Iminoguanidines as allosteric inhibitors of the iron-regulated heme oxygenase (HemO) of pseudomonas aeruginosa

Article abstract of DOI:10.1021/acs.jmedchem.6b00757

New therapeutic targets are required to combat multidrug resistant infections, such as the iron-regulated heme oxygenase (HemO) of Pseudomonas aeruginosa, due to links between iron and virulence and dependence on heme as an iron source during infection. Herein we report the synthesis and activity of a series of iminoguanidine-based inhibitors of HemO. Compound 23 showed a binding affinity of 5.7 μM and an MIC₅₀ of 52.3 μg/mL against P. aeruginosa PAO1. An in cellulo activity assay was developed by coupling HemO activity to a biliverdin-IXα-dependent infrared fluorescent protein, in which compound 23 showed an EC₅₀ of 11.3 μM. The compounds showed increased activity against clinical isolates of P. aeruginosa, further confirming the target pathway. This class of inhibitors acts by binding to an allosteric site; the novel binding site is proposed in silico and supported by saturation transfer difference (STD) NMR as well as by hydrogen exchange mass spectrometry (HXMS).

Full text of DOI:10.1021/acs.jmedchem.6b00757

Article
pubs.acs.org/jmc
Iminoguanidines as Allosteric Inhibitors of the Iron-Regulated Heme
Oxygenase (HemO) of Pseudomonas aeruginosa
Geoffrey A. Heinzl, Weiliang Huang, Wenbo Yu, Bennett J. Giardina, Yue Zhou,
Alexander D. MacKerell, Jr., Angela Wilks,* and Fengtian Xue*
University of Maryland Computer-Aided Drug Design Center, Department of Pharmaceutical Sciences, School of Pharmacy,
University of Maryland, Baltimore, Maryland 21201, United States
S
* Supporting Information
ABSTRACT: New therapeutic targets are required to combat
multidrug resistant infections, such as the iron-regulated heme
oxygenase (HemO) of Pseudomonas aeruginosa, due to links
between iron and virulence and dependence on heme as an iron
source during infection. Herein we report the synthesis and
activity of a series of iminoguanidine-based inhibitors of HemO.
Compound 23 showed a binding affinity of 5.7 μM and an
MIC50 of 52.3 μg/mL against P. aeruginosa PAO1. An in cellulo
activity assay was developed by coupling HemO activity to a
biliverdin-IXα-dependent infrared fluorescent protein, in which
compound 23 showed an EC50 of 11.3 μM. The compounds
showed increased activity against clinical isolates of P.
aeruginosa, further confirming the target pathway. This class
of inhibitors acts by binding to an allosteric site; the novel
binding site is proposed in silico and supported by saturation transfer difference (STD) NMR as well as by hydrogen exchange
mass spectrometry (HXMS).
Gram-negative pathogens.20−22 Although these compounds
ince the introduction of antimicrobials into the clinic,
Sresistant strains of pathogenic microbes have developed achieve cellular uptake, they rely on chelated ferric iron to
rapidly regardless of the mechanism of action of the drug class.1
promote active transport. As a result, these compounds would
prove ineffective against pathogens that may have adopted to
utilize ferrous iron and/or heme as a primary iron source within
the host.
The major approved antimicrobial drug classes target essential
bacterial cell functions including cell wall formation,2 protein
synthesis,3 and DNA synthesis.4 Targeting such essential
cellular processes places the bacterial cell under significant
selective pressure to acquire mutations, accelerating the
development of drug resistance.5,6 To combat the decline in
viable antibiotics and growing antimicrobial resistance, an
alternative hypothesis suggests targeting virulence factors that
are not required for survival but are essential for pathogenesis
within the host. Potential antivirulence targets include quorum
sensing,7 secretion systems,8 and production of exotoxins and
biofilms.9 Inhibition of these virulence factors would render
pathogens less infectious while preventing the stress for survival
that leads to resistance.10−13
Iron is an essential micronutrient for survival and patho-
genesis of almost all bacterial pathogens and is tightly regulated
by the iron-regulatory protein Fur.14−16 Gram-negative bacteria
acquire iron via secretion and uptake of high-affinity iron
chelators called siderophores. Previous research in the area of
antivirulence development focuses on the inhibition of
siderophore-mediated iron-uptake;17−19 while initial therapies
proved ineffective at inhibiting iron availability, current “Trojan
horse” strategies using siderophore−β-lactam conjugates show
improved uptake and antimicrobial activity in a wide range of
P. aeruginosa, a common nosocomial and drug-resistant
pathogen, encodes a well-defined heme uptake and utilization
system including the outer-membrane receptor PhuR, the
periplasmic transfer protein PhuT, the inner membrane ABC
transporter complex PhuUV, and the cytoplasmic chaperone
PhuS.23,24 Heme uptake concludes with the oxidative cleavage
of heme by the iron-regulated heme oxygenase (HemO),
releasing Fe3+, CO(g), and β- and δ-biliverdin.25 The importance
of heme utilization by chronic infections of P. aeruginosa has
been validated by recent studies showing that during chronic
infection of the lung longitudinal clonal isolates adapt to
become more efficient at heme uptake.26 Furthermore, genetic
analyses of isolates from patients with chronic lung infection
have identified mutations within the promoter of the PhuR
transporter that upregulate its expression.27 Additionally, we
have recently shown in a PAO1 ΔhemO deletion strain that a
catalytically active HemO is required to drive heme flux into the
cell.28 Taken together, the experimental evidence suggests that
Received: May 18, 2016
© XXXX American Chemical Society
A
DOI: 10.1021/acs.jmedchem.6b00757
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
HemO represents a viable target for the development of
antivirulants targeting P. aeruginosa.
Previous work in the Wilks lab has explored HemO as a
target for inhibiting heme utilization. Multiple scaffolds have
been identified via a virtual screen that produced compounds
with inhibitory activity both in vitro and in vivo,29 including the
iminoguanidine compound 1 (Table 1).30 In addition to a
different areas including anticoagulation,35 pain management,36
cancer therapy,37 and management of alcohol dependence.38
Current work explores inhibitory and antimicrobial activities of
iminoguanidines (analogues of 1) and their activities in clinical
isolates of P. aeruginosa. Further, binding modes of analogues of
1 are suggested by in silico methodologies and experimentally
validated structure−activity relationships are confirmed through
saturation transfer difference (STD) NMR and hydrogen−
deuterium exchange mass spectrometry (HXMS).
Table 1. Dissociation Constants, KD, and Effective
Concentrations, EC50, for Compounds 1−25
RESULTS AND DISCUSSION
■
compd
R1
R2
R3
KD (μM)
EC50 (μM)
Chemical Synthesis. Target compounds 1−25 (Table 1)
were synthesized using a one-step condensation reaction
(Scheme 1) of aminoguanidine hydrochloride and selected
1
H
F
H
H
F
N(CH3)2
8.8 0.8
11 1.4
12 1.5
14 2.0
11 1.6
8.4 0.6
7.0 0.7
8.4 0.7
6.7 0.4
12 1.2
15 3.1
9.2 0.8
21 2.2
4.8 0.5
8.0 1.3
8.0 0.7
123
2
H
>200
45.1
77.5
11.9
19.4
122
3
H
H
H
4
H
H
Cl
H
Cl
H
Br
H
H
OH
H
F
Scheme 1. Synthesis of Inhibitors 1−25
5
Cl
H
H
6
H
7
H
Cl
Cl
H
8
H
20.3
35.3
11.2
35.6
102
9
Br
H
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
H
H
Br
H
OH
H
H
>200
>200
>200
60.3
72.7
benzaldehyde derivatives. The products were isolated as
hydrochloride salts. Although synthesis of compound 1 was
reported as early as 1933,39 more recent methodologies
perform the reaction in refluxing methanol followed by
filtration, resulting in a monosalt and reducing the appearance
of the reported biguanide side product.40 This reaction provides
good yields for most aromatic substitutions, but product may
be lost upon filtration for more lipophilic compounds such as
compounds 22−25.
H
OH
H
a
H
OH, OH
OH
OCH3
H
Br
H
b
H
H
ND
b
b
OCH3
H
H
ND
ND
H
OCH3
H
7.8 0.9
12 1.8
77.4
52.4
43.1
21.9
11.3
34.3
64.4
c
c
Naph
Naph
c
c
b
H
H
H
H
H
Naph
Naph
ND
H
Ph
i-Pr
H
6.7 0.4
5.7 1.0
Computational Analysis by Site Identification by
Ligand Competitive Saturation (SILCS) and Docking
Studies. In order to instruct future inhibitor design, HemO
was modeled using SILCS.41 Fragment maps, or FragMaps,
which represent the binding affinity pattern of functional
groups with the entire protein surface, were generated based on
MD simulations of HemO in aqueous solution containing a
variety of small organic solutes, such as benzene, propane, and
methanol. As shown in Figure 2, when contoured at grid free
energy (GFE) cutoffs of −1.2 kcal/mol, regions of significant
affinity for functional group binding can be seen both in the
heme-binding pocket and on the back side of the HemO.
Probes of high hydrophilicity, as indicated by hydrogen-bond
donor and acceptor maps, show fewer areas of favorable
binding compared to their hydrophobic counterparts. The
heme-binding pocket shows two distinct binding areas for
hydrophobic compounds, one of which overlaps with the
binding areas previously suggested for small molecules by NMR
shifts.30 This area contains the catalytic site where the α-meso
carbon of heme is hydroxylated and later removed as CO
during the degradation of heme. The second site near Trp-158
suggests a small area appropriate for binding inhibitors; while
this small site might be sampled by inhibitors, as also suggested
by previous NMR studies showing perturbations of Phe-117
and Val-118,30 the utility for further inhibitor design is limited
based on its small size. Another site of interest appears near
Arg-188 and Trp-115 (Figure 2, right). The area of this
potential alternative pocket suggests an alternative and
previously unexplored mode of binding to HemO and
modulation of HemO activity. The apolar FragMap highlights
H
b
OBn
H
ND
NO2
6.2 0.6
a
b
c
3,5-Disubstituted. Not determined. Indicates bridging carbons.
modest binding affinity for HemO (31.2 μM) and antimicrobial
activity against P. aeruginosa (MIC50 = 250 μg/mL), compound
1 also exhibited curing activity in a Caenorhabditis elegans
infection model.30 Iminoguanidines have found application in a
number of therapeutic agents. For example, iminoguanidine-
based α-2 agonist guanabenz (Figure 1) is approved by the
FDA for the treatment of hypertension;31 another iminogua-
nidine-containing compound, semapimod, has been developed
as an anti-inflammatory and antiparasitic agent.32−34 Iminogua-
nidines have also been evaluated for their biological activities in
Figure 1. Molecular structures of guanabenz, an FDA-approved
compound for the treatment of hypertension, and semapimod, an
investigational compound with anti-inflammatory and antiparasitic
properties. Both compounds contain the iminoguanidine moiety found
in 1 and its analogues.
B
DOI: 10.1021/acs.jmedchem.6b00757
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Figure 2. Fragment maps from SILCS. Potential small-molecule binding sites of HemO are identified in silico using small molecule probes. Apolar,
hydrogen bond donor and acceptor, negative and positive charged FragMaps are shown in green, blue, red, orange, and cyan colors and contoured at
−1.2, −1.2, −1.2, −1.5, and −1.5 kcal/mol, respectively. The heme-binding face of HemO (indicated by His-26, panel a) and the back face of HemO
(indicated by Asp-99 and Arg-188, panel b) show putative hydrophobic binding sites.
the high hydrophobic character of compounds that could bind
to that site, and the nearby salt bridge between Arg-188 and
Asp-99 suggests areas of hydrogen bonding or electrostatic
interactions for polar moieties, such as the iminoguanidyl group
present in compounds 1−25.
In order to further examine the ability of the compounds to
bind to the proposed allosteric site of HemO, compounds 1−
25 were docked onto both the heme-binding site and the
allosteric site using the Monte Carlo site identification by ligand
competitive saturation (MC-SILCS) protocol.42 Figure 3 shows
the predicted binding orientation of compound 23, as well as
overlap with selected FragMaps for both the heme-binding site
(upper right) and the allosteric site (upper left). Overlap with
the FragMaps in the heme-binding pocket is poor, while strong
overlap in the allosteric site indicates preferred binding at the
putative site. Overlap of the aromatic ring and isopropyl groups
with the apolar FragMaps and of the guanidinium moiety with
the positive FragMaps show these moieties to make favorable
contributions to binding in the site. The preference for the
putative site is quantified by the ligand-grid free energy (LGFE)
scores of the ligands calculated for both the heme-binding site
and the allosteric site (Figure 3, center). All compounds except
compound 24 are predicted to have greater affinity for the
allosteric site over the heme-binding pocket. Additionally, when
the ligand efficiencies (LE, the LGFE divided by the number of
non-hydrogen atoms) are calculated for the ligands in both
sites, all compounds except compound 24 are again predicted
to interact more favorably with the allosteric site (Figure 3,
bottom). Compound 24 also shows the least efficient binding,
suggesting a lack of substantial contributions from its benzyloxy
substitution. Validation of these computational results will
provide additional evidence of an alternative interaction with
HemO instead of competitive inhibition at the heme-binding
pocket as previously proposed.
Binding Studies by Fluorescence and STD NMR.
Inhibitors 1−25 were tested for binding to HemO by intrinsic
fluorescence quenching, as performed previously.29 Optical
interference in these measurements due to high absorptivity of
the inhibitors around 250−350 nm was corrected as suggested
using experimentally determined extinction coefficients.43 As
shown in Table 1, all compounds showed binding affinities
between 4 and 20 μM regardless of aromatic substitution,
except for compounds 17, 18, 21, and 24, which exhibited
Figure 3. Computational docking and scoring of compounds binding
to HemO on the back site and the heme site. Compound 23 is shown
docked to the proposed alternative binding site (upper left) as well as
the heme binding site (upper right) overlaid with SILCS FragMaps
generated for HemO. FragMaps are colored and contoured at −1.2
kcal/mol for the apolar and negative FragMaps and at −0.6 kcal/mol
for the positive, hydrogen bond donor, and hydrogen bond acceptor
FragMaps. LGFE scores (kcal/mol), based on the Monte Carlo
docking of the ligands into the SILCS FragMaps, were calculated as an
estimate of the affinity of the ligands for the two binding sites
(middle). LE (kcal/mol/atom), which are calculated as the LGFE per
non-hydrogen atom, are also shown (bottom).
C
DOI: 10.1021/acs.jmedchem.6b00757
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Figure 4. Binding epitopes of selected compounds [(A) 12; (B) 9; (C) 17; (D) 24] determined by STD NMR. For each compound, traditional
pulse proton spectra are shown below in black and the difference spectra are shown above in red.
small-molecule fluorescence that interfered with the assay.
Previous STD NMR studies30 suggested that aromatic protons
contributed most to the inhibitor−HemO interaction; however
all mono- and disubstituted compounds tested showed similar
binding by fluorescence quenching. If the compounds bind in
the active site cavity and given the relatively larger size of heme,
the native substrate, it was predicted that substitutions allowing
for more van der Waals contacts or aromatic stacking
interactions (compounds 17−24) would show increased
binding affinity compared to compound 1. Without any
observed increase or decrease in binding affinity caused by
aromatic substitution, it can be assumed that an alternative
binding mode contributes to the binding of these compounds
to HemO.
In order to further study this series of compounds with
regard to binding mode, analysis of direct interaction between
select compounds and HemO was performed by STD NMR.30
Selected compounds included those containing hydrophilic or
lipophilic substitutions and individually resolved protons (9, 12,
and 17) as well as the largest substitution, compound 24.
Integration of the difference peaks allows for comparison of
relative magnetization transfer, and therefore degree of
interaction, of each proton with HemO (Figure 4). Compound
12 (part A) appears to interact most strongly at the para-
position of the benzyl ring, and the relative interaction
decreases around the ring to the benzylimine proton at 59%
that of the para-proton. However, compounds 9, 17, and 24
(parts B, C, and D, respectively) display the strongest
interaction at the benzylimine proton. Compounds 9 and 17
both show good interactions with the aromatic protons, with
equal relative interaction at the meta-position of compound 9
to the benzylimine proton. Additionally, the methoxy protons
of compound 17 show much less interaction with HemO than
the aromatic protons, much like the N-methyl protons of
compound 1.30 The largest compound, 24, contains two sets of
aromatic protons with overlapping signals that cannot be
resolved. The two unique signals, for the benzylimine proton
and the para-position proton, show the largest relative
interactions at 100% and 82%, respectively. The methylene
protons could not be detected due to the Watergate pulse
sequence used to reduce the background water signal. Still, the
available relative interactions suggest that the benzylimine ring
interacts more directly with HemO than the benzyloxy ring,
much like the other compounds studied in this series. Little
change in both binding affinity and interaction via STD NMR
for all compounds regardless of substitution(s) suggests that
the compounds are not interacting in the heme-binding pocket,
as previously proposed. The variety of functional groups in
compounds 1−25, chosen for their hydrogen bonding,
lipophilic, or steric properties, should interact differently with
the mostly hydrophobic heme-binding pocket of HemO. If
instead the iminoguanidyl moieties of the compounds
contribute a majority of the interaction with HemO, then any
sufficiently tolerated aromatic substitution will allow the
compound to bind and give the results seen in Table 1, as
well as the lack of substitution contributions to binding by STD
NMR seen in Figure 3. The additional benzyloxy ring of
compound 24 could also make contacts with the secondary
binding area in the heme-binding site, increasing its preference
for the heme-binding site over the allosteric site. These SILCS
and docking studies propose an allosteric site of binding that
matches the relatively flat SAR suggested by binding and STD
NMR studies while also matching the NMR data previously
shown upon binding of compound 1 to HemO in which
residues Arg-188 and Trp-115 are significantly shifted.30
HXMS of HemO with Selected Compounds. We further
investigated the interaction of compounds 1 and 22 by HXMS.
Peptide coverage following hydrogen−deuterium exchange and
pepsin digestion was approximately 98% with 7-fold redun-
dancy (Figure S1 in Supporting Information). HXMS, in
D
DOI: 10.1021/acs.jmedchem.6b00757
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Figure 5. Color coded protein structure of HemO representing deuterium uptake change upon binding of compound 1. Deuterium incorporation
was mapped onto the HemO crystal structure (PDB code 1SK7) as viewed from front (heme-binding pocket, upper left) and from back (upper
right). Peptide regions of holo-HemO that were more protected from deuterium exchange in the presence of 1 are colored in blue, whereas regions
that became more prone to deuterium exchange are colored in red. Relative deuterium uptake of peptides showed differences between holo-HemO
and holo-HemO in the presence of 1 at 30 min (bottom). Two peptides (53−62 and 130−138) were significantly more protected from deuterium
exchange upon binding of 1 (Student’s t-test, p < 0.05, n = 2), while other peptides showed significantly increased deuterium uptake (p < 0.05). Error
bar indicates standard deviation.
addition to providing confirmation of the ligand binding site on
reduced hydrogen exchange, can also give insight on the global
effects of ligand binding that may include deuteration
enhancement.44 Observation of increased deuterium exchange
is often a result of ligand binding causing unfavorable structural
change in the protein. As previously observed by chemical shift
perturbations of apo-HemO on binding of compound 1, the
surface loop containing Asp-99, which forms a salt bridge with
Arg-188 and the helices packing over the distal helix (residues
76−84 and 92−102), shows reduced hydrogen exchange
indicative of protection upon compound binding (Figure S2).
The data are consistent with the SILCS analysis and previous
STD NMR that compound 1 most likely binds at an alternative
hydrophobic site on the protein surface.
Interestingly, compound 1 in holo-HemO shows differences
in hydrogen exchange within several of the same regions but
where the overall effect is to increase hydrogen exchange within
the distal helices (Figure 5). Much of the same hydrogen
exchange profile on holo-HemO is also seen with compound
22 (Figure 6), whereas little-to-no change in hydrogen
exchange is seen with compound 22 on apo-HemO. These
data agree with the alternative binding site identified by SILCS
and the compounds’ proposed preferences for binding to the
allosteric site, marked by the signification protection of peptide
53−62. Further, the protection of this peptide compared to
holo-HemO shows interactions with HemO when the heme
site is occupied, pointing again to the allosteric binding site.
The addition of lipophilic groups in compounds 20−24 showed
an overall improvement across the spectrum of biological assays
suggesting these inhibitors are causing global effects on protein
stability. Extended interaction of the lipophilic substituents with
the hydrophobic pocket may perturb the binding pocket
adjacent to the salt bridge. Although we cannot be certain that
the iminoguanidine group disrupts the salt bridge, mutation of
either Asp-99 or Arg-188 results in a similar pattern of
hydrogen exchange (data not shown), highlighting the
importance of this region in maintaining structural integrity
and enzymatic activity. The binding of ligands to the allosteric
site appears to “loosen up” the distal pocket, which possibly
results in disruption of the hydrogen bond network critical in
stabilizing the activated oxygen intermediate required for initial
hydroxylation of the heme.45
In Cellulo HemO Inhibitory Activity of Compounds. In
order to assess the abilities of compounds 1−25 to inhibit the
activity of HemO, it was necessary to develop a new, high-
throughput assay. Current methods for measuring HO catalytic
activity rely on spectroscopic measurements of bilirubin.46 As
the Gram-negative HemO enzymes do not release biliverdin to
eukaryotic biliverdin reductase enzymes, it is not possible to
perform multiple turnover reactions in vitro. Instead, inspired
by the work with bacterial phytochromes by Filonov47 and
Shu,48 an in cellulo system coexpressing HemO and an
E
DOI: 10.1021/acs.jmedchem.6b00757
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Figure 6. Color coded protein structure of HemO representing deuterium uptake change upon binding of compound 22. Deuterium incorporation
perturbation data were mapped onto the HemO crystal structure (PDB code 1SK7) as viewed from front (heme-binding pocket, upper left) and
from back (upper right). Peptide regions of 22-bound holo-HemO that were more protected from deuterium exchange are colored in blue, whereas
regions that became more prone to deuterium exchange were colored in red. Relative deuterium uptake of peptides showed differences between
holo-HemO and 22-bound holo-HemO at 30 min (bottom). One peptide (53−62) was significantly more protected from deuterium exchange upon
binding of 22 (Student’s t-test, p < 0.05, n = 2), while other peptides showed significantly increased deuterium uptake (p < 0.05). Error bar indicates
standard deviation.
engineered infrared fluorescent protein (IFP) was developed to
assay inhibition of HemO activity in bacterial cells. The
advantages of the assay are several including (i) a measure of
the ability of the compounds to cross the bacterial cell
membrane, (ii) ability to inhibit HemO activity, and (iii) lack of
toxicity to E. coli cells that do not require a HemO for survival.
Inhibition of HemO activity prevents the formation of
biliverdin, which is measured spectrophotometrically as a
decrease in fluorescence of the biliverdin-dependent IFP1.4.
The in cellulo assay was optimized so that expression of IFP1.4
is not rate limiting (Figure S3A) and a linear range of
fluorescence is obtained (Figure S3B). Utilizing the in cellulo
assay, the concentration-dependent inhibition of HemO was
measured for compound 1 as a function of decreased
fluorescence over time, corrected for OD600 to account for
differences in growth (Figure S3B). In addition the in cellulo
assay provides a mechanism to screen out inhibitors with
nonspecific cell toxicity versus HemO inhibitory activity, an
important consideration for developing inhibitors specific to
the invading pathogen versus the host microbial flora. We
confirmed the concentration-dependent inhibition of HemO by
compound 1 was not a consequence of cell toxicity (the cells
grew at a similar rate when treated with compound 1) by
microscopic analysis of the untreated and treated cultures
(Figure 7). For routine screening of HemO inhibition the assay
was adapted to a 96-well plate format and the fluorescence
Figure 7. In cellulo fluorescence quenching of HemO activity. (A)
Concentration dependent inhibition of HemO activity by compound 1
as measured by biliverdin-dependent IFP1.4 fluorescence. Experiments
were performed as described in Experimental Section. All experiments
were performed in triplicate and averaged. Relative fluorescence was
corrected for OD600, and values represent the average of three separate
experiments. (B) In situ detection of the biliverdin-IFP1.4 within live
bacteria imaged on the Cy5 channel of a fluorescence microscope in
the absence (upper left panel) or presence of 120 μM compound 1
(upper right panel). Phase contrast images of the bacterial cells are
shown in the lower panels.
emission at 700 nm measured at mid log phase (16 h). The
EC50 for the compounds was calculated as described in the
methods, and the EC50 values for compounds 1−25 are shown
in Table 1.
F
DOI: 10.1021/acs.jmedchem.6b00757
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Unlike the binding constants, the EC50 data comprise a wide
range from 11.9 μM up to >200 μM, the highest concentration
tested. Generally, more lipophilic compounds showed increased
potency compared to the parent compound 1, while more
hydrophilic compounds showed decreased potency. In addition
to the change in polarity of the various functional groups tested,
the position on the benzylidene ring also contributed to
potency. For example, compounds 5 and 6 show potent
inhibition with chloro substitutions on the 2′ and 3′ positions,
whereas compound 7, the 4′-chloro substitution, shows
inhibition similar to parent compound 1. Potency is restored
with the 3′,4′ disubstituted compound 8. The reverse trend is
seen in the hydroxyl substituted compounds. Compounds 13
and 14, substituted at the 3′ and 4′ positions, respectively, show
poor potency (>200 μM), whereas the 2′ substituted
compound 12 maintains activity similar to the parent
compound 1. The inhibitory activity of compound 12 in
contrast to 13 and 14 may be due to an internal hydrogen bond
between the hydroxyl substituent and the imine nitrogen from
the iminoguanidine moiety of 12, masking its hydrophilic
character and increasing its ability to cross the Gram-negative
cell membrane. Inhibitory activity is further increased in
compound 16 with an additional bromo substitution at the 3′
position. Thus, the in cellulo assay allows a rapid ranking of
compounds based on inhibitory activity and the ability to cross
the Gram-negative cell membrane, confirming earlier data
showing reduced biliverdin levels in HemO overexpressing E.
coli treated with compound 1.29
Antimicrobial Activity of Compounds. Compounds 1−
25 were tested for antimicrobial activity against PAO1, a
laboratory strain of P. aeruginosa, as well as JSRI-1 and JSRI-2,
longitudinal clinical isolates of P. aeruginosa from a cystic
fibrosis patient.26 The minimum inhibitory concentration for
50% growth (MIC50) showed great variation depending upon
the substitutions of the aromatic ring as shown in Table 3.
Generally, increased lipophilicity led to more potent inhibition
of growth, as exemplified by compounds 20−24, whereas
increased hydrophilicity (13−15) greatly reduced the com-
pounds’ abilities to inhibit growth. Compounds 12 and 16
showed moderate activity, much like in the in cellulo assay
shown in Table 1. Halogen substitutions showed mildly
increased activity, and the dichloro-substituted inhibitor 8
showed equal potency to the lipophilic compounds 20−24.
The relationship between MIC50 and lipophilicity (as
determined by log P(o/w)) for these compounds is shown in
Figure 8. Correlations are seen indicating that physical
properties of the compounds related to membrane permeability
affecting MIC50, as well as EC50, are more important for activity
than the in vitro binding affinities given the weak correlation
between KD and MIC50.
Figure 8. Correlation between MIC50 and log P(o/w) (top) as well as
molecular weights (bottom) for compounds with MIC50 lower than
200 μg/mL for PAO1. Correlation coefficients R2 are also shown.
the heme utilization pathway and shows sustained activity in
otherwise resistant strains.
CONCLUSIONS
■
Various analogues of iminoguanidine 1 were explored
computationally, synthesized, tested for binding to HemO,
and tested for antimicrobial activity. Although little differences
were seen in binding by fluorescence and STD NMR for many
of the compounds, structure−activity relationships for this
series were drawn from a combination of SAR methods with in
cellulo activity and antimicrobial assays. From this series,
compound 23 showed the most improved overall activity, with
a binding affinity of 5.7 μM, an EC50 of 11.3 μM, and an MIC50
of 52.3 μg/mL against PAO1. Unfortunately the pulses used in
STD NMR do not allow for analysis of compound 23 due to its
upfield isopropyl group. Compound 23 should demonstrate
good selectivity for HemO over α-2 adrenergic receptors
according to the known structure−activity relationship of the α-
2 adrenergic agonists.49 Nguyen and co-workers report that α-2
adrenergic activity is maintained only with the 2′,6′-dicholoro
substitution found in guanabenz (see Figure 1) and altered
chloro substitutions abrogate agonistic activity.49 It was also
seen that more lipophilic compounds 20−24 proved more
active against all strains of P. aeruginosa than the hydrophilic
compounds 13−15, which correlates well with their inhibitory
activity in the E. coli IFP assay. Correlations shown in Figure 8
confirm the greater role of lipophilicity and cell membrane
permeability than binding affinity for in vivo activity. Addition-
ally, many compounds displayed increased activity against the
clinical isolates JSRI-1 and JSRI-2, which were previously
characterized as highly efficient heme-utilizing strains.
Following MIC50 growth conditions, new cultures were
inoculated to classify growth inhibition as bacteriostatic or
bactericidal. Compounds with the best activity against growth
via MIC50 were also shown to act in a bactericidal manner, as
growth was not recovered upon greater than 1000-fold dilution
of culture into new media. Again, compounds 8 and 20−24
showed the most potent bactericidal activity. Further, most
compounds showed higher potency against the JSRI-2 isolate,
which was previously shown to more efficiently utilize heme as
an iron source,26 as well as being more resistant to antibiotics
such as tobramycin and gentamycin. While bactericidal activity
was not anticipated from these compounds, increased activity
against the JSRI isolates versus PAO1 supports their targeting
G
DOI: 10.1021/acs.jmedchem.6b00757
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Table 2. Contributions of Selected Moieties to the LGFE Scores for Selected Compounds
a
LGFE contributions (kcal/mol)
total
b
c
compd
substituent
KD (μM)
Ph
Guan
Ph + Guan
subst
LGFE
LE
8
3,4-dichloro
2-hydroxyl
2-methoxyl
1,2-naphthyl
8.4 0.7
9.2 0.8
ND
−13.2
−12.7
−12.3
−12.3
−11.9
−4.9
−6.4
−6.3
−5.5
−4.4
−18.1
−19.1
−18.7
−17.9
−16.3
−2.1
−0.8
−1.5
−6.8
−5.1
−20.2
−19.9
−20.1
−24.7
−21.4
−1.44
−1.53
−1.44
−1.54
−1.43
12
17
20
23
12 1.8
5.7 1.0
4-isopropyl
a
b
c
Ligand grid free energy. Phenyl. Guanidinium.
A somewhat unexpected result was the relative insensitivity
from the LGFE by dividing by the number of non-hydrogen
atoms, the ligand efficiency of compound 20 falls in the range
of the other compounds. This indicates that the more favorable
LGFE is an artifact of the SILCS method associated with the
large size of the substituent. Thus, the similarity of the affinities
is indicated to be due to a “push−pull” mechanism where
favorable interactions of the substituents lead to less favorable
interactions of the parent compound moieties, yielding a flat
SAR profile.
of the KD values to the different functional groups, with the
maximum and minimum affinities of 4.8 and 21 μM obtained,
respectively. Consistent with the KD values, the LGFE scores
were also relatively insensitive to the studied modifications as
seen in Figure 3. To understand the molecular origins of this
phenomenon, the LGFE scores determined for the allosteric
site were partitioned into the contributions of different moieties
in the ligands, based on the sum of the atom-GFE scores for
each moiety. The allosteric-site analysis is shown in Table 2 for
Structure−activity relationships from chemical shift pertur-
bation NMR and HXMS studies, together with the computa-
tional SILCS methodology, suggested a novel allosteric binding
site from the active site for compounds 1−25. This new
binding site comprises a shallow hydrophobic cleft formed by
the two helices running along the backside of the distal helix,
which are further stabilized by a salt bridge formed between
Asp-99 and Arg-188. The lack of improvement in binding
affinities of the various compounds is consistent with a push−
pull mechanism in the binding site. However, as shown by
HXMS, it is possible to expand on the inhibitory activity of 1 by
generating back side instability of HemO upon binding to the
pocket adjacent to the Asp-99/Arg-188 salt bridge, as
demonstrated by compound 22. Consistent with the proposed
binding of the inhibitors to the newly identified allosteric site,
we have recently shown that mutation of either Asp-99 or Arg-
188 to an Ala results in poorly active HemO mutants when
overexpressed in E. coli (data not shown). Finally, the
alternative binding site is unique to the bacterial HemO
providing for selectivity against the human HO-1 and HO-2
enzymes.
Table 3. MIC50 and MBC of Compounds 1−25 in PAO1 and
Clinical Isolates
a
b
MIC50 (μg/mL)
MBC (μg/mL)
compd
PAO1
JSRI 1
JSRI 2
PAO1
JSRI 1
JSRI 2
1
121
32.5
28.6
70
15.5
72.9
68.2
69.4
35
1000
1000
1000
1000
500
1000
>1000
1000
1000
500
≤250
≤250
250
2
131.2
66.6
162
3
4
87.4
16.3
19.2
10.7
24.1
62.9
23
500
5
47.3
52.4
49
250
6
33.5
41.6
16.4
33.4
19.4
13.4
79.1
617
121
564
45.6
125
93.4
38
250
500
250
7
500
250
≤62.5
125
8
32.3
50.1
51.3
52.5
123
125
125
9
500
500
250
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
250
500
≤125
≤62.5
250
19.7
58.5
391
443
338
145
38.4
48.6
30.5
30.4
31.7
34.6
20.6
25.5
61.4
250
250
>1000
>1000
c
c
c
750
ND
ND
ND
408
>1000
>1000
>1000
c
c
c
1140
80.5
99.5
134
ND
ND
ND
500
1000
>1000
1000
1000
250
250
EXPERIMENTAL SECTION
■
1000
>1000
500
500
General Procedures. All reagents and solvents were of analytical
grade and used without further purification. NMR spectra were
obtained on a Varian INOVA 400 MHz NMR spectrometer at 25 °C.
Chemical shifts are reported as δ values (parts per million) using the
500
113
500
32.1
48.4
59.1
52.3
23.2
72.8
36.1
75.6
38
250
125
1
residual solvent peak as an internal reference. Data for H NMR are
250
250
125
reported in the following order: chemical shift, multiplicity (s, singlet;
d, doublet; t, triplet; sept, septuplet; dd, double doublet; dt, double
triplet; m, multiplet), number of protons, coupling constant (Hz).
Data for 13C NMR are reported as δ values (parts per million). UV
spectra were obtained on a Nanodrop 2000c spectrophotometer.
High-resolution mass spectra (HRMS) were obtained on a JEOL
AccuTOF with ESI/APCI ion sources coupled to an Agilent 1100
HPLC system. HPLC analysis was performed on a Shimazdu HPLC
fitted with a C-18 reversed-phase column (Phenomenex, 4.6 mm ×
250 mm) with a flow rate of 0.5 mL/min using CH3OH−H2O 3:1
with 0.1% NH4OAc mobile phase. The purity of final products are
>95%.
General Procedure for the Synthesis of Compounds 1−25.
To a solution of aminoguanidine hydrochloride (110 mg, 1.0 mmol) in
methanol (5 mL) was added the corresponding benzaldehyde (1.1
mmol). Solution was heated to reflux and stirred for 6 h. Solvent was
evaporated. Residue was stirred in CH2Cl2 (20 mL) and filtered.
125
125
≤62.5
≤125
62.5
≤125
81
125
125
33.9
47.2
125
125
1000
1000
a
Minimum inhibitory concentration for 50% growth (MIC50).
Minimum bactericidal concentration (MBC). Not determined.
b
c
selected compounds. Results show that the sum of the GFEs
for the phenyl and guanidinium groups becomes less favorable
as the GFE contribution of the substituent becomes more
favorable. This indicates a situation in which favorable
interactions of the substituents lead to less favorable
interactions of the parent phenyl and guanidinium moieties.
With compound 20, the LGFE score is significantly more
favorable; however, when the ligand efficiencies are extracted
H
DOI: 10.1021/acs.jmedchem.6b00757
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Precipitate was washed with additional CH2Cl2 (50 mL) to generate
the desired product.
(E)-2-(3,4-Dichlorobenzylidene)hydrazinecarboximidamide
Hydrochloride (8). Yield, 58%; mp 221−223 °C. 1H NMR (DMSO-
d6) δ 7.84 (dd, 1H, J1 = 8.4 Hz, J2 = 1.6 Hz), 8.17 (s, 1H), 8.29 (d, 1H,
J = 0.8 Hz), 12.27 (s, 1H). 13C NMR (DMSO-d6): 128.0, 128.6, 130.9,
131.8, 132.6, 134.3, 144.1, 155.5, 159.1. UV λmax (log ε) 286 (4.47).
HRMS (ESI) m/z: calculated 231.0199 for [C8H9Cl2N4]+, found
231.0206. HPLC analysis: retention time = 12.86 min; peak area,
95.0%; eluent A, CH3OH; eluent B, NH4OAc solution (0.1%);
isocratic (3:1) over 60 min with a flow rate of 0.5 mL/min and
detection at 254 nm.
(E)-2-(2-Bromobenzylidene)hydrazinecarboximidamide Hy-
drochloride (9). Yield, 52%; mp 220−221 °C. 1H NMR (DMSO-d6)
δ 7.39 (t, 1H, J = 7.6 Hz), 7.46 (t, 1H, J = 8.0 Hz), 7.69 (d, 1H, J = 8.0
Hz), 8.26 (d, 1H, J = 7.6 Hz), 8.52 (s, 1H), 12.12 (s, 1H). 13C NMR
(DMSO-d6): 124.1, 125.5, 128.4, 132.4, 132.7, 133.5, 145.7, 155.5. UV
λmax (log ε) 281 (4.56). HRMS (ESI) m/z: calculated 241.0083 for
[C8H10BrN4]+, found 241.0087. HPLC analysis: retention time = 9.31
min; peak area, 99.9%; eluent A, CH3OH; eluent B, NH4OAc solution
(0.1%); isocratic (3:1) over 60 min with a flow rate of 0.5 mL/min and
detection at 254 nm.
(E)-2-(4-(Dimethylamino)benzylidene)hydrazine-
carboximidamide Hydrochloride (1). Yield, 80%; mp 216−217
1
°C. H NMR (DMSO-d6) δ 2.93 (s, 6H), 6.68 (d, 2H, J = 8.8 Hz),
7.60 (d, 2H, J = 8.0 Hz), 7.96 (s, 1H), 11.56 (s, 1H). 13C NMR
(DMSO-d6): 111.4, 120.5, 128.8, 147.4, 151.6, 155.0. UV λmax (log ε)
346 (4.47). HRMS (ESI) m/z: calculated 206.1400 for [C10H16N5]+,
found 206.1404. HPLC analysis: retention time = 7.82 min; peak area,
99.9%; eluent A, CH3OH; eluent B, NH4OAc solution (0.1%);
isocratic (3:1) over 60 min with a flow rate of 0.5 mL/min and
detection at 254 nm.
(E)-2-(2-Fluorobenzylidene)hydrazinecarboximidamide Hy-
1
drochloride (2). Yield, 86%; mp 193−195 °C. H NMR (DMSO-
d6) δ 7.29 (m, 2H), 7.52 (dt, 1H, J1 = 6.8 Hz, J2 = 6.4 Hz), 8.23 (t, 1H,
J = 7.6 Hz), 8.39 (s, 1H), 12.17 (s, 1H). 13C NMR (DMSO-d6): 115.8,
116.0, 124.7, 127.0, 132.4, 132.5, 139.3, 155.3, 174.8. UV λmax (log ε)
273 (4.45). HRMS (ESI) m/z: calculated 181.0884 for [C8H10FN4]+,
found 181.0891. HPLC analysis: retention time = 7.70 min; peak area,
98.8%; eluent A, CH3OH; eluent B, NH4OAc solution (0.1%);
isocratic (3:1) over 60 min with a flow rate of 0.5 mL/min and
detection at 254 nm.
(E)-2-(3-Bromobenzylidene)hydrazinecarboximidamide Hy-
1
drochloride (10). Yield, 81%; mp 225−229 °C. H NMR (DMSO-
d6) δ 7.39 (t, 1H, J = 7.2 Hz), 7.62 (d, 1H, J = 8.0 Hz), 7.79 (d, 1H, J =
8.0 Hz), 8.14 (s, 1H), 8.22 (s, 1H), 12.10 (s, 1H). 13C NMR (DMSO-
d6): 122.7, 127.7, 129.7, 131.2, 133.4, 136.3, 145.6, 155.9. UV λmax
(log ε) 281 (4.56). HRMS (ESI) m/z: calculated 241.0083 for
[C8H10BrN4]+, found 241.0080. HPLC analysis: retention time = 9.61
min; peak area, 99.0%; eluent A, CH3OH; eluent B, NH4OAc solution
(0.1%); isocratic (3:1) over 60 min with a flow rate of 0.5 mL/min and
detection at 254 nm.
(E)-2-(3-Fluorobenzylidene)hydrazinecarboximidamide Hy-
1
drochloride (3). Yield, 77%; mp 202−203 °C. H NMR (DMSO-
d6) δ 7.28 (dd, 1H, J1 = 8.4 Hz, J2 = 1.6 Hz), 7.49 (q, 1H, J = 6.4 Hz),
7.61 (d, 1H, J = 7.6 Hz), 7.85 (d, 1H, J = 10 Hz), 8.19 (s, 1H), 11.87
(s, 1H). 13C NMR (DMSO-d6): 113.4, 113.6, 117.6, 117.9, 125.0,
131.2, 131.3, 136.3, 136.3, 146.0, 155.6, 161.6, 164.1. UV λmax (log ε)
278 (4.56). HRMS (ESI) m/z: calculated 181.0884 for [C8H10FN4]+,
found 181.0898. HPLC analysis: retention time = 7.86 min; peak area,
98.9%; eluent A, CH3OH; eluent B, NH4OAc solution (0.1%);
isocratic (3:1) over 60 min with a flow rate of 0.5 mL/min and
detection at 254 nm.
(E)-2-(4-Bromobenzylidene)hydrazinecarboximidamide Hy-
1
drochloride (11). Yield, 60%; mp 156−159 °C. H NMR (DMSO-
d6) δ 7.61 (d, 2H, J = 8.0 Hz), 7.79 (d, 2H, J = 8.8 Hz), 8.11 (s, 1H),
8.74 (s, 1H), 12.06 (s, 1H). 13C NMR (DMSO-d6): 129.9, 132.1,
133.2, 146.0, 155.8, 159.5. UV λmax (log ε) 285 (4.53). HRMS (ESI)
m/z: calculated 241.0083 for [C8H10BrN4]+, found 241.0088. HPLC
analysis: retention time = 9.84 min; peak area, 99.9%; eluent A,
CH3OH; eluent B, NH4OAc solution (0.1%); isocratic (3:1) over 60
min with a flow rate of 0.5 mL/min and detection at 254 nm.
(E)-2-(2-Hydroxybenzylidene)hydrazinecarboximidamide
Hydrochloride (12). Yield, 69%; mp 226−228 °C. 1H NMR
(DMSO-d6) δ 6.82 (t, 1H, J = 7.2 Hz), 6.91 (d, 1H, J = 8.4 Hz),
7.23 (t, 1H, J = 7.2 Hz), 7.93 (d, 1H, J = 7.6 Hz), 8.41 (s, 1H), 10.14
(s, 1H), 11.94 (s, 1H). 13C NMR (DMSO-d6): 116.6, 119.6, 120.0,
127.1, 132.3, 143.8, 155.6, 157.0. UV λmax (log ε) 276 (4.50). HRMS
(ESI) m/z: calculated 179.0927 for [C8H11N4O]+, found 179.0930.
HPLC analysis: retention time = 10.24 min; peak area, 99.9%; eluent
A, CH3OH; eluent B, NH4OAc solution (0.1%); isocratic (3:1) over
60 min with a flow rate of 0.5 mL/min and detection at 254 nm.
(E)-2-(3-Hydroxybenzylidene)hydrazinecarboximidamide
Hydrochloride (13). Yield, 35%; mp 153−156 °C. 1H NMR
(DMSO-d6) δ 6.88 (t, 1H, J = 8.0 Hz), 7.24 (m, 3H), 8.08 (d, 1H,
J = 2.4 Hz), 9.71 (d, 1H, J = 3.2 Hz), 11.99 (s, 1H). 13C NMR
(DMSO-d6): 114.5, 118.1, 119.0, 130.1, 135.1, 147.5, 155.8, 158.1,
159.5. UV λmax (log ε) 277 (4.41). HRMS (ESI) m/z: calculated
179.0927 for [C8H11N4O]+, found 179.0929. HPLC analysis: retention
time = 6.69 min; peak area, 99.9%; eluent A, CH3OH; eluent B,
NH4OAc solution (0.1%); isocratic (3:1) over 60 min with a flow rate
of 0.5 mL/min and detection at 254 nm.
(E)-2-(4-Fluorobenzylidene)hydrazinecarboximidamide Hy-
1
drochloride (4). Yield, 49%; mp 195−197 °C. H NMR (DMSO-
d6) δ 7.26 (t, 2H, J = 8.8 Hz), 7.91 (dt, 2H, J1 = 5.6 Hz, J2 = 2.8 Hz),
8.14 (s, 1H), 12.03 (s, 1H). 13C NMR (DMSO-d6): 116.3, 130.4,
146.0, 155.9, 162.6, 165.0. UV λmax (log ε) 277 (4.50). HRMS (ESI)
m/z: calculated 181.0884 for [C8H10FN4]+, found 181.0894. HPLC
analysis: retention time = 7.91 min; peak area, 98.2%; eluent A,
CH3OH; eluent B, NH4OAc solution (0.1%); isocratic (3:1) over 60
min with a flow rate of 0.5 mL/min and detection at 254 nm.
(E)-2-(2-Chlorobenzylidene)hydrazinecarboximidamide Hy-
drochloride (5). Yield, 72%; mp 198−200 °C. 1H NMR (DMSO-d6)
δ 7.42 (dt, 2H, J1 = 20.4 Hz, J2 = 1.2 Hz), 7.50 (d, 1H, J = 6.8 Hz),
8.27 (d, 1H, J = 7.2 Hz), 8.52 (s, 1H), 12.26 (s, 1H). 13C NMR
(DMSO-d6): 127.9, 128.1, 130.3, 131.1, 132.4, 133.7, 143.1, 155.7. UV
λmax (log ε) 279 (4.56). HRMS (ESI) m/z: calculated 197.0589 for
[C8H10ClN4]+, found 197.0596. HPLC analysis: retention time = 8.61
min; peak area, 97.6%; eluent A, CH3OH; eluent B, NH4OAc solution
(0.1%); isocratic (3:1) over 60 min with a flow rate of 0.5 mL/min and
detection at 254 nm.
(E)-2-(3-Chlorobenzylidene)hydrazinecarboximidamide Hy-
drochloride (6). Yield, 73%; mp 227−229 °C. 1H NMR (DMSO-d6)
δ 7.49 (m, 2H), 7.77 (d, 1H, J = 7.6 Hz), 8.10 (s, 1H), 8.18 (s, 1H),
12.18 (s, 1H). 13C NMR (DMSO-d6): 126.8, 127.3, 130.5, 131.0,
134.2, 136.1, 145.6, 155.9. UV λmax (log ε) 277 (4.57). HRMS (ESI)
m/z: calculated 197.0589 for [C8H10ClN4]+, found 197.0602. HPLC
analysis: retention time = 9.23 min; peak area, 98.3%; eluent A,
CH3OH; eluent B, NH4OAc solution (0.1%); isocratic (3:1) over 60
min with a flow rate of 0.5 mL/min and detection at 254 nm.
(E)-2-(4-Chlorobenzylidene)hydrazinecarboximidamide Hy-
drochloride (7). Yield, 21%; mp 146−149 °C. 1H NMR (DMSO-d6)
δ 7.53 (d, 2H, J = 8.4 Hz), 7.91 (d, 2H, J = 9.2 Hz), 8.16 (s, 1H), 11.86
(s, 1H). 13C NMR (DMSO-d6): 128.7, 129.2, 132.3, 134.9, 145.4,
155.3. UV λmax (log ε) 285 (4.53). HRMS (ESI) m/z: calculated
197.0589 for [C8H10ClN4]+, found 197.0593. HPLC analysis:
retention time = 9.42 min; peak area, 96.0%; eluent A, CH3OH;
eluent B, NH4OAc solution (0.1%); isocratic (3:1) over 60 min with a
flow rate of 0.5 mL/min and detection at 254 nm.
(E)-2-(4-Hydroxybenzylidene)hydrazinecarboximidamide
Hydrochloride (14). Yield, 83%; mp 251−253 °C. 1H NMR
(DMSO-d6) δ 6.79 (d, 2H, J = 8.0 Hz), 7.64 (d, 2H, J = 8.4 Hz),
8.01 (s, 1H), 10.03 (s, 1H), 11.68 (s, 1H). 13C NMR (DMSO-d6):
116.0, 124.8, 129.8, 147.5, 155.6, 160.3. UV λmax (log ε) 287 (4.58).
HRMS (ESI) m/z: calculated 179.0927 for [C8H11N4O]+, found
179.0933. HPLC analysis: retention time = 6.52 min; peak area,
99.3%; eluent A, CH3OH; eluent B, NH4OAc solution (0.1%);
isocratic (3:1) over 60 min with a flow rate of 0.5 mL/min and
detection at 254 nm.
( E ) - 2 - ( 3 , 5 - D i h y d r o x y b e n z y l i d e n e ) h y d r a z i n e -
carboximidamide Hydrochloride (15). Yield, 45%; mp 258−260
I
DOI: 10.1021/acs.jmedchem.6b00757
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
1
°C. H NMR (DMSO-d6) δ 6.40 (s, 1H), 6.68 (d, 2H, J = 1.6 Hz),
(s, 1H). 13C NMR (DMSO-d6): 126.4, 126.7, 126.8, 126.9, 127.1,
128.0, 128.2, 129.0, 129.0, 132.6, 141.9, 146.3, 155.4, 159.1. UV λmax
(log ε) 302 (4.42). HRMS (ESI) m/z: calculated 213.1291 for
[C14H15N4]+, found 213.1296. HPLC analysis: retention time = 11.79
min; peak area, 97.9%; eluent A, CH3OH; eluent B, NH4OAc solution
(0.1%); isocratic (3:1) over 60 min with a flow rate of 0.5 mL/min and
detection at 254 nm.
(E)-2-(4-Isopropylbenzylidene)hydrazinecarboximidamide
Hydrochloride (23). Yield, 20%; mp 116−121 °C. 1H NMR
(DMSO-d6) δ 1.21 (d, 7H, J = 6.8 Hz), 2.92 (sept, 1 H, J = 6.8
Hz), 7.32 (d, 2H, J = 8.0 Hz), 7.77 (d, 2H, J = 8.0 Hz), 8.13 (s, 1H),
11.88 (s, 1H). 13C NMR (DMSO-d6): 23.6, 33.4, 126.7, 127.7, 131.1,
146.9, 151.2, 155.3. UV λmax (log ε) 284 (4.67). HRMS (ESI) m/z:
calculated 205.1448 for [C11H17N4]+, found 205.1457. HPLC analysis:
retention time = 11.98 min; peak area, 96.0%; eluent A, CH3OH;
eluent B, NH4OAc solution (0.1%); isocratic (3:1) over 60 min with a
flow rate of 0.5 mL/min and detection at 254 nm.
7.98 (s, 1H), 9.52 (s, 2H), 11.89 (s, 1H). 13C NMR (DMSO-d6):
105.4, 106.2, 135.4, 148.0, 155.7, 159.0, 159.5. UV λmax (log ε) 286
(4.50). HRMS (ESI) m/z: calculated 195.0877 for [C8H11N4O2]+,
found 195.0878. HPLC analysis: retention time = 6.25 min; peak area,
99.4%; eluent A, CH3OH; eluent B, NH4OAc solution (0.1%);
isocratic (3:1) over 60 min with a flow rate of 0.5 mL/min and
detection at 254 nm.
(E)-2-(3-Bromo-2-hydroxybenzylidene)hydrazine-
carboximidamide Hydrochloride (16). Yield, 74%; mp 263−265
°C. 1H NMR (DMSO-d6) δ 6.89 (t, 1H, J = 7.6 Hz), 7.62 (d, 1H, J =
8.0 Hz), 7.83 (d, 2H, J = 8.0 Hz), 8.43 (s, 1H), 9.94 (s, 1H), 12.13 (s,
1H). 13C NMR (DMSO-d6): 111.8, 121.4, 121.8, 128.2, 134.8, 145.4,
152.6, 155.1. UV λmax (log ε) 282 (4.55). HRMS (ESI) m/z: calculated
257.0032 for [C8H10BrN4O]+, found 257.0030. HPLC analysis:
retention time = 8.81 min; peak area, 99.9%; eluent A, CH3OH;
eluent B, NH4OAc solution (0.1%); isocratic (3:1) over 60 min with a
flow rate of 0.5 mL/min and detection at 254 nm.
(E)-2-(3-(Benzyloxy)benzylidene)hydrazinecarboximidamide
1
Hydrochloride (24). Yield, 37%; mp 170−172. H NMR (DMSO-
(E)-2-(2-Methoxybenzylidene)hydrazinecarboximidamide
Hydrochloride (17). Yield, 85%; mp 244−246 °C. 1H NMR
(DMSO-d6) δ 3.86 (s, 3H), 7.01 (t, 1H, J = 6.8 Hz), 7.11 (d, 1H, J
= 8.4 Hz), 7.44 (t, 1H, J = 8.0 Hz), 8.09 (d, 1H, J = 7.2 Hz), 8.47 (s,
1H), 12.08 (s, 1H). 13C NMR (DMSO-d6): 55.7, 111.8, 120.6, 121.2,
126.3, 132.1, 142.0, 155.3, 157.7. UV λmax (log ε) 276 (4.52). HRMS
(ESI) m/z: calculated 193.1084 for [C9H13N4O]+, found 193.1095.
HPLC analysis: retention time = 7.75 min; peak area, 99.9%; eluent A,
CH3OH; eluent B, NH4OAc solution (0.1%); isocratic (3:1) over 60
min with a flow rate of 0.5 mL/min and detection at 254 nm.
(E)-2-(3-Methoxybenzylidene)hydrazinecarboximidamide
Hydrochloride (18). Yield, 99%; mp 135−138 °C. 1H NMR
(DMSO-d6) δ 3.80 (s, 3H), 7.03 (t, 1H, J = 6.8 Hz), 7.37 (d, 2H, J
= 4.8 Hz), 7.48 (s, 1H), 8.14 (s, 1H), 11.70 (s, 1H). 13C NMR
(DMSO-d6): 55.8, 112.1, 117.1, 121.2, 130.3, 135.1, 147.4, 155.5,
160.0. UV λmax (log ε) 278 (4.57). HRMS (ESI) m/z: calculated
193.1084 for [C9H13N4O]+, found 193.1093. HPLC analysis: retention
time = 7.88 min; peak area, 97.8%; eluent A, CH3OH; eluent B,
NH4OAc solution (0.1%); isocratic (3:1) over 60 min with a flow rate
of 0.5 mL/min and detection at 254 nm.
d6) δ 5.15 (s, 2H), 7.10 (m, 1H), 7.39 (m, 4H), 7.48 (m, 2H), 7.63 (s,
1H), 8.14 (s, 1H), 8.77 (s, 1H), 12.00 (s, 1H). 13C NMR (DMSO-d6):
69.4, 112.6, 117.2, 121.0, 128.0, 128.5, 129.8, 134.8, 136.8, 146.6,
155.4, 158.7. UV λmax (log ε) 278 (4.35). HRMS (ESI) m/z: calculated
269.1397 for [C15H17N4O]+, found 269.1401. HPLC analysis:
retention time = 12.37 min; peak area, 99.9%; eluent A, CH3OH;
eluent B, NH4OAc solution (0.1%); isocratic (3:1) over 60 min with a
flow rate of 0.5 mL/min and detection at 254 nm.
(E)-2-(4-Nitrobenzylidene)hydrazinecarboximidamide Hy-
1
drochloride (25). Yield, 34%; mp 243−247 °C. H NMR (DMSO-
d6) δ 8.12 (d, 2H, J = 8.8 Hz), 8.25 (m, 3H), 12.35 (s, 1H). 13C NMR
(DMSO-d6): 124.2, 129.0, 140.2, 144.9, 148.5, 156.0. UV λmax (log ε)
315 (4.46). HRMS (ESI) m/z: calculated 208.0829 for [C8H10N5O2]+,
found 208.0835. HPLC analysis: retention time = 7.82 min; peak area,
99.8%; eluent A, CH3OH; eluent B, NH4OAc solution (0.1%);
isocratic (3:1) over 60 min with a flow rate of 0.5 mL/min and
detection at 254 nm.
Protein Purification. P. aeruginosa HemO was purified as
previously reported with slight modification.50 Protein was bound to
a Q Sepharose Fast Flow column (2.5 cm × 6 cm) equilibrated with
20 mM tris (pH 8.0), 50 mM NaCl, and 50 mM 1,3-diaminopropane
(DAP). Residual biliverdin was retained on the column, and
apoprotein was eluted with a 50−250 mM NaCl gradient. Apo-
HemO fractions (determined by SDS−PAGE) were pooled and
dialyzed against 20 mM Tris-HCl (pH 8.0).
Determining Binding Affinities (KD) by Intrinsic Fluores-
cence Quenching. Fluorescence experiments were performed on a
Synergy H1 hybrid microplate reader using flat-bottom black 96-well
plates. Protein concentration was 1 μM HemO in 20 mM Tris-HCl
(pH 8.0). Compounds were dissolved in DMSO, and final
concentrations ranged from 0.5 to 1000 μM. Solutions were excited
at 295 nm, and emission spectra were recorded from 300 to 400 nm.
Decrease in maximum emission (332 nm) was corrected using
experimentally determined extinction coefficients for each com-
pound.43 Binding was determined using eq 1 and fit to a one-site
binding model.51
(E)-2-(4-Methoxybenzylidene)hydrazinecarboximidamide
1
Hydrochloride (19). Yield, 79%; mp 79−81 °C. H NMR (DMSO-
d6) δ 3.80 (s, 3H), 7.00 (d, 2H, J = 8.8 Hz), 7.79 (d, 2H, J = 8.4 Hz),
8.11 (s, 1H), 11.60 (s, 1H). 13C NMR (DMSO-d6): 55.8, 114.6, 126.3,
129.7, 147.3, 155.5, 161.6. UV λmax (log ε) 287 (4.56). HRMS (ESI)
m/z: calculated 193.1084 for [C9H13N4O]+, found 193.1090. HPLC
analysis: retention time = 7.56 min; peak area, 99.9%.
( E ) - 2 - ( N a p h t h a l e n - 1 - y l m e t h y l e n e ) h y d r a z i n e -
carboximidamide Hydrochloride (20). Yield, 46%; mp 153−154
°C. 1H NMR (DMSO-d6) δ 7.65 (m, 3H), 8.04 (m, 2H), 8.24 (d, 1H,
J = 6.8 Hz), 8.45 (d, 1H, J = 8.4 Hz), 9.01 (s, 1H), 12.05 (s, 1H). 13C
NMR (DMSO-d6): 123.6, 125.9, 126.7, 127.1, 127.6, 127.9, 129.0,
129.3, 130.8, 131.4, 133.8, 146.2, 155.7, 159.5. UV λmax (log ε) 320
(4.23). HRMS (ESI) m/z: calculated 213.1135 for [C12H13N4]+, found
213.1139. HPLC analysis: retention time = 10.22 min; peak area,
99.9%; eluent A, CH3OH; eluent B, NH4OAc solution (0.1%);
isocratic (3:1) over 60 min with a flow rate of 0.5 mL/min and
detection at 254 nm.
(F − F∞)L0
KD + L0
0
F − F =
0
( E ) - 2 - ( N a p h t h a l e n - 2 - y l m e t h y l e n e ) h y d r a z i n e -
(1)
carboximidamide Hydrochloride (21). Yield, 74%; mp 237−241
1
°C. H NMR (DMSO-d6) δ 7.58 (m, 2H), 7.96 (m, 3H), 8.20 (m,
STD NMR. Preparation of samples and collection of STD spectra
were conducted as previously reported with slight modifications.30
Briefly, to a 950 μL solution of 50 mM sodium phosphate (pH = 7.4)
in D2O was added 50 μL of desired compound dissolved in DMSO-d6
and 10 μL of purified HemO such that final concentration of HemO
was between 3 and 5 μM. After 4 h incubation at room temperature
the mixture was centrifuged at 6000g for 3 min to remove residual
precipitate. After preparation of 600 μL sample was added 3 μL of 50
mM 4,4-dimethyl-4-silapentane-1-sulfonic acid (DSS) dissolved in
D2O as internal standard.
2H), 8.34 (s, 1H), 12.13 (s, 1H). 13C NMR (DMSO-d6): 123.0, 126.8,
127.4, 127.8, 128.3, 128.4, 129.5, 131.2, 132.7, 133.9, 146.8, 155.4,
159.1. UV λmax (log ε) 270 (4.55). HRMS (ESI) m/z: calculated
213.1135 for [C12H13N4]+, found 213.1136. HPLC analysis: retention
time = 10.26 min; peak area 95.3%; eluent A, CH3OH; eluent B,
NH4OAc solution (0.1%); isocratic (3:1) over 60 min with a flow rate
of 0.5 mL/min and detection at 254 nm.
(E)-2-([1,1′-Biphenyl]-4-ylmethylene)hydrazine-
carboximidamide Hydrochloride (22). Yield, 40%; mp 179−182
1
°C. H NMR (DMSO-d6) δ 7.41 (m, 2H), 7.50 (t, 2H, J = 8.0 Hz),
In Cellulo Activity Assay. We have developed an E. coli cell based
7.61 (t, 3H, J = 8.0 Hz), 7.97 (d, 2H, J = 8.0 Hz), 8.24 (s, 1H), 12.13
assay for the measurement of HemO activity. The assay is based on the
J
DOI: 10.1021/acs.jmedchem.6b00757
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
coexpression of a HemO mutant with biliverdin-IXα regioselectivity
and the IFP1.4.48 E. coli BL21 (DE3) cells were transformed with
pET28a expressing IFP 1.4 and pBAD expressing HemO. Cells were
plated on LB-agar containing 30 μg/mL kanamycin and 15 μg/mL
tetracycline. A single colony was used to inoculate a 25 mL culture of
LB medium containing 30 μg/mL kanamycin and 15 μg/mL
tetracycline. The culture was incubated with shaking (200 rpm) at
37 °C overnight and used to inoculate a fresh culture treated to a final
OD600 of 0.1. Cultures were further incubated until reaching an OD600
= 0.7 at which point IFP 1.4 expression was induced with IPTG (final
concentration 1 mM). The HemO and IFP 1.4 proteins were allowed
to express at 25 °C induced with 0.02% arabinose (uninduced wells
were used for negative control) and aliquoted (200 μL) in 96-well
plates. Wells were treated with 0.5−200 μM compound (in a final
concentration 1% DMSO) or 1% DMSO in the positive control.
Cultures were incubated at 25 °C for 16 h following which the OD600
and fluorescence (ex 630, em 700) were measured on a Synergy H1
hybrid microplate reader. Background fluorescence in the absence of
HemO expression was measured as 0% HemO activity (negative
control). Maximum fluorescence on expression of HemO was
considered 100% activity (positive control). The percent inhibition
was calculated using eq 2 following correction of the fluorescence
solutes with different chemical functionalities (benzene, propane,
acetaldehyde, methanol, formamide, imidazole, acetate, and methyl-
ammonium) were added into the system at ∼0.25 M concentration, to
probe the functional group requirements of the protein.
Ten such systems with different initial fragment positions were
created to expedite the convergence of the simulations. Each system
was minimized for 5000 steps with the steepest descent (SD)
algorithm54 in the presence of periodic boundary conditions (PBC)
and was followed by a 250 ps MD equilibration. During SILCS
simulations, weak restraints were applied on the backbone Cα carbon
1
atoms with a force constant (k in /2kδx2) of 0.12 kcal mol−1 Å−1 to
prevent the rotation of the protein in the simulation box. Ten GCMC/
MD simulations were run for 100 cycles where each cycle has 200 000
steps of GCMC and 0.5 ns of MD, yielding a cumulative 200 million
steps of GCMC and 500 ns of MD. During GCMC, solutes and water
are exchanged between their gas-phase reservoirs; the simulation
system and the excess chemical potential used to drive such exchange
are periodically fluctuated over every 3 cycles to yield the average value
to be close to the hydration free energy of that fragment. The
configuration at the end of each GCMC run is used as the starting
́
configuration for the following MD. During MD, the Nose−Hoover
method55,56 was used to maintain the temperature at 298 K, and
pressure was maintained at 1 bar using the Parrinello−Rahman
barostat.57 CHARMM36 protein force field,58 CHARMM general
force field (CGenFF),59,60 and modified TIP3P water model61 were
used to describe protein, fragments, and water during the simulation,
respectively. GCMC was performed by in-house code, and MD was
conducted using GROMACS program.62
intensity as a function of OD600
.
F
− F
− F
inhibitor
0% activity
percent inhibition = 100 ×
F
100% activity
0% activity
(2)
The EC50 for each compound was calculated from the concentration-
dependent curve at 50% fluorescence inhibition. Fluorescence images
of live bacteria were obtained on a Nikon eclipse Ti fluorescence
microscope using NIS-Elements. Cells were imaged through a 100×
objective by phase contrast and through a Cy5 filter for detection of
near-infrared emissions from the biliverdin-IFP1.4 chromophore.
Growth Inhibition Assay. P. aeruginosa growth inhibition was
conducted as previously described.30 Briefly, a culture (25 mL) of each
strain was grown from a freshly plated colony for 8−12 h at 37 °C in
LB medium. Cultures were diluted back to an OD600 of 0.05 and
aliquoted into 96-well plates (200 μL) in either LB medium alone or
containing 8−1000 μg/mL of compound. The OD600 was measured
following 12 h of growth on a Synergy H1 hybrid microplate reader
and subtracted from LB media containing 50 μg/mL gentamycin to
prevent bacterial growth. MIC50 values were calculated from the
average of three experiments as previously described. Cultures from
wells containing 62.5−1000 μg/mL of compound were used to
inoculate fresh cultures in 5 mL of LB medium. Cultures were grown
for 12 h, and the lowest concentration that showed no growth by
measurement of OD600 was considered to be the minimum bactericidal
concentration (MBC).
SILCS. SILCS is a novel computer-aided drug design (CADD)
protocol that uses all-atom explicit-solvent molecular dynamics (MD)
simulations that include small organic solutes, such as benzene,
propane, methanol, and others, to identify spatial functional-group
binding patterns on the target. Benefiting from the use of explicit
solvent MD, SILCS intrinsically incorporates target flexibility and
desolvation effects and has more advantages over other CADD
methods which usually only consider limited target flexibility and
desolvation effects.
The current SILCS run was performed using the new grand
canonical Monte Carlo (GCMC)/MD protocol for SILCS. Compared
to the MD-alone technique, the GCMC/MD protocol has the benefit
of quickly targeting occluded pockets and decreasing the false binding
pattern caused by low fragment exchange rate at specific positions.52
The protein structure in the complex crystal structure (PDB code
1SK7) was used to initialize the SILCS simulation with removal of
cofactors such as heme and sulfate ions. The protein structure was
processed by Reduce53 to determine the most physical protonation
state of histidine residues and optimal side chain orientations of
asparagine and glutamine residues. The protein was solvated in a water
box, the size of which is determined to have the protein extrema
separated from the box edge by 12 Å on all sides. Eight representative
FragMaps Preparation and Ligand Docking. 3D probability
distributions of the selected atoms from the small molecules, called
“FragMaps”, from the SILCS simulations were constructed and
combined to obtain both specific and generic FragMap types as
previously described.41 Atoms from snapshot outputs every 10 ps from
each SILCS simulation trajectory were binned into 1 Å × 1 Å × 1 Å
cubic volume elements (voxels) of a grid spanning the entire system to
acquire the voxel occupancy for each FragMap atom type being
counted. The voxel occupancies computed in the presence of the
protein were divided by the value in bulk to obtain a normalized
probability. Normalized distributions were then converted to GFE
based on a Boltzmann transformation for quantitative use.
Ligand docking was conducted under the Monte Carlo based SILCS
(MC-SILCS) framework for both the heme binding site and the
allosteric site.41 The MC sampling was guided by LGFE, which is the
summation of the GFE contributions of the classified ligand atoms in a
molecule, and it represents the overlap of the functional groups in a
molecule with corresponding types of FragMaps. Five independent
MC-SILCS runs were performed for each ligand and each run was
repeated in two-step cycles. The first step of each cycle is 10 000
Metropolis MC steps that sample a wide range of different binding
poses, and the second step is 40 000 steps of MC simulated annealing
(SA) slow cooling designed to find a local minimum on the LGFE
surface. The initial ligand conformation is randomized by randomly
adjusting the torsion angels around all single bonds in the molecule,
and its potential energy was checked to ensure that unphysical
conformations are rejected. CGenFF was used to describe the ligands
during the MC-SILCS simulations. The generated conformation was
then placed at the desired binding site with a randomized orientation,
and its LGFE was checked to remove bad starting orientations. The
five MC SILCS runs involved repeated MC cycles that were repeated
until the best three LGFE scores were with 0.5 kcal/mol, with a
minimum of 50 cycles performed. The docking pose with the most
favorable LGFE was reported as the predicted binding mode.
HXMS. Deuterium exchange reactions were performed as described
by Chen et al. (2007).62 HemO protein samples were prepared by
diluting the protein to a final concentration of 100 μM with 20 mM
HEPES buffer in H2O (pH 7.4). Deuterium exchange was initiated by
diluting the HemO protein 50-fold with 20 mM HEPES buffer in D2O
(pD 7.4, deuteration buffer) at 23 °C. 100 pmol of protein was
removed from the reaction at 0 s, 10 min, and 30 min, and the
deuteration reaction was immediately quenched by lowering the pH to
K
DOI: 10.1021/acs.jmedchem.6b00757
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
2.5 with ice-cold HCl. Quenched samples were frozen on dry ice prior
to analysis.
funded by a predoctoral fellowship from the ACS Division of
Medicinal Chemistry and a predoctoral fellowship from the
American Foundation for Pharmaceutical Education to G.A.H.;
NIH Grant T32GM066706 to G.A.H.; and NIH Grant
AI102883 to A.W.
For reactions containing holo-HemO, the heme required for 95%
saturation was estimated using a KD of 1 μM. 100 μM apo-HemO was
reconstituted with 114 μM heme during the sample preparation, and
heme was added to the deuteration buffer to a final concentration of
21 μM. For reactions of inhibitor bound HemO, the inhibitor (1 or
22) required for 95% binding was estimated using a KD of 10 μM.
Apo- or holo-HemO was incubated with 285 μM inhibitor during the
sample preparation, and the inhibitor was added to the deuteration
buffer to a final concentration of 192 μM.
ABBREVIATIONS USED
■
CHARMM, chemistry at Harvard molecular mechanics;
GCMC, grand canonical Monte Carlo; GFE, grid free energy;
HemO, heme oxygenase; HEPES, (4-(2-hydroxyethyl)-1-
piperazineethanesulfonic acid; HO, human heme oxygenase;
HXMS, hydrogen−deuterium exchange mass spectrometry;
IFP, infrared fluorescent protein; LB, Luria−Bertani; LGFE,
ligand-grid free energy; MBC, minimum bactericidal concen-
tration; MIC50, minimum inhibitory concentration for 50%
growth; PBC, periodic boundary conditions; SA, simulated
annealing; SD, steepest descent; SDS−PAGE, sodium dodecyl
sulfate−polyacrylamide gel electrophoresis; SILCS, site identi-
fication by ligand competitive saturation; STD NMR, saturation
transfer difference nuclear magnetic resonance; UPLC, ultra-
performance liquid chromatography
Quenched samples were thawed and immediately injected into a
nanoACQUITY UPLC system with HDX manager (Waters). The
protein was digested online at 10 °C using an Enzymate BEH pepsin
column (2.1 mm × 30 mm, Waters) in 1 min. The digest was trapped
and desalted online on an ACQUITY Vanguard BEH C18 precolumn
(2.1 mm × 5 mm, Waters) at 0 °C for 4 min at a flow rate of 125 μL/
min in 0.1% formic acid. Peptides were separated on an ACQUITY
UPLC BEH C18 column (1.7 μm, 1 mm × 100 mm, Waters) at 0 °C
by a 15 min linear acetonitrile gradient (5−50%) with 0.1% formic
acid at a flow rate of 40 μL/min. The eluent was directed into the ion
source of a coupled SYNAPT G2 HDMS mass spectrometer (Waters).
Mass spectra were acquired in the MSE mode over the m/z range of
50−2000. Mass spectrometer parameters were as follows: electrospray
ionization positive (ESI+) mode, capillary voltage 3 kV, collision
energy 20−30 eV, sampling cone voltage 30 V, source temperature 80
°C, desolvation temperature 175 °C, and desolvation gas flow 500 L/
h. To generate a peptide list for ion search, 100 pmol of undeuterated
protein in 2 mM HCl in H2O was injected. The undeuterated peptides
were identified using Waters ProteinLynx Global Server software. The
peptide list generated was imported into Waters DynamX software to
guide the search of deuterated peptides, and the relative deuterium
incorporation levels for each deuterated peptide were calculated using
the 0 s sample as reference.
REFERENCES
■
(1) Clatworthy, A. E.; Pierson, E.; Hung, D. T. Targeting Virulence:
A New Paradigm for Antimicrobial Therapy. Nat. Chem. Biol. 2007, 3,
541−548.
(2) Page, M. G.; Heim, J. New Molecules from Old Classes:
Revisiting the Development of Beta-Lactams. IDrugs 2009, 12, 561−
565.
(3) Fair, R. J.; Hensler, M. E.; Thienphrapa, W.; Dam, Q. N.; Nizet,
V.; Tor, Y. Selectively Guanidinylated Aminoglycosides as Antibiotics.
ChemMedChem 2012, 7, 1237−1244.
ASSOCIATED CONTENT
* Supporting Information
■
S
(4) Fab
of and Resistance to Quinolones. Microb. Biotechnol. 2009, 2, 40−61.
(5) Guenard, S.; Muller, C.; Monlezun, L.; Benas, P.; Broutin, I.;
Jeannot, K.; Plesiat, P. Multiple Mutations Lead to MexXY-OprM-
̀
rega, A.; Madurga, S.; Giralt, E.; Vila, J. Mechanism of Action
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jmed-
chem.6b00757.
́
́
Dependent Aminoglycoside Resistance in Clinical Strains of
Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 2014, 58,
221−228.
(6) Voor, A. F.; Severin, J. A.; Lesaffre, E. M.; Vos, M. C.
Carbapenem Use and Medical Devices Highest Risk Factors for
Carbapenem-Resistant Pseudomonas aeruginosa: A Systematic Review
and Meta-Analyses. Antimicrob. Agents Chemother. 2014, 58, 2626−
2637.
Additional figures depicting the HXMS peptide coverage
following pepsin digestion, HXMS profile of apo-HemO
with compound 1, and Western blot and fluorescence
emission profiles of the E. coli HemO IFP expression
system (PDF)
Molecular formula strings (CSV)
(7) Costabile, G.; d’ Angelo, I.; Rampioni, G.; Bondí, R.; Pompili, B.;
Ascenzioni, F.; Mitidieri, E.; d’ Emmanuele di Villa Bianca, R.;
Sorrentino, R.; Miro, A.; et al. Toward Repositioning Niclosamide for
Antivirulence Therapy of Pseudomonas aeruginosa Lung Infections:
Development of Inhalable Formulations through Nanosuspension
Technology. Mol. Pharmaceutics 2015, 12, 2604.
(8) Alekshun, M. N.; Levy, S. B. Targeting Virulence to Prevent
Infection: To Kill or Not to Kill? Drug Discovery Today: Ther. Strategies
2004, 1, 483−489.
(9) Rasko, D. A.; Sperandio, V. Anti-Virulence Strategies to Combat
Bacteria-Mediated Disease. Nat. Rev. Drug Discovery 2010, 9, 117−128.
(10) Limoli, D. H.; Rockel, A. B.; Host, K. M.; Jha, A.; Kopp, B. T.;
Hollis, T.; Wozniak, D. J. Cationic Antimicrobial Peptides Promote
Microbial Mutagenesis and Pathoadaptation in Chronic Infections.
PLoS Pathog. 2014, 10, e1004083.
AUTHOR INFORMATION
Corresponding Authors
*A.W.: phone, 410-706-2537; fax, 410-706-5017; e-mail,
awilks@rx.umaryland.edu.
*F.X.: phone, 410-706-8512; fax, 410-706-0886; e-mail, fxue@
rx.umaryland.edu.
Author Contributions
All authors contributed to the writing of this manuscript. All
authors have given approval to the final version of the
manuscript.
■
Notes
The authors declare the following competing financial
interest(s): A.D.M., Jr., is cofounder and CSO of SilcsBio LLC.
(11) Jeukens, J.; Boyle, B.; Kukavica-Ibrulj, I.; Ouellet, M. M.; Aaron,
S. D.; Charette, S. J.; Fothergill, J. L.; Tucker, N. P.; Winstanley, C.;
Levesque, R. C. Comparative Genomics of Isolates of a Pseudomonas
aeruginosa Epidemic Strain Associated with Chronic Lung Infections of
Cystic Fibrosis Patients. PLoS One 2014, 9, e87611.
ACKNOWLEDGMENTS
■
The authors thank Angela Nguyen and Amanda Oglesby-
Sherrouse for their generous donations of the JSRI-1 and JSRI-
2 strains and guidance for their handling. This research was
L
DOI: 10.1021/acs.jmedchem.6b00757
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(12) Balasubramanian, D.; Kumari, H.; Mathee, K. Pseudomonas
aeruginosa AmpR: An Acute-Chronic Switch Regulator. Pathog. Dis.
2015, 73, 1−14.
(13) Roux, D.; Danilchanka, O.; Guillard, T.; Cattoir, V.; Aschard,
H.; Fu, Y.; Angoulvant, F.; Messika, J.; Ricard, J.-D.; Mekalanos, J. J.
Fitness Cost of Antibiotic Susceptibility during Bacterial Infection. Sci.
Transl. Med. 2015, 7, 297ra114.
(14) Mittal, R.; Sharma, S.; Chhibber, S.; Harjai, K. Iron Dictates the
Virulence of Pseudomonas aeruginosa in Urinary Tract Infections. J.
Biomed. Sci. 2008, 15, 731−741.
(15) Hauser, A. R. Pseudomonas aeruginosa Virulence and
Antimicrobial Resistance. Crit. Care Med. 2014, 42, 201−202.
(16) Fillat, M. F. The FUR (ferric Uptake Regulator) Superfamily:
Diversity and Versatility of Key Transcriptional Regulators. Arch.
Biochem. Biophys. 2014, 546, 41−52.
(17) Lowy, F. D.; Pollack, S.; Fadl-Allah, N.; Steigbigel, N. H.
Susceptibilities of Bacterial and Fungal Urinary Tract Isolates to
Desferrioxamine. Antimicrob. Agents Chemother. 1984, 25, 375−376.
(18) Collins, H. L.; Kaufmann, S. H.; Schaible, U. E. Iron Chelation
via Deferoxamine Exacerbates Experimental Salmonellosis via
Inhibition of the Nicotinamide Adenine Dinucleotide Phosphate
Oxidase-Dependent Respiratory Burst. J. Immunol. 2002, 168, 3458−
3463.
(19) Kontoghiorghes, G. J.; Kolnagou, A.; Skiada, A.; Petrikkos, G.
The Role of Iron and Chelators on Infections in Iron Overload and
Non Iron Loaded Conditions: Prospects for the Design of New
Antimicrobial Therapies. Hemoglobin 2010, 34, 227−239.
(20) Ji, C.; Miller, P. A.; Miller, M. J. Iron Transport-Mediated Drug
Delivery: Practical Syntheses and In Vitro Antibacterial Studies of Tris-
Catecholate Siderophore−Aminopenicillin Conjugates Reveals Selec-
tively Potent Antipseudomonal Activity. J. Am. Chem. Soc. 2012, 134,
9898−9901.
(21) Baco, E.; Hoegy, F.; Schalk, I. J.; Mislin, G. L. A. Diphenyl-
benzo[1,3]dioxole-4-Carboxylic Acid Pentafluorophenyl Ester: A
Convenient Catechol Precursor in the Synthesis of Siderophore
Vectors Suitable for Antibiotic Trojan Horse Strategies. Org. Biomol.
Chem. 2014, 12, 749−757.
́ ́
(22) Ji, C.; Juarez-Hernandez, R. E.; Miller, M. J. Exploiting Bacterial
Iron Acquisition: Siderophore Conjugates. Future Med. Chem. 2012, 4,
297−313.
(23) Benson, D. R.; Rivera, M. Heme Uptake and Metabolism in
Bacteria. In Metallomics and the Cell; Springer, 2013; pp 279−332.
(24) Wilks, A.; Barker, K. D. Mechanism of Heme Uptake and
Utilization in Bacterial Pathogens. Handb. Porphyrin Sci. 2011, 15,
357−398.
(25) Wilks, A.; Heinzl, G. Heme Oxygenation and the Widening
Paradigm of Heme Degradation. Arch. Biochem. Biophys. 2014, 544,
87−95.
(26) Nguyen, A. T.; O’Neill, M. J.; Watts, A. M.; Robson, C. L.;
Lamont, I. L.; Wilks, A.; Oglesby-Sherrouse, A. G. Adaptation of Iron
Homeostasis Pathways by a Pseudomonas aeruginosa Pyoverdine
Mutant in the Cystic Fibrosis Lung. J. Bacteriol. 2014, 196, 2265−
2276.
(31) Baum, T.; Eckfeld, D. K.; Metz, N.; Dinish, J. L.; Rowles, G.;
Van Pelt, R.; Shropshire, A. T.; Fernandez, S. P.; Gluckman, M. I.;
Bruce, W. F. 2, 6-Dichlorobenzylidene Amino Guanidine Acetate (Wy-
8678). A New Hypotensive Agent. Experientia 1969, 25, 1066−1067.
(32) Specht, S.; Sarite, S. R.; Hauber, I.; Hauber, J.; Gorbig, U. F.;
̈
Meier, C.; Bevec, D.; Hoerauf, A.; Kaiser, A. The Guanylhydrazone
CNI-1493: An Inhibitor with Dual Activity against Malariainhibition
of Host Cell pro-Inflammatory Cytokine Release and Parasitic
Deoxyhypusine Synthase. Parasitol. Res. 2008, 102, 1177−1184.
(33) Lowenberg, M.; Verhaar, A.; van den Blink, B.; ten Kate, F.; van
̈
Deventer, S.; Peppelenbosch, M.; Hommes, D. Specific Inhibition of c-
Raf Activity by Semapimod Induces Clinical Remission in Severe
Crohn’s Disease. J. Immunol. 2005, 175, 2293−2300.
(34) Ulloa, L. The Vagus Nerve and the Nicotinic Anti-Inflammatory
Pathway. Nat. Rev. Drug Discovery 2005, 4, 673−684.
(35) Soll, R. M.; Lu, T.; Tomczuk, B.; Illig, C. R.; Fedde, C.;
Eisennagel, S.; Bone, R.; Murphy, L.; Spurlino, J.; Salemme, F. R.
Amidinohydrazones as Guanidine Bioisosteres: Application to a New
Class of Potent, Selective and Orally Bioavailable, Non-Amide-Based
Small-Molecule Thrombin Inhibitors. Bioorg. Med. Chem. Lett. 2000,
10, 1−4.
(36) Gaubert, G.; Bertozzi, F.; Kelly, N. M.; Pawlas, J.; Scully, A. L.;
Nash, N. R.; Gardell, L. R.; Lameh, J.; Olsson, R. Discovery of
Selective Nonpeptidergic Neuropeptide FF2 Receptor Agonists. J.
Med. Chem. 2009, 52, 6511−6514.
(37) Carniato, D.; Gourvest, J.-F.; Ruxer, J.-M.; Knolle, J.; Peyman,
A.; Bodary, S. C.; Gadek, T. R. Iminoguanidine derivatives, preparation
method, use as medicines. U.S. Patent 6,391,904, May 21, 2002.
(38) Barron, S.; Lewis, B.; Wellmann, K.; Carter, M.; Farook, J.; Ring,
J.; Trent Rogers, D.; Holley, R.; Crooks, P.; Littleton, J. Polyamine
Modulation of NMDARs as a Mechanism to Reduce Effects of Alcohol
Dependence. Recent Pat. CNS Drug Discovery 2012, 7, 129−144.
(39) Conard, V. A.; Shriner, R. L. Aminoguanidine Derivatives. J. Am.
Chem. Soc. 1933, 55, 2867−2870.
(40) Ring, J. R. Synthetic Aromatic Agmatine Analogs as Allosteric
Modulators of the N-Methyl-D-Aspartate (NMDA) Receptor
Channel. Ph.D. Dissertation, University of Kentucky, Lexington, KY,
2006.
(41) Raman, E. P.; Yu, W.; Lakkaraju, S. K.; MacKerell, A. D.
Inclusion of Multiple Fragment Types in the Site Identification by
Ligand Competitive Saturation (SILCS) Approach. J. Chem. Inf. Model.
2013, 53, 3384−3398.
(42) Yu, W.; Lakkaraju, S. K.; Raman, E. P.; Fang, L.; MacKerell, A.
D., Jr Pharmacophore Modeling Using Site-Identification by Ligand
Competitive Saturation (SILCS) with Multiple Probe Molecules. J.
Chem. Inf. Model. 2015, 55, 407−420.
(43) Epps, D. E.; Raub, T. J.; Caiolfa, V.; Chiari, A.; Zamai, M.
Determination of the Affinity of Drugs toward Serum Albumin by
Measurement of the Quenching of the Intrinsic Tryptophan
Fluorescence of the Protein. J. Pharm. Pharmacol. 1999, 51, 41−48.
(44) Sowole, M. A.; Konermann, L. Effects of Protein−ligand
Interactions on Hydrogen/deuterium Exchange Kinetics: Canonical
and Noncanonical Scenarios. Anal. Chem. 2014, 86, 6715−6722.
(45) Friedman, J.; Lad, L.; Li, H.; Wilks, A.; Poulos, T. L. Structural
Basis for Novel Δ-Regioselective Heme Oxygenation in the
Opportunistic Pathogen Pseudomonas aeruginosa. Biochemistry 2004,
43, 5239−5245.
(27) Marvig, R. L.; Damkiær, S.; Khademi, S. H.; Markussen, T. M.;
Molin, S.; Jelsbak, L. Within-Host Evolution of Pseudomonas aeruginosa
Reveals Adaptation toward Iron Acquisition from Hemoglobin. mBio
2014, 5, e00966-14.
(28) O’Neill, M. J.; Wilks, A. The P. aeruginosa Heme Binding
Protein PhuS Is a Heme Oxygenase Titratable Regulator of Heme
Uptake. ACS Chem. Biol. 2013, 8, 1794−1802.
(29) Furci, L. M.; Lopes, P.; Eakanunkul, S.; Zhong, S.; MacKerell, A.
D.; Wilks, A. Inhibition of the Bacterial Heme Oxygenases from
Pseudomonas aeruginosa and Neisseria meningitidis: Novel Antimicrobial
Targets. J. Med. Chem. 2007, 50, 3804−3813.
(30) Hom, K.; Heinzl, G. A.; Eakanunkul, S.; Lopes, P. E.; Xue, F.;
MacKerell, A. D., Jr; Wilks, A. Small Molecule Antivirulents Targeting
the Iron-Regulated Heme Oxygenase (HemO) of P. aeruginosa. J. Med.
Chem. 2013, 56, 2097−2109.
(46) Rucker, H.; Amslinger, S. Identification of Heme Oxygenase-1
̈
Stimulators by a Convenient ELISA-Based Bilirubin Quantification
Assay. Free Radical Biol. Med. 2015, 78, 135−146.
(47) Filonov, G. S.; Piatkevich, K. D.; Ting, L.-M.; Zhang, J.; Kim, K.;
Verkhusha, V. V. Bright and Stable near-Infrared Fluorescent Protein
for in Vivo Imaging. Nat. Biotechnol. 2011, 29, 757−761.
(48) Shu, X.; Royant, A.; Lin, M. Z.; Aguilera, T. A.; Lev-Ram, V.;
Steinbach, P. A.; Tsien, R. Y. Mammalian Expression of Infrared
Fluorescent Proteins Engineered from a Bacterial Phytochrome.
Science 2009, 324, 804−807.
(49) Ratliff, M.; Zhu, W.; Deshmukh, R.; Wilks, A.; Stojiljkovic, I.
Homologues of Neisserial Heme Oxygenase in Gram-Negative
M
DOI: 10.1021/acs.jmedchem.6b00757
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Bacteria: Degradation of Heme by the Product of the pigA Gene of
Pseudomonas aeruginosa. J. Bacteriol. 2001, 183, 6394−6403.
(50) Rawel, H. M.; Frey, S. K.; Meidtner, K.; Kroll, J.; Schweigert, F.
J. Determining the Binding Affinities of Phenolic Compounds to
Proteins by Quenching of the Intrinsic Tryptophan Fluorescence. Mol.
Nutr. Food Res. 2006, 50, 705−713.
(51) Lakkaraju, S. K.; Raman, E. P.; Yu, W.; MacKerell, A. D., Jr
Sampling of Organic Solutes in Aqueous and Heterogeneous
Environments Using Oscillating Excess Chemical Potentials in
Grand Canonical-like Monte Carlo-Molecular Dynamics Simulations.
J. Chem. Theory Comput. 2014, 10, 2281−2290.
(52) Word, J. M.; Lovell, S. C.; Richardson, J. S.; Richardson, D. C.
Asparagine and Glutamine: Using Hydrogen Atom Contacts in the
Choice of Side-Chain Amide Orientation. J. Mol. Biol. 1999, 285,
1735−1747.
(53) Levitt, M.; Lifson, S. Refinement of Protein Conformations
Using a Macromolecular Energy Minimization Procedure. J. Mol. Biol.
1969, 46, 269−279.
́
(54) Nose, S. A Molecular Dynamics Method for Simulations in the
Canonical Ensemble. Mol. Phys. 1984, 52, 255−268.
(55) Hoover, W. G. Canonical Dynamics: Equilibrium Phase-Space
Distributions. Phys. Rev. A: At., Mol., Opt. Phys. 1985, 31, 1695−1697.
(56) Parrinello, M.; Rahman, A. Polymorphic Transitions in Single
Crystals: A New Molecular Dynamics Method. J. Appl. Phys. 1981, 52,
7182−7190.
(57) Best, R. B.; Zhu, X.; Shim, J.; Lopes, P. E.; Mittal, J.; Feig, M.;
MacKerell, A. D., Jr Optimization of the Additive CHARMM All-Atom
Protein Force Field Targeting Improved Sampling of the Backbone Φ,
Ψ and Side-Chain χ1 and χ2 Dihedral Angles. J. Chem. Theory Comput.
2012, 8, 3257−3273.
(58) Vanommeslaeghe, K.; Hatcher, E.; Acharya, C.; Kundu, S.;
Zhong, S.; Shim, J.; Darian, E.; Guvench, O.; Lopes, P.; Vorobyov, I.;
MacKerell, A. D. CHARMM General Force Field: A Force Field for
Drug-like Molecules Compatible with the CHARMM All-Atom
Additive Biological Force Fields. J. Comput. Chem. 2010, 31, 671−690.
(59) Yu, W.; He, X.; Vanommeslaeghe, K.; MacKerell, A. D.
Extension of the CHARMM General Force Field to Sulfonyl-
Containing Compounds and Its Utility in Biomolecular Simulations.
J. Comput. Chem. 2012, 33, 2451−2468.
(60) Durell, S. R.; Brooks, B. R.; Ben-Naim, A. Solvent-Induced
Forces between Two Hydrophilic Groups. J. Phys. Chem. 1994, 98,
2198−2202.
(61) Hess, B.; Kutzner, C.; Van Der Spoel, D.; Lindahl, E.
GROMACS 4: Algorithms for Highly Efficient, Load-Balanced, and
Scalable Molecular Simulation. J. Chem. Theory Comput. 2008, 4, 435−
447.
(62) Chen, S.; Brier, S.; Smithgall, T. E.; Engen, J. R. The Abl SH2-
Kinase Linker Naturally Adopts a Conformation Competent for SH3
Domain Binding. Protein Sci. 2007, 16, 572−581.
N
DOI: 10.1021/acs.jmedchem.6b00757
J. Med. Chem. XXXX, XXX, XXX−XXX