Article

pubs.acs.org/jmc

Siderophore Receptor-Mediated Uptake of Lactivicin Analogues in

Gram-Negative Bacteria
Jeremy Starr,#,† Matthew F. Brown,#,† Lisa Aschenbrenner,§ Nicole Caspers,¶ Ye Che,⧧
Brian S. Gerstenberger,† Michael Huband,§ John D. Knafels,¶ M. Megan Lemmon,§ Chao Li,†
Sandra P. McCurdy,§ Eric McElroy,† Mark R. Rauckhorst,† Andrew P. Tomaras,§ Jennifer A. Young,†
Richard P. Zaniewski,§ Veerabahu Shanmugasundaram,⧧ and Seungil Han*,#,¶
†
Medicinal Chemistry, ⧧Computational Chemistry, §Antibacterials Research Unit, and ¶Structural Biology, Pfizer Global Research and
Development, Eastern Point Road, Groton, Connecticut 06340, United States
*
S Supporting Information

ABSTRACT: Multidrug-resistant Gram-negative pathogens

are an emerging threat to human health, and addressing this
challenge will require development of new antibacterial agents.
This can be achieved through an improved molecular
understanding of drug−target interactions combined with
enhanced delivery of these agents to the site of action. Herein
we describe the first application of siderophore receptor-
mediated drug uptake of lactivicin analogues as a strategy that
enables the development of novel antibacterial agents against
clinically relevant Gram-negative bacteria. We report the first
crystal structures of several sideromimic conjugated compounds bound to penicillin binding proteins PBP3 and PBP1a from
Pseudomonas aeruginosa and characterize the reactivity of lactivicin and β-lactam core structures. Results from drug sensitivity
studies with β-lactamase enzymes are presented, as well as a structure-based hypothesis to reduce susceptibility to this enzyme
class. Finally, mechanistic studies demonstrating that sideromimic modification alters the drug uptake process are discussed.

■ INTRODUCTION
Since Fleming’s landmark publication of the discovery of
penicillin in 1929,1 the β-lactam antibiotic class has produced
more drugs than any other chemotype and has had a profound
impact on extending human life. Despite the development of
new anti-infective drugs, bacterial infections continue to
represent a major global health threat, especially infections
with serious Gram-negative pathogens such as Pseudomonas
aeruginosa, Acinetobacter baumannii, Klebsiella pneumoniae, and
Escherichia coli.2 Particularly vulnerable are hospitalized patients
with comorbidities or weakened immune systems who are at
high risk of infection with multidrug-resistant variants. β-
Lactams continue to be utilized as the foundation of treatment
for multiple conditions arising from these infections, such as
ventilator-associated pneumonia, intra-abdominal infections,
and bacteremia,3−5 but their effectiveness continues to diminish
due to ever increasing prevalence of drug-resistant strains.6,7
There is a critical need for new therapeutic options for these
difficult to treat infections.8
Penicillin binding proteins (PBPs) are a large class of
enzymes ubiquitously expressed across the spectrum of
prokaryotic pathogens and are the biochemical targets of β-
lactam drugs.9 Most PBPs are bound in the peptidoglycan layer
and, in the case of Gram-positive bacteria, are readily accessible
to drugs capable of transiting the relatively permeable cell wall.
By contrast, an inhibitor must pass through the formidable
outer membrane of Gram-negative bacteria to gain access to its
target in the periplasmic space. Classically, a β-lactam with
Gram-negative activity exhibits specific structural and phys-
icochemical properties carefully tuned to enable passage
through porin channels while not engaging efflux trans-
porters.10 Inhibition of PBPs then leads to the introduction
of flaws in the peptidoglycan layer, resulting in loss of structural
integrity, osmotic control, morphological changes, and eventual
cell lysis. In response to drug pressure, bacteria utilize a variety
of drug-neutralizing mechanisms, including the production of
β-lactamase (BLA) enzymes. To combat this resistance
mechanism clinically, β-lactams are often administered with
BLA inhibitors. However, inhibitor-resistant BLA variants are
prevalent and contribute to significant treatment failure.
Therefore, novel PBP inhibitors with reduced susceptibility to
BLAs are critical to the development of next-generation
antibacterial therapies. It is noteworthy that while decades of
research across the pharmaceutical industry has generated
dozens of marketed β-lactam drugs, no other PBP-inhibiting
chemotype has thus far reached the market.
Bacteria have developed a highly effective mechanism to
acquire iron, a critical element, from their host organism. The
process involves the release of siderophores with extremely
Received: February 10, 2014
Published: April 2, 2014

© 2014 American Chemical Society 3845 dx.doi.org/10.1021/jm500219c | J. Med. Chem. 2014, 57, 3845−3855
Journal of Medicinal Chemistry
Figure 1. Lactivicin (1) and comparator agents.
high affinity for iron, which can effectively compete with the
host’s endogenous iron binding proteins. Following secretion
and chelation of iron, the siderophore complexes are actively
retrieved through binding to specialized bacterial receptors. β-
Lactams conjugated to structurally simple, small molecule
siderophore mimics (hereafter referred to as “sideromimics”)
such as catechols and hydroxypyridones have been described
which utilize this internalization process, leading to active
cellular uptake. Despite extensive industrial and academic
research efforts utilizing this “Trojan horse” approach to
enhance intracellular delivery of β-lactams,11−20 a similar
approach has not been reported for non-β-lactam PBP
inhibitors. Here we describe the application of the sideromimic
conjugation strategy to lactivicins, a non-β-lactam natural
product class of PBP inhibitors of microbial origin (Figure
1).21−23 We report that lactivicin−sideromimic conjugates
provide an improved PBP profile relative to aztreonam, with
potent inhibition of PBP3 and 1a as well as enhanced potency
vs PBP1b. This expanded PBP inhibitory profile may eventually
prove to be an advantage for the lactivicin drug class. We report
the first X-ray crystal structures of sideromimic-conjugated
lactivicin analogues bound to Pseudomonas aeruginosa PBP3
(Pae PBP3) and PBP1a (Pae PBP1a), as well as the related β-
lactam sideromimic-conjugate 4 (BAL30072) (Figure 1)16,17
bound to Pae PBP3 and discuss novel insights gained from the
effectiveness of these compounds. We report the sensitivity of
the lactivicin sideromimic conjugates to β-lactamase enzymes,
and based on structure-derived knowledge, we propose that
optimal attachment and positioning of the sideromimic moiety
enhances intrinsic activity and could lead to reduced
susceptibility to drug-degrading BLA enzymes. In addition, to
aid analogue design and improve the molecular understanding
of covalent bond formation, relative reactivities of lactivicin and
monobactam analogues are characterized using a computational
method which defines the activation barrier of a transition-state
structure derived from the reaction of the electrophilic carbonyl
with a simple model nucleophile, CH3O−. We have also
characterized the role of bacterial siderophore receptors in the
uptake of the new phthalimide-conjugated lactivicin analogue
17 and have discovered that it utilizes a broader set of Ton-B-
dependent siderophore receptors as compared to related β-
lactam sideromimic conjugates. Taken together, these results
3846
Article
could positively influence the discovery of novel, next-
generation Gram-negative antibacterial agents.
■ RESULTS AND DISCUSSION
Relative Reactivity of Lactivicin and Monobactam
Analogues. Critical to the mechanism of β-lactam drugs is the
presence of an electrophilic carbonyl that covalently modifies a
highly conserved PBP active site serine hydroxyl group leading
to the formation of an acyl enzyme complex that is significantly
lower in energy due to relief of ring strain. By comparison, a
monocyclic five-membered lactam or larger homologue would
not be expected to exhibit a strain-induced irreversibility due to
the exceptional stability of such lactams, while a highly strained
three-membered ring lactam would be too reactive in water to
survive en route to its bacterial target. However, the primacy of
four-membered ring lactams as PBP inhibitors has been
challenged.24−26 For example, the lactivicin class is unusual
because it is the only known naturally occurring PBP inhibitor
class that does not contain a β-lactam. Instead of a β-lactam
ring, lactivicin (1, Figure 1) and its derivatives contain
cycloserine (a five-membered lactam) and γ-lactone (a five-
membered cyclic ester) rings. Recently published X-ray
crystallographic data supports the covalent mechanism of
action of lactivicins wherein the carbonyl of the cycloserine ring
reacts with the Gram-positive Streptococcus pneumoniae active
site serine hydroxyl to form a covalent bond analogous to that
formed by a typical β-lactam drug (Figure 2A).27
Nucleophilic substitution at the carbonyl group of an amide
usually occurs in a stepwise manner initiated by the formation
of a tetrahedral intermediate as the rate-determining step
(Figure 2A). Here the relative reactivity of covalent inhibitors
such as β-lactams and lactivicins was theoretically characterized
by molecular orbital calculations as the activation barrier (ΔG⧧)
of the transition state structure in an aqueous solution with a
simple nucleophile (CH3O−) representing the active site serine.
While this approach does not factor in the multiple hydrogen-
bonding interactions which stabilize the transition state in the
oxyanion hole of the protein, it can be useful in the design of
covalent inhibitors. For example, previously reported semi-
empirical calculations (CNDO/2) by Boyd et al. described an
optimal reactivity range for β-lactams which provides effective
covalent inhibition of PBPs combined with the hydrolytic
stability needed to provide antibacterial activity in an aqueous
dx.doi.org/10.1021/jm500219c | J. Med. Chem. 2014, 57, 3845−3855
Journal of Medicinal Chemistry Article

environment.28 Here, we compare ΔG⧧ values, derived from

Figure 2. (A) Proposed mechanisms of acylation of lactivicin
analogues. (B) The activation barrier (ΔG⧧) of forming the
transition-state structure derived from the reaction of the electrophilic
carbonyl with a simple nucleophile CH3O− (DFT calculations).
high-level DFT calculations, for a number of reported lactivicin
analogues to evaluate the influence of ring strain and electron-
withdrawing substituents on reactivity and subsequent
antibacterial activity (Figure 2B).
While the expanded lactam ring size of lactivicins relative to
β-lactams such as aztreonam (Figure 1) may be expected to
lead to reduced reactivity, the calculated activation barriers for
each are quite similar (ΔG⧧ for 1 and monobactam 9 = 31.3
and 30.7 kcal/mol, Figure 2B). Our assumption is that the
reduced ring strain of lactivicins relative to monobactams is
overcome by the electronic activation provided by the
appended lactone moiety and the cycloserine ring oxygen,
thus providing similar ΔG⧧ values, sufficient PBP reactivity, and
the resulting antibacterial activity. In addition, lactivicins are
known to undergo a ring-chain tautomerization (i.e., 5 to 6)
which leads to equilibration of the γ-lactone chiral center
(Figure 2A).29 The ring-opened tautomer 6 may be expected to
be a highly reactive species, and calculations support this
statement as virtually no barrier exists, suggesting that a
spontaneous reaction would occur with the nucleophile
CH3O−. Nevertheless, the fact that lactivicin analogues exhibit
sufficient chemical stability to provide antibacterial activity in an
aqueous environment suggests this may not be the case. Harada
et al. provide an explanation suggesting that the imminium ion
6 may be stabilized by the tethered carboxylate(s).29 So while
the existence of tautomer 6 is necessary to enable the reported
lactone equilibration process, its impact on overall chemical
stability and reactivity with the PBP active site serine remains
unknown. The potential of tautomer 6 to react with PBP is
captured in Figure 2a; however, the calculations described
above are based solely on the transition of the ring-closed
tautomer 5 to 7.
A number of research groups have reported efforts to modify
the lactivicin core, most of which lead to a significant or
complete loss of antibacterial activity. These results can be
rationalized with calculated ΔG⧧ values. For example, the
expanded lactam ring analogue 10 was reported to be inactive

Table 1. MICs and Pae PBP IC50 Data for Lactivicin Analogues

MIC (μg/mL)a Pae PBP IC50 (μM)

a
compound Eco EC-28 Pae PA01 Pae 1091-05 Kpn KP-3700 Aba AB-3167 1a 1b 2 3 nb
aztreonam 0.125 4 4 >64 32 3.34 ± 0.57 2.87 ± 0.64 >300 0.008 ± 0.004 8
13 NTc 32 32 4 32 0.046 1.23 33.3 0.092 1
14 2 8 4 32 0.5 0.046 2.46 22.2 0.14 1
15R >64 >64 >64 >64 >64 0.11 ± 0.032 2.44 ± 0.03 47.1 ± 23.5 0.11 ± 0.03 2
15S >64 >64 >64 >64 >64 0.065 ± 0.033 1.74 ± 0.87 33.3 0.065 ± 0.033 2
16R 1 16 2 16 0.5 0.046 0.82 6.41 ± 5.23 0.065 ± 0.033 2
16S 1 2 64 8 0.5 0.046 0.82 3.7 0.092 2
17 0.25 0.5 0.5 0.5 0.06 0.030 0.27 ± 0.39 3.7 ± 1.8 0.046 2

a
Eco: E. coli, Pae: Pseudomonas, Kpn: Klebsiella pneumoniae, Aba: Acinetobacter baumannii. bn = number of replicates for PBP IC50 determination.
c
NT: not tested.

3847 dx.doi.org/10.1021/jm500219c | J. Med. Chem. 2014, 57, 3845−3855

Journal of Medicinal Chemistry
Figure 3. Interactions of compound 14 and 4 in the active site of the Pae PBP3. (A) Active site of Pae PBP3 bound to compound 14 (navy). (B)
Active site of Pae PBP3 bound to 4 colored in green. (C) Molecular surface of the Pae PBP3 in the active site region in complex with compound 4
(green). The residues of hydrophobic aromatic wall (Tyr-Tyr-Phe) and Val333 are shown in orange. (D) Molecular surface of the Pae PBP3 active
site in complex with aztreonam in cyan (PDB code: 3PBS). (E) Molecular surface of the Pae PBP3 active site in complex with 3 in pink (PDB code:
3PBT).
vs a panel for Gram-positive and negative bacteria.30 Ring
expansion would be expected to reduce reactivity, and this is
reflected in the 2.7 kcal/mol increase in the ΔG⧧ value for 10 vs
1 (Figure 2B). In addition, a number of reports describe efforts
to replace the lactone N-activating group. For example, 11
contains the N-sulfate activating group common to the
monobactam drug class. The significantly lower ΔG⧧ of this
analogue (24.6 kcal/mol) as compared to 1 (31.3 kcal/mol) or
monobactam 9 (30.7 kcal/mol) is suggestive of high level
reactivity and poor hydrolytic stability, which may explain the
reported lack of antibacterial activity.31 Similarly, the low
calculated ΔG⧧ (19.3 kcal/mol) for compound 12 explains the
reported lack of antibacterial activity and poor aqueous
stability.32 While the utilization of calculated ΔG⧧ values has
great potential to enable the rational design of next-generation
lactivicin (or β-lactam) core structures, our initial efforts
maintained the cycloserine-lactone core structure found in the
lactivicin natural product (vide infra).
Initial Application of Sideromimic-Conjugated Lacti-
vicins: Oxime-Linked Dihydroxypyridone 14. Lactivicin
lead optimization efforts reported by Takeda describe a number
of potent analogues, including 13 (Table 1), possessing a fairly
balanced spectrum of activity toward Gram-negative pathogens
and in vivo efficacy.33−35 However, when evaluated against a
contemporary panel of clinically relevant Gram-negative
bacteria, weak whole cell activity (MIC) was observed for 13
vs P. aeruginosa and A. baumannii strains (Table 1). As
discussed earlier, conjugation of sideromimics such as catechols
and hydroxypyridones to β-lactams has been shown to impart
improved cellular potency via utilization of the bacterial iron
uptake process. Recent examples include 3 (MC-1) and 4, both
of which employ hydroxypyridones as the sideromimics (Figure
1).16,18 Incorporation of a hydroxypyridone moiety in lactivicin,
as in compound 14, quickly demonstrated the potential of the
siderophore receptor uptake strategy, providing improved
MICs in P. aeruginosa and A. baumannii strains while retaining
PBP inhibitory potency similar to comparator analogue 13
which lacks a sideromimic (Table 1). Furthermore, the
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sideromimic-containing C4 side chain of compound 14 is
identical to that found in the monocyclic β-lactam analogue 4,
which is currently in clinical evaluation for the treatment of
Gram-negative infections. Compound 14 provided our first
opportunity to examine the covalently bound conformation of a
lactivicin−sideromimic analogue with its biological target and
enable comparison to the related monocarbam analogue 4.
Complexes of Pae PBP3 with 14 and 4. The 2.3 Å
resolution crystal structure of Pae PBP3 with 14 revealed that
both the L-cycloserine and γ-lactone rings are open and the
antibiotic is covalently linked to the nucleophilic hydroxyl
group of Ser294 (Figure 3A). The carboxylic acid group of 14
resulting from the opening of the lactone ring is stabilized by a
H-bonding interaction with conserved residues Ser349, Ser485,
and Thr487. The interaction of the core of 14 with Pae PBP3 is
similar to the published S. pneumoniae PBP1b−lactivicin
complex which involves all three signature motifs (SXXK,
SXN, and KSGT).27 Importantly, in the structure of the
complex of 14, the electron densities corresponding to the
ethylene carboxylate moiety resulting from the opening of the
lactone ring and that of the dihydroxypyridone sideromimic
moiety are weak and discontinuous, suggesting that these side
chains are not only flexible but also do not form strong
interactions with Pae PBP3 (Figure S1, Supporting Informa-
tion). These flexible side-chain conformations suggested that
the potency of 14 could be further improved by structural
modifications to enable interaction with the unique Tyr-Tyr-
Phe aromatic wall present in Pae PBP3 (vide infra).
Given the structural similarities between 14 and 4, a 2.0 Å
resolution crystal structure of the Pae PBP3 in complex with 4
was prepared to enable direct comparison of the two series
(Figure 3B). While not well-resolved, the 4-dihydroxypyridone
moiety is positioned near a hydrophobic pocket formed by
Tyr503, Tyr532, and Phe533 and the N-hydroxy group appears
to be within hydrogen bonding distance of the phenolic
oxygens in both Tyr532 and Tyr503. The low resolution in this
region of the structure limits the ability to confidently define
meaningful drug−protein interactions. This is different from
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Journal of Medicinal Chemistry Article

Figure 4. Crystal structure of Pae PBP1a in complex with compound 17 and comparison of the structures with Pae PBP3 and Aba PBP3. (A)
Overall structure of Pae PBP1a complexed with compound 17. The bound compound 17 is shown as spheres. (B) Active site of Pae PBP1a. The
catalytic Ser461 is shown in red. The loop connecting β3 and β4 is shown in cyan. (C) Overlap of proteins from 3−Pae PBP3 (orange) and 17−
PBP1a (cyan) structures, illustrating that PBP3 Phe533 occupies the same general space as PBP1a Tyr733. (D) Comparison of the extended loop
connecting β3 and β4 in apo-Pae PBP3 (green) and apo-Aba PBP3 (pink).

the published complexes of aztreonam and monocarbam 3 with
Pae PBP3 in which the C4-linked gem-dimethyl group common
to these compounds clearly interacts favorably with the
hydrophobic pocket composed of Tyr503-Tyr532-Phe533 and
the oxime-linked carboxylate forms a salt bridge with Arg489
(Figure 3C−E).36 Based on these favorable interactions, a
design strategy was developed to incorporate this isobutyric
acid side chain into the lactivicin analogues while exploring an
alternate attachment point of the sideromimic group.
Linkage of the Sideromimic to the α-Lactone
Position. Of particular interest were α-amido-γ-lactones such
as 15−17 (Table 1). A sideromimic attachment at the lactone
α-position would potentially position this substituent in the
semiopen channel distal from the active site serine where
additional ligand−protein interactions might be possible. This
channel has been exploited previously in the design of
monocarbams such as 336 (Figure 3E) and related mono-
bactams.19,20 A priori, predicting the optimal stereochemistry of
the lactone α-amido connection was challenging. The available
data from the crystal structure of P. aeruginosa PBP3 covalently
bound to 14 showed only the ring-opened adduct, as expected,
and could not inform about a preferred binding orientation of
the ring-closed species. This analysis was further complicated
by the dynamic stereochemistry of the tertiary γ-carbon of the
lactone resulting from the ring-opening equilibration process
described in Figure 2A. Therefore, exemplars of both
configurations of the α-lactone substituent were prepared and
evaluated. The resulting compounds (15R and 15S) bearing a
benzyloxycarbonyl (Cbz) group (not a sideromimic) exhibited
3849
no meaningful whole cell activity against any of the Gram-
negative pathogens of interest despite both having PBP
inhibitory potency comparable to the Takeda prototype
compound 13 as well as the hydroxypyridone-conjugate 14
(Table 1). The increased size and lipophilicity of the Cbz group
was likely deleterious to passive transit through porins and
suggested that structurally similar compounds would gain little
exposure to the periplasm without facilitated transport.
Furthermore, on the basis of the similar PBP activity, there
was little apparent preference for one configuration of the
amido substituent over the other.
Replacement of the Cbz groups in 15R and 15S with a
hydroxypyridone sideromimic provided the diastereomeric
compounds 16R and 16S. With siderophore receptor facilitated
transport now enabled, both analogues exhibited improved
MICs relative to 15R and 15S, despite only limited differences
in PBP inhibition relative to the comparators lacking a
sideromimic, providing additional evidence that sideromimics
play a critical role in cell entry in this series. Again, despite a
difference in stereochemistry of sideromimic attachment, the
PBP inhibitory activity exhibited little differentiation between
the diastereomers. However, fairly significant differences in P.
aeruginosa cellular potency were observed for the 16R/S isomer
pair. The reason for this is currently unknown.
In an effort to further optimize drug PBP3 enzyme
interactions, a design strategy to exploit potential aryl−aryl
interactions between the sideromimic and the Tyr503, Tyr532,
and Phe533 aromatic residues led to analogues bearing phenyl
rings. From this effort, compound 17 was found to exhibit both
dx.doi.org/10.1021/jm500219c | J. Med. Chem. 2014, 57, 3845−3855
Journal of Medicinal Chemistry Article

the lowest MICs and PBP IC50s to date for the series, perhaps of 17 bound to Pae PBP3 was not prepared, comparison of the
due to an improved interaction between the phthalimide previously reported 3−Pae PBP3 structure36 with the 17−Pae
phenyl ring and the PBP active sites and to the competence of PBP1a structure demonstrates a similar positioning of the PBP3
the phthalimide as a substrate for active transport via Phe533 and PBP1a Tyr733 side chains, suggesting that
siderophore receptors (vide infra). Interestingly, attachment analogous π-stacking interactions with the phthalimido group
of a 4,5-dihydroxyphthalimide to a cephalosporin core has been of 17 may occur in both PBP1a and PBP3 (Figure 4C).
reported to provide potent antibacterial analogues as well, Evidence Supporting Siderophore Receptor-Medi-
suggesting that this sideromimic may have general utility for ated Drug Uptake. P. aeruginosa strains are able to express
drug transport.37 an impressive range of Ton-B-dependent siderophore recep-
Monobactam antiobiotics such as aztreonam typically tors.39 This high level of functional redundancy is critical to
demonstrate potent inhibition of PBP3, with lesser activity vs ensure that adequate levels of iron can be obtained in a variety
PBP1a and 1b and no relevant activity vs PBP2 (Table 1). of environments. Previous work from our group has suggested a
While selective PBP3 inhibitors like aztreonam can successfully role for the siderophore receptors PiuA and PirA for the uptake
treat susceptible infections, enhanced potency vs other PBPs of a variety of hydroxypyridone-conjugated β-lactams.20,40
could lead to improved antibacterial performance. On the basis Because analogues 14 and 16R/S incorporate the same
of initial data, it would appear that the lactivicin drug class hydroxypyridone sideromimics, our assumption is that they
provides an improved PBP profile relative to aztreonam, utilize a similar uptake process; however, this remains to be
providing potent inhibition of PBP3 and 1a as well as enhanced determined. Because lactivicin analogue 17 incorporates a
potency vs PBP1b and 2 (Table 1). While the impact of this structurally unique phthalimide sideromimic, a study was
expanded PBP inhibitory profile remains to be determined, it conducted to determine which receptors are involved in
may eventually prove to be an advantage for the lactivicin drug cellular drug uptake. Whole cell activity was recorded for 17 vs
class. the wild-type P. aeruginosa strain PAO1 as well as an isogenic
Class A Pae PBP1a Structure and Its Complex with a panel of strains lacking one or more siderophore receptors
Phthalimide Sideromimic, Compound 17. The potent (Table 2). The study was conducted with both standard
PBP1a inhibitory activity of phthalimide 17 catalyzed crystallo-
graphic studies to investigate its binding pose with this class A Table 2. Pae PAO1 Isogenic Siderophore Receptor Mutant
PBP. Following an extensive effort with multiple constructs, Panel
Pae PBP1a crystals were obtained after controlled proteolysis
MICs (μg/mL)a
with trypsin (see Experimental Section for details). The
crystallized Pae PBP1a is composed of four major polypeptides, 17
including: Pro47-Met64, Thr256-Leu494, Thr506-Val609, MHB/low- aztreonam low-
Glu652-Glu792. In Pae PBP1a, the initial transmembrane strains MHB low-Fe Fe Fe
helix is followed by an N-terminal transglycosylase (TG) PAO1 0.5(1) ≤0.03(1) 16 4(1)
domain connected through a β-rich linker to a C-terminal piuA 8(16) 0.125(≥4) 64 4(1)
transpeptidase (TP) domain. The entire TG domain is absent pirA 0.5(1) ≤0.03(1) 16 4(1)
in the Pae PBP1a structure due to intrinsic flexibility and tryptic f pvA 16(32) ≤0.03(1) 512 2(0.5)
cleavage. The interdomain linker contains a six-stranded β- f ptA 4(8) 0.06(≥2) 64 4(1)
sheet where four strands are from TG and TP domains. The pfeA 0.5(1) ≤0.03(1) 16 4(1)
TP domain in the center of Pae PBP1a (residues 292−762) piuA fpvA 16(32) 0.25(≥8) 64 4(1)
shares a similar overall fold with other transpeptidases and piuA fptA 8(16) 0.5(≥16) 16 4(1)
serine β-lactamases. In addition to TP and TG domains that are piuA pirA 16(32) 0.25(≥8) 64 4(1)
commonly found in bifunctional PBPs, Pae PBP1a contains an piuA pfeA 8(16) 0.125(≥4) 64 4(1)
OB (oligonucleotide/oligosaccharide binding)-fold domain pirA pfeA 0.5(1) ≤0.03(1) 16 4(1)
inserted in the TP domain between the first α-helix and the piuA pfeA pirA 16(32) 0.25(≥8) 64 4(1)
first β-strand, also seen in the A. baumannii PBP1a (Aba a
Data in parentheses = strain MIC/PAO1 MIC.
PBP1a) structure (Figure 4A).38 The overall Pae PBP1a
structure can be superimposed onto the Aba PBP1a structure Mueller Hinton Broth (MHB) as well as a modified broth
with an rmsd of 1.5 Å for 384 Cα atoms. depleted of iron to mimic the low iron in vivo environment to
In complex with 17, the electron density of the P. aeruginosa determine if the drug uptake mechanism varies based on
PBP1a active site unambiguously revealed a covalent acyl- availability of iron. A significant shift (>4×) in MIC for a
enzyme interaction (Figure S1, Supporting Information). The receptor deficient strain vs the parent strain PAO1 is indicative
phthalimide ring and the isobutyrate groups of 17 are well of the involvement of the deleted receptor(s) in drug uptake.
defined, and the electron density is continuous between the For example, under standard conditions (MHB), PiuA, FpvA,
active site Ser461 hydroxyl group and the connecting lactivicin and FptA single mutants all provided an 8-fold or greater shift
acyl carbon, with the acyl carbonyl oxygen lying in the oxyanion in MIC relative to PAO1 when treated with 17, as did a number
hole defined by the Ser461 and Thr698 main chain amides. The of double mutant strains. However, under low iron conditions,
carboxylate at the γ-lactone position in 17 is anchored by H- only the PiuA single mutant showed a modest MIC shift (4-
bonds with the side chains of Thr696 and Thr698, and a water- fold), with no relevant shift observed for the FpvA or FptA
mediated H-bond with the Gly735 backbone. Though the single mutants, suggesting an adaption in receptor expression
ethylene carboxylate resulting from lactone ring opening does and/or function has occurred with differing iron levels, which is
not show a clear interaction with PBP1a, the group does a known phenomenon.41 More robust MIC shifts, especially in
provide rigidity to the phthalimide ring, facilitating a π-stacking the low iron media, were observed for a number of the double
interaction with Tyr733 (Figure 4B). While a crystal structure mutant strains, suggesting that multiple receptors including
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Table 3. Isogenic Library of E. coli Expressing β-Lactamases

MIC (μg/mL)
BLA-expressed ampicillin cefepime 3 4 13 14 17
DH5α (parent strain) 2 0.03 0.125 0.125 0.25 1 0.03
pUCP26 (empty vector) 2 0.03 0.125 0.125 0.25 0.5 0.03
SHV-5 >64 4 0.5 >64 0.5 32 0.03
SHV-12 >64 0.5 0.25 >64 0.25 16 0.015
TEM-1 >64 0.125 0.03 0.03 0.25 1 0.06
TEM-24 >64 0.5 0.125 >64 0.5 4 0.06
CTX-M-15 >64 8 0.25 1 0.5 4 0.015
VIM-2 >64 0.06 0.015 0.03 0.25 0.5 0.015
NDM-1 >64 8 0.03 0.06 0.5 1 0.03
OXA-24/40 32 0.06 0.25 0.125 1 1 0.03
OXA-58 >64 0.03 0.125 0.125 0.25 1 NTa
KPC-3 64 0.125 0.06 0.125 0.5 1 0.03
a
NT: not tested.

PiuA, PirA, FpvA, FptA, and perhaps others are involved in the
uptake of 17. The PiuA and PirA receptors are known to
recognize catechol-containing siderophores, such as enter-
obactin, while FpvA and FptA are involved in the uptake of the
major P. aeruginosa siderophores pyoverdine and pyochelin,
respectively. In comparison to the previously reported results
for hydroxypyridone-containing β-lactams,20,40 compound 17
appears to utilize a broader set of siderophore receptors, which
could translate to a therapeutic advantage in vivo where
siderophore receptor expression and function is expected to
vary depending on the availability of iron. In addition, 17
appears to provide a relative potency advantage in the low iron
media, and the magnitude of the activity difference observed in
normal vs low-iron conditions for 17 (16−512-fold) is
significantly greater than that reported for the pyridone-
conjugated β-lactams. (0.5−32-fold).20,40
While these preliminary in vitro results with phthalimide-
conjugated lactivicin analogue 17 may appear promising, a
recent report demonstrates that the gold standard in vitro
potency (i.e., MIC) and frequency of resistance (FOR)
methodologies utilized ubiquitously in antibacterial drug
research fail to predict in vivo efficacy for the related
monobactam−sideromimic conjugate, 2 (MB-1) (Figure 1).42
The lack of efficacy appears to involve an adaptive response of
bacteria when exposed to 2, leading to modified expression and
utilization of siderophores and siderophore receptors, thus
reducing the effectiveness of 2 in murine P. aeruginosa infection
model studies. The authors describe modified in vitro
methodologies which correlate nicely with 2 in vivo outcomes,
and it remains to be determined if the modified siderophore
receptor repertoire of phthalimide 17 conveys any advantage
over 2 in these assays.
Profiling β-Lactamase Susceptibility. Given that lactivi-
cins inactivate PBPs in a fashion analogous to β-lactams, it is
not surprising that they have been reported to be ineffective in
killing bacteria harboring certain BLA enzymes.23,43 Therefore,
to assess the relative BLA susceptibility for new lactivicin
analogues, an isogenic panel of E. coli strains was assembled
wherein each strain was engineered to express a single, specific,
commonly occurring BLA.40 While this panel was not
developed to match wild-type expression levels of BLAs or
assess the impact of BLAs working in concert, evaluation of
whole cell activity (i.e., MICs) against this panel as compared to
the parent E. coli strain harboring the empty vector (pUCP26)
provides a snapshot of the relative risk of BLA susceptibility of
3851
any β-lactam or related drug. Table 3 reports the data generated
for 3, 4, cefepime, ampicillin, and lactivicin analogues 13, 14,
and 17. Results are reported as MICs, and susceptibility to a
particular BLA can be inferred from a >4-fold MIC shift of the
corresponding mutant E. coli strain relative to the empty vector
control strain. As expected, the positive control drug, ampicillin,
was hydrolyzed and thus inactivated by the full range of BLAs,
and the more robust cephalosporin control, cefepime, displayed
susceptibility to SHV-5, CTX-M-15, and NDM-1 while
resisting hydrolysis by the other BLAs in the panel. As
previously reported, the monocarbam 3 exhibited significant
stability toward inactivation by a broad range of BLAs with only
borderline susceptibility to SHV-5.40 In general, the lactivicin
analogues also provide broad stability to BLAs similarly to 3,
including the strain expressing the New Delhi metallo-β-
lactamase (NDM-1). The original Takeda compound 13
lacking a sideromimic moiety exhibited good stability across
the entire panel, with only the MIC vs the OXA-24/40
expressing strain being 4-fold higher than the empty vector
control strain. The phthalimide-conjugated lactivicin 17 also
performed well, consistently inhibiting the BLA mutant strains
at MICs within 2-fold of the empty vector control including the
OXA-24/40 BLA-expressed E.coli strain. In contrast, both 4 and
the related lactivicin derivative 14 displayed liabilities, notably
with respect to SHV-5 and SHV-12, and to a lesser extent,
CTX-M-15 and TEM-24. Given the significantly different core
structure of 14 compared to 4, the similar BLA liabilities may
reside with the common placement of the dihydroxypyridone
moiety shared by these compounds. When considered
alongside the original Takeda lead 13, the dramatic increase
in susceptibility of 14 to SHV-5 and SHV-12 further suggests
that this placement of the sideromimic enhances susceptibility
to some BLAs and may potentially limit utility against
Enterobacteriaceae carrying these common extended spectrum
β-lactamases (ESBLs). By installing the known isobutryic acid
C4 group found in 3, 13, and aztreonam, and by modifying the
sideromimic point of attachment, compound 17 demonstrates a
significant advantage over 4 and 14 with regard to BLA
susceptibility in this panel. In addition, the 64-fold improve-
ment in potency for 17 vs 14 when tested against the K.
pneumoniae strain KP-3700 may provide additional evidence for
the BLA susceptibility discussion above as this strain is known
to express SHV-5 (Table 1). Bonomo et al. recently published a
crystal structure of a preacylation complex of the BLA inhibitor
sulbactam bound to a S70C variant of the SHV-1 enzyme.44
dx.doi.org/10.1021/jm500219c | J. Med. Chem. 2014, 57, 3845−3855
Journal of Medicinal Chemistry
Modeling of the ring-closed analogues 14 and 17 in this crystal
structure lends further support to the hypothesis that
sideromimic placement could impact BLA susceptibility, as
14 can be accommodated in the active site with the
sideromimic moiety pointing toward solvent, while preserving
key interactions such as proximity to nucleophile and
positioning in the oxyanion hole. However, the phthalimide
group of compound 17 appears to clash with the protein due to
the limited space available in this region, while maintaining
other key interactions and thereby leading to poor binding
(Figure 5). While a more definitive BLA susceptibility
Figure 5. Proposed models of compound 14 and 17 in the active site
of crystal structure of S70C SHV-1 β-lactamase. The S70C mutation
was designed to affect the reactivity of the catalytic residue to allow for
capture of the preacylation complex. (A) Model of S70C SHV-1 β-
lactamase with compound 14. The volume occupied by the ring-closed
form of the analogue is shown as a blue wire surface and the active site
of SHV-1 as an indigo solid surface. (B) Model of compound 17. The
volume occupied by the ring-closed form of the analogue is shown as a
pink wire surface and the active site of SHV-1 as an indigo solid
surface. The arrow points to a region in the active site where there is
significant steric clash with compound 17.
assessment would require evaluation against an expanded
panel of clinical isolates known to express specific BLAs, these
initial results may provide useful guidance for future drug
optimization efforts.
■
CONCLUSION
We have successfully applied the sideromimic-conjugation
strategy to lactivicins, providing new analogues with enhanced
in vitro Gram-negative activity. Analogue design was signifi-
cantly aided by learnings derived from structures of inhibitors
3852
Article
bound to Pae PBP3 and Pae PBP1a. In the course of this work,
we identified compound 17, a novel phthalimide-conjugated
lactivicin analogue. Efforts to understand the Ton-B-dependent
siderophore receptors involved in the uptake of compound 17
suggest that it may utilize a broader set of receptors than related
hydroxypyridone-conjugated β-lactams, potentially providing a
unique advantage in uptake and Gram-negative effectiveness.
Further profiling of 17 for susceptibility against BLAs indicates
the potential for generally low susceptibility, and evaluation vs
PBPs suggests the potential for a broader inhibitory profile
relative to monobactams. Overall, the results demonstrate that
the lactivicin−sideromimic conjugate class merits additional
research in the quest to find new effective antibacterial agents.
■ EXPERIMENTAL SECTION
Cloning, Expression, and Purification of Pae PBP3 and
PBP1a. Pae PBP3 was prepared as discussed previously.36 PBP1a
(residues 36−822) from PAO1 genomic DNA was cloned into the
pET28a vector (Novagen) with an N-terminal His6 fusion tag. Protein
was expressed in E. coli BL21 (Gold) cells grown in autoinduction
media (Novagen) overnight at 25 °C. Cell pellets were lysed in 5
volumes (w/v) of B-PER protein extraction reagent (Thermo
Scientific Pierce) supplemented with 0.4 M NaCl, 10 mM MgCl2,
Complete EDTA-free protease inhibitor tablets (Roche), and
Benzonase (Novagen). The lysate was clarified by centrifuging at 4
°C for 1 h in a Sorvall SS-34 rotor at 30 000g and loaded onto two
tandem 5 mL HisTrap FF crude columns (GE Healthcare)
equilibrated in buffer A (25 mM TrisHCl, 0.4 M NaCl, pH 8.0)
with 20 mM imidazole pH 8.0 (Emerald Bio) added. After the
columns were washed with 20 column volumes of buffer, PBP1a was
eluted with a linear gradient to 250 mM imidazole over 20 column
volumes. The eluted peak was concentrated and loaded onto a HiLoad
Superdex 200 16/60 gel filtration column (GE Healthcare)
equilibrated in buffer A. The peak corresponding to monomeric
PBP1a (data not shown) was pooled and concentrated to 20 mg/mL
using an Amicon Ultra 30 kDa MWCO centrifugal concentrator.
Aliquots were flash-frozen in liquid nitrogen and stored at −80 °C.
PBP1a was digested in a series of 84 μL reactions at a concentration of
2.67 mg/mL in 100 mM Tris-HCl, 100 mM NaCl, 10 mM CaCl2, and
1.1 μg of TPCK trypsin (Thermo Scientific Pierce). After 2 h at 4 °C,
the digestions were stopped by adding PMSF to 10 mM. Ten
digestion reactions were pooled and loaded onto a Superdex 200 HR
10/30 column equilibrated in buffer A. Fractions with digested PBP1a
were pooled, concentrated to 14 mg/mL, flash-frozen in liquid
nitrogen, and stored at −80 °C.
Crystallization and Data Collection of Pae PBP3 and PBP1a.
Crystals of apo-PBP3 were obtained with a reservoir solution
containing 30% PEG 4000, 0.2 M MgCl2, and 0.1 M Tris pH 8.5.
The compound was soaked into the crystals. Soaks were performed
with 1 mM compound in reservoir solution for 1 day. Crystals were
cryoprotected by dragging the crystals through MiTeGen’s LV
CryoOil (MiTeGen, LLC) and flash frozen in liquid nitrogen.
PBP1a was crystallized by sitting drop vapor diffusion at room
temperature using equal volumes of 10 mg/mL protein and well
solution. Crystallization conditions were screened using Crystal Screen
HT (Hampton Research). Crystals grew from condition 11 (0.1 M
sodium citrate tribasic dihydrate pH 5.6, 1.0 M ammonium phosphate
monobasic) and reached their full size (∼100 μm) in 2−3 weeks.
Crystals were soaked for 4 h in a solution containing 0.1 M sodium
citrate tribasic dihydrate pH 5.6, 1.2 M ammonium phosphate
monobasic, and 2 mM compound 17. The soaked crystals were
cryoprotected in 0.07 M sodium citrate tribasic dihydrate pH 5.6, 0.7
M ammonium phosphate monobasic, and 35% glycerol and flash-
frozen in liquid nitrogen.
Structure Determination. Data were processed using the
HKL2000 software suite.45 The structures of PBP3− and PBP1a−
inhibitor complexes were solved by molecular replacement methods
with the CCP4 version of PHASER46 using Acinetobacter baumannii
dx.doi.org/10.1021/jm500219c | J. Med. Chem. 2014, 57, 3845−3855
Journal of Medicinal Chemistry
PBP1a (PDB code: 3UDF) and Pae PBP3 (PDB code: 3PBN) as a
search model. After molecular replacement, maximum likelihood-based
refinement of the atomic position and temperature factors were
performed with autoBUSTER47 and the atomic model was built with
the program COOT.48 The stereochemical quality of the final model
was assessed with PROCHECK.49 Crystallographic statistics for the
final models are shown in Table S1, Supporting Information. Figures
were prepared with PYMOL (www.pymol.org).
Reactivity Modeling. Density functional theory (DFT) calcu-
lations, full geometry optimizations, and frequency analyses were
carried out with B3LYP hybrid functional and triple-ζ 6-311+G(d,p)
basis set using the Gaussian 09 package.50 A scaling factor of 0.9877
was used to correct zero-point vibrational energies.51 Implicit solvation
using SMD polarizable continuum model of Cramer and Thrular was
utilized.52 Transition state optimizations were conducted for additions
of a simple model nucleophile (CH3O−), as a computational surrogate
for the catalytic serine residue, to the electrophilic carbonyl. The
nature of stationary points was checked by means of frequency
calculations, and transition states were further verified by IRC
calculations.53 The activation barrier (ΔG⧧) determined at room
temperature (298.15 K) was used for ranking relative reactivity.
MICs, PBP IC50s, PAO1 Isogenic Siderophore Receptor
Mutant Panel, and E. coli Isogenic BLA Panel. The minimum
inhibitory concentration (MIC) values were determined using the
broth microdilution protocol according to the methods of the Clinical
and Laboratory Standards Institute (CLSI).54 The experimental
methods utilized to generate the P. aeruginosa siderophore receptor
KO data as well as the E. coli isogenic BLA panel have been described
in detail,40 as has the method utilized to generate PBP IC50 values.20
Chemistry. Detailed synthetic procedures and spectral character-
ization for all compounds are provided in the Supporting Information.
The methods of purification and purity determination of compounds
13, 14, 15R/S, 16R/S, and 17 are as follows:
Method A. Phenomenex Max-RP, C18, 150 × 21.2 mm, 5 μm;
mobile phase A: 0.1% formic acid in water (v/v); mobile phase B:
0.1% formic acid in methanol (v/v); gradient: 5% B for 1.50 min, 5%
to 100% B over 8.5 min 100% B for 1 min; flow rate: 28 mL/minute.
Temperature: not controlled; detection: DAD 210−360 nm; MS (+)
range 150−700 m/z; injection volume: 10 μL.
Method B. Phenomenex Gemini-NX, 4.6 mm × 50 mm, C18, 3 μm,
110A; mobile phase A: 0.1% formic acid in water (v/v); mobile phase
B: 0.1% formic acid in acetonitrile (v/v); gradient: 0% to 100% B over
4.1 min, 100% B 0.4 min; flow rate: 1.5 mL/min. Temperature: 60 °C.;
detection: 200−450 nm; MS (+) range 100−1200 m/z; injection
volume: 5 μL; instrument: column oven from Agilent Technologies,
Wilmington, DE; autosampler and MS detector from Waters
Corporation, Milford, MA; ELS detector from Varian medical devices,
Palo Alto, CA.
Method C. Waters Acqity HSS T3, C18, 2.1 × 50 mm, 1.7 μm;
mobile phase A: 0.1% formic acid in water (v/v); mobile phase B:
0.1% formic acid in acetonitrile (v/v); gradient: 5% B over 0.1 min, 5%
to 95% B over 2.5 min, 95% B 0.35 min; flow rate: 1.25 mL/min.
Temperature: 60 °C.; detection: 200−450 nm; MS (+) range 100−
2000 m/z; injection volume: 5 μL; instrument: Waters Acquity UPLC.
Compound 13. Purification method A, purity determination
method B. The compound was found to have a purity > 95%.
Compound 14. Crude product was triturated with diethyl ether
followed by 4:1 dichloromethane: diethyl ether. Purity determination
method B. The compound was found to have a purity > 95%.
Compound 15R. Purification method A, purity determination
method B. The compound was found to have a purity = 87%.
Compound 15S. Purification method A, purity determination
method B. The compound was found to have a purity = 94%.
Compound 16R. Purification method A, purity determination
method B. The compound was found to have a purity > 95%.
Compound 16S. Purification method A, purity determination
method B. The compound was found to have a purity = 84%.
Compound 17. Purification method A, purity determination
method C. The compound was found to have a purity > 95%.
3853
■
Article
ASSOCIATED CONTENT
*
S Supporting Information
The experimental details regarding the synthesis of PBP
inhibitors and reactivity modeling. This material is available
free of charge via the Internet at http://pubs.acs.org.
Accession Codes
The atomic coordinates and structure factors have been
deposited in the Protein Data Bank with entry codes 4OOL,
4OOM, and 4OON.
■ AUTHOR INFORMATION
Corresponding Author
*Phone: (860)-686-1788. E-mail: seungil.han@pfizer.com.
Author Contributions
#
J.S, M.F.B., and S.H. contributed equally.
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
We thank Mark Plummer, Mark Mitton-Fry, John Mueller, Paul
Miller, and Mark Noe for helpful discussions. Our appreciation
is also extended to Jinshan (Mike) Chen for managing external
chemistry resources. Finally, we thank Juergen Bulitta for
providing helpful comments regarding this manuscript.
■
ABBREVIATIONS USED
MIC, minimum inhibitory concentration; PBP, penicillin
binding protein; MHB, Mueller−Hinton broth; Pae, Pseudo-
monas aeruginosa; Kpn, Klebsiella pneumoniae; Eco, Escherichia
coli; Aba, Acinetobacter baumannii; BLA, β-lactamase; FOR,
frequency of resistance; TG, transglycosylase; TP, trans-
peptidase
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