference (STD) NMR spectroscopy
Experimental details are presented in Supplementary Information.
Synthesis and preparation of analogs
Experimental details of the synthetic procedures developed to prepare compounds to be tested as
inhibitors of the BfrB-Bfd interaction, as well as the corresponding 1H and 13C NMR spectra, are presented
in Supplementary Information.
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Crystallization, ligand soaking and data collection
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Crystallization screening was conducted in Compact 300 (Rigaku Reagents) sitting drop vapor
diffusion plates at 18 °C using equal volumes (0.5 µL) of BfrB and crystallization solution equilibrated
against 75 L of the latter. Three different BfrB constructs were investigated to grow crystals of BfrB
suitable for soaking experiments with the different fragments and analogs. BfrB crystals were observed in
1-2 days as follows: C89S/K96C BfrB: Red prismatic crystals were obtained from Wizard 1-2 (Rigaku
Reagents) condition E2 (35% (v/v) 2-methyl-2,4-pentanediol, 100 mM MES 6.5, 200 mM Li2SO4). Apo-
BfrB (BfrB devoid of heme): Colorless prismatic, or light yellow crystals, were obtained from the Wizard
3-4 screen (Rigaku Reagents) condition B1 (8% (w/v) PEG 8000, 100 mM Na acetate pH 4.6). BfrB: Red
plates grew from the Cryo 1-2 HT screen (Rigaku Reagents) condition H6 (30% (v/v) PEG 200, 100 mM
Na acetate pH 4.5, 100 mM NaCl). To prepare for soaking experiments, a stock solution (100 mM in
DMSO) of each fragment or analog (compound) was mixed with crystallization solution to obtain a 20 mM
compound solution to be used in soaking experiments. Crystals were transferred to these soaking solutions
and incubated for 3.0 to 3.5 h before harvesting directly from the drop and storing in liquid nitrogen. Analog
13 was soaked in a 25 mM compound solution for 2 h, and analog 16 was soaked in a 10 mM compound
solution for 3 h. The compound soaking solutions, which also served as the cryoprotectant, contained 80%
crystallization solution and 20% DMSO. Structures of compounds bound to BfrB were obtained from the
following compound/BfrB crystal combinations: Fragment 1/C89S/K96C BfrB, analog 12/apo-BfrB, and
analogs 11, 13, 14, 15, 16/BfrB. X-ray diffraction data were collected at the Advanced Photon Source
beamline 17-ID using a Dectris Pilatus 6M pixel array detector.
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Structure solution and refinement
Intensities were integrated using XDS,36 via Autoproc37 and the Laue class analysis and data scaling
were performed with Aimless.38 Structure solution was conducted by molecular replacement with Phaser39
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and structure refinement/manual model building were performed with Phenix40 and Coot,41 respectively.
Electron density omit maps for the ligands were calculated using the Polder omit routine42 with the Phenix
software suite. Structure validation was carried out with Molprobity43 and figures were prepared with
CCP4mg.44 The search models used for molecular replacement were as follows: C89S/K96C BfrB (PDB:
4TOF)26 and BfrB (PDB: 5D8O).31 Apo-BfrB: Structure solution was carried out using a single subunit of
a previously determined structure of BfrB as the search model (PDB: 3IS7).28 The top solution was obtained
in the space group C2221 with 12 molecules in the asymmetric unit.
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Growth curves and IC50 determination.
Pre-cultures (5 mL LB media) were grown for 12 h at 37 °C and 220 rpm in 50 mL conical tubes
(VWR International, PA) covered with an air permeable membrane. The cells were then centrifuged at
4,000 rpm for 10 min, the resultant cell pellets washed twice in M63 media and then diluted in M63 media
to OD600 = 0.01. Stock solutions (100 mM) of analog in DMSO were prepared weekly and stored at 4 °C.
Prior to initiating experiments in 96 well plates, the analog stock solution was serially diluted to make 10
mM or 1 mM working solutions in DMSO. A small volume of the appropriate working solution was
transferred to a glass vial, diluted with DMSO to 30 µL, and then diluted to a final volume of 1.5 mL with
pre-culture cell suspension with an OD600 = 0.01. The resultant cell suspension (200 µL) was transferred to
a clear-bottom polystyrene 96 well plate (VWR), and incubated at 35 °C and 205 cpm for 13 h in an Epoch
2 microplate spectrophotometer (Biotek Instruments, Inc., Vermont). The growth curve in the presence of
Ciprofloxacin was obtained in the same plate. To this end, 11.2 µL of a Ciprofloxacin working solution
(100 µg/mL) was mixed with 30 µL of DMSO prior to diluting to a final volume of 1.5 mL with cell
suspension having an OD600 = 0.01 and transferring to the plate. The resultant concentration of ciprofloxacin
in the wells was 0.75 µg/mL, which is equivalent to 3 times the reported MIC.45 Each condition was
replicated in 5 wells of the plate, so the growth curves shown in Figure 5 and S5 are constructed from the
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average and standard deviation of 5 replicates. The growth % was estimated using the OD600 values obtained
13 h post-inoculation and equation 1, where ODT is the optical density of the culture treated with analog,
ODU is the optical density of the untreated control, and ODCip is the optical density of the culture treated
with ciprofloxacin. To calculate the IC50 values, the growth % was plotted as a function of log[analog (µM)],
and fitted to equation 2, where the terms are defined as above and b is the slope factor.46
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―
growth % = ((
))x100
ODT ODCip
(1)
―
ODU ODCip
ODU ― ODCip
(2)
growth % = ODCip
+
―푏
푙표푔[analog (μM)]
1 +
(
)
IC
50
Analysis of secreted pyoverdin
These experiments were carried out in 96 well plates, as described above. P. aeruginosa cells treated
with analog 11 or 16 (125 µM final concentration) were cultured for 13 h before the contents of each well
was serially diluted in phosphate buffered saline (PBS, pH 7.4) and then plated on PIA plates to enumerate
viable cells. The 500-fold diluted solution was centrifuged and the cell-free supernatant was analyzed for
pyoverdin by fluorescence spectrophotometry in a Synergy H1 microplate reader (Bioteck) with excitation
at 400 nm and emission at λmax = 455 nm. Full emission spectra (430-550 nm) were also recorded using a
Perkin Elmer LS50B spectrophotometer.
Imaging of iron stored in BfrB and analysis of total iron levels
Pre-cultures were grown as described above and cells were diluted in M63 media supplemented
with 4 µM Fe (6 µM total Fe) to OD600 = 0.1 in 50 mL conical tubes. The resultant cell suspensions (5 mL)
were mixed with a small volume of a 10 mM working solution of analog 16 in DMSO to give a 125 µM
solution and 2% DMSO, or simply 2% DMSO (control). The conical tubes were covered with an air
permeable membrane and the cultures incubated at 35 °C and 120 rpm for 6, 9, 12, 15, 18, 21 and 24 h.
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Prior to separating the cells by centrifugation (4,000 rpm, 15 min), a 100 µL aliquot was withdrawn from
each conical tube and serially diluted for plating and enumerating viable cells. The separated pellet was
washed with 5 mL of PBS and frozen at -20 °C for subsequent analysis. The corresponding cell free
supernatants were used for iron analysis in the spent media, as described below. The cell pellets were used
to image iron stored in BfrB according to a previously described method19 with some minor modifications.
Briefly, cell pellets were suspended in 300 µL of lysis buffer (50 mM Tris pH 8.0, 20% glycerol, 20 mg/mL
lysozyme, 0.2 mg/mL DNAse, 100 mM NaCl, 10 mM MgCl2, and 1% Triton X) and freeze-thawed twice
using liquid N2. The resultant suspensions were incubated at 25 °C for 90 min in a rocker and then
centrifuged at 12,500 rpm for 15 min at 4 °C. Lysate solutions (100 µL) were each mixed with 10 µL of the
loading dye and the samples (100 µL) loaded onto a 3 mm-thick native polyacrylamide gel (8% resolving
gel and 4% stacking gel) for separation. Electrophoretic separation was carried out at 60 V for 7 h at 4 °C
and the gels were stained with Ferene S47 for 10 min in a solution containing 0.049 g of Ferene S, 250 µL
of thioglycolic acid, 2.5 mL of acetic acid and 100 mL of water. The scanned images were processed and
compared using Image J.48
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To determine the total iron levels of cells treated with analog 16 or DMSO only (control), 5 mL
cultures were grown in M63 media supplemented with 4 µM iron, as described above, and analyzed at 12,
15, 21, 24, and 28 h. Prior to separating the cells by centrifugation (4,000 rpm, 15 min), a 100 µL aliquot
was withdrawn from each conical tube and serially diluted for plating, in order to enumerate viable cells.
Cell pellets were washed twice with 10 mL of PBS and the total levels of cellular iron were measured using
a published protocol.19, 49 In brief, cell pellets were mixed with 500 µL of freshly prepared digestion reagent
(1:1 v/v 1.2 N HCl and 4.5% w/v KMnO4 in water), thoroughly mixed by vortexing, and incubated at 65
°C for 4 h. The digested solutions were cooled to 25 °C, mixed with 500 µL of iron chelating agent (6.5
mM Ferene S, 13.1 mM neocuproine, 2 M ascorbic acid, 5 M ammonium acetate) and then incubated at 25
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°C for 30 min. The iron concentration of the resultant solution was measured from the absorbance of the
Fe2+-Ferene S complex at 593 nm (ε = 34,500 M-1 cm-1)50 using a Cary 60 UV-vis spectrophotometer,
normalized by cell count and reported as Fe atoms per CFU (colony forming unit). The colorimetric
determination of total intracellular iron offers a sensitive, accurate and low cost analytical technique, which
has been shown to produce results similar to those obtained by atomic inductively coupled plasma-mass
spectrometry.51
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Effect of analogs on the potency of fluoroquinolone antibiotics
Cultures were treated with (i) analog 16 only, (ii) ciprofloxacin 0.25 µg/mL, levofloxacin 0.5
µg/mL, or norfloxacin 0.9 µg/mL only, and (iii) both fluorquinolone (concentration as above) and analog
16. To treat cells with analog only, pre-cultures were grown as described above; the cells were then diluted
in M63 media supplemented with 4 µM Fe to OD600 = 0.1 in 50 mL conical tubes. The resultant cell
suspensions (5 mL) were mixed with a small volume of a 10 mM working solution of analog 16 in DMSO
to give the desired analog concentration (75, 100, or 125 µM) and 2% DMSO, or simply with DMSO
(untreated control). The conical tubes were covered with an air permeable membrane and the cultures were
incubated for 18 h at 35 °C and 120 rpm. The resultant cultures were serially diluted in PBS and plated on
PIA for enumeration of viable cells. To treat cells with ciprofloxacin only, the procedure was identical
except that the cell suspensions (5 mL) were mixed with a small volume of ciprofloxacin working solution
(100 µg/mL) to give the desired antibiotic concentration and 2% DMSO. The combined treatment was
carried out similarly, ensuring that the final concentration of DMSO was 2%.
Measurement of dissociation constants (Kd)
Experimental details are presented in Supplementary Information.
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RESULTS
Screening and detection of fragment binding to the Bfd binding site on BfrB
6
7
8
9
Structural information obtained from the BfrB-Bfd complex was used to design a fragment library
to screen for molecules that bind BfrB at the Bfd-binding site. Bfd residues M1, Y2, and L5 and BfrB
residues L68 and E81 dominate the buried surface area at the protein-protein interface (Figure 1C) and
contribute significantly to the binding energy of the BfrB-Bfd complex.31 Consequently, the fragment
library was focused on fragments that may bind at the sites occupied by Y2 and L5 from Bfd and included
groups with chemical properties similar to the aromatic and aliphatic side chains of Tyr and Leu, also
utilizing standard fragment criteria (MW < 300 Da, clogP <3, and total count of hydrogen bond
acceptors/donors <3 each). In addition, fragments capable of π-π stacking were included with both electron
rich and deficient aromatic rings. To screen the library in search of fragments that bind BfrB at the Bfd-
binding site we developed a competition assay that utilizes saturation transfer difference (STD) NMR
spectroscopy. This technique is ideally suited to screen fragments that bind to the large BfrB (~440 kDa)
because protein resonance assignments are not necessary and very low protein concentrations are required.
In addition, the large rotational correlation time (τc) of BfrB enhances spin diffusion and therefore saturation
transfer within the protein and to the ligand.52 Two solutions were prepared for each fragment: a solution
of the fragment alone, and a solution of the fragment and BfrB. Three spectra were obtained for each
fragment, as illustrated in Figure S1 of the Supplementary Information for fragment 1, which binds BfrB at
the Bfd-binding site. The 1H spectrum of the fragment alone was used to determine fragment integrity and
solubility, the 1H spectrum of the fragment in the presence of BfrB was used to corroborate that fragment
integrity and solubility is not affected by BfrB, and the STD spectrum of the solution containing the
fragment and BfrB was used to assess fragment binding. This strategy uncovered 18 compounds that bind
BfrB. The specificity criterion for fragment binders, however, is that these bind BfrB specifically at the
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Bfd-binding site. To eliminate non-specific binders, a displacement strategy was implemented that utilizes
Bfd as a specific competitor. For this purpose, a STD spectrum was acquired from a solution containing
fragment, BfrB and Bfd. Nearly complete disappearance of the STD signal indicates that the fragment binds
BfrB at the Bfd-binding site. With the aid of this competitive displacement strategy, it was determined that
of the 18 fragments that bind BfrB, the 6 molecules shown in Chart 1 bind at the Bfd-binding site. These
fragments were advanced to the next stage, which was focused on uncovering structural information of
fragment binding. Results from these experiments are presented below.
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Chart 1. Structures of fragments identified to bind BfrB at the Bfd-binding site using the screening strategy
described in the text.
Structure-based optimization of fragment binders
A structure-guided approach was used to synthetically elaborate fragments discovered to bind BfrB
at the Bfd-binding site into analogs capable of binding with higher affinity. The affinity and selectivity of
the fragments and analogs for the Bfd-binding site on BfrB were investigated by X-ray crystallography,
surface plasmon resonance (SPR) and fluorescence polarization methods. The structures of fragments and
derived analogs which have been demonstrated to bind BfrB at the Bfd-binding site using X-ray
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crystallography are shown in Figure 3B. During the course of these experiments, crystals obtained from
three different BfrB constructs were tested in ligand soaking experiments, in an effort to identify the “best”
crystals for the study. Hence, crystals of BfrB (PDB 5D8O), C98S/K96C BfrB (PDB 4TOF), and apo-BfrB
were soaked in crystallization solution containing the various analogs. The structure of 5-
hydroxyisoindoline-1,3-dione (fragment 1) bound to BfrB was obtained by soaking crystals of C98S/K96C
BfrB in a solution of the fragment, as described in the Experimental Section. In subsequent experiments,
crystals of apo-BfrB were used because this protein formed robust, highly reproducible crystals. These
experiments culminated in the structure of analog 12 bound to BfrB. It is important to underscore that the
structure of apo-BfrB is nearly identical to that of BfrB, at the subunit level (RMSD = 0.18 Å), as well as
the biological assembly level, including the Bfd-binding sites (Figure S2A). Additional evidence indicating
that the Bfd-binding sites on apo-BfrB are unaffected relative to the holo-protein was obtained in the
dissociation constant for the interaction between Bfd and apo-BfrB (Kd = 3.1 µM) (Figure S2B and C),
which is nearly identical to that previously reported for the interaction between Bfd and holo-BfrB (Kd =
3.4 µM).31, 53 Finally, the BfrB protein also produced reproducible crystals that were isomorphs with those
formed by apo-BfrB. Consequently, the structures of analogs 11 and 13-16 bound to BfrB were obtained
by soaking crystals of BfrB in solutions of each of the analogs.
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Figure 2A depicts a portion of the BfrB-Bfd complex interface and illustrates how Y2 and L5 in Bfd
(cyan cylinders) contribute to anchor Bfd to the BfrB surface. A prior study indicated that interactions
between L68 and E81 in BfrB and Y2 and L5 in Bfd contribute significantly to the binding energy of the
BfrB-Bfd complex.31 It was therefore surmised that fragments capable of binding to this region of the BfrB
surface would be good candidates for subsequent structure-guided synthetic elaboration aimed at
discovering inhibitors of the BfrB-Bfd interaction. To obtain the structures of fragments bound to BfrB,
crystals of the protein were soaked in crystallization solution containing each of the 6 fragments found to
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bind BfrB at the Bfd-binding site with the aid of the competitive displacement STD NMR strategy described
above. These efforts culminated in a 1.5 Å resolution co-crystal structure of 5-hydroxyisoindoline-1,3-dione
(1) bound to BfrB (Figures 2B and 2C and Table S1), which showed that 1 binds BfrB at the Bfd-binding
site, in the same pocket where L5 from Bfd would bind. The fragment rests on a platform at the base of a
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Figure 2. The protein-protein interaction interface of the BfrB-Bfd complex. Subunits A and B of a BfrB
subunit dimer are colored gray and green respectively. A) Residues Y2 and L5 of Bfd (cyan cylinders)
are positioned within pockets at the BfrB subunit dimer interface. B) Fo-Fc omit map (orange mesh)
contoured at 3σ showing 5-hydroxyaminoisoindoline-1,3-dione (fragment 1) bound within the cleft
occupied by L5 in the BfrB-Bfd complex. C) Hydrogen bond interactions (dashed lines) between fragment
1 and BfrB. Water mediated contacts are indicated by the solid lines.
77
79
shallow depression on the BfrB surface formed by the side chains of
and
(the subscript denotes
IB
LB
subunit and the superscript denotes residue number), surrounded by a semicircular wall comprised by the
68
70
side chain and backbone atoms of
,
,
72, and 77. One of the carbonyl-oxygen atoms of 1 accepts a
LA NA QA LB
69
H-bond from the backbone NH of 71, and its N-H group forms a H-bond to the backbone carbonyl of
LA
.
PA
Additional stabilization for binding probably stems from the network of H-bonded waters linking a carbonyl
oxygen in 1 and 67 in BfrB. This structural information suggested a strategy to grow 1, or its analogs 4-
GA
aminoisoindoline-1,3-dione (8) and 5- aminoisoindoline-1,3-dione (5), by branching from the isoindoline
ring carbons C4 or C5 to engage the cleft formed by the side chains of L68 and E81 in BfrB, where Y2
from Bfd anchors.
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A general synthetic approach (Figure 3A) was formulated to generate a series of ether analogs
represented by 4, amine analogs represented by 6 or 9, and amide analogs represented by 7 or 10.
Preparation of the ether analogs of 4 started with the esterification of the acid groups in 4-hydroxyphthalic
acid 2, followed by alkylation of the phenolic oxygen to produce 3 and subsequent base cleavage of the
8
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Figure 3. (A) Schematic summary of the synthetic procedures employed to prepare analogs of fragments
1, 5 and 8 to form ethers 4 derived from 5-hydroxyisoindoline-1,3-dione (1); amines 6 or amides 7 derived
from 5-aminoisoindoline-1,3-dione (5); or amines 9 or amides 10 derived from 4-aminoisoindoline-1,3-
dione (8). Descriptions of the synthetic procedures, as well as the characterization of the analogs are
presented in Supplementary Information. (B) Structures of fragment 1 and analogs prepared from fragment
8, which have been shown to bind BfrB at the Bfd binding site by X-ray crystallography.
esters and cyclization to produce the isoindoline-1,3-dione ethers 4. Synthesis of the amine analogs
represented by 6 and 9 was carried out by reductive amination of 5 or 8, respectively, with a series of
aldehydes, whereas amide analogs 7 and 10 were obtained from 5 or 8, respectively, via reactions with a
series of acid chlorides. The collection of ether, amine and amide analogs prepared for this study, as well
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as the details of their synthetic preparation and characterization, are presented in the Supplementary
Information.
7
8
9
Although all of the compounds synthesized for each of the analog types shown in Figure 3A were
tested in crystal soaking experiments, structures of analog-bound BfrB were obtained only for analogs 11
– 16 (Figure 3B), which are derivatives of 4-aminoisoindoline-1,3-dione (8) with –(CH2) – and –(CH2)3–
linkers. The pose of each of these analogs in the Bfd-binding site of BfrB is shown in Figures 4 and S3.
Inspection reveals that the isoindoline-1,3-dione moiety in all the compounds invariably binds BfrB in a
10
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manner identical to that described above for fragment 1 binding to BfrB (see Figure 2). In addition, and as
68
was expected, the benzyl portion of the analogs extends to engage the cleft formed by the side chains of
LA
81
and
in BfrB via hydrophobic packing interactions. The interactions experienced by analog 11 at the
EB
Bfd-binding site on BfrB are illustrated in Figure 4A-C. Strong electron density consistent with the
compound can be observed in 9 of the 12 subunits in the asymmetric unit; the 3 subunits in which compound
11 was not modeled displayed electron density not associated with the protein at the Bfd-binding site, but
this electron density was too weak to model the ligand. The o-hydroxyphenyl ring of 11 is observed in two
orientations that differ by a 180° rotation of the ring; one orientation places the hydroxyl group pointing
toward the base of the cleft, where it forms a hydrogen bond with 80, while the second orientation places
GB
the hydroxyl group toward the solvent and enables packing of the o-hydroxyphenyl ring and the –(CH2)–
31
moiety bridging the phenyl and isonindoline rings against the side chains of
and 79, respectively.
IB
MB
Additional stabilization for binding probably stems from the network of H-bonded waters linking a carbonyl
oxygen in 11 and 67, similar to the network of H-bonded water molecules observed in the structure of 1
GA
bound to BfrB (see Figure 2C). The structure of a similar analog (12) bound to BfrB shows identical
interactions of the isoindoline-1,3-dione moiety with BfrB and similar interactions of the phenyl ring with
the cleft formed by the side chains of 68 and 81 on the BfrB surface (Figure S3 A-C).
LA
EB
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Figure 4. Binding modes of analogs 11 and 16 at the Bfd-binding site on BfrB. Subunits A and B of a
BfrB subunit dimer are colored gray and green, respectively. A) Fo-Fc omit map (orange mesh)
contoured at 3σ from analog 11 bound at the Bfd-binding site on BfrB. B) Same as panel A but showing
a different perspective to illustrate the two orientations modeled for the o-hydroxyphenyl ring of analog
11. C) Hydrogen bond interactions (dashed lines) between analog 11 and BfrB. Water mediated contacts
are indicated by the solid lines. D) Fo-Fc omit map (orange mesh) contoured at 3σ from analog 16
bound at the Bfd-binding site on BfrB. E) Same as panel D but rotated to illustrate the m-hydroxyphenyl
68
81
ring of analog 16 positioned between the cleft formed by the side chains of
and
in BfrB. F)
EB
LA
Hydrogen bond interactions (dashed lines) between analog 16 and BfrB. Water mediated contacts are
indicated by the solid lines.
A similar close view of compound 16 bound at the Bfd binding site on BfrB (Figure 4D-F) illustrates
how the isoindoline-1,3-dione ring presents the same pose and set of interactions as those described above
for fragment 1 and analogs 11 and 12. Clear electron density consistent with the compound can be observed
in all 12 subunits in the asymmetric unit, but the m-hydroxyphenyl ring was partially disordered in several
of the subunits. The m-hydroxyphenyl ring and the –(CH ) – linker pack against the 68 and hydrophobic
LA
2 3
portion of the 81 side chains, with additional stabilization probably stemming from a network of H-bonded
EB
waters connecting a carbonyl oxygen in the isoindoline-1,3-dione ring, the hydroxyl group on aromatic ring
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and the carbonyl oxygen in 68. The structures of four additional compounds similar to 16 bound to BfrB
LA
5
6
7
8
(Figure S3), which show a nearly identical pose of the isoindoline-1,3-dione moieties and very similar
interactions of the linkers and phenyl rings, demonstrate the specificity with which the series of 4-amino
derivatives listed in Table 1 engage the Bfd-binding site on BfrB.
9
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11
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Prior to testing the effect that the 4-substituted isoindoline-1,3-dione derivatives might exert on P.
aeruginosa cells, the strength of their interaction with BfrB was evaluated in vitro with a fluorescence
polarization assay developed based on the intrinsic fluorescence of the isoindoline-1,3-dione moiety.
Because initial fluorescence spectroscopic measurements revealed that the heme groups in BfrB interfere
with the signal of the fluorescent ligand, we utilized apo-BfrB for these measurements, capitalizing on our
earlier findings that the Bfd-binding sites in apo-BfrB are nearly identical to those in BfrB, and that the Kd
for the interaction between apo-BfrB and Bfd is very similar to that measured for the interaction between
BfrB and Bfd (see above and Figure S2). Hence, the Kd values were measured by titrating apo-BfrB into a
fixed concentration of the appropriate fluorescent 4-aminoisondoline-1,3-dione ligand while analyzing
fluorescence polarization and intensity near the emission λmax (Figure S4). The Kd values (Table 1) show
that analogs 11-16 exhibit significantly higher affinity than fragment 8, observations that are in agreement
with the structural insights, which revealed that growing the fragments from the isoindoline ring carbon C4
makes it possible to engage the cleft formed by the side chains of L68 and E81 in BfrB. The two derivatives
with a –(CH2)– linker exhibit binding affinities approximately 2 to 5-fold lower than derivatives with a –
(CH2)3– linker, also in agreement with a relatively more efficient hydrophobic packing facilitated by the
longer linker.
4-Aminoisoindoline-1,3-dione derivatives elicit a growth retardation phenotype in P. aeruginosa cells
The binding affinity and structural information indicate that most of the analogs in Table 1 bind
BfrB at the Bfd binding site with a strength comparable to that of the BfrB-Bfd association (Kd = 3 µM),
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Table 1. Structure, binding affinity and IC50 of 4-aminoisoindoline-1,3-dione derivatives
4
5
6
7
8
9
Analog
8
Structure
Kd (µM)
IC50 (µM)
O
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
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45
46
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50
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60
NH
300 ± 50
not active
O
NH2
O
O
N
H
11
12
13
14
15
16
11 ± 1
15 ± 2
3 ± 1
4 ± 2
5 ± 2
6 ± 1
258 ± 23
not active
201 ± 18
143 ± 8
NH
NH
OH
O
O
N
H
HO
N
H
O
O
NH
NH
HO
O
N
H
HO
HO
OH
O
O
N
H
O
227 ± 10
121 ± 4
NH
O
OCH3
N
H
NH
O
OH
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therefore suggesting that these compounds may be capable of interfering with the BfrB-Bfd interaction in
the P. aeruginosa cytosol. As will be described below, this idea was investigated, first by demonstrating
that the analogs elicit a growth phenotype in P. aeruginosa, and then by showing that one of the most potent
analogs inhibits the mobilization of iron from BfrB in the bacterial cytosol.
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Figure 5. Analogs of 4-aminoisoindoline-1,3-dione elicit a growth defect in P.aeruginosa. Panels (A)
and (B) show the time dependent growth retardation of P. aeruginosa cultures treated with analogs 16
and 11, respectively. The black circles correspond to untreated cells (DMSO control), and the open circles
to cells treated with ciprofloxacin (0.75 µg/mL). The concentrations of analog 16 in (A) are: 25 µM (red),
50 µM (green), 75 µM (yellow), 100 µM (blue) , and 125 µM (magenta); the concentrations of analog
11 in (B) are: 25 µM (red), 75 µM (yellow), 125 µM (magenta), 175 µM (purple), and 250 µM (brown).
The corresponding IC50 values (C and D) were obtained by calculating the % growth using OD600 values
at 13 h and fitting to equation 2, as indicated in the Experimental Section. Each of the growth curves was
constructed from the average and standard deviation of 5 replicate wells. The IC50 values (see Table 1)
are the average and standard deviation from three independent experiments.
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To investigate the effect of the analogs on cell growth, cultures of P. aeruginosa in M63 media were
challenged with 4-aminoisoindoline-1,3-dione derivatives, and their growth was monitored in 96 well plates
by following the OD600. Figures 5A and 5B illustrate the dose-dependent growth inhibition observed upon
challenging the cultures with analogs 11 and 16, respectively; the highest concentration of analog used was
determined by its solubility in PBS buffer, 250 µM for analog 11, and 125 µM for analog 16. Growth curves
from similar experiments conducted with the other analogs in Table 1 are shown in Figure S5. In order to
facilitate a quantitative comparison of the effect exerted by the analogs on P. aeruginosa growth, IC50 values
were calculated. To this end, the growth inhibition caused by each of the analogs was compared to that of
the untreated culture (100% growth) and to the growth in the presence of ciprofloxacin present at a
concentration equivalent to 3 times the reported MIC (0% growth). Hence, the relative growth in the
presence of a 4-aminoisoindoline-1,3-dione analog, measured 13 h post-inoculation, is defined by equation
1 (see Experimental Section). The IC50 values, obtained from fitting the plots relating growth % and analog
concentration to equation 2 (Figures 5C, 5D and S5), are listed in Table 1. Comparison of these values
shows that analog 16 is the most efficacious at inhibiting cell growth, despite a very similar binding affinity
for BfrB when compared to similar analogs such as 13, 14, and 15. The reasons for the higher efficacy of
analog 16 to inhibit cell growth are not yet evident, but it is possible to speculate that these may be related
to their relative ability to penetrate the P. aeruginosa cell and/or their reactivity/stability in cell culture.
To obtain initial insights on the selectivity of the analogs for their intended target in P. aeruginosa,
mutant cells with a deletion of the bfrB gene (ΔbfrB) were challenged with analog 11 or 16. To this end,
wild type and ΔbfrB cells were cultured in M63 media supplemented with 4 µM Fe, in the presence of
different concentrations of analog. The effect of the analogs on cell growth was evaluated 15 h post-
inoculation by plating and enumerating viable cells and comparing the cell growth of each strain to the
corresponding untreated control (Figures 6A and B). As expected, the analogs elicit a monophasic dose-
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dependent growth defect on the wild type cells. In comparison, the analogs induce a biphasic growth
response on the ΔbfrB mutant cells, which consists of a shallow first phase (0 - ~100 µM) that is nearly
independent of analog concentration, followed by a steep second phase where the analogs become rapidly
toxic. Since BfrB is not essential, it is likely that the cell can compensate for the absence of the iron storage
protein. Nevertheless, the nearly independent growth defect observed in the first phase, which is in good
agreement with the absence of BfrB, supports the idea that analogs 11 and 16 exhibit significant selectivity
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Figure 6. Deleting the bfrB gene (ΔbfrB) changes the monophasic dose-dependent growth response
exhibited by the wild type P. aeruginosa cells to analogs 16 and 11 into a biphasic response, which is nearly
independent of analog until ~100 µM and rapidly toxic thereafter. The plots illustrate the growth % of the
ΔbfrB (○) and wild type (●) cells relative to untreated control as a function of analog 16 (A) and analog 11
(B) concentration. The values at each point are the average and standard deviation from three independent
experiments.
for BfrB in the P. aeruginosa cell. The sudden onset of toxicity observed in the second phase, which is not
observed with the wild type cells, is probably related to off-target effects that expose a fitness vulnerability
caused by the absence of BfrB. Consequently, it is possible to conclude that the dose-dependent growth
defect elicited by 11 or 16 on wild type P. aeruginosa cells is largely a consequence of the interaction
between the small molecule inhibitors and BfrB in the bacterial cell. This issue was investigated in
additional detail by probing the phenotypic and biochemical response of wild type cells treated with the
small molecule inhibitors. The results of these studies are presented below.
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4-Aminoisoindoline-1,3 dione derivatives engage their target (BfrB) in P. aeruginosa cells
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Studies conducted to determine whether the analogs are capable of engaging BfrB and inhibiting
iron mobilization from the bacterioferritin in the P. aeruginosa cytosol were conducted mainly with the
most efficacious analog (16), and when practical, also with analog 11, which is the most active of the two
compounds harboring a –(CH2)– linker. As indicated above, genetic manipulations were used in prior work
to delete the bfd gene (Δbfd) and evaluate the consequences of inhibiting the BfrB-Bfd interaction in P.
aeruginosa.19 Preventing the BfrB-Bfd interaction in the Δbfd mutant cells dysregulates iron homeostasis
by causing the irreversible accumulation of iron in BfrB and the concomitant depletion of free iron levels
in the cytosol. The resultant phenotype is overproduction of the siderophore pyoverdin, which is ~4-fold
larger than that secreted by wild type cells.19 If the 4-aminoisoindoline-1,3-dione analogs are capable of
binding BfrB, blocking the BfrB-Bfd interaction and consequently inhibiting iron mobilization from BfrB
in the P. aeruginosa cytosol, then they would be expected to elicit a similar pyoverdin hyper-production
phenotype. To investigate whether 4-aminoisoindoline-1,3-dione derivatives indeed elicit such a
phenotype, P. aeruginosa cells cultured in M63 media were treated with analog 16 at a concentration of
125 µM. As expected, cell cultures treated with the analog exhibited ca. 30% of viable cells relative to cells
in the untreated control (Figure 7A). To analyze the levels of secreted pyoverdin, cells were pelleted and
the supernatant was diluted 500-fold prior to measuring the fluorescence intensity at 455 nm (Figure 7B).
Normalizing the intensity of pyoverdin fluorescence to cell density (CFU/mL) shows that cells treated with
16 secrete ~4-fold more pyoverdin than the untreated control (Figure 7C). To demonstrate that the intrinsic
fluorescence of 16, which is significantly weaker than that of pyoverdin, does not interfere with the
measurement, the fluorescence spectrum from a solution of analog 16 (125 µM) after a 500-fold dilution in
PBS is shown in the green trace of Figure 7B. Similar observations are made when cell cultures are treated
with analog 11 (Figure S6), albeit with a less pronounced phenotype, which is in agreement with the lower
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affinity of the analog for BfrB and correspondingly higher IC50. These findings, which indicate that the
analogs elicit the anticipated pyoverdin hyper-production phenotype in P. aeruginosa, suggest that they
bind BfrB in the P. aeruginosa cytosol, block the BfrB-Bfd interaction and inhibit iron mobilization from
BfrB.
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Figure 7. P. aeruginosa cells treated with analog 16 overproduce pyoverdin. (A) P. aeruginosa cultures
treated with analog 16 (125 µM) for 13 h exhibit approximately 30% of the viable cells in the untreated
DMSO control. (B) Fluorescence spectra obtained from cell free supernatants (13 h post-inoculation)
after a 500-fold dilution in PBS buffer, pH 7.4. The black trace is the spectrum from pyoverdin present
in cell-free supernatant from untreated cells (DMSO control), and the red trace is the spectrum from
pyoverdin in cell-free supernatant from samples treated with analog 16 (125 µM). The green trace was
obtained after a 125 µM solution of analog 16 in M63 media was diluted 500-fold in PBS, to show that
the relatively weak intrinsic fluorescence of the analog does not interfere with the strong fluorescence
response from pyoverdin. (C) Fluorescence intensity normalized to the number of viable cells (CFU/mL)
show that cells treated with analog 16 secrete ~4.5-fold more pyoverdin than cells in the DMSO control.
Error bars represent standard deviations from three independent experiments.
To obtain additional evidence that 16 blocks the BfrB-Bfd interaction and inhibits iron mobilization
from BfrB in the P. aeruginosa cytosol, we resorted to visualizing BfrB-iron in native PAGE gels stained
with Ferene S. A similar approach has been used to demonstrate that the Δbfd mutant irreversibly
accumulates iron in BfrB.19 To this end, cells cultured in M63 media supplemented with 4 µM iron were
treated with 16 (125 µM) or with an equivalent volume of DMSO (control). At different time points cells
were harvested by centrifugation after a small aliquot had been sampled to enumerate cell density
(CFU/mL). The growth curves (Figure 8A) show that at every time point the number of viable cells in the
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untreated cultures is approximately 2.5-fold larger than in cultures treated with 16. To visualize iron stored
in BfrB, the cells harvested at different time points were lysed and the clarified supernatants loaded onto
native PAGE gels for separation and subsequent staining with Ferene S (Figure 8B); recombinant BfrB
mineralized with an iron core of approximately 400 iron ions was used as a standard for the electrophoretic
mobility of BfrB. Lanes loaded with lysate from untreated cells (DMSO) show that iron accumulating in
BfrB reaches a maximum at ca. 15 h, and then is mobilized. A similar trend is observed if the lysate
solutions of untreated cells are diluted 2-fold prior to loading the gels (0.5x DMSO in Figure 8B), in order
to account for the nearly 2-fold larger CFU/mL observed at each time point relative to the treated culture.
In contrast, the lanes loaded with lysates obtained from cultures treated with 16 show only iron
accumulation in BfrB. The distinct trends of iron accumulation in BfrB observed with the treated vs
untreated (0.5x DMSO) cultures can be readily visualized in the plot of Figure 8C, which was constructed
with the aid of densitometry analysis of the gel bands. It is evident that the untreated cells store iron in BfrB
during the logarithmic growth phase and then mobilize the stored reserves during the stationary phase. In
contrast, when cells are treated with 16, the flow of iron into BfrB appears to be mostly unidirectional, with
much slower (inhibited) mobilization of iron from BfrB. Consistent with the nearly irreversible
accumulation of iron in BfrB when cultures are treated with 16, measurements of total cellular iron levels
normalized to viable cell counts show that P. aeruginosa cells harbor approximately twice as much iron in
the treated cultures relative to the untreated control (Figure 8D). Taken together, these observations strongly
support the idea that blockade of the BfrB-Bfd interaction by 16 inhibits iron mobilization from BfrB and
leads to nearly an irreversible accumulation of unusable iron in the bacterial cell.
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4-Aminoisoindoline-1,3-dione derivatives enhance the killing activity of fluoroquinolones
Previous studies demonstrated that intact iron homeostasis is essential for bacterial cell survival
under antibiotic stress, which suggests that bacterial iron homeostasis may be a potential target for boosting
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Figure 8. Analog 16 inhibits iron mobilization from BfrB in P. aeruginosa. Cells cultured in M63 media
supplemented with 4 µM Fe were treated with analog 16 (125 µM) or with an equivalent volume of DMSO
(control). (A) Enumerating viable cells shows that cultures treated with analog 16 (open circles) have
approximately 2.5-fold fewer cells than untreated control (black circles) at all time points. (B) The iron
stored in BfrB was visualized with the aid of native PAGE gels stained with Ferene S, which stains the
iron stored in the interior cavity of BfrB blue. Recombinant BfrB (Rec. BfrB) was used to show the
electrophoretic mobility of BfrB in the native PAGE gels. Lanes loaded with lysates of untreated control
(DMSO) show iron stored in BfrB, with maximum accumulation in late exponential growth (ca. 15 h) and
subsequent mobilization in the stationary phase. Lanes loaded with lysates of untreated control diluted 2-
fold (0.5x DMSO) to account for the 2-fold larger number of viable cells in the untreated cultures show a
similar trend. Lanes loaded with lysates of cells treated with 16 show significant inhibition of iron
mobilization from BfrB. (C) Plot of peak areas obtained from densitometry analysis of the native PAGE
gels in panel B. The open circles track the peak area in lanes loaded with lysates of cells treated with 16,
while the black circles track the peak area from the untreated control (0.5x DMSO). (D) Analysis of total
iron levels normalized to viable cell count (CFU/mL) shows approximately 2-fold higher iron levels in
cells treated with 16 (white bars), in agreement with nearly irreversible iron accumulation in BfrB. Error
bars represent the standard deviation of three independent experiments. Panels B and C show results from
a representative experiment.
the action of antibiotics.54-55 Consequently, we asked if the 4-aminoisoindoline-1,3-dione probes developed
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to disrupt bacterial iron homeostasis by blocking the BfrB-Bfd interaction would also potentiate the killing
activity of antibiotics. The idea was initially tested by treating P. aeruginosa PAO1 cultures with (i) analog
16, (ii) ciprofloxacin at the reported MIC45 (0.25 µg/mL), and (iii) a combination of ciprofloxacin and
analog 16. The effect was evaluated 18 h post treatment by plating, enumerating viable cells (CFU/mL) and
comparing the results to untreated cells (Figure 9A). Cultures treated with only analog 16 (125 µM)
experienced the anticipated ~30% survival, and cultures treated with only ciprofloxacin experienced
approximately 10% survival relative to untreated control. In comparison, cultures treated with a
combination of ciprofloxacin and analog 16 experienced significantly lower survival, in an analog-
concentration dependent manner, such that when analog 16 was present at 125 µM the % survival was ~50-
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Figure 9. Analog 16 potentiates the activity of fluoroquinolones. (A-C) P. aeruginosa PAO1 cultures in
M63 media supplemented with 4 µM iron were treated with analog 16 only, fluoroquinolone at the reported
MIC only, and a combination of analog 16 and fluoroquinolone at the MIC. The fluoroquinolones studied
are: (A) ciprofloxacin, 0.25 µg/mL, (B) levofloxacin, 0.5 µg/mL, and (C) norfloxacin 0.9 µg/mL. (D) P.
aeruginosa clinical isolate MR3B cultures in M63 media supplemented with 4 µM iron were treated with
analog 16 only, or ciprofloxacin only (0.2 µg/mL), or a combination of 16 and ciprofloxacin. (E) P.
aeruginosa clinical isolate MR60 cultures in the same media as above were treated with analog 16, or
ciprofloxacin (1.0 µg/mL), or a combination of 16 and ciprofloxacin. Error bars represent the standard
deviation from three independent experiments.
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